Symposium Organizers
Mei Wei University of Connecticut
Jordan Green The Johns Hopkins University
Xinqiao Jia University of Delaware
James Olson Teleflex Medical
KK3: Poster Session: Scaffold Design and Cell Biology
Session Chairs
Tuesday AM, November 29, 2011
Exhibition Hall C (Hynes)
KK1: Advanced Scaffold Design
Session Chairs
Monday PM, November 28, 2011
Room 102 (Hynes)
9:30 AM - **KK1.1
Poly(Lactide-co-glycolide)-Hydroxyapatite Composites: The Development of Osteoinductive Scaffolds for Bone Regenerative Engineering.
Cato Laurencin 1 2 3 , Emily Cushnie 1 , Meng Deng 1 2 , Bret Ulery 1 2
1 Institute for Regenerative Engineering, University of Connecticut Health Center, Farmington, Connecticut, United States, 2 Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut, United States, 3 Department of Chemical, Materials and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, United States
Show AbstractRegenerative Engineering has been defined as “the integration of tissue engineering with advanced materials science, stem cell science and developmental biology toward the regeneration of complex tissues, organs, or organ systems”. One goal of regenerative engineering is the design of materials capable of inducing associated cells toward highly specialized functions. For example, the interaction of cells with calcium phosphate surfaces has proven to be an important signaling modality in promoting osteogenic differentiation. A biodegradable polymer-ceramic composite system has been developed from poly(lactide-co-glycolide) and in situ synthesized hydroxyapatite based on the three-dimensional sintered microsphere matrix platform. We have systematically optimized scaffold physico-chemical, mechanical, and structural properties for bone tissue regeneration applications by varying several parameters such as solution pH, polymer:ceramic ratio, sintering time and sintering temperature. The bioactivity of composite scaffolds is attributed to their ability to deliver calcium ions to surrounding medium and allow for reprecipitation of calcium phosphate on the scaffold surface. Furthermore, the composite scaffolds have demonstrated increased loading capacity of osteoinductive growth factor (BMP-2) and a more sustained release profile due to a greater number of adsorption sites provided by the ionic calcium and phosphate groups as well as a larger matrix surface area. In vitro cell studies were performed to investigate the efficacy of this composite system to induce osteogenic differentiation of human adipose-derived stem cells. Cells cultured on the ceramic containing scaffolds exhibited significantly higher expression of osteoblastic markers and greater extracellular matrix mineralization than non-ceramic containing scaffolds, indicating the potential for the ceramic phase to promote osteogenic differentiation. In addition, loaded BMP-2 retained its bioactivity as a mitogen and osteoinductive agent during the differentiation of adipose-derived stem cells into mature osteoblasts. In vivo evaluation using a critical size defect model in New Zealand white rabbits demonstrated the ability of composite scaffolds to support cellular infiltration throughout the scaffold pore structure and vascularization of new tissue, as well as facilitate formation of newly mineralized bone tissue. The work described herein provides strong evidence for the potential of polymer-ceramic composite scaffolds to function as osteoinductive bone graft substitutes, and paves the way for future development of advanced tissue-inducing materials.
10:00 AM - KK1.2
Nanostructured Synthetic Hydrogels as Scaffolds for Cell and Gene Delivery.
Li Yan 1 2 , Chuan Yang 2 , Shaoqiong Liu 2 , Majad Khan 2 , James Hedrick 3 , Yi Yan Yang 2 , Pui Lai Rachel Ee 1
1 Pharmacy, National University of Singapore, Singapore Singapore, 2 , Institute of Bioengineering and Nanotechnology, Singapore Singapore, 3 , IBM Almaden Research Center, San Jose, California, United States
Show AbstractEffective delivery of DNA to modulate cell behavior in well-defined three dimensional scaffolds offers a superior approach in tissue engineering. In this study, we aimed to synthesize nanostructured hydrogels with tunable mechanical properties and injectability for cell and gene delivery. The hydrogels were formed via Michael addition chemistry using four-arm acrylate-terminated PEG and four-arm thiol-functionalized PEG. By using the same chemistry, nanosized micelles self-assembled from an amphiphilic diblock copolymer, vinyl sulfone-PEG-b-polycarbonate, were incorporated into the hydrogel networks at contents varying from 0 to 80%. The use of Michael addition chemistry allows for in situ hydrogel formation under physiological conditions. Analysis of the mechanical property of the nanostructured hydrogels revealed a correlation between the content of micelles and the storage modulus of the hydrogels. Morphology of hydrogels was observed using a field emission scanning electron microscope (FESEM). It was observed that the number and size of the pores in the hydrogel increased with increasing micelle content. MTT assays demonstrated an increase in cell viability as a function of the micelle content. Importantly, the gene expression level in hMSCs in the hydrogel with the optimized micelle content was significantly higher than that in the hydrogel without micelles. Therefore, incorporating the nanostructures into the hydrogel is a good strategy to control cellular behavior in 3-D through changes in mechanical properties and porosity of the microenvironment.
10:15 AM - KK1.3
Controlling Cell Function Using Engineered, Hyaluronic Acid-Based Hydrogel Matrices.
Xinqiao Jia 1
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States
Show AbstractWe have established various hyaluronic acid (HA)-based hydrogel particles (HGPs) and macroscopic gels suitable for the controlled release of growth factors (GFs) and 3D culture of cells of mesenchymal origin. HA HGPs with an average diameter of 10 µm (HGP10) and 0.9 µm (HGP 0.9) were synthesized by inverse emulsion polymerization techniques. To improve the biological functions of HA HGPs, perlecan domain I (PlnDI), a proteoglycan that binds various heparin binding GFs (HBGFs), was successfully conjugated to the HGPs through the core protein via a flexible PEG linker. The immobilized PlnDI can bind HBGFs and modulate in vitro release and function. Similar, sustained release of HBGFs was achieved using HA HGPs with covalently integrated heparin (HP). By varying the amount HP incorporated in HGPs, the release kinetics can be tuned. HA-based doubly crosslinked networks (DXNs) were synthesized via the covalent integration or physical entrapment HA HGPs in a HA-based secondary network. These hybrid matrices are hierarchical, consisting of densely crosslinked HGPs integrated in a loosely connected secondary matrix. Their mechanical properties and degradation kinetics are readily tuned by varying particle size, functional group density, intra- and interparticle crosslinking. Cells entrapped in the matrix proliferate readily and produce ECM. Cell-adhesive HA DXNs were fabricated by encapsulating HA HGPs decorated with gelatin (HA-gelHGP) or collagen-like peptide (HA-clpHGP) in a secondary HA matrix. Human mesenchymal stem cells (MSCs) adhered to the composite matrix through focal adhesion sites clustered on particle surface. The cell-adhesive composite matrices support MSC proliferation and induce their osteogenic differentiation in the absence of osteogenic factors. In summary, the modified HA-based hydrogel matrices are hierarchically structured, mechanically robust and enzymatically stable, capable of mediating cellular functions through the spatial and temporal presentation of defined biological cues.
10:30 AM - KK1.4
Designing Enzyme Triggered Injectable Hydrogels for 3D Cell Culture and Tissue Engineering Applications.
Jean-Baptiste Guilbaud 1 , Laura Szkolar 1 , Aline Miller 2 , Alberto Saiani 1
1 School of Materials, The University of Manchester, Manchester United Kingdom, 2 School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester United Kingdom
Show AbstractMolecular self-assembly has emerged as a powerful tool for the fabrication of molecular materials with a wide variety of properties. In recent years, considerable advances have been made in using simple oligopeptides as building blocks for the production of novel biomaterials due to their propensity to self assemble into ordered supramolecular structures. Of particular interest is being able to trigger the self assembly of these small molecules using an external stimulus (e.g.: enzyme, light, pH and/or ionic strength) for the reversible fabrication of hydrogels for cell culture and tissue engineering applications. Recently, we have reported the enzyme catalysed synthesis and gelation of ionic complementary oligopeptides from a non-gelling tetrapeptide precursor via reverse hydrolysis (J.-B. Guilbaud et al., Langmuir, 26(13), 11297-11303, 2010). We showed that for an initial tetra-peptide concentration C0 ≥ 100 mg mL-1 octa-peptides were the dominant product of the reverse hydrolysis reaction. These longer sequences self-assembled into β-sheet rich fibrils leading to the development of dense fibrillar networks and hydrogels.In the present work we investigated further the early stage kinetics of fibril formation and subsequent gelation of this enzymatic system, in particular the effects of tetrapeptide/enzyme ratio. Real-time small angle X-ray scattering and TEM showed that for all samples the same overall network topology is obtained and that the rate of self-assembly play a key role in determining the final properties of the materials. The kinetic and structural information obtained allowed the elucidation of the relationships between self-assembly behavior, local nanostructure and final physical properties of the materials. This information was then used to design of a simple protocol to create injectable hydrogels for cell culture and tissue engineering applications. Hydrogels (including cells: human dermal fibroblasts - HDF) were directly prepared in syringes and then injected into cell culture wells. Microscopy showed that cells survived the injection and were uniformly distributed within the hydrogels. Cell counting and live/dead staining showed proliferation of HDF with limited cell death over 7days.
10:45 AM - KK1.5
Morphology and Crystallinity Control of Novel Spider Silk-like Block Copolymer.
Wenwen Huang 1 , Sreevidhya Krishnaji 2 3 , David Kaplan 3 , Peggy Cebe 1
1 Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, United States, 2 Department of Chemistry, Tufts University, Medford, Massachusetts, United States, 3 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Show AbstractWe synthesized and characterized a new family of di-block copolymers based on the amino acid sequences of Nephila clavipes major ampulate dragline spider silk, which have a strong potential for applications in tissue regeneration and drug delivery. The co-polymers have the form HABn and HBAn (n=1-6), comprising an alanine-rich hydrophobic block, A, a glycine-rich hydrophilic block, B, and a histidine tag, H, and self–assemble to form periodic nanostructures. Using scanning electron microscopy we observed that HBA forms fibrillar structure in methanol solution, whereas HBA2, HBA3 and HBA6 formed 2um diameter hollow micelles in both water and methanol solution. Biomaterials in the form of fibrils and spherical microstructures with hollow cores may serve as templates either for tissue regeneration or as vehicles for drug delivery. To provide further insight into the relationships among peptide amino acid sequence, block length, and self assembled structural features, we assessed the secondary structure of water-cast spider silk-like block copolymer films by Fourier transform infrared spectroscopy (FTIR). The crystallinity was determined by Fourier self-deconvolution of amide I spectra and confirmed by wide angle X-ray diffraction (WAXD). Results indicate that we can control the self-assembled morphology and the crystallinity by varying the block length, and a minimum of 3 A-blocks are required to form beta sheet crystalline regions in water-cast spider silk block copolymers. We also developed a model to calculate the reversing heat capacity, Cp(T), and obtained excellent agreement between the theoretical value and the Cp(T) determined by temperature modulated differential scanning calorimetry. This method is generally applicable to any amino-acid based biomaterial, and thus can serve as a standard by which to assess the thermal properties and the crystallinity for other biologically inspired block copolymers. Aside from the fundamental perspective, we also anticipate that these results will provide a roadmap for the design and synthesis of precise materials having some of the same properties of spider silks, or other valuable rare material, using well controlled protein sequences.Support was provided from the National Science Foundation, Division of Chemical, Bioengineering, Environmental, and Transport Systems, through CBET-0828028 and the MRI Program under DMR-0520655 for thermal analysis instrumentation.
11:30 AM - **KK1.6
Biomaterials That Heal v. Self-Healing Biomaterials. Where is the Materials Science?
William Reichert 1 , Alice Brochu 1
1 Biomedical Engineering, Duke University, Durham, North Carolina, United States
Show AbstractThe goal of this talk is to introduce the emerging field of self-healing materials, and also to illustrate how one could utilize and modify self-healing approaches to develop new classes of biomaterials. A brief discussion of the in vivo mechanical loading and resultant failures experienced by biomedical implants is followed by presentation of the self-healing methods for combating mechanical failure. If conventional composite materials that retard failure may be considered zeroth generation self-healing materials, then taxonomically speaking, first generation self-healing materials describe approaches that ‘‘halt’’ and ‘‘fill’’ damage, whereas second generation self-healing materials strive to ‘‘fully restore’’ the prefailed material structure. In spite of limited commercial use to date, primarily because the technical details have not been suitably optimized, it is likely from a practical standpoint that first generation approaches will be the first to be employed commercially, whereas second generation approaches may take longer to implement. For self-healing biomaterials the optimization of technical considerations is further compounded by the additional constraints of toxicity and biocompatibility, necessitating inclusion of separate discussions of design criteria for self-healing biomaterials.
12:00 PM - KK1.7
Nanoscale Characterization of Electromechanical Properties from Natural and Assembled Collagenous Tissues.
Denise Denning 1 , Mohammad Abu-Rub 2 , Dimitrios Zeugolis 2 , Stefan Habelitz 3 , Abhay Pandit 2 , Andrzej Fertala 4 , Brian Rodriguez 1
1 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin Ireland, 2 Network of Excellence for Functional Biomaterials, National University of Ireland Galway, Galway Ireland, 3 Department of Preventative and Restorative Dental Sciences, University of California , San Francisco, California, United States, 4 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
Show AbstractMany biopolymers, including collagen, are piezoelectric (i.e., generate charge under mechanical strain and deform under applied electric field). Piezoelectricity in collagen is directly related to the dipole (arising from amine and carboxyl termini) molecular orientation of the collagen monomer building block. It has been suggested that electromechanical coupling in piezoelectric biopolymers could play a role in bone remodelling. In order determine the possible functional role of piezoelectricity in biomaterials we must study both the piezoelectric properties and molecular order of biological tissues. Piezoresponse force microscopy (PFM) is a variant of atomic force microscopy (AFM) which measures bias-induced surface deformations at the nanoscale. This technique is widely used to study ferroelectrics materials and has recently been employed to investigate electromechanical coupling in biosystems. In this study, PFM has been used to determine the existing architecture and molecule orientation in porcine eye tissues, rat tail tendon, and isoelectrically focused collagen. Piezoelectricity was evident in both the natural collagenous tissues and the assembled isoelectrically focused collagen hydrogel. The molecular orientation of individual collagen fibrils within the tissues was also obtained from the PFM phase images, which has allowed the polar architecture of natural and assembled tissues to be visualized with some evidence of systematic organization observed. Studying piezoelectricity and the effect of the polar orientation in tissues could be useful in improving our understanding of cell signalling and mechanotransduction.This research was supported by SFI (grant no. 10/RFP/MTR2855).
12:15 PM - KK1.8
Human Mesenchymal Stem Cells (hMSCs) Interaction with Nanoporous Titania Substrates.
Dattatri Nagesha 1 , James Maniscalco 1 , Daniela Guarnieri 2 , Valentina Belli 2 , Paolo Netti 2 , Srinivas Sridhar 1
1 Physics, Northeastern University, Boston, Massachusetts, United States, 2 Interdisciplinary Research Centre on Biomaterials , University of Naples, Naples Italy
Show AbstractNanoporous titania (TiO2) are increasingly being investigated for biomedical applications ranging from coatings on cardiovascular stents to dental implants and artificial metallic joints. An elegant and established method to fabricate nanoporous TiO2 is through the controlled anodization of Ti in an electrolyte containing fluoride or chloride ions. The dimension of TiO2, pore size and thickness can be precisely controlled by varying the experimental conditions. The pore lengths in the order of several microns can be uniformly fabricated using chloride ions in the electrolyte. The nanoporous feature of these TiO2 surface allows for their use as drug reservoir for localized drug delivery. In addition, the nano-topographical features have remarkable influence on the proliferation and adhesion of cells. In this work, we studied the interaction of human mesenchymal stem cells (hMSCs) with nanoporous TiO2 surface of different sizes. The cell morphology and cytoskeletal organization was followed by observing the stained actin microfilaments. When compared to bare TiO2 surface, nanoporous TiO2 showed an enhanced cell adhesion and a striking difference in cell stretching and polarization. Furthermore, it was observed that nanofeatures on TiO2 surface also influence the migration paths of hMSCs suggesting the presence of topological contact guidance skin. This work was supported by IGERT Nanomedicine Science and Technology Program (NSF 0504331) and Northeastern University.
12:30 PM - KK1.9
Saccharide-Peptide Hydrogels as Biocompatible Scaffolds for Tissue Engineering.
Zhibin Guan 1 , Kanika Chawla 1 , Sophia Liao 1 , Ting-Bin Yu 1
1 , University of California, Irvine, California, United States
Show AbstractA new class of functional saccharide–peptide copolymer–based hydrogels was de novo synthesized and investigated as synthetic extracellular matrices for regenerative medicine applications. The polymer was composed entirely of natural building blocks, namely galactaric acid and lysine on backbone, with various cross-linking moieties grafted onto the side chain as a handle for mild hydrogelation. The resulting hydrogels are degradable under physiological conditions and exhibit minimal cytotoxicity on a wide range of cell lines including fibroblast, PC–12, smooth muscle cells (SMCs), and stem cells. The de novo synthesis of the system offers great versatility in tuning various properties including the chemical, mechanical, and other functional properties. The hydrogels are applicable for both 2-D cell culture and 3-D cell encapsulation. It was exciting to observe that simple change in hydrogel physical properties could induce direct phenotypic change in cell adhesion and proliferation. For example, in 2-D cell culture, depending on the substrate mechanical modulus, cell morphology changed and proliferation rate differed by an order of magnitude for different cell lines. These data suggest our saccharide–peptide hydrogels as promising synthetic extra–cellular matrices for cell culture and tissue regeneration. In this presentation, we will discuss about results of application of our saccharide-peptide hydrogels as scaffolds for various tissue engineering applications including the control of the differentiation of stem cells.
KK2/V3: Joint Session: Cell-Biomaterial Interactions
Session Chairs
Andreas Lendlein
Kaiming Ye
Monday PM, November 28, 2011
Room 312 (Hynes)
2:30 PM - **KK2.1/V3.1
Micro- and Nanoscale Technologies for Stem Cell Bioengineering and Tissue Regeneration.
Ali Khademhosseini 1
1 Harvard-MIT HST, Harvard Medical School, Cambridge, Massachusetts, United States
Show AbstractMicro- and nanoscale technologies are emerging as powerful tools for mimicking cell microenevironment by controlling the interaction between cells and their surroundings and have been applied to biological studies, tissue engineering, and cell-based screening. In addition, hydrogel biomaterials have been increasingly used in various tissue engineering applications since they provide cells with a hydrated 3D microenvironment that mimics the native extracellular matrix. In this talk, I will discuss this emerging research area with a focus on our laboratory’s work on the application of microfabrication techniques to the manipulation of hydrogel biomaterials for directing stem cell differentiation and engineering 3D tissue with controlled microarchitecture. In addition, I will describe the fabrication and the use of microscale hydrogels for tissue engineering by using a ‘bottom-up’ and a ‘top-down’ approach. Top-down approaches for fabricating complex engineered tissues involve the use of miniaturization techniques to control cell-cell interactions or to recreate biomimetic microvascular networks within mesoscale hydrogels. Our group has also pioneered bottom-up approaches to generate tissues by the assembly of shape-controlled cell-laden microgels (i.e. tissue building blocks), that resemble functional tissue units. Using this approach, we have demonstrated the use of the directed assembly of cell-laden microgels to create complex tissue structures consisting of multiple cellular components.
3:00 PM - KK2.2/V3.2
Tissue and Disease-Specific Adhesive Materials.
Natalie Artzi 1 2 , Maria Carcole 1 3 , Nuria Oliva 1 , Sagi Shitreet 1 , Elazer Edelman 1 4
1 Health Sciences and Technology, MIT, Cambridge, Massachusetts, United States, 2 Anesthesiology , Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States, 3 Chemistry, Institut Quiimic de Sarria`, Universitat Ramon Llull, Barcelona Spain, 4 Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
Show AbstractTo avoid stress concentrations and tissue damage, nature often employs physical property gradients at interfaces between tissues with different functionality through subtle chemical variations. We hypothesized that within the three regions of the small intestine, variation in chemical composition serves as a mean to support the diverse functionality of these regions. Disease type and state would further alter tissue properties and alter the interaction of such tissue surfaces with implanted materials. Existence of chemical gradients takes increasing importance when designing adhesive materials that chemically interact with tissue surfaces to prevent leakage after surgeries by sealing and by providing mechanical support to the wounded tissue. Leakage of gut content is a frequent surgical complication that results in high morbidity and mortality. We designed tissue responsive adhesive materials by matching material and tissue properties. We used aldehyde-amine chemistry to determine if alterations in tissue surfaces (amine density in duodenum, jejunum and ileum) could affect interactions with materials of varied (aldehyde content and density) composition. A two component material based on dextran aldehyde and dendrimer amine provides cohesive gel through aldehyde-amine crosslinking, and adhesive interface by dextran aldehyde selective reaction with tissue amines. At the same time, material amines absorb excess non-reactive aldehydes and thus prevent toxicity. Adhesive strength varied dramatically with material chemistry and is tissue-specific. Tissue:material interfacial region changed when the same material composition was applied to the three small intestinal regions. These data support the notion that both tissue and material functional groups influence aldehyde-mediated adhesive interactions, providing a functional basis for tissue-specific sealant design. As disease state often alters tissue properties, we have analyzed the effect of colon inflammation and colon cancer on the interaction with our adhesive materials. The severity of the disease as well as type of disease have differently affected tissue properties, and in our case amine density, modifying both adhesion and cohesion strength of the materials employed. Amine groups are ubiquitous on tissue surfaces and their density varies from tissue to tissue and in diseased tissues to a different extent. By providing chemical key in the form of tethered aldehydes for specific tissue locks (tissue amines), we modulated adhesion while maintaining biocompatibility.
3:15 PM - KK2.3/V3.3
Self-Assembled Peptide-Based Hydrogels as Scaffolds for In Vitro Cell Culture.
Ayeesha Mujeeb 1 3 , Jie Gao 1 3 , Kate Meade 1 , Julie Gough 1 , Catherine Merry 1 , Aline Miller 2 3 , Alberto Saiani 1
1 School of Materials, University of Manchester, Manchester United Kingdom, 3 Manchester Interdisciplinary Biocentre, University of Manchester, Manchester United Kingdom, 2 School of Chemical Egineering and Analytical Sciences, University of Manchester, Manchester United Kingdom
Show AbstractNature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. One such approach is self-assembly, which represents a simple and efficient route to the construction of large, complex structures. De novo designed peptides are in particular attracting considerable interest due to their structural simplicity, diverse functionality and their ability to self-assemble into a variety of structures and form hydrogels.We have recently investigated the self-assembling and gelation properties of a series of ion-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptides: FEFEFKFK (F: phenylalanine, E: glutamic acid, K: lysine) which has been shown to self-assemble in solution and form β-sheet rich nanofibres which, above a critical gelation concentration (CGC), entangle to form self-supporting hydrogels. (A. Saiani et al. Soft Matter, 5, 193, 2009). Here we show that this system can be used for the in-vitro culture of a variety of cells with different requirement: chondrocytes, fibroblast and stem cells showing the flexibility and versatility of the materials developed.Chondrocytes were cultures over 21 days in 3D conditions. Light microscopy and ESEM images revealed that the cells adopted a rounded morphology. Live-dead staining and collagen antibody-staining results show the presence of living chondrocytes and the production of mainly collagen II. The cell proliferation results demonstrated the scaffolds to be cytocompatible with the cells showing varying metabolic activity.Pluripotent embryonic stem (ES) cells have the unique capacity to form any adult cell phenotype. However, effective differentiation into organised tissue constructs is limited by current two dimensional (2D) culture techniques. In this work mouse ES cells containing an Oct4-GFP reporter construct were seeded within the octapeptide gels in 3D conditions. The cell grew into rounded aggregates and could be retrieved and directly passaged into fresh octapeptide gels and after a fifteen day culture period, consisting of three passages. Levels of Oct4-positive cells remained elevated through the passaging showing that the cells remained pluripotent.Fibroblast were culture on non-functionalised and “RGD”-functionalised hydrogel. Live/dead stained and cell counting showed an extensive proliferation of the cells in the functionalised hydrogel. Collagen and F-actin stained show the extensive production of matrix by the cells.By exploiting the simple self-assembly of short peptides we have developed a platform that allows to design “simple” 3D hydrogels for the culture of a variety of cell. The physical, in particular mechanical, properties and functionality of the hydrogels can be tailored by design to suit the cell cultured.
3:30 PM - KK2.4/V3.4
Biological Applications of Nanoparticle Modified Polyelctrolye Capsules.
Susana Carregal-Romero 1 , Markus Ochs 1 , Pilar Rivera Gil 1 , Wolfgang Parak 1
1 FB Physics, Biophotonics, University of Marburg, Marburg Germany
Show AbstractThe fabrication and engineering of three-dimensional delivery vehicles is one of the issues of the bio-nanotechnology field attracting increasing interest for a variety of different applications, ranging from drug delivery systems and targeted gene therapy to biosensor devices. Engineered polyelectrolyte multilayer (PEM) microcapsules offer a unique opportunity to combine surface multifunctionality with design flexibility for the delivery of encapsulated macromolecules into designated compartments and cells. Though several methods exist for delivering molecules inside cells, still the number of carrier systems which allow for controlled release of the molecules is limited. One major requirement is that the molecules which are going to be delivered are released only inside the target cells but not in the extracellular space. For this purpose these molecules can be for example encapsulated in a shell of a material which is enzimatically degraded inside the cell. Alternatively, the molecules to be delivered can also be encapsulated in a shell of a material which is responsive to external triggers. In this context, plasmon assisted photothermal processes provide a powerful tool for controlled release. Gold nanoparticles can be easily incorporated in PEM microcapsules. Due to their plasmonic resonance, metallic nanoparticles are able to absorb light and dissipate this energy into heat. The hereby created heat locally melts or ruptures the wall of the PEM microcapsules and the encapsulated molecules can be ralease. Release of different macromolecules conjugated with organic fluorophores from so-modified capsules has been studied via fluorescent microscopy and electron microscopy imaging.
3:45 PM - KK2/V3:CelMat
BREAK
4:15 PM - KK2.5/V3.5
Development of a Multifunctional Synthetic PEG-Based Hydrogel with Independent Control of Mechanical and Bioadhesive Properties to Probe Stem Cell Behavior.
Anirudha Singh 1 , Jianan Zhan 1 , Jennifer Elisseeff 1 2 3
1 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 2 Jules Stein Chair in Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States, 3 Department of Orthopedic surgery, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractMesenchymal stem cells (MSCs) hold great promise in tissue engineering because of their proliferative capacity and ability to differentiate into several phenotypes, and ultimately built new tissues. However, to enable translation of stem cell therapies, there is a significant need to generate materials that can be used to study fundamentals in biology and modulate or enhance differentiation. Poly(ethylene glycol) (PEG)-based hydrogels are frequently utilized to encapsulate cells in a 3D environment. Unfortunately, PEG based biomaterials lack functionality to incorporate multiple chemical and biological moieties as signals for stem cells to probe behavior and guide development. Conventional methods to modify PEG hydrogels include copolymerization and chain extension, which leads to changes in mechanical properties of the material. We therefore developed an advanced multifunctional hydrogel by combining PEG- and α-Cyclodextrin (α-CD). α-CD is a six membered oligosaccharide ring, which threads onto the PEG chains.[1] The hydroxyl groups of α-CD provide necessary functional sites to introduce various moieties, such as integrin-binding peptide RGD by chemical modifications. This simple strategy provides a unique ability to independently control mechanical and bioadhesive properties of the hydrogels to probe stem cell behavior. MSCs were seeded onto PEG-CD hydrogels with varying stiffness and adhesive peptide concentrations. We found that cell spreading and morphology are dependent on both stiffness[2] and concentrations of adhesive peptide. On the stiffer substrate, MSCs adopted elongated morphology at all the concentrations of RGD. Interestingly, on the softer substrate MSCs adopted elongated morphology at higher concentrations of RGD. In summary, we developed an interesting and simple biomaterial platform for probing stem cell behavior, such as proliferation and lineage specific differentiation by independently controlling the mechanical and bioadhesive properties of the material.References:1.A. Harada, J. Li, M. Kamachi, Nature, 1992, 356, 325-327.2.A.J. Engler, S. Sen, H.L. Sweeny, D.E. Discher, Cell, 2006, 126(4), 677-689.Acknowledgements:Maryland Stem Cell (TEDCO) fellowship, Dr. Mooney O. Uy, Cindy Berlinicke, JHE lab members.
4:30 PM - KK2.6/V3.6
Organic Conducting Polymers: A Multifunctional Stimulus Platform for Mammalian Cells.
Gordon Wallace 1
1 Intelligent Polymer Research Institute, University of Wollongong, Wollongong, New South Wales, Australia
Show AbstractOrganic conductors have emerged as being excellent candidates as platforms for mammalian cell culturing (1-4). Our recent studies demonstrate that this compatibility is dependent on the inherent nanostructure of ICP surfaces (5). Furthermore, the introduction of nanostructure via templates has been shown to enhance the ability of ICPs to provide electrically stimulated release of bioactive molecules at the interface (6).Organic conducting polymers provide a platform capable of delivering direct electrical stimulation to excitable cells such as nerve or muscle. In addition, they are capable of responding to electrical stimulation to provide triggered drug or growth factor release. They can also be configured to provide electromechanical effects, enabling the modulus to be tuned in response to electrical stimulation, and/or movement/force to be generated in response to electrochemical stimulation.This multifunctional behaviour is complex, being determined by the initial polymer composition, the nature of the electrochemical structures used and the operational environment (7-10). Understanding and controlling the different dimensions of this multifunctional behaviour is critical to the development of efficient platforms for mammalian cell culturing and then to the use of these materials in implantable medical bionic devices such as in electrodes for the bionic ear implant or as conduits for nerve or muscle regeneration.References1.Wallace, G.G., Moulton, S.E., Clark, G.M. Science 2009, 324 (5924), 185-186.2.Wallace, G.G., Moulton, S.E. Chemistry in Australia 2009, 76 (5), 3-8.3.Quigley, A.F., Razal, J.M., Thompson, B.C., Moulton, S.E., Kita, M., Kennedy, E.L., Clark, G.M., Wallace, G.G., Kapsa R.M.I. Advanced Materials 2009, 21 (43), 4393-4397.4.Razal, J.M., Kita, M., Quigley, A.F., Kennedy, E., Moulton, S.E., Kapsa, R.M.I., Clark, G.M., Wallace, G.G. Advanced Functional Materials 2009, 19 (21), 3381-3388.5.Gelmi, A., Higgins, M.J., Wallace, G.G. Biomaterials 2010, 31 (8), 1974-1983.6.Thompson, B.C., Chen, J., Moulton, S.E., Wallace, G.G. Nanoscale 2010, 2 (4), 499-501.7.Thompson, B.C., Moulton, S.E., Richardson, R.T., Wallace, G.G. Biomaterials 2011, 32, 3822-3831.8.Foroughi, J., Spinks, G.M., Wallace, G.G. Sensors and Actuators B: Chemical 2011, 155, 278-284.9.Halldorsson, J.A., Wu, Y., Brown, H.R., Spinks, G.M., Wallace, G.G. Thin Solid Films (In Press).10.Wagner, K., Byrne, R., Zanoni, M., Gambhir, S., Dennany, L., Breukers, R., Higgins, M., Wagner, P., Diamond, D., Wallace, G.G., Officer, D.L. Journal of the American Chemical Society 2011, 133 (14), 5453-5462.
4:45 PM - KK2.7/V3.7
Photocrosslinked Co-Networks with Multifunctional Properties Based on Glycidylmethacrylated Gelatin and Poly(Ethylene Glycol) Methacrylates.
Benjamin Pierce 1 , Axel Neffe 1 , Friedrich Jung 1 , Andreas Lendlein 1
1 Center for Biomaterial Development and Berlin-Brandenburg Center for Regenerative Therapies, Institute of Polymer Research, Helmholtz-Zentrum Geesthacht, Teltow Germany
Show AbstractBiopolymer-based systems with adjustable macroscopic properties, which can be varied in a wide range by only small changes in their chemical composition are promising candidate materials for biomaterial-induced autoregeneration. Such systems may be formed by functionalizing biopolymers with synthetic components to enable hybrid materials that can perform a variety of functions, such as cell attachment, which is enabled by the biopolymer component, segmented hydrolytic degradation kinetics, which is due to the presence of degradable biopolymers and non-hydrolyzable components, and dynamic load support, which is due to the synergistic properties of both components. To achieve such an array of functions, glycidylmethacrylated gelatin was photopolymerized with the addition of poly(ethylene glycol) (PEG) mono- or dimethacrylate to form co-networks in pH 7.4 PBS buffer solution. The crosslinking step was performed by employing an Excimer laser so that an addition of a potentially toxic photoiniator was not necessary. The tailorability and potential as a class of biomaterials were investigated by rheology, WAXS, tensile tests, DMTA, as well as water uptake and swelling properties. The networks exhibited Young’s moduli E = 1 – 192 MPa and helical contents <3% in the dry state at room temperature, Q = 243 – 1077 vol% and G’ = 0.7 – 145 kPa in 37 °C pH 7.4 PBS buffer solution. The mechanical properties of these materials in the swollen state at 37 °C were tailorable and in the low kPa range, which are applicable for biomaterials in regenerative therapeutic applications. An initial screening of their degradability was performed for the homonetwork and representative co-networks, which showed that the homonetwork degraded completely within 24 h, while the co-networks only partially degraded to materials that still contained gelatin fragments. While the eluates from the co-networks based on PEGMA (<25wt%) induced some morphological changes in L929 mouse fibroblasts, the eluates of the homonetwork and PEGDMA-based co-networks were well-tolerated by the cells, which indicated that this class of materials may be further studied as biomaterials.
5:00 PM - KK2.8/V3.8
Gelation Characteristics and Encapsulation of Stromal Cells in Star Acrylate-Functionalized Poly (Ethylene Glycol-co-lactide) Macromonomers.
Seyedsina Moeinzadeh 1 , Danial Barati 1 , Esmaiel Jabbari 1
1 Chemical Engineering, University of South Carolina, Columbia, South Carolina, United States
Show AbstractIntroduction: In situ crosslinkable hydrogels coupled with minimally invasive arthroscopic techniques are an attractive alternative for treating irregularly shaped or inaccessible defects. Polyethylene glycol (PEG) hydrogels have been used extensively to study the effect of bioactive factors in the microenvironment on cell function because PEG gels do not illicit an immune response and biomolecules retain their activity in PEG gels. However, PEG gels are not degradable, thus limiting their use for minimally invasive applications. In an attempt to develop degradable PEG-based hydrogels for cell encapsulation, multi-arm PEG was used as the polymerization initiator in ring-opening polymerization of L-lactide to produce degradable polyethylene glycol-co-lactide (SPEL) macromonomers. Next, the chain ends of the macromonomer are functionalized with reactive acrylate groups for is situ crosslinking. The objective of this work was to investigate gelation characteristics of star and linear acrylate-functionalized polyethylene glycol-co-lactide (SPELA and LPELA) macromonomers and viability of marrow stromal cells encapsulated in those macromonomers.Experimental: SPEL and LPEL were synthesized by ring-opening polymerization of L-lactide with 4-arm and 2-arm PEG, respectively, with 5 kDa molecular weight. Next, the chain ends were acrylated by the reaction of acryloyl chloride with the hydroxyl end-groups of the macromonomer. The macromonomers were crosslinked in aqueous solution by UV polymerization. The hydrogels were characterized with respect to gelation by rheometry, sol fraction, water content, degradation, and viability and osteogenic differentiation of bone marrow stromal (BMS) cells. BMS cells were isolated from the bone marrow of young adult male Wistar rats. The sol fraction of the 4-arm SPELA was significantly less than that of the linear LPELA for all concentration. Results: The numbers of lactides on each arm of the linear and star macromonomers was 3 and 3.7, respectively. The shear modulus of the hydrogels increased with the concentration of linear and star macromonomers. Both macromonomers had gelation times of < 60 s but the gelation time of SPELA was lower than that of LPELA. The shear modulus of the SPELA increased by 2.2 fold from 28 to 60 kPa as the macromonomer concentration was increased from 10 to 25%. For example, as concentration was increased from 10 to 25%, sol fraction of the star SPELA decreased from 13 to 5% while that of Linear LPELA decreased from 32 to 19%. Encapsulation experiments demonstrated that the SPELA hydrogel supports viability and osteogenic differentiation of BMS cells. Conclusions: Star SPELA macromonomer, due to the higher functionality and lower viscosity, produced hydrogels with higher modulus and lower sol fraction compared to the linear LPELA. SPELA macromonomer is potentially useful as a degradable carrier in cell-based minimally-invasive therapies.
5:15 PM - KK2.9/V3.9
Live Detection of Neural and Glioma-Derived Stem Cells by an Oligothiophene Derivative.
Shirin Ilkhanizadeh 1 , Rozalyn Simon 2 , Andreas Aslund 2 , Markus Back 2 , Ana Teixeira 3 , Peter Konradsson 2 , Johan Holmberg 3 , Per Uhlen 4 , Bertrand Joseph 5 , Peter Nilsson 2 , Ola Hermanson 1
1 Dept Neuroscience, Karolinska Institutet, Stockholm Sweden, 2 IFM, Dept Chemistry, Linkoping University, Linkoping Sweden, 3 CMB, Karolinska Institutet, Stockholm Sweden, 4 MBB, Karolinska Institutet, Stockholm Sweden, 5 CCK, Karolinska Institutet, Stockholm Sweden
Show AbstractThe development of molecular probes for non-invasive live detection of specific cell types is a critical issue in cancer and stem cell biology. Here we report the synthesis of a luminescent conjugated oligothiophene (LCO), named p-HTMI, which could be used in conventional microscopy for near instant real-time detection of live embryonic neural stem cells, but not other types of stem cells, differentiated cells, or cancer cells investigated. Interestingly, whereas p-HTMI stained only a fraction of glioma cells in culture, 100% of glioma-derived stem cells were stained. The completely opposite result was obtained with a LCO having an alternative side chain functionalization. Cell sorting experiments proved that neural and glioma-derived stem cells could be specifically detected in samples of mixed cell types. p-HTMI is functionalized with a methylated imidazole moiety resulting in a structure similar to methylated histidine/histamine and importantly non-methylated analogues did not show the same characteristics. We propose that LCOs with distinct and defined side chain functionalities represents a novel generation of molecular probes for immediate and specific detection of specific stem and cancer cell types.
5:30 PM - KK2.10/V3.10
Injectable Solid Peptide Hydrogel as Cell Carrier: Effects of Shear Flow on Hydrogel and Cell Payload.
Congqi Yan 1 , Michael Mackay 1 , Joel Schneider 2 , Darrin Pochan 1
1 Materials Science and Engineering, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States, 2 Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States
Show AbstractPeptides were designed to fold into β-hairpins once they are exposed to physiological conditions and then subsequently self-assemble into a rigid hydrogel with a network structure of branched and entangled, 3nm-wide fibrils. These physical hydrogels can be injected as preformed solids, because they can shear-thin and consequently flow under an appropriate shear stress but immediately recover back into solids on removal of the stress with gel stiffness restoring over time. These properties suggest that it is possible to deliver the hydrogel construct with a desired encapsulated therapeutic payload toward an in vivo site by syringe injection. In this work, mechanisms of gel shear-thinning and immediate recovery were elucidated by investigating gel behavior during and after flow via mechanical and structural characterizations. Importantly, hydrogel flow behavior was studied in a capillary geometry that mimicked the actual situation of syringe injection. Hydrogel flow profiles were obtained via fluorescent particle tracking and the profile shape was found dependent on flow rate and gel stiffness. Also living cells were 3D encapsulated in hydrogel and then injected via the same capillary geometry. Flow profile for cellular delivery was investigated and live-dead assay was performed to evaluate effects of flow rate on encapsulated cells. The results demonstrate that these hydrogels can be excellent candidates for tissue regeneration substrates and injectable therapeutic delivery vehicles.
KK3: Poster Session: Scaffold Design and Cell Biology
Session Chairs
Tuesday AM, November 29, 2011
Exhibition Hall C (Hynes)
9:00 PM - KK3.10
Preparation of Nanoparticle-Containing Aligned Collagen Fibers for Dense Connective Tissue Repair and Regeneration.
Xingguo Cheng 1 , Sapna Desai 1
1 , Southwest Research Institute, San Antonio, Texas, United States
Show AbstractTo overcome the limitations of tendon/ligament autografts and allografts, synthetic natural graft materials are pursued. The optimal tendon/ligament graft material should have good biomechanical properties, biodegradability, and cell-supporting properties. Since collagen constitute more than 90% of the dry weight of the tendon/ligament and it is biodegrdable and biocompatible, it holds great promises for tendon/ligament repair. However, tranditonal FDA-approved collagen grafts have weak biomechanical properties (an order of magnitude lower compared to natural tendon) and lack delivery of growth factors in a controlled and gradual manner. Previously, we have demonstrated that highly-aligned collagen fibers can be fabricated by a novel aqeuous electrochemical process. The aligned collagen fiber has improved biomechanical properties and can support and guide the growth of tendon-derived fibroblasts as well as bone marrow stem cells. However, to further enhance the cell-supporing propoerties and promote tendon wound healing, there is a urgent need to load growth factors from such aligned collagen fibers and release the active factors such as Platelet-Derived Growth Factor (PDGF). In the current study, we have fabricated PDGF-containing PLGA nanoparticles (NPs) by using a water-oil-water double emulsion technique. The size and charge of NPs were characterized by BrookHaven Particle analyzer and Zeta-potential analyzer. These drug-containing NPs were mixed with dialyzed collagen molecules, and then were co-assembled into macroscopic aligned collagen fibers by using an electrochemical process at 4 Volts. The NP-containing fibers were collected after 1 hrs,crosslinked with EDC, and dried for further chracterization. After hydrolysis of PLGA nanoparticles in 1N NaOH, the PDGF loading inside the NPs was determined by an ELISA assay. Fluorescence microscopy imaging indicated that NPs were efficiently loaded inside the collagen fibers.Since both collagen and PLGA can be degraded in vivo by collagenase and hydrolysis, these NP-containing aligned collagen fibes will be able to support cell growth and deliver PDGF for long periods of time(e.g., 4-6 weeks). Our future study will be focused on investigation of the cell-supporting properties of this novel biomaterial using adipose-derived stem cells and implantation in a tendon gap-defect model for tedon repair and regneration.
9:00 PM - KK3.11
Effect of Diameter and Orientation of Electrospun PMMA Fibers on Fibroblast Cell Migration.
Sisi Qin 1 , Richard Clark 2 , Miriam Rafailovich 1
1 Department of Materials Sciences and Engineering, Stony Brook University, Stony Brook, New York, United States, 2 Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractThe mechanism of cell migration is a continuous process that enables cells to move forward. The interactions between cells and their extracellular matrix (ECM) are crucial in determining cell migration attachment and motility. Many of the studies about cell migration are based on planar surfaces or imprinting patterns. However, in this study, we used elecrospun PMMA fibers because they well imitated fibrillar structure as in vivo situation. Three different sizes (1um, 4um, 8um) of well aligned PMMA fibers were electrospun. It was found that cell migration velocity and distance increased with decreasing fiber diameters after 24 hours, which can be explained by more vinculin staining (one of the focal adhesion sites) on thicker fibers. Besides fiber sizes, cell migration dynamics could also be affected by changing substrate pattern angles which was done by altering 2-layer fiber orientations. On large angle substrates, SEM analysis indicated that fibroblasts had fewer anchorage points and that the fiber forced the cells to stretch their shape abnormally so that they moved faster at the junction than on small angle substrates. Morphology of 1-layer, 2-layer and 3-layer fibers had also been studied, and the studies indicate that fibroblasts had a preference of following fibers regardless of pattern orientations and that the chance for them to migrate towards top layers was not related with angle between fibers. The 3-layer study shows that fibroblasts had the potential of diffusing through 3-D electropsun PMMA fiber structure with time, which means that such fibroblasts could be widely used in wound healing.
9:00 PM - KK3.12
Quantification of Protein Incorporated into Electrospun Polycaprolactone Nerve-Tissue Engineering Scaffolds.
Nicole Zander 1 2 , Joshua Orlicki 1 , Adam Rawlett 1 , Thomas Beebe 2
1 Macromolecular Science and Technology Branch, U.S. Army Research Lab, APG, Maryland, United States, 2 Chemistry & Biochemistry, University of Delaware, Newark, Delaware, United States
Show AbstractThe modification of synthetic tissue engineering scaffolds is essential to improve their hydrophobicity and cellular compatibility. In this work, polycaprolactone (PCL) electrospun fibers were modified by air-plasma treatment followed by the covalent attachment of laminin. The amount of protein incorporated into the fiber mat was controlled by varying the soaking time and protein solution concentration. The protein and the total carboxylic acid content were quantified using X-ray photoelectron spectroscopy (XPS), solid-state ultra-violet visible spectroscopy (UV-VIS) and two fluorescence-based assays. XPS results showed a nearly linear increase in protein coverage with increasing protein soaking concentration until a monolayer was formed. Results from the UV-VIS and fluorescence assays revealed multilayer protein coverage at protein solution concentrations above 25 µg/mL. The effect of protein concentration on the neurite outgrowth of PC12 cells was evaluated, and outgrowth rates were found to be strongly correlated to protein concentration.
9:00 PM - KK3.13
The Role of Physical Modulation of Substrate Properties on Osteoblast Differentiation.
Kathryn Dorst 1 , Xia Zhao 2 , Aaron Stein 3 , Michael Hadjiargyrou 2 , Yizhi Meng 1 4
1 Materials Science and Engineering, Stony Brook University, Stony Brook, New York, United States, 2 Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States, 3 Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, New York, United States, 4 Chemical and Molecular Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractA crucial step in the process of bone formation is mineralization of the extracellular matrix (ECM), which provides structural support for cells, regulates cell-to-cell communication, and is essential for the development and regeneration of tissues. The proper formation of the ECM depends on the material properties of the pre-existing bone and greatly influences the initial adhesion, migration, alignment and cytoskeletal organization of osteoblasts, as these cells produce the new osteoid. The expression of osteoblast-specific genes has been shown to correlate to these early events that occur at the cell-materials interface, although the exact underlying mechanism(s) remain unknown. Thus, by perturbing the physical and/or chemical microenvironment of the cell, it may be possible to elucidate how the differentiation potential of osteoblasts may be controlled by external factors such as the physical properties of the substrate. Using standard soft lithography techniques, we fabricated polydimethylsiloxane (PDMS) substrates containing “wide” micropatterns (20µm width ridges, 30 µm pitch, 2 µm height), “narrow” micropatterns (2µm width, 10µm pitch, 2 µm height), or no patterns (flat). Plasma etched surfaces were functionalized with fibronectin, an ECM protein, and were seeded with murine pre-osteoblasts (MC3T3-E1 subclone 4). Comparison over 21 days showed no significant differences in cell proliferation, spreading area, or late stage differentiation (via regulation of osteocalcin and mineral nodule formation). However, considerable differences were seen in directional actin filament alignment, migration speed, and early stage differentiation (alkaline phosphatase level). These results suggest that micro-scale topographical variations can influence mechanisms that control proper bone mineralization.
9:00 PM - KK3.14
Parametric Study of Fibroblast Attachment Kinetics on Fibronectin-Coated Polystyrene Tissue Culture Plates.
Shawn Regis 1 , Sina Youssefian 1 , Nima Rahbar 1 , Sankha Bhowmick 1
1 , University of Massachusetts - Dartmouth, North Dartmouth, Massachusetts, United States
Show AbstractCell adhesion is mediated by specific interaction between receptors and ligands. This research presents the results of a numerical and experimental study on cellular attachment to fibronectin-coated 24-well polystyrene plates. This specific attachment process relies on the interaction between the α5β1 integrin on the NIH/3T3 fibroblast surface and the fibronectin ligands on the tissue culture plate. This project focuses on the generation of insightful models to accurately represent fibroblast ability to attach to fibronectin. Based on existing literature and experimental parameters, a dynamical model of fibroblast attachment kinetics on fibronectin coated tissue culture plates was developed. This was accomplished using a phase plane analysis of a system of nonlinear ordinary differential equations, which govern the changes in free receptor density and bond density within the contact area with time. It was determined that the variables with the most dramatic effect on cell attachment were attachment time, ligand density, forward rate constant, and angular velocity of the cell. Furthermore, experimental data of receptor-ligand bond density was used to analyze the accuracy of the devised model. These experimental results were verified by the proposed model to investigate the effects of parameters such as attachment time and ligand density.
9:00 PM - KK3.15
MMSSC Chemotaxis near Porous Surface of Biocompatible NiTi Scaffolds, Synthesized via SLS Method.
Igor Shishkovsky 1 , Stanislav Volchkov 2 , Olga Tumina 2
1 Laboratory of Technological lasers, Lebedev Physics Institute of Russian Academy of Sciences, Samara branch, Samara Russian Federation, 2 , Regional Clinical Cell Technology Centre, Samara Russian Federation
Show AbstractMultipotential mesenchymal stromal stem cells (MMSSC) are the excellent model for the testing on the toxicity and biocompatibility as artificial, as natural tissue-engineering scaffolds (extracellular matrix). Such study allows predicting behaviour of an implanted material in the human. In our study we conducted a testing of the three dimensional prototype of smart material – nitinol (intermetallic phase NiTi) and nitinol with hydroxyapatite for the evaluation of their chemotaxis and biocompatibility. The porous samples, synthesized via selective laser sintering (SLS) method, with different surface conditions were prepared. The surface microstructure and roughness were observed by the scanning electron and optical microscopy. The results reveal a clear influence of the surface roughness on the stem cell proliferation, morphology and adhesion. The nitinol and hydroxyapatite composite was well tolerated by the cells, but the number of focal contacts was lower than in the pure nitinol samples, account of the high porosity of the first. The proliferation speed consisted 0,694 doubling/day in control group, 0.532 doubling/day for NiTi group and 0.292 doubling/day for NiTi + HA. It was detected, that near of an external irritant (i.e. the NiTi scaffold) the stem cells are growing up to enormous sizes (i.e. quickly get old!) and those fission activity are fallen sharply in compare with the young and actively divided stem cells in control group.
9:00 PM - KK3.16
End Grafted Thermally Responsive Polymers - Effect of Polymer Morphology on Cell-Release Characteristics.
Sandip Argekar 1 , Yan Zhang 1 , Girish Kumar 2 , Dale Huber 3 , Dale Schaefer 1
1 , University of Cincinnati, Cincinnati, Ohio, United States, 2 , National Institute of Standards and Technology, Gaithersburg, Maryland, United States, 3 , Center for Integrated Nanotechnologies , Albuquerque, New Mexico, United States
Show AbstractPoly (N-isopropylacrylamide) (PNIPAm) is a well-known thermally responsive polymer exhibiting an aqueous lower critical solution temperature (LCST) around 32°C. The convenient LCST is used as a thermally activated switch facilitating the recovery of entire cell sheets using surface-grafted PNIPAm nanolayers. The intact sheets of cells can be used to repair and regenerate new tissues. However the mechanism of cell release is not clear. Release of cell sheets from PNIPAm grafted substrates is unpredictable and often very slow, affecting sheet integrity. On the contrary, cells may not adhere properly in the adhesive state. Understanding the physico-chemical basis for cell-sheet release is important to improve cell adhesion and release characteristics.We explain the cell-release potential of PNIPAm on the basis of the changes in swelling behavior. We synthesized well-defined, end-tethered PNIPAm nanolayers using surface-initiated atom-transfer radical polymerization and free-radical polymerization schemes. The schemes enable control over polymer graft density and molecular weights. Human bone marrow stromal cells (hBMSCs) adhesion and release was correlated to PNIPAm morphology as determined by X-ray reflectivity and neutron reflectivity under swollen conditions. We find cell adhesion and release are strongly dependent on PNIPAm molecular weight. By relating PNIPAm swelling to cell-release a ‘sweet spot’ is desired to optimize both cell adhesion and release behavior.
9:00 PM - KK3.17
Shear Induced Fibrillogenesis of FN Nanofibers by Rotary Jet-Spinning.
Holly McIlwee 1 , Mohammad Badrossamay 1 , Leila Deravi 1 , Josue Goss 1 , Kevin Kit Parker 1
1 Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractWithin the extracellular matrix (ECM), the protein, fibronectin (FN), is a critical component for maintaining the structural integrity and spatial organization of cells and tissues.[1] FN is a disulfide-linked dimer secreted by cells in a soluble, globular conformation before binding to cell surface receptors, unfolding by cell traction forces exposing cryptic FN-FN binding sites, and inducing fibrillogensis.[2] Fabrication of insoluble FN nanofibers in vitro remains a challenge. The Rotary Jet-Spinning (RJS) system[3] is a technique developed for producing nanofibers by elongating polymer jets through centripetal and viscous shear forces, as they are ejected from a capillary perforated reservoir attached to a high-speed rotating motor. We hypothesized shear forces induced during RJS may be used to unfold globular FN, exposing protein-protein binding sites, forming FN nanofibers with mechanical and physiological integrity. To test this hypothesis, shear forces within the RJS were first modeled using the Poiseuille equation, and then experimentally studied as a function of rotation speed and solution viscosity. Aligned, continuous FN fibers produced by RJS had an average diameter of 232.6 ± 59 nm. The effect of shear forces on FN molecules within formed nanofibers was then studied by measuring protein conformation using two techniques probing secondary structure: fluorescence resonance energy transfer (FRET) and Raman spectroscopy. Results indicated that FN molecules within the collected fibers were extended due to the shear forces within the RJS. Over 24 hours, FN molecules relax into a compact conformation, as solvent evaporates from the fibers as indicated by an increase in FRET fluorescence and a decrease in Raman signal. Bioactivity of FN nanofibers is demonstrated by robust attachment of vascular smooth muscle cells to FN nanofiber scaffolds. FN nanofibers were insoluble in physiological media, demonstrating that in vitro shear-induced fibrillogenesis has occurred. We demonstrate rapid, cell-free, shear force driven assembly of fibronectin nanofibers useful for the in vitro study of tissue development and disease.References: 1. Feinberg, A.W. et al. Nano Letters, 2010. 10(6): p. 2184-2191.2. Mao, Y. et al. Matrix Biology, 2005. 24(6): p. 389-399.3. Badrossamay, M.R., et al., Nano Letters, 2010. 10(6): p. 2257-2261.4. Schwarzbauer et al. Cur Opinion in Cell Bio. 1999, 11, 622-627.
9:00 PM - KK3.18
Ultra-Thin, Functionalized Biohybrid Gels for Virus-Free Cell Reprogramming.
Suzanne Balko 1 , Yixin Zhang 2 , Elly Tanaka 3 , Jens-Uwe Sommer 4 5 , Carsten Werner 1 3
1 Max Bergmann Center of Biomaterials Dresden (MBC), Leibniz Institute for Polymer Research Dresden e.V. (IPF), Dresden Germany, 2 Innovation Center for Molecular Bioengineering (B CUBE), Technische Universität Dresden, Dresden Germany, 3 Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden Germany, 4 Institute of Theoretical Physics, Technische Universität Dresden, Dresden Germany, 5 , Leibniz-Institut für Polymerforschung Dresden e.V. (IPF), Dresden Germany
Show AbstractRecent advances in molecular biology have made it possible to induce the transformation of differentiated cells into pluripotent stem cells. For example, transcription factors fused to cell-penetrating peptides (CPPs) have successfully immortalized human fibroblasts[1]. Taking this work as a starting point, we merge the cell reprogramming power of transcription factors with matrix engineering, via ultra-thin, biohybrid gels, to exogenously trigger cellular fate decisions. Using a novel, heparin-polyethylene glycol hydrogel system, functionalized with CPP-fused transcription factors, we study the translocation of these functional constructs across model cell membranes. The surface of these hydrogels is easily modified with adhesion ligands and growth factors to tailor interactions with cells [2]. The composition of the hydrogel may be modified, thus producing a tunable and responsive biocompatible material [3]. We have characterized the mechanical and surface properties of our hydrogel system and present protein loading studies of the hydrogels. We conclude with an outlook onto experimental and computer simulation studies of model lipid membrane and peptide translocation as a result of interactions with the hydrogels and comparisons to more conventional polyelectrolyte polymer layers[4].REFERENCES[1] D. Kim, C.H. Kim, J.I. Moon, Y.G. Chung, M.Y. Chang, B.S. Han, S. Ko, E. Yang, K.Y. Cha, R. Lanza, K.S. Kim. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell (2009) 6: 472-6.[2] U. Freudenberg, A. Hermann, P. Welzel, K. Stirl, S. Schwarz, M. Grimmer, A. Zieris, W. Panyanuwat, S. Zschoche, D. Meinhold, A. Storch, C. Werner. A starPEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases. Biomaterials (2009) 30: 5049-5060.[3] M.V. Tsurkan, K.R. Levental, U. Freudenberg, C. Werner. Enzymatically degradable heparin-polyethylene glycol gels with controlled mechanical properties. Chem Commun (Camb) (2010) 7: 1141-3.[4] L. Renner, T. Osaki, S. Chiantia, P. Schwille, T. Pompe, C. Werner. Supported lipid bilayers on spacious pH responsive polymer cushions with varied hydrophilicity. J Phys Chem B (2008) 112: 6373-6378.
9:00 PM - KK3.19
Nanomechanical Characterization of Peptide Nanofibers by Double-Pass Force-Distance Mapping.
Turan Erkal 1 , Aykutlu Dana 1 , Mustafa Guler 1
1 Materials Science and Nanotechnology, Bilkent University UNAM-Institute of Material Science and Nanotechnology, Ankara Turkey
Show AbstractRecent studies have shown that the peptide nanofibers used to mimic the extracellular matrix have had a noteworthy influence on regenerative medicine. Due to the bioactive and physical characteristics of peptide nanofibers, they provide mechanical support and suitable chemical environment for cells. These bioactive nanofibers provide signaling cues that enables convenient manipulation of cellular fate, including adhesion, spreading, proliferation, and differentiation. In addition to the bioactivity imparted by the bioactive signal-carrying nanofibers, like RGD, REDV, and KRSR, the mechanical properties of the network formed by such nanofibers have a considerable impact on different cells types. For example, neurogenic cells prefer less stiff microenvironment (~0.1-1kPa) compared to myogenic cells (10-20kPa), and osteogenic cells (25-40kPa). Here, we demonstrate a novel approach inspired by the structure and mechanics of mussel byssus to control the mechanical properties of peptide nanofibers. We developed peptide nanofibers conjugated with lysine and 3,4-dihydroxy-L-phenylalanine (L-Dopa) molecules, which are found in mussel adhesive proteins for attachment of mussels in aqueous environment. With the help of Dopa molecule, the peptide nanofibers can be crosslinked to improve and control the mechanical properties of peptide nanofibers by changing the crosslinking density of the material. Furthermore, mechanical characterization of these peptide nanofibers is essential for correlating the cell behaviour with strength of peptide nanofibers. To determine the nanomechanical property of peptide nanofibers, we have developed a new atomic force microscopy (AFM) technique inspired by double-pass imaging which has been used to characterize electrostatic and magnetic forces. The double-pass force-distance mapping use minimal indentation forces which enable mechanical characterization of fragile molecules like biomacromolecules. Additionally, this technique delivers rapid data acquisition, thus reducing susceptibility to mechanical drifts and providing more detailed information regarding sample.
9:00 PM - KK3.2
A Comparison of Two Common Polycaprolactone-Based Biomaterials.
P. Warren 2 , Dominic McGrath 1 , Jonathan Vande Geest 3 4 5
2 Graduate Interdisciplinary Program in Biomedical Engineering, University of Arizona, Tucson, Arizona, United States, 1 Department of Chemistry, University of Arizona, Tucson, Arizona, United States, 3 Department of Aerospace and Mechanical Engineering, University of Arizona, Tucson, Arizona, United States, 4 Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, United States, 5 BIO5 Institute for Biocollaborative Research, University of Arizona, Tucson, Arizona, United States
Show AbstractRecently there has been a significant increase in publications regarding the use of polycaprolactone (PCL) as a resorbable biomaterial due to its mechanical advantages over other aliphatic polymers. PCL is FDA approved, inexpensive, and can be easily incorporated into a substantial range of long term (3-4 years) devices for implant and/or drug delivery. As a result of this renewed interest PCL has also been combined with other materials (e.g. polyurethane) to make it more suitable for other applications, for example, where a device may see cyclical loading. To this end, two main trends seem to dominate the literature. One such process simply combines the PCL and another material in solvent to make a blend of the two materials. The other process requires slightly more work to actually covalently link PCL to another material to produce a copolymer. Examples of covalently linked PCL products include lactide based and amino acid based poly(ester-urethane-urea)s utilizing diisocyanates. In this work, a common blend of PCL and a commercially available linear polyurethane, Tecoflex, were compared to a poly(ester-urethane-urea) copolymer based on lysine, both in the diisocyanate and in the chain extender. The materials were chemically and physically characterized including degradation experiments, while measuring crystallinity, to better understand the role of PCL crystallinity in governing physical properties. The end goal of these analyses was to conceive of the best direction for future research on fabricating biomaterials for cardiovascular implant devices.
9:00 PM - KK3.20
Development of an Electrospun Chitosan Scaffold for Wound Dressing Application.
Abdelhafid Aqil 1 , Victor Tchemtchoua 2 , Alain Colige 2 , Ganka Atanasova 3 , Christine Jerome 1
1 CERM, University of Liege, Liege Belgium, 2 LCTB, University of Liege, Liege Belgium, 3 FUNDP, University of Namur , Namur Belgium
Show AbstractWound dressing is one of the most promising medical applications for chitosan, due to its adhesive nature, together with some biological properties including bacteriostatic and fungistatic properties that help in faster wound healing. In this work we propose a chitosan biomimetic scaffolds and methods for modulating their intrinsic properties such as rigidity, elasticity, resistance to mechanical stress, porosity, biodegradation and absorbance of exudates. Therefore, the chitosan scaffold comprising at least two fused layers, wherein the advantage of a first fused layer composed of a chitosan electrospun nanofiber membrane are oxygen-permeability, high porosity, variable pore-size distribution, high surface to volume ratio, and most importantly, morphological similarity to natural extracellular matrix in skin, which promote cell adhesion migration and proliferation. The advantages of a second fused layer comprising a porous chitosan support layer are improving mechanical property, good absorption capacities to remove excess exudates and good water and gas exchange. Moreover, the scaffold was characterized by (i) a good adhesion between the porous and nanofiber layers, (ii) a tuneable porosity of the nanofiber layer by tuning the distance between the nanofibers, (iii) a stable nanofibers and porous morphology even when immersed in water. Finally, the scaffolds have shown tremendous promise as a wound dressing, in tissue engineering. The three main human cell types fibroblasts, endothelial cells and keratinocytes was cultured in vitro on electrospun nanofibers scaffold. Properties of electrospun chitosan scaffold and chitosan sponges obtained by lyophilization were also compared in vivo, in order to evaluate importance of the 3D-architecture of the biomaterial.
9:00 PM - KK3.21
Semi-Degradable Porous Hydrogels for Controlled Release of IGF-1 for Cartilage Tissue Engineering.
Claire Martin 1 2 , Sun Hengyun 3 , Kara Spiller 1 2 , Guangdong Zhou 3 , Wei Liu 3 , Anthony Lowman 2
1 School of Biomedical Engineering, Drexel University, Philadelphia, Pennsylvania, United States, 2 Chemical and Biological Engineering - Biomaterials and Drug Delivery Laboratory, Drexel University, Philadelphia, Pennsylvania, United States, 3 Shanghai Key Tissue Engineering Laboratory - Plastic and Reconstructive Surgery, Shanghai Jiao Tong University School of Medicine, Shanghai China
Show AbstractRepair or regeneration of articular cartilage remains one of the most difficult challenges in orthopedic medicine. Biodegradable scaffolds or hydrogels, comprised of naturally-derived or synthetic polymers, have shown promise in supporting the proliferation of cartilage tissue. Poly(vinyl alcohol)(PVA) hydrogels have shown potential in their ability to provide superior mechanical support, porosity, and swelling properties that accurately replicate mature cartilage. The controlled release of growth factors from PVA hydrogels has facilitated cellular proliferation and cartilage production, resulting in a hybrid cartilage-hydrogel construct. However, the major disadvantage of these implants is their poor integration with surrounding tissue, due to limited chondrocyte migration. The controlled release of IGF-1 from a hydrogel has been shown to stimulate chondrocytes from the surrounding cartilage to migrate into the implant and integrate the implant with the surrounding tissue. In this study, IGF-1 was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles embedded in non-degradable PVA hydrogels in a single step based on a double emulsion technique. Hydrogels had interconnected pores with a size of 200um and greater. Chondrocyte seeded hydrogels were implanted subcutaneously into the backs of nude mice and harvested after two and five weeks in vivo in order to study the controlled release of IGF-1 on cartilage tissue engineering in characterized hydrogels over time. The introduction of high levels of porosity to PVA hydrogels resulted in a reduction in mechanical properties. The increased porosity also allowed for mature cartilage growth within the pores of the hydrogel, but did not provide compensation for decreased mechanical properties. This study shows that the sustained release of IGF-1 can enhance tissue formation within the pores of a non degradable hydrogel and improve implant integration with surrounding tissue.
9:00 PM - KK3.22
Injectable Biomimetic Materials Using Carbon Nanofibers, PolyHEMA and HRNs for Cardiovascular Applications.
Xiangling Meng 1 , Thomas Webster 2 3 , Hicham Fenniri 4 5
1 Chemistry, Brown University, Providence, Rhode Island, United States, 2 Orthopaedics, Brown University, Providence, Rhode Island, United States, 3 School of Engineering, Brown University, Providence, Rhode Island, United States, 4 Chemistry, University of Alberta, Edmonton, Alberta, Canada, 5 National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada
Show AbstractIntroduction:Heart disease or cardiovascular disease is a critical cause for death. Currently, there is a significantly high and increasing rate of cardiovascular disease, which clearly has led to a significant public health problem. Myocardial infarction results in a large scale loss of cardiac muscle. Therefore, the purpose of this in vitro experiment was to develop a strategy for cardiac repair based on the self-assembly properties of helical rosette nanotubes (HRNs), the conductive properties of carbon nanofibers (CNFs), and the biocompatible properties of hydrogels (specifically, poly (2-hydroxyethyl methacrylate), pHEMA).Methods:Scaffold PreparationThe material fabricated and tested here were combinations of three different materials: helical rosette nanotubes (HRNs), poly (2-hydroxyethyl methacrylate)-pHEMA (Polysciences Inc), and carbon nanofibers (CNFs) modified with –OH and – COOH (Pyrograf Products Inc).For cardiomyocyte adhesion, CNFs were added into a HEMA monomer, 2, 2-azobisisobutyronitrile (free radical initiator, Sigma-Aldrich) and a HRN solution to give a 0.2 wt% composite. The concentrations of HRN were 0mg/ml, 0.01mg/ml, 0.02mg/ml and 0.05mg/ml. To obtain well dispersed CNF hydrogel composites, the CNFs were first sonicated in the HEMA solution for 30 min. 1 mg/ml HRN-K and 2, 2-azobisisobutyronitrile were then added into the above mixtures, mixed thoroughly, and heated at 70°C for around 2 h to polymerize. Finally, all of the samples were sterilized in 70% ethanol and 100% ethanol for 5 min. Then, the samples were evaporated and sterilized under UV light in the biohood overnight before cell experiments.Cardiomyocyte AdhesionHuman cardiomyocytes at population numbers 6 to 7 (Celprogen Stem Cell Research and Therapeutics), were cultured in Human Caridomyocyte Complete Media supplemented with 10% fetal bovine serum (FBS, Hyclone) under standard cell culture conditions (a humidified, 5% CO2/95% air environment at 37 °C). Cardiomyocytes were seeded onto the composites at a density of 3500 cells cm−2 and were cultured under standard cell culture conditions for 4 h. Adherent cells were counted using the MTT assay. Experiments were run in triplicate and repeated three separate times for each substrate.Materials Characterization A scanning electron microscope (SEM) operating at 10kV accelerating voltage was used to image the dispersed CNF in HRN and pHEMA hydrogels. SEM images were taken from the top of the samples.Results and Discussion:This study demonstrated that HRNs play a positive role in improving cardiomyocyte density and should be further studied for cardiac applications. Moreover, CNFs are well dispersed in the HRN and pHEMA hydrogels and also improved cardiomyocyte density.Acknowledgements:We would like to acknowledge the Hermann Foundation for fundings.
9:00 PM - KK3.23
Composition-Dependent Sol-Gel Transition of Diblock and Triblock PEG Amphiphiles for 3-D Cell Culture Scaffolds.
Akihiro Aso 1 , Kazutaka Taki 2 , Hitoshi Tamiaki 2 , Kazunori Toma 3 , Atsushi Hotta 1
1 Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan, 2 Institute of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga, Japan, 3 , Asahi Kasei Corporation, Fuji, Shizuoka, Japan
Show AbstractLow-molecular-weight gelators, particularly the amphiphiles with hydrophobic and hydrophilic moieties, have been widely investigated for 3-D cell culture scaffolds in the tissue engineering field. The amphiphiles can self-assemble in an aqueous solution to form micellar structures presenting sol or gel features by changing the dispersed states of the micelles through molecular interactions such as hydrogen bonding and hydrophobic interaction in response to temperature. It is, however, often difficult to adjust the sol-gel transition temperature for the cell-culture applications simply by the molecular design of the gelators. In this work, the sol-gel transitions of the aqueous solution of diblock and triblock poly(ethylene glycol) (PEG) gallamide amphiphiles were studied using dynamic mechanical analysis, cryogenic-transmission electron microscopy (cTEM), dynamic light scattering and 1H-NMR measurements. The diblock amphiphile consisted of long alkyl chains, PEG and a gallamide linker, while the triblock amphiphile possessed the alkyl chains and the gallamide linkers at both ends of PEG. It was found that the 10 wt% aqueous solution of the diblock amphiphiles was in the sol state, and that the 5 wt% triblock solution was solid gel over a wide range of temperature. The diblock and the triblock amphiphiles were mixed at different ratios in 15 wt% solution, and the mixture showed a thermoreversible sol-gel transition upon heating at ~37 °C, indicating that the blend was potentially useful for 3-D cell culture. The solutions with the molar ratios of the diblock to the triblock amphiphiles at 52.5:47.5 and 50:50 showed sol to gel transitions at around 40 and 37 °C, respectively, upon heating, and hysteresis was observed in both cases. From the cTEM observation, dispersed spherical micelles of ~50 nm in diameter and their large aggregates of ~200 nm or ~1.2 μm in diameter were observed in the sol state. Pearl necklace-like network structures, where spherical micelles of 50~100 nm aggregated, were observed in the gel state through the sol-gel transition. It was considered that the composition of the diblock and the triblock amphiphiles governed the sol-gel transition, and that the micelles and the aggregates observed in the sol got connected through the bridges of the triblock amphiphiles to form hydrogel. It was found that the dynamic moduli and the sol-gel transitions could be effectively controlled by changing the mixing ratio of the amphiphiles. The thermoreversible sol-gel transition behaviors around 37 °C suggested that the system could be suitable for in–vitro 3-D cell culture scaffolds.
9:00 PM - KK3.24
Chitin Nanofiber Microstructures for Neuron Growth: Topographical Effects on Neuronal Behavior.
Chao Zhong 1 , Ashleigh Cooper 1 , Adnan Kapetanovic 1 , Miqin Zhang 1 , Marco Rolandi 1
1 Materials Science & engineering, University of Washington, seattle, Seattle, Washington, United States
Show AbstractMicro/nanopatterned surfaces/structures for neuronal cell culture are essential in understanding surface topographical effects on neuron behavior, including cell migration, morphological changes, and neurite development. Despite a considerable amount of ongoing research, patterning nanostructures that mimic the 3-D fibrous textures in the native ECM environment is still a difficult task. Chitin, with N-acetylglucosamine chemical unit and typical fibril morphology closely resembling natural ECM, is an ideal ECM-mimic for neuronal cell culture. Self-assembled chitin nanofibers (3nm) are nontoxic to Schwann cells. When these nanofibers are coupled with poly-d, lysine (PDL), cortical cell attachment and neural network formation is greatly enhanced. In this study, we create patterned chitin nanofiber microstructures via a “chitin nanofiber ink” approach. Microcontact printing, replica molding, and inkjet printing produce 1, 2 and 3-D micro/nano structures including lines, squares, circles, and pillars. We use these microstructures to assess topography effects on neuronal cell behavior. Preliminary data on topography-directed neuronal cell growth will be presented.
9:00 PM - KK3.25
Solid Free-Form Fabrication of Tissue Engineering Scaffolds with HA-PLGA Conjugate Encapsulating Intact BMP-2/PEG Complex.
Jung Kyu Park 1 , Jin-Hyung Shim 2 , Kyung Shin Kang 2 , Junseok Yeom 1 , Jong Young Kim 3 , Keum Hong Lee 4 , Tae-Ho Kim 5 , Shin-Yoon Kim 4 , Dong-Woo Cho 2 , Sei Kwang Hahn 1
1 Department of Materials Science and Engineering, POSTECH, Pohang, Kyungbuk, Korea (the Republic of), 2 Department of Mechanical Engineering, POSTECH, Pohang, Kyungbuk, Korea (the Republic of), 3 Department of Mechanical Engineering, Andong National University, Andong, Kyungbuk, Korea (the Republic of), 4 Kyungpook National University Hospital, Kyungpook National University, Daegu Korea (the Republic of), 5 Department of Medicine, School of Medicine, Kyungpook National University, Daegu Korea (the Republic of)
Show AbstractDespite wide applications of bone morphogenetic protein - 2 (BMP-2), there are few methods to incorporate BPM-2 within polymeric scaffolds maintaining the biological activity. In this work, solid free-form fabrication (SFF) of tissue engineering scaffold was successfully carried out with poly(lactic-co-glycolic acid) grafted hyaluronic acid (HA-PLGA) encapsulating intact BMP-2/poly(ethylene glycol) (PEG) complex. HA-PLGA conjugate was synthesized in DMSO by the conjugation reaction between adipic acid dihydrazide modified HA (HA-ADH) and PLGA activated with N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). BMP-2 was complexed with PEG, which was encapsulated within the PLGA domain of HA-PLGA conjugate by SFF to prepare tissue engineering scaffolds. In vitro release tests confirmed the sustained release of intact BMP-2 from the scaffolds for up to a month. After confirmation of the enhanced osteoblast cell growth, and high gene expression levels of alkaline phosphatase (ALP), osteocalcin (OC), and osterix (OSX) in the cells, the HA-PLGA/PEG/BMP-2 scaffolds were implanted into the calvarial bone defects of Sprague Dawley (SD) rats. Microcomputed tomography (μCT) and histological analyses with Masson’s trichrome, and hematoxylin and eosin (H&E) staining revealed the effective bone regeneration on the scaffolds of HA-PLGA/PEG/BMP-2 blends.
9:00 PM - KK3.26
Modular Resilin-like Polypeptides as Tissue Engineering Scaffold for Mechanically Active Tissues.
Christopher McGann 1 , Kristi Kiick 1 2
1 Materials Science & Engineering, University of Delaware, Newark, Delaware, United States, 2 , Delaware Biotechnology Institute, Newark, Delaware, United States
Show AbstractA recombinant protein polymer based upon a motif sequence found in the elastomeric protein, resilin, has been expressed, purified and cross-linked into hydrogels via Michael-type addition reaction. Resilin is an important structural protein found in the mechanically active tissues of insects and is renowned for its resilience and elasticity. The recombinant protein polymer described in this work utilizes repeated resilin domains to confer those mechanical properties, but also includes additional sequences for enzymatic degradation, cell adhesion and heparin binding. Previously, we have demonstrated that this protein polymer can form elastic and highly resilient hydrogels by cross-linking lysine residues using a hydroxymethyl phosphine cross-linker (THPP). The current work investigates hydrogels cross-linked through cysteine residues using multi-arm star PEG terminated with vinyl sulfone or maleimide moieties; a strategy which keeps the lysine-rich heparin binding domains intact. Expression and purification of the protein polymer was verified through SDS-PAGE, mass spectrometry and amino acid analysis. Oscillatory rheology was employed to demonstrate the formation of elastic hydrogels with storage moduli in the 0.1-1 kPa range. Additionally, higher molecular weight resilin-like polypeptide protein polymers were created through the recursive ligation of the original gene. These modular resilin-like protein polymer hydrogels potentially can serve as elastomeric and cell-instructive scaffolds for tissue regeneration.
9:00 PM - KK3.28
Synthesis and Characterization of Photocrosslinkable Poly(Glycerol-co-sebacate)-cinnamate Elastomer.
Congcong Zhu 1 , Suze Ninh 1 , Christopher Bettinger 1 2
1 Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 2 Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States
Show AbstractPoly(glycerol-co-sebacate) (PGS) is a tough biodegradable elastomer which shows biocompatibility both in vivo and vitro. However, high temperature is needed to synthesize crosslinked PGS networks, which limits its biomedical use. In this report, we modified PGS with cinnamate groups and obtained a photocrosslinkable elastomer based on the [2+2] photocycloaddition of cinnamate. The prepolymer of poly(glycerol-co-sebacate)-cinnamate (PGS-CinA) was synthesized by bulk polycondensation of PGS followed by modification with cinnamoyl chloride. Thin films of polymer were prepared using melt-pressed method and spin-coating of dichloromethane solution and crosslinked using UV irradiation. Elastomeric networks can be formed using two approaches: (1) exposure to ultraviolet light with wavelength longer than 280 nm; (2) additional bulk polycondensation using thermal curing. Photocrosslinking of cinnamate groups is reversible. Retrocycloaddition can be induced by UV irradiation at a wavelength of 254 nm. FTIR was used to verify the incorporation of cinnamate group, which has the typical C=C characteristic bond at 1637 cm-1. The degree of substitution of cinnamate groups was measured by NMR. Kinetic parameters were determined by measuring absorption at 282 nm. Crosslinking density was assessed by measuring load-displacement curve using nanoindentation. Thermal properties of PGS-CinA prepolymer and crosslinked networks were measured by DSC. In vitro degradation was performed by measuring the mass loss of films in hydrolytic medium consisting of sodium acetate in water at 37 °C. The crosslinked PGS-CinA showing good mechanical properties is promising as a new biomaterial for tissue engineering applications.
9:00 PM - KK3.29
Preparation and In Vitro Characterization of Polycaprolactone and Demineralized Bone Matrix Scaffolds.
Titilayo Moloye 1
1 Biomedical Engineering, University of Florida, Gainesville, Florida, United States
Show AbstractThere is a growing need to address the thousands of soldiers returning from war with traumatic bone injury. Currently, the gold standard for these non-union fractures is autografting. However, this introduces the patient to donor site morbidity, which can lead to a new fracture site. The goal of this study is to develop a biocompatible bone replacement scaffold from polycaprolactone (PCL) and demineralized bone matrix (DBM) in which the matrix is embedded within the PCL. In this particular study, the in vitro bioactivity of the scaffold is presented. 1 cm cylindrical polycaprolactone scaffolds consisting of 25%, 35%, and 50% DBM were fabricated using a salt particle leaching method. PCL and Glacial PCL pellets were dissolved in acetic acid at a concentration of 40% (by weight). 25%, 35%, and 50% (by weight) DBM was added to equal amounts of polymer solution in a 1 cm cylindrical mold. Apatite deposition on these scaffolds was systematically monitored by collecting supernatants and analyzing for levels of ionized calcium and phosphorous at time periods of 5, 10, 15, 20 and 25 days post-immersion in Simulated Body Fluid (SBF) and DI water at 37 °C. Cell proliferation and alkaline phosphate production were also assessed. Biochemical assays of inorganic calcium and phosphate revealed no measurable apatite precipitation manifested by scaffolds incubated in either DI or SBF. However, scaffolds incubated in SBF did consistently have a higher concentration of ionized calcium and phosphorous. While SBF contains phosphate and calcium ions, SBF was not replenished and therefore was subject to insufficient ionic activity SEM micrographs consistently showed calcium precipitates of a globoid and needle-like shape on the surface of those scaffolds incubated in SBF. Cell proliferation was not significantly affected by scaffold composition. However, Alkaline Phosphate activity was significantly higher in scaffolds with a greater amount of DBM.
9:00 PM - KK3.3
Biocompatibility of Single Crystalline Fe70Pd30 Ferromagnetic Shape Memory Films for Cell Actuation.
Mareike Zink 1 , Uta Allenstein 1 , Ariyan Arabi-Hashemi 2 , Yanhong Ma 2 , S. Mayr 2 1 3
1 Department of Physics and Earth Sciences, University of Leipzig, Leipzig Germany, 2 , Leibniz-Institut für Oberflächenmodifizierung e.V., Leipzig Germany, 3 Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig Germany
Show AbstractFerromagnetic shape memory alloys (FSMAs) have received great attention recently as an exciting class of smart functional materials. They exhibit large reversible strains of several percent at moderate stresses due to an external magnetic field induced reorientation of twin variants in the martensitic phase. External controllability at constant temperatures and sufficiently high strains thus make them excellent candidates for biomedical actuation devices, such as surgical implant materials, applying for bone prostheses or drug delivery systems. In comparison to conventional shape memory alloys, FSMA bear the significant potential for miniaturized devices for single cell actuation which is capable of yielding magnetically controllable shear strains and/or volume dilations of several percent, thus perfectly matching the requirements of cell investigations. However, biocompatibility of this material remains to be confirmed. Thus, our work focuses on the interaction of fibroblast cells with single crystalline Fe70Pd30 FSMA films on MgO substrates. Additionally, corrosion resistance of the films was obtained employing simulated body fluid (SBF) tests. Calcium-phosphate aggregates with granular microstructure were detected on the film surface after soaking in SBF. Cell viability and biocompatibility tests with NIH 3T3 cells revealed that the cells adhered and proliferated on the surface of the FSMA. Additionally, the cytoskeletal arrangement, as well as focal contacts of the cells were examined using confocal laser scanning microscopy. It turned out that Fe70Pd30 FSMA films are biocompatible and bioactive, while adhesion of the cells to the substrate can be tuned by employing different coating materials such as fibronectin.
9:00 PM - KK3.30
Diels-Alder Reversible Cross-Linking of Biodegradable Poly(Glycerol-co-sebacate) Elastomer.
Chi Ninh 1 , Christopher Bettinger 1 2
1 Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 2 Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States
Show AbstractBiodegradable elastomers that have mechanical properties similar to those of soft tissues have proven to play an important role in biomedical engineering. Poly(glycerol-co-sebacate) (PGS) has been studied as a tough, biodegradable elastomer that exhibits suitable biocompatibility and mechanical properties for tissue regeneration. However, current cross-linking techniques are irreversible and often require harsh temperature curing conditions or other additives such as photoinitiator. In order to design a simple thermally reversible network, PGS was cross-linked via Diels-Alder chemistry of furan-modified PGS prepolymer with 1,4-Di(maleimido)butane. Furan modification of PGS was achieved via coupling of low molecular weight PGS with furfuryl chloride in anhydrous dichloromethane. After solvent was evaporated, the mixture was washed with ethyl acetate to obtain a viscous product. Cross-linking was done in 4 different ratios of Furan group to Maleimido group (1:1, 2:1, 4:1, 8:1). The forward reaction occurs at room temperature up to 60 °C. Elastomeric networks undergo retro Diels-Alder when heated to 110 °C. Furan-modified PGS and 1,4-Di(maleimido)butane were melt processed into thin films at 110 °C for 3 hours. The films were then cooled to 25 °C for 24 hours to form the crosslinked network. Cross-linking kinetics were examined by UV spectroscopy and verified by NMR. The increase of the area under the peak corresponding to the proton of the Diels-Alder adduct over time shows efficient reaction conversion. In addition, rheology study proves that the material still retains the desired mechanical properties. These results demonstrate that Diels-Alder chemistry is a suitable and easy cross-linking technique for PGS elastomer. The ability to undergo reversible cross-linking at relatively low temperatures will allow for a wide variety of architecture and promising use in biomedical engineering.
9:00 PM - KK3.31
Biocompatible Polyelectrolyte Fibers as Host for Ceria Nanoparticles.
Astha Malhotra 2 1 , Lei Zhai 1 2
2 Chemistry, University of Central Florida, Orlando, Florida, United States, 1 Nanoscience Technology Center, University of Central Florida, Orlando, Florida, United States
Show AbstractCeria nanoparticles have the ability to switch their oxidation states which makes them ideal candidates as radical scavengers, comparable to other antioxidants. Such property of ceria nanoparticles can be used for the protection from reactive oxygen species and radiation damage. For relevant medical applications, ceria nanoparticles should be in a medium/matrix compatible to physiological conditions. PAA-PAH fibers have been used as hosts for ceria nanoparticles. These polyelectrolyte spun fibers have combined the properties of hydrogels and large surface area, and have high capability to accommodate nanoparticles. The ceria nanoparticles are synthesized in situ in the fiber as fast stimuli-responsive systems for various applications such as tissue engineering, drug delivery, engineered anti-oxidants, etc.
9:00 PM - KK3.32
Immobilization of Adhesion Peptides on PEG-Based Thin Films to Define the Stem Cell Microenvironment.
Samantha Schmitt 1 , Daniel Sweat 2 , William Murphy 3 1 , Padma Gopalan 1 2
1 Department of Materials Science and Engineering, University of Wisconsin - Madison, Madison, Wisconsin, United States, 2 Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States, 3 Department of Biomedical Engineering, University of Wisconsin - Madison, Madison, Wisconsin, United States
Show AbstractWe have developed a model platform that will allow for stable presentation of cell adhesion motifs over an extended timeframe, to clearly address the hypothesis: ECM-derived ligand concentration strongly influences human mesenchymal stem cell (hMSC) adhesion and viability. Our research exploits the attributes of crosslinked thin-films which include exceptional stability, application to atypical substrates, chemical tailorability, and photo-patternability. Mechanically stable and substrate independent biointerfaces were created. Atom transfer radical polymerization (ATRP) was utilized for the synthesis of polyethylene glycol (PEG) based copolymers, which includes a low density of crosslinkable groups and 2-20 % of functionalizable moieties. The PEG provides a cytophobic background to prevent non-specific adsorption which is switched upon the introduction of RGDSP. The polymer was crosslinked thermally. Crosslinked thin films were then functionalized with RGDSP, a fibronectin derived peptide, using a mild iodination method. This method has not previously been explored for surface modification and is described here in detail. X-ray photoelectron spectroscopy was utilized for peptide quantification. Our focus is on using surface characterization techniques to quantify the density of the peptides on the film surface and hence understand the hMSC response.
9:00 PM - KK3.33
Comparative Biodegradation Study of Polymer Foams.
Courtney LeBlon 1 , Sabrina Jedlicka 2 3 4
1 Mechanical Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Materials Science and Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 3 Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania, United States, 4 Center for Advanced Materials & Nanotechnology, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractBiodegradable polymer scaffolds for tissue engineering often require a biodegradation rate that closely matches that of the tissue ingrowth. While the biodegradation kinetics of most biocompatible polymers have been characterized, scaffold morphology will play a role in these dynamics, and is not well characterized comparatively between polymers. Here, we examine polymer foams with variable elasticities and biodegradation rates: poly(D,L-lactic-co-glycolic acid) (PLGA) (85:15), poly(D,L-lactide-co-caprolactone) (PLCL) (50:50), thermoplastic polyurethane (PU, non-biodegradable), and poly(glycerol sebacate) (PGS). To assess porous scaffold degradation, scaffolds were immersed in simulated body fluid (SBF) and incubated in a controlled, asceptic environment (37°C, 5% CO2). Every two weeks, samples were analyzed using for chemical, morphological, and mechanical changes using Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), gel permeation chromatography (GPC), scanning electron microscopy (SEM), and compressive loading. A functional analysis of the scaffolds was examined using two test cell models: P19 embryonal carcinoma cells and human mesenchymal stem cells, which were both induced to undergo cardiomyogenic differentiation using the hanging drop method. Proliferation rate and protein expression for cardiac markers was analyzed. PLCL and PLGA undergo bulk degradation, while PGS and PU degrade by surface erosion. PLCL and PLGA have a rapid decrease in the compressive modulus (CM), while PU and PGS generally have a more linear decrease in CM. In both PLGA and PLCL, polymer chain scission can be tracked by the O-H stretch (3200-3400 cm-1). While no chemical changes were observed by ATR-FTIR for PU, surface cracks were observed by SEM, indicating surface damage. Shifts in glass transition and melting temperatures were observed with DSC, and decreases in molecular weights were examined with GPC. Cell analysis revealed that cells were able to adhere and proliferate on each of the scaffolds, but cardiac protein markers were expressed only on PGS and PU, as expected, given the compressive modulus of the materials. Understanding biodegradation dynamics is an important element in scaffold design, as the byproducts of biodegradation may have localized effects on adherent cells. This study shows that all scaffolds undergo marked changes in mechanical and/or chemical properties during degradation.
9:00 PM - KK3.34
Lubricin as a Novel Protein Coating to Prevent Bacterial Biofouling.
George Aninwene 1 , Erik Taylor 1 , Amy Mei 1 , Gregory Jay 2 , Thomas Webster 1 3
1 School of Engineering , Brown University, Providence, Rhode Island, United States, 2 Emergency Medicine, Brown University, Providence, Rhode Island, United States, 3 Orthopaedics, Brown University, Providence, Rhode Island, United States
Show AbstractStatement of purpose: Yearly, there are over 6 million cataract surgeries worldwide that involve intraocular lenses (IOLs)(1). However, preventing post operative biofouling of these lenses remains a challenge. A major complication is bacterial infection (2). Surface modification of the IOLs may provide a solution. This study proposes the use of the anti-adhesive protein lubricin (LUB), a glycoprotein found in the synovial fluid, as a means to make polymer surfaces less prone to bacterial adhesion and proliferation, thus reducing the opportunity for post operative infection(3). Methods:Proteins: LUB used in this study was extracted from bovine synovial fluid (Pel-Freez) (4). The center domain of the lubricin protein is similar to mucin (5). Previous studies by Shi et al. indicated that mucin surface coatings could decrease bacterial adhesion by reducing the hydrophobic binding forces, and by providing a barrier, which prevents the bacteria from making close contact with the surfaces (6). Thus, bovine submaxillary mucin (BSM) (Sigma-Aldrich) was also used in these studies to determine the role this central mucin like domain of LUB may play in bacterial suppression.Bacteria Study: Staphylococcus aureus (S. aureus) (25923) and Staphylococcus epidermidis (S. epi), obtained from the ATCC (35984), were separately seeded in 96 well plates in tryptic soy broth (TSB) (Sigma Aldrich). The seeding densities of the bacteria were diluted to 1 × 10^7 bacteria/mL (as estimated by the McFarland scale). Bacteria were co-cultured with LUB or BSM. The proteins were used at a concentration of 200μg/ml. Optical density measurements were taken every 4 minutes for 24 hours, while the temperature was maintained at 37°C. Statistics: All of the above trials were performed at least in duplicate with a minimum of 3 trials. Student t-tests were used to determine statistical significance.Results and Discussion: When LUB was present in the bacteria growth media, it resulted in a decrease in S. aureus proliferation. Additionally, trials showed that BSM was able to significantly reduce bacterial proliferation of S. aureus and S. epi. Conclusions: These trials showed that both LUB and BSM in a growth solution suppressed the growth curve of S. aureus and S. epi. This study indicates that additional trials should be preformed to understand how LUB and BSM alters bacterial interactions.References: 1.L. Werner, Journal of Cataract & Refractive Surgery 33, 713 (2007).2.L. Kodjikian et al. (Assoc Research Vision Ophthalmology Inc, 2002), pp. 3717-3721.3.B. Zappone, M. Ruths, G. W. Greene, G. D. Jay, J. N. Israelachvili, Biophys. J. 92, 1693 (2007).4.G. D. Jay, D. A. Harris, C.-J. Cha, Glycoconjugate Journal 18, 807 (2001).5.G. D. Jay, J. R. Torres, M. L. Warman, M. C. Laderer, K. S. Breuer, Proc. Natl. Acad. Sci. U. S. A. 104, 6194 (Apr, 2007).6.L. Shi, R. Ardehali, K. D. Caldwell, P. Valint, Colloids and Surfaces B: Biointerfaces 17, 229 (2000).
9:00 PM - KK3.35
Bioactive Supramolecular Twin Based Linker Nanotubes/Poly(2-hydroxyethyl Methacrylate)/Nano-hydroxyapatite Composites as Orthopedic Implants.
Linlin Sun 1 , Hicham Fenniri 3 4 , Thomas Webster 1 2
1 School of Engineering , Brown University, Providence, Rhode Island, United States, 3 National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada, 4 Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, 2 Department of Orthopaedics, Brown University , Providence, Rhode Island, United States
Show AbstractLearning from the art of natural molecular structures, a variety of amphiphilic small molecules are able to form supramolecular architectures with one or two dimensions at the nanoscale. With multifunctionality and nanometer structure, DNA based supramolecules exhibit unique biological and mechanical properties, making them promising to serve as a new generation of implants. Twin-based linker molecules (TBLs), based on the guanine and cytosine motif, can also display a self-assembly process with a combination of hydrogen bonding, π-π stacking and van der Waals interactions. In aqueous solution, six small molecules can organize spontaneously into a hierarchical structure, which then, in a subsequent process, self-assemble into nanotube with a 1.1 nm inner diameter and several micrometers in length. Due to the hydrophobic inner structure and several modifiable sites, TBLs can be incorporated with small molecules or growth factors, such as dexamethasone and BMP-7 short peptides. Previously, TBLs have been demonstrated to enhance the osteoblast cell adhesion and proliferation. The optimal implant composite have adequate mechanical strength and excellent bioactivity. Especially for injured femurs or intervertebral discs, the mechanical properties of pure pHEMA are inadequate. Previous work has shown that the incorporation of inorganic materials into pHEMA improves mechanical properties. In this study, TBLs served as a bioactive addition in composites of poly(2-hydroxyethyl methacrylate) (pHEMA) and hydroxyapatite (HA) nanoparticles (TBLs/HA/pHEMA composite) for orthopedic applications. Moreover, adding HA nanoparticles is able to enhance composite compressive strength and decreased tensile strength to more closely match those properties of bone itself. The properties of this composite were characterized after the solidification process including surface morphology, mechanical properties, and cytocompatibility properties. The solidification properties of these composites were optimized. The mechanical properties of the composites increased with HA nanoparticles and decreased with water content. The addition of HA nanoparticles can achieve high mechanical strength and improve implant-bone bonding. Particularly, the composites with the highest ratio of HA nanoparticles had a compressive strength similar to that of the natural vertebral disc. TBLs and HA nanoparticles were effective in increase the bioactivity of the composites. Thus, this study provided evidence that TBL/HA/pHEMA composites are very promising injectable orthopedic implants which should be further studied. AcknowledgementThe authors acknowledge Audax Medical, Inc. for financial assistance.
9:00 PM - KK3.36
Molecular Defined Functionalized Polyurethane (PU) Surfaces - Towards a New Approach for Materials in Regenerative Medicine.
Sebastian Kruss 1 , Tobias Wolfram 2 , Joachim Spatz 1
1 New Materials and Biosystems, MPI for Metals Research, Stuttgart Germany, 2 Research and Development, KLS Martin Group, Muehlheim Germany
Show AbstractPolyurethane materials are used in a wide variety of implantable systems and technologies, e.g. stents, breast augmentation, nose surgery, and bladder reconstruction. Despite the excellent chemically control for manufacturing the bulk material and the frequently observed good biocompatibility, a major concern remain with the interface of polyurethane with the biological environment. A chemically controlled surface engineering approach could improve desired protein adsorption processes and cellular interactions within different tissues, preventing uncontrolled events of the implant-body interface especially in early stages shortly after operational procedures.To gain a better control over the polyurethane surfaces we designed different bulk polyurethane materials and developed a transfer-nanolithography technique to deposit inorganic nanoparticles with defined structural features on the polyurethane surface. Transfer of metallic nanoparticles to the polyurethane surfaces were analyzed on the optical level with electroless gold deposition on polyurethane substrates. Different nanoparticle patterns were transferred and analyzed by scanning electron microscopy (SEM). Topographical features of PU substrates were investigated by high resolution transmission electron microscopy (HR-TEM) and atomic force microscopy (AFM). Transferred metallic nanoparticles showed a high stability on polyurethane substrates even under extreme sonication conditions. In a final step, those nanoparticles were functionalized with chemical or biological entities to facilitate cellular adhesion under physiologically relevant conditions. As a proof of concept, rat embryonic fibroblast cells were cultured on a peptide functionalized polyurethane interface and investigated by scanning electron microscopy.In conclusion, we showed a feasible method for a chemically controlled manufacturing procedure of a surface-functionalized polyurethane which leads to an inorganic/organic hybrid material. This molecular functionalized construct showed good stability characteristics and in vitro biocompatibility in cell culture assays. Next steps will include improvement of the biomimetic interface and assays towards more in vivo approaches like organotypic culture conditions.
9:00 PM - KK3.37
Physical and Biological Characterization of PEGylated Peptide Modified Silica Nanoparticles.
Emily Geishecker 1 , Sabrina Jedlicka 1 2 3
1 Department of Materials Science and Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania, United States, 3 Center for Advanced Materials and Nanotechnology, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractCellular membranes contain surface receptors capable of detecting extracellular signaling molecules. Activation of these cell membrane receptors triggers signal transduction pathways that may alter cell migration, differentiation, and other events. Nanomaterials, such as nanoparticles, have been researched for use in nanomedicine, however, little is currently known about the way nanomaterials interact with cellular receptors. The objective of this work is to design, synthesize, and characterize novel nanomaterials to investigate ligand-receptor binding. In the study that will be presented, peptide functionalized PEGylated silica nanoparticles are conjugated with molecules that mimic the natural receptor ligand in order to provide selective targeting to integrin receptors of interest. Silica nanoparticles with a narrow particle size distribution (40nm) were synthesized by hydrolysis of tetraethylorthosilicate in an aqueous L-lysine solution. This method is more benign (lower pH) than the traditional Stöber process, allowing for the addition of bioactive peptides. Nanoparticle size and morphology were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). RGD, a 10-mer peptide containing Arg-Gly-Asp from fibronectin type III, was synthesized using 9-fluorenylmethyloxycarbonyl (Fmoc) solid state synthesis. Peptide molecules are further modified with polyethylene-glycol bis(amine), molecular weight 10,000, to be used as a flexible spacer, through crosslinking with 1,5-Difluoro-2,4-dinitrobenzene. The PEG-peptide linkage was confirmed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI–TOF MS) and gel permeation chromatography. PEGylated-peptide molecules are then linked to (3-aminopropyl)trimethoxysilane (APTMS) to allow conjugation to the silica nanoparticle. Nanoparticle surfaces are decorated with the APTMS-PEG-peptide at a density of 1:1000 peptides per PEG. Peptide functionalized PEGylated nanoparticles are characterized used TEM, DLS, and atomic force microscopy. Analysis of these PEGylated peptide nanoparticles show that they have increased in size over bare nanoparticles and are compressible using AFM. Biologically, the NPs are demonstrated to be active using modified ELISA techniques, competitive binding assays and in vitro cell analysis.
9:00 PM - KK3.45
Antiadhesion Superhydrophobic Plasma-Functionalized Polyurethane Membranes and Thin Films as Anti-Infective Surfaces for Biomedical Applications.
Adriana Moraes 1 , Daniel Paptista 2 , Marcelo Costa 3 , Gabriel Soares 2 , Alexandre Macedo 4
1 Institute of Biophysics - IBCCF, Federal University of Rio de Janeiro, Rio de Janeiro Brazil, 2 Institute of Physics, Federal University of Rio Grande do Sul, Porto Alegre Brazil, 3 Department of Physics, PUC-Rio, Rio de Janeiro Brazil, 4 Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre Brazil
Show AbstractThe possibility of modifying polymers surface properties through hydrophilic/hydrophobic balance as well as by biologically active species such anticoagulants or biorecognizable groups opens up the possibility of exploiting these materials in dynamic biomedical applications, such as Guide Tissue Regeneration. In this way, anti-infective surfaces are required, reducing the bacteria biofilm formation and consequent infections. In the present contribution, a detailed investigation of the structural and physicochemical modifications of plasma-functionalized polyurethane (PU) membranes and its behavior concerning cell-material interaction is presented.The synthesis of superhydrophobic PU membranes and thin films treated by CF4 plasma is reported. The PU membranes were prepared from a 15% solution of PU in Tetrahydrofuran (THF). Transparent, cloudy and white non-transparent membranes were produced via direct drying (transparent) or phase inversion method by immersion precipitation process using nonsolvents. Thin PU films were also produced using spin coating technique. The PU membranes and the films were then subjected to CF4 plasma for 10 min. It was used 4 sccm of CF4 and a self-bias voltage of -190 V, controlled by a low input power of ~4W. The chemical structure of the bulk and surface material was characterized by attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS), respectively. The wettability of the PU surfaces was investigated through contact angle measurements. The surface roughness analyses were performed by atomic force microscopy (AFM). The results show a high improvement in the hydrophobicity of all PUs, reaching contact angle values of 130 degrees for the cloudy one. When compared with the transparent PU membrane, the cloudy and nontransparent ones presented both higher contact angle and RMS roughness, before and after the plasma treatment. The content of fluorine species attached to the surface after plasma treatment was analyzed through the C1s XPS spectrum. The CF, CF2 and CF3 species were detected after plasma functionalization. The roles of both roughness and chemical functionalization on the wettability of the PU membranes and thin films are described through a theoretical model. Finally, the antiadhesion performance against Staphylococcus epidermidis is presented.
9:00 PM - KK3.46
Peptide-Nanotube Reinforced Polymers: A Two-Component System for Tunable, Composite Materials.
Daniel Rubin 1 , Jia Jia Zhang 1 , Neel Joshi 1
1 School of Engineering and Applied Sciences, Harvard University, Somerville, Massachusetts, United States
Show AbstractThe ability to make biodegradable materials whose mechanical, physical, and chemical properties can be precisely tuned at the nano-scale is highly relevant to advances in technologies for tissue engineering, wound dressings, medical sutures, filtration devices, and textiles. The following work showcases a biologically inspired approach to synthesizing mechanically strong and moldable poly(caprolactone) (PCL) fibers from the in situ self-assembly of peptide nanotubes within the polymer matrix. High-modulus peptide nanotubes were composed of supra-molecular assemblies of D,L-cyclic peptides (DLCPs), which were then co-spun with PCL via a rotary jet spinning apparatus. Upon fiber formation, partial alignment of the polymer chains induced peptide nanotube anisotropy, resulting in a fiber that is mechanically reinforced by the high-stiffness nanotube additive. The fibers have been characterized to determine their composition, response to various mechanical deformations, and the results are correlated with the structural characterizations to assess the degree of nanotube assembly and orientation within the fiber. As the fibers are biodegradable, and therefore suitable for in vivo applications, their ability to support cell growth in vitro has been established. It is expected that the same basic methodology developed in these studies will be widely applicable to the mechanical reinforcement of a variety of polymer fibers with straightforward, scalable chemistry, and furthermore, as a building block to functional biocompatible fibers with the ability to conduct electricity, mineralize inorganics, or display biologically relevant epitopes.
9:00 PM - KK3.47
Mechanical Properties of Nanofiber Hydrogel Composites for Nucleus Pulposus Tissue Engineering.
Daniel Strange 1 , Michelle Oyen 1
1 Department of Engineering, Cambridge University, Cambridge United Kingdom
Show AbstractTissue engineering offers a paradigm shift in the treatment of back pain. Engineered intervertebral discs could replace degenerated tissue and overcome the limitations of current treatments, which substantially alter the biomechanical properties of the spine. The center of the disc, the nucleus pulposus, is collagen-proteoglycan composite with a significant bound water content. It can resist substantial compressive loads. The nucleus pulposus is a mechanically functional tissue and a successful tissue engineered construct will need to demonstrate similar mechanical properties. Hydrogels, which have a significant water content, have frequently been considered as substitutes for the nucleus pulposus but they lack much of the structural complexity of the native tissue. Here, randomly aligned polycaprolactone electrospun nanofibers are mixed into gelatin and agar hydrogels to form novel composite materials that mimic the fiber-reinforced structure of the nucleus pulposus. These composites are swollen in saline for 24 hours and the time-dependent behavior of the composites is characterized using compression and indentation testing and compared to that of pure hydrogels. Agar and gelatin were shown to demonstrate significantly different time dependent and swelling behavior. Composite models predict that fiber reinforcement can restrict the swelling of the hydrogel and as well as increasing its stiffness. These composites offer a new method of creating biomimetic scaffolds that more closely mimics the composite structure of native tissue.
9:00 PM - KK3.5
Synthesis and Characterization of Polycaprolactone Grafted Gelatin Copolymers.
Zichen Fang 1 , Yakai Feng 1 , Heyun Wang 1 , Jintang Guo 1
1 , tianjin university, Tianjin China
Show AbstractPolycaprolactone (PCL) is an excellent biomedical polymeric material which has low biodegradability, unique biocompatibility and permeability. PCL [1] has been widely used in drug delivery systems, controlled release matrix, polymer blending and polymer networks due to its high compatibility with other polymers. Gelatin [2] is a natural biopolymer which is extensively used in medical products. Gelatin [2, 3] has been widely applied because of not only high biocompatibility, biodegradability and bioactivity[3], but also reactive -OH groups. However, gelatin is lack of toughness and plasticity. In this work, a new grafting route was used to modify gelatin with the hydrophobic synthetic biodegradable polymer. The grafted gelatin with PCL was prepared using isophorone diisocyanate(IPDI) as intermediate [4]. Firstly, PCL with hydroxyl group was synthesized via ring-opening polymerization with n-butyl alcohol as initiator. Secondly, PCL was reacted with IPDI to generate PCL with isocyanate group (PCL-NCO). PCL-NCO was reacted with hydroxyl/amino groups of gelatin [5]. The grafting reaction was carried out in homogeneous system and yielded copolymers in anhydrous DMSO solvent. The obtained gelatin-graft-PCL copolymers were characterized by means of FTIR, DSC and XRD. The results of DSC and XRD analysis demonstrated the conjugation of PCL onto gelatin chains. The copolymers were expected as an amphoteric nature/synthetic hybrid material to have special properties and potential applications in many fields because of their biocompatibility, biodegradability and functionality. [1] E. J. Chong, T. T. Phan, I. J. Lim, Y. Z. Zhang, B. H. Bay, S. Ramakrishna and C. T. Lim. Acta Biomaterialia, 2007, 3, 321. [2] H. Nagahama, H. Maeda, T. Kashiki, R. Jayakumar, T. Furuike, H. Tamura, Carbohydrate Polymers, 2009, 76, 255. [3] M. Li, Y. Guo, Y. Wei, A. G. MacDiarmid, P. I. Lelkes. Biomaterials, 2006, 27, 2705. [4] L. Liu,Y. Li, H. Liu, Y. E. Fang. European Polymer Journal 40, 2004, 2739. [5] X. K. Li, S. X. Cai, B. Liu, Z. Xu, X. Z. Dai, K. Ma, S. Q. Li, L. Yang, K. L. Paul Sung and X. B. Fu. Colloids and Surfaces B: Biointerfaces, 2007, 57, 198.
9:00 PM - KK3.6
Improved Cellular Functions on Carbon Nanofiber Cardiac Patches.
David Stout 1 , Thomas Webster 1 2
1 School of Engineering, Brown University, Providence, Rhode Island, United States, 2 Department of Orthopaedics, Brown University, Providence, Rhode Island, United States
Show AbstractIntroduction:
In recent research, it was demonstrated that the use of nano-materials can promote the growth of cardiomyocytes compared to conventional or micro-structured materials. One such nano-material consisted of poly-lactic-co-glycolic acid (PLGA) with carbon nanofibers (CNFs) where it was shown that cardiomyocytes proliferated faster on the composites compared to a pure PLGA film after 5 days.
The objective of this study was to better understand the cytocompatibility properties of this composite material for myocardial applications by using electrical stimulation which mimics that of the heart. For this reason, an in vitro continuous electrical stimulation model was used to determine cardiomyocyte functions on PLGA: CNF composite materials.
Methods:
A 4-point conductivity method, scanning electron microscopy (SEM), Raman Spectroscopy, atomic force microscopy (AFM) and X-Ray Diffraction were used to characterize the materials after preparation. This consisted of using purified CNFs (99.9% by weight %, Catalytic Materials, MA) with a diameter of 100 nm and sonicating them in 20 ml of chloroform at 20W for 30 minutes. After the PLGA and CNF solutions were prepared, various PLGA:CNF weight percent ratios were created (100:0, 75:25, 50:50, 25:75, 0:100) by adding the appropriate amount of CNF to PLGA in 20 ml disposable scintillation vials.
To investigate cytocompatibility properties, human cardiomyocytes (Celprogen, Cat #36044-15, USA) were seeded onto PLGA:CNF composites in complete growth media supplemented with fetal bovine serum and antibiotics (Celprogen, Cat #M36044-15S, USA) at a density of 10 x 104 cells/cm2 and were continuously stimulated (rectangular, 2 nm, 5 (V/cm) , 1 Hz) for 1, 3, and 5 days. MTT, Triponan I, Triponin T, and PDGF-BB ELISA assays were completed to analyze cytocompatibility and cell viability on the composites. All experiments were performed at least in triplicate and results were compared to their non-electrically stimulated PLGA:CNF composite counterparts. When data were compared, ANOVA software and a student T-test were used. A p-value of < 0.05 was considered to be significant.
Results and Discussions:
Compared to non-electrical stimulation, results of this study provided evidence that electrical stimulation increased cardiomyocyte density on all PLGA:CNF composite ratios as well as the synthesis of the cardiac protein biomarker Troponin I (with the 50:50 [PLGA:CNF (wt:wt) composite having the highest cell density and Troponin I levels). This indicated that PLGA:CNF composites promoted cardiomyocyte proliferation and differentiation in an electrically stimulated human heart environment as well as increased heart cell viability via increasing Troponin I protein synthesis.
Acknowledgments
The authors would like to thank the Hermann Foundation and The National Science Foundation (NSF) Graduate Student Fellowship (NSF#09603)
9:00 PM - KK3.7
A New Photopolmerizable Hyaluronan-Based Hydrogel for Stem Cell Culture and Bioprinting.
Thomas Zarembinski 1 , Nathaniel Doty 1 , Hynes Richard 2 , Nathaniel Cady 2 , Michael Toepke 3 , Michael Schwartz 3 , William Murphy 3 , Caitlin Amin 1 , Sarah Atzet 1
1 , BioTime, Inc, Alameda, California, United States, 2 College of Nanoscale Science & Engineering, University of Albany, Albany, New York, United States, 3 Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States
Show AbstractAn emerging theme in 3D cell culture is its miniaturization which has important implications in future stem-cell based drug discovery and tissue engineering. The small size of each 3D cell droplet in an array enables drug screening in 3D using automated microscopy and imaging techniques. Miniaturization also enables layer-by-layer biofabrication of tissues by depositing stem or progenitor cells in small volumes in specific 3D patterns. The important component that enables these developments is the appropriate hydrogel matrix. It must be customizable for each cell type. In addition, it is ideally composed of hyaluronan which is important during embryogenesis. Finally, the user must have high spatio-temporal control of gelation to prevent non-uniform spotting, clogging of the spotting apparatus, or spot dehydration. Herein we describe the development of a new hyaluronan-based hydrogel system based on a commercially available hydrogel (HyStem®-C) and radical-mediated thiol-ene step growth photopolymerization. UV light exposure causes gelation to occur in less than one minute with shear elastic modulus G’ greater than 2 kPa. The resulting hydrogels support stem cell growth in 2D and 3D for eventual drug discovery or tissue engineering applications.
9:00 PM - KK3.9
Silk Hydrogels and Nanoparticles as Bio-Functional Materials.
Keiji Numata 1
1 , RIKEN, Saitama Japan
Show AbstractSilk fibroins have been successfully used in the biomedical field as sutures for several decades, and have also been explored as biomaterials for cell culture, tissue engineering, and drug delivery systems, earning Food and Drug Administration approval for such expanded utility because of their excellent mechanical properties, versatility in processing and low cytotoxicity [1]. In the present study, silk hydrogel and nanoparticles were developed as new types of protein-based biomaterials [2]. Hydrogel is an attractive biomaterial for regenerative medicine and tissue engineering because of its excellent biocompatibility, which is attributed to high water content of over 90%. The role of water molecules in hydrogels has been investigated by many researchers, with the result that bound, bulk, and intermediate water have been shown to exist in hydrogels. In the present study, we developed a facile and quick method to prepare silk hydrogel with ethanol, and also analyzed the gelation behavior, state of water, secondary structure and mechanical properties of the resulting hydrogel. The average molecular weight between crosslinks of the silk hydrogel prepared at different polymer concentrations was calculated and compared with the network structure of the silk hydrogels. Further, the cell proliferation and viability of human mesenchymal stem cells (hMSC) on the silk hydrogels was characterized, and then the relationship among the state of water, molecular structure and cytotoxicity of the silk hydrogel was discussed. The bulk water content of the silk hydrogel was found to be readily regulated by the concentration of silk proteins, which is helpful to investigate effects of the state of water of polymeric hydrogel on the other properties. The influence of the state of water in the silk hydrogel on the cytotoxicity was recognized by means of differential scanning calorimetry and cell viability assay. Based on the results, the bound water is considered to support cell-adhesion proteins in the cellular matrix to interact with the surface of the silk hydrogels. On the other hand, the bulk water would disturb the cell-adhesion proteins to adhere on the surface of the silk hydrogels, due to relatively higher mobility of water. This new insight into the state of water of hydrogels provides options to design hydrogel-based biomaterials to form cell-interactive biointerfaces. To add a new function to release several kinds of chemicals and peptides, silk nanoparticles were prepared and incorporated into the networks of the silk hydrogel. The diameter and secondary structures of silk particles significantly influenced on the release behaviors, which indicates that the combination of silk hydrogels and nanoparticles shows a potential of multi-step releases of bioactive molecules from silk-based materials.[1].Numata, K.; Kaplan, D. Adv. Drug. Deliv. Rev. 62, 1497, 2011.[2].Numata, K.; Katashima, T.; Sakai, T. Biomacromolecules in press.
Symposium Organizers
Mei Wei University of Connecticut
Jordan Green The Johns Hopkins University
Xinqiao Jia University of Delaware
James Olson Teleflex Medical
KK4: Soft Tissue Regeneration
Session Chairs
Tuesday AM, November 29, 2011
Room 102 (Hynes)
9:30 AM - **KK4.1
Silks as Fusion Proteins and Carriers as New Functional Biomaterials.
David Kaplan 1
1 Biotechnology Ctr Rm 153, Tufts University, Medford, Massachusetts, United States
Show AbstractSpider and silkworm silks provide the basis for self-assemblying and robust materials. As such these novel proteins from nature provide an important starting point for the design of new material systems that offer functional utility in many areas of materials science and engineering, including strong and tough material systems, optical and electronic platforms, and biomaterials for implants for regenerative medicine. Two emerging opportunities that build upon these features include expanded functional features in the silk polymers, and the use of the silk nanoassembled structures to entrap and stabilize labile molecules. Genetic control of silk sequence and chemistry allow for the directed addition of new features to the protein as fusion or chimeric systems. The challenge is to add these new capabilities without disrupting native silk material properties. Examples of recent success toward this challenge include metal binding domains, antimicrobial peptides and cell membrane targeting domains. The utilization of the silk protein to entrain labile substrates has also been found to lead to remarkable stabilization of these compounds. This approach exploits the unique block copolymer nature and low water content of silk, resulting in the ability to sequester, stabilize and store a range of bioactive compounds with remarkable robustness. In both of these approaches, the unique structure and chemistry of silks is exploited for basic materials understanding and foundation, while new functions are added to the silk material systems.
10:00 AM - KK4.2
Tissue Engineering the Retina: Soft Lithography-Patterned Microchannel PLGA Scaffolds Guide the Morphogenesis of Dissociated Newborn Mouse Retinal Cells and Human Embryonic Stem Cell-Derived Retinal Cultures.
Andrew McUsic 1 3 , Thomas Reh 2 3
1 Department of Bioengineering, University of Washington, Seattle, Washington, United States, 3 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States, 2 Department of Biological Structure, University of Washington, Seattle, Washington, United States
Show AbstractRetinal photoreceptor degenerations cause blindness for millions worldwide. In vitro models of these diseases lag behind advances in high-throughput screening methods, which, given an adequately representative model of photoreceptor degeneration, may identify anti-degenerative pharmaceuticals. While the advent of stem cell technologies and subsequent application of retinal differentiation protocols to derive photoreceptors promises a cell source for enhanced in vitro models, a valid “disease-in-a-dish” requires the construction of photoreceptors and Müller glia into an array of three-dimensionally aligned units that more faithfully mimics the in vivo outer retina. We report the first attempt to solvent-process poly(lactic-co-glycolic acid) (PLGA), a common biomaterial, into a microchannel scaffold format using soft lithography to achieve this retinal geometric constraint. We compared the effect of PLGA concentration on channel array morphology and, along with other culture conditions, on the infiltration of dissociated newborn Nrl-GFP mouse retinal cells into the channels. PLGA was nontoxic to dissociated photoreceptors, causing apoptosis in less than 1% of rods. When the cell-seeded microchannel scaffolds were cultured submerged under media, cell infiltration was low, with 1.45 +/- 0.40 cells per channel populating the 5% PLGA scaffolds after three days of culture, decreasing to 0.89 +/- 0.08 cells per channel after seven days. This effect was exacerbated by higher PLGA concentrations, suggesting compromised availability of dissolved oxygen. By contrast, culturing seeded scaffolds on filter inserts at the gas-liquid interface significantly increased infiltrated cell viability, resulting in 12.3 +/- 1.70 cells per channel after three days and 6.61 +/- 0.36 cells per channel after seven days. Rod photoreceptors and Müller glia aligned and extended processes parallel to the microchannel walls. Otx2+ and Pax6+ subpopulations from dissociated retina migrated to different depths inside the scaffolds, recapitulating in vivo retinal lamination behavior. Next, we illustrated the feasibility of constructing photoreceptor-loaded scaffold/retinal pigment epithelium (RPE) explant sandwiches and observed rods extending rhodopsin-positive processes toward RPE cells. Finally, human embryonic stem cell-derived photoreceptors exhibited infiltration and morphological characteristics similar to mouse retinal cells inside the scaffolds. These findings further efforts to generate tissue-level retinal models from dissociated cells for use in a variety of applications. [This work was supported by the Foundation Fighting Blindness Wynn/Gund Translational Award, National Institutes of Health Grants R01 EY013475, R01 EY021482, 1P01GM081619, and by a National Science Foundation Graduate Research Fellowship. Part of this work was conducted at the University of Washington NanoTech User Facility, a member of the NSF National Nanotechnology Infrastructure Network (NNIN).]
10:15 AM - KK4.3
Encoding Cell-Instructive Cues to PEG-Based Hydrogels via Triple Helical Peptide Assembly.
Patrick Stahl 1 , S. Michael Yu 1
1 Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractOne of the challenges in the design of successful artificial cell/tissue scaffolds is the incorporation of insoluble bioactive molecules that induce cell adherence, proliferation, and differentiation. PEG-based hydrogels, produced by photocrosslinking PEG diacrylate (PEGDA) macromers into a covalently linked network, is commonly used for 3D cell encapsulation. However, hydrophilic PEGDA exhibits almost no biological activity on its own, and incorporating bioactive components to these scaffolds is essential for culturing various mammalian cells. In this work, we employed collagen mimetic peptide’s (CMP) strong propensity to form a triple helix as a source of binding to incorporate cell adhesive peptides to PEGDA hydrogels. PEGDA hydrogels displaying CMPs were synthesized by co-photopolymerizing PEGDA and CMP-conjugated PEG acrylate, and the cell adhesive peptide (RGD sequences) were encoded into these hydrogels by addition of CMP-RGD to preformed hydrogels. We demonstrated successful adhesion and proliferation of both human fibroblasts and endothelial cells seeded on the PEGDA-CMP gels modified with CMP-RGD. This CMP-mediated physical assembly strategy more closely mimics extracellular matrix protein interactions found in nature. Furthermore, unlike conventional hydrogels in which RGD sequences are permanently photocrosslinked into the PEGDA gel, our encoding approach creates opportunities to use the same base polymerized PEGDA-CMP gel and to subsequently modify the scaffold with different types,concentrations, or locations of cell adhesive CMPs. These capabilities may lay the foundation for materials that promote generation of complex and spatio-temporally defined artificial tissues for regenerative medicine.
10:30 AM - KK4.4
Spatially Defined Microenvironments to Study and Engineer Vasculature in Health and Disease.
Laura Dickinson 1 , Matthew Moura 1 , Sharon Gerecht 1
1 Chemical and Biomolecular Engineering, Johns Hopkins Universtiy, Baltimore, Maryland, United States
Show Abstract Engineering functional vascular networks is essential in the field of regenerative medicine as a vital component in developing successful, integrated tissue constructs. Indeed, spatial control in guiding cell adhesion and growth is critical in tissue engineering applications. The extracellular matrix (ECM) provides biochemical and physical cues that dynamically influence tissue formation and vascular growth. To clarify the signaling pathways that regulate and control vascular network formation, we study the effects of spatio-temporal microenvironmental cues, specifically ECM components, during endothelial progenitor cell (EPC) maturation. Fibronectin (Fn) is one ECM protein known to enhance endothelial cell growth, adhesion, and tubulogenesis during vascular development. We previously demonstrated that optimized, patterned Fn surfaces guide the ordered adhesion of human EPCs, support their elongation and assemble into unidirectional chains/tubular structures within a three-dimensional fibrin gel. Angiogenesis is also a critical component of tumor progression. The ability of a tumor to achieve sustained vascularization is one of essential hallmarks of cancer biology that dictates malignancy. Tools for interrogating the pathways of vascular network formation associated with tumor progression are needed to better define the mechanisms that control tumor cell growth and invasion. To further elucidate the mechanisms of tumor angiogenesis, we have developed both 2D and 3D in vitro systems to study the intercellular interactions between endothelial and cancer cells. A dual patterning approach allows the discrete molecular presentation of both Fn and hyaluronic acid in defined. Hyaluronic acid (HA) is one stromal component of the ECM that has been specifically associated with facilitating tumor cell growth by enhancing cancer cell motility, invasion, and angiogenesis. Preferential adhesion to the 2D patterned surfaces (EPCs to Fn; cancer cell phenotypes to HA) allows a unique opportunity to characterize the dynamic interactions between the two cell types. In an extension of this co-culture approach, 3D intercellular interactions are also being investigated. To do this, cancer cells are embedded in patterned arrays of HA hydrogels adjacent to patterned EPCs.
10:45 AM - KK4.5
Nanoceria Interactions with Cardiac Progenitor Cells: Protection against Oxidative Stress.
Enrico Traversa 1 , Francesca Pagliari 2 , Corrado Mandoli 1 , Stefania Pagliari 2 , Giancarlo Forte 1 , Paolo Di Nardo 1
1 MANA, NIMS, Tsukuba, Ibaraki, Japan, 2 Laboratory of Cellular and Molecular Cardiology, Univ. Rome Tor Vergata, Roma Italy
Show AbstractOver the last few years, regenerative medicine is taking advantage of nanotechnology. The extensive use of nanoparticles, standing alone or as fillers of polymeric scaffolds for tissue engineering, has disclosed a new generation of nano-biomaterials for medical applications. Consequently, studying how cells interact with the novel nano-biomaterials is crucial for tailoring reproducible and promising bioactive properties. The trend is towards the development of bioactive rather than bio-inert materials, with materials directly triggering or participating to cellular reaction pathways.Cerium oxide nanoparticles have been recently reported to show outstanding biomedical activity [1] and potential pharmacological applications [2] due to redox changes in the Ce oxidation state (Ce4+/Ce3+) that trigger the abatement of intracellular reactive oxygen species (ROS), hindering the oxidative stress cytotoxic effects [3]. These properties have attracted the use of nanoceria in bone tissue engineering [4]. We recently demonstrated that ceria nanoparticles embedded into PLGA scaffolds favor and direct cardiac progenitor cell (CPC) adhesion and proliferation, as compared with PLGA alone [5]. Here we will present the Linneg/Sca-1pos cardiac progenitor cell response in terms of cyto-compatibility, cell morphology and phenotype, and cell differentiation and multipotency to ceria nanoparticle exposure. In particular, we investigated the possibility that nanoceria could protect CPCs from oxidative stress resulting by ROS production. The study showed that 24 hour exposure to 10, 25, and 50 μg mL-1 of nanoceria did not affect CPC survival and function. On the contrary, all concentrations were effective in protecting CPCs from water peroxide insults even after 7 days from the nanoceria exposure, which could be ascribed to the efficient antioxidant mechanism elicited by ceria nanoparticles. These results unravel enormous possibilities for the use of nanoceria in cardiac tissue regeneration therapies. References[1] A. Karakoti, S. Singh, J. Dowding, S. Seal, W. Self, Chem. Soc. Rev., 39 (2010) 4422.[2] I. Celardo, J.Z. Pedersen, E. Traversa, L. Ghibelli, Nanoscale, 3 (2011) 1411.[3] I. Celardo, J.Z. Pedersen, E. Traversa, L. Ghibelli, ACS Nano, DOI: 10.1021/nn200126a.[4] A. Karakoti, O. Tsigkou, S. Yue, P.D. Lee, M.M. Stevens, J. Jones, S. Seal, J. Mater. Chem., 20 (2010) 8912.[5] C. Mandoli, F. Pagliari, S. Pagliari, G. Forte, P. Di Nardo, S. Licoccia, E. Traversa, Adv. Funct. Mater., 20 (2010) 1617.
11:30 AM - **KK4.6
Building 3D Tissue Engineered Scaffolds for Soft Tissue Regeneration.
Sha Jin 1 , Kaiming Ye 1
1 Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas, United States
Show AbstractThe success in directed differentiation of human embryonic stem (hES) cells into clinically relevant cell lineages raises new hope for cell replacement therapy for treating many diseases including diabetes. Nevertheless, the realization of these potentials, in many cases, relies upon the generation of biologically functional, i.e,, mature cells. For example, the cell-based diabetes treatment requires the transplantation of mature pancreatic cells that can form soft tissues such as islets in vivo. The generation of these soft tissues in vitro has not become possible yet. All strategies being developed so far, including stepwise hES cell differentiation strategies have failed to generate mature pancreatic cells in vitro, although maturation and physiological success can be achieved by transplantation of late-stage differentiated hES cells in mouse. While many factors may contribute to these failures, the lack of tissue niches in the current differentiation systems play a big role in impairing hES cell maturation to soft tissue cells such as beta cells. So far, most works have been focused on one or two types of tissue components such as cells, hormones, or signaling molecules. However, in vivo tissue functionality relies upon (at least) three properties: i.e., cell type, extracellular matrix (ECM) or scaffolds, hormones and/or other signaling molecules. Thus, a three rather than two-dimensional environment appears essential for progenitor differentiation and maturation. We have developed a 3D ES differentiation system and demonstrated that the maturation of pancreatic cells differentiated under 3D conditions can be significantly elevated. Our experimental results suggest that 3D scaffolds offer adequate physiochemical stimulations that encourage cell-cell and cell-matrix interaction in the stepwise pancreatic endoderm differentiation process. Such physiochemical niches promote pancreatic cell maturation.
12:00 PM - KK4.7
Direct Laser Writing of Polylactide 3D Scaffolds for Neural Tissue Engineering Applications.
Vaso Melissinaki 2 , Andrew Gill 1 , Ilida Ortega 1 , Maria Vamvakaki 2 , Anthi Ranella 2 , Costas Fotakis 2 , John Haycock 1 , Maria Farsari 2 , Frederik Claeyssens 1
2 Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Heraklion Greece, 1 Materials Science and Engineering , University of Sheffield, Sheffield United Kingdom
Show AbstractWe report on the fabrication of high resolution 3D scaffolds of polylactide-based materials using direct laser writing and we explore their use as neural tissue engineering scaffolds. In this study we investigate the relationship between scaffold topology and cell growth of neural cells on 3D scaffolds fabricated using Direct fs Laser Writing (DLW) of a poly-lactide-based material.[1] DLW has been demonstrated as a technology for the fabrication of 3D structures with high resolution. The technique is based on the phenomenon of multi-photon polymerization. When the beam of an ultrafast infrared laser is tightly focused into the volume of a photosensitive material, the polymerization process can be initiated by nonlinear absorption within the focal volume. By moving the laser focus three-dimensionally through the photosensitive material, 3D structures can be fabricated. Recently, we explored DLW for use in the construction of biodegradable scaffolds using a poly-caprolactone based photopolymer.[2]Here, we describe the synthesis and use of a polylactide-based (PLA) photopolymer and we show that it can be structured accurately in three-dimensions via DLW. We report the fabrication of high-resolution 2.5D and 3D structures and we study neuronal cell growth on these structures, to specifically assess neurocompatibility of the used materials. Additionally, we highlight the use of these structures for studying aligned neuronal growth and neuronal cell ingrowth in 3D microstructures. This study indicates that DLW is a feasible fabrication route for manufacturing 3D tissue engineering scaffolds with reproducible feature shape and sizes.[1] A.A. Gill and F. Claeyssens:“3D structuring of biocompatible and biodegradable polymers via stereolithography.” Methods in molecular biology (Clifton, NJ), 695:309-321 (2010)[2] F. Claeyssens, et al. "Biodegradable Structures Fabricated by Two-Photon Polymerization," Langmuir 25, 3219-3223 (2009)
12:15 PM - KK4.8
Modification of Natural Collagen Scaffolds with Insoluble VEGF Mimetic Peptide for Localized Endothelial Cell Activation.
Tania Chan 1 2 , S. Michael Yu 1 2
1 Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractThe control of angiogenesis is vital to tissue engineering. Long term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial cell differentiation and vasculature formation are dependent on spatio-temporal cues in the extracellular matrix that dynamically interact with cells, a process difficult to reproduce in artificial systems. Here, we present a novel bifunctional peptide that mimics insoluble vascular endothelial growth factor (VEGF) and can encode spatially controlled angiogenic signals in collagen substrates. The peptide, QKCMP, is comprised of a VEGF mimetic domain with pro-angiogenic activity, and a collagen binding domain that can bind to type I collagen through a unique hybridization mechanism. We first confirm the triple helical structure and the collagen binding affinity of the collagen mimetic domain through circular dichroism and collagen binding studies, and we then demonstrate the peptide’s ability to induce endothelial cell morphogenesis and network formation as an insoluble factor in 2D and 3D collagen scaffolds. Furthermore, we show spatial modification of collagen substrates with the peptide, which induces localized endothelial cell activation and network formation in pre-defined areas within wider cell constructs. These results demonstrate that the peptide can be used to present spatially directed angiogenic cues in collagen scaffolds, which may be useful for engineering organized microvasculature that are more akin to natural tissues.
12:30 PM - **KK4.9
Biomaterials as Scaffolds and Bilogics Carriers for Tissue Regeneration Applications.
Timothy Sargeant 1 , Arpan Desai 1 , Saumya Banerjee 1 , Angela Throm 1 , Yves Bayon 2 , Michael Fehlings 3 4 5 , Steven Bennett 1 , Kevin Lavigne 1 , Nathaniel Mast 1 , Marisha Godek 1
1 R&D, Covidien Surgical Devices, North Haven, Connecticut, United States, 2 R&D, Covidien Surgical Devices, Trevoux France, 3 Genetics and Development, Toronto Western Research Institute, Toronto, Ontario, Canada, 4 Krembil Neuroscience Centre, University Health Network, Toronto, Ontario, Canada, 5 Division of Neurosurgery, University of Toronto, Toronto, Ontario, Canada
Show AbstractIn recent years tissue regeneration strategies have been pursued as an approach to solve a number of clinical diseases and injuries in which the body cannot heal itself. Biomaterials play a critical role in the regenerative medicine paradigm, acting as scaffolds for cells to reproduce extracellular matrix and carriers for cells and biologically-active factors. There are many clinical applications for these constructs each with unique and differing requirements, including functionality, mechanical loads, cell population, and extent of vascularity, to name a few. Consequently, a variety of materials and technologies have been developed and coupled to unique strategies to design prototypes that may be used to regenerate and heal cartilage, spinal cord injuries, complex abdominal wall defects, and other soft tissue defects among others. Herein we discuss several efforts to translate unique and novel technologies in the regenerative medicine space to develop products that may address some of the injuries and defects mentioned above and potentially improve patient outcomes. In particular, we present initial results towards developing therapeutic injectable hydrogels for spinal cord injury, biosynthetic adhesive materials to facilitate tissue adhesion and regeneration in soft tissue repair applications, and in situ forming hydrogels with and without interpenetrating networks with different gelation mechanisms to create tailored structures for complex defects and drug delivery. These results will include robust mechanical and chemical characterization, standard and novel benchtop in vitro analysis, and pre-clinical evaluation of these prototype devices.
KK5: Hard Tissue Regeneration
Session Chairs
Xinqiao Jia
Chris Sorrell
Tuesday PM, November 29, 2011
Room 102 (Hynes)
2:30 PM - **KK5.1
Murine Models for Evaluating Scaffold-Directed Bone Repair.
David Rowe 1 , Xi Jiang 1 , Liping Wang 1 , Seung-Hyun Hong 2 , Dong-Guk Shin 2
1 Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut, United States, 2 Computer Science, University of Connecticut School of Engineering, Storrs, Connecticut, United States
Show AbstractOur objective is to develop a rapidly performed, high content and relatively low cost histology for assessing skeletal repair that can be used by a tissue engineering research team that iteratively uses the information to make modifications to their repair strategy. The platform employs multiplexed GFP reporter mice in which the fluorescent signal can be interpreted as a cell at a specific stage of differentiation utilizing a frozen, non-decalcified histology that has minimal background fluorescence. Highly automated scanning fluorescence microscopy has been adapted to capture a high power image of the entire repair field that allows the user to examine the outcome of the repair on their PC as if they were examining the section on the fluorescent microscope. A bilateral calvarial defect and segmental long bone repair model has been established in which the host mice carry a green reporter while the donor cells carry a blue reporter. One day prior to sacrifice, the animals are injected with alizarin complexone (AC) to label active bone forming surfaces. The histological analysis will identify active bone matrix forming cells on the bone surface and whether they are of host or donor origin. Additional fluorescent stains for alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) as well as a traditional hematoxylin stain can be layered over the GFP/AC image to provide greater context in the interpretation of the repair.Most of our effort with these models has focused on identifying a source of osteoprogenitor cells that reliably leads to bone healing of a critical sized defect. These studies have utilized a commercial collagen/hydroxyapatite scaffold (Healos) that is permissive to bone formation by inoculated progenitor cells and during the repair process the scaffold is resorbed. Progenitors derived from bone marrow stromal cultures generate a cortical like bone with ample marrow that integrates with the host bone periosteal cells that bridge a long bone segmental defect. In contrast, neonatal calvarial derived or bone chip out growth progenitors make a disorganized membraneous bone that integrate poorly with host bone. Fresh bone marrow and adipocytic stromal cells lack inherent osteogenic activity and no evidence for circulating progenitors can be demonstrated.Because we have been using the Healos as the scaffold for developing a reliable progenitor population, we can use it as a reference against which other scaffolds are compared when inoculated with a common source of progenitors. We have begun to provide this comparison to collaborators with various scaffolds designs. An image analysis program has been developed, based on principles of dynamic bone histomorphometry, to provide a quantitative comparison of the test scaffold relative to the reference. We hope this common imaging and quantitative platform will be of value to the science of tissue engineering in assessing competing bone repair strategies.
3:00 PM - KK5.2
Self-Powered Fluidic Supply Channels in Bone Implant.
Hae Lin Jang 1 , Kyoungsuk Jin 1 , Ki Tae Nam 1 , Jimmy Xu 2 1 , Kug Sun Hong 1
1 WCU program, Material Science Engineering, Seoul National University, Seoul Korea (the Republic of), 2 Engineering and physics, Brown university, Providence, Rhode Island, United States
Show AbstractWe present the design and operation of a micro to nano scale fluidic system built into in a ceramic bone implant. The system is inspired by the capillary force and transpiration of the plant, which can pull and transport water to the highest points of its xylem. Channels in trees decrease their diameter with height, finally approaching nano-meter sizes and enabling strong capillary force. Amazingly, the human circulatory system also shows similar organization. In very thin capillaries, cells create vacuum instantaneously via suction to circulate body fluids through capillary forces. This attractive mechanism can be applied to bone implants to create self-powered supply that can pump body fluid and nutrients throughout their structures. Interestingly, pores in living bone show a decrease in their diameter from the inner to outer wall. To realize self-powered fluidic supply structures in bone implants, a gradient in mechanical pressure was exerted on hydroxyl apatite (HAP) and PEG mixtures during sintering. As a result, networks of pores were formed in the composite with a continuous variation in pore sizes, developing a pore diameter gradient that was found to be opposite to the applied mechanical pressure gradient. This counter-intuitive anti-correlation between the pore size and mechanical pressure gradient may be attributed to phase separation between HAP and PEG, where applied pressure during the sintering process was found to be critical in the formation of the porous channels. Fluidic tests were performed on both conventional solid HAP samples and HAP with built-in pore gradients. The results confirm that bone implant sample with pore gradients draw up fluids fastest and highest when the end of the sample with the larger pore sizes of the gradient was immersed in the liquid compared to solid and inverted pore gradient samples. This study sheds light on a function inherently available in the porosity of bone, one that can facilitate healing and regeneration as well as improves toughness-weight ratio, to inspire new implant design and improved utility. We thank the WCU program and AFOSR for supporting this work.
3:15 PM - KK5.3
In Vitro Cellular Response of Osteoblast Cells on Bioactive Alumina Fibrous Scaffolds.
Santanu Dhara 1 , Saralasrita Mohanty 1 , Arun Prabhu Rameshbabu 2 , Pallab Datta 1 , Paulomi Ghosh 1
1 School of Medical Science and Technology, IIT Kharagpur, Kharagpur, West Bengal, India, 2 School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu, India
Show AbstractBone aging has been associated with osteoporosis and it is often considered as a disease of cancellous bone. The loss of bone density is mainly due to osteogenic cell depletion. Autografts, homografts and xenografts are generally used to repair the bone tissue. But due to the risk of disease transmission, complicated multistage surgery and limited availability of materials, there is a need for the development of synthetic porous bioactive scaffold. In this context, ceramics fibers are the preferred choice for the fabrication of porous scaffold for bone tissue engineering application due to their biocompatibility, etchability, and mechanical stability. Alumina based slurries were prepared in 2 vol% aqueous acetic acid medium up to 33.3 wt% powder loading at pH ~ 6.5. The slurries were blended with 4 wt% chitosan in 2 vol% acetic acid. The slurries were extruded into ceramic fibers using a spinneret of 100 µm pore size in sodium tripolyphosphate (STPP) bath due to ionotropic gelation. Green fibers of equal mass were packed into propylene moulds and then sintered at 1550 °C to obtain porous cylindrical scaffolds with average crushing strength of 0.9 MPa for density of 0.9 g/cc. The sintered scaffolds were found to have open, interconnected pores favourable for tissue ingrowth with a compressive strength similar to cancellous bone. The response of MG-63 cells, to these scaffolds was evaluated using assays of MTT and alkaline phosphatase (ALP) activity. Rhodamine phalloidin and DAPI staining were performed to visualise the cells attached to the scaffold. Scanning electron micrographs showed a closely adhering, well-spread morphology of MG-63 cells seeded on the scaffold. These results indicate that alumina fibrous scaffolds are a favourable substrate for the growth and differentiation of osteoblasts cells and a promising material for bone repair applications in vivo.
3:30 PM - KK5.4
Influence of CaP on Bone Formation and Vascularization.
Samantha Polak 1 , Amy Wagoner Johnson 2 1
1 Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 2 Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
Show AbstractCalcium phosphate (CaP) based materials are currently used as scaffolds to help restore bone in dental, craniofacial, and orthopedic procedures. CaP scaffolds can be fabricated with well controlled macroporosity (>100µm) and microporosity (<20µm). The necessary scaffold macroporosity for transport of nutrients and removal of waste from the defect site is well established. The optimum micropore fraction and diameter are less clear. Microporosity has been shown to positively influence bone formation, though the mechanism behind the influence is unknown. In a recent in vivo study, we demonstrated that CaP scaffolds containing 50% microporosity (MP) implanted in porcine mandibles resulted in an increase in both bone volume and uniformity of bone distribution over non-microporous (NMP) scaffolds. This suggested that a vascular network had formed throughout the MP scaffolds to support the bone tissue. In order to explore and understand the effects of CaP and material characteristics, multiple systems must be investigated. We have investigated the effect of microporosity and CaP both in terms of bone formation and vascularization.To investigate the effect of CaP on vascularization, we performed chorioallantoic membrane (CAM) assays using white leghorn chicken embryos. The ex ovo method was used to allow for longitudinal observation of vessel formation. CaP was applied in various patterns to assess its influence on both vessel quantity and the directionality. An automatic segmentation program was developed to aid in quantification and analysis. Results indicate migration of vessels to the CaP. Additionally, samples were visualized using SEM and histological techniques. Results also show strong adhesion of vascularized CAM to the CaP.
3:45 PM - KK5.5
Bioengineering Single Crystal Growth.
Ching-Hsuan Wu 1 , Regina Knapp 1 , Derk Joester 1
1 Materials Science, Northwestern University, Evanston, Illinois, United States
Show AbstractWhile the production of food, biochemicals, and pharmaceuticals by biotechnological means has reached a high level of sophistication, the development of (bio)materials by similar approaches is still in its infancy. Biomineralization is a “bottom-up” synthesis process that results in the formation of inorganic/organic nano-composites with unrivaled control over structure, superior mechanical properties, adaptive response, and the capability of self-repair. While de novo design of such highly optimized materials, for example for reconstructive medicine, may still be out of reach, engineering of the biosynthetic machinery may offer an alternative route to design advanced materials.
Herein, we present an approach using microcontact-printed lectins for patterning sea urchin embryo primary mesenchyme cells (PMCs) in vitro.[1] We demonstrate that PMCs cultured on these substrates not only show attachment to wheat germ agglutinin (WGA) and concanavalin A (ConA) patterns, but more importantly, that the deposition and elongation of calcite (CaCO3) spicules occurs cooperatively by multiple cells and in alignment with the printed pattern. This allows us to control the placement and orientation of smooth, cylindrical calcite single crystals where the crystallographic c-direction is parallel to the cylinder axis and the underlying line pattern. Preliminary data indicates an exiting new role of VEGF in controlling PMC mineralization and we will report on use of recombinant sea urchin VEGF to control the growth of single crystals.
(1) C.-H. Wu, A. Park, and D. Joester, J. Am. Chem. Soc. 2011, 33, 1658–1661.
4:30 PM - **KK5.6
Reaction Bonded Silicon Nitride Intervertebral Spacers: Results of 10 Year Clinical Study.
Charles Sorrell 1 , Philip Hardcastle 2 , Ross Druitt 3 , Rolfe Howlett 1 , Eric McCartney 1
1 School of Materials Science and Engineering, University of New South Wales, Sydney, New South Wales, Australia, 2 Retired, Formerly Hardcastle Pty. Ltd., Wembley, Western Australia, Australia, 3 None, Sialon Ceramics Pty. Ltd., Doyalson, New South Wales, Australia
Show AbstractDuring 1986 to 1988, 30 patients with long-term spinal degeneration problems underwent anterior interbody fusion of the lower spine (L3/4, L4/5, L5/S1) using reaction bonded silicon nitride intervertebral spacers. The patients consisted of 16 males and 14 females of ages ranging from 25 to 64 years. Patient reviews were undertaken after 1, 5, and 10 years. A control population of 10 underwent the same procedure using autologous bone.The only post-operation issue consisted of slippage of the implant in 2 patients and dislodgement in 1 patient. No rectification was performed on the former 2 patients. The implant was removed from the latter patient and posterior fusion was performed.The 1-year review of 25 patients involved pain assessment only, which consisted of a subjective ranking from 0 (no pain) to 10 (worst pain imaginable). Of these, 14 patients reported substantial pain reduction, 7 reported more modest pain reduction, and 4 reported no change. No patient reported an increase in pain.The 5-year review of 22 patients showed further slippage in only 1 patient, subsidence in 2 cases, indication of reaction with the implant in 2 cases, and loosening in 1 case. Interbody bone fusion was observed in every case except 1, which was uncertain. Subjective pain assessments on a scale of 0 to 10 indicated pain decrease in all cases but 3, with 2 patients perceiving no change and 1 reporting an increase in pain. There were no significant differences in patient satisfaction or rates of union between the autologous bone grafts and ceramic implants, but there was a significant reduction in interspace collapse with the silicon nitride implants. Overall satisfaction was very high, with 15 happy, 4 uncertain, and 3 unhappy patients.The 10-year review of 16 patients showed no slippage, subsidence, or reaction. Interbody bone fusion was maintained in all cases. Pain perception remained approximately constant compared to the 5-year review, with 1 patient reporting a significant increase and 2 cases reporting significant decreases. Overall patient satisfaction decreased somewhat compared to the 5-year review, with 9 happy, 3 uncertain, and 4 unhappy patients. However, progressive degeneration was observed in 9 of 13 cases assessed. Further, degeneration was observed at levels adjacent to L4/5 in 13 of 16 cases assessed. Although the reason for these degenerations cannot be demonstrated, it is likely to be the result of stress shielding due to elastic modulus mismatch.
5:00 PM - KK5.7
Development of Biomimetic Collagen/Apatite Scaffolds for Bone Regeneration.
Zengmin Xia 1 , Xiaohua Yu 1 , Liping Wang 2 , David Rowe 2 , Mei Wei 1
1 Department of Chemical, Materials & Biomolecular Engineering, University of connecticut, Storrs, Connecticut, United States, 2 Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, United States
Show AbstractCollagen/apatite composite scaffold has attracted much attention as collagen and apatite are the two main components in natural bone. However, the correlation between scaffold structure and new bone formation remains unclear. The objective of the current study was to design collagen/apatite scaffolds which mimic both hierarchical architecture and chemical composition of natural bone. Meanwhile, freeze casting was applied to create scaffold with tunable structure to meet the needs of different tissue engineering applications.Briefly, collagen/apatite precipitates were formed by a one-step co-precipitation method in a collagen-containing modified simulated body fluid. The collagen and apatite suspension was then frozen at a constant cooling rate followed by lyophilization. It was found that such a procedure produced a hierarchical mineralized structure by simply controlling the rheological behavior of m-SBF without using any additives. The scaffolds exhibit a homogeneous equiaxed cellular structure, and their pore sizes range from 60 µm to 200 µm where cells can migrate and ingrow. Moreover, the chemical composition of the scaffold can be precisely controlled by varying the collagen concentration in m-SBF, and composite scaffolds with an apatite content of 0-60 wt% were successfully attained. The biological activity of the collagen/apatite scaffolds were also tested in vitro using MC3T3-E1 cells and in vivo using a mouse femoral critical-size defect model. It was found that the composite scaffolds well support osteoblast activities in vitro. In combination with mesenchymal stem cells, significant new bone formation was observed in all tested group. Moreover, it was discovered that the group with higher HA content demonstrated earlier bone formation and better integration with host bone. Thus, these results collectively indicate that our collagen/apatite scaffold is a promising candidate for bone repair and regeneration.
5:15 PM - KK5.8
Extracellular Matrix Based Biomimetic Scaffold for Bone Tissue Engineering.
Sriram Ravindran 1 , Qi Gao 1 , Mrignayani Kotecha 2 , Anne George 1
1 Oral Biology, University of Illinois at Chicago, Chicago, Illinois, United States, 2 Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United States
Show AbstractThe gold standard in clinics for bone repair is autografts and demineralized bone matrix (DBM). Autografts induce donor site morbidity and DBMs poorly induce regeneration of native tissue. Alternative strategy is to use allografts that come with the inherent risk of immune rejection. These drawbacks in the current methodology can be resolved by utilizing tissue engineering based approaches for bone repair. All tissues consist of cells embedded within their custom designed extracellular matrix (ECM). This ECM dictates cellular behavior, secretion of growth factors and tissue functionality. In the present study, we have developed biomimetic ECM based scaffolds consisting of cell secreted ECM incorporated within a co-polymer matrix of collagen and chitosan for bone tissue engineering. We show that this matrix supports the nucleation of hydroxyapatite both under physiological and high concentrations of calcium and phosphorus ions. Using magnetic resonance imaging techniques and nano-indentation, we show that the ECM incorporated scaffold possesses better mechanical properties with respect to the control scaffold. Real time PCR data shows that the ECM incorporated scaffold has the potential to induce differentiation of mesenchymal stem cells into osteoblasts without the need for external addition of growth factors or differentiating agents. Developing such ECM based biomaterials will eliminate the need for growth factor delivery in tissue engineering applications. This scaffold has the potential to replace the traditional triad of cells, scaffolds and growth factors with a duo of just cells and scaffolds.This study is supported by NIH grant DE11657 and the Brodie Endowment Fund.
5:30 PM - KK5.9
Dental Pulp Stem Cell Differentiation Regulated by Mechanics of Polybutadiene Rubber Films.
Chungchueh Chang 1 , Aneel Bherwani 2 , Vladimir Jurukovski 1 , Marcia Simon 2 , Miriam Rafailovich 1
1 Materials Science and Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 2 Oral Biology and Pathology, SUNY at Stony Brook, Stony Brook, New York, United States
Show AbstractDental Pulp Stem Cells (DPSCs) are known to differentiate in either bone, dentine, or nerve tissue through different environmental signals. In this study we explored whether differentiation could occur in the absence of chemical induction and through mechanical stimuli only. This was accomplished by coating HF etched silicon wafers with monodisperse Polybutadiene (mw=205,000 mw/mn=1.49) films with thicknesses ranging from 20 to 300 nm. The films were then annealed in a vacuum of 10-8 Torr at 150C in order to adsorb the film onto the Si substrate. Atomic force microscopy in the Scanning Modulation Force, indicated that the moduli of the films increased monotonically with decreasing film thickness due to confinement of the polymer chains within a few Rg of the surface. In this manner, mechanical modulation, by more than a factor of seven, could be achieved with identical surface chemistry. The modulus of the thickest film was measured with micro-indentation to be 1MPa. In order to probe the effects of mechanics on DPSC differentiation, cells were then plated on substrates of different film thickness and cultured with and without dexamethasone induction media. The moduli of the cells were measured after 7 day incubation and the result showed that the cell moduli tracked those of the underlying films indicating that the DPSCs have the ability to sense the mechanics of surfaces. After 21 days of incubation, SEM analysis indicated that cells had produced large amount of calcium phosphate deposits only on the thinner films, below 150nm, while those cultured on the thicker substrates did not biomineralize. GIXD was also performed on these substrates, where we observed crystalline peaks corresponding to crystalline forms of hydroxyapatite. To further determine if DPSC differentiation occurs on for cells in direct contact with the surfaces, the confocal microscopy was employed to investigate the distribution of osteocalcin stain in multi-layer cell samples. Osteocalcin, a differentiation marker, was found in all cells, regardless of surface contact, indicating that the substrate mechanics could induce differentiation across the entire sample.All research was supported in part by the NSF-MRSEC program.
5:45 PM - KK5.10
Bioactive Bi-Layered Poly(Vinyl Alcohol) Scaffolds for Periodontal Tissue Regeneration.
Rachael Oldinski 1 , Kristen Shibuya 1 , Naseeba Khouja 2 , James Bryers 1 2
1 Bioengineering, University of Washington, Seattle, Washington, United States, 2 Oral Biology, University of Washington, Seattle, Washington, United States
Show AbstractPeriodontal disease (PD) is related to osteoimmunology, in which inflammatory mediators activate pathways leading to bone and soft tissue resorption. A major goal of periodontal therapy is the restoration of damaged tissues to their original form and function. For the purpose of regenerating periodontal tissue, a bi-layered poly(vinyl alcohol) (PVA) scaffold was fabricated where surfaces of pores in one layer presented methacrylated hyaluronan (HA-MA) while pore surfaces in the second layer presented hydroxyapatite (HAp). HA was complexed with an ammonium salt and dissolved in dimethyl sulfoxide and reacted with methacrylic anhydride for 24 h. The HA-MA was hydrolyzed and characterized by 1H-NMR. Scaffolds were fabricated by freeze-drying various polymer blends of PVA and HA-MA, and slurries of PVA and HAp. Solutions of 2 and 3% PVA were subjected to 4, 6 and 8 freeze-thaw cycles. PVA/HA-MA scaffolds were crosslinked by UV radiation. Bi-layered scaffolds were fabricated by joining two scaffolds together in solution and running through additional freeze-thaw cycles. Scaffolds were characterized by microscopy, swell ratio and weight loss calculations, and unconfined compression testing. Toluidine Blue O and Alizarin Red S stains were used to confirm HA-MA and HAp within the scaffolds. Scaffolds and controls were cultured with human mesenchymal stem cells (hMSCs) for 21 days under standard culture conditions in standard growth medium (alpha-MEM, 10% fetal bovine serum, 1% antibiotic/antimycotic). The methacrylation conjugation efficiency was 80% for the modified HA. Freeze-drying resulted in porous scaffolds that swelled upon hydration. Increasing the molecular weight of PVA, increasing PVA concentration, and increasing the number of freeze-thaw cycles increased pore diameter (30-80µm), decreased swell ratio (500-1000%), and increased compression modulus (4-39 kPa). Durable bi-layered scaffolds with two different pore diameters were fabricated. Retention of HA-MA and HAp was verified by colorimetric staining. 3% PVA samples with and without HA-MA were slightly cytotoxic (~80% viability) compared to the 2% PVA scaffolds (~110% viability) normalized to untreated controls. Cells cultured on scaffolds that contained HA-MA displayed higher levels of glycosaminoglycans (GAGs) and the concentration of GAGs increased with time. The alkaline phosphatase (ALP) concentration also increased with time, suggesting both HA and HAp were initiating osteogenic differentiation.Pore size was optimized by varying freeze-thaw cycles and polymer density; pore size was shown to affect compression modulus. PVA scaffolds consisting of two distinct pore size regions, unique in composition and structure, were fabricated. The bioactive PVA scaffolds displayed low cytotoxicity and demonstrated the potential to direct stem cell differentiation. Future in vitro experiments will examine periodontal ligament adult stem cell differentiation on bi-layered scaffolds.
KK6: Poster Session: Tissue Engineering
Session Chairs
Wednesday AM, November 30, 2011
Exhibition Hall C (Hynes)
9:00 PM - KK6.1
Osseomimetic Porous Structure for Optimal Implant Fixation.
Gautam Gupta 1 , Thomas Vanasse 1 , Clinton Kehres 1 , Chirag Shah 2
1 , Biomet, Warsaw, Indiana, United States, 2 , Exova, Glendale Heights, Illinois, United States
Show AbstractPorous Ti6Al4V structures have been used in orthopedic applications for their excellent biocompatibility. However, the pore structure and mechanical properties of such structures have to be optimized to support bone ingrowth and improve implant fixation. Traditional manufacturing methods to create porous structures often do not allow flexibility in design of complex geometries. The advances with additive manufacturing techniques like electron beam melting (EBM) provide flexibility in selecting design features to achieve this. In this study, a high resolution micro-CT scan of human cancellous bone was obtained from cadaver humeral head. This data was converted into STL file, and porous Ti6Al4V structures that mimic cancellous bone were produced in a vacuum chamber by consolidation of thin layers of metal powder. The manufacturing parameters were controlled to optimize the pore-size, strength and modulus of the porous structure. SEM image analysis showed that the pores were in the size range of 200-900 microns, with an average pore-size of 580 microns. The average porosity, as determined by Archimedes principle was about 65%. Mechanical testing showed that compressive strength and modulus were 92 MPa and 0.72 GPa, respectively. EDX analysis confirmed that the final chemical composition of the Ti6Al4V alloy was within the acceptable limit per ASTM F136. To determine the bone ingrowth within the porous structure, cylindrical Ti6Al4V samples (10 mm diameter X 20mm long) containing the cancellous bone like structure were implanted in sheep tibiae and femora. Porous Ti6Al4V structures manufactured with traditional methods like sintering were used as control. The 12 week long study is ongoing. At the end of the study, histological analysis will be conducted to evaluate the efficacy of bone ingrowth into the porous metal with bone-like structure. The advances in manufacturing technologies provide freedom of design that can create new opportunities for improved implant fixation.
9:00 PM - KK6.11
Polycaprolactone Scaffolds with Controlled Nano/Microstructures for Annulus Fibrosus Tissue Engineering.
Laura Koepsell 1 , Hao Fong 2 , Ying Deng 1
1 Biomedical Engineering Program, University of South Dakota, Sioux Falls, South Dakota, United States, 2 Department of Chemistry, South Dakota School of Mines and Technology, Rapid City, South Dakota, United States
Show AbstractDamage of the annulus fibrosus (AF) is implicated in common spinal pathologies. One solution for remedying the AF damage is to fabricate scaffolds that are designed to support the growth of annulus fibrosus cells. In tissue engineering, it is important to fabricate a scaffold that resembles extra cellular matrix (ECM) and topographical appearance of the native tissue. To address this issue, polycaprolactone scaffolds with different topographical features were fabricated to mimic the nano/microstructures of the native tissue. The testing hypothesis was that varying the nano/microstructures of the electrospun scaffolds by manipulating the morphology of fiber ends or the relative degree of fiber alignment would influence the behaviors of porcine annulus fibrosus cells. A scaffold with novel round-ended fibers and five types of partially aligned scaffolds with controllable degree of fiber alignment were prepared and investigated. The scaffold nano/microstructures were morphologically determined by scanning electron microscopy. In vitro biocompatibility experiments have been carried out to further assess cell-material interaction, cell proliferation, and ECM production within the scaffolds. Porcine fibrochondrocytes were harvested, seeded onto the electrospun scaffolds, and cultured for 1, 7 and 14 days for the biocompatibility evaluation. The resulting cell/scaffold constructs were then collected at each time point for morphology, cell number (normalized by DNA content), and ECM content analyses. The results reveal that the innovative scaffold with round-ended fibers outperformed all of the partially aligned scaffolds in cell initial adhesion. The partially aligned scaffolds with oriented fibers provide strong guidance to the cell orientation and ECM distribution. In addition, the mechanical test data suggest that the partially aligned scaffolds could potentially mimic both the anisotropic and nonlinear mechanical properties of the native AF. In conclusion, the nano/microstructures of the electrospun scaffolds can influence cell behavior and may guide in vitro formation of functional constructs for AF tissue engineering. This research was partially supported by the joint Biomedical Engineering (BME) Program between the University of South Dakota and the South Dakota School of Mines and Technology. The authors would also acknowledge the South Dakota Board of Regents Competitive Research Grant Award (No. SDBOR/USD 2011-10-07) for the financial support.
9:00 PM - KK6.13
Biohybrid ECM/Polymer Scaffolds for Valve Tissue Engineering.
Kartik Balachandran 1 , Guillaume Rousseau 1 , Mohammad Badrossamay 1 , Kevin Parker 1
1 Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractCurrent synthetic tissue engineered scaffolds for heart valves are made of biodegradable polymers with good mechanical properties, but are not conducive for cell adhesion due to their hydrophobic nature and lack of bio-recognition sites. We developed a novel biohybrid ECM/polymer scaffold for cardiac valve tissue engineering and hypothesized that the scaffold will have superior biomimetic and cell adhesion properties without compromising its mechanical properties compared to pure polymer scaffolds. To test this hypothesis, we first prepared a hybrid polymer solution by dissolving polycaprolactone (PCL) and gelatin at different composition ratios in hexafluoroisopropanol and spin-coated the hybrid solution onto coverslips and stretchable elastomer membranes to form a layer of 2-4μm thickness. Fibronectin was micropatterned onto the substrates to provide cues for cell attachment and spreading.Via confocal and scanning electron microscopy, we were able to demonstrate formation of anisotropic tissues of valve interstitial cells (VICs) on the hybrid structures. The tissue engineered biohybrid VIC tissues can be stretched to mimic the in vivo mechanical environment, promoting increased cell alignment and proliferation. We are now investigating via mechanical testing the tensile modulus and the basal tone of these biohybrid VIC tissues. In conclusion, we fabricated VIC tissues using a biohybrid ECM/polymer material that shows promise for a tissue engineered valve replacement.
9:00 PM - KK6.16
Neuronal Outgrowth and Differentiation on Poly(Glycerol Sebacate).
Meghan Casey 1 , Sabrina Jedlicka 1 2 3
1 Bioengineering, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Materials Science and Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 3 Center for Advanced Materials and Nanotechnology, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractExtensive recent work has indicated the importance of the extracellular mechanical environment in stem cell differentiation. In neuronal cell differentiation, much of the work performed has correlated matrix stiffness and neurite outgrowth, highlighting the impact of matrix effects on neuronal cell morphology. In addition, on materials that approach the physiological mechanical properties of brain tissue, neurons from mixed phenotype primary cultures will prevail. However, if the same mixed culture is grown on polystyrene, glial populations are more prevalent. Developing an understanding of these differentiation processes will expand our abilities to design materials for neuronal implants that are conducive to neuronal survival, resist glial scarring and promote axonal outgrowth and cell connectivity. Specifically, elastomers such as Poly(glycerol sebacate) (PGS) hold promise in neuronal tissue engineering, due to their mechanical tunability. PGS is biocompatible, biodegradable and possesses mechanical properties similar to that of living tissue. To study the effects of this elastomeric polymer on neuronal cell differentiation, we used P19 embryonic carcinoma cells, which can be differentiated into a neuronal phenotype using retinoic acid. The P19 cells are grown on varying cure temperatures of PGS including 120°C, 140°C and 165°C. The elastic modulus of the PGS material increases with cure temperature. The substrate effect on the cells is characterized via immunocytochemistry and flow cytometry. With the 120°C PGS grown cells, limited neuronal differentiation and outgrowth was observed. The population of cells was primarily astrocytic. The PGS 140°C grown cells resulted in expanded neurite outgrowth and increased the neuronal population percentage. The best results were observed in the cell population grown on the 165°C PGS. These samples had large populations of neurons, with long neurite outgrowths. These results indicate that neuronal differentiation is influenced by underlying substrate mechanical properties, but that a material with a Young’s modulus similar to that of neuronal tissue (PGS 120°C) may not necessarily be the most conducive to in vitro differentiation.
9:00 PM - KK6.17
Kinetics of the Induced-Polarization Decay in Poly (L-lactic) Acid for Bone Regeneration.
Paula Vilarinho 1 , Nathalie Barroca 1 , Helena Fernandes 1 , Pankaj Sharma 2 , Alexei Gruverman 2
1 Department of Ceramics and Glass Engineering, University of Aveiro, Aveiro Portugal, 2 Department of Physics and Astronomy, University of Nebraska–Lincoln, Lincoln, Nebraska, United States
Show AbstractElectromechanical phenomena in bone are thought to be responsible for its organization and growth processes. The combination of mechanical strain stimuli and endogenous electrical currents has been shown to control osteogenic growth. In the clinical cases where bioengineering is required to treat a bone defect induced by fracture, aging processes or bone diseases, the use of materials, which can sustain and promote the native processes for bone growth, can be a reliable strategy to enhance the healing process. Therefore, piezoelectric materials are good candidates to mimic bone tissue due to the piezo effect, i.e. generation of an electric current in response to the pressure induced by skeletal weight and motion. Poly (L-lactic) acid (PLLA), a semi-crystalline synthetic polymer exhibits piezoelectricity, biodegradability and biocompatibility. Macroscopically, its piezoelectric properties depend on dispersion and orientation degree of its crystallites in the amorphous phase that can be controlled by stretching. In vivo experiments have shown that uniaxially stretched PLLA films were able to enhance bone growth. Mechanisms underlying the facilitated bone growth by piezoelectric implants are far from being understood. We recently presented the experimental evidence of the protein adsorption process’s dependence on the surface polarization of PLLA [1]. PLLA can be used as a replacement for bone living tissue for the remodeling functions. But for these applications, stability of the polarization state in PLLA is critically important. In the present study the kinetics of the decay of the electrically-induced polarization of PLLA films is addressed via piezoresponse force microscopy (PFM). PLLA thin films were prepared by spin coating using a 5 (wt %) PLLA solution on Pt/TiO2/SiO2/Si substrates and subsequently submitted to isothermal annealing treatments to obtain different crystallized states. After melting the films at 190 degree C for 15 minutes, some of them were maintained at the isothermal crystallization at 80 degree C and others at 140 degreeC temperature for 30 minutes. Poling of the films was performed locally by applying a DC field through the PFM tip at variable temperatures. The samples poled at room temperature experienced a rapid decay of the polarization. After a few hours, the orientation of dipoles was lost. Within this group, films annealed at 80 degree C present a faster decay that can be related to the higher content of amorphous phase in the poled area. The polarization above glass transition temperature allowed the freezing of the dipoles in the oriented state. Under these conditions the polarization was imaged by PFM for up to 12 days. Present results are an important proof of the suitability of PLLA as piezoelectric substrates for tissue engineering devices.[1] N. Barroca, P. M. Vilarinho, A. L. Daniel-da-Silva, A. Wu, M. H. Fernandes, A. Gruverman, Applied Physics Letters, 98, 133705, 2011
9:00 PM - KK6.18
Crosslinking of Electrospun Hyaluronic Acid Nanofibers for Wound Healing Applications.
Laura Toth 1 2 , Caroline Schauer 1
1 Materials Engineering, Drexel University, Philadelphia, Pennsylvania, United States, 2 Biology, Temple University, Philadelphia, Pennsylvania, United States
Show AbstractBurn trauma is one of the most common forms of injury treated in hospitals world-wide. Mortality nears 75% in cases where burns cover more than 40% of the body surface. Proper maintenance of the burn wound area, which includes the ability of the burned tissue to heal without getting infected, is crucial to patient survival. In order to find improved coverings for burn wound treatment we investigated electrospinning with the extracellular matrix component hyaluronic acid (HA). We developed methods to spin robust HA nanofibers within a neutral solvent system and crosslink them, in order to prevent degradation within aqueous solutions, which has been a problem with this technology. These HA fibrous assemblies were analyzed using SEM, FTIR, XPS, and solubility tests. These new methods of electrospinning will develop a covering that should enhance wound healing and prevent bacterial infection for the burn victim.
9:00 PM - KK6.2
The Effect of Substrate Mechanics on Differentiation of Dental Pulp Stem Cells.
Divya Bhatnagar 1 , Aneel Bherwani 2 , Vladimir Jurukovski 2 3 , Rafailovich Miriam 1 , Marcia Simon 2
1 Materials Science and Engineering, Stonybrook University, Stony Brook, New York, United States, 2 School of Dental Medicine, Stony Brook University, Stony Brook, New York, United States, 3 Chemical and Molecular Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractDental Pulp Stem Cells (DPSCs) are known to differentiate in bone, dentine, or nerve tissue through different environment signals. This work investigates whether differentiation could occur in the absence of chemical induction and through mechanical stimuli only or just by the conformational change due to cross-linking. For this study, we chose enzymatically cross-linked gelatin hydrogels as our substrates. The stiffness of the substrates was varied by varying the concentration of the enzyme (microbial transglutaminase, mTG). Rheological studies carried out by oscialltory shear rheometry indicated that the modulus of the hardest hydrogel was of the order of 8kPa where as the medium and the softest hydrogel had modulus of the order of 1kPa and 100Pa respectively. DPSC were then plated on all three substrates and cultured with and without dexamethasone induction media. After 21 days of incubation, SEM and EDX analysis indicated that the cells cultured produced huge amounts of biomineralized deposites on all the gels irrespective of the stiffness and chemical inducer. AFM analysis modulli of the cells was independent of the chemical inducer as well as the stiffness of the hydrogels. The Real-Time Polymerase Chain Reaction (RT-PCR) assays performed on the cells after 21 days of incubation indicated that cells expressed more osteocalcin when cultured in non-induction media and harder substrate. Further tests also indicated that the enzyme and gelatin alone did not trigger biomineralization and that the conformational change due to the crosslinking of gelatin with mTG could be the reason for this large amount of biomineralization.
9:00 PM - KK6.20
Functionalisation of Polycaprolactone (PCL) Scaffold and Phosphonate Polymer for Bone Regeneration.
Shuangwu Li 1 , Sandra Downes 2
1 , Queen Mary, University of London, London United Kingdom, 2 , The University of Manchester, Manchester United Kingdom
Show AbstractBisphosphonates has been used as a drug to treat osteoporotic patients. A phosphonate polymer (PP) which has an active moiety of C-P is employed in this study to mimic the backbone of P-C-P of drug bisphosphonates to support bone regeneration. In this study we established a new method to functionalise poly (ε-caprolactone) (PCL) with this phosphonate polymer to link these two polymers by chemical binding. Results have proven that we have successfully bound PP with PCL using this method. Further elemental release study has shown that the level of the phosphonate polymer using this method is much higher comparing to the element weight percentage from other methods; this is promising as the increased phosphonate polymer level is very helpful for further cell attachment and bone regeneration.
9:00 PM - KK6.21
Synthesis and Characterization of Iron and Cobalt Substituted Hydroxyapatite Prepared by a Simple Ion Exchange Soaking Procedure.
Erica Kramer 1 , Menka Jain 2 , Joseph Budnick 2 , Mei Wei 1
1 Chemical, Materials and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, United States, 2 Physics Department, University of Connecticut, Storrs, Connecticut, United States
Show AbstractHydroxyapatite (HA), Ca10(PO4)6(OH)2, is the main mineral component of natural bone. As such, HA has excellent biocompatibility and bioactivity and is widely studied for bone regeneration applications. It is known that a wide variety of substitutions can be made into the HA lattice by anions, cations, and functional groups, and thereby tailor the physical, chemical, mechanical, and biological properties of HA suitable for broader biomedical applications. Magnetic ion substituted apatites have attracted enormous attention due to their potential applications in bone replacement therapies, drug delivery, medical imagining, or hyperthermia-based cancer therapies. However, most work was done either during the apatite synthesis, or via ion exchange procedures which employ harsh preparation conditions such as high temperature and pH. In the current study, a new approach has been employed where magnetic ion substituted HA was synthesized at a mild temperature (room temperature) via a simple ion exchange procedure.Briefly, HA was first synthesized via a wet precipitation procedure. The resulting HA powder was then soaked in dilute iron or cobalt containing solutions at room temperature under moderate stirring for an hour. This simple soaking procedure yielded both iron-substituted apatite (FeHA) and cobalt-substituted apatite (CoHA) samples. EDX spectra confirmed the presence of iron and cobalt in the samples, and XRD patterns showed that the substituted samples maintained a single phase apatite structure. Furthermore, VSM measurements indicated that both FeHA and CoHA powders have magnetic properties, while pure HA powder is diamagnetic. These results collectively suggest that single-phase, magnetic apatite powders have been successfully prepared under mild fabrication conditions. Finally, the sintering behavior and mechanical properties of HA, FeHA and CoHA were also compared.
9:00 PM - KK6.22
Greater Structural Control of Osteogenic Collagen-Hydroxyapatite Scaffolds by Unidirectional Freeze Casting.
Max Villa 1 , Mei Wei 1
1 Chemical, Materials & Biomolecular Engineering Department, University of Connecticut, Storrs, Connecticut, United States
Show AbstractBiomimetic collagen-hydroxyapatite scaffolds combined with bone marrow cells represent a promising approach for therapeutically regenerating bone. In our experience with mouse calvarial defect models, collagen-hydroxyapatite scaffolds are highly osteogenic, degradable and biocompatible. The composite is fabricated by a novel biomimetic method previously developed in our lab. This process involves the self-assembly of collagen fibers in the presence of precipitating hydroxyapatite nanocrystals from a concentrated physiological saline solution. The resultant material is a highly intermingled collagen-hydroxyapatite composite. The composite material is rehydrated and frozen to introduce a pore structure templated by ice crystals. However, robust control of scaffold pore structure formed by ice templating is currently a challenge.Here we present the fabrication of biomimetic collagen-hydroxyapatite scaffolds by unidirectional freeze casting to enable greater control of scaffold pore directionality and size. The structure of unidirectional scaffolds is compared with nondirectional scaffolds produced by freezing samples in a culture dish on a freeze dryer shelf. The culture dish method is relatively cheap and simple, but lacks control over freezing direction and pore size. Building on the extensive theoretical and experimental freeze casting work in the literature, we have successfully produced collagen-hydroxyapatite composite scaffolds with directional structure following the cooling direction. To achieve this, we constructed a two-sided tunable temperature freezing system to allow for control over the freezing front velocity. Examined by histology, 2-photon microscopy and scanning electron microscopy, it was found that the unidirectionally frozen scaffolds we have produced contain large interconnected pores, ranging from 200 microns to over 1 millimeter on a side. This morphology may better promote cell loading, viability, migration and infiltration; ultimately improving angio- and osteogenesis within the scaffold-cell construct. Additionally, the collagen fibers in the scaffold generate a signal that is the second harmonic of the long wavelength excitation light typically used in 2-photon imaging. This allows us to generate 3-dimensional reconstructions of scaffold surfaces and thereby gain a deeper understanding of scaffold microstructure while in a hydrated state. Finally, multiphoton imaging of collagen-hydroxyapatite scaffolds can be extended to real-time monitoring of physiological processes in vitro and in vivo. Future work will examine the in vivo performance of directional collagen-hydroxyapatite scaffolds in a calvarial model.
9:00 PM - KK6.23
Short Term Cellular Responses to Stainless Steel Fibre Networks.
Rose Spear 1 , Vera Malheiro 1 , Roger Brooks 2 , Athina Markaki 1
1 Department of Engineering, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom, 2 Orthopaedic Research Unit, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom
Show AbstractPhysiotherapeutic exercise following orthopaedic implant operations is commonly prescribed on the basis of mechanical stimulation-induced bone remodelling [1]. An alternative approach would be to use an implant material with the ability to induce therapeutic strains directly into bone tissue [2]. Therapeutic levels of strain might be achieved by application of an external magnetic field to ferromagnetic fibre networks, composed of a material such as 444 ferritic stainless steel, during in-growth of bone tissue [2].A primary requirement for such an implant material is healthy cellular interactions. Recent work by the authors [3] demonstrated favourable cellular responses of human osteoblasts and monocytes to two-dimensional sheets of 444 ferritic stainless steel surfaces. Building on these results, the current work examines cellular responses to sintered fibre networks composed of 444 ferritic stainless steel fibres. Two variants of the material have been studied experimentally. They were made from 444 coil-shaved fibres with either 60 x 100 µm2 or 80 x 100 µm2 cross-sectional areas. In both variants, the fibres occupy about 15 % of the volume.CD-14 positive human monocytes and human osteoblasts were used to evaluate cellular responses to the fibre networks. Using CD-14 positive monocytes, inflammatory and cytotoxicity potentials of the samples were investigated as a function of TNF-α and lactate dehydrogenase release, respectively. Using human osteoblasts, cellular proliferation, metabolic activity, morphology and early osteogenic differentiation were examined, using the CyQuant® assay, AlamarBlue® assay, scanning electron microscopy and fluorescence imaging, and an alkaline phosphatase activity assay, respectively.The results indicated that 444 ferritic stainless steel fibre networks do not cause significant inflammatory or cytotoxic responses from human monocytes compared to thermanox. Additionally, human osteoblast cells responded successfully, in terms of attachment, proliferation and osteogenic differentiation, when in contact with these materials. Taken together, these results suggest that 444 ferritic stainless steel fibre networks can potentially be used for magneto-mechanical stimulation of human bone tissue.[1] J. Kerner, R. Huiskes, G.H. van Lenthe, H. Weinans, B. van Riertbergen, C.A. Engh, A.A. Amis. Journal of Biomechanics, 1999, 32(7): 695-703.[2] A.E. Markaki and T.W. Clyne. Biomaterials, 2004, 25(19): 4805-4815.[3] V.N. Malheiro, R.L. Spear, R.A. Brooks, A.E. Markaki. Biomaterials, 2011, in press.ACKNOWLEDGEMENTS: This research was supported by the European Research Council (Grant No. 240446). Financial support for VNM and RAB has been provided via the Portuguese Foundation for Science and Technology (FCT - SFRH/BD/60445/2009) and the National Institute for Health Research (NIHR), respectively.
9:00 PM - KK6.25
Nanoscale Elasticity and Surface Charge Investigations of Planar and Nanofibrillar Tissue Scaffolds for Rat Central Nervous System.
Volkan Mujdat Tiryaki 1 , Virginia Ayres 1 , Ijaz Ahmed 2 , David Shreiber 2
1 Electrical and Computer Engineering, Michigan State University, East Lansing, Michigan, United States, 2 Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States
Show AbstractAstrocytes are cellular bridges between the neurons and capillaries in the blood brain barrier (BBB). It was recently suggested that nanophysical properties of the basement membrane at BBB can influence astrocyte and neuron response. In this work, cerebral cortical astrocytes were cultured on standard poly-L-Lysine coated glass substrates, and on electrospun polyamide nanofibers whose properties may recapitulate those of the basement membrane [1]. The polyamide nanofibers were electrospun on ACLAR substrates for use as cell culture surfaces.The nanoscale elasticity and charge states of each culture environment were investigated and compared. The elasticity of bare and PLL coated glass substrates, and of bare ACLAR substrates were investigated by force volume imaging. The elasticity of the nanofibrillar surfaces was investigated by SPRM [1]. The charge states of the poly-L-Lysine coated glass substrates, and the polyamide nanofibers electrospun on ACLAR substrates were investigated by electric force microscopy. Variations in elasticity and charge state were correlated with astrocyte responses. [1] R. Delgado-Rivera, S.L. Harris, I. Ahmed, A.N. Babu, R. Patel, V.M. Ayres, D.A. Flowers, and S. Meiners “Increased FGF-2 Secretion and Ability to Support Neurite Outgrowth by Astrocytes Cultured on Polyamide Nanofibrillar Matrices”, Matrix Bio. 28:137-147 (2009).[2] V.M. Ayres, Q. Chen, Y. Fan, D.A. Flowers, S.A. Meiners, I. Ahmed, R. Delgado-Rivera, “Scanning Probe Recognition Microscopy Investigation of Neural Cell Prosthetic Properties”, Int. J. Nanomanufacturing 6: 279-290 (2010).
9:00 PM - KK6.26
Biocompatibility Evaluation of Fish Scale Collagen Intermingled Chitosan Based Nano-Fibers for Skin Tissue Engineering Application.
Soumi Dey Sarkar 1 , Falguni Pati 1 , Brooke Farrugia 2 , Preetham Guha Ray 1 , Tim Dargaville 2 , Jyotirmoy Chatterjee 1 , Santanu Dhara 1 , Pallab Datta 1
1 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, Kharagpur, West Bengal, India, 2 Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
Show AbstractCollagen type I, a major extra cellular matrix (ECM) protein, finds wide usage in tissue engineering due to its low antigenecity, wound healing potential and cell adhesive properties. However high cost of the protein, restricts its wide applicability. The present research explores the potential of a cost effective Type I collagen, extracted from waste fish scale, intermingled with chitosan based nano-fibers for skin tissue engineering application.The chitosan/polyethylene oxide (PEO) nano-fibres (CP) were fabricated by electro-spinning followed by sodium tripolyphosphate (STPP) treatment to retain its fibrous architecture. Furthermore, Type I collagen was isolated from fish scale using a two-step demineralisation and salting out technique. The STPP treated electro-spun fibers were freeze-dried with Type-I collagen to form a composite structure (CP-Col). Morphologies of CP and CP-Col scaffolds were assessed using scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FT-IR), mechanical properties, and swelling behavior of both scaffolds were studied. Cell attachment/proliferation assays were performed on CP and CP-Col scaffolds using 3T3 fibroblast cells and observed under fluorescent microscope by 4', 6-diamidino-2-phenylindole (DAPI) nuclear staining. Cell viability on scaffolds was evaluated using Live/Dead assay and quantified using MTT assay. The scaffolds were further tested in a bio-engineered human skin equivalent (HSE) model, as an alternative to in vivo animal testing SEM micrographs revealed formation of electro-spun chitosan/PEO nano-fibrous (50-100 nm diameter) mat which further attained a composite architecture with self-assembled, freeze-dried collagen. Mechanical and swelling properties justify use of both CP and CP-Col scaffolds as support matrix for cell seeding. CP and CP -Col scaffolds both show cell attachment and proliferation, however quantitative evaluation reveals the better cell adhesion on CP-Col scaffolds. The viability of cells is also significantly high on CP-Col scaffolds in comparison to CP scaffolds. The application of CP-Col scaffolds on HSE wound model shows keratinocytes migration along the scaffold’s surface which is a prerequisite for re-epithelisation and wound healing.
9:00 PM - KK6.27
Three Dimensional Chemical Scaffolds for Cellular Organization.
Yevgeniy Kalinin 1 , Jatinder Randhawa 1 , David Gracias 1
1 Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractChemical scaffolds are spatio-temporal patterns of chemicals such as chemoattractants or growth factors that can guide the motion and organization of living cells. We present a strategy that enables the creation of precise 3D patterns of chemicals via diffusion through lithographically patterned polyhedral containers. Using a combination of numerical simulations and experiments we highlight considerable spatial and temporal versatility. We show that a number of conceptually different experimental strategies can be utilized for spatial control of chemical patterns. In one such strategy, the overall shape of the container can be chosen to closely match the desired 3D spatial profile. As a part of a different strategy, spatial control is achieved by specific placement of pores on the polyhedral containers. Temporal control is achieved by varying the pore sizes. To demonstrate applicability of our concept to in-vitro organization of living cells in specific 3D geometries, we describe chemotactic self-organization of bacterial cells in a variety of well-defined shapes and space curves. Using E. coli bacteria as an example we describe some of the guiding principles (such as dependence of the self-organized pattern on characteristics of bacterial motion, chemoattractant concentrations and other environmental conditions. These principles are important to generate precise 3D spatio-temporal chemical patterns for cellular organization into precise 3D architectures.
9:00 PM - KK6.28
Mechanically Strong Cell-Laden Double-Network Hydrogel.
Hyeongho Shin 1 2 , Bradley Olsen 3 , Ali Khademhosseini 2 4
1 Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts, United States, 3 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 4 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractAmong the various natural tissues in human bodies, load-bearing tissues exhibit exceptional mechanical properties. For example, cartilage can resist repeated compressive stresses of several MPa over several years without fatiguing. Hydrogels are leading candidates for tissue engineering scaffolds due to their resemblance to natural tissues, however, the lack of mechanical strength is a major challenge in applications for the load-bearing tissues. In this study, we used the double-network (DN) strategy to develop mechanically strong hydrogels that can encapsulate cells in three dimensions to better mimic the natural environment for cells and facilitate tissue formation. We improved upon previously developed DN hydrogel formulations by developing processing conditions compatible with cell survival. We made the cell-laden DN formation possible by a two-step photo-crosslinking of two macromolecules, gellan gum and gelatin. Gellan gum was chosen as the first component because of its high molecular weight (~1 MDa) and feasibility for high degree of functionalization to result in a stiff, highly crosslinked network, while gelatin was chosen as the second component because it can form a more flexible, loosely crosslinked network. After being modified to be photo-reactive, gellan gum was photo-crosslinked to form the first network that acts as the rigid backbone, and subsequently, gelatin was photo-crosslinked in the gellan gum network to form the second network that absorbs the crack energy. Examination of the diffusion of gelatin molecules into the gellan gum network and increase in mechanical properties of the resulting hydrogels over the second crosslinking time confirmed the formation of DN. The resulting DN hydrogels exhibited higher strength than single-network hydrogels, displaying as high compressive failure stress as up to ~7 MPa. There was optimal range in the crosslink density of the second network that most improves the strength of the DN hydrogels, and the concentration of each network also had great effect on the mechanical properties of the DN hydrogels. Three-dimensional encapsulation of NIH-3T3 fibroblasts and the following viability assay proved that the whole process of DN formation was cell-compatible. Given the high mechanical strength and the ability to encapsulate cells, our DN hydrogels from photo-crosslinkable polymers have great potential in applications as scaffolds for the load-bearing tissues.
9:00 PM - KK6.30
Role of Nanoporosity on the Performance of Bioactive Nano-Macro Dual-Porous Glass Scaffolds.
Shaojie Wang 1 , Tia Kowal 2 , Ahmed Rashad 3 , Mona Marei 3 , Mathias Falk 2 , Himanshu Jain 1
1 Department of Materials Science and Engineering , Lehigh University, Bethlehem, Pennsylvania, United States, 2 Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States, 3 Faculty of Dentistry, Alexandria University, Alexandria Egypt
Show Abstract Recently, nano-macro dual porous bioactive glass (BG) scaffolds have been developed in our laboratories by a modified sol-gel process for tissue regeneration. In these Tailored Amorphous Multi-Porosity (TAMP) structures, interconnected macropores with diameter of tens to one hundred micrometers are created allowing for the in-growth of tissue and blood vessels. In addition, interconnected nanopores ranging from several to one hundred nanometers are created for enhancing nutrient circulation and the degradation rate to match the tissue growth rate. Now we have investigated the role of nanopores on the performance of TAMP scaffolds by conducting both in vitro and in vivo tests. The in vivo animal tests, conducted in New Zealand rabbits, show that soft tissue ingrowth significantly depends on the size and volume fraction of nanopores within the scaffold. Large fraction of nanopores seems to promote in vivo cell penetration. In parallel, in vitro tests are performed with MC3T3-E1 pre-osteoblasts, and the results are compared with the observations on in vivo tests. Our results show that there is an intermediate nanopore size for which the cell response is optimal. The processing conditions for obtaining such optimized nanostructure of TAMP BG scaffolds will be discussed.
9:00 PM - KK6.31
Investigation of Nanophysical Properties of Aging Nanofibrillar Tissue Scaffolds by SPRM, TEM, and FTIR and Raman Spectroscopies.
Virginia Ayres 1 , Kan Xie 1 , Volkan Mujdat Tiryaki 1 , Ijaz Ahmed 2 , David Shreiber 2
1 Electrical and Computer Engineering, Michigan State University, East Lansing, Michigan, United States, 2 Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States
Show AbstractRecent research indicates that when injury sites are supplied with scaffold-based environments that are physically and biochemically mimetic for an extracellular matrix or basement membrane, endogenous cell populations can regenerate and re-establish functional connections with healthy surrounding tissue. It is further emerging that the nanophysical properties of such scaffolds including elasticity, surface roughness and surface chemistry can be directive for multiple direct and indirect cellular responses. The polyamide nanofibrillar surfaces have recently shown promising results for spinal cord repair. Tissue scaffold physical properties can evolve over time. In the present work, the nanoscale physical properties of the electrospun polyamide nanofibrillar matrices that are newly electrospun versus those that are > 2 year old are investigated using scanning probe recognition microscopy [1], transmission electron microscopy [2] and FTIR and Raman Spectroscopies. [1] V.M. Ayres, Q. Chen, Y. Fan, D.A. Flowers, S.A. Meiners, I. Ahmed, R. Delgado-Rivera, “Scanning Probe Recognition Microscopy Investigation of Neural Cell Prosthetic Properties”, Int. J. Nanomanufacturing 6: 279-290 (2010).[2] V.M. Tiryaki, V.M. Ayres, A.A. Khan, D.A. Flowers, I. Ahmed, R. Delgado-Rivera, S. Meiners, “Investigation of Nanofibrillar Influence on Cell-Cell Interactions of Astrocytes by Atomic Force Microscopies”. In Mater. Res. Soc. Symp. Proc. Volume 1316E: Nanofunctional Materials, Nanostructures, and Nanodevices for Biomedical Applications II, edited by L A Nagahara, R Sinclair, R Bashir, T Thundat, W Lin, Cambridge University Press, Cambridge, UK (2011), 1316-QQ09-16.
9:00 PM - KK6.32
Micronail Substrates for Vascular Differentiation and Assembly.
Laura Dickinson 1 , Danielle Rand 2 , Lauren Chinn 1 , Joanna Tsao 1 , Wolfgang Eberle 2 , Sharon Gerecht 1
1 Chemical and Biomolecular Engineering, Johns Hopkins Universtiy, Baltimore, Maryland, United States, 2 , Interuniversity Microelectronics Center, Leuven Belgium
Show AbstractThe extracellular matrix (ECM) is topographically complex environment that provides a structural platform, as well as presenting specific proteins for vascular tube morphogenesis of both endothelial progenitor cells (EPCs) and mature endothelial cells (ECs). Many of these interactions occur at the micron and sub-micron scale. Previously we have demonstrated that optimized, patterned Fn surfaces guide the ordered adhesion of human EPCs, support their elongation and assemble into unidirectional chains/tubular structures within a three-dimensional fibrin gel. However, micro- and nano-structured surfaces, which more closely mimic the native vasculature ECM environment, may have a more profound effect on endothelial cell responses, including vascular tube formation. Through a collaborative effort with IMEC (Interuniversity Microelectronics Center) we have investigated and compared the cellular responses of mature ECs (human umbilical vein endothelial cells) and EPCs on periodic micro and submicron strutures. The IMEC supplied substrates, oxide nail beds, provide an array of micron and sub-micron environments at varying heights from 1um to 8um. Using soft lithography, we have successfully patterned these substrates with fibronectin (Fn), an ECM protein known to enhance endothelial cell growth, adhesion, and tubulogenesis during vascular development, and compared the results from these topographically rich environments, to our previous results on flat surfaces. Preliminary data reveals that the adhesion, spreading kinetics, and tubular formation of EPCs is greatly dependent on the height and dimensions of the nail beds. Further analysis will elucidate the biochemical and physical cues that dynamically control vascular network formation.
9:00 PM - KK6.34
Effect of BMP-2 Derived Peptide Grafted to Nanoparticles on Differentiation of Stromal Cells.
Esmaiel Jabbari 1 , Angel Mercado 1 , Junyu Ma 1 , Xuezhong He 1
1 Chemical Engineering, University of South Carolina, Columbia, South Carolina, United States
Show AbstractIntroduction: rhBMP-2 plays a major role in differentiation of bone marrow stromal (BMS) cells. Peptides based on the active domains of rhBMP-2 have been proposed as an alternative to the protein, reducing side effects associated with high dose of rhBMP-2. The peptide LYLTSIASLETPVSSAKPIK (BMP peptide), has been shown to induce osteogenesis in vivo. We hypothesized that immobilization of the peptide on the surface of nanoparticles (NPs) can enhance interaction with cell surface receptors, leading to enhanced osteogenic differentiation of BMS cells. The objective of this work was to determine the osteogenic activity of the BMP peptide grafted to the surface of poly(lactide fumarate) (PLAF) NPs.Materials and methods: A cysteine-terminated BMP peptide was synthesized in the solid-phase and grafted to PLAF NPs by linking the cysteine residue on the peptide to fumarate groups on the NPs. As the control group, rhBMP-2 was grafted to succinimide-functionalized PLAF NPs. Bone marrow stromal BMS cells were isolated from adult Male Wistar rats and seeded at a density of 5×104 cells/mL with six different treatments: no NPs, NPs, free BMP peptide, free rhBMP-2, NP-BMP peptide, and NP-rhBMP-2. BMP concentration was kept at 200 ng/mL. Samples were collected at 4, 7, 14 and 21 days and used for determination of DNA content, Ca concentration, ALPase activity, mRNA expression, and protein levels.Results and Discussion: The decrease in cell number after 21 days was consistent with an increase in mineral content and a decrease in proliferation with BMS cell differentiation. ALPase activity peaked at 14 days, consistent with the start of the osteogenic cascade. A slightly higher mineral content was observed in the BMP-grafted NP groups after 14 days, but there was a comparable extent of mineralization for all groups after 21 days. The expression of transcription factors Runx2 and Dlx5 was significantly higher for the NP-protein (peaked at 7 days) and for the NP-peptide (14 days) groups. This trend was confirmed by Western Blot analysis. Earlier activation of Dlx5 and Runx2 by the BMP protein resulted in earlier expression of the proteins downstream in the signaling pathway. The mRNA analysis of osteogenic markers osteopontin (OP) and osteocalcin (OC) and the vasculogenic peptide PECAM-1 showed a more pronounced expression for the BMP protein and BMP peptide grafted to the NPs after 21 days. Immunostaining showed that cells in the NP-BMP peptide group had a higher content of OP and OC osteogenic proteins after 21 days. Results suggest that the BMP peptide grafted to NPs has greater affinity to BMP-2 cell surface receptors, leading to a stronger activation of the signaling pathway.Conclusion: Use of the BMP peptide and subsequent enhancement of the osteogenic cascade by NP grafting is an attractive alternative to rhBMP-2 protein for clinical application.
9:00 PM - KK6.35
Toxicity Screening with Neural Progenitor Cells on Compliant Hydrogel Surfaces.
Stephanie Hume 1 , Tammy Oreskovic 1 , Kavita Jeerage 1
1 Materials Reliability Division, National Institute of Standards and Technology (NIST), Boulder, Colorado, United States
Show AbstractNeurite outgrowth measurements, which quantify the extension of axons and dendrites, have been proposed for in vitro neurotoxicity screening. Neurite outgrowth is sensitive to known neurotoxins in monocultures grown on plastic surfaces. However these cultures may not adequately represent the in vivo environment, given the lack of astrocytes and the extreme stiffness of plastic compared to native tissue. Neural progenitor cells (NPCs) are an emerging model with which it is possible to assess multiple aspects of neural development, including differentiation and neurite outgrowth. In addition, NPCs are sensitive to substrate modulus; neuronal differentiation is favored on compliant gels, whereas glial differentiation is favored on stiffer gels. Since astrocytes provide trophic support to neurons and promote neurite outgrowth, substrate modulus could impact the result of toxicity screening by changing the astrocyte to neuron ratio. Here we investigate whether substrate modulus impacts the toxicity profile of NPCs exposed to lithium chloride (LiCl). Immunocytochemical staining shows that NPCs seeded onto plastic surfaces differentiate into neurons (beta-III-tubulin) and astrocytes (glial fibrillary acidic protein) after 10 days in culture. Exposure to 30 mmol/L and 10 mmol/L LiCl was cytotoxic, as determined by adenosine triphosphate content, whereas exposure to 3 mmol/L, 1 mmol/L, or 0.3 mmol/L LiCl was not. However neurite outgrowth was inhibited at these lower concentrations. PEG hydrogels were photopolymerized from solutions containing 5% to 30% poly(ethylene glycol) dimethacrylate. Swelling studies indicate that mesh size, which is inversely related to modulus, ranges from about 10 nm to greater than 100 nm. NPCs seeded onto these surfaces are being used to assess the ratio of neurons to astrocytes and total neurite outgrowth as a function of LiCl exposure and PEG hydrogel compliance.
9:00 PM - KK6.36
Bio-Hybrid Materials for Cardiac Tissue Engineering: Bio-Functionality and In Vitro Degradation.
Mohammad Badrossamay 1 , Megan McCain 1 , Kevin Parker 1
1 Disease Biophysics Group, Wyss Institute for Biologically-Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractSuitable biomaterials for tissue engineering should mimic the bioactivity, biocompatibility, mechanical stability, and three-dimensional fibrillar structure of native extracellular matrix [1]. In spite of the availability of various synthetic and natural polymers, no single-component polymeric material can satisfy bioactivity, biocompatibility, mechanical stability and the three-dimensional fibrillar structure of native extracellular matrix [2]. Therefore, it is advantageous to design multi-component polymer systems to include different components into a hybrid material as an innovative, multi-functional biomaterial. We have developed several bio-hybrid materials based on combinations of polycaprolactone (PCL) and ECM proteins such as gelatin and collagen. Thin films of bio-hybrid materials were prepared by conventional spin coating. Fibronectin was micro-patterned onto the substrates to provide cues for cell attachment and spreading. Anisotropic cardiac tissues were built by culturing neonatal rat ventricular cardiomyocytes on composite patterned films. In addition, 3-dimensional well-aligned fibrous scaffolds of bio-hybrid materials were produced through the rotary-jet spinning (RJS) process [3] to demonstrate the possibility of building bio-hybrid fibrous scaffolds. Thin film bio-hybrid cardiac tissues were characterized through contractility assays and confocal imaging. Cardiomyocyte cell culture on fibrous hybrid constructs based on the type and amount of ECM component were verified through electron and confocal laser microscopy. The results show that myocytes self-organized with respect to external cues provided by the fibronectin pattern on thin films or aligned fibers on fibrous constructs as indicated by sarcomere alignment. In-vitro degradation studies of the bio-hybrid scaffold revealed rapid degradation of the rich-content composition while low ECM content hybrid scaffold showed slow hydrolytic cleavage. These data suggest that low ECM content (≤ 25%) bio-hybrid constructs have the potential to be used as possible biomaterials for in-vivo cardiac tissue engineering. References:1.Chen, Q. et.al, Materials Science and Engineering: R: Reports, 2008, 59 (1-6), Pages 1-37.2.Cipitria, A., et.al, Journal of Materials Chemistry, 2011, Advance Article, DOI: 10.1039/C0JM04502K.3.Badrossamay, M.R., et al., Nano Letters, 2010, 10(6), 2257-2261.
9:00 PM - KK6.37
Nanostructured Surfaces through Directed Irradiation Synthesis for Improved Biocompatibility.
Emily Walker 1 , Zhangcan Yang 1 , Brandon Holybee 1 , Brian Epp 2 , Emily Gordon 1 , Christy Cooper 3 , Lisa Reece 4 , Jean Paul Allain 1
1 Nuclear Engineering, Purdue University, West Lafayette, Indiana, United States, 2 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States, 3 Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, United States, 4 Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, United States
Show AbstractBiocompatibility of medical implants can be improved through nanostructuring or nanopatterning of their surfaces. Nanoscale surface morphology has been shown to play an important role in cell regulation, differentiation, adhesion and proliferation [1-5]. These nanostructures can be created through directed irradiation synthesis (DIS) on metal and semiconductor surfaces. Directed irradiation synthesis provides a novel method for tuning the surface morphology, surface chemistry and mechanical properties in a single step process through a variety of parameters. These include varying the beam energy, in this study from 200 eV to 4 keV, using various ion species including Ar, Kr and Xe, and varying the angle of incidence of irradiation from near normal to grazing incidence. This technique can be used for a variety of surfaces, including metals, semiconductors and polymers [6-7]. In this study, the response of human umbilical vein endothelial cells (HUVEC) to bare unpatterned silicon wafers will be compared to their response to nanopatterned silicon, as well as a variety of nanopatterned metal thin films. The viability of the cells was assessed using the Comet Assay, as well as a live cell/dead cell assay. Cell adhesion was measured through biological atomic force microscopy (bioAFM). The topography of the samples was examined through atomic force microscopy (AFM) and scanning electron microscope (SEM) imaging while surface chemistry was examined through x-ray photoelectron spectroscopy (XPS). [1] Stevens MM. Science. 2005:310:1135-1138[2] Curtis AS. Nanomedicine. 2006:1:67-72[3] McNamara LE. J. Tissue Eng. 2010:2010:1-13[4] Dalby MJ. Nature Materials 2007:6: 997-1003[5] Dalby MJ. Biomaterials. 2002:23:2945-2945[6] Facsko S. Science. 1999:285:1551[7] Frost F. Phys. Rev. Lett. 2000:85:411
9:00 PM - KK6.38
Aligned Carbon Nanotube Substrates Enhance the Growth of Motor Neurons.
Megan Roberts 1 , Michelle Leach 1 , Eric Meshot 1 , Sameh Tawfick 1 , Youssef Naim 1 , Joseph Corey 1 2 , A. John Hart 1
1 , University of Michigan, Ann Arbor, Michigan, United States, 2 , Veterans Affairs Ann Arbor Health System, Ann Arbor, Michigan, United States
Show AbstractIn pursuit of better ways to repair the damaged nervous system, researchers have explored extensively how physical and chemical cues guide regenerating neurons. Surface topography has been shown to guide the growth of several types of neurons including hippocampal neurons and dorsal root ganglia, however relatively few studies explore the influence of synthetic topographies on motor neurons, the type of neuron required for motor function. Our goal is to study how developing motor neurons interpret anisotropic surface texture on the order of 1-10 nm. We cultured primary (E15) rat motor neurons on thin films of horizontally aligned carbon nanotubes (HACNTs) and flat glass substrates in serum free media. After 1-4 days in vitro, cell morphology was compared qualitatively and by quantitative analysis of fluorescent light and electron microscopic images. We have demonstrated the survival of motor neurons on HACNT sheets for up to 8 days. We find that although neuritogenesis is slower on HACNTs, longer culture durations show that neurites grow longer on HACNTs compared to glass (p-value < .05). In separate experiments, we show that neurites grow directionally along the edges of CNT films, and that longer neurites prefer to grow perpendicular to aligned CNTs. Given the advantages of the electrical conductivity and chemical functionality of CNTs, these findings suggest potential promise in the use of CNT-based materials in tissue engineering of motor circuits and in the development of neural interfaces.
9:00 PM - KK6.39
Resorbable Elastomeric Networks Based on Trimethylene Carbonate Polymers for Tissue Engineering Applications.
Erhan Bat 1 , Theo van Kooten 2 , Gustavo Higuera 3 , Clemens van Blitterswijk 3 , Jan Feijen 1 , Dirk Grijpma 1 2
1 Polymer Chemistry and Biomaterials, University of Twente, Enschede, Overijssel, Netherlands, 2 Biomedical Engineering, University of Groningen, Groningen, Groningen, Netherlands, 3 Tissue Regeneration, University of Twente, Enschede, Overijssel, Netherlands
Show AbstractOwing to their flexible and elastic nature that closely mimics that of native tissues; synthetic resorbable elastomers are increasingly utilized for controlled release applications and soft-tissue engineering. Poly(trimethylene carbonate) (PTMC) is a resorbable polymer that degrades relatively rapidly by surface erosion in vivo without releasing acidic degradation products. Crosslinking of PTMC is required to obtain form-stable elastomeric networks as PTMC has an amorphous structure and a low glass transition temperature of approximately -17 oC. We have previously shown that gamma irradiation can be used to obtain form-stable networks based on high molecular weight PTMC. Although the use of gamma irradiation is advantageous as it allows simultaneous crosslinking and sterilization, the crosslinking process is not very efficient. This leads to the formation networks with relatively low gel contents and crosslink densities and also to partial deterioration of the mechanical properties. A more efficient and practical way of crosslinking of PTMC may lead to better elastic properties and probably tuneable (slower) erosion. This would allow utilization of PTMC in a broader range of applications. Photocrosslinking by ultraviolet (UV)light irradiation is highly efficient for preparing networks at ambient temperatures. In this study, we prepared resorbable elastomeric networks in a facile manner by photocrosslinking of high molecular weight PTMC in the presence of a crosslinking aid. As crosslinking aids, synthesized two-arm and three-arm trimethylene carbonate based oligomers that were end-functionalised with methacrylate groups as well as commercially available multifunctional (meth)acrylates were used. These crosslinking aids were found to be very effective as densely crosslinked networks having very high gel contents (>90 %) were obtained upon irradiation with 254 nm ultraviolet light. The networks had elastic moduli below 10 MPa and showed excellent elastic properties in cyclic creep tests. Biodegradation of the networks were evaluated in macrophage cultures as well as in cholesterol esterase and superoxide containing aqueous solutions. These biodegradation assays revealed that the erosion rate of the networks could be tuned by varying their crosslink density. The wettability and erosion rate of the networks could be tuned also by incorporating block copolymers of PTMC with poly(ethylene glycol) or poly(ε-caprolactone). Fused deposition modelling was used to fabricate tissue engineering scaffolds based on trimethylene carbonate polymers containing the crosslinking aids. Upon ultraviolet irradiation, form-stable and flexible scaffolds were obtained. The fabricated scaffolds were found to be compatible with human mesenchymal stem cells The cells adhered well and proliferated in the scaffolds. These resorbable and elastomeric may find a broad range of applications in the fields of tissue engineering and controlled release.
9:00 PM - KK6.40
Effect of Surface Roughness of 45S Bioactive Glass on Adhesion, Proliferation and Differentiation of Pre-Osteoblast Cells.
Raina Jain 1 , Jutta Marzillier 1 , Shaojie Wang 2 , Hassan Moawad 2 , Matthias Falk 1 , Himanshu Jain 2
1 Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Materials Science and Engineering, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractBioglass 45S is a promising bone implant material, yet little is known about the role of surface topology on its use as an implant. Therefore, we have investigated the effect of surface roughness (Ra~ 0.01 to 1.8 μm) of Bioglass 45S on the response of MG63 osteoblast-like and MC3T3-E1 pre-osteoblast cells. Their adhesion and proliferation were determined by fluorescent microscopy. Morphology and focal adhesion sites were detected after staining for the adhesion protein, vinculin, the F-actin cytoskeleton, and the cell nucleus. The expression profile of a set of bone-cell specific (RunX2, BSP1, BGLAP) and of other osteoblast-relevant marker proteins (Col1A, ALP, Cx43) known to be up-regulated during different stages of bone cell differentiation and mineralization, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The housekeeping gene GAPDH was used as expression reference. We found that over time, smoother surfaces (Ra~0.01-0.2 μm) promoted cell adhesion and proliferation better than rougher (Ra~0.4-1.8 μm) surfaces. This difference was indicated by a flat, spread-out cell morphology (including distinct filopodial and lamellipodial cell-extensions), the formation of pronounced focal adhesions, and an increased proliferation rate on smoother surfaces. Cells adhering to rougher surfaces appeared rounded, lacked pronounced focal adhesions, and exhibited a slower proliferation rate. Taken together, these results indicate that smoother Bioglass surfaces provide a better surface topology for cell attachment and proliferation, probably by allowing cells to more efficiently establish and maintain cell-surface junctions, and to more efficiently migrate on the smoother surfaces. The effect of surface microarchitecture on intracellular structure is observed through the formation of the actin cytoskeleton on only one intermediate surface roughness. Interestingly, cells tended to align along deeper scratches, when the scratch width was approximately equivalent to cell size.In our analyses, surface roughness of Bioglass exhibited only a relatively small effect on the expression level of the analyzed genes, suggesting that the surface topology of 45S Bioglass has only a minor effect on osteoblast differentiation. To our surprise, control borosilicate coverglass stimulated up-regulation of the analyzed genes and MC3T3-E1 pre-osteoblast differentiation much more efficiently than any of the analyzed 45S Bioglass samples. Notwithstanding, these observations are particularly relevant to recent reports that borate ions and borate glasses are conducive to osteogenesis, as well as wound healing.
9:00 PM - KK6.42
The Role of Moderate Static Magnetic Fields and Susbtrate Interactions on Biomineralization of Osteoblasts.
Miriam Rafailovich 1 , Marcia Simon 4 , Ba Xiaolan 1 , Elaine DiMasi 3 , Yizhi Meng 1 , Zhongkui Ta 1 , Michael Hadjiargyrou 2
1 Materials Science and Engineering, stony brook university, Stony Brook, New York, United States, 4 Oral Biology, Stony Brook School of Dental Medicine, Stony Brook, New York, United States, 3 , Brookhaven National Laboratory, Upton, New York, United States, 2 Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractWe have investigated the effects of moderate Static Magnetic Fields (SMFs) on the process of biomineralization of mouse MC3T3-E1 osteoblasts, cultured on plastic tissue culture petri dish plastic and partially sulfonated polytstyrene ( SPS, degree of sulfonation~0.28). We found that in both cases the SMF influenced biomineralization, with the effect being much larger for the cells on the SPS substrate. We had shown previously that SPS substrates enhanced biomineralization and that the ECM proteins secreted by the cells self assembled on these substrates into a fibrillar network, which templated the biomineralization process. We found that SMFs had a profound influence, producing much larger ECM fibers during the first seven days. We also found that only the ECM fibers produced in the presence of the SMF were able to template mineral deposition during the early stages of the biomineralizing process. The rapid response was attributed to orientation of diamagnetic ECM proteins already present in the plasma, which could then initiate further cellular signalling. Cells cultured in the presence of the SMF exhibited a larger increase in the rate of osteocalcin production and gene expression, after 15 days, which correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite with a high degree of crystalinity. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.
9:00 PM - KK6.43
Fabrication of Multicompartment Hydrogel Scaffold Using Density Gradient Method.
Yogesh Ner 1 , Jerome Karpiak 1 , Adah Almutairi 1
1 , University of California San Diego, La Jolla, California, United States
Show AbstractWe report a new method, a density gradient method (DGM), as a simple and versatile technique to fabricate multicompartment hydrogels. Multicompartment hydrogels with distinct layers of mechanical/chemical cues can mimic complex 3D structural organization or provide a gradient of cues existing in native tissue. However, the requirement of integrating heterogeneous materials in a mechanically robust composite provides a challenge for fabrication. Unlike conventional methods, DGM allows integration of heterogeneous layers before gelation thereby resulting in improved cohesion between layers. We will highlight the DGM technique with respect to formation of a number of layers, their discreteness and the mechanical properties of the composite. Furthermore, we will discuss the ability of DGM to produce discrete and cross gradients of a variety of proteins based on diffusion in multilayers. Finally, we will show the ability of multilayered hydrogels to produce a chemically distinct environment by allowing cell growth in specified layers.
9:00 PM - KK6.45
Angiogenic Microfiber/Microparticle Patches for Directed Blood Vessel Growth.
Ross DeVolder 1 2 , Harim Bae 3 , Jonghwi Lee 3 , Hyunjoon Kong 1 2
1 Chemical and Biomolecular Engineering, University of Illinois, Urbana, Illinois, United States, 2 Institute of Genomic Biology, University of Illinois, Urbana, Illinois, United States, 3 Chemical Engineering and Materials Science, Chung-Ang University, Seoul Korea (the Republic of)
Show AbstractIntroduction: Therapeutic angiogenesis has emerged as a promising strategy to treat various acute and chronic vascular diseases, and to enhance tissue repair and regeneration. Common revascularization therapies include the administration of angiogenic factors, such as vascular endothelial growth factor (VEGF). These therapies greatly rely on the ability to engineer mature and functional neovessels, uniformly distributed within target tissues. The objective of this study was to develop an angiogenic patch that releases angiogenic growth factors and ultimately regulates the directional growth of mature and functional blood vessels.
Materials and Methods: Angiogenic microfiber patches were prepared by integrating electrospinning and electrospraying processes. Poly(lactic acid) (PLA) microfibers with negative surface chargers were extruded through an electrohydrodynamic nozzle, and VEGF encapsulating poly(lactic-co-glycolic acid) (PLGA) microparticles with positive surface charges were simultaneously sprayed over the fiber surfaces. The microparticle covered fibers were collected on Teflon frames in a directionally aligned manner. The proangiogenic patches were individually implanted on the chorioallantoic membrane (CAM) of chicken embryos and examined over a week for blood vessel formation. The CAMs were excised and histological CAM cross-sections were analyzed for blood vessel densities, distributions, and alignment.
Results and Discussion: The electrospinning/electrospraying technique produced an angiogenic patch that sustainably released VEGF from degrading PLGA microparticles, while the PLA microfibers act as a stable structure preventing the dislocation of microparticles at the delivery site. The temporal control of electrostatic interactions using this technique has the advantage of improving bond strength between fibers and particles compared to previously reported techniques. Cells adhered to the patches, were anisotropically directed by the alignment of the microfiber patches. The implantation of angiogenic patches on CAMs of chicken embryos resulted in a significant increase in the size and density of mature blood vessels compared to control samples. The formation of blood vessels was evenly distributed and aligned parallel to the microfiber alignment. We suggest that the VEGF released from the microparticles locally distributes around and associates with the PLA microfibers, subsequently presenting concentration gradients that direct blood vessel formation along the microfibers.
Conclusion: We have demonstrated that the angiogenic patch fabricated by integrating an electrospinnning and elecrospraying process allows the control of both the density and spatial organization of blood vessels at an implant site. Overall, the angiogenic patch developed in this study will be broadly useful for improving the quality of treatments for various vascular diseases and tissue defects.
9:00 PM - KK6.46
Induction of Dental Pulp Stem Cell Differentiation by Non-Degradable Polymers.
Aneel Bherwani 1 , Giulia Suarato 2 , Sisi Qin 2 , Chung-Cheh Chang 2 , Aaron Akhavan 3 , Joseph Spiegel 2 , Vladimir Jurukovski 2 4 , Miriam Rafailovich 2 , Marcia Simon 1
1 Oral Biology and Pathology, Stony Brook University, Stony Brook, New York, United States, 2 Materials Science, Stony Brook University, Stony Brook, New York, United States, 3 , Ramban Mesizta High School, Cedarhurst, New York, United States, 4 Biology, Suffolk Community College, Selden, New York, United States
Show AbstractIn vitro, dental pulp stem cells (DPSC) can undergo odontoblast, osteoblast, neuronal and adipogenic differentiation dependent upon substrate and soluble mediators. We have previously shown that substrate mechanics plays a role in DPSC differentiation on elastomeric substrates. In the current study we evaluated the impact of rigid but biocompatible polymers, polymethylmethacrylate (PMMA) and poly (4-vinylpyridine) (P4VP) using different biophysical cues. The following substrates were prepared: [1] 200 nm films spun cast on HFX-Si wafers, annealed for 24-hours at 170°C under a vacuum of 10-4 Torr, [2] 5 µm and 500nm electrospun PMMA onto 200 nm films, annealed for an additional 30-minutes to secure the fiber onto the PMMA surface [3] The P4VP was dissolved in dimethylformamide at a concentration of 7 mg ml−1 and spun cast onto the freshly cleaned Si wafers at 2.05 × 103 r.p.m. The samples were then annealed in a vacuum oven (P~103 Torr, T=170 °C) overnight [4] 5 µm and 500nm electrospun P4VP onto 200 nm films, annealed for an additional 30-minutes to secure the fiber onto the P4VP surface.DPSC were obtained from pulp tissues of extracted wisdom teeth under protocols approved by the Stony Brook University Internal Review Board. Primary cells were isolated by enzymatic digestion and used from 3rd-5th passage. For experiments on differentiation, cultures were grown in alpha-MEM supplemented with 10% fetal bovine serum, 0.2 mM L-ascorbic acid 2-phosphate, 2 mm glutamine, 10 mM beta-glycerol phosphate either with or without 10 nM dexamethasone. After 21-days samples were examined using confocal microscopy of cells stained with Alexafluor 488-linked phalloidin and propidium iodide, and by scanning electron microscopy (SEM) and Energy dispersive X-ray Analysis (EDAX). Using SEM, biomineralization was observed on all the substrates in the presence of dexamethasone. In the case of P4VP extensive biomineralization was observed in the absence of dexamethasone, where the deposits were templated along the fibers. Minimal biomineralization was observed on PMMA fibers. EDAX spectra indicated that the deposits on both P4VP and PMMA fibers were comprised primarily of Ca and phosphorous, in a ratio consistent with hydroxyapatite. On the P4VP additional carbonaceous fiber deposits were observed in the vicinity of CaP deposits. Odontoblast and osteoblast markers are being evaluated by RT-PCR. The effect of fiber diameter and morphology on the differentiation of the DPSC will be discussed.
9:00 PM - KK6.5
Structure and Stability of Pol(L-lysine)/Hyaluronan Thin Films as Reservoirs for the Bone Morphogenetic Protein-2.
Flora Gilde 1 , Ofelia Maniti 1 , Raphael Guillot 1 , Catherine Picart 1
1 Department of Bioengineering, Grenoble Institute of Technology, Grenoble France
Show AbstractPolyelectrolyte multilayer films (PEM) made of poly(L-lysine) and hyaluronan (PLL/HA) have been recently investigated for their ability to trap the osteoinductive recombinant human Bone Morphogenetic protein-2 (rhBMP-2) (Crouzier et al., Small 2009). However, there are several tests to be performed prior to their possible use in clinical applications as bioactive coatings of dental or orthopaedic implants. First, all medical devices should sustain a sterilization process before in vivo implantation and they should also sustain storage for at least several months. In view of future applications of the PEM films, we first aimed here to investigate the stability, shelf life-time and sterilization of (PLL/HA) film. Secondly we aimed to investigate the secondary structure of rhBMP-2 trapped hydrated or dry (PLL/HA) films, as compared to the protein in solution at different pHs. To this end, we used Fourier Transform Infrared Spectroscopy (FTIR) to investigate the structure of (PLL/HA) films in dry state, their stability over time, the effects of sterilization by gamma irradiation and the structure of rhBMP-2 trapped in the films. We found that the (PLL/HA) films were very stable over the 2-month period, with less than 2% variation in the absorbance intensity of the amide I band. We also proved that the film can withstand the sterilization process without any major change in their structure. We quantified the secondary structure of rhBMP-2 trapped in a hydrated film and observed that it exhibited a similar structure to that of the protein in solution at pH 3 (representing the optimal conditions for BMP-2 loading the film). The preservation of the secondary structure of the film when trapped and confined in the PEM film may explain why rhBMP-2 remained bioactive. Interestingly, when the protein was trapped in dry films, a significant change in the protein structure was visible with the appearance of intermolecular beta-sheets. We attributed these changes to protein/protein interactions or to protein/PLL interactions.
9:00 PM - KK6.7
Fabrication of Dental Restorative Composites from the Mixture of Bis-GMA Alternatives and Spiro Orthocarbonates and Their Characteristics.
Sunhwa Yoo 1 , Sangchul Roh 1 , Chang Keun Kim 1
1 School of Chemical Engineering & Materials Science, Chung-ang University, Seoul Korea (the Republic of)
Show AbstractThe characteristics of resin matrices containing 2,2-bis[4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl]propane (Bis-GMA) alternatives and spiro orthocarbonate (SOC) were examined to produce dental restorative composites exhibiting lower curing shrinkage and better mechanical strength than the commercially available dental composites. Bis-GMA alternatives were prepared by replacing the hydroxyl groups in Bis-GMA with alkoxy groups. Various SOCs that showed the expected volume expansion during the curing reaction caused by the ring opening reaction were added to Bis-GMA alternatives for a further decrease in curing shrinkage. The curing shrinkage of the dental composites containing Bis-GMA alternative and SOC was always lower than that of the dental composite used as the control, which contained 70 wt% Bis-GMA and 30 wt% triethylene glycol dimethacrylate (TEGDMA) as the resin matrix. Furthermore, the mechanical strength of the dental composites prepared from the 2,2-bis[4-(2-methoxy-3-methacryloyloxy propoxy)phenyl]propane (Bis-M-GMA) mixture containing approximately 10 wt% SOC was higher than that of the dental composite used as the control.
9:00 PM - KK6.8
Photobiological Assays Using ClAlPc-Nanoemulsion and Portable Light Source with Multiple-Wavelengths on 3D-Human Stem Cell Dermal Equivalent to Wound Healing Trials.
Fernando Primo 1 , Leonardo de Paula 1 , Camila Amantino 1 , Antonio Tedesco 1
1 Chemistry, São Paulo University, Ribeirão Preto, São Paulo- Rtibeirão Preto, Brazil
Show AbstractIn recent years scientific advances combining nanotechnology and tissue engineering converged in several studies that reported the development of biological models useful for the understanding of in vitro wound healing process and extracellular matrix activity [1-2]. Nanotechnology have been showed many new interesting results in the multidisciplinary fields especially as a promising candidates for improving superior biocompatibility of many drugs, and development of materials with different optical, catalytic and magnetic properties compared to conventional materials (micron-structured) [3]. In order in this work was developed a nanobiomaterial to controlled and sustained release of the photosensitizer drug chloroaluminum phthalocyanine (ClAlPc) [1;3] loaded on biodegradable oil/water nanoemulsion. Photobiological assays were carried out using as in vitro model the stem cells dermal equivalent (SCDE), a 3D-human dermal-skin model to evaluation of the biological response after photoactivation with multiple wavelengths at 810 nm, 660 nm and 560 nm respectively. Results showed that is possible a modulation after photoactivation using appropriate low-level laser combination (infrared and visible red) which leads to differentiated SCDE kinetics contraction after ClAlPc-SCDE cellular uptake. Besides, the electronic micrographs showed that effect in the fibroblasts/stem-cells network with density differentiated to each laser-dose and wavelengths applied in these assays. Wound healing process will be evaluated in the next stages by matrix metaloproteases (MMP-2 and MMP-9) activity secreted in the SDCE culture medium from zymography gel and specific polyclonal antibodies tests. Thereby, the use of combined infrared and visible low-level laser is a promise therapeutic strategy useful to skin care trials and photorejuvenation procedures. The authors acknowledge the FAPESP post-doc project 2009/15363-9 and 2008/53719-4 (F.L.P.), and the CNPq PhD project 140998/2011-0 (L.B.P), Brazilian agencies to research financial support.[1] F.L. Primo, M.B. da Costa Reis, M.A. Porcionatto, A.C. Tedesco, Current Medicinal Chemistry, 18, in press, (2011).[2] L. Zhang and T.J. Webster, Nano Today, 4, 66 (2009). [3] M. P. Siqueira-Moura, F. L. Primo, A.P.F. Peti and A.C. Tedesco, Pharmazie, 65, 1 (2010).
9:00 PM - KK6.9
Development of New Sol-Gel Derived Ag-Doped Biomaterials for Dental Applications.
Xanthippi Chatzistavrou 1 , Eleana Kontonasaki 2 , Athina Bakopoulou 2 , Anna Theocharidou 2 , Afroditi Sivropoulou 3 , Konstantinos Paraskevopoulos 4 , Petros Koidis 2 , Aldo Boccaccini 5 , Toshihiro Kasuga 1
1 Department of Frontier Materials, Nagoya Institute of Technology, Nagoya Japan, 2 School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki 54124 Greece, 3 Biology Department, Aristotle University of Thessaloniki, Thessaloniki 54124 Greece, 4 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki 54124 Greece, 5 Department of Materials Science and Engineering, Institute of Biomaterials, University of Erlangen-Nuremberg 91058 Erlangen, Erlangen Greece
Show AbstractIn the field of dental repair and restoration the sol-gel derived bioactive glass-ceramic materials hold an important key position. The fabrication of a new sol-gel derived bioactive glass-ceramic in the system SiO2 58.6%-P2O5 7.2%-Al2O3 4.2%-CaO 24.9%-Na2O 2.1%-K2O 3% (wt.%), with attractive physicochemical and mechanical properties, has already been presented [1]. The successful application of this composite as coating on dental ceramic substrates, enhancing the attachment and proliferation of two cell types (human gingival fibroblast (HGF) and human periodontal ligament (HPDL)) was confirmed. An additional important property, for the optimization of the prepared composite and its further introduction to the clinical practice, is the development of an antibacterial action. It is critical for the materials to inhibit inflammatory reactions caused by the presence of bacteria, which can finally lead to the failure of the tooth/restoration complex. The aim of our work is to introduce antibacterial properties in the new composite material by incorporating silver ions in the ceramic structure and to prepare new Ag-doped composite materials with biocompatible and at the same time antimicrobial behavior. Two systems with different concentrations in Ag2O (2.1 and 4.2 wt. %) were prepared by the sol-gel method, which allows the synthesis of materials at low sintering temperature (700oC), enabling the preparation and tailoring of the glass-ceramics in special compositions with specific microstructural characteristics. The successful fabrication of colorless, homogenous and chemically durable materials, which can slowly release silver ions for a long period, was achieved. The prepared glass-ceramics show the formation of hydroxyapatite and wollastonite phases. Aluminum ions remain in the amorphous phase forming [AlO4]- tetrahedra with negative charge which is compensated by the Ag+ ion. These developed microstructures lead to the slow releasing ability of Ag+ ions during the degradation process, allowing a bioactive behavior in an antimicrobial environment. The antibacterial properties of the Ag-doped glass-ceramics were tested against a bacterial colony (Staphylococcus aureus) and the material-cell interaction was monitored for the HGF and HPDL cells. The implementation of the new materials in the field of dental restorations’ construction might potentially lead to the development of new surfaces even on restored dental implants, with strong bioactive, bactericidal and non-toxic behaviour. These conditions can consequently enhance the bonding and the regeneration of the surrounding tissues, sealing the marginal gap between tooth and restoration and thus preventing the failure of the tooth/restoration complex.
Symposium Organizers
Mei Wei University of Connecticut
Jordan Green The Johns Hopkins University
Xinqiao Jia University of Delaware
James Olson Teleflex Medical
KK7: Advances in Tissue Engineering
Session Chairs
Wednesday AM, November 30, 2011
Room 102 (Hynes)
9:30 AM - **KK7.1
Advances in Tissue Engineering.
Robert Langer 1
1 David H. Koch Institute Professor, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractBy combining mammalian cells, including stem cells, with synthetic polymers, new approaches for engineering tissues are being developed that may someday help repair tissues for patients with burns, damaged cartilage, paralysis and vascular disease. Various technological advances in controlled drug delivery, nanotechnology and other areas may aid in the development of these approaches. These advances include controlled delivery of bioactive agents including DNA and siRNAs, the development of new biomaterials and the creation of new materials-based tools for understanding stem cell growth and differentiation.
10:00 AM - KK7.2
The Design of Hydrogel Cell Carriers to Improve Stem Cell Viability during Transplantation by Direct Injection.
Brian Aguado 2 , Sarah Heilshorn 1 2
2 Bioengineering, Stanford University, Stanford, California, United States, 1 Materials Science & Engineering, Stanford University, Stanford, California, United States
Show AbstractCell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells (hASC), rat mesenchymal stem cells (rMSC), and mouse neural progenitor cells (mNPC). Two types of hydrogel designs were investigated: alginate hydrogels with plateau storage moduli (G’) ranging from 0.33 to 58.1 Pa and protein-engineered hydrogels with G' ranging from 30 to 100 Pa. In both systems, compliant hydrogels with G’ ~ 30 Pa yielded significantly higher cell viability compared to cells in Newtonian solutions (e.g., media alone). Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Furthermore, cells within non-crosslinked, viscoelastic solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biopolymer chemistry. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell membrane rupture and cell death. Physical hydrogels that undergo shear-thinning may be able to undergo plug-flow through the syringe needle, whereby bands of shear-thinned polymer are located at the needle walls and lubricate the flow of individual gel "plugs" through the needle. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies.
10:15 AM - KK7.3
High Conductivity PEDOT as a Substrate for Cell Adhesion and Proliferation.
Manrico Fabretto 1 2 , Elise Stewart 3 , Mischa Mueller 2 , Rob Short 2 , Gordon Wallace 3
1 Ian Wark Research Institute, University of South Australia, Mawson Lakes, South Australia, Australia, 2 Mawson Institute, University of South Australia, Mawson Lakes, South Australia, Australia, 3 Intelligent Polymer Research Institute, University of Wollongong, Wollongong, New South Wales, Australia
Show AbstractThe use of conducting polymers as a biomaterial platform has been steadily increasing in recent years. This is due to their stability, ease of synthesis, tuneable conductivity and redox state. The polymer, poly(3,4-ethylenedioxythiophene) (PEDOT) has emerged as a promising material capable of interacting with cell systems [1, 2] and has even been polymerised around living cells [3]. The synthesis and material characterisation of high conductivity PEDOT and its suitability with respect to cell interactions is described. Vapor phase polymerised (VPP) PEDOT, using the oxidant Fe(III)Tosylate, was optimised with respect to conductivity. This was achieved by incorporating a glycol tri-block polymer, poly(ethylene glycol-propylene glycol-ethylene glycol), into the oxidant. The resulting conductivity varied from 400 to 1500 S.cm1, as the amount of glycol was changed. Contact angle measurements, XPS and ToFSIMS confirmed the presence of embedded glycol on the surface. AFM imaging down to the nanometre scale did not show any phase contrast, indicating that the glycol was incorporated within the PEDOT structure at smaller length scales. The expectation was that changes in PEDOT morphology, and in particular glycol concentration, would have an adverse impact on protein adsorption, and hence cell adhesion and proliferation. QCM was utilised to compare fibronectin adsorption on different PEDOT samples. The amount adsorbed did not vary significantly as glycol content was increased. Furthermore, the monolayer of protein formed at the PEDOT surface had similar viscoelastic properties regardless of glycol addition. Cell adhesion and proliferation was also investigated using mouse fibroblasts (3T3) and human keratinocyte (HaCaT) cell lines. The initial adhesion after 2 hr did not vary significantly between various PEDOT samples. Keratinocytes, however, proliferated best on the highest conductivity PEDOT. The incorporation of high conductivity processable PEDOT into biomedical devices allows for the opportunity to exploit this property for the delivery of electrical stimulus in-situ.[1] Bolin MH, Svennersten K, Wang X, Chronakis IS, Richter-dahlfors A, Jager EWH, et al. Nano-fiber scaffold electrodes based on pedot for cell stimulation. Sensors And Actuators B: Chemical 2009;142:451-6.[2] Svennersten K, Bolin MH, Jager EWH, Berggren M, Richter-Dahlfors A. Electrochemical modulation of epithelia formation using conducting polymers. Biomaterials 2009;30:6257-64.[3] Richardson-Burns SM, Hendricks JL, Foster B, Povlich LK, Kim D-h, Martin DC. Polymerization of the conducting polymer poly(3,4-ethylenedioxythiophene) (pedot) around living neural cells. Biomaterials 2007;28:1539-52.
10:30 AM - KK7.4
Dynamic Micromolding Method.
Halil Tekin 1 2 3 , Tonia Tsinman 2 4 , Jefferson Sanchez 2 5 , Brianna Jones 2 6 , Robert Langer* 3 5 7 , Ali Khademhosseini* 2 7
1 Department of Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States, 3 David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 5 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 6 Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 7 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractMicroengineered hydrogels were utilized to encapsulate living materials and chemical compounds for a number of applications such as tissue engineering and drug delivery. Spatial immobilization of these materials within different compartments of hydrogels is critical either to mimic biological complexity or to create multi-compartment drug carriers. Sequential patterning with photolithographic methods is useful to create multi-compartment hydrogels. However, these methods do not apply for non-photocrosslinkable materials. Furthermore, previously offered micromolds possess a static nature, which inhibits molding of subsequent hydrogels. Herein, we introduce a dynamic micromolding technique to sequentially pattern hydrogel microstructures. Dynamic micromolds fabricated from poly(N-isopropylacrylamide) exhibited increased patterned areas with increased temperature, allowing sequential molding of hydrogels at different temperature points. We generated multi-compartment hydrogel microstructures with different geometries such as stripes, cubes, and cylinders by using a dynamic micromolding method. Fluorescent microbeads were encapsulated within different compartments of hydrogel microstructures to model the immobilization of chemical compounds within multi-compartmental hydrogel microstructures for drug delivery applications. Living materials were also encapsulated to show that this dynamic micromolding method can be useful to spatially immobilize different cell types within different compartments of a single hydrogel microstructure for mimicking biological complexity. This dynamic micromolding method can be utilized to spatially organize the positions of living materials and chemical compounds within a single microgel, which may potentially be useful for various applications in life sciences, materials science and engineering, biology, and chemistry.
10:45 AM - KK7.5
Biofunctionalized Gold Nanostructures for Controlling Cell Adhesion.
Christine Selhuber-Unkel 1 , Julia Reverey 1 , Janosch Deeg 2 , Ilia Louban 2 , Horst Kessler 3 , Joachim Spatz 2
1 Institute for Materials Science, University of Kiel, Kiel Germany, 2 New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Stuttgart Germany, 3 Center of Integrated Protein Science Munich, Technical University of Munich, Munich Germany
Show AbstractIn nature, cells are constantly in touch with micro- and nanostructures; an example is collagen, which has a periodic surface structure in the nanometer regime. By mimicking these structures we might have the opportunity to control cellular properties and parameters that are important for tissue regeneration, such as cell motility, cell differentiation and cell adhesion. With block-copolymer micelle nanolithography (BCMN) it is possible to produce self-assembled structures of gold nanoparticles that provide anchorage points for single cell adhesion receptors (e.g. integrins) in a hexagonal lattice with nanometer resolution. The spacing between the anchorage points can be varied in a wide range between approx. 20 and 400 nm. It has been shown that in this way cellular parameters such as spreading and motility can be controlled. In order to characterize the recognition of nanostructures by cells as a function of time and to quantify cell adhesion on a sub-cellular level, we are using an AFM-based method to measure adhesion strength. This method is capable of quantifying the adhesion forces of cells to surfaces at arbitrary time-scales and over large force regimes. We found that the nano-scale spacing between individual adhesion receptor proteins in the cell membrane cooperatively controls the strength of cell adhesion and the mechanical properties of cells. Furthermore, it turned out that the local density of anchorage points is the controlling factor rather than their global density. Funding from the DFG Emmy Noether Program is gratefully acknowledged. References: Deeg, J., Louban, I., Aydin, D., Selhuber-Unkel, C., Kessler, H., Spatz, J. Impact of local versus global ligand density on cellular adhesion. Nano Letters (2011), 11(4), 1469-1476. Selhuber-Unkel, C., Erdmann, T., López-García, M., Kessler, H., Schwarz, U. S. and Spatz, J. P. Cell adhesion strength is controlled by intermolecular spacing of adhesion receptors. Biophysical Journal (2010), 98, 543-551.
11:30 AM - **KK7.6
Tailoring Degradable Polymers for Biofunctional Implants Inducing Tissue Regeneration.
Andreas Lendlein 1
1 Center for Biomaterial Development and Berlin-Brandenburg Center for Regenerative Therapies, Institute of Polymer Research, Helmholtz-Zentrum Geesthacht, Teltow Germany
Show AbstractResearch in the field of degradable biomaterials is driven by the aim to create temporary implants, which can substitute certain tissue/organ functions, induce the autoregeneration of defects, or deliver drugs in a controlled manner. When the implant is not needed anymore it degrades so that an additional surgical procedure for its explantation can be avoided [1-4, 8]. The design of degradable polymers is complex [3-5, 8]. The change of material properties during degradation, e.g. changes in elasticity, and the degradation products are important characteristics to be considered. The non-toxicity of the original and the partly degraded material as well as of the degradation products and the targeted biofunctionality of the polymer are essential prerequisites for its applicability as implant material. It still is a challenge to reliably predict the degradation behavior of biomaterials in physiological environments. In this context experimental as well as computational tools are being developed to evaluate the mechanisms of polymer degradation [3]. In many clinical applications, it became apparent that one single function like degradability is not sufficient, but multifunctionality is required [4-8]. The use of biopolymers as basis for novel biomaterials in this context is highly desirable in terms of structurally-mimicking the extracellular matrix, which plays a crucial role in tissue regeneration. However, this is challenging due to difficulties in tailoring and controlling the material properties [9, 10].In this presentation, design principles for multifunctional, degradable biomaterials are outlined on different levels of hierarchical organization. Finally, examples are given for dual and triple functional polymers and potential applications are outlined.References[1] A. Lendlein, A.T. Neffe, B.F. Pierce, J. Vienken, Int. J. Artif. Organs 34, 71-75 (2011).[2] A. Lendlein, A. Sisson, Handbook of Biodegradable Polymers, Wiley-VCH, Weinheim (2011).[3] D. Hofmann, M. Entrialgo-Castaño, K. Kratz, A. Lendlein, Adv. Mater. 21, 3237-3245 (2009).[4] V.P. Shastri, A. Lendlein, Materials in Regenerative Medicine, Adv. Mater. 21, 3231-3234 (2009).[5]A.T. Neffe, B.D. Hanh, S. Steuer, A. Lendlein, Adv. Mater. 21, 3394-3398 (2009).[6] A. Lendlein, M. Behl, B. Hiebl, C. Wischke, Expert Rev Med Devic 7(3), 357-379 (2010).[7] M. Behl, M. Razzaq, A. Lendlein, Adv. Mater. 22, 3388–3410 (2010).[8] F. Jung, C. Wischke, A.Lendlein, MRS Bull. 35 (8), 607-613 (2010).[9] A. Neffe, A. Zaupa, B. Pierce, D. Hofmann, A. Lendlein, Macomol. Rapid Comm. 31(17), 1534-1539 (2010).[10] G. Tronci, A.T. Neffe, B.F. Pierce, A. Lendlein, J. Mater Chem. 20(40), 8875-8884 (2010).
12:00 PM - KK7.7
Micropatterning and Covalent Immobilization of Multiple Bioactive Molecules for Regenerative Medicine Applications.
Erhan Bat 1 , Jordi Cabanas Danes 1 , Pascal Jonkheijm 1 , Jurriaan Huskens 1
1 Molecular Nanofabrication, University of Twente, Enschede, Overijssel, Netherlands
Show AbstractIncorporating bioactive molecules in biomaterials is increasingly preferred to instruct the cells and to obtain a specific and desired biological response. Surface tethered growth factors offer a better control of their spatial and temporal availability in the extracellular environment in contrast to soluble proteins. Due to challenges in simultaneous patterning of multiple biomolecules, only few studies have investigated the effect of co-patterning multiple different molecules to (stem-) cells. Such co-patterns would be very instrumental in studying the synergistic- or antagonistic activity of different bioactive molecules such as growth factors. Although inkjet printing allows arraying of multiple biomolecules, no control is available over the shape and size (>100 µm). Parallel patterning with micrometer level resolution and with desired shapes can be provided using microcontact printing and combined with its low fabrication costs, simplicity and reproducibility, however more wide spread implementation is challenged by the necessity of patterning multiple biomolecules with a single stamp. Here, we present hydrogel-filled silicon stamps having individually addressable ink reservoirs with which multiplexicity can be achieved while avoiding the need for re-inking. We used these stamps to micropattern and covalently immobilize multiple different bioactive molecules on surfaces of biocompatible polymers for investigating the interaction of stem cells with multiple bioactive molecules. We fabricated silicon microstructures having separate wells (320×320×380 µm) and each well having a 25 µm thick membrane (144 microchannels measuring 5 µm in diameter) on the printing side. The reservoirs and microchannels of the silicon microstructures were filled with macroporous poly(2-hydroxyethyl methacrylate-co-ethylene glycol dimethacrylate) hydrogels that were covalently bound to the surface of the silicon. After filling the separate reservoirs of the stamps with different growth factors (VEGF, EGF, bFGF, TGF-β, and BMP-6) and fibronectin simultaneously a multi-content micropatterns were generated on epoxy-functionalized poly(dimethylsiloxane) (PDMS) or poly(trimethylene carbonate) substrates. Moreover, up to twenty times pattern replication was possible without re-fill. The integrity of the multi-content micropatterns was verified with immunofluorescence stainings and currently the bioactivity of these surfaces for the differentiation of mesenchymal stem cells towards different lineages is studied. These engineered bioactive surfaces may become very instructive tools in designing future biomaterials for regenerative medicine.
12:15 PM - KK7.8
Fabrication of Porous Hydrogels with Embedded Microchannels Using Living Sacrificial Porogens.
Feng Xu 1 , Banupriya Sridharan 1 , Ahmet Yavuz 1 , Shuqi Wang 1 , Naside Durmus 1 , Umut Gurkan 1 , Utkan Demirci 1
1 Harvard-MIT Health Sciences and Technology, Harvard-MIT Health Sciences and Technology, Cambridge, Massachusetts, United States
Show AbstractPorous hydrogel scaffolds have emerged as viable tissue mimics with widespread applications in tissue engineering, regenerative medicine and drug discovery. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. We developed an innovative approach for fabricating three-dimensional hydrogel scaffolds with microchannels by integrating printing technology and living sacrificial porogens (e.g., bacteria) with microscale spatial control over the size and density of microchannels. he concept is based upon the ability to print E.coli in an interconnected three-dimensional (3D) network configuration, culture the bacteria, decellularize the resulting network structure and then introduce cells. The developed method has potential to enable fabrication of porous microfluidic hydrogel scaffolds with microscale resolution for various regenerative medicine and pharmaceutical drug discovery applications.
12:30 PM - **KK7.9
Modulation of Cell-Biomaterial Interactions for Tissue Regeneration.
Mario Barbosa 1 2
1 , INEB-Instituto de Engenharia Biomédica, Universidade do Porto, Porto Portugal, 2 , Instituto Ciências Biomédicas Abel Salazar, UPorto, Porto Portugal
Show AbstractAdsorbed proteins play a key role in many biological processes, including the interaction of cells with biomaterials for tissue regeneration. For instance, protein adsorption mediates cell adhesion, proliferation and differentiation. In general, the biomaterial surface is not selective towards protein adsorption. However, selective protein adsorption is crucial to guide biological behavior on engineered materials, particularly those used for tissue regeneration. Selectivity is fundamental for cell behavior and has become a major goal for biomaterials science, namely for guiding stem cell differentiation, inducing tissue regeneration, avoiding clot formation an controlling the inflammatory reaction.Surface chemistry, particularly if designed at the nanoscale, is an essential parameter through which selective adsorption can be controlled. Various strategies have been proposed to induce and guide tissue regeneration, including the mimicking of the interactions between cells and the components of the extracellular matrix (ECM). Grafting of functional groups into polymeric materials, formation of polyelectrolyte polysaccharide- or protein-based complexes have been widely investigated. Exploiting the ability of some of these structures to self-assembly in the biological medium has been proposed in order to produce matrices that resemble the natural ECM.To understand the fundamental processes occurring in some of these new materials self-assembled monolayers (SAMs) of alkanethiols produced on gold substrates have shown to be an ideal model, particularly when the objective is to study the influence of specific functionalities on protein adsorption and how it controls cell behavior. For tissue engineering 3D porous and nonporous (hydrogels) scaffolds can also be designed with the purpose of modulating cell attachment and differentiation, matrix synthesis and degradation (e.g. by metalloproteases) and intracellular cross-talk. This presentation will cover the above aspects in the context of molecular interactions that take place between cells and substrates.
KK8: Applications and Medical Devices
Session Chairs
Wednesday PM, November 30, 2011
Room 102 (Hynes)
2:30 PM - **KK8.1
ECM-Supported Tissue Regeneration: Matrix Biological and Structural Integrity as Key Factors for Tissue Repair and Regeneration.
Wendell Sun 1
1 , LifeCell Corporation, Branchburg, New Jersey, United States
Show AbstractAcellular tissue matrices (ECM) derived from human and several animals have been in use to support human tissue regeneration for over three decades. The first wave of commercially available products currently commands a market value over 1 billion US dollars, and in many cases, these products are demonstrated to perform significantly better than alternative synthetic biomaterials. The natural tissue matrices modulate tissue regeneration via in situ cell repopulation, revascularization and remodeling with patient’s own cells, although the specific mechanisms of action have not been fully understood.This presentation will discuss (1) the effect of the ECM biological and structural integrity in the positive body/implant recognition and successful tissue regeneration, and (2) the importance of understanding and measuring the ECM biological and structural integrity for future product development. The creation of regenerative acellular tissue matrices is dependent upon the preservation of the ECM functions as demonstrated by the robust host-cell repopulation, induction of stem cell differentiation, absence of significant foreign body response and active ECM remodeling. The retention of ECM biological and structural integrity during the process of cell removal, sterilization and preservation is a key factor for the positive body/implant recognition and successful tissue regeneration. ECM materials that are produced under the conditions that effectively removed cellular and selected matrix components while preserving its functional and structural integrity are able to support, when tested in vitro, the growth of fibroblasts and bone marrow derived stem cells while inducing only minimal lymphocytic proliferation and macrophage activation. The implantation of ECM as tissue scaffolds in various animal models demonstrated clinical acceptance of the grafts, restoration of functional defects and remodeling. In contrast, structurally and chemically modified ECM materials resulted in macrophage activation in vitro and a significant inflammatory response, not an active tissue remodeling process, when tested in vivo. One of the major obstacles to elucidate the mechanisms of action is the complexity of ECM biochemistry and structure. Various matrix elements are arranged in a functional 3-D structure, where the relation between its function and structural attributes remains to be understood.
3:00 PM - KK8.2
Optimized Deep Partial Thickness Swine Burn Model for the Evaluation of Keratin Hydrogel Treatments.
Carmen Gaines 1 , Deepika Poranki 1 , Mark Van Dyke 1
1 WFIRM, Wake Forest Institute for Regenerative Medicine, Winston-Salem, North Carolina, United States
Show AbstractThe porcine model is the preferred method in which to study progressive healing of thermal contact burns in vivo. Of the numerous studies cited in the literature, little emphasis is placed upon minimizing lateral and depth wound variability in porcine burn models. This is especially problematic in the evaluation of experimental burn treatments, as inconsistent wounds make it difficult to accurately quantify their wound healing capabilities. While drawbacks such as variance in subcutaneous tissue structure along the animal’s body and inefficient heat transfer from source to skin for burn creation may affect wound creation, our lab has employed standard materials science principles in developing a novel method to create deep partial thickness burns that are significantly consistent and highly reproducible.Burn wound creation was facilitated through the use of a custom made, spring loaded burn instrument, allowing for the preservation of lateral wound size and depth consistency when used by various operators. Insertable brass blocks, 3 cm in dia., served as the actual wound mechanism. An azeotropic mixture of boiling 80:20 poly(ethylene) glycol (PEG):water proved more effective than water alone in raising the block temperature to an optimal range of 99 to 103°C and creating an interface between skin and metal for effective heat transfer. This model was used to evaluate the efficacy of human hair extracted keratin hydrogel treatments in a large scale, preclinical swine study. Under general anesthesia, 12 deep partial thickness wounds were created, 6 on each side of the shaved dorsal midline between the shoulder and hip. The wounds were covered with occlusive dressings and allowed to recover for 10 hrs prior to experimental treatment application. Treatments included crude and fractioned human hair keratin proteins, extracted via oxidative chemistries and reconstituted in saline to hydrogel form, along with silver sulfadiazine cream (standard of care) and a collagen-based hydrogel as controls. Every 3 days, the dressings were removed, wounds cleaned with saline soaked gauze and digital images taken. Treatments were then reapplied and wounds rebandaged. At designated time points, a pair of animals was euthanized and tissue collected for further histological analysis. Over the course of 30 days, the keratin-treated wounds exhibited a faster healing rate in comparison to those treated with the standard of care. Similarly, histological evaluation of excised tissue showed a greater degree of reepithelialization in wounds treated with keratin, indicative of the degree of healing that had occurred. The execution of a highly reproducible, consistent burn model in combination with the regenerative capabilities of human hair keratins may lead to the production of a bioactive, biocompatible, inexpensive topical burn therapy option to enhance the healing progression of deep partial thickness burns.
3:15 PM - KK8.3
Presentation of BMP-2 from a Soft Biopolymeric Film Unveils Its Activity on Cell Adhesion and Migration.
Thomas Crouzier 1 , Laure Fourel 1 2 , Thomas Boudou 1 , Corinne Albiges-Rizo 2 , Catherine Picart 1
1 Department of Bioengineering, Grenoble Institute of Technology, Grenoble France, 2 , INSERM, Grenoble France
Show AbstractCell biologists and biomaterials scientists are used presenting cell adhesive ligands as they are in vivo: that is, in their insoluble form and in proximity to the cell adhesion site (1-2). Intriguingly, this approach has barely been adopted for growth factors, usually presented to the cell in solution or as releasable molecule, whereas growth factors in vivo are known to strongly interact and to bind with the components of the extracellular matrix(3-4). In addition to these biochemical signals, the mechanical properties of the cell’s microenvironment have also been described to greatly impact cell behavior. To date, there is no study aimed to investigate the interplay between the mechanical properties of a biomaterial and the presentation of growth factor by the same material. Here, we design a new biomimetic nanoassembled film combining both matrix-bound presentation of the growth factor bone morphogenetic protein 2 (BMP-2) and modulation of the material’s mechanical properties. Both cell adhesion and migration were investigated on C2C12 cells seeded on films with controlled stiffness and BMP-2 was presented as matrix-bound or in solution. Bound BMP-2 was able to induce cell adhesion and migration in the case of films with low stiffness. Thus, this simple design strategy allowed us to employ a well-characterized biomimetic film to vary simultaneously two major material’s properties: its stiffness and matrix-bound presentation of BMP-2. With this innovative tool, we demonstrated the importance of film’s mechanical properties to unravel specific effects of matrix-bound BMP-2 (5). Our results highlight that a soft film with controlled spatial organization and restricted diffusion of BMP-2 can regulate signaling sensitivity and cytoskeleton dynamics, likely in synergy with adhesion receptors. 1. Morgan M. R., M. J. Humphries, and M. D. Bass. 2007. Synergistic control of cell adhesion by integrins and syndecans. Nat Rev Mol Cell Biol 8:957-69.2. Huebsch N., and D. J. Mooney. 2009. Inspiration and application in the evolution of biomaterials. Nature 462:426-32.3. Taipale J., and J. Keski-Oja. 1997. Growth factors in the extracellular matrix. Faseb J. 11:51-9.4. Hynes R. O. 2009. The extracellular matrix: not just pretty fibrils. Science 326:1216-9.5. Crouzier T., L. Fourel, T. Boudou, C. Albiges-Rizo, and C. Picart. 2011. Presentation of BMP-2 from a soft biopolymeric film unveils its activity on cell adhesion and migration. Adv. Mater. 23:H111-8.
3:30 PM - KK8.4
Mussel-Inspired Functionalization of Metal Surfaces with Bioactive Self-Assembled Peptide Nanofibers.
Hakan Ceylan 1 , Ayse Tekinay 1 , Mustafa Guler 1
1 UNAM-Institute of Materials Science and Nanotechnology, Bilkent University, Ankara Turkey
Show AbstractMetal implant surfaces, such as cardiovascular stents and dental/orthopedic implants, needs to be functionalized with bioactive cues so that they could efficiently support rapid and sustained regeneration of the native tissue surrounding them. For example, rapid endothelialization over the luminal stent surface plays an indispensible role in preventing restenosis and in-stent thrombosis over the long term treatment of the cardiovascular diseases. Likewise, successful osseointegration in dental and orthopedic implants highly depends on the proliferation and migration of mineral deposing cells over the implant site. In this work, we developed bioactive peptide nanofibers which were conjugated with bioactive peptide sequences, REDV (an endothelial cell specific adhesion sequence), KRSR (an osteoblast specific adhesion sequence), and mussel-inspired adhesive molecule, 3,4-dihydroxy-L-phenylalanine (Dopa). These nanofibers can mimic the structure and function of the native tissue extracellular matrix and can be safely immobilized onto stainless steel and titanium surfaces through Dopa-mediated adhesion. We analysed behaviours of endothelial and smooth muscle cells on stainless steel surface functionalized with REDV/Dopa nanofibers and of osteoblasts and fibroblasts on titanium surface functionalized with KRSR/Dopa nanofibers. In vitro results demonstrated that endothelial cells and osteoblasts could specifically adhered, spread, and proliferated on REDV/Dopa and KRSR/Dopa nanofibers, respectively. On the other hand, the growth of smooth muscle cells and fibroblasts was inhibited on REDV/Dopa and KRSR/Dopa nanofibers, respectively. Overall, our design nanofibers, REDV/Dopa and KRSR/Dopa, delivered adaptive and selective microenvironment for endothelial cells and osteoblast, respectively, thereby providing a convenient platform for these materials to be used in clinical applications as stent and orthopedic implant coatings.
4:15 PM - KK8.5
Enhanced T2 Shortening by Polymeric Entrapment and Cellular Internalization of MRI Contrast Agents.
Chenjie Xu 1 2 , David Miranda-Nieves 3 , James Ankrum 2 1 , Isaac Roes 1 2 , Matthias Nahrendorf 4 , Jeffrey Karp 1 2
1 Medicine, Brigham & Women's Hospital, Cambridge, Massachusetts, United States, 2 Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, United States, 3 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States, 4 Center for Systems Biology, Massachusetts General Hospital , Boston, Massachusetts, United States
Show AbstractCellular therapy promises to revolutionize medicine by restoring tissue and organ function. However, to develop effective cell therapies, the location, distribution and long-term viability of these cells must be evaluated in a noninvasive manner. Magnetic resonance imaging (MRI) of cells labeled with contrast agents after either local injection or systemic infusion has the potential to fulfill this goal. Currently, the most widely used cell labeling agents for MRI based tracking are iron oxide based magnetic nanoparticles (IO-NPs) ranging from 10 nm to 200 nm. However, emerging evidence indicates that labeling agents can be transferred to neighboring cells or to the extracellular matrix, confounding the interpretation of results. This happens because of the exocytosis of IO-NPs from transplanted cells to neighboring cells. In addition, division of labeled cells leads to dilution of signal between daughter cells reaching undetectable levels within only a few cell divisions. We aim to develop an MRI sensitive contrast formulation that exhibits minimal exocytosis and minimal signal loss during cell division. Specifically, we aim to synthesize a biocompatible and biodegradable IO-NPs based particle that has reduced exocytosis rate and permit single particle detection. We have previously shown that 1-2 µm sized poly(lactide-co-glycolide) (PLGA) particles are readily internalized by mesenchymal stem cells (MSC), yet exhibit minimal exocytosis for at least 7 days. Taking advantage of the intracellular stability of micron-sized PLGA particles, we developed the magnetic PLGA particles encapsulating IO-NPs. The purpose of this study was to study the T2 shortening effect as a function of entrapment in PLGA and as a function of cell internalization.Micron-sized (~1 µm) and nano-sized (~200 nm) magnetic PLGA particles were synthesized via the encapsulation of IO-NPs using a single-emulsion and double-emulsion evaporation, respectively. Human MSCs were used to study the cellular uptake of the different particles. Both labeled cells and pure particles were imaged using a 4.7 T MRI scanner to evaluate the T2 relaxation time. The T2 signal of each sample in the MRI phantom picture was quantified to derive a T2 intensity figure, which revealed that the micron-sized PLGA particles produced similar T2 enhancement at every concentration as the other two particles, given that the intensity difference between them is less than 20%. However, after cell internalization, the micron-sized PLGA particles produced much stronger T2 enhancement in cells compared to 200 nm or 20 nm particles, given that the intensity difference between them is more than 100%.
4:30 PM - KK8.6
Iridium Oxide Hybrids as Electrodes for the Neural System.
Nieves Casan-Pastor 1 , Nina Carretero 1 , Javier Moral 1
1 , Institut de Ciencia de Materiales de Barcelona, CSIC, Bellaterra Spain
Show AbstractIridium oxide, IrOx, as well as titanium nitride, platinum or another phases emerging such as PEDOT (poly(ethylenedioxythiophene)), are being applied for neural stimulation and recording electrodes [1,2]. However, IrOx and some conducting polymers show much larger charge capacity than noble metals, such as platinum, mainly because it undergos redox reactions themselves during the electron-ionic exchange at the interface, before reaching the potentials for radical formation from oxygen and water reactions, that raises its charge delivery capacity. To date, electrodeposited IrOx is the best materials, with PEDOT nearby in terms of specific charge.[2]In addition to functional stimulation, electric fields of different magnitudes could induce tissue repair in the nervous system through the use of implanted electrodes in scaffold configurations. The candidate materials are expected to have larger charge capacities than functional electrostimulation electrodes, and therefore a significant improvement is needed in the electroactive materials. But not only the electrical, but the mechanical properties of the electrodes need to be improved. A significant study is being done through the design of hybrid phases that join the best of both, inorganic and organic biocompatible conductive phases. The formation of hybrids with single wall carbon nanotubes (SWCNTs) has been thought as a possible outcome. CNTs have been proved to be highly biocompatible and apart from their unique physical, chemical and electrical properties, many applications in biological media have been published [3], [4]. So, using methods similar to those of electrodeposited IrOx , the synthesis including carbon nanotubes will improve the electric conductivity and the mechanical properties of the material, avoiding in this sense the degradation processes, as for example, delamination observed when the electrode is in contact with biological media in vivo studies. In this line of work, hybrids of IrOx with SWCNTs have been developed. The new composites have been prepared by an electrochemical process that yields thin films of the hydrated iridium oxide with the SWCNTs forming a scaffold supporting the oxide. Iridium oxide is growing on the carbon nanotube, in several steps, what can be an indication of the presence of a nucleation points on the surface. Moreover, a new hybrid material containing iridium oxide, PEDOT and SWCNTs has been developed by a similar electrochemical process. The chemical, structural and surface characterization of the different samples has been done by XPS, SEM, HR-TEM and EDX. Also, the electrochemical properties of the materials have been studied, as well as preliminary toxicological tests.References:[1] S. F. Cogan., Annu. Rev. Biomed. Eng. 2008. 10, 275[2] D. Martin et al, Adv. Funct. Mat. 2009, 19, 573[3] A. V. Liopo et al, J. Nanosci. Nanotechnol. 2006, 6, 1365[4] G. G. Wallace, J Biomed Mater Res Part B: Appl Biomater 2007, 82B, 37
4:45 PM - KK8.7
Alginate-Based Muscular Thin Films for In Vitro Cardiac Contractility Assays.
Yohan Farouz 1 2 , Ashutosh Agarwal 1 , Megan McCain 1 , Kevin Parker 1
1 Disease Biophysics Group, Harvard University, Cambridge, Massachusetts, United States, 2 , Ecole Polytechnique ParisTech, Palaiseau France
Show AbstractIn vitro assays are under development that measure the contractility and the electrophysiology of single cardiomyocytes and engineered cardiac tissues. Simultaneously, cardiac patches are under investigation for surgical implantation in victims of heart failure. Although the requirements are different, both technologies are based on cell sheet engineering for the controlled assembly of contractile layers of cardiomyocytes. In this study we present a new method for the fabrication of muscular thin films that can be applied for the development of either in vitro contractility assays or implantable patches for cardiac regeneration. The polysaccharide alginate has been established as a good candidate because of its previous successes in biomedical applications and its simple yet powerful chemistry available. The use of alginate hydrogels as the main material allowed for the fabrication of thin films having mechanical properties close to the extracellular matrix as well as sustained drug release capabilities. The chemical properties of alginate allowed us to create a topographical micropattern at the surface of the films but also to pattern fibronectin through biotin-streptavidin interactions, thus providing an environment suitable for the attachment and alignment of cardiomyocytes. Indeed, cardiomyocytes successfully attached and aligned on the patterned surface of the films, leading to synchronous and anisotropic contraction of the construct. When field stimulated, alginate muscular thin films started contracting at the stimulation frequency. Also, when the films had been loaded with fluorescent beads, it has been possible to study patterned single cell contractility using traction force microscopy.After being integrated into a microfluidic system, this technology could provide a cost effective device to study in vitro the combined effect of drugs delivered through the culture medium and drugs released in a sustained fashion through the alginate porous structure. For in vivo studies, the technology of alginate thin films could lead to the fabrication of patches for cardiac regeneration after myocardial infarction, providing topographical cues as well as delivering developmental factors for the transdifferentiation of fibroblasts from the scar tissue into functional cardiomyocytes.
5:00 PM - KK8.8
Control of BMP2-Engineered Mesenchymal Progenitor Cell Differentiation by Polymeric Substrates.
Aneel Bherwani 1 , Chungchueh Chang 2 , Manideep Chavali 2 , Gadi Pelled 3 4 , Zulma Gazit 3 4 , Dan Gazit 3 4 , Elaine Dimasi 5 , Miriam Rafailovich 2 , Marcia Simon 1
1 Oral Biology and Pathology, Stony Brook University, Stony Brook, New York, United States, 2 Materials Science, Stony Brook University, Stony Brook, New York, United States, 3 Skeletal Biotech Laboratory, Hebrew University-Hadassah , Jerusalem Israel, 4 Regenerative Medicine Institute-Department of Surgery, Cedars Sinai Medical Center, Los Angeles, California, United States, 5 National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York, United States
Show AbstractStem cell differentiation is influenced by matrix chemistry and mechanics, as well as by soluble factors. To test the combined effects of these biochemical and biophysical cues, we used C3H10T1/2-derived progenitor cells genetically engineered to express the differentiation inducer rhBMP-2 under control of the Doxycycline (Dox)-repressible promoter, Tet-Off. Cells were grown on different polymeric substrates in DMEM, supplemented with 10% fetal bovine serum, 0.2 mM L-ascorbic acid 2-phosphate, 2 mm glutamine, 10 mM beta-glycerol phosphate 100 U/ml Penicillin, and 100ug/ml Streptomycin with or without 1 µg/mL Dox. The substrates compared included 20 & 200 nm thick Polybutadiene (PB) and 200 nm Sulfonated Polystyrene (SPS) films spun cast on HF treated Si wafers and annealed in a vacuum oven (P~103 Torr, T=170 °C) overnight. Differentiation was monitored over a 21 day period. Cells that were grown on PB (20 or 200 nm) with or without Dox failed to show significant biomineralization even up to 21 days, as measured by scanning electron microscopy (SEM) and Energy dispersive X-ray Analysis (EDAX). In contrast, cells grown on SPS (200 nm) in the absence of Dox (rhBMP-2 expressing as determined by qPCR with human specific primers), showed biomineralization as early as day 14. Differentiation on PB was further analyzed by qPCR and revealed the rhBMP-2 driven expression of chondrogenic markers (Aggrecan, COLX, and COLIIA2) as well as enhanced expression of rhBMP-2, Osterix, and Runx2. Scanning force microscopy was also used to determine the relative modulii of the cells on both PB and SPS, while on SPS we also measured the stiffness of the ECM fibers. The correlation between the mechanical properties of both cells and ECM with protein expression at different stages in the differentiation process will be discussed.