Elizabeth Orwin Harvey Mudd College
Brenda Mann Sentrx Animal Care
Ben Wu University of California-Los Angeles
Anthony Ratcliffe Synthasome, Inc.
DD1: Novel Materials in Tissue Engineering
Monday PM, December 01, 2008
Hampton A/B (Sheraton)
9:30 AM - DD1.1
Hyaluronic Acid Hydrogels that Degrade via both Enzymatic and Hydrolytic Mechanisms to Control Stem Cell Behavior.
Cindy Chung 1 , Sudhir Khetan 1 , Michael Beecham 1 , Sujata Sahoo 1 , Jason Burdick 1 Show Abstract
1 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Our laboratory is interested in designing hydrogels with controlled properties (both chemical and physical) that can influence the behavior of cells that interact with the material. Others have shown the importance of material chemistry and mechanics on the differentiation and behavior of encapsulated stems cells. Our work is motivated by the use of mesenchymal stem cells (MSCs) towards the regeneration of a wide range of tissues. Towards tissue engineering applications, we have been designing hydrogels based on hyaluronic acid (HA) that interact with cells via surface receptors (e.g., CD44) and degrade via hyaluronidases. When MSCs are encapsulated in HA hydrogels, enhanced chondrogenesis (upregulation of type II collagen, aggrecan) is noted compared to inert hydrogels (e.g., PEG) and chondrogenesis is observed even without growth factors present. When CD44 receptors were blocked with antibodies, this enhancement was diminished, illustrating the importance of these receptor interactions. Also, the mechanical loading of HA hydrogels leads to enhanced ECM and hyaluronidase gene expression. To improve control over network temporal properties, we recently synthesized a novel HA macromer with either lactic acid or caprolactone between the backbone and reactive groups. When polymerized, this hydrogel degrades via both enzymatic and hydrolytic mechanisms and can be used for control over growth factor delivery and ECM distribution. For instance, one composition with lactic acid degrades in ~3 days, one composition with caprolactone degrades in ~3 weeks, and little degradation in hydrogels without hydrolytically degradable units is observed without hyaluronidases. Through the copolymerization of these macromers, uniform ECM distribution was possible. Our recent efforts are towards HA hydrogels containing MMP degradable units formed through Michael-type reactions, where cells readily spread when encapsulated as long as adhesion sites (i.e., RGD) are present. If the Michael-type reaction and photopolymerization are used sequentially, this spreading can be spatially controlled in the hydrogels, leading the way to more complex tissue structures. Overall, these advanced HA hydrogels allow us the opportunity to investigate diverse and controlled material properties on MSC interactions.
9:45 AM - DD1.2
Microstructure and Mechanical Properties of Mineralized and Unmineralized Collagen-Glycosaminoglycan Scaffolds.
Biraja Kanungo 1 2 3 , Lorna Gibson 1 Show Abstract
1 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States, 2 Materials Science & Engineering, The Ohio State University, Cambridge, Massachusetts, United States, 3 Metallurgical Engineering, Indian Institute of Technology, Chennai, Tamil Nadu, India
Mineralized (50 wt% mineral content) and non-mineralized collagen-glycosaminoglycan scaffolds have been synthesized for hard and soft tissue regeneration, respectively. Scaffolds were synthesized with four different relative densities: 1×, 2×, 3× and 4×; where 1× = 0.042 g/ml and 0.005 g/ml for the mineralized and non-mineralized scaffolds respectively. Here, we characterize the microstructural and mechanical properties (Young’s modulus, collapse strength and collapse strain) of these scaffolds as a function of their relative density. Scaffolds had uniform pore structure throughout; however, pore size decreased with increase in density of the scaffold. The compressive stress–strain response of the scaffolds was characteristic of low-density open-cell foams with distinct linear elastic, collapse plateau and densification regimes. The elastic modulus and strength of the scaffolds increased with relative density. The elastic modulus and strength of individual struts in the scaffolds were measured using an atomic force microscopy cantilevered beam bending technique and nanoindentation, respectively. Cellular solids models give a good description of the bulk mechanical properties of scaffolds.
10:00 AM - **DD1.3
Novel Biomaterial Design Inspired by an Improved Understanding of the Chemical and Mechanical Properties of the Extracellular Matrix.
Andrew Putnam 1 2 Show Abstract
1 Biomedical Engineering, University of California, Irvine, Irvine, California, United States, 2 Chemical Engineering and Materials Science, University of California, Irvine, Irvine, California, United States
Research in the field of biomaterials has experienced a renaissance in the past two decades. Initially driven by the need for biocompatible, bio-inert materials for orthopedic and cardiovascular applications, a significant amount of research in this area is now focused on engineering “cell-instructive” materials designed to interact with biological macromolecules, cells, and tissues in a specific fashion. Efforts to design such new materials are largely inspired by the native extracellular matrix (ECM), a complex 3-D network of proteins and polysaccharides that surrounds cells in the majority of tissues in the human body. Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is widely recognized that the ECM provides both chemical and mechanical instructive cues to the cells with which it interacts. With respect to mechanical signals, extensive 2-D cell culture studies now support the hypothesis that ECM mechanical properties govern cell fate. However, the impact of ECM mechanics on normal and pathologic morphogenesis in 3-D remains unclear, due in large part to the fact that substrate mechanical properties cannot be tuned independently from adhesion ligand density and proteolytic sensitivity using native biopolymers systems (e.g., collagen, fibrin, Matrigel). To address the independent and coordinated effects of ECM chemistry and mechanics on cell phenotype in both 2-D and 3-D cultures, we have adapted several polymeric biomaterial platforms, including those based either on natural biopolymers or synthetic polymers. In this presentation, specific vignettes with respect to our findings regarding angiogenesis, myogenesis, and osteogenesis will be discussed. Focusing on the mechano-chemical functionality of the ECM in these morphogenetic processes has inspired our efforts to engineer novel hybrid biomaterials containing both synthetic and natural building blocks. Such versatile materials would have enormous implications for applications in tissue engineering and regenerative medicine, and as more realistic in vitro platforms in which to study fundamental morphogenetic mechanisms associated with numerous pathologies.
10:30 AM - DD1.4
Self-Assembly Mechanism of Short Aromatic Peptide Derivatives into Nanostructured Hydrogels.
Claire Tang 1 2 , Andrew Smith 1 2 , Rein Ulijn 1 2 , Alberto Saiani 1 Show Abstract
1 School of Materials, The University of Manchester, Manchester United Kingdom, 2 Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester United Kingdom
A class of peptide based scaffolds formed from peptides modified with a N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group has recently been developed . The aromatic moieties play a key role in the self-assembly behaviour of such peptides through π-π interactions, while peptide components are stabilised via hydrogen bonding. The main focus of our work to date has been to characterise the self-assembling behaviour of modified dipeptides. For example Fmoc-diphenylalanine (Fmoc-FF) has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture [1,2]. A molecular model has subsequently been proposed for the self-assembled structure formed by this system. Fmoc-FF peptide was shown to arrange in antiparallel β-sheets that self-assemble through the π-stacking of fluorenyl groups and phenyl rings of the amino acids side chains. The twisted nature of the β-sheets causes the formation of cylindrical fibres. By investigating the pH effect on the molecules’ self-assembly we have shown that this process results in two dramatic pKa shifts at four and seven units above the theoretical pKa of Fmoc-FF (3.5) . Different structures displaying significant property changes have been observed around these two transitions although the peptides were shown to arrange in antiparallel β-sheets over the pH range studied. Non-gelling rigid and flat ribbons form at low pH, while an entangled network of flexible fibrils, forming a weak hydrogel, dominates at intermediate pH values. In this presentation, we will provide evidence for the self-assembly mechanism of aromatic short peptide derivatives using rheology, FT-IR and TEM.  Jayawarna, V. et al. Adv. Mater., 2006, 18, 611-614.  Smith, A. M. et al. Adv. Mater., 2008, 20, 37-41.  Tang, C. et al. Soft Matter, 2008, under review.
10:45 AM - DD1.5
Spatio-Temporal Modification of Natural Collagen Mediated by Collagen Mimetic Peptides for Multi-Functional Tissue Scaffolds.
Michael Yu 1 , Allen Wang 1 , Shirley Leong 1 , Xiao Mo 1 Show Abstract
1 , Johns Hopkins University, Baltimore, Maryland, United States
Biomaterials based on purified animal collagen have made a major impact in the field of soft-tissue engineering and repair. Recent widespread interest in the development of engineered tissue for organ replacement therapies has prompted demand for new approaches to immobilize exogenous components to natural collagen. Functionalized collagen may offer new and improved applications for collagen-based biomaterials, but passively adsorbed molecules readily diffuse out from collagen matrix and conventional chemical reactions on collagen prove difficult to control and can compromise the biochemical feature of natural collagen. We developed a new collagen modification technique that utilizes associative chain interaction between synthetic collagen mimetic peptide (CMP) and natural type I collagen. We discovered that collagen mimetic peptides of sequence -(Pro-Hyp-Gly)x- exhibit specific affinity to type I collagen under controlled thermal conditions. The length of the CMP that determines the associative strength of CMP triple helix influences both its level of adhesion to collagen film and release characteristics from CMP-loaded collagen films and gels. This discovery led to a revolutionary concept, strand invasion/exchange mechanism in collagen and a number of CMP derivatives that can i) image collagens of human tissue, ii) enhance tubulogenesis of endothelial cells, and iii) promote chondrocyate maintenance. Our CMP-based collagen modification methods show great promise in spatial and temporal display of biological cues in collagen that can potentially aid in functional tissue formation.
11:30 AM - **DD1.6
Drug Discovery and Cell Biology in Three Dimensions
Glenn Prestwich 1 Show Abstract
1 Medicinal Chemistry, University of Utah, Salt Lake City, Utah, United States
12:00 PM - DD1.7
Virus Based Tissue Engineering Materials.
Seung-Wuk Lee 1 2 , Anna Merzlyak 1 2 Show Abstract
1 Bioengineering , University of California, Berkeley, Berkeley, California, United States, 2 Physical Bioscience Division, Lawrence Berkeley National Lab, Berkeley, California, United States
We have demonstrated that genetically engineered M13 bacteriophage can be utilized to construct a novel tissue engineering material that is able to both support and influence cell growth. Several characteristics of the filamentous M13 phage make it an attractive candidate for use as a building block in tissue engineering scaffolds. Long-rod shape and monodispersity of the phage enable them to self-assemble into directionally ordered liquid crystalline structures. Through genetic engineering, a high-density array of peptide-based signaling molecules and therapeutic materials can be simultaneously displayed on its major and minor coat proteins. Using such techniques we have constructed M13 phage to display various peptides that promote cell interaction (IKVAV, RGD) on all 2700 copies of major coat proteins. Through viability assays we have verified that these viruses are biocompatible with neuronal cells. Via microscopy studies and immunostaining we have shown that neural progenitor cells can both proliferate and differentiate when grown on viral surfaces and that there is a preference of cell interaction with the genetically engineered over wildtype phage. Lastly utilizing SEM and bright-field microscopy we have demonstrated that such engineered phage can self assemble into directionally organized structures, which in turn can dictate the alignment and direction of cell growth. This exploratory project has shown that engineered virus-based materials can be used as promising novel substrates for neural cell growth. Further design and study of these materials might enable them with greater control over cell behavior at the molecular level, regeneration of various tissues, and potentially help in the research leading to a cure of challenging conditions such as spinal cord injuries.
12:15 PM - **DD1.8
New View of an Old Material: Collagen as an Intelligent, Self-organizing, Load-responsive Structural Building Block.
Jeff Ruberti 1 Show Abstract
1 Mechanical and Industrial Eng, Northeastern University, Boston, Massachusetts, United States
12:45 PM - DD1.9
Development of a Novel Hydrolytically Degradable PEG Hydrogel with Tunable Degradability and Protein Release.
Silviya Petrova 1 , Jennie Leach 1 Show Abstract
1 Chemical and Biochemical Engineering, UMBC, Baltimore, Maryland, United States
A degradable poly(ethylene glycol) (PEG) hydrogel with tunable physical properties is developed. A 3D scaffold is made by covalently cross-linking 4-arm PEG vinyl sulfone and PEG-dithiol ester. The hydrogel is then characterized in terms of degradation time and diffusion profiles and aimed towards protein delivery applications. This is one of the few degradable PEG-based scaffolds that does not utilize copolymers (e.g. PEG-polylactic acid), does not rely on UV exposure for polymerization (e.g. PEG-diacrylate), or have a non-specific cross-linking chemistry (e.g. PEG-amine). The hydrogel is biocompatible, inert, and has tunable mechanical properties. Protein diffusion from hydrogels is greatly influenced by the degradability of the polymer, the mesh size of the network, and the protein size. Therefore, the focus of our characterization is on these three parameters. The degradability of the hydrogel is controlled by three distinct strategies: by varying the molecular weight of the cross-linker, by varying polymer density, and by using cross-linkers with different number of methylene groups between the ester and thiol groups. It should be noted that degradation gradually increases the mesh size and decreases stiffness until the network is highly disrupted and the degradation complete. Thus the change of mesh size is monitored continuously. The change in mechanical properties (i.e., shear modulus) and the protein release are also tested as a function of degradation time. Furthermore, three proteins of different molecular weight are used to test the dependence of protein release on the size of the protein. The developed degradable hydrogel has a variety of applications in the area of drug delivery and tissue engineering due to its unique properties, tunability, and ability to form 3D matrices under physiological conditions.
DD2: Cell Responsive Materials
Monday PM, December 01, 2008
Hampton A/B (Sheraton)
2:30 PM - DD2.1
Toxicity and Enhanced Extra Cellular Matrix Expression of Primary Osteoblast Cells on Single Walled Carbon Nanotube Scaffolds.
Wojtek Tutak 1 , KiHo Park 2 , Giovanni Fanchini 1 , Anatoly Vasilov 2 , Nicola Partridge 2 , Federico Sesti 2 , Manish Chhowalla 1 Show Abstract
1 Materials Science and Engineering, Rutgers University, Piscataway, New Jersey, United States, 2 Physiology & Biophysics , Robert Wood Johnson Medical School , Piscataway, New Jersey, United States
Carbon nanotubes are being considered for many biological applications due to their unique properties. Unfortunately, a survey of the existing knowledge on the osteoblast cells response to carbon nanotube scaffolds reveals that some results are inconsistent. That is, reported biological studies describe nanotubes either as being toxic or enhancing cellular growth. In this study, very well characterized single walled carbon nanotube (SWNT) scaffolds in the form of thin films or networks have been used to host cell cultures. Total protein, alkaline phosphatase and toxicology assays were used to investigate long term effects. Cellular development was evaluated using scanning electron microscopy and complementary western blot analysis was employed to quantify extra cellular matrix (ECM) expression. Cellular toxicity in form of nanotube uptake in time dependent manner was recorded using transmission electron microscopy. Our study reveals complex interactions between initial cellular toxic response and enhanced ECM protein expression. We found that the initial SWNT uptake is related to high cellular toxicity levels causing sudden cellular death as indicated by sharp decline in proliferation rates. However, cellular destruction leads to the release of proteins and organelles into local environment which stimulates surviving cells and directly mediates short and long term cell proliferation and enhances ECM production levels. In summary, our results reconcile seemingly inconsistent results that indicate that carbon nanotubes can be both toxic and lead to an enhancement in ECM levels.
2:45 PM - DD2.2
Morphology of Aggrecan Produced By Equine Mesenchymal Stem Cells and Chondrocytes in Self-Assembling Peptide Hydrogels.
Hsu-Yi Lee 1 , Paul Kopesky 2 , Laura Daher 3 , Ana Mosquera Pelegrina 4 , David Frisbie 5 , John Kisiday 5 , Alan Grodzinsky 1 2 6 , Christine Ortiz 7 Show Abstract
1 Electrical Engineering and Computer Science, MIT, Cambridge, Massachusetts, United States, 2 Biological Engineering, MIT, Cambridge, Massachusetts, United States, 3 Chemistry, MIT, Cambridge, Massachusetts, United States, 4 Mechanical Engineering, University of Puerto Rico-Mayagüez, Mayagüez, Puerto Rico, United States, 5 Equine Orthopaedic Research Center, Colorado State University, Fort Collins, Colorado, United States, 6 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States, 7 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States
Mesenchymal stem cells (MSCs) are multipotent with the potential to differentiate into cells of the chondrocyte lineage. For this reason, they are a candidate cell source for cartilage regeneration therapies. Marrow-derived stem cells undergoing chondrogenesis in vitro can synthesize aggrecan similar in structure to that synthesized by chondrocytes under the same conditions. To more accurately identify which cell sources are superior for cartilage regeneration, detailed knowledge of the molecular structure and mechanical properties of synthesized extracellular matrix molecules, such as aggrecan, is necessary. In this study, high resolution tapping mode atomic force microscopy (AFM) imaging was used to visualize the structure of individual aggrecan molecules produced by four equine cell types: adult MSCs, adult chondrocytes, foal MSCs, and foal chondrocytes seeded into self-assembling peptide hydrogel scaffolds cultured in chondrogenic media. Animal-matched chondrocytes and bone marrow-derived MSCs were harvested from foal (2-4 month old) and adult (2-4 yr old) horses. Isolated cells were seeded in (KLDL)3 self-assembling peptide hydrogel and cultured in TGF-β1 and IGF-1 supplemented medium for 21 or 42 days. Aggrecan was isolated from the constructs and used in AFM imaging. Aggrecan was deposited on 3-aminopropyltriethoxysilane functionalized mica substrates. Tapping mode AFM imaging proceeded in ambient conditions with Si cantilevers (k=45 N/m, tip radius < 10 nm.) Aggrecan core protein contour length, Lc and CS-GAG chain length, LCS-GAG, were measured. Effective persistence length, Lp, was calculated using the worm-like-chain model. The Lc for foal MSC, adult MSC, foal chondrocyte and adult chondrocyte were 503±149 nm, 487±165 nm, 437±137nm, and 412±166 nm (mean±SD; n=110~231), respectively. The LCS-GAG for foal MSC, adult MSC, foal chondrocyte and adult chondrocyte were 63±11 nm, 73±24 nm, 40±8 nm, and 46±18 nm (n=28~35) respectively. MSC-produced aggrecan had significantly larger Lc and LCS-GAG than chondrocyte-produced aggrecan (p<0.05). No effect of animal age (in the 2-month to 4-year range tested) on aggrecan monomer properties was found (two-way ANOVA, p<0.05 for cell type). All cell types were also found to have similar effective persistence length Lp (239~254 nm, p>0.05). Regardless of the animal age, MSC-produced aggrecan had not only similar morphology, but also larger molecular size than chondrocyte produced aggrecan. In contrast, recent reports showed that aggrecan extracted directly from native cartilage was larger for young animals. The similar structural dimension for aggrecan produced by MSCs and chondrocytes seeded in peptide hydrogel scaffolds, from both young and adult horses, suggests the potential use of MSCs as a cell source for mechanically-functional replacement tissue. Ongoing research aims to compare the nanomechanical properties of adult MSC produced aggrecan with that of aggrecan extracted from native cartilage.
3:00 PM - **DD2.3
Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates Collagen Fibrillogenesis.
Alyssa Panitch 1 , John Paderi 1 Show Abstract
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States
3:30 PM - DD2.4
A Library of Polymers to Probe the Effects of Chemical Composition, Mechanical Stiffness and Adhesivity of Biomaterials on Cell Proliferation and Differentiation.
Abraham Joy 1 , Daniel Cohen 2 , Emmanuel Anim-Danso 1 , Colette Shen 2 , Christopher Chen 2 , Joachim Kohn 1 Show Abstract
1 NJ Center for Biomaterials, Rutgers University, Piscataway, New Jersey, United States, 2 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Introduction Understanding the interactions between the material and the ECM is essential for the design of biologically responsive materials for regenerative medicine. A number of material properties affect cellular responses, including the chemical composition, the mechanical stiffness and the presence of adhesive ligands. Our aim is to understand the effects of chemical composition, mechanical stiffness and adhesivity on cellular functions like proliferation and differentiation. A library of methacrylate polymers of low modulus was employed for the current study. Each polymer was synthesized from three monomers by AIBN mediated radical polymerization. Varying the ratio of the monomers provides a family of polymers of varying mechanical stiffness. Subsequently surface modification gives a family of polymers of varying stiffness but with the same cell adhesiviness. This methodology provides a means to understand the effects of chemical composition, mechanical stiffness and cellular adhesivity of biomaterials on cell functions. Results and Discussion As a representative example of the library of polymers, a terpolymer of hydroxyethylmethacrylate (HEMA), triethyleneglycol monoethylmethacrylate (TEGMA), and glycidyl methacrylate (GMA) was synthesized by AIBN-mediated radical polymerization. The mechanical stiffness of this family of polymers is determined by the ratio of the HEMA and TEGMA in the terpolymer whereas the surface density of functionalized peptide is determined by the amount of GMA. Surface functionalization was achieved by coupling azide terminated RGD peptides to surfaces containing propargyl functional groups. NIH 3T3 cell proliferation and hMSC differentiation were examined on spin coated polymers. It was seen that the cell proliferation was greatly affected by the chemical composition and mechanical stiffness of the materials. Surface functionalization with adhesive peptides substantially increases the proliferation rates.Differentiation of mesenchymal stem cells (hMSC) on this library of polymers is currently being examined. Preliminary results show that alkaline phosphatase expression is greatly affected by the chemical composition and mechanical stiffness of the polymers. Conclusion: A library of methacrylate polymers that enable us to examine the interactions of chemical composition, mechanical stiffness and adhesivity of biomaterials on cell proliferation and differentiation was developed. The design of the library allows us to alter the composition, stiffness and adhesivity of the library of polymers with a great degree of flexibility. The proliferation (NIH 3T3 cells) and differentiation (hMSC) can be controlled by the composition, stiffness and adhesivity of these polymers.
3:45 PM - DD2.5
Transforming Orthopedic Biomaterials Into Bone Cancer Inhibiting Implants: The Role of Selenium Nanoclusters.
Phong Tran 1 , Love Sarin 2 , Hurt Robert 2 , Thomas Webster 2 3 Show Abstract
1 Physics Department, Brown University, Providence, Rhode Island, United States, 2 Divisions of Engineering, Brown University, Providence, Rhode Island, United States, 3 Department of Orthopedic Sugery, Brown University, Providence, Rhode Island, United States
It is estimated that 2,380 individuals will be diagnosed with bone and joint cancers and 1,470 individuals will die from primary bone and joint cancers in 2008 in the U.S. . Primary bone cancer is rare as usually bone cancer is a result of the spread of cancer from other organs (such as the lungs, breasts and prostate) . A common technique to treat bone cancer is the surgical removal of the cancerous tissue followed by insertion of an orthopedic implant to restore patient functions. Therefore, it would be beneficial to have implants specifically designed to prevent the occurrence and reoccurrence of bone cancer and promote healthy bone tissue growth. Unfortunately, current materials used as an orthopedic implant do not possess anti-cancer properties and, thus, have no inherent mechanisms to keep cancer from reoccurring. The objective of the present in vitro study was to create an orthopedic implant with anti-cancer chemistry to simultaneously: (i) promote healthy bone cell functions and (ii) inhibit cancerous bone cell functions. Elemental selenium was chosen as the biologically active agent in this effort because of its known chemopreventive properties. Nanostructured surface features in selenium were employed to promote bone cell functions since numerous studies have shown greater osteoblast (bone-forming cells) functions on nanostructured surfaces than on conventional, micron structured surfaces. To demonstrate the versatility of using selenium nanoclusters to transform orthopedic implant materials which do not inhibit cancer growth to cancer inhibiting ones, selenium nanoclusters were coated on three common orthopedic implant materials: titanium, stainless steel, and ultra high molecular weight polyethylene (UHMWPE). Three different selenium cluster densities were prepared on each type of substrate. Compared to uncoated surfaces, substrate surfaces with greater amounts of selenium nanoclusters inhibited cancerous bone cell functions while promoting healthy bone cell functions. Thus, this study provided for the first time a coating material (selenium) for the orthopedic cancer community which may inhibit bone cancer growth and promote healthy bone growth.
4:30 PM - DD2.6
The Effect of Surface Mechanics on Cell Function, Mechanics and Mobility.
Miriam Rafailovich 1 , Richard Clark 1 , Glenn Prestwich 2 , Ying Liu 1 , Pan Zhi 1 , Kaustabh Ghosh 3 Show Abstract
1 Materials Science and Engineering, Stony Brook University, Stony Brook, New York, United States, 2 , Harvard University, Cambridge, Massachusetts, United States, 3 , University of Utah, Salt Lake City, Utah, United States
4:45 PM - DD2.7
Engineering Two- and Three-dimensional Cellular Aggregates by Electrochemical Reaction.
Rina Inaba 1 , Hiroaki Suzuki 1 , Junji Fukuda 1 Show Abstract
1 Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki Japan
5:00 PM - **DD2.8
Design of Biomaterial Environments to Study Disease Mechanisms and Potential Treatments.
Kristyn Masters 1 Show Abstract
1 Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States
Traditional tissue engineering techniques focus primarily on the construction of healthy neo-tissues, wherein optimization of the biomaterial system is performed to promote maintenance of a healthy cell phenotype. However, an emerging application of tissue engineering is the recreation or replication of diseased human tissues, which may help to uncover disease mechanisms and etiology, as well as provide advanced, physiologically-relevant in vitro platforms for testing disease treatments. To this end, we have generated 2-D and 3-D models of calcific heart valve disease. The etiology of valvular disease is poorly understood, and there are currently no treatments available to stop the progression of valvular disease. Our work in creating calcific valvular disease in vitro has revealed information about the roles of extracellular matrix components, growth factors, peptide-receptor interactions, and intracellular signaling pathways in valve calcification. By tailoring both 2-D and 3-D environments to regulate the diseased phenotype of these cells, we can create defined systems that mimic elements of native valvular disease, thereby providing insight into the mechanisms of disease progression and potential targets for its prevention.Investigation of how the scaffold environment controls the disease phenotype of cells is also important in creating healthy neo-tissues, as engineered tissue replacements will be implanted into in vivo environments that are rich in disease-inducing factors. However, because optimization of these scaffold environments is normally performed solely in healthy culture conditions, the reaction of this neo-tissue to native disease stimuli is often not tested. For example, in the case of myocardial repair, a tissue-engineered cardiac patch is likely to be placed on heart tissue that is significantly stiffer than normal and is flooded with molecules that induce pathological hypertrophy. Work performed in our lab has shown that cardiomyocytes in different scaffolds respond differently to such pro-hypertrophic factors in ways that could not have been predicted had we evaluated solely ‘healthy’ culture conditions. The scaffold that best maintains a functional cell phenotype under healthy culture conditions is not necessarily the same scaffold that maintains a functional cell phenotype under diseased culture conditions. This work illustrates the crucial role of the scaffold microenvironment in regulating the cellular response to various stimuli and emphasizes the importance of optimizing the scaffold system in more complex, physiologically-relevant conditions such that engineered tissues do not readily succumb to the same fate as the original diseased tissue.
5:30 PM - DD2.9
Development of Elastin-Heparin Matrices for Vascular Tissue Engineering: Characterization and In Vitro Blood Compatibility Testing.
Aditee Kurane 1 , Naren Vyavahare 1 Show Abstract
1 , Clemson University, Clemson, South Carolina, United States
Cardiovascular diseases are the leading cause of death worldwide. Blood vessel replacement is a common treatment for vascular diseases such as atherosclerosis, restenosis and aneurysm, with over 300,000 bypass procedures performed each year. Autologous vein grafts are limited due to their availability. Although synthetic vascular replacements have been successful for large diameter arteries, they have shown minimal success in arteries with diameters <6mm. This is because most synthetic materials induce thrombus formation which, within a few months of implantation, causes failure of the vascular graft due to occlusion. Tissue engineering provides a promising approach to create non-thrombogenic vascular grafts. We have created pure elastin (EL) tubes from porcine carotid arteries as scaffolds for tissue engineered blood vessel replacements. These scaffolds are biodegradable, non-immunogenic and have mechanical properties that match the native arterial tissue. In the present study, we investigated a method to covalently immobilize heparin onto the surface of the elastin scaffolds to improve their blood compatibility. Different molar ratios of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were investigated to identify the most appropriate combination that would immobilize the maximum amount of heparin. Having identified this optimal combination (molar ratio of 1; 10.43mM) we attempted to immobilize increasing amounts of heparin using this ratio. The maximum amount of heparin that could be immobilized was 1.84μg heparin/mg dry elastin. Blood compatibility of the heparinized elastin scaffolds was evaluated using plasma recalcification time (PRT) and platelet adhesion. The anticoagulant activity of the immobilized heparin was determined by its’ ability to mediate the inactivation of thrombin by binding antithrombin. Platelet adhesion was significantly suppressed and PRT was significantly prolonged when the maximum amount of heparin was immobilized. Also, increasing amounts of heparin progressively increased anticoagulant activity as measured by thrombin inhibition. The results of this study suggest that heparin immobilization to elastin scaffolds by EDC/NHS chemistry may improve the in vivo blood compatibility of this material.
5:45 PM - DD2.10
Cell Modification in 3D: Osteogenic Stimulation of Human Bone Marrow Stromal Cells after Hydroxyapatite Coating in the Absence of Chemical Cues.
Lauren Hails 1 , Jodie Babister 2 , Rosanna Gonzalez-McQuire 1 , Stephen Mann 1 , Richard Oreffo 2 , Sean Davis 1 Show Abstract
1 School of Chemistry, University of Bristol, Bristol United Kingdom, 2 Developmental Origins of Health and Disease, University of Southampton, Southampton United Kingdom
Osteoinductive, living biomaterial composites have been created comprising human bone marrow stromal cells (HBMSCs) coated with biocompatible, amino acid and peptide-functionalised hydroxyapatite (HAP) nanoparticles. 3D encapsulation of HBMSCs suspended in media was achieved via electrostatic interaction with positively charged, biomolecularly stabilised HAP nanoparticles. Subsequent maintenance of these constructs under a variety of in vitro culture conditions invariably resulted in osteogenic induction even in the absence of the chemical cues typically employed to achieve differentiation towards the osteoblastic lineage. Biochemical analysis of cells coated with arginine and alanine-functionalised HAP and cultured in monolayer for up to 21 days show significantly elevated alkaline phosphatase (ALP) activity in comparison to HBMSCs cultured in basal conditions. Importantly, these studies demonstrate elaboration of the nanoparticle functionality by addition of the cell-adhesion peptide sequence arginine-glycine-aspartic acid (RGD), which at an optimised concentration results in ALP activity comparable to that of uncoated cells cultured in osteogenic media. HAP/cell constructs assembled into pellets and maintained under 3D culture conditions for 21 days, showed elevated levels of ALP activity and substantial mineralisation of the extracellular matrix in contrast to control cultures.In addition to the modelling of skeletal cell differentiation and function, it is anticipated that these novel biomaterial composites will be particularly relevant in osteoregenerative applications. In this regard, in vivo experiments have been conducted involving subcutaneous implantation of these pellets in immunocompromised mice. Immunocytochemistry of samples recovered after 3 weeks, demonstrated the formation of extensive areas of fibrous collagen both within peripheral and central regions, indicating areas of osteoid formation, again not observed within uncoated 3D constructs. Reference: R. Gonzalez-McQuire, D.W. Green, K.A. Partridge, R.O.C. Oreffo, S. Mann, S.A. Davis, Adv. Mater. 2007, 19, 2236.
DD3/HH6: Joint Poster Session: Materials in Tissue Engineering
V. Prasad Shastri
Tuesday AM, December 02, 2008
Exhibition Hall D (Hynes)
9:00 PM - DD3.1/HH6.1
Segmented Polyurethane Implants For Regeneration Of Esophagus.
Yerkesh Batyrbekov 1 , Nurzhan Bubeev 2 , Bulat Zhubanov 1 Show Abstract
1 , Institute of Chemical Sciences, Almaty Kazakhstan, 2 , South Kazakhstan State Medicinal Academy, Chimkent Kazakhstan
9:00 PM - DD3.10/HH6.10
In Situ Mineralization of Block Copolymer Hydrogels.
David Griffin 1 , Surita Bhatia 1 Show Abstract
1 Chemical Engineering, University of Massachusetts, Amherst, Massachusetts, United States
Nanocomposites of hydroxyapatite in polymeric matrices have a number of important applications in bone and cartilage tissue engineering. Most studies to date have focused on systems where the polymeric matrix is a homopolymer gel or block copolymer solution. Use of a block copolymer hydrogel may offer additional control over crystallite size and morphology. Here we report on our efforts to synthesize hydroxyapatite in situ in block copolymer gels. Inorganic ceramic nanocomposites were formed by diffusion of calcium ions into a phosphate-containing Pluronic® F127 hydrogel. Initial pH of the gel prior to nucleation strongly influenced the final crystal structure and morphology. Nucleation at near physiological pH preferentially produced a highly crystalline calcium phosphate phase on the millimeter scale whereas mineral growth at alkaline conditions was found to produce hydroxyapatite crystals in the micrometer size range. Calcium phosphate mineral phases were determined by powder x-ray diffraction (XRD) and substantiated through energy dispersive x-ray spectroscopy (EDS). Morphology of the composites was examined using scanning electron microscopy (SEM). Rheological studies on these systems show that higher elastic moduli are obtained with composites prepared using the in situ synthesis technique as compared to conventionally prepared polymer-hydroxyapatite composites. The biomimetic nature of our investigation suggests that composites formed by this in situ technique my have significant biomaterial and drug delivery applications.
9:00 PM - DD3.11/HH6.11
Porous Hydroxyapatite/gelatin Scaffolds via Freeze-drying: the Effect of Bioceramic Nanoparticles on the Scaffold’s Microarchitecture and Properties.
Andrei Stanishevsky 1 , Sonda Sengupta 1 , Erin Ellis 1 Show Abstract
1 , University of Alabama at Birmingham, Birmingham, Alabama, United States
9:00 PM - DD3.12/HH6.12
Biomimetic Chitosan/nano-hydroxyapatite Composite Scaffolds for Bone Tissue Engineering.
Wah Wah Thein-Han 1 , Jinesh Shah 1 , Devesh Misra 1 Show Abstract
1 Center for Structural and Functional Materials, University of Louisiana at Lafayette, Lafayette, Louisiana, United States
We describe here three dimensional biodegradable chitosan-nanohydroxyapatite (nHA) composite scaffold with improved mechanical, physico-chemical, and biological properties compared to pure chitosan scaffolds for bone tissue engineering. High and medium molecular weight chitosan scaffolds with 0.5, 1, and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposite scaffolds were characterized by a highly porous structure with interconnected pores and the pore size was similar for the scaffolds with varying content of nHA. The nanocomposite scaffolds exhibited greater compression modulus, slower biodegradation rate and reduced water uptake, but the water retention ability was similar to pure chitosan scaffolds. Favorable biological response of pre-osteoblast (MC 3T3-E1) on nanocomposite scaffolds includes improved cell adhesion, higher proliferation, and well spreading morphology in relation to pure chitosan scaffold. The study underscores chitosan-nHA composite as a potential scaffold material for bone regeneration.
9:00 PM - DD3.13/HH6.13
Towards Functional Biomaterials: Elucidating Design Principles of Enzyme Activated Hydrogelation.
Andrew Hirst 1 2 , Rein Ulijn 1 2 3 Show Abstract
1 School of Materials and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, Lancashire, United Kingdom, 2 School of Materials, University of Manchester, Manchester United Kingdom, 3 School of Chemistry, University of Strathclyde, Glasgow United Kingdom
The use of well-designed molecular building blocks, capable of forming self-assembled structures, is a fundamental construction principle for biological materials, with this approach being employed in various systems, ranging from double stranded DNA to complex structures such as the tobacco mosaic virus. The appeal that nature holds, is that molecular scale information guides the organisation of complexity, expressed at the ‘system’ or materials level in terms of a specific function. Intense activity has recently been devoted to the application of molecular recognition processes to control the formation of functional gel-phase materials, from simple molecular building-blocks. For example, regenerative medicine and tissue engineering using small molecule hydrogels as nanostructured scaffolds have been demonstrated to be genuine possibilities – for example, the regrowth of nerve cells has been recently demonstrated in vivo. Therefore, to develop new technologies based on self-assembly, the ideal self-assembling material should have a simple synthesis, which is amenable to molecular design and can potentially be tailored for a broad range of applications. This presentation will focus on elucidating the self-assembly and hydrogelation properties of a small library of aromatic short peptide derivatives using microscopy, rheometry and thermal measurements. These systems are based on several combinations of amino acid residues including Tyrosine, Valine and Leucine. Additionally, the peptides are modified with aromatic stacking ligands based on fluorenylmethoxycarbonyl (Fmoc). In each case, molecular self-assembly, which underpins macroscopic gelation is driven by exploiting an enzymatic hydrolysis process. Furthermore, the ability of the gel network structure to be modified by simply varying enzyme concentration will also be discussed. This offers an interesting perspective of fixing the chemical composition of the system, whilst modulating the gel materials properties. This strategy may offer a potential route to access new nanostructured materials applicable as three-dimensional (3D) scaffolds in which different morphological systems exert control over cell behaviour.  (a) K. van Workum, J. F. Douglas, Phys. Rev. E 2006, 73, 031502; (b) G. M. Whitesides, B. Grzybowski, Science 2002, 295, 2418-2421. (a) M. Boncheva, G. M. Whitesides, MRS Bull. 2005, 30, 736-742; (b) I. W. Hamley, V. Castelletto, Angew. Chem. Int. Ed. 2007, 46, 4442-4455. R. G. Ellis-Behnke, Y.-X. Liang, S.-W. You, D. K. C. Tay, S. Zhang, K.-F. So, G. E. Schneider, Proc. Natl. Acad. Sci. USA 2006, 103, 5054-5059. (a) S. Toledano, R. J. Williams, V. Jayawarna, R. V. Ulijn, J. Am. Chem. Soc, 2006, 128, 1070-1071; (b) A. K. Das, R. Collins, R. V. Ulijn, Small, 2008, 4, 1279-1287.
9:00 PM - DD3.14/HH6.14
Hybrid Biomaterials with Tunable Elastic Modulus Through Sol-gel Functionalization of Peptidic Hydrogels.
Aysegul Altunbas 1 , Nikhil Sharma 1 , Radhika Nagarkar 2 , Joel Schneider 2 , Darrin Pochan 1 Show Abstract
1 Materials Science & Engineering, University of Delaware, Newark, Delaware, United States, 2 Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware, United States
Sol-gel chemistry was used to mineralize a pre-assembled peptide hydrogelscaffold for bone tissue engineering applications. The 20 amino acid peptideused in this study, MAX8, consisted mostly of alternating hydrophilic(lysine) and hydrophobic (valine) residues flanking a four amino acid turnsequence in the center (VKVKVKVKVDPLPTKVEVKVKV-NH2). After correctlyintramolecularly folding into a beta-hairpin conformation on addition of adesired solution stimulus, this peptide intermolecularly self-assembled intoa three dimensional network of interconnected fibrils rich in beta-sheetwith a high density of lysine groups exposed on the fibril surfaces.Polyamines are known to catalyze the polycondensation of silicic acid inwater. Therefore, the lysine-rich surface chemistry was utilized to createa silica shell around the fibrils. The mineralization process of the fibrilswas initiated by adding the silica precursor, tetramethyl orthosilicate, tothe pre-assembled hydrogel, which results in a rigid, porous silicate meshthat retains the microscale and nanoscale structure of the fibrillar peptidenetwork. Structural, mechanical, and in vitro biological properties of thesilicified hydrogels will be presented.
9:00 PM - DD3.16/HH6.16
Novel Composite Biomaterials Made from Resilin and Cellulose.
Shaul Lapidot 1 , Mara Dekel 1 , Sigal Meirovitch 1 , Sigal Roth 2 , Daniel Siegel 2 , Noa Lapidot 2 , Oded Shoseyov 1 2 Show Abstract
1 The Faculty of Agriculture, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Israel, Rehovot Israel, 2 , CollPlant ltd 2 Pekeris St. POB 2310 , Rehovot Israel
Despite the significant advance of tissue engineering and regenerative medicine, to date, resilient biomaterials that support load bearing tissue are still absent. Some of the top-performing natural bio-composite materials combine polymeric proteins with polysaccharides via Carbohydrate Binding Modules (CBMs) that enable introduction of both molecular order and interfacing between the composite components.The high affinity of carbohydrate binding modules (CBMs) proteins to cellulose allows their utilization for cellulose fiber modification and for cross-bridging between cellulose and other molecules. These properties led to the development of CBM technology that was applied in a wide variety of biotechnological applications. Previous work in our laboratory demonstrated the utilization of CBMs for both in-vitro and in-vivo cellulose fiber modification (Shoseyov et al, 2006). Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Its unique mechanical properties allow the outstanding jumping ability of fleas, up to 30 cm high equivalent to a human high jump of 400 meters. Resilin binds to the cuticle polysaccharide chitin via a Chitin Binding Domain and further polymerized through oxidation of the tyrosine residues resulting in formation of di-tyrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We report for the first time of the production of recombinant resilin-Cellulose Binding Domain (CBD) fusion protein that enable binding and polymerization of resilin on cellulose to obtain a new composite biomaterial. Assembly of novel composites made from resilin-CBD and different cellulosic materials such as whiskers (cellulose nano-crystals), regenerated cellulose films and fibers and their application as scaffolds for tissue engineering will be discussed. Reference:Shoseyov O, Shani Z, Levy I. (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev. 70: 283-295.
9:00 PM - DD3.17/HH6.17
Modified Alginate for Biomedical Applications.
Soumitra Choudhary 1 , Surita Bhatia 1 Show Abstract
1 Chemical Engineering, University of Massachusetts, Amherst, Amherst, Massachusetts, United States
Hydrophobically modified alginate (HMA) was synthesized by condensation reaction with different alkyl amine (C8-C12) at low pH. At or above critical concentration, HMA forms a physical gel in aqueous media by hydrophobic interaction. Unreacted guluronic units of alginate can be further crosslinked with divalent cations, such as Ca2+. Interplay of the two different gelation mechanisms, hydrophobic association and chemical crosslinking, enables us to tune the rheological and mechanical properties of the system. Uniform gels were obtained by slow release of calcium ions from calcium-ethylene diamine tetra acetic acid complex with the addition of D-glucono-δ-lactone. Solubility of lipophilic drugs was found to be greatly improved compared to neat alginate presumably due to preferential uptake of the drugs by micelles formed by hydrophobic moiety. Controlled and extended release rate was observed for HMA due to stronger crosslinked alginate units surrounding the hydrophobic rich domains. Small angle scattering analysis and optical microscopy will be presented along with rheological data to determine the structure-property relationship of the resultant gels. Effect of hydrophobic chain length on structure and properties will also be presented.
9:00 PM - DD3.19/HH6.19
Hyaluronic Acid-gelatin Nanofibrous Scaffold Produced by Electrospinning of Their Aqueous Solution for Tissue Engineering Applications.
Ying Liu 1 , Richard Clark 2 , Lei Huang 3 , Miriam Rafailovich 1 Show Abstract
1 Department of Materials Science and Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 2 Department of Biomedical Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 3 Department of Physics and Astronomy, SUNY at Stony Brook, Stony Brook, New York, United States
9:00 PM - DD3.2/HH6.2
Synthetic Platelets’ Role in Vascular Trauma and Their Interactions with Platelets.
James Bertram 1 , Nolan Flynn 2 , Erin Lavik 1 Show Abstract
1 Biomedical Engineering, Yale University, New Haven, Connecticut, United States, 2 Chemistry, Wellesley College, Wellesley, Massachusetts, United States
Under normal circumstances the body is able to maintain hemostasis. However, this intrinsic system is often insufficient due to pathologies or severe vascular trauma. Current treatments for aiding hemostasis are either limited by storage requirements, supply, or the nature of the injury. This brings to light the need for a non-immunogenic synthetic platelet substitute. An ideal platelet substitute would have minimal storage requirements, but more importantly, its hemostatic efficacy would not induce indiscriminant thrombosis. To address this need, we created a synthetic platelet substitute consisting of a nanosphere core comprised of a poly(lactic-co-glycolic acid)-poly(ε-carbobenzoxy-L-lysine) block copolymer (PLGA-PLL). Polyethylene glycol (PEG) terminated with the cell recognition motif arginine-glycine-aspartic acid (RGD) was conjugated to the core surface to finalize the synthetic platelet structure. Synthetic platelets’ interactions with rat platelets were analyzed in vitro. This platelet aggregation assay aided in the structural optimization of our synthetic platelet. To determine efficacy in vivo, we analyzed bleed/clot times in the rat vasculature following an intravenous injection of our synthetic platelet and its constituents. These injury models included a mechanical perturbation to the macrovasculature (femoral artery/vein) or a light-dye injury in the microvasculature (arterioles/venules). We observed that by varying the molecular weight of our PEG tether, thus varying the proximity of our RGD motif to the nanosphere surface, we were able to mitigate bleed times in the rat femoral artery in vivo as well as vary platelet affinity in vitro. This effect was found to be dose dependent as well. Our findings suggest that in an injured environment, where platelet aggregation is not the primary means of hemostasis (i.e. vasoconstriction is prominent in an artery/arteriole), synthetic platelets significantly augment clotting. These results imply that a synthetic platelet may be a feasible substitute for current treatments in facilitating hemostasis in an injured or pathological state.
9:00 PM - DD3.20/HH6.20
Iron Oxide Magnetic Nanoparticles for Treating Bone Diseases.
Nhiem Tran 1 , Thomas Webster 2 Show Abstract
1 Department of Physics, Brown University, Providence, Rhode Island, United States, 2 Division of Engineering and Orthopaedics, Brown University, Providence, Rhode Island, United States
Magnetic drug delivery systems have drawn great interest from the medical research community in recent years. In this method, magnetic nanoparticles are prepared with modified surfaces and drug embedded coatings. These nanoparticles are later focused to the disease site by either an external magnetic field or an internal magnetic implant and, thus, results in improved drug effectiveness compared to other non-direct drug delivery methods. Our research goal is to prepare a magnetic drug delivery system that is capable of treating bone diseases such as osteoporosis. Results from this study provided evidence of increased osteoblast (bone forming cells) density in the presence of various coated magnetic nanoparticles compared to without nanoparticles. Magnetic nanoparticles of Fe3O4 and γ-Fe2O3 were synthesized via co-precipitation with ferrous (Fe2+) and ferric (Fe3+) ions by a base (NaOH) in an aqueous solution. Nanoparticles were characterized by transmission electron microscopy (TEM) and dynamic light scattering. All particles were magnetic with sizes ranging from 10nm to 20nm in diameter. The particles were further coated with calcium phosphate (CaP: the main inorganic component of bone) to tailor them to treat osteoporosis. To reduce nanoparticle agglomeration, a common problem encountered when using nanoparticles that decrease their effectiveness, these particles were coated in the presence of surfactants citric acid (CA), bovine serum albumin (BSA) and dextrant. Coated crystallites of CaP were controlled thermally to obtain highly crystalline hydroxyapatite or less crystalline CaP with and without hydrothermal treatment, respectively. TEM images showed that iron oxide nanoparticles were successfully embedded in the CaP particles. Osteoblast proliferation tests conducted after 1, 3 and 5 days showed that Fe3O4 particles coated in the presence of BSA significantly increased osteoblast density compared to the controls (no particles). While the mechanism for this is unclear at this time, it is important evidence showing that CaP coated magnetic nanoparticles increased osteoblast density, and, thus, could be useful to treat osteoporosis. For future studies, cell experiments with CaP, iron oxide and surfactants separately will be performed to understand the contribution of each factor to the osteoblast proliferation process.
9:00 PM - DD3.21/HH6.21
Drug Loaded Polypyrrole Coatings on Titanium for Supporting Bone Growth and Inhibiting Fibrosis.
Sirinrath Sirivisoot 1 , Rajesh Pareta 1 , Thomas Webster 2 Show Abstract
1 Division of Engineering, Brown University, Providence, Rhode Island, United States, 2 Division of Engineering and Orthopaedics, Brown University, Providence, Rhode Island, United States
Electrically conductive polymers provide potentially interesting surfaces for bone implants because their surface properties can be versatile even releasing drugs immobilized by redox reactions. Specially, our previous research has demonstrated that carbon nanotubes grown out of nanotubular anodized titanium (Ti) can determine whether bone is occurring; such materials can also apply a voltage and increase bone formation. In this study, electropolymerization of pyrrole with drugs on Ti substrates were accomplished, providing a new cytocompatible surface for bone implants. Penicillin/streptomycin (P/S) and dexamethasone (Dex) were individually incorporated within the PPY thin film by cyclic voltammetry. The release of Dex was prolonged further by coating PPY film with poly(D,L-lactic-co-glycolic acid) (PLGA). X-ray photoelectron spectroscopy monitored and compared the reaction effectiveness and the yield of electropolymerization. Polypyrrole thin films with P/S and Dex, and even further coated with PLGA, all possessed nanometer scale roughness, as analyzed by atomic force microscopy. Preliminary in vitro results with human osteoblasts demonstrated greater adhesion after 4 hours on PPY-drugs embedded films, while the number of fibroblasts that adhered decreased compared to conventional Ti. A further step of this study was to evaluate drug release profiles. Such release of P/S and Dex from the polypyrrole film may inhibit infection and inflammation around hip/joint implants after surgery and the successful lyses of bacteria under a biofilm formed around a bone implant.
9:00 PM - DD3.22/HH6.22
Surface Modification Effect of Carbon Nanotubes-based Scaffolds on Human Embryonic Stem Cells Adhesion and Differentiation.
Tzu-I Chao 1 , Wei-Chun Chin 1 , Jennifer Lu 1 , Fang Lu 1 Show Abstract
1 , University of California at Merced, Merced, California, United States
9:00 PM - DD3.23/HH6.23
Mechanical Behavior of Individual Bovine Marrow-derived Stem Cells Undergoing Chondrogenesis.
BoBae Lee 1 , Paul Kopesky 2 , Eric Vanderploeg 3 , Bodo Kurz 6 , Christine Ortiz 1 , Alan Grodzinsky 2 4 5 Show Abstract
1 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States, 2 Biological Engineering, MIT, Cambridge, Massachusetts, United States, 3 Center for Biomedical Engineering, MIT, Cambridge, Massachusetts, United States, 6 Anatomisches Institut, Christian-Albrechts-Universität zu Kiel, Kiel Germany, 4 Electrical Engineering and Computer Science, MIT, Cambridge, Massachusetts, United States, 5 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States
Stem cell-based tissue engineering holds great potential for the regeneration and/or replacement of damaged cartilage. Mechanical studies of individual mesenchymal stem cells (MSCs) cultured in vitro can provide valuable insights into changes in intracellular morphology that accompany differentiation to the chondrocyte phenotype, as well as the synthesis of the cartilage-like neo-tissue during chondrogenesis. Here, we probe the temporal evolution of the single cell mechanical properties of bovine MSCs cultured within 3-D alginate scaffolds and stimulated to undergo chondrogenesis using dexamethasone and TGF-β1 for up to 10 days. Quasistatic indentation and force relaxation was carried out on each cell placed in a microfabricated silicon well using a spherical colloidal probe tip (end radius~ 2.5 μm) in an atomic force microscope in DMEM culture medium. Elastic moduli were calculated using a Hertzian contact mechanical model and time-dependent mechanical properties were estimated using a viscoelastic 5-element Maxwell-Weichert model. Dimethyl methylene blue (DMMB) dye binding and hydroxyproline assays were performed to quantify glycosaminoglycan and total collagen accumulation within the beads over time, respectively. The elastic modulus of bovine MSCs was found to be 575±56 Pa on Day 0 and stayed relatively constant up to day 10, and was lower than that of bovine primary chondrocytes (1000±112 Pa). Time-dependent mechanical properties of MSCs depended significantly on culture duration (ANOVA, p<0.05). At Day 3, MSCs exhibited instantaneous and quasi-equilibrium moduli which were significantly different from MSCs at Day 0 and 10. MSCs at Day 10 showed shorter τ1 (initial relaxation time constant) and longer τ2 (final relaxation time constant) compared to MSCs at Day 0 and 3. Notably, the final relaxation time constant for MSCs undergoing chondrogenesis was distinctly longer than that of primary chondrocytes even by 10 days, suggesting unique differences in intracellular organization and/or PCM that have not reached the final chondrocyte-like state. Variations in elastic modulus of MSCs during chondrogenesis were not detectable up to Day 10, while biochemical assays indicated that MSCs in alginate scaffolds synthesized and accumulated GAG and collagen in amounts comparable to MSCs in agarose and self-assembling peptide scaffolds as well as primary chondrocytes in alginate. This may be due to the fact that newly developing PCM of primary chondrocytes exhibits lower stiffness at initial culture duration. Further studies are being carried out on long-term culture of MSCs in alginate scaffolds up to 1 month to include mechanical testing using dynamic oscillatory compression.
9:00 PM - DD3.25/HH6.25
Cell Patterning Using Nanostructured Surfaces.
ChiungWen Kuo 1 , Jau-Ye Shiu 1 , Peilin Chen 1 Show Abstract
1 Research Center for Applied Sciences, Academia Sinica, Taipei Taiwan
The understandings of the cell-substrate interactions are important in many aspects including biocompatibility, cell culture, cell spreading and tissue engineering. It is recognized that the adhesion of cells on materials depends on surface characteristics such as hydrophobicity, surface charge, surface chemistry and roughness. Here we report a cell adhesion study on the nanostructured surfaces. Using a combination of nanosphere lithography and nanoimprinting, we were able to create nanostructures on various polymer surfaces, such as Teflon and polystyrene, without changing the surface chemistry of these polymers. In our approach, the nanosphere lithography was used to create close-packed well-ordered patterns. The size of the polymeric colloidal nanoparticles was trimmed by oxygen plasma treatment and followed by metal deposition. After the lift-off and deep etching process, nanohole arrays with desired depth could be obtained, which were then used the stamp in the nanoimprint process. By controlling the oxygen plasma etching time, the separation distance and the diameter of the holes could be adjusted, which in turn produced various types of polymeric nanopillars with surface water contact angle ranging from 120 degree to 160 degree. When these nanostructured surfaces were used to culture HeLa, 3T3, CHO and PC12 cells. It was found that all cells adhered preferentially on the roughened area allowing selective growth of cells on the desired area. Such nanostructured materials could be used as new materials for tissue engineering.
9:00 PM - DD3.26/HH6.26
Neural and Glial Cell Adhesion and Proliferation on Carbon Nanotube and Nanoparticle Zinc Oxide Polymer Composites.
Justin Seil 1 , Thomas Webster 1 Show Abstract
1 , Brown University, Providence, Rhode Island, United States
9:00 PM - DD3.27/HH6.27
Photocurable Biodegradable Elastomers as Tissue Engineering Scaffolds.
Cody Schoener 1 , Christopher Perry 1 , Ranjini Murthy 1 , Melissa Grunlan 1 Show Abstract
1 Biomedical Engineering, Texas A & M University, College Station, Texas, United States
There is currently a deficiency of biodegradable tissue engineering scaffolds which exhibit the elastic nature of many soft tissues and which degrade homogeneously with subsequent linear loss in mechanical properties. Conventional thermoplastic biodegradable polymers such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA) and their copolymers are generally brittle at physiological temperatures and degrade in a non-homogeneous fashion such that mechanical properties are dramatically diminished prior to significant loss of mass. Thus, thermoset elastomeric biodegradable polymers are promising candidates for scaffolds with mechanical properties more closely paralleling soft tissues and which degrade homogeneously. We have developed novel photo-crosslinked inorganic-organic elastomers as a new class of thermoset elastomeric biodegradable scaffolds for the regeneration of engineered soft tissues. Elastomers were prepared by photo-crosslinking of diacrylated triblock copolymers consisting of a central inorganic poly(dimethylisiloxane) (PDMS) block and terminal organic poly(caprolactone) (PCL) blocks. The impact of number average molecular weight (Mn) and PDMS:PCL ratio on mechanical properties, degradation behavior, and surface properties of the resulting elastomer were examined. The analogous porous elastomeric scaffolds were also formed in using a particle/leaching technique.
9:00 PM - DD3.28/HH6.28
Cytotoxicity and Biological Effects of Functional Nanomaterials Delivered to Various Cell Lines.
Meena Mahmood 1 Show Abstract
1 Applied Science, UALR, Nanotechnology Center, Little Rock, Arkansas, United States
Nanostructured materials have been found to be uptaken by various cell lines and highly affect their biological behavior. In this work, gold and silver metal nanoparticles as well as single wall carbon nanotubes were incubated separately and with apoptotic agents (Dexamethasne and Etoposide), in cell cultures of mouse long murine osteocytic bone cells (MLO-Y4 cells) and human cervical cancer cell line (Hela cells). The incubation of the nanomaterials with the cell cultures was carried out at two concentrations (0.5 X 10-9 M and 10-12 M) for 24 hours and the apoptotic agents (10-5 M and 75 X 10-6 M) were introduced for six hours. The cytotoxicity data revealed that the Au-NPs had a significant lower cytotoxic effect than the Ag-NPs and CNTs, values reflected by the percentage of the dead cells vs. living cells and that the combination of the nanomaterials and the apoptotic agents had a combinatorial effect, resulting in a significantly larger number of cells that died. The results highlighted by this study could represent a major development for the delivery of specific drug molecules into cancer cells and tumors by nanomaterials.
9:00 PM - DD3.29/HH6.29
Platelet Response on Poly (lactide-co-glycolic-acid) (PLGA) Film with Nano-structured Fillers.
Li Buay Koh 1 , Isabel Rodriguez 2 , Subbu S Venkatraman 1 Show Abstract
1 School of Materials Science and Engineering, Nanyang Technological University, Singapore Singapore, 2 , Institute of Materials Research & Engineering, A*STAR (Agency for Science, Technology and Research), Singapore Singapore
Thrombosis is a frequent complication associated with blood-contacting devices such as catheters and artificial stents. Current control of thrombus formation via the use of anticoagulant therapies is clearly not the best option, as complications can arise due to their usage. For that reason, a material with high level of blood compatibility is highly desirable. Platelet adhesion and activation on to the implant surface are crucial events in the formation of thrombus resulting from the interaction between the flowing blood and the foreign material. One of the parameters that have been shown to influence the adhesion of platelets is surface topography. In this work, we showed evidence that a nano structured polymer surface significantly reduces platelet adhesion as compared to pristine films. Nano-structured fillers were prepared on poly (lactide-co-glycolic-acid) (PLGA) films by infiltrating the PLGA solution into a nano porous anodized alumina (PAA) template. The inter-spacing between the nano-sized fillers and the aspect ratio proved to be the important parameters influencing the amount of platelet adhesion. The results illustrate that nanotopographic modifications of surfaces can elicit desired interfacial platelet response which is significant in the development for new polymeric blood-contacting materials with low thrombogenicity.
9:00 PM - DD3.3/HH6.3
Skin-external Device Integration using Expandable Cationic Poly(DMAA-co-AMTAC) Hydrogels.
Antonio Peramo 1 , Joong Hwan Bahng 2 , Cynthia Marcelo 3 , Nicholas Kotov 2 1 4 , David Martin 1 4 5 Show Abstract
1 Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan, United States, 2 Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 3 Surgery, University of Michigan, Ann Arbor, Michigan, United States, 4 Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 5 Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan, United States
There has been a steady increase in the number of medical procedures with permanently implanted transcutaneous devices, which range from glucose sensors and catheters to more complex biointegrated prosthetics. A typical complication is the break-down of the skin around the device, but the most prevalent problem producing device failure arise from infection processes. These devices show high variability in their intrinsic properties, with a disparate number of material composition, surface structure, porosities and topologies. This variability hampers the investigation of a general solution for this unresolved problem.For this application, we were seeking to use a soft, biocompatible material inducing dermal and epidermal integration that could be produced as hydrogels with the form of scaffolds or deposited as coatings on a variety of substrates. We have been investigating the use of N,N-Dimethylacrylamide (DMAA) copolymerized with (3-Acrylamidopropyl)-trimethylammonium chloride (AMTAC) (poly(DMAA-co-AMTAC)) for its use in the integration of human skin with transcutaneous devices. This material has the interesting property that induces cell adhesion without the need of scaffold surface modification.Poly(DMAA-co-AMTAC) hydrogel scaffolds, fabricated using the inverted colloid crystal method, were used to observe their integration with human skin. Full thickness human breast skin explants discarded from surgeries were cultured from 5 to 10 days at the air–liquid interface using a Transwell culture system. Cylindrical, disks or other shaped hydrogels were placed inside the skin explants fitting the punctures produced by punch biopsies and full section histological analysis of the skin explants with the inserted hydrogel was then performed. In addition, separated hydrogel scaffolds were cultured up to seven days with either human fibroblasts or keratinocytes.Results indicate that poly(DMAA-co-AMTAC) hydrogels induce substantial extracellular matrix deposition by fibroblasts, maintain excellent dermal integrity in the contact areas with the skin and induce dermal fibers to completely integrate into the pores. Different types of cells remaining in the explants migrated into the scaffold pores, including red blood cells and fibroblasts and fibroblasts and keratinocytes adhered and colonized the separately cultured hydrogel scaffolds. Our results suggest that this type of soft, biodegradable material could be used as a general interface that induces skin integration with transcutaneous devices in contact with skin.
9:00 PM - DD3.30/HH6.30
Designing an Equivalent of the Corneal Extracellular Matrix by Investigating Corneal Fibroblast Response to Aligned Collagen Scaffolds.
Donna Phu 1 , Lindsay Wray 1 , Elizabeth Orwin 1 Show Abstract
1 Engineering and Biology, Harvey Mudd College, Claremont, California, United States
The extracellular matrix (ECM) of the native corneal stroma is comprised of highly-organized collagen nanofibers and corneal keratocyte cells. Our project aims to recreate the microenvironment of the corneal stroma by electrospinning aligned collagen type I fibers. Corneal stromal cells undergo specific phenotypic changes in response to wound healing. The quiescent keratocyte differentiates reversibly into a myofibroblast, characterized by the expression of alpha smooth muscle actin (α-sma), which is undesirable due to its contribution to corneal haze (Jester 1999). Preliminary studies in our lab have shown that corneal fibroblasts cultured in monolayer on aligned collagen mats express less α-sma than unaligned samples. The overall goal of this project is to test the ability of the monolayer scaffolds to build a three-dimensional construct that will support cell proliferation and maintain the transparent phenotype. In this study, we compare aligned and unaligned electrospun collagen substrates to standard substrate materials for application to recreating a native corneal stroma. Substrate materials were seeded with rabbit corneal fibroblasts and were assessed for α-sma expression by immunoflourescence staining and Western Blotting, and for cell stratification and overall transparency with Optical Coherence Microscopy (OCM). In addition to scaffold type, we investigated the ability of 2-O-α-D-Glycopryranosyl-L-ascorbic acid (G-Asc) to build a thicker scaffold. To assess the viability of this method, we measured intracellular protein expression, cell stratification, and construct transparency. Preliminary studies in our lab have shown that G-Asc may promote cell stratification and increase the number of cell layers better than cells cultured in normal media (DMEM, 10% fetal bovine serum and 1% antibiotic/antimycotic). However, G-Asc also upregulates α-sma expression in the cells and decreases the overall transparency regardless of scaffold type. For the aligned scaffolds, cells cultured in G-Asc media expressed more α-sma than cells cultured in normal media. However, cells cultured on aligned scaffolds still exhibited less α-sma than on other scaffold types relative to both media conditions. This suggests that culturing cells in G-Asc on aligned collagen scaffolds may provide a promising method to simultaneously obtain thicker scaffolds and downregulate α-sma expression.
9:00 PM - DD3.31/HH6.31
Bone Tissue Induction, using a BC-PCL Composite Scaffold Material.
Maria Cortes 1 , Filipe A. F. Macedo 1 , Ruben Sinisterra 2 , Alfonso Gala-Garcia 1 Show Abstract
1 Restorative Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 2 Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
An artificial scaffold is required to be used in tissue engineering applications that can restore diseased or damaged bone to its natural state and function. Although autografts have all the desired characteristics with almost no disadvantage, the amount of autogenous bone that can be dissected from another host site is limited and difficult to shape. The aim of this study was prepared, characterized and evaluated the in vivo and in vitro response of composites of poly (ε-caprolactone) (PCL) and a bioceramic matrix (BC). The composites were prepared during the dissolution of the polymer phase in weight ratio 1:5 (polymer:bioceramic). Scanning electron microscopy (SEM) analysis revealed a characteristic surface of the interconnected porous scaffold. The X-ray diffraction shows pattern of BC-PCL composite where typical, PCL and BC peaks appeared in it. There were no others peaks nor peak shifts in the composite, suggesting that no chemical reactions occurred. The structure of the composite coating was analyzed using infrared spectroscopy, and characteristic structural bands of both BC and PCL were observed. In the current work, we investigated cellular viability, proliferation and morphology changes of rat primary culture osteoblasts in contact with BC-PCL composite. We observed that cell viability was increased in the composite when comparing with BC and control. We further found that collagen and alkaline phosphatase production was higher in osteoblasts cultured in the presence of BC-PCL compared to BC and control. In vivo experiments were performed after the analysis in vitro. In vivo analysis we used males Wistar rats, each animal received two different tablets implanted in a dorsal region under aseptic conditions following a triplicate assay. The mice were killed after 2 months and subcutaneous tissue treated with BC-PCL showed biocompatibility and all implants were surrounded by osteoblasts-like cells. In conclusion, BC-PCL had crystallinity characteristics with strong correlation between structure-activity and it was biocompatible in vitro and in vivo.
9:00 PM - DD3.32/HH6.32
Fabrication of Three-Dimensional Microfluidic Hydrogels by X-ray Lithography.
Chang Mo Jeong 1 , Ji Tae Kim 1 , Jung Ho Je 1 , Yeukuang Hwu 2 , Giorgio Margaritondo 3 Show Abstract
1 X-ray Imaging Center, Department of Materials Science and Engineering, Pohang University of Science and Technology, Pohang Korea (the Republic of), 2 Institute of Physics, Academia Sinica, Taipei Taiwan, 3 School of Basic Sciences, Ecole Polytechnique Fadarale de Lausanne, Lausanne Switzerland
Microfluidic hydrogels have been widely studied for tissue-engineering scaffolds because of their advantages such as mechanical strength, permeability, and biocompatibility (1). In particular, alginate-based microfluidic hydrogels are known to show a good transport of gases and nutrients through continuous networks in channel and therefore capable of tissue generation and growth in three dimension with appropriate permeation of biological growth factors (2). Particularly three-dimensional (3D) structures of microfluidic hydrogels with pervasive, interconnected channels are essentially required for biomimetic applications (2-3). The construction of 3D features however is still a challenge in conventional lithography such as photolithography and soft-lithography (4). In this study we present a novel strategy to fabricate 3D alginate-based microfluidic hydrogels using X-ray lithography. We demonstrate a successful fabrication of 3D microchannels with a few centimeter depths by an irradiation of X-rays (10-60 keV) from a synchrotron source. The 3D microchannel formation is attributed to fast scission of alginate hydrogels by X-ray irradiation. The microchannel depth is proportional to the X-ray irradiation time at a rate of 2 mm/min. We suggest that the X-ray-based strategy may provide a feasible method to fabricate 3D microfluidic hydrogels that would be potentially useful for widespread applications of tissue-engineering, microfluidic, and biotechnological systems.References:(1) J. A. Rowley, et al. Biomater. 20, 45 (1997).(2) M. Cabodi, et al. J. Am. Chem. Soc. 6, 908 (2007).(3) N. W. Choi, et al. Nature Mater. 6, 908 (2007).(4) D. Therriault, et al. Nature Mater. 2, 265 (2003).
9:00 PM - DD3.33/HH6.33
Fabrication of a Crosslinked Hydrogel Using Dense Gas Technology.
Nasim Annabi 1 , Suzanne Mithieux 2 , Anthony Weiss 2 , Fariba Dehghani 1 Show Abstract
1 School of Chemical and Biomolecular Engineering, Sydney University, Sydney , New South Wales, Australia, 2 School of Molecular and Microbial Biosciences, Sydney University, Sydney , New South Wales, Australia
The aim of this study was to fabricate a biopolymeric hydrogel using dense gas technology for tissue engineering applications. The hydrogel was synthesized through coacervation followed by crosslinking. Dense gas process facilitated coacervation, expedited cross-linking reaction, and induced porosity through the hydrogel matrix. The properties of fabricated hydrogel including pore sizes, pore interconnectivity, mechanical properties, and swelling ratio were tailored by pressure, temperature, processing time, and depressurization rate. The hydrogels fabricated using dense gas process exhibited superior properties compared with hydrogels produced at atmospheric pressure. The results of micro-CT scan and SEM images demonstrated that pore interconnectivity was substantially enhanced for fabricated hydrogel using dense gas process. Dense gas CO2 reduced the wall thickness and size of the pores. The cell adhesion and proliferation were remarkably increased for the samples fabricated using dense gas process. The cells were proliferated into the hydrogel matrix processed by the dense gas CO2 due to the formation of channels in the microstructure and nanosized fibrous features that were similar to collagen structures.
9:00 PM - DD3.34/HH6.34
Direct-Write Assembly of 3D Microperiodic Hydrogel Scaffolds for Tissue Engineering Applications.
Sara Parker 1 , Jennifer Hanson 1 , Robert Shepherd 1 , Robert Barry 1 , Roy Rotstein 1 , Pierre Wiltzius 1 , Ralph Nuzzo 2 , Jennifer Lewis 1 Show Abstract
1 Materials Science and Engineering, University of Illinois, Urbana, Illinois, United States, 2 Chemistry, University of Illinois, Urbana, Illinois, United States
We have fabricated three-dimensional microperiodic scaffolds by direct-write assembly of a concentrated ink, which is composed of poly(2-hydroxyethyl methacrylate) (HEMA) chains, HEMA monomer, crosslinker, photoinitiator, and water. The ink exhibits a viscoelastic response that enables it to flow readily during printing, yet retain its filamentary shape even as it spans gaps in the underlying layer(s). The scaffolds are patterned by extruding this ink through a fine deposition nozzle to form micron-sized hydrogel filaments that are periodically arrayed in three dimensions. After assembly, the printed scaffolds are cross-linked by UV exposure. 3D hydrogel scaffolds with features as small as 2 μm have been constructed. Poly(HEMA) scaffolds are then seeded with developing rat hippocampal neurons and imaged with fluorescence microscopy. We are currently investigating the cellular response to scaffold feature size and mechanical properties.
9:00 PM - DD3.35/HH6.35
Electrospun Poly(Vinilidene Fluoride) Nano Fibers for Electroactive Scaffolds.
Vitor Sencadas 1 2 , Jose Gomez Ribelles 2 3 4 , Manuel Monleon Pradas 2 3 4 , Senentxu Lanceros-Mendez 1 Show Abstract
1 , University o Minho, Braga Portugal, 2 Centro de Biomateriales, Universidad Politecnica de valencia, Valencia Spain, 3 Regenerative Medicine Unit. , Centro de Investigación Príncipe Felipe, Valencia Spain, 4 , CIBER en Bioingeniería, Biomateriales y Nanomedicina, Valencia Spain
9:00 PM - DD3.37/HH6.37
Fabrication of Agarose Porous Scaffolds by X-ray Lithography.
Kyu Hwang Won 1 , Byung Mook Weon 1 , Jung Ho Je 1 , Yeukuang Hwu 2 , Giorgio Margaritonto 3 Show Abstract
1 X-ray Imaging Center, Department of Materials Science and Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea (the Republic of), 2 Institute of Physics, Academia Sinica, Taipei Taiwan, 3 School of Basic Sciences, Ecole Polytechnique Fadarale de Lausanne, Lausanne Switzerland
Porous scaffolds play a significant role in tissue engineering by preserving tissue volume, providing temporary mechanical function, and delivering biofactors (1). In particular agarose gels, which are chemically and electrically neutral materials, have been widely used in biomedical or biological research because of their biocompatibility and biomimetic capabilities in terms of water content, soft rubbery consistency, and low interfacial tension. Agarose-based porous scaffolds have extensively studied for potential applications of pancreas tissue engineering, cell-gel hybrids, nerve-guiding scaffolds, microcarriers for drug delivery or cell culture, and micropatterned stamping arrays (2). The applications require improved properties such as high porosity, proper pore size, biodegradability, mechanical strength, and toxicity. However optimizing all requirements remains a difficult challenge. A longstanding challenge is to design porous architectures with optimum porosity and mechanical strength. In this study we present a novel fabrication method that enables to easily build complicated agarose porous architectures using X-ray lithography. We reveal that a large volume of agarose hydrogels rapidly collapses by an irradiation of X-rays (10-60 keV). Using the X-ray-reactive volume collapse, we are able to fabricate agarose porous scaffolds of well-defined porous, interconnected structures with various channel widths and depths, which are respectively controlled by mask size and X-ray-reactive rate (~1mm/min). We suggest that X-ray-based strategy may provide a feasible way to easily fabricate complicated porous scaffolds for tissue engineering and biotechnology.References:(1) S. J. Hollister, Nature Mater. 4, 518 (2005).(2) J. Roman, et al. J. Biomed. Mater. Res. A 84, 99 (2008).
9:00 PM - DD3.4/HH6.4
Chemical Engineering of a Mesenchymal Stem Cell Homing Response.
Debanjan Sarkar 1 2 , Praveen Vemula 1 2 , Grace Teo 1 , Jeffrey Karp 1 2 Show Abstract
1 Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States, 2 Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts, United States
Stem cell based therapies offer enormous hope for patients suffering from a wide range of diseases and disorders. Recently there has been significant interest in the clinical use of adult mesenchymal stem cells (MSCs), which are connective tissue progenitor cells. MSCs are currently in pre-clinical and clinical trials to treat several diseases. One of the greatest challenges in traditional stem cell therapy is to deliver a large quantity of viable stem cells with high engraftment efficiency. Unfortunately, MSCs home at a low efficiency; typically less than 1% of the infused cells reach the targeted site. The predominant reason why MSCs are thought to have low engraftment efficiency is due to the lack of relevant adhesion molecules on their surface Leukocytes, whose homing mechanisms have been well elucidated, depend on a range of adhesion molecules in a multistep homing process. The first step in the homing process involves reversible, adhesive interactions between glycoprotein receptors on specific circulating cells and ligands expressed on the surface of of the vascular endothelium. Several efforts have been made to modify MSCs by enzymatic and genetic methods to increase their homing efficiency but the efficacy of these modifications are limited due to the complexity and the potential safety concerns.To engineer a mesenchymal stem cell homing response, we employed a simple chemical approach. Specifically, the sialyl Lewisx (SLeX) moiety, found on the surfaces of leukocytes representing the active site of the P-selectin glycoprotein ligand (PSGL-1), was covalently immobilized to the cell surface by biotin-streptavidin conjugation to improve the homing response. Modified MSCs exhibited velocities of 2µm/sec at a wall shear stress of 0.366 dynes/cm2 which is 96% lower than the unmodified MSCs on P-selectin surface in a parallel plate flow chamber assay. Moreover, the flux value of the modified MSCs was 75 cells/mm2.sec whereas the unmodified cells displayed a value of 20 cells/mm2.sec. The lower rolling velocity and high flux value indicates that the MSCs modified covalently with SLeX have increased homing efficiency. Moreover, the MSCs’ native phenotype including its ability to proliferate and differentiate into multi-lineages was retained after the modification. Hence, MSCs with covalently conjugated SLeX moiety can potentially be targeted to inflammatory sites while preserving the normal cell phenotype. This platform approach offers a simple method to target potentially any cell type to specific tissues within the body through conjugation of specific targeting agents to the cell surface.
9:00 PM - DD3.5/HH6.5
Three-dimensional Hydrogel Structures with Submicron Scale for Biomedical Applications via Phase Mask Interference Lithography.
Ji-Hyun Jang 1 , Shalin Jhaveri 2 , Boris Rasin 1 , Christopher Ober 2 , Edwin Thomas 1 Show Abstract
1 Institute for Soldier Nanotechnologies, Department of Materials Science and Engineering, , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Materials Science and Engineering, Cornell University, Ithaca, New York, United States
A random terpolymer of poly(HEMA-co-MMA-co-MAA) has been synthesized and used as a negative-tone photoresist to generate bicontinuous 3D hydrogel structures at submicron scale. Phase mask interference lithography (PMIL) has been employed to fabricate three-dimensional hydrogel structures with high surface area. We have shown that the fully opened 3D hydrogel structures can be used as pH-responsive patterned polymer in drug-delivery system for the delivery of neurotrophins to enhance the performance of neural prosthetic devices.
9:00 PM - DD3.6/HH6.6
Electrical Stimulation of Nerve Cells: Image Analysis of Cell Behavior.
Kwang-Min Kim 1 , Julie Richardson 2 , Sung-Yeol Kim 1 , Celinda Kofron 2 , Diane Hoffman-Kim 1 2 , G. Tayhas Palmore 1 2 Show Abstract
1 Division of Engineering, Brown University, Providence, Rhode Island, United States, 2 Division of Biology and Medicine, Brown University, Providence, Rhode Island, United States
Neurons of the central nervous system (CNS) encounter both permissive and inhibitory cues after injury. Conflicting results have been reported as to the benefits of electrical stimulation on neuronal outgrowth. To assess the therapeutic value of electrical stimulation, we aim to quantify how long and in which direction neuron processes extend when subjected to electrical stimulation. Toward this end, we have designed a platform with which to characterize the behavior of nerve cells during electrical stimulation. In this talk, the fabrication of this platform and its use in the study of electrically stimulated nerve cells will be described. The platform consists of two parallel chambers, which permits live-cell imaging of dorsal root ganglia (DRG) in the presence or absence (control) of electrical stimulation simultaneously. Electrical stimulation was applied to one of the two chambers containing adhered DRGs for 10 minutes at three different time points after initial cell plating. DRGs in both chambers were imaged during the electrical stimulation of one chamber and after removal of the electric field for one hour. Images were analyzed using MATLAB programming and FFT to compare the behavior of the neurons with and without electrical stimulation. Results from this analysis indicate that the mobility of both cell soma and neurites increases when electrically stimulated and tend to move in the direction of the applied field and neighboring cells. In addition to live-cell image analysis, cells were fixed and stained 25h after initial cell plating. Both length and angle of neurites were analyzed. Results from this analysis indicate electrical stimulation at an early stage of neuron culture had a negative impact on neurite outgrowth while electrical stimulation at an intermediate stage of the neuron culture had a positive impact on neurite outgrowth. Furthermore, neurites from these time points preferentially extended parallel to the direction of the electric field. Neurites stimulated at a late stage of culture, however, did not exhibit a significant difference in length and angle when compared to cells that were not electrically stimulated. This result suggests that some lag time post-electrical stimulation may be required to observe a difference in the length and direction of neurite extension. These results will be discussed in the context of a timeline of biochemical responses and their importance to nerve regeneration.
9:00 PM - DD3.7/HH6.7
Immunoreactivity of Self-Assembling Peptide-Polymer Biomaterials.
Jai Rudra 1 , David Hildeman 2 , Joel Collier 1 Show Abstract
1 Department of Surgery, University of Chicago, Chicago, Illinois, United States, 2 Department of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
Monday, 12/1New Presenter - Poster HH6.7/DD3.7Immunoreactivity of Self-Assembling Peptide-Polymer Biomaterials. Jai S. Rudra
9:00 PM - DD3.8/HH6.8
Different Macrophage Responses on Hydrophilic and Hydrophobic Carbon Nanotubes.
Young Wook Chun 1 , Dongwoo Khang 1 , Thomas Webster 1 Show Abstract
1 , Brown Univ., Providence, Rhode Island, United States
Purified carbon nanotubes (with removed toxic catalytic particles) have been considered as novel materials for drug delivery and for generating artificial organs more efficiently due to their unique surface features. Traditionally, the surface chemistry of carbon nanotubes has been modified through various functionalization strategies to increase biocompatibility. Importantly, modulating the intrinsic material surface energy of carbon nanotubes (without functionalization, thus, establishing permanent, non degradable chemical, and physical surface properties) can potentially reduce an immune response mediated by macrophages. Herein, we report macrophage responses on different surface energy carbon nanotubes while keeping their nanoscale surface roughness. Specifically, interactions of ultra hydrophobic (bare or unmodified) and hydrophilic carbon nanotubes (due to the formation of oxide layers) with macrophages (including adhesion, proliferation, lipopolysaccharide (LPS) stimulation, hydrogen peroxide production and cytokine secretion (IL-1alpha, IL-1beta)) were investigated. All results clearly support the evidence that tailoring the surface energy of carbon nanotubes mediates a macrophage-dependent immune response while maintaining their ability to increase tissue growth, such as bladder tissue regeneration.
9:00 PM - DD3.9/HH6.9
Protein Adsorption can be Controlled by Varying PEG Block Length in PEG-containing Copolymers.
Arnold Luk 1 , Sanjeeva Murthy 1 , Wenjie Wang 2 , Hak-Joon Sung 1 , Joachim Kohn 1 Show Abstract
1 New Jersey Center for Biomaterials, Rutgers University, Piscataway, New Jersey, United States, 2 Department of Physics, University of Vermont, Burlington, Vermont, United States
Controlling the protein adsorption process on biomaterial surfaces is an important challenge for biomedical engineering. The performance of a medical device may be linked to the way proteins adsorb to its surface once it is implanted. It has been well documented that in random copolymer systems composed of hydrophobic units and PEG blocks, increasing the amount of PEG reduces protein adsorption and subsequent cell attachment. However, it is currently poorly understood how variations in the PEG block size affect polymer morphology and the subsequent protein adsorption process. To address this question, we prepared a system of three model polymers comprised of a hydrophobic monomer (desaminotyrosyl-tyrosine ethyl ester, DTE) which was copolymerized with PEG blocks of different molecular weights (100, 1000 and 35,000). In all polymers, we kept the total weight percentage of PEG constant at 40 weight %. Therefore, the only difference between these polymers was the PEG block length and the associated changes in polymer morphology. As expected for a PEG-rich polymer, we find that fibrinogen is repelled when the PEG block length is 1000. However, when PEG100 and PEG35k were used, fibrinogen adsorbs to the corresponding polymer surfaces readily. Analysis of the bulk materials using small angle neutron scattering (SANS) indicates that the copolymer with PEG35k blocks contains PEG domains with radii of about 10 nm separated by 33 nm between domains. In contrast, the copolymer with PEG1000 blocks had PEG domains with radii of about 5 nm separated by 15 nm between domains. Although the polymer containing PEG100 could have PEG-rich domains separated by > 70 nm, a distance that was at the limit of instrument resolution, it is most likely that PEG is homogeneously distributed at ~ 1 nm length scales. Since the dimensions of a molecule of fibrinogen are 47 nm x 4.5 nm x 4.5 nm, we speculate that the modulation of protein adsorption is caused by the spatial distribution of PEG domains: If PEG domains are sufficiently far apart, proteins will adsorb onto the hydrophobic DTE regions. This condition was met only for copolymers having PEG35k and PEG100 blocks. The data presented provide new insights into the mechanisms of surface-protein interactions and how these interactions can aid the rational design of biomaterials for tissue engineering.
Elizabeth Orwin Harvey Mudd College
Brenda Mann Sentrx Animal Care
Ben Wu University of California-Los Angeles
Anthony Ratcliffe Synthasome, Inc.
DD4: Applied Tissue Engineering: Tissue Engineered Products and Materials Issues in Industry
Tuesday AM, December 02, 2008
Hampton A/B (Sheraton)
9:30 AM - DD4.1
Biomimetic Approaches for Tissue Engineering of Vocal Fold Lamina Propria.
Alexandra Farran 1 , Fang Jia 1 , Amit Jha 1 , Randall Duncan 2 , Xinqiao Jia 1 Show Abstract
1 Materials Science & Engineering, University of Delaware, Newark, Delaware, United States, 2 Biological Sciences, University of Delaware, Newark, Delaware, United States
To engineer a functional vocal fold lamina propria, the proper combination of primary porcine vocal fold fibroblasts (PVFF), three dimensional biomimetic hydrogel matrices, appropriate biological cues and biophysical stimulation is required. In our initial studies, PVFF were encapsulated in collagen/hyaluronan composite hydrogels in situ, and were subsequently cultured under static conditions for up to 28 days. Cell morphology, cell proliferation, and ECM production were analyzed at different time points using fluorescent confocal microscopy and standard biochemical and histological assays. We found that encapsulation of the PVFF does not cause significant cell death. Upon encapsulation, PVFF attached to the matrix and adopted a spindle-shaped morphology. The cell number increased steadily over 28 days. The hyaluronan content in the constructs decreased, while the collagen content remained essentially unchanged. The mechanical properties of the constructs were evaluated, at human phonation frequencies, using a torsional wave apparatus. The results indicate that the elastic modulus of the constructs reaches a plateau value of approximately 530 Pa at the end of the culture. We have developed a vocal fold bioreactor that is capable of generating high frequency vibrational stress as well as low frequency elongational tension to the encapsulated PVFF. The stresses imposed to the constructs were monitored using a Doppler Laser Vibrometer. Different modes of mechanical stimulations were systematically evaluated, and their effects on PVFF were compared to the static culture. It is clear that defined mechanical stimulations are essential for the successful engineering of functional vocal fold lamina propria.
9:45 AM - DD4.2
Development of Polymer-Based Heart Valve Tissue Engineering Scaffolds with Growth Factor Delivery.
Meghan Smith 1 , Yakov Elgudin 2 , Olivier Arnoult 3 , Nicholas Greco 2 , Mary Laughlin 2 , Steven Emancipator 4 , Brian Cmolik 2 , Gary Wnek 3 Show Abstract
1 Chemical Engineering, Case Western Reserve University, Cleveland, Ohio, United States, 2 Medicine, Case Western Reserve University, Cleveland, Ohio, United States, 3 Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, Ohio, United States, 4 Pathology, Case Western Reserve University, Cleveland, Ohio, United States
Purpose: Heart valve disease affects a substantial population, and while prosthetic valve replacement is common, tissue engineering presents the opportunity to restore the autologous tissue. Scaffolding is a key component in tissue engineering, as the matrix must provide for cell growth and delivery, while also providing comparable biomechanical properties to the native tissue until remodeling had occurred. For heart valve tissue engineering, the mechanical properties of the scaffold are of utmost importance, due to the high level of hemodynamic stress the valve is subjected to. Electrospinning is commonly used to develop tissue engineering scaffolds, and we have employed a variation of this process to develop a tissue engineering matrix capable of controlled delivery of multiple growth factors. The purpose of this study is to evaluate the incorporation and delivery of growth factors from electrospun scaffolds, using biocompatible and biodegradable polymers, as well as to evaluate the effect of the encapsulated domains on the mechanical properties of the scaffolds, and compare them to native valve tissue. Methods: Electrospinning was carried out with a series of polymers including poly(ε-caprolactone) and poly(lactic-co-glycolic acid), as well as blends of these polymers, all in organic solvents. An aqueous suspension of the growth factors within the polymer solution was formed, and the resulting suspension was electrospun to create solid polymer fibers with encapsulated aqueous domains containing the protein. Mats were characterized with scanning electron microscopy for fiber diameters and porosity. Fluorescent labeling and microscopy were used to locate the growth factors within the polymer fibers. Dynamic mechanical analysis was performed to evaluate the tensile mechanical properties of the biphasic scaffolds, and compared to scaffolds of the pure polymer, as well as to stabilized porcine valve tissue. ELISA was performed to quantify the delivery of the growth factors from the scaffolds, and to evaluate kinetic release profiles.Results and Conclusions: Tissue engineering scaffolds containing growth factors were successfully produced via suspension electrospinning. The encapsulated proteins were found to be localized within aqueous pockets within the fiber. Fiber diameters were found to average around 1 um. Release of the growth factor from the scaffolds was readily achieved, with some burst effect seen in the first 12 hours. Mechanical properties were not significantly impacted by the inclusion of aqueous domains compared to scaffolds spun from pure polymer phase. In comparison with porcine heart valve tissue, the scaffolds were found to not have the “J-shaped” stress-strain profile characteristic of native tissue, although numerically comparable tensile properties were observed. Approaches to tailor mechanical properties of synthetic scaffolds to more closely mimic those of natural heart valve tissue will be discussed.
10:00 AM - **DD4.3
Applied Tissue Engineering-Tissue Engineered Products and Materials Issues in Industry.
Gabriele Niederauer 1 Show Abstract
1 , ENTrigue Surgical Inc., San Antonio, Texas, United States
10:30 AM - DD4.4
The Design of Better Vascular Stents Through Nanotechnology.
Jing Lu 1 , Dongwoo Khang 1 , Thomas J. Webster 2 Show Abstract
1 Division of Engineering, Brown University, Providence, Rhode Island, United States, 2 Division of Engineering and Orthopaedics, Brown University, Providence, Rhode Island, United States
Bio-inspired nano-materials might provide a promising alternative for current vascular stents. There are three important factors to be considered to fabricate such materials: topography, roughness and surface energy. In case of topography, it has been shown that highly controllable nanostructured surface features could greatly enhance vascular endothelial cell functions. For roughness and surface energy, previous studies indicated that both sub-micron (features larger than 100nm in the lateral scale) and nanometer (feature less than 50nm in the lateral and vertical scales) surface features on titanium vascular stents increased vascular endothelial cell density and surface energy. Compared with sub-micron surface features, nano surface features on titanium vascular stents were more effective for increasing both cell adhesion and surface energy collectively. However, it has been unclear about the higher efficiency of nano surface features on accelerating endothelization and whether they could also promote hyperproliferation of vascular smooth muscle cells in the case of atherosclerosis. Therefore, the objective of this in vitro study was to investigate the mechanism of nano and submicron surface features on titanium in enhancing endothelial cell adhesion and their behavior in the situation of atherosclerosis with the help of a vascular endothelial/smooth muscle cell co-culture model. Furthermore, different titanium surface structures were aligned to determine the possible selective and spatial control of different cell adhesion. Three different titanium roughness values: flat, nano and sub-micron were created using E-beam evaporation. Meanwhile, a masking technique was applied to fabricate highly patterned titanium surface features, which generated two different titanium surface roughness values. Samples were characterized for surface chemistry and topography as well as surface energy and wettability. Co-culture adhesion and initial protein adsorption assays were conducted. The protein adsorption assays showed that primary extracellular matrix (ECM) proteins, fibronectin and vitronectin, preferentially adsorbed onto nano structured titanium surfaces and sub-micron structured titanium surfaces compared to flat titanium surfaces, with more ECM protein adsorption on nano surfaces although their surface energy was lower than sub-micron structured titanium surfaces. However, fibrinogen adsorption was not promoted by either surface features, which might indicate that nano and sub-micron surface might not activate platelets. Moreover, in the competitive co-culture system, with the existence of vascular smooth muscle cells, vascular endothelial cells still adhered more on both nano and sub-micron titanium features. In contrast, smooth muscle cell adhesion was not influenced by titanium surface feature. All these results showed that nano and sub-micron structured surfaces have great potential in replacing convention vascular stents.
12:00 PM - DD4.7
Nanotube Surface for Significantly Accelerated Bone Healing and Tissue Engineering
Karla Brammer 1 , Seunghan Oh 1 , Sungho Jin 1 Show Abstract
1 Materials Science and Engineering, University of California, San Diego, La Jolla, California, United States
Titanium (Ti) is considered exceptionally biocompatible, provides sufficient load-bearing strength, and is one of the most widely used materials for all bone implants. However, the surface properties needed for firm and permanent bone-implant integration is lacking as the titanium implants in patients tend to fail due to loosening at the bone-implant interface causing painful and costly resurgery. Improving this structural connection between living bone and implant (i.e. osseointegration) is one focus of our research objectives. We have modified Ti implant surfaces by introducing a nano-structured TiO2 nanotube porous topography fabricated by anodization. The Ti nanotube surface accelerates bone growth by a factor of ~3 and significantly enhances the interface bonding strength. The novel nano-structure actually allows for structural components (filopodia) of growing cells to go into the nanotube pores for increased spreading and propagation. The nanotube configuration, unlike a simple pored configuration, also provides pathway for continuous supply and exchange of cellular fluids containing essential ions, nutrients, proteins, and cell signaling molecules beneath the growing cells for a healthier environment and improved functioning.We have found that the TiO2 nanotube surface topography also provides desirable tissue scaffolding characteristic that enhances the cellular response in many types of mammalian cells, not just osteoblast cells. The combined nanotopography and nano cues of the TiO2 nanotube surface structure led to an improved endothelial response with primary bovine aorta endothelial cells . The beneficial effects of the TiO2 nanotube structure included increased extra cellular matrix formation, and substantially raised level of nitric oxide-endothelin ratio, which can be useful for design of a vascular stent material with a reduced probability of late stent thrombosis (LST). In the area of heart tissue engineering, cardiomyocyte adhesion on moving substrates (simulating a heart beating) with the nanotube topography was significantly improved over flat Ti controls because the nanotubes allow for cellular interlocking and superior bonding at the cell/surface interface between the heart cells and the scaffold.  K.S. Brammer, S. Oh, J. O. Gallagher, S. Jin, Enhanced cellular mobility guided by TiO2 nanotube surfaces. Nano Lett, 2008. 8(3): p. 786-793.
12:15 PM - DD4.8
Nanocomposite Materials for Vascular Grafts Made of Collagen, Chitosan and Elastin.
Krishna Madhavan 1 , Min Li 2 , Wei Tan 1 2 Show Abstract
1 Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, United States, 2 Department of Pediatric Cardiology, Institute of Health Sciences & Children’s Hospital, University of Colorado, Denver, Colorado, United States
The need for vascular grafts to replace small caliber vessels (< 6 mm) is still exceedingly high. Compliance mismatch between current vascular graft materials and blood vessels is believed to be a major cause of graft failure because it induces flow disturbance at the graft – vessel junction. Blood vessels have a non-linear response to dynamic pulse caused by pressure and flow in arteries. Designing a material, that demonstrates a similar stress – strain curve as arteries, may produce a new vascular graft with improved in vivo performance. To develop grafts that mimic arterial compliance, we are engineering a series of materials made from naturally derived materials, collagen, chitosan and elastin. We demonstrate the capability of tuning tensile stress – strain curve with varied compositions. Similar to the structure of the extracellular matrix, collagen fibers form the basis of the matrix material. We demonstrate that the addition of chitosan and elastin into the collagen matrix influences the shape of stress – strain curves. While the collagen – chitosan – elastin constructs exhibit higher elasticity within the range of low stress, collagen and collagen – chitosan constructs exhibit lower elasticity within a similar range of stresses. This demonstrates the successful integration of elastin into collagen – chitosan construct. Also, this demonstrates that elastin may affect the material response to low – stress (or low – pressure) while collagen may affect the response to relatively higher stresses. The structure of the nanofibers is analyzed under field emission scanning electron microscope. From structural characterization, it is identified that the addition of chitosan causes fiber binding and, the bundled collagen fibers may more efficiently recruit fibers when being stretched. Additionally, effects of crosslinkers on various collagen – based constructs are studied. Crosslinking is done using three different crosslinkers – N-ethyl-N’-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDAC), paraformaldehyde and genipin. Samples crosslinked with EDAC and genipin have similar tensile properties. The effects of polymerization time and crosslinking time on the construct strength and elasticity are included in the strategy for improving mechanical strength. Besides the characterizations with a tensile tester, compressive and shear modulus of the matrix gel construct are also obtained using dynamic mechanical analyzer and rheometer. Bovine vascular endothelial cells are seeded on the gels and their compatibility with the crosslinked materials is tested using a live – dead assay staining method. The samples crosslinked with genipin showed best cellular biocompatibility among all the tested samples. By changing the concentration of the materials, we may further tune the mechanical and biological properties and develop materials with superior functionality suitable for making small – caliber vascular grafts.
12:30 PM - DD4.9
Transcutaneous Biomedical Device with a Regenerative Materials Interface.
Antonio Peramo 1 , Cynthia Marcelo 2 , Steven Goldstein 5 4 , David Martin 1 3 4 Show Abstract
1 Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan, United States, 2 Surgery, University of Michigan, Ann Arbor, Michigan, United States, 5 Orthopedic Surgery, University of Michigan, Ann Arbor, Michigan, United States, 4 Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 3 Macromolecular Science and Envineering, University of Michigan, Ann Arbor, Michigan, United States
The use of medical devices passing through the skin has continued to rise. In conjunction with an increased use of catheterization and a general tendency toward minimally invasive procedures in medicine, the need for rapid and reproducible testing methods to evaluate the interfaces between devices and internal tissues and organs has dramatically increased. In the particular case of permanently implanted percutaneous devices, complications ranging from break-down of the skin to serious infections have substantially limited their success, mainly because they do not permit the formation of a stable dermal and epidermal sealing around the implant.We have developed an in vitro culture system composed of organotypic human skin explants interfaced with titanium rods or stainless steel fixator pins. The use of this regenerative interface provides a model to evaluate strategies for creating a stable, long-term connection with living skin and chronic percutaneous devices. Our hypothesis is that the delivery of specific biomaterials at this interface will create a dynamic, slowly flowing matrix for epidermal integration and a means for the local administration of drugs.This system was used to observe changes in tissue morphology of the skin in contact with the rods or pins in the presence or absence of a mixture of hyaluronic acid and dermatan sulfate (HA+DS) or physiological saline. Full thickness human breast skin explants discarded from surgeries were cultured from 5 to 15 days at the air–liquid interface. The skin specimens were punctured to fit at the bottom of hollow cylindrical titanium rods or stainless steel fixator pins. The HA+DS mixture or physiological saline were delivered to the specimens thorough the rods or fixator pins either discontinuously or continuously by using an attached fluid pump. Different concentrations of the mixture and pumping rates were tested and histological analysis of the skin explants was then performed.Results indicate that that during short culture periods (five days), specimens interfaced with fixator pins treated with HA+DS had better epidermal architecture and dermal structure than control specimens or specimens treated with physiological saline. Non treated specimens had a deteriorating basal lamina and a disappearing stratum spinosum. During longer culture periods (fifteen days), the presence of these percutaneous materials induced apoptosis and hyperproliferation in the areas close to the materials. The delivery of HA+DS resulted in a moderate reduction of apoptosis and proliferation. These results suggest that this type of treatment may be applicable to tissue areas in contact with fixator pins and other percutaneous devices in vivo with the expectation that these two polysaccharides may maintain a better epidermal architecture, tissue regeneration and wound healing. Our model system makes it possible to perform rapid, repeatable studies of living skin response to chronically implanted materials.
12:45 PM - DD4.10
Independent Control of Fracture Toughness and Stress Relaxation of Hydrogels with Hybrid Crosslinking.
Xuanhe Zhao 1 , Nathaniel Huebsch 1 2 , David Mooney 1 , Zhigang Suo 1 Show Abstract
1 School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, United States, 2 2.Harvard-MIT Division of Health Sciences and Technology, Harvard and MIT, Melbourne, Victoria, Australia
In tissue engineering, hydrogels have been widely used as matrices to grow new tissues and organs, and as delivery vehicles of growth factors and cells. For reconstruction of tissues or organs, hydrogels are generally embedded in a mechanically dynamic environment, subject to forces from bones, muscles and blood vessels. In order to protect the encapsulated components (e.g. cells and growth factors), it is necessary to maintain the structural integrity of hydrogels. In addition, controlling the mechanical properties of the hydrogels is an important way in regulating the adhesion and gene expression of the cells.Previous studies have demonstrated that various mechanical properties of hydrogels including rigidity, fracture toughness, swelling ratios, and large strain hysteresis can be independent controlled. Here, we focus on another mechanical property critical to hydrogels’ application in tissue engineering, i.e. stress relaxation.We use alginate gel as an example hydrogel. Alginate gel can form via either ionic or covalent crosslinkings of polymer chains. We show that the crosslink types strongly affect the stress relaxation property of hydrogels. The stress relaxation in covalently crosslinked hydrogel is mainly due to the migration of water molecules. The reversible crosslinking/decrosslinking of polymer chains account for the stress relaxation of ionically crosslinked hydrogel. We form a hydrogel in a hybrid way with both ionic and covalent crosslinkings. By varying the ratio of ionical and covalent crosslinks in a hydrogel, we can independently tune its stress-relaxation behavior while maintaining its rigidity and swelling ratio unchanged. In addition, the hybrid hydrogel has a fracture toughness much higher than the corresponding hydrogel with pure covalently crosslinks.
DD5: Scaffold Fabrication Methods
Tuesday PM, December 02, 2008
Hampton A/B (Sheraton)
2:30 PM - DD5.1
Development of 3D Elastomeric Scaffolds with Aligned and Interconnected Pore Structures by use of Modular Cell-Laden Micropatterned Hydrogels.
Jason Nichol 1 , Joost Bruggeman 1 , Bong Geun Chung 1 , Berend-Jan De Bruin 1 , Ali Khademhosseini 1 , Robert Langer 1 Show Abstract
1 Health Sciences and Technology, Harvard-MIT, Cambridge, Massachusetts, United States
2:45 PM - DD5.2
Electrospun Fibroin-based Nanocomposites as New Vascular Graft Materials.
Walter Bonani 1 2 , Antonella Motta 1 , Wei Tan 2 3 , Claudio Migliaresi 1 Show Abstract
1 Department of Materials Engineering and Industrial Technologies, University of Trento, Trento Italy, 2 Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, United States, 3 Department of Pediatrics – Cardiology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado, United States
Vascular grafting materials currently used in the medical field are often made from strong, stiff, bioinert synthetic materials such as polytetrafluoroethylene (PTFE) and Dacron. The high failure rate of these materials in the replacement of small vessels is related to their mechanical and biological properties which lead to adverse hemodynamic, inflammatory or coagulatory conditions. In natural tissues, cells are surrounded by nanofibrous extracellular matrix (ECM) whose function is to support and organize cells in 3-D space and to provide them with suitable signals. Silk fibroin is a highly promising natural biopolymer with demonstrated properties that promote cell growth, cell proliferation and anti-thrombogenicity. Electrospinning is an attractive process for fabricating scaffolds for tissue engineering or other biomedical applications due to its simplicity and its capability of generating nanofiber structure that mimics the natural ECM. A variety of polymers have been electrospun for biomedical applications proposed in a wide range of areas including skin, cartilage, bone and vascular tissues. In the present study, we demonstrated an electrospinning process to fabricate robust nanofibrous fibroin-based composites. To achieve electrospun vascular grafts, tubular scaffolds were produced by means of a rotational mandrel. The influence of rotational velocity of the mandrel on the anisotropic mechanical properties of tubular scaffolds has been determined. Furthermore, in order to improve mechanical properties and biological functions, synthetic biodegradable materials such as poly-(D,L)-lactic acid, poly-ε-caprolactone and poly(L-lactide-co-ε-caprolactone) copolymer have been added into the electrospinning process with a newly established double-electrospinning apparatus. We have demonstrated a variety of nanocomposites with interpenetrating nanofibrous networks composed of precisely tailored proportions among different polymers. Additionally, the nanocomposites are formed into multi-layered structure with heterogeneous composition and actively functional surface. The developed techniques allows one to achieve nanocomposite mats with controllable degradation kinetics as well as to combine the advantage of bioactive properties of natural-derived biopolymers with the stability and mechanical performances of a large number of synthetic polymers. Structural, physiochemical, mechanical and biological characterizations have been carried out on the resultant nanocomposites. The morphology of non-woven nets is investigated with scanning electron microscopy. Electrospun mats are also characterized by means of thermal analysis and infrared spectroscopy. The mechanical properties are evaluated with tensile tests. Cell adhesion and gene expression of vascular endothelial cells seeded on the electrospun nanocomposites are also investigated in comparision to current graft material PTFE. The achieved results and the implications will be discussed in detail.
3:00 PM - **DD5.3
Additive Layered Manufacturing Methods for Complex 3D Scaffold Fabrication.
Ryan Wicker 1 2 Show Abstract
1 Mechanical Engineering, University of Texas El Paso, El Paso, Texas, United States, 2 W.M. Keck Center for 3D Innovation, University of Texas El Paso, El Paso, Texas, United States
Additive layered manufacturing (ALM) technologies also known as rapid prototyping, direct digital manufacturing, solid freeform fabrication, and other names are technologies that allow for fabrication of complex three-dimensional (3D) shapes from computer models by successively manufacturing thin slices of the desired object and stacking them together one layer at a time. Commercial ALM systems, originally introduced in the mid 1980s, have been traditionally used for prototyping in the automotive, medical device, aerospace, space, toy and other industries. Since their introduction, considerable advancements in processing speed, accuracy, resolution and capacity have been achieved and the materials available for use with ALM technologies have expanded a great deal, enabling customized end-use products to be directly manufactured in a wide range of applications. Many new ALM technologies have been released over the past two decades that use different processes for fabricating the individual layers from a wide variety of liquid, solid, and powder-based materials ranging from photoreactive polymers to metals.In parallel, researchers have used and developed new ALM technologies to take advantage of the layer-based manufacturing method and access to individual layers during fabrication to manufacture unique, multi-material 3D devices. One particular focus of our group is in the area of manufacturing 3D spatially complex bioactive scaffolds, and developing the systems and methods required to fabricate these scaffolds. Over the past five years, we have used commercial line-scan stereolithography (SL), an ALM process that selectively crosslinks a photoreactive liquid polymer contained in a vat using a ultraviolet laser, to fabricate a variety of multi-material 3D scaffolds out of poly(ethylene glycol). SL allows for control over scaffold characteristics (through multiple material fabrication) as well as placement of cells and bioactive agents within the scaffold during construction. A number of complex scaffold architectures have been demonstrated and will be described during this presentation. In addition, commercial SL systems are limited to feature resolutions down to ~150 μm but more typically on the order of ~500 μm, which has led us to develop a projection-based microstereolithography (MSL) system capable of fabricating features down to ~10 μm. The capabilities of this system will also be described as well as several other systems that have been developed and used to fabricate unique scaffold architectures.
3:30 PM - DD5.4
A Novel Microfluidic Method to Fabricate Regenerated Bombyx Mori Silk Fibers for Tissue Engineering Applications.
Michelle Kinahan 1 , Emmanouela Filippidi 1 , Sarah Koester 2 , Heather Evans 2 , Thomas Pfohl 2 , David Kaplan 3 , Joyce Wong 1 Show Abstract
1 Biomedical Engineering, Boston University, Boston, Massachusetts, United States, 2 , Max Plank Institute for Dynamics and Self-Organization, Göttingen Germany, 3 Department of Biomedical Engineering Science and Technology Center, Tufts University, Medford, Massachusetts, United States
3:45 PM - DD5.5
Combined Nano- and Micro-fiber Electrospun Scaffolds to Maximize Stem Cell Function.
Sherif Soliman 1 , Giancarlo Forte 2 , Stefania Pagliari 2 , Antonio Rinaldi 1 , Paolo Di nardo 2 , Enrico Traversa 1 Show Abstract
1 Chemical Science and Technology, University of Rome “Tor Vergata”, Rome, Rome, Italy, 2 Internal Medecine, University of Rome “Tor Vergata”, Rome, Rome, Italy
Electrospinning is recognized as an efficient technique for manufacturing polymeric fibers that are of substantial interest for potential application in tissue engineering, due to the tuneability of the fiber material and morphology. The efficacy of this approach is likely to depend on the interaction between cells and the physico-chemical composition of the substrate. In this respect, the fiber diameter is crucial, as it is responsible of scaffold porosity, pore size, and mechanical properties. Nanofiber matrices display a structure similar to the natural extracellular matrix (ECM). However, culture of stem cells on nanofiber matrices has been reported to result in a monolayer of cells on its surface, because the small diameter of the pores obtained between the nanofibers inhibits cellular infiltration. On the contrary, microfibers usually give raise to pores large enough to allow cell migration, while not providing a physical ECM mimicry for cells. To determine how fiber diameter can affect mesenchymal stem cell (MSC) adhesion, spreading and scaffold colonization, we produced electrospun poly(ε-caprolactone) scaffolds with different fiber diameter (between 0.2 and 6 µm) by optimizing both solution and processing parameters. To capitalize on both nano and microscale features in a single structure, a mixed electrospun fiber mesh composed of 0.2 µm and 2 µm fibers has been also fabricated. For this aim, two different polymer solutions were electrospun simultaneously, and the fibers were collected on a custom-made target rotating underneath the two syringes under the actuation of a high-speed step motor. Scanning electron microscopy was used to characterize the morphology and geometry of the fibers and tensile tests were performed to investigate their mechanical properties. The results indicated that both the stiffness and pore size of the nonwoven structures increased with fiber diameter, while the average porosity was not significantly influenced by the fiber diameter. Interestingly, the nanofiber structure alone showed a low stiffness, but, when combined with the microfibers, was able to enhance the overall structure stiffness. The scaffolds were evaluated for their potential to affect MSC viability. After 1, 3 and 5 days in culture, the results showed that cell function can be regulated by varying the fiber diameter. In particular, cells appeared to align on 6 µm fibers, while growing randomly on the other scaffolds considered. Importantly, functional cell adhesion and proliferation on the scaffolds made of nano- and microfibers was observed in comparison with the single nano- or microfiber structures, as evidenced by MTT assay and hemathoxylin/eosin staining. The scaffold design presented in this study coupled the positive effect of microfibers in favouring cellular colonization of the inner layers and that of the nanofibers as a physical mimicry of ECM.
4:30 PM - DD5.6
Investigation of Spatially Controlled Neural Stem Cell Interactions using Laser-Machined Elastomeric Microstencils.
Karen Ellison 1 2 , Douglas Chrisey 3 , Deanna Thompson 1 2 Show Abstract
1 Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States, 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institite, Troy , New York, United States, 3 Materials Science and Engineering, Rensselaer Polytechnic Institite, Troy , New York, United States
Injury and disease to the nervous system is devastating. Neurodegenerative diseases, such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), and Alzheimer’s, as well as spinal cord injury result in loss of function affecting a patient’s long term quality of life. Unfortunately, current treatments are limited and complete functional recovery is not possible. Rare multi-potent populations of neural stem cells (NSC) are present in adult brain tissue and possess the ability to self renew or differentiate into neurons or glia. NSC are a potential patient-specific cell source for cell-mediated therapies to treat such neurodegenerative diseases and injury. However, isolation, expansion ex vivo, and controlled differentiation pose critical challenges toward the development of a clinically relevant therapy.NSC reside in a specialized microenvironment (niche), where proliferation and differentiation are regulated in vivo by environmental factors. Deconstructing this cellular microenvironment may provide insight on the mechanisms that affect NSC fate. By spatially controlling cell placement, the influences of cell-cell interactions on self-renewal and differentiation can be systematically investigated. Spatial control of cell placement can be achieved using microstencils, a soft lithographic technique used for masking specific regions of a substrate. Thin 50 micrometer poly(dimethylsiloxane) membranes were created by spin-coating uncured PDMS onto Petri dishes and transfering the cured membranes onto glass slides for laser machining. CAD patterns with dimensions 20-750 micrometers in diameter were created and imported into the CAM computer software to control automated pattern formation to generate microstencils. The laser-machined stencils were cleaned, sterilized and applied to glass coverslips and unmasked regions were selectively treated with poly-l-ornithine. NSC were dissociated and cultured on the substrate. Cell attachment was localized to the unmasked regions after removal of the stencil, leaving micropatterned NSC. NSC self-renewal and differentiation will be investigated over 14 days in culture by immunofluorescent staining of markers for self-renewal (LeX, Nestin) and differentiation (β-III tubulin, O4). In summary, the fabrication of microstencils using laser machining is an attractive alternative to traditional rapid prototyping methods. The microstencils were repeatedly used to pattern protein and NSC, demonstrating their functionality and durability. NSC self-renewal and differentiation will be investigated as a function of time and geometry. This platform allows one to systematically vary homotypic NSC interactions (NSC-NSC) while holding initial NSC density constant. The ability to control of NSC self-renewal ex vivo is important for patient-specific cell source for cell mediated therapies and the creation of in vitro models of neurodegenerative disease.
4:45 PM - DD5.7
Fibrous Biomimetic Hydrogels for Tissue Engineering.
Jean Altus 1 , Pamela Sundelacruz 1 , Maude Rowland 1 , Jennifer West 1 Show Abstract
1 Department of Bioengineering, Rice University, Houston, TX, Texas, United States
Hydrogels are a commonly used class of materials for tissue engineering applications, wound healing, and drug delivery because of their highly hydrophilic nature and generally high biocompatibility. We investigate the use of photopolymerizable hydrogels with tunable mechanical properties in which extracellular matrix (ECM) mimetic components can readily be incorporated to control cell attachment, proliferation, and differentiation. Examples of the ECM-mimetic components that have been incorporated into these hydrogels include adhesive ligands, proteolytically degradable sequences, and growth factors. Also, the ECM can be thought of as a hydrated network of proteins that is a fibrous hydrogel-like system. Thus, in a continuing effort to develop biomimetic materials for the advancement of tissue engineering and the investigation of events in cell biology we are investigating techniques to incorporate microstructured features into the photopolymerizable hydrogels. Microstructured materials have been shown to enhance cell response by directing cell migration and tissue regeneration by replicating features that emulate the shapes and structures of native tissues. Fibrous hydrogels have been created via electrospinning. Electrospinning is a technique that generates submicron and nanometer diameter fibers by applying a high voltage (5-20 kV) to a polymer solution; the voltage overcomes the surface tension of the solution and emits a fiber jet that is collected on a counter electrode a set distance away. The hydrogel fibers are a mixture of photoactive poly(vinyl alcohol) (pPVA) and polyethylene glycol diacrylate (PEGDA). The electrospun fibers are crosslinked using long wavelength ultra violet (UV) light and 2,2 dimethoxy-2-phenolacetophenone in 1-vinyl-2-pryyolidone as the photoinitiator. The fibrous hyrogels have fiber diameters between 500 nm and 1 µm when dry. The fibrous gels are stable under cell culture conditions for at least several weeks with a fibrous mesh that is visible via optical microscopy. An adhesive ligand (RGDS) has been incorporated into the fibrous electrospun membranes by a using monoacrylate-PEG-RGDS. The incorporation of PEG-RGDS was conformed by fluorescently labeling the RGDS and imaging via confocal microscopy. Initial cell studies, using 3T3 fibroblasts, indicate strong cell attachment and cell spreading on the fibrous hydrogels with RGDS as compared to electrospun fibrous hydrogels with no RGDS and non-fibrous PEGDA hydrogels with RGDS.
5:00 PM - **DD5.8
Microengineered Hydrogels for Tissue Engineering.
Ali Khademhosseini 1 Show Abstract
1 Department of Medicine, Brigham and Women's Hospital, Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, United States
5:30 PM - DD5.9
Preparation of Core-Shell Conducting Nanofibers and Their Potential Application in Neural Tissue Engineering.
Jingwei Xie 1 , Matthew MacEwan 1 , Stephanie Willerth 1 , Daniel Moran 1 , Shelly Sakiyama-Elbert 1 , Younan Xia 1 Show Abstract
1 Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, United States
Integration of biochemical signal, topographic feature, and electrical stimulation is a desired engineering strategy for neural tissue engineering. However, few studies have been fulfilled this challenge so far. In the present work, core-shell conducting nanofibers were prepared through a combination of electrospinning and in situ polymerization to solve this issue. Specifically, poly(ε-caprolactone) (PCL) and poly(L-lactide) (PLA) nanofibers serve as template cores upon which in situ polymerization deposition was used to form smooth polypyrrole (PPy) sheaths of even thickness. Core-shell structures of fibers were proved by scanning electron microcopy (SEM) and transmission electron microscopy (TEM) studies. Fluorescence microscopy images show that dorsal root ganglia (DRG) explants were adhered to the core-shell conducting nanofibers and neurite outgrowth was observed in the presence of nerve growth factors. Furthermore, aligned core-shell conducting nanofibers not only enhance but also guide neurites extension, demonstrating the great potential in applications for neural tissue engineering. The conducting microtubes generated in this study could also be used in applications such as transistor and switch, supercapacitor, and electrochromic device. In addition, the nanotubes can be converted to carbon nanotubes by heat treatment.
5:45 PM - DD5.10
Bio-inspired Tissue Engineering Scaffolds for Heart Valves.
Olivier Arnoult 1 , Yakov Elgudin 2 , Meghan Smith 3 , Nicholas Greco 2 , Mary Laughlin 2 , Steven Emancipator 4 , Brian Cmolik 2 , Gary Wnek 1 Show Abstract
1 Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, Ohio, United States, 2 School of Medecine, Case Western Reserve University, Cleveland, Ohio, United States, 3 Chemical Engineering, Case Western Reserve University, Cleveland, Ohio, United States, 4 Pathology, Case Western Reserve University, Cleveland, Ohio, United States
While the lifetime of a heart valve implant is 15 years in average for a patient of 60 years or older, it is greatly reduced when implanted in children or young adults, due to calcification which leads to mechanical failure of the valve. Although decellularized allografts or xenografts for heart valve replacement lead to quasi similar biomechanical properties as native valves, they cannot be re-colonized by host cells. Work has previously been carried out by several research groups to create tissue engineering scaffolds made of various biocompatible and biodegradable polymers which have been successfully used for cell growth. However, these scaffolds fail to reproduce the true structure of the leaflets, being composed of three layers: the fibrosa, spongiosa and ventricularis. Each layer has distinctive function enabling the valve to operate mechanically. Electrospinning is commonly used to develop tissue engineering scaffolds, and we have employed a variation of this process to develop a multi-layer bio-inspired scaffold that more closely resembles the native heart valve tissue structure.Electrospinning of a series of polymers including poly(ε-caprolactone), poly(vinyl alcohol), poly(ethylene oxide), collagen, as well as blends of these polymers, all in organic solvents was used to create a multilayered microfibrous scaffold. A media solution containing the fibroblasts cells was electrosprayed simultaneously fibers were electrospun to optimize scaffold colonization. Each layer was developed separately to match the properties of the corresponding layer in the native tissue. A high speed rotation drum enabled us to obtain quasi-aligned fibers that characterized the fibrosa of the natural valves. The spongiosa, a sponge-like buffer zone, was mimicked with highly swellable polymer scaffold. The ventricularis is a scaffold of co-electrospun collagen and elastin. A multilayer scaffold was made combining these three layers through consecutive electrospinning of each polymer onto the same rotating drum. Scaffolds were characterized with optical and scanning electron microscopy for morphology studies and fluorescent cell viability studies. Dynamic mechanical analysis was performed to quantify the mechanical properties (tensile, elastic and flexural) of each layer in dry and wet state, and compared to stabilized porcine valve tissue. Several tissue engineering scaffolds mimicking the properties of each layer of a heart valve were successfully made. The multilayer scaffold does not exhibit any delamination due to the continuous electrospinning of the different layers. The cells were viable in the single layer as well we in the multi layer scaffolds. Scaffolds without cells were made and it appears that the presence of the cells does not influence the mechanical properties of the scaffolds at an early stage. Mechanical tests are ongoing to compare the porcine heart valve tissue preserved in gluteraldehyde or alcohol/epoxy solution.