Symposium Organizers
Sujata Bhatia Dupont Corporation
Stephanie Bryant University of Colorado
Jason A. Burdick University of Pennsylvania
Jeffrey M. Karp Harvard-MIT Division of Health Sciences and Technology
Katie Walline CeraPedics, Inc.
RR1: Bio-Inspired Scaffolds
Session Chairs
Jason Burdick
Bill Murphy
Monday PM, November 30, 2009
Back Bay C (Sheraton)
9:30 AM - **RR1.1
Biomaterial Regulation of Molecular Signaling in Engineered Tissues.
John Fisher 1
1 Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States
Show AbstractSoluble signaling molecules determine cell phenotype and thus tissue function. For example, growth factors are well known regulators of cell proliferation, migration, and differentiation, typically leading to anabolic tissue growth. While the role of signaling molecules has been well examined in normal tissue biology as well as abnormal states, such as the uncontrolled cell proliferation associated with tumor development, there has been a relative lack of investigation of endogenously expressed signaling molecules in engineered tissues. Rather, the development of engineered tissues has largely focused upon the viability of cell populations within artificial matrices, or the augmentation of cell function by the delivery of exogenous signaling molecules. This presentation plans to build upon these well explored strategies by considering the overall hypothesis that cell encapsulation within synthetic scaffolds alters the expression and regulation of endogenous signaling molecules, therefore affecting cell phenotype and tissue function. The engineering of specific tissues, particularly bone and articular cartilage, is discussed, with an emphasis on the role of biomaterials in regulating molecular signaling within these engineered tissues. For example, we consider the endogenous expression of insulin like growth factor-1 by alginate embedded chondrocytes, and the role of cell density, matrix density, and exogenous signal delivery upon growth factor signaling. In addition, we discuss coculture systems where endogenously expressed factors are utilized to induce specific cellular responses, including mesenchymal stem cell differentiation. Finally, we consider the development of cyclic acetal based biomaterials which have properties specifically developed for facilitated molecular signaling for the regeneration of craniofacial bone. The presentation aims to integrate biomaterials development into cell signaling studies so as to initiate new strategies for the engineering of tissues.
10:00 AM - RR1.2
Culture and Preservation of Pancreatic Islets Through Biomaterial Interactions and The Reestablishment of The Three-Dimensional Islet Microenvironment In Vitro.
Jamal Daoud 1 , Jean-Phillipe Coutu 1 , Maria Petropavlovskaia 2 , Lawrence Rosenberg 2 , Maryam Tabrizian 1
1 Biomedical Department, McGill University, Montreal, Quebec, Canada, 2 Department of Surgery, McGill University, Montreal, Quebec, Canada
Show AbstractPancreatic islet transplantation requires successful isolation and in vitro survival; however, studies have shown that islet isolation exposes the islet to a variety of cellular stresses, destroys the basement membrane (BM) and disrupts the cell-matrix relationship, leading to apoptosis. The rationale behind this research study is to identify factors responsible for islet preservation and survival in vitro. This will lend to emulate the basement membrane of native islet tissue through proper cell-substrate interactions. Pancreatic islet preservation and regeneration is dependent on the application of tissue engineering principles which allow for the post-isolation reestablishment of the islet-matrix relationship as well as the maintenance of islet functionality and encouragement of survival and differentiation in vitro. This study has shed light into important factors that promote human islet adhesion, morphology, survival and functionality in vitro. Collagen I/IV and fibronectin functionalized surfaces, compared to other ECM components such as laminin, were shown to encourage adhesion levels of more than 50% after 12 hours. However, collagen I surfaces showed the greatest strength of adhesion while fibronectin-modified surfaces maintained maintain islet morphology and structural integrity. Furthermore, glucose-induced insulin release was optimal for fibronectin cultured islets, in contrast to its ECM counterparts. Islet gene expression of insulin, glucagon, somatostatin, pancreatic polypeptide and PDX1 were also elevated relative to islets cultured on BSA-control surfaces. These results were then transferred to three-dimensional studies on gels and geometrically-specific PLGA scaffolds designed through solid freeform fabrication. The tailored PLGA scaffolds were seeded with collagen gel embedded islets along with the appropriate ECM components identified via the surface interaction studies. Analysis was conducted through electron microscopy, immunofluorescence, gene expression and insulin functionality studies. This yielded favorable results promoting islet culture and function in an in vitro three-dimensional environment. Furthermore, we demonstrated the fabrication of microstructure-controlled PLGA scaffolds using rapid prototyping techniques and their subsequent characterization via the utilization and interpretation of complex permittivity measurements (CP). It is important to be able to detect cell behaviour, which is marked by morphological changes, such as proliferation and differentiation, using non-invasive methodologies. Therefore, the successful reestablishment of a favourable microenvironment for isolated pancreatic islets is the first step towards achieving three-dimensional functionalized scaffold materials that promote islet culture and regeneration in a non-invasively monitored tissue bioreactor setting.
10:15 AM - RR1.3
Engineered Extracellular Matrix for Soft Tissue Regeneration.
Amit Jha 1 , Weidong Yang 2 , Catherine Kim-Safran 2 , Randall Duncan 2 , Mary Farach-Carson 2 , Xinqiao Jia 1
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 Biological Sciences, University of Delaware, Newark, Delaware, United States
Show AbstractWe have engineered a new class of hyaluronic acid (HA)-based, artificial extracellular matrices that contain HA hydrogel particles (HGPs) embedded in and covalently cross-lined to a secondary network. Two types of HA HGPs were synthesized using an inverse emulsion polymerization technique, and the resulting HGPs exhibited varying overall sizes and surface functionalities. Hierarchically structured, doubly cross-linked networks (DXNs) were engineered using HA HGPs as the building blocks and a water-soluble HA derivative as the secondary cross-linker. These hydrogels are soft and elastic; their viscoelastic properties can be readily modulated by varying the particle size, surface functional group, inter-particle and intra-particle crosslinking. In vitro cell proliferation assays showed that HA HGPs did not adversely affect the proliferation of the cultured fibroblasts. To further enhance the biological activities of HA HGPs, perlecan domain I (PlnDI) was covalently conjugated to the particles via its core protein through a flexible poly(ethylene glycol) (PEG) linker. The immobilized PlnDI maintains its ability to bind bone morphogenetic protein (BMP-2) specifically. PlnDI conjugated HA HGPs allow for sustained release of BMP-2 and stimulate the chondrogenic differentiation of mesenchymal stem cells (MSC) in vitro. The repair potential of these bioactive hydrogel matrices is currently being tested in vivo using an intra-articular injection approach in mouse osteoarthritic knees.
10:30 AM - RR1.4
A Synthetic Strategy for Mimicking the Extracellular Matrix Provides a New Tool for Studying Cancer Biology.
Michael Schwartz 1 2 , Robert Rogers 1 2 , Benjamin Fairbanks 1 2 , Lydia Everhart 4 , Rajagopal Rangarajan 3 , Muhammad Zaman 3 , Kristi Anseth 1 2
1 , Howard Hughes Medical Institute, Boulder, Colorado, United States, 2 , University of Colorado at Boulder, Boulder, Colorado, United States, 4 , University of Dayton, Dayton, Ohio, United States, 3 , University of Texas at Austin, Austin, Texas, United States
Show AbstractUnderstanding the role of the tumor microenvironment in cancer progression and metastasis is complicated by highly complex interactions between cells, biomolecules, and the physical and biochemical characteristics of the extracellular matrix (ECM). We have developed poly(ethylene glycol) (PEG) hydrogels and PEG/fibrin composites to control the ECM environment as a means of studying cancer biology, but also to provide a general strategy for studying a broad range of biological phenomena. Hydrogels are synthesized using a thiol-ene photopolymerization mechanism to copolymerize ene-functionalized PEG precursors with thiol-containing peptides. We specifically incorporate matrix metalloproteinase (MMP)-degradable crosslinkers and adhesion molecules to form hydrogels that are permissive towards cell spreading, migration, and remodeling of the extracellular environment. Through the use of various techniques, such as cell encapsulation and invasion assays, we have been able to study cancer biology in a quantitative manner, leading to new insights about cancer progression and metastasis. The strategy discussed here has broad potential as both a tool for studying biology and for medical applications.
10:45 AM - RR1.5
A Mimetic Peptide Approach to the Spatio-Temporal Modification of Natural Collagen Scaffold for Microvasculature Engineering.
Tania Chan 1 2 , S. Michael Yu 1 2
1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 2 Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractThe control of angiogenesis is vital to the success of tissue engineering. Long term survival and success of artificial tissue constructs depend greatly on adequate vascularization. Endothelial cell differentiation and morphology are dependent on 3D environmental cues, and currently there is no method available to reproduce a spatial and temporal signaling gradient in tissue scaffolds. We have developed a novel bifunctional peptide with a vascular endothelial growth factor (VEGF) mimetic domain and a collagen mimetic domain aim to control angiogenesis for tissue engineering application. We present a new peptide construct that contains a VEGF mimetic domain of known angiogenic activity [1], and a collagen mimetic domain that can physically attach to natural collagen through triple helical interaction as previously reported by our group [2]. This bifunctional peptide exhibits binding affinity to type I collagen gels and has the ability to activate endothelial cell tubulogenesis. These results show that the bifunctional peptide can be used to present spatial and temporal morphogenic cues in natural collagen scaffold which may provide a major breakthrough in angiogensis engineering and in the study of endothelial cell signaling. [1] D’Andrea, L. et al. PNAS. 2005, 102, 14215-14220. [2] Wang, A. et al. Biomacromolecules. 2008, 9, 1755-1763
11:00 AM - RR1: Bio
BREAK
11:30 AM - **RR1.6
Materials To Program Cells In Situ.
David Mooney 1 2
1 , Harvard, Cambridge, Massachusetts, United States, 2 , Wyss Institute for Biologically Inspired Engineering, Cambridge, Massachusetts, United States
Show AbstractThere are hundreds of clinical trials of cell therapy currently underway, with the goal of curing a variety of diseases, but simple cell infusions lead to large-scale cell death and little control over cell fate. We propose a new approach, in which material systems are first used either as cell carriers or attractors of host cell populations, and in either case the material then programs the cells in vivo and ultimately disperses the cells to surrounding host tissues or organs to participate in tissue regeneration or destruction.
12:00 PM - **RR1.7
Cells in Gels: Understanding Cellular Morphogenesis and Differentiation in 3-D Culture.
Dror Seliktar 1 , Iris Mironi-Harpaz 1 , Yonatan Shachaf 1 , Keren Shapira 1 , Maya Gonen-Wadmany 1 , Andrei Yosef 1 , Offra Sarig-Nadir 1
1 Biomedical Engineering, Technion - Israel Institute of Technology, Haifa Israel
Show AbstractThe regulation of cellular morphogenesis and differentiation via the physical properties of the extracellular matrix (ECM) is poorly understood and our group has been working towards elucidating the dominant physical factors of the ECM that influence cell spreading, migration and differentiation in 3-D culture. We apply a biosynthetic PEG-protein hydrogel as an ECM-analog for cell culture, with highly defined and precisely controllable density, microarchitecture, proteolytic susceptibility, compliance and biofunctionality. The matrix is used to encapsulate mesenchymal cells while pseudo-independently altering biochemical and physical properties of the microenvironment using simple compositional modifications to the bio-synthetic constituents. We have shown that the proteolytic resistance and compliance of the matrix have a profound influence on the regulation of cell morphogenesis and phenotype determination. Beyond the control over the intrinsic physical attributes of the hydrogel, our laboratory has recently developed an optical 3-D micro-patterning approach to non-invasively create any prescribed geometrical feature having submicron spatial resolution in situ, anywhere within the PEG-protein hydrogel biomaterial. The micropatterns are made using a simple but highly effective application of computer-guided laser micro-ablation that creates localized imperfections in the hydrogel architecture. These imperfections are used to guide anisotropic cellular development within the amorphous material, including preferentially guiding neural cellular development in the hydrogels based on contact guidance and differential mechanical resistance of the scaffolding. Precisely controlled bulk material properties and custom 3-D landscaping with micropatterning are collectively used to elucidate the dominant and influential physical factors affecting morphogenesis patterns, phenotypic states, and differentiation of various cell types.
12:30 PM - RR1.8
Bioorthogonal Click Chemistries for Synthesizing and Patterning the 3D Cell Niche.
Cole DeForest 1 , Evan Sims 1 , Kristi Anseth 1 2
1 Chemical & Biological Engineering, University of Colorado, Boulder, Colorado, United States, 2 , Howard Hughes Medical Institute, Boulder, Colorado, United States
Show AbstractSince its conception by Sharpless et al. in 2001, “click chemistry” has promised extremely selective and fully orthogonal reactions that proceed with high efficiency and under a variety of mild conditions. Functional molecules can be easily synthesized via these independently modular reactions and ultimately incorporated into materials with highly defined properties. Though these versatile click reactions have been broadly exploited in many fields including drug discovery, material science, and bioconjugation, the intrinsic toxicity of their synthetic schemes has limited their application in biologically-based systems. This work introduces a robust synthetic strategy where multifunctional macromolecular precursors react via a copper-free click chemistry, enabling the direct encapsulation of cells within click hydrogels for the first time. Specifically, a four-arm poly(ethylene glycol) tetraazide was reacted with a bis(difluorinated cyclooctyne) matrix metalloproteinase cleavable peptide (GPQGILGQ) to yield a load-bearing network. The step-growth nature of this polymerization process provides ideal network structures with minimal defects and local heterogeneities, ensuring that each cell experiences initially identical material properties. These local properties can be then altered at user-dictated locations in space and time via an orthogonal thiol-ene photocoupling click chemistry that facilitates patterning of biological functionalities within the gel, ultimately providing tailorability of the physical and chemical properties of the cell culture niche in situ. Photofunctionalization is achieved using conventional photolithographic, single-photon, and multiphoton techniques, each affording a higher degree of patterning specificity than the last. These local manipulations of the gel microenvironment provide an avenue to introduce chemical cues that direct cell function and/or assay cell behavior throughout specific regions within the material. For example, by selectively patterning in the RGDS sequence, altered morphology and increased proliferation of NIH 3T3s was confined to user-dictated 3D locations within our gels. These chemical cues were sequentially introduced to create multifunctional gels with regions of distinct peptide functionalities. These functionalization reactions are completed in minutes, allowing for the real-time manipulation of the cell microenvironment.
12:45 PM - RR1.9
Dynamic Studies on Injectable Beta-hairpin Peptide Hydrogels: Mechanisms of Shear-thinning and Immediate Self-healing.
Congqi Yan 1 3 , Radhika Nagarkar 2 3 , Joel Schneider 2 3 , Darrin Pochan 1 3
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 3 , Delaware Biotechnology Institute, Newark, Delaware, United States, 2 Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States
Show AbstractBy exposure to physiological conditions, properly designed peptides can be triggered to fold into β-hairpins, and then subsequently self-assemble into a rigid fibrillar gel network. The resulting rigid, physical hydrogels are highly responsive to mechanical shear because they shear-thin and flow when exposed to a proper shear stress. But once the stress is removed, the gels immediately self-heal into solids and restore their original rigidity with time relative to the shear rate and duration applied. This unique shear-reversibility indicates the possibility of in vivo delivery by syringe injection of a solid gel construct with a desired therapeutic payload. In this work, the flow of the hydrogels through a channel were tracked and studied with confocal microscopy. Rheometric experiments were performed to characterize gel self-restoration under various shear treatment conditions. Also, rheo-SANS (small angle neutron scattering combined with oscillatory rheology in a Couette geometry) was adopted to monitor gel network morphology change under shear flow. The results explain network structure evolution during shear-thinning and during the restoration process. The mechanisms of gel shear-thinning and rehealing will be discussed.
RR2: Stem Cell/Material Interactions
Session Chairs
Stephanie Bryant
Rob Mauck
Monday PM, November 30, 2009
Back Bay C (Sheraton)
2:30 PM - **RR2.1
Bioinspired Materials that Regulate Growth Factor Signaling and Stem Cell Behavior.
William Murphy 1 , Gregory Hudalla 1 , Jae Sam Lee 1 , Jae Sung Lee 1 , Justin Koepsel 1
1 , University of Wisconsin, Madison, Wisconsin, United States
Show AbstractSchemes to mimic tissue development and engineer functional tissues are likely to benefit from control over the cell’s local signaling environment. This concept is particularly important in stem cell-based applications, in which local signaling can dictate self renewal and differentiation. Recent studies have demonstrated that the characteristics of the local microenvironment – including substrate mechanics and cell adhesion – can significantly impact stem cell fate. Collectively, these studies suggest that engineered biomaterials may be useful as platforms to actively regulate the microenvironment and, in turn, stem cell behavior. We are interested in assembling biomaterials that actively regulate the presence and activity of soluble proteins in the local stem cell microenvironment. Specifically, we have used non-covalent interactions to build biomaterials that interact non-covalently with soluble growth factors. The affinity and specificity of these interactions can be tailored to regulate local growth factor signaling in a manner that mimics the natural extracellular matrix. This presentation will detail two specific approaches we have used to regulate local growth factor signaling: i) the use of engineered protein-peptide interactions to regulate FGF-2-mediated stem cell proliferation; and ii) the use of engineered protein-mineral interactions to promote BMP-2-mediated stem cell differentiation into bone-forming cells. These approaches can be generalized to multiple soluble growth factors, and may therefore be broadly applicable in stem cell biology and bioengineering.
3:00 PM - RR2.2
Extracellular Matrix Mechanics Affect Stem Cell Lineage in 3D by Controlling Integrin Binding.
Nathaniel Huebsch 1 2 , Praveen Arany 1 , Angelo Mao 1 , Jose Rivera-Feliciano 1 , David Mooney 1 3
1 SEAS, Harvard University, Cambridge, Massachusetts, United States, 2 , Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, United States, 3 , Wyss Institute for Biologically Inspired Engineering, Cambridge, Massachusetts, United States
Show AbstractStem cell therapies hold great clinical promise, but control of transplanted cell fate remains a significant challenge. (Mooney and Vandenburgh 2008). Material-based systems offer a promising means to program stem cells (Silva 2008), and in 2D cell culture, cell fate can be manipulated by changing either the biochemical composition or rigidity of the adhesion substrate (Kong 2005, Engler 2006, Klees 2005). However, the extent to which ECM mechanics affect stem cell fate in physiologically-relevant 3D micro-environments, and the biophysical mechanisms underlying mechanosensing are unclear. Here, we demonstrate that the rigidity of cell-encapsulating 3D biomaterials (RGD-modified alginate hydrogels) can change lineage specification in primary and clonally-derived mesenchymal stem cells (MSC). However, in contrast to 2D studies that suggest matrix rigidity directs stem cell fate by altering morphology (Engler 2006, McBeath 2004), ECM rigidity had very little effect on cell shape in these 3D cultures. Instead, matrix stiffness regulated integrin ligation by the adhesion ligand RGD in a biphasic manner, and matrices with optimal stiffness for integrin-RGD binding elicited the highest degree of osteogenic lineage specification in stem cells. Integrin-RGD binding correlated with cells' ability to reorganize adhesion ligands presented from the matrix, on the nanometer scale, via acto-myosin mediated traction forces. In 2D cell culture, MSC, like other cell types, used αV-integrins to ligate RGD when presented via surface adsorbed vitronectin, or from 2D RGD-modified hydrogel substrates. Strikingly, however, α5-integrins acted as RGD receptors in the same MSC when this adhesion ligand was presented, without PHSRN synergy sites, from a 3D, cell-encapsulating hydrogel α5-integrin-RGD bonds acted as 3D-mechanosensors, and inhibiting the formation of these bonds with function blocking antibodies diminished osteogenic lineage specification in MSC. Altogether, these data demonstrate that cells interpret changes in the physical properties and dimensionality of substrates as though they were chemical changes in adhesion ligand presentation. As cells themselves, by interplay between acto-myosin mediated traction forces and extracellular matrix mechanics, played a significant role in determining the structure of the cell-biomaterial interface, both in terms of the type and total number of bound integrins, this work suggests a paradigm to engineer living materials: namely, that cells can be harnessed as tools to process simple, scalable materials into complex structures that feedback to manipulate stem cell fate.References: Engler et al. Cell 2006. Kong et al. Proc Natl Acad Sci USA 2005. Mooney and Vandenburgh Cell Stem Cell 2008. Silva et al. Proc Natl Acad Sci USA 2008. Klees et al. Mol Biol Cell 2005. McBeath et al. Dev Cell 2004.
3:15 PM - RR2.3
Regulating Stem Cell Fate by Using Combinatorial Micro-/nanoarrays of Microenvironmental Cues.
KiBum Lee 1 , Aniruddh Solanki 1 , Shreyas Shah 1 , Sung Young Park 2 , Seunghun Hong 2
1 Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States, 2 Physics and Astronomy, Seoul National University, Seoul Korea (the Republic of)
Show AbstractThis talk will focus on the interface of micro-/nanoscale science and stem cell biology. Stem cells hold great potential for treating a number of devastating injuries and damage caused by degenerative diseases (e.g. Alzheimer’s/Parkinson’s disease, and spinal cord injury). However, harnessing the therapeutic potential of stem cells requires an extensive knowledge of extrinsic microenvironmental cues that dynamically interact with and control stem cell fate. For this purpose, developing nanotechnology-based combinatorial approaches for regulating stem cell fate and studying the functions of multiple microenvironmental cues that regulate stem cell behaviors would be critical. To address the aforementioned challenges, two important research projects will be presented: i) development of bio-surface engineering methods to generate combinatorial arrays of microenvironmental signal molecules; and ii) application of the combinatorial platforms to understand the temporal/spatial effects of microenvironmental cues on growth, migration, and differentiation of stem cells (e.g. neural stem cells and embryonic stem cells). It has been reported that not only soluble signal molecules but also insoluble/physical signals have significant effect on stem cell behaviors. To investigate the complex behaviors of stem cells (e.g. adhesion, growth, and differentiation), we first patterned extracellular matrix (ECM) and signal molecules in combinatorial ways (e.g. ECM compositions, pattern geometry, pattern density, and gradient patterns) by using soft micro-/nanolithographic methods. We then investigated the responses of stem cells to multiple cues at the single cell level. Moreover, for more successful transplantation therapies, those patterning approaches was extended to biodegradable ECM-coated nanofibers and biodegradable substrates. Our combinatorial signal arrays allowed us to selectively control stem cell differentiation into specific neuronal lineages in an efficient and reproducible way. In this talk, I will briefly summarize the results of our efforts and discuss future directions.
3:30 PM - RR2.4
Engineered Microenvironments to Interrogate Extrinsic Determinants of Stem Cell Function.
Vanessa Lundin 1 , Anna Herland 1 , Edwin W.H. Jager 2 , Magnus Berggren 2 , Urban Lendahl 3 , Ana Teixeira 1
1 Department of Neuroscience, Karolinska Institute, Stockholm Sweden, 2 Department of Science and Technology, Linköping University, Norrköping Sweden, 3 Department of Cell and Molecular Biology, Karolinska Institute, Stockholm Sweden
Show AbstractStem cell function during development and in the adult is governed by the integration of cell-intrinsic factors with extrinsic cues from the stem cell niche. These include chemical signals arising within the extracellular matrix, diffusion of soluble factors, or through direct cell-cell contact by membrane-bound receptors. Additionally, microenvironmental mechanical properties are increasingly regarded as relevant stem cell stimuli. Specifically, accumulating evidence suggests that matching substrate elasticity with in vivo tissue elasticity facilitates stem cell differentiation (1).The Notch signaling pathway is used repeatedly throughout development to control stem cell lineage decisions in numerous tissues. Activation of Notch signaling requires cell-cell contact and specific binding between the Notch receptor and DSL ligand present on the surface of an adjacent cell. Functional endocytosis in the ligand cell is required for Notch activation in the Notch receptor expressing cell. It has been proposed that endocytosis generates a tension between the ligand and the receptor, required for Notch signaling activation to take place. This issue is being addressed by making use of neural stem cells, that show endogenous Notch activity, and stable cell lines expressing full-length Notch1 and Jagged1, in which endocytosis has been inhibited in the ligand expressing cell. Furthermore, taking advantage of the volume changes of the conducting polymer polypyrrole upon electrical activation (2), the mechanical force generated by endocytosis can be mimicked. Preliminary data showed poor biocompatibility of neural stem cells on polypyrrole, which was unexpected since polypyrrole is known to have good biocompatibility properties. However, a significant improvement was observed by coating the polymer with a thin layer of gelatin. In addition, electrical activation of polypyrrole substrates showed no negative effects on neural stem cell viability. A previously described fluorescent protein-based reporter construct allows for real-time detection of Notch signaling activation with single-cell resolution (3). We have validated the use of this reporter assay as a readout for Notch activity in the neural stem cell culture system. We report on the development of a device to electroactively control the tension between Notch receptor and DSL ligand by using polypyrrole microactuators. These studies aim at gaining a fundamental understanding of the effects of the mechanical properties of the microenvironment on Notch signaling activation and its impact on neural stem cell state and fate. (1) Teixeira, A.I., Ilkhanizadeh, S. et al. 2009, Biomaterials(2) Jager, E.W.H. et al. 2000, Science(3) Hansson, E. et al. 2006, Developmental Neuroscience
3:45 PM - RR2.5
Influence of Hydrogel Mechanical Properties on Differentiation of Encapsulated Human Embryonic Stem Cells.
Max Salick 2 , Richard Boyer 3 , Chad Koonce 4 , Kristyn Masters 3 , Tim Kamp 4 , Sean Palecek 5 , Wendy Crone 1 2 3
2 Engineering Mechanics, University of Wisconsin - Madison, Madison, Wisconsin, United States, 3 Biomedical Engineering, University of Wisconsin - Madison, Madison, Wisconsin, United States, 4 Medicine, University of Wisconsin - Madison, Madison, Wisconsin, United States, 5 Chemical and Biological Engineering, University of Wisconsin - Madison, Madison, Wisconsin, United States, 1 Engineering Physics, University of Wisconsin - Madison, Madison, Wisconsin, United States
Show AbstractThe inability for the human heart to replace damaged cells has resulted in high demand of a method to regenerate this lost tissue. It is believed that new, healthy cardiomyocytes may be developed by encapsulating human embryonic stem cells (hESC) within biocompatible hydrogels. Controlling the differentiation of these embedded hESC’s is critical in producing the desired type of cells for tissue engineering applications. Previous studies on two-dimensional cultures have shown that the mechanical properties of hydrogels greatly impact the differentiation of stem cells. To further simulate the natural environment of stem cells, as well as to develop a more plausible means of stem cell delivery, three-dimensional cultures are used in this research to better understand how topology and mechanical properties of a surrounding matrix impact stem cell differentiation. For this experiment, H9 hESC’s were embedded in hydrogels of varying Young’s moduli. The amount of contractile behavior was quantified, and polymerase chain reaction (PCR) tests were conducted at various time points.
4:30 PM - **RR2.6
Artificial Niches for Neural Stem Cell Differentiation.
Gregory Christopherson 1 , Shawn Lim 2 , Hongjun Song 3 , Hai-Quan Mao 1 2
1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 3 Institute of Cell Engineering and Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
Show AbstractIt has long been recognized that the in vivo extracellular matrices, e.g. basal lamina, exhibit characteristic micro to nanoscale fibrous topography. Ample experimental evidence demonstrates that artificial matrix topographical features, including islands, pillars, grooves and fibers, significantly influence the adhesion, survival, proliferation and differentiation of stem cells. Electrospun fiber matrices have been explored as artificial substrates for stem cell culture to regulate stem cell adhesion, proliferation and differentiation in a cell-type specific manner. We have developed a series of electrospun fibrous matrices with fiber diameter ranged from 230 nm to 2 µm, and functionalized fiber surface with poly(L-ornithine)/laminin to promote the adhesion of rat adult neural stem/progenitor cells (NSCs). We have shown that both fiber diameter and alignment significantly influence rat NSC adhesion, proliferation and differentiation. Cell adhesion was at the highest level on fibers with smaller diameter (283 ± 45 nm); however, NSCs proliferated at significantly slower rates on all fibrous matrices than that cultured on the 2-D control in the presence of fibroblast growth factor-2 (FGF-2). Upon growth factor withdrawal and supplementation of serum and retinoic acid, fibers with smaller diameter (283 ± 45 nm) favored oligodendrocyte differentiation of rat NSCs in comparison to a similarly modified 2-D substrate. In contrast, fibers with larger diameter (745 ± 153 nm) preferentially differentiated towards neuronal lineage. In addition, aligned fibrous matrix favored neuronal differentiation of rat NSCs. Previously, insulin-like growth factor 1 (IGF-1) and noggin have been shown to increase NSC oligodendrocyte differentiation when supplemented in the medium. When we supplement IGF-1 and noggin in differentiation medium, rat NSCs cultured on nanofiber matrix (228 ± 44 nm) preferentially differentiated towards oligodendroglial lineage with over 95% RIP+ cells after 5 days of culture, compared to 45% among cells cultured on the TCPS substrates. In addition, cells cultured on nanofiber substrates expressed RIP at a much higher intensity than their TCPS counterparts. This work provided initial evidence that a combination of biochemical and topographical cues is more efficient in directing cellular fate and raises important questions regarding fate-specification mechanisms enhanced by substrate topography.
5:00 PM - RR2.7
Covalently Immobilized Growth Factors for Control of Progenitor Cell Differentiation and Vascularization of Engineered Tissues.
Loraine Chiu 1 , Katherine Chiang 1 , William Stanford 1 , Milica Radisic 1
1 , University of Toronto, Toronto, Ontario, Canada
Show AbstractVascularization of engineered tissues in vivo and in vitro remains one of the key problems. Here we describe a novel approach to promote vascularization of engineered tissues using angiogenic growth factors covalently immobilized onto scaffolds for tissue engineering. We covalently immobilized vascular endothelial growth factor-165 (VEGF) and angiopoietin-1 (Ang1) onto three-dimensional porous collagen scaffolds using 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) chemistry. VEGF and Angiopoietin 1 co-immobilized onto porous collagen scaffold promoted H5V endothelial cell proliferation, tube formation and in vivo angiogenesis better than the immobilized single growth factors. Notably, the group with co-immobilized VEGF and Ang1 showed significantly higher cell number (P=0.0079), lactate production rate (P=0.0044) and glucose consumption rate (P=0.0034) at Day 3, compared to its corresponding soluble control for which growth factors were added to culture medium. By Day 7, hematoxylin and eosin, live/dead, CD31, and von Willebrand factor staining all showed improved tube formation by ECs when cultivated on scaffolds with co-immobilized growth factors compared to single growth factors. Immobilized growth factors were also more effective in promoting cell proliferation than soluble growth factors applied to the scaffolds with identical tensile moduli. Remarkably, even the growth factor immobilized scaffolds aged for 28 days in PBS at 37oC retained their ability to promote angiogenesis in vivo (chicken CAM assay), thus illustrating significant stability of the immobilized growth factors. In addition, immobilized and patterned growth factors were utilized to control differentiation of vascular progenitors derived from mouse embryonic stem cells. Mouse ESCs engineered to express eGFP under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ vascular progenitros were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF165 was covalently immobilized onto Collagen IV using EDC chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF-ColIV was stamped as 100um wide lanes on top of the chitosan layer and the Flk1+ progenitros were seeded for site specific differentiation. Lanes stamped with ColIV only served as controls. The results demonstrate that cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53+/-13% CD31 positive and 17+/-2% smooth muscle actin positive); whereas areas without VEGF yielded primarily vascular-like smooth muscle cells (26+/-17% CD31 positive and 38+/-9% smooth muscle actin positive). Thus, immobilzied growth factors can be used for spatial control of progenitor cell differentiation as well as vascularization of scaffolds for tissue engineering.
5:15 PM - RR2.8
The Effects of Hypoxia on Differentiation and the Extracellular Matrix in Human Embryonic Stem Cells.
Renita Horton 1 , Eleftherios Sachlos 2 , Debra Auguste 1
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 2 , McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario, Canada
Show AbstractHuman embryonic stem (hES) cells are pluripotent, having the ability to differentiate into all cell lineages. One typical differentiation method involves the generation of 3D cell clusters called embryoid bodies (EBs), which undergo spontaneous differentiation and are able to recapitulate embryogenesis. The extracellular matrix (ECM) plays an important during early embryonic development regulating stem cell fate decisions, migration, and proliferation. The ECM is composed of proteins that provide both structure and signaling, e.g. collagens, laminin, and fibronectin. Hypoxia (5% oxygen tension) has also been considered a key regulator in stem cell decision making processes. We show that the ECM proteins, in particular collagens, laminin, and fibronectin, are affected by hypoxia. We also show the effects of hypoxia on EB differentiation. We monitored gene expression for markers for vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 (HIF1), extracellular matrix proteins (collagen I, collagen IV, laminin, fibronectin), and mesodermal markers (Brachyury, KDR, NK2.5). Our results demonstrate the effects of hypoxia on the extracellular matrix and cardiogenic differentiation within EBs. EBs cultured under hypoxic conditions decrease collagen expression and increase fibronectin expression. Cardiomyocyte differentiation is enhanced relative to EBs cultured at normoxic conditions. We have investigated temporal as well as long term exposure of hypoxia on ECM production and differentiation. We examined EB topography using scanning electron microscopy to examine the effects of hypoxia on ECM deposition on the EB surface as a function of time. This study provides insight into the role of the ECM in EB differentiation and the effects of hypoxia on cardiogenesis.
5:30 PM - RR2.9
Investigating the Function of Microenvironmental Cues on Stem Cell Fate via Microfluidic Combinatorial Analysis.
KiBum Lee 1 , Aniruddh Solanki 1 , Shreyas Shah 1 , Ken-ichiro Kamei 2 , Shuling Guo 2 , Zeta Tak For Yu 2 , Minori Ohashi 2 , Owen Witte 2 , Hsian-Rong Tseng 2
1 Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States, 2 Molecular & Medical Pharmacology, UCLA, Los Angeles, California, United States
Show AbstractThis talk will focus on developing combinatorial microfluidic methods for stem cell assays and utilizing the tools to study the functions of microenvironmental cues on stem cell fate such as self-renewal and differentiation. Although stem cells hold great potential for regenerative medicine, the control of stem cell fate and complete knowledge of the underlying mechanisms of microenvironmental cues are the most important issues to address before the therapeutic potential of stem cells is fully realized. However, the function of stem cell microenvironments that are comprised of soluble signals, cell-cell interactions, and insoluble/physical signals, are extremely complex to investigate and thereby only a few methods have been successful so far. Development of combinatorial signal assays for high throughput screening of stem cell responses to each stimulus would be beneficial to address the current limitations. Conventional experimental studies on human embryonic stem cell (hESC) responses toward microenvironmental cues are typically conducted on a large cell population, which inevitably produces data measured from inhomogeneous distribution of cellular responses. Unless hESC behaviors and processes are isolated from inhomogeneous signals at the single cell level, it would be extremely difficult to elucidate the intricate hES cellular systems and analyze their complex dynamic signaling transduction. To address the aforementioned problems, we have developed a microfluidic assay platform to identify the optimal conditions for screening hESC behaviors (e.g. self-renewal and differentiation). Our microfluidic approach provides unique control, both spatially and temporally, over insoluble and soluble signal molecules. For high throughput screening in microfluidics, we used Oct4-GFP reporter hESC lines, where EGFP expression levels are driven by the endogenous Oct4 promoter, and the microfluidic assays were based on two different methods: a phenotype assay and a cell signaling assay. Moreover, we have cultured hESCs in chemically defined culture conditions and demonstrated feasibility of culturing dissociated hESCs in chemically defined culture conditions using Rho Kinase (ROCK) inhibitors. The capability of performing single stem cell fate mapping using microfluidics provides valuable information for determining other stem cellular behaviors. Moreover, the microfluidic assay tools for stem cells will allow for better control over microenvironmental cues and intrinsic cellular regulators simultaneously. In this talk, a summary of the results from these efforts and future directions will be discussed.
5:45 PM - RR2.10
Hydrogel Wrinkling Patterns for Controlling Stem Cell Behavior.
Murat Guvendiren 1 , Shu Yang 2 , Jason Burdick 1
1 Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Materials Science and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractStem cells respond to many microenvironmental cues towards their decisions to spread, migrate, and differentiate and these cues can be incorporated into materials for regenerative medicine. In this work, we investigated the effect of hydrogel pattern morphology and feature size on stem cell shape and fate. Importantly, the mechanical properties of hydrogels closely approximate those of soft tissues and are more relevant than rigid substrates. Poly(hydroxyethyl methacrylate) (PHEMA) hydrogels were cross-linked from partially polymerized, viscous prepolymer solutions with ethyleneglycol dimethacrylate (EGDMA). The hydrogel swelling was constrained normal to the surface by covalently attaching them to a much stiffer glass substrate, which generated a biaxial compressive stress. A modulus gradient such that the degree of crosslinking increased with depth was created (verified with FTIR, AFM, confocal microscopy) due to oxygen inhibition during photocrosslinking. Osmotically driven surface wrinkles emerged ranging from a highly ordered hexagonal pattern in transit to peanut shape, lamellar and random worm-like structures. The gradient, and consequently pattern morphology, was controlled by EGDMA concentration and the pattern size was linearly proportional to the initial film thickness. The various wrinkle patterns, ranging in size and morphology, were replicated using standard techniques to PHEMA gels with a uniform modulus (~200 kPa with AFM) to decouple hydrogel mechanics and morphology.Human mesenchymal stem cells (hMSCs, Lonza) were seeded onto the templated PHEMA films with hexagonal and lamellar patterns with 90 and 180 micron feature size. Samples were pre-incubated with fibronectin to encourage cell adhesion. When quantified, short term (1-day) studies indicated that cells remained rounded (low aspect ratio, low cell area) on the hexagonal patterns for the smaller feature size and were elongated on the lamellar patterns with the larger feature size (high aspect ratio, medium cell area). Long term studies (14-days) in osteo-adipo (1:1) mixed media indicate that this control over cell morphology influences fate decisions in the cells. For the hexagonal case, the cells that were in the wells (i.e., rounded) differentiated into the adipogenic lineage (staining for lipids), whereas elongated cells on the lamellar patterns differentiated into the osteogenic lineage (staining for alkaline phosphatase). There was a direct correlation between the ability of the cell to spread and to differentiate into the different lineages. Our results indicate that it is possible to control cellular interactions and differentiation by simply changing the substrate morphology and size and these cues could potentially be included into tissue engineering approaches.
RR3: Poster Session
Session Chairs
Stephanie Bryant
Jason Burdick
Tuesday AM, December 01, 2009
Exhibit Hall D (Hynes)
9:00 PM - RR3.1
Single-Walled Carbon Nanotube-Enhanced Pulse-Laser Photoacoustic Stimulation Differentiates Multipotent Marrow Stromal Cells towards Osteoblasts.
Danielle Green 1 , Jon Longtin 2 , Balaji Sitharaman 1
1 Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States, 2 Mechanical Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractMarrow stromal cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts when exposed to certain stimuli (physical or chemical) which can be beneficial for bone remodeling and regeneration applications. Here we have used a Nd:YLF laser (200 ns pulse duration,10 Hz repetition rate and 10 mJ pulse energy) along with single-walled carbon nanotubes (SWCNTs) to generate photoacoustic waves. The generated photoacoustic waves stimulated MSCs for 10 minutes a day for 4, 9, and 16 days and differentiated them primarily into osteoblasts. Alkaline phosphatase content, calcium matrix deposition, and osteopontin expression were markers used to quantify osteogenesis. The calcium matrix deposited for MSCs undergoing photoacoustic stimulation after 16 days was 612% greater than static MSCs cultured in osteogenic supplemented media (supplemented with β-glycerophosphate, l-ascorbic acid, and dexamethasone). Non-stimulated samples cultured in media with and without SWCNTs had no detectable levels of calcium implying that they did not deposit a calcified matrix. The MSC groups with SWCNTs and undergoing photoacoustic stimulation showed up to a 100% greater amount of calcium compared to the stimulated group without SWCNTs after 16 days. The results indicate that there is a synergistic optical and acoustic effect assisting osteogenesis which is greatly enhanced by SWCNTs. Further development of this nanoparticle enhanced biophysical stimulus should lead to potential applications for tissue engineering and regenerative medicine.
9:00 PM - RR3.10
Development of Calcium Alginate Sub-Microparticles for Controlled Gene Delivery.
Rachael Oldinski 1 , Kathleen Jee 2 , Connie Cheng 1 , James Bryers 1
1 Bioengineering, University of Washington, Seattle, Washington, United States, 2 , University of Maryland, College Park, Maryland, United States
Show AbstractThe overall goal of this project is to develop DNA vaccine delivery for the efficient transfection of dendritic cells. Our objective here is to fabricate and characterize novel calcium alginate sub-microparticles for the packaging and release of either naked plasmid DNA (pDNA) or cationic polymer condensed (polyplexed) pDNA. POLYPLEX FORMATION: Aqueous solutions of polyethylenimine (PEI, branched, MW=25kDa) and pDNA (pCMV-luciferase) were mixed at a N:P=5 at room temperature for 20 min. PARTICLE FORMATION: Solutions of 2% sodium alginate and either naked pDNA or polyplexes were mixed together then blended into a 5% Span 80 isooctane solution for 3 min. Next, a 90mM calcium chloride solution was added to the emulsion and mixed for 3 min. An excess volume of isopropanol was then added to harden the particles. The particles were collected by centrifuging, rinsed with isopropanol 3 times then freeze-dried. CHARACTERIZATION: The shape of the particles was assessed via optical microscopy. The average diameter of the particles at 37°C was determined by dynamic light scattering. The encapsulation efficiency (EE) was determined by dissolving a known mass of particles in a 3% sodium citrate solution; EE was calculated by dividing the measured pDNA content in the particles by the amount of pDNA added to the reaction and multiplying by 100. In vitro release profiles were determined by placing a known mass of particles in PBS supplemented with 0.05% polyvinyl alcohol at 37°C; after 1h, 4h, 24h the particles were collected by centrifuging, and the supernatant was collected and replaced with fresh solution. The pDNA content from the EE and in vitro experiments was measured via a PicoGreen® assay. The in vitro release products were also analyzed via agarose gel electrophoresis. Finally, particles were incubated with a macrophage (MØ) cell line (murine RAW 264-7) for 24h at 37°C and 5% CO2 ; cytotoxicity was assessed by a lactate dehydrogenase assay. RESULTS: Alginate particles were spherical and uniform in shape. The average diameters of the pDNA and polyplex-loaded particles were 504±66 and 622±62 nm. The EE and in vitro release experimental results are shown in the table. Gel electrophoresis indicated that the naked pDNA released from the particles was intact. Further, polyplex-loaded particles released intact pDNA:PEI polyplexes. The alginate particles, with naked pDNA or polyplexed pDNA were not cytotoxic to MØ over 24 hrs.Alginate particles made by water/oil emulsion resulted in uniform sub-micron sized particles. The majority of the encapsulated pDNA and polyplexes were released within 1 h of incubation; the burst release of the pDNA may account for the low EE of the two groups. The alginate particles are not cytotoxic and the fabrication process is not harmful to the pDNA.
9:00 PM - RR3.12
De Novo Regeneration of the Hierarchical Extracellular Matrix with Protein nanoFabrics.
Adam Feinberg 1 , Kevin Parker 1
1 School of Enigneering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractThe multi-component, hierarchical extracellular matrix (ECM) that surrounds cells is critical to the structure and function of all tissues and organs within the body. However, to date there has been a limited ability to engineer the complex ECM de novo because matrix assembly is a cell-mediatedm process. Our vision is to engineer scaffolds for tissue regeneration directly from ECM proteins with complex fibrillar architectures that recapitulate the structure of the ECM in vivo. To do this we have developed a novel, surface-initiated fibrillogenesis process that utilizes surface chemistry to unfold matrix proteins and expose cryptic protein-protein binding domains required for them to undergo supramolecular assembly. Initially we sought to mimic the integrin-mediated assembly of fibronectin (FN) dimers into fibrils, but have since extended this cell-free fibrillogenesis capability to laminin, fibrinogen and collagens type I and type IV. We can integrate one or more of these ECM proteinsinto free-standing, fabric-like structures with complex, interconnected architectures. The exact spatial structure and composition is controlled by altering the features of the polydimethylsiloxane (PDMS) stamp used for microcontact printing and/or by printing multiple proteins, multiple times at different angles. In proof-of-concept experiments, we have engineered protein nanoFabrics with uniquematerials properties and tissue regeneration capabilities. As a high-performance material, FN nanoFabrics can be stretched 15-fold due to reversible unfolding of type III repeats, exceeding the capability of synthetic fabrics. For tissue engineering, FN nanoFabrics support cell binding, migration and contractile forces. We have demonstrated the ability to generate highly anisotropic strands of myocardium composed of neonatal rat ventricular cardiomyocytes. These myocardial fibers are typically ~20 μm wide and millimeters long and have uniaxial, synchronized contraction similar to papillary muscle. We have extended this further by engineering arrays of nanoFabric fibers in 3D to build thicker muscle and are working towards larger-scale, anisotropic sheets of myocardium similar to the lamellar layers of the ventricular wall. In total, protein nanoFabrics are microstructured, ECM matrices with mechanical properties comparable to cell-assembled matrix, can regenerate functional cardiac muscle and are actively being developed to regenerate larger, more complex tissue systems.
9:00 PM - RR3.16
Biomimetic Nanofilament-integrated Multicellular Spheroids and their Bioactive Microstructure-assisted Assembly for Vascularized Adipose Tissue Formation.
Taek Gyoung Kim 1 , Tae Gwan Park 1
1 Department of Biological Sciences, Korea Advaced Institute of Science and Techonology, Daejeon Korea (the Republic of)
Show Abstract The nanofibers assembled into the 3D architecture are ideal for creating biomimetic environments to direct cellular behaviors, holding promise for various biomedical applications due to their extracellular matrix (ECM)-mimicking size and format. Recently, we fabricated the biodegradable fragmented fibrous nano-materials from electrospun polymeric nanofibers via aminolysis method. The length and diameter of the polymeric nanomaterials could be controlled via electrospinning and aminolysis parameters, and the produced nanomaterials were shown to be well-dispersed in aqueous liquid, indicative of the feasibility of the solution-phase 3D assembly with various cells in bio-friendly condition. The variety of mammalian cells have been employed for forming multicellular spheroids aiming at targeted differentiation and phenotypic stabilization, as well as the simulation of 3D physiological condition. Especially, the multicellular spheroid used as a building unit in engineered tissue construction afford the possibility for their promising application. Human mesenchymal stem cell (hMSC) is promising source for regenerative medicine due in large part to their self-renewal capacity and their multipotency. Microfabrication techniques based on rapid prototyping (RP) methods entails the tailor-made design of scaffold with 3D anatomical shape along with desired biological and mechanical performance. Herein, we provide novel hierarchical and multifunctional scaffold design concept via two-step process consisting of the preparation of biomimetic nanofilament-integrated multicellular hMSC spheroids and subsequent spheroids-based assembly aided with precisely controlled microstructure as a solid structural framework. The 3D construction is realized through interlocking each spheroids-microstructure hybrid by additive layer-by-layer manner. This fabrication strategy permits the hierarchical assembly through the cellular adhesion making the connection between the nanostructure and microstructure. In addition, due to the non-vascularized nature of the assembled spheroids, the blood vessel formation is crucial for attaining the bulk functional tissue. Thus, the microfabricated framework was allowed to deliver angiogenic growth factor for vasculized tissue formation.
9:00 PM - RR3.17
In Vitro Comparison of SiHA and HA Coatings under Different VPS Conditions.
Qian Tang 1 , Roger Brooks 2 , Serena Best 1
1 Materials Science and Metallurgy, University of Cambridge, Cambridge Centre for Medical Materials, Cambridge United Kingdom, 2 , Orthopaedic Research Unit, University of Cambridge, Cambridge United Kingdom
Show AbstractIntroduction: Plasma spraying is widely used in industry to produce hydroxyapatite (HA) coatings on metallic prostheses for major load-bearing applications. The implants combine the mechanical properties of metals with the bioactivity of HA. Incorporation of silicon into HA can increase bone apposition rate [1]. The coating properties are dependent on the spraying parameters. One of the most important parameters is the plasma gun input power. In this study, both HA and SiHA powders produced in-house were coated on Ti-6Al-4V substrates using vacuum plasma spraying (VPS). The bioactivity of HA and SiHA coatings produced under different VPS conditions are compared in vitro. Materials and Methods: Phase pure HA and 0.8wt% SiHA were produced through a wet precipitation method and sprayed onto 10 mm diameter Ti-6Al-4V discs in a Plasma Technik P1800 vacuum plasma spray unit. 4 samples were prepared as below.SamplePowdersPlasma gun power/kwHAC37HA37HAC40HA40SiHAC37SiHA37SiHAC40SiHA40chamber pressure: 200 mbar, stand-off distance: 260mm, primary plasma gas (Ar) flow rate: 50 slpm.Human osteoblast-like cells (HOB) were seeded on the discs at the concentration of 20,000cells/cm2 and incubated at 37oC, in a 5% CO2 atmosphere. Cell and coating morphology at day 1, 6 and 12 was observed using a JEOL 6340F Field Emission Gun Scanning Electron Microscope. At each time point, the samples were critical point dried and then coated with a thin layer of palladium for SEM observation. Type I collagen synthesized by the cells was determined using a specific enzyme-linked immunoassay (MetraTM CICP Enzyme Immunoassay Kit, Quidel, Dorking, UK) on Day 1, 3, 6, 9, and 12.Results and Discussion: SEM images showed that cells spread and grew well on all samples with filopodia anchoring onto the surfaces. Extracellular matrix (ECM) and a CaP crystal layer were also observed on all surfaces after 12 days incubation. Cells on HAC37 showed more pronounced cell spreading with HAC40 at the same time point. Cells on SiHA coatings grew more rapidly and developed better than those on phase pure HA coatings produced under the same conditions. Collagen synthesis increased with culture time. Coatings produced at lower plasma gun input power were associated with increased level of collagen synthesis. Higher collagen levels detected for SiHA coatings suggest that silicon stimulated collagen synthesis. Higher plasma gun input power resulted in a higher impurity content and amorphous phases, which led to higher solubility. Coatings produced at 40 kW appeared to dissolve too fast to support cell proliferation and differentiation.Conclusions: All HA and SiHA coated discs produced at the selected parameters were supportive for HOB cell growth and collagen synthesis. Comparing the four samples, SiHA coatings produced at lower plasma gun power (37kw) showed the best bioactivity. Reference: 1. Patel et al. J. Mater. Sci. Mater. Med. (2005), Vol. 16, p. 429.
9:00 PM - RR3.18
Effect of Substrate Composition and Organization on Cultured Cardiomyocyte Viscoelastic Properties.
Sandra Deitch 1 , Bruce Gao 1 , Delphine Dean 1
1 Bioengineering, Clemson University, Clemson, South Carolina, United States
Show AbstractCardiomyocyte phenotype changes significantly in 2D culture systems depending on the substrate composition and organization. Given the variety of substrates that are used both for basic cardiac cell culture studies and for regenerative medicine applications, there is a critical need to understand how the different matrices influence cardiac cell mechanics. Clean glass slides were coated with thin layers of fibronectin and collagen (1 mg/ml solutions), in aligned and unaligned orientations. A modified inkjet printer was utilized to align the substrate fibers within thin printed lines. Cardiomyocytes were obtained from day 3 neonatal rat hearts and seeded at 50,000 cells/cm2. At various time points between 1 and 15 days in culture, mechanical properties were measured using AFM. On each sample, 15-20 cells were each indented 5 times to approximately 1 micron depth at 1 micron/sec using a borosilicate spherical probe (radius ~2.5 microns). The elastic modulus was estimated by fitting the Hertz model to the first 500 nm of indentation. Cells were also subjected to 1 micron step indentation and 60 sec hold (stress-relaxation) experiments to characterize their viscoelastic behavior. The resulting curves were fit to the Quasilinear Viscoelastic (QLV) and Standard Linear Solid (SLS) models. It was observed that the cells stiffened over the first 5 days in culture before reaching a plateau. After 5 days, the cells on aligned fibronectin were stiffest, followed by those on unaligned fibronectin, aligned collagen, and finally unaligned collagen (p<0.1, t-test). These results correlate with the observed changes in cytoskeletal architecture associated with culture on the different substrates. The QLV model fit the stress-relaxation data very well. On all substrates, the rate of stress relaxation decreased over the first 5 days in culture before leveling off. No significant changes in relaxation were observed for cells on different matrices. This research illustrates the dependence of cellular mechanics on matrix composition and organization. These results should be taken into consideration when choosing a specific matrix for a given experiment.
9:00 PM - RR3.19
Dual-Gelling Biomaterials for Inkjet Printing and Cell Seeding.
Manuela Di Biase 1 2 , Rachel Saunders 1 , Nicola Tirelli 2 , Brian Derby 1
1 School of Materials, University of Manchester, Manchester United Kingdom, 2 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester United Kingdom
Show AbstractInkjet printing is an attractive route for the fabrication of devices from biomaterials. However, the use of inkjet printing restricts the range of biomaterials that can be codeposited with cells. The materials must be deliverable in liquid form and preferably in aqueous solution and the phase change to solid after printing must also be cytocompatible. Finally the range of fluid viscosity, density and surface tensions are limited to those compatible with inkjet delivery and can be defined by the following limits of the Reynolds and Weber numbers 1 < √We/Re < 10, or 1 < √(γρd)/η < 10, where γ, ρ, and η are the fluid surface tension, density and viscosity respectively, and d is a characteristic length. Because of the constraints listed above, the majority of work that has used inkjet printing to deliver biomaterials in parallel with cells has used Na alginate, crosslinked by Ca2+ ions as the structural material. As an alternative, we propose to use a dual gelling system that incorporates both physical and chemical gellation mechanisms. A physical gellation mechanism is used to ensure rapid phase change on printing, followed by a secondary chemical crosslinking to increase the stability of the gelled deposit. This combination of cross-linkable polymers and hydrogels will provide a robust tissue engineering solution to the manufacture of tissue scaffolds and their co-seeding with cells. Specifically, we use photopolymerization reactions for this purpose, because of a double advantage: a) easy control of the process; the cross-linking reaction does not depend on any other variable, such as mixing time of reagents, and can be switched on at any time in the process; b) by using appropriate masks, the resolution attainable with the process can be improved and further morphological features can be added to ink-jet printed scaffolds. Here we present a study of the inkjet printing of a cross-linking physical gelating system based on di-functionalised Pluronic materials. These have been used to fabricate 3-dimensional structures that have been seeded with co-printed mammalian cells.
9:00 PM - RR3.2
Gene Delivery Mediated by Recombinant Silk Protein Block Copolymers Containing Cationic and Cell Binding Motifs.
Keiji Numata 1 , David Kaplan 1
1 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Show AbstractSilk proteins are biodegradable, biocompatible, self-assemble, and can also be tailored to contain other design features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and the cell-binding motif, RGD, to enhance cell interactions and transfection efficiency. Ionic complexes of these silk-polylysine-RGD based block copolymers with pDNA were studied for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentiometer, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide ratio. pDNA complexes with 30-lysine residues and 10 RGD sequences prepared at a polymer/nucleotide ratio of 200 showed the highest efficiency for transfection (24±10%). These systems were 10 mV positively charged and exhibited a solution diameter of 80 nm. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailorable gene delivery systems.
9:00 PM - RR3.20
Physical Properties of Regenerated/Liquid Silk Fibroin Blend Nanofiber Mats.
Taiyo Yoshioka 1 , Yutaka Kawahara 2 , Andreas Schaper 1
1 Material Sciences Center, Philipps University, Marburg Germany, 2 Department of Biological and Chemical Engineering, Gunma University, Kiryu Japan
Show AbstractSilk fibroin has been expected to be one of the exceptional biomaterials, which covers the wide range of requirements as scaffold material due to its naturally high biocompatibility and its excellent mechanical properties. In many trials of fabrication of fibroin-based scaffolds, regenerated fibroins obtained by dissolving native cocoon fibers are used. However, those products are known to have seriously poor mechanical properties because of high brittleness compared with their original cocoon fibers. Especially the products with high amounts of beta-crystalline modification, which is generally induced by treatments with ethanol, methanol or water vapor, show serious brittleness although the treatment is preferably used to endow the high strength. We have found that blending liquid fibroin with regenerated fibroin improves the mechanical behavior of regenerated fibroin-based products dramatically. We have investigated this effect in detail and could show the significant improvement of strength and fracture strain, and a considerable reduction of the modulus in fibers blended with a small amount of liquid fibroin. This modification lends regenerated silk a more viscoelastic behavior. On the other hand, the blending also enabled to fabricate the electrospun nanofiber mats with well controlled crystalline structure and with improved mechanical properties. In addition to the mechanical properties, crystal structure and thermal properties were investigated by transmission electron microscopy (TEM) and X-ray diffraction (XRD) measurement, and differential scanning calorimetry (DSC), respectively.
9:00 PM - RR3.21
Bio-functionalization of Materials for Implants Using Engineered Peptides.
Dmitriy Khatayevich 1 , Mustafa Gungormus 1 , Christopher So 1 , Sibel Cetinel 2 , Hong Ma 1 , Alex Jen 1 , Candan Tamerler 2 1 , Mehemt Sarikaya 1
1 Materials Science and Engineering, Univ. of Washinginton, Seattle, Washington, United States, 2 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey
Show AbstractUncontrolled interactions between synthetic materials and living systems are a major concern in implant and tissue engineering. The most successful approaches to resolving this issue involve modification of the implant or scaffold surface with various functional molecules, such as anti-fouling polymers or cell growth factors. To date, such techniques have relied on surface immobilization methods that are often applicable only to a limited range of materials and require the presence of specific functional groups, synthetic pathways, or biologically hostile environments. We report using peptide motifs that have been engineered to bind to gold, platinum and silica glass to modify surfaces with poly(ethylene glycol) anti-fouling polymer and RGD integrin-binding sequence. The peptides have several advantages over conventional molecular immobilization techniques because they require no hostile environments for binding, are inherently non-toxic, are specific to their materials, and could be designed to carry various active entities. In addition, their material selectivity properties could be used in designing complex devices simply by relying on conventional inorganic manufacturing techniques and self-assembly. We have successfully developed anti-fouling properties on gold and platinum using 3GBP1 and PtBP1 peptide motifs, respectively, in conjunction with PEG. These surfaces perform as well as those functionalized through thiol chemistry in vitro, by significantly diminishing adhesion of fibroblast and osteoblast cells. We also induced a 3.5-fold increase in the number and a 1.6-fold increase in the spreading of osteoblast cells on glass using the QBP1-RGD peptide construct. We achieved comparable improvements in the adhesion and spreading of fibroblast cells on glass. The ability to use material specific solid binding peptides for functionalization of surfaces provide a promising outlook in medical implants and tissue engineering. This research is supported by NSF-MRSEC, NSF-BioMat, and NSF-IRES programs.
9:00 PM - RR3.22
Solid-binding Peptide-based Antibacterial Implants.
Hilal Yazici 1 2 , Mary Rood 1 3 , Brandon Wilson 1 , Mustafa Gungormus 1 , Candan Tamerler 1 2 , Mehmet Sarikaya 1 2
1 Genetically Engineered Materials Science and Engineering Center, MSE, University of Washington, Seattle, Washington, United States, 2 Molecular Biology-Biotechnology and Genetics, Istanbul Technical University , Istanbul Turkey, 3 Cellular and Molecular Biology , University of Washington , Seattle , Washington, United States
Show AbstractImplant-associated infections are a primary cause of early implant failures. Such infections have been difficult to treat due to the unique (and complex) biomicroenvironment inside the human body. The success of implants depends not only on the bone–implant integration, but also on the presence of a sterile environment around the implant that will prevent bacterial infection. The generally prescribed oral antibiotics, e.g., for dental implants, are not always effective in combating implant-associated infections for a variety of reasons including the inability to reach the infection site in bone tissue, and an increase in bacterial resistance. A novel class of peptides, the antimicrobial peptides (AMPs), is useful for their utility as therapeutic agents mainly because of the difficulty for microorganisms to develop resistance towards them. In the present study, we use a novel bi-functional peptide based approach for implant surface functionalization. Specifically, we use Titanium-binding peptides that have been selected using biocombinatorial approach, via a flagella display method, and well characterized in binding and material selectivity properties. We designed a bifunctional peptide that exhibits both titanium-binding (TiBP1) and antimicrobial (AMP) properties. The efficiency of TiBP1-AMP bi-functional was evaluated in vitro against infection by several bacteria, common in oral cavity, and the growth was analyzed in solution by optical density measurement. Bacterial infection was also evaluated on the functionalized titanium surface with fluorescence microscopy (FM) and cell count was established by scanning electron microscopy (SEM) analysis at various time points. For example, Streptococcus mutans adhesion was reduced on the TiBP1-AMP peptide- based functionalized substrate compared to both positive and negative controls. The approach is general and applicable to various biomaterial and implant surfaces and may be a candidate for the prevention of implant infections. This research is supported by GEMSEC, an NSF-MRSEC at the University of Washington, and NSF-IRES/TUBITAK programs.
9:00 PM - RR3.23
Shape Memory Behavior of Side-chain Liquid Crystalline Copolymers Bearing Cholesterol.
Suk-kyun Ahn 1 , Rajeswari Kasi 1 2
1 Institute of Materials Science, University of Connecticut, Storrs, Connecticut, United States, 2 Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States
Show AbstractShape memory polymers (SMPs) are a class of stimuli-responsive materials which have capability to fix their temporary or meta-stable shape and to recover their original shape under external stimulus, such as temperature. Recently, the SMPs are gaining great attention because of their potential applications including self-repairing materials, biomedical devices, sensors and actuators. There have been numerous efforts in the last two decades to develop these SMPs using different types of triggering temperature such as glass transition temperature (Tg), melting temperature (Tm), and clearing temperature (Tcl).Herein, we report new side-chain liquid crystalline copolymers bearing cholesterol mesogens and butylacrylate side-chains which exhibits shape memory behavior upon temperature change. These copolymers were prepared by ring opening metathesis polymerization (ROMP) with polynorbornene as their main-chain. The butylacrylate composition of copolymers was varied from 5 to 51 weight percent in order to tune glass transition temperature as well as clearing temperature. Smectic A phase of the copolymers was identified from two dimensional wide-angle X-ray diffraction patterns. The acrylate functional groups were further cross-linked either by thermally or by using UV light.The shape memory (SM) behavior of the cross-linked polymers were qualitatively characterized by immersing polymers in hot (50 oC) and cold water (5 oC) bath to deform, fix and recover their shape. Furthermore, the quantitative analysis of SM behaviors was carried out using dynamic thermomechanical analysis to evaluate strain fixity and strain recovery rate.The prepared shape memory polymers are expected to use for biomedical devices due to i) low cytotoxicity of norbornene derivates, ii) biocompatibility of cholesterol moieties and iii) possible shape memory behavior around body temperature.
9:00 PM - RR3.25
Synthesis and Characterization of Ultrananocrystalline Diamond (UNCD) Films as Substrate/Scaffold Material for Developmental Biology: Regenerative Tissue/Organ Development.
Bing Shi 1 , Qiaoling Jin 2 , Liaohai Chen 2 , Orlando Auciello 1 3
1 Materials Science Division, Argonne National Laboratory, Argonne, Illinois, United States, 2 Biosciences Division, Argonne National Laboratory, Argonne, Illinois, United States, 3 Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois, United States
Show AbstractBioengineering development provides novel treatment techniques for patients who suffer from tissue/organ damages and face the limitation of donation sources. Extensive studies have been performed on regenerative tissue/organ approaches. One key theme on this research focused on regenerative medicine is the investigation of implantable substrates for supporting cell growth. The research discussed in this paper focuses on exploring a novel form of diamond films developed at Argonne National Laboratory, named ultranacrystalline diamond (UNCD), as biocompatible substrates/scaffolds. UNCD films are grown via exposure of substrates to Ar-rich microwave plasmas including minority percentage of CH4 with or without addition of N2 or O2 to chemically treat the surface of the UNCD layer. The UNCD films exhibit a unique microstructure with 2-5 nm grains and ~ 0.5 nm wide grain boundaries, which provide the outstanding biocompatible properties. Different types of UNCD films have been synthesized and cell culture done on their surface using UNCD coated culture dishes. Results showed that all types of UNCD films: plain UNCD, H-doped (i.e., hydrogen inserted into grain boundaries) UNCD, H-doped/H-plasma treated (i.e., exposure of the UNCD surface to a hydrogen plasma to achieve H-terminated UNCD surface), N-doped (i.e. nitrogen inserted into grain boundaries) UNCD, N-doped/H-plasma treated UNCD films, all of them produced with different doping percentages for systematic studies, support cell growth, although there are differences in coverage that will be discussed in terms of the indicated parameters. Studies involved growth of different types of cells, such as mouse embryonic fibroblasts (MEFs), neurons, osteoblasts, and stem cells (Figure 1). SEM, XPS, MALDI, FTIR, and Raman were done on the UNCD, cells, and cell cultured UNCD surface. Mechanisms of cell adhesion on the surface of UNCD films will be discussed, in view of supporting evidence obtained also from cell culture on diamond powder seeded culture dishes.This work was supported by the US Department of Energy, BES-Materials Sciences, under Contract DE-AC02-06CH11357.
9:00 PM - RR3.26
Topographic Effect of Zinc Oxide Nanoflower Structure onGrowth and Proliferation of MC3T3-E1 Osteoblast Cells.
Ju Hyeong Jeon 1 , Yong-Jin Kim 1 , Gyu-Chul Yi 1 , Jong Heo 1 , Sei Kwang Hahn 1
1 Biomedical Nanomaterials Laboratory, Pohang University of Science and Technology, Pohang , Kyungbuk, Korea (the Republic of)
Show AbstractCell-material interactions are important factors to be considered for the application of biomaterials to various tissue engineering fields. It has been reported that the topography of biomaterial surfaces has great influences on cellular behaviors such as adhesion, proliferation, and differentiation. In this work, we investigated the topographic effect of zinc oxide nanoflower structure on the growth of MC3T3-E1 osteoblast cells and their adhesion to the zinc oxide surface. Single crystalline ZnO nanoflowers were prepared on glass substrates by chemical solution deposition method. According to in vitro culture tests with MC3T3-E1 osteoblast cells, ZnO nanoflowers resulted in enhanced DNA content and alkaline phosphatase activity compared to the controls of silicone and ZnO substrates. Furthermore, we could confirm the improved adhesion strength of the cells to ZnO nanoflowers from the centrifugation tests to measure the adhesion force between them. When the cells anchored to the surface, ZnO nanoflowers appeared to promote osteoblast proliferation and differentiation. All these results demonstrated that nanoflower structure of metal oxide might be advantageous to improve the osteoblast adhesion strength to the surface as well as enhancing the cell adhesion, proliferation, and differentiation. This novel approach would be successfully exploited to improve the osteoblast cell adhesion to the dental implant system using titanium oxide.
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Effect of Porosity of Unidirectional Porous Hydroxyapatite/collagen Bone-like Nanocomposite on Regeneration of Critical Tibial Defect of Dog.
Masanori Kikuchi 1 , Koji Aoki 2 , Kazuya Edamura 2 , Yoshihisa Koyama 3 , Kazuo Takakuda 3 , Shigeo Tanaka 2
1 Biomaterials Center, National Institute for Materials Science, Tsukuba, Ibaraki, Japan, 2 College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan, 3 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan
Show AbstractWe have developed novel hydroxyapatite/collagen (HAp/Col) nanocomposite with bone-like nanostructure and chemical composition. The HAp/Col is incorporated into bone remodeling process and new bone is regenerated when it is implanted into bone defect. To enhance cell migration in the material, we also have developed unidirectional porous materials from the HAp/Col membrane. In the present study, we reported the effect of pore structures and porosity on regeneration of critical tibial defect of dog.The long and short fibers of HAp/Col nanocomposite (HAp:Col=7:3 in mass ratio) were synthesized by a simultaneous titration method. Mixture of long and short fiber at 1:4 volume ratio was filtered, vacuum dried then pressed at 30 MPa into thin membrane. The membrane was placed onto wavy mold, moisturized and pressed at 140 kPa into wavy membrane. The wavy membrane was rolled up to be a unidirectional porous scaffold 15 mm in diameter and 20 mm in height. For low porosity scaffold, grooves on one side of the wavy membrane were filled up with the HAp/Col paste (HAp:Col mass ratio was 4:1) before rolling up. After the rolling up, the edge was adhered with collagen. The scaffold obtained was crosslinked by vacuum heating at 140 °C for 12 h. The scaffold was implanted into tibial segmental 20 mm defect of mongrel dog up to 20 weeks. The implanted site was periodically observed by X-ray and histologically observed after extraction.The practical porosities of high and low porosity HAp/Col were 85.1% and 48.1%, respectively. In the X-ray observation, regenerated bone tissue was observed from both ends of the defect at 2 weeks after implantation and bone defect was filled by bone at 12 weeks. Image analysis of bone formation area including surrounding callus showed 90% and 109% new bone formation in average were observed for the high porosity and low porosity HAp/Col. Further, the low porosity one showed stable staying at the defect site with maintenance of its shape. In the histological observation, cancellous-like neogenesis bone tissue was formed in bone defects. The HAp/Col scaffold was absorbed even the low porosity one.In general, interconnected high porosity is recommended for bone regeneration. In the present study, however, no significant differences in bone regeneration and scaffold resorption were observed or even better trend in low porosity scaffold. The reason could be stable existence of scaffold till cell proliferation and competent extracellular matrices formation was required for regeneration of huge critical defect.In conclusion, the HAp/Col scaffold revealed comfortable regeneration in dog’s critical tibia defect. In addition, optimal “low” porosity could be required for much stable regeneration in scaffold materials with biological resorption rate, such as the HAp/Col nanocomposite.
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A PVA_PCL_Bioglass Composite with Potential Implications for Osteochondral Tissue Engineering.
Prabha Nair 1 , Lakshmi Mukundan 1 , Remya Nirmal 1 , Neethu Mohan 1
1 Division of Tissue Engineering & Regeneration Technologies, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Trivandrum, Kerala, India
Show AbstractA Bioglass of composition SiO2 (67.12 mol %), CaO (28.5 mol %), and P2O5 (4.38 mol %) was synthesized and stabilized by a novel technique using ethanol. Bioactive glasses have a wide range of application in the field of biomaterials promoting bone bonding( 1) as well as bonding to soft tissue (2) Recent studies have shown the potential of bioactive glass in stimulating the gene expression (3) and growth factor production in osteoblasts (4). Applications of bioactive glass in lung tissue engineering were also reported by Tan et al(5).Richard found that the bioactive glass can be effective in inducing angiogenesis by stimulating the expression and release of VEGF and bFGF when smaller quantities are used (6). Earlier our lab developed a novel PVA_PCL Semi IPN porous and 3D scaffold that was found to favour chondrogenesis (7). In the present study, a composite of this polymer and bioglass is prepared by an emulsion freeze drying process, as a porous 3 dimensional scaffold. The bioglass composites allowed for loading of upto 30 wt% of bioglass without any phase separation. The scaffolds were characterized for their physicochemical properties and ability to support tissue regeneration. The composite scaffolds were observed to be noncytotoxic by direct and indirect contact cytotoxic assays using chondrocytes. Chondrocyte cells cultured in vitro for a month on the composite scaffolds regenerate cartilaginous tissue, secreting GAGs and collagen in amounts nearly comparable to the amounts on the control PVA_PCL Semi IPN scaffold. The composite scaffold is also biomimetic and bioactive and favours mineralization by forming a hydroxycarbonate apaptite layer, when immersed in simulated body fluid for a 14 day period. The composite PVA_PCL – bioglass composite is hence expected to have potential implications as a scaffold for osteochondral tissue engineeringReferences :1.Hench L L, Splinter R J and Allen W C. Bonding mechanisms at the interface of ceramic prosthetic materials. J. Biomed. Mater Res, 2, 117, 19712.Wilson J, Nicolletti D. Bonding of soft tissues to bioglass. In: Yamamuro T, Hench L L, Wilson J, editors. Handbook of bioactive ceramics vol I. Boca Raton, FL CRC Press, 1990, 283-302.3.Xynos I D, Edgar A J, Buttery L D K, Hench L L. Polak J M. J Biomed mater Res 55, 151-7, 2001.4.Xynos I D, Edgar A J, Buttery L D K, Hench L L. Polak J M. Biochem Biophys Res Commun 276, 461-465, 2000.5.Tan A, Romanska H M, Lenza R, Jones J, Polak J M, Bishop A E. The effect of 58S bioactive sol- gel derived foams on the growth of murine lung epithelial cells. Key Eng Mater 719-24, 240-242, 2003. 6.Richard M D. Bioactive glass stimulates the secretion of angiogenic growth factors and angiogenesis in vitro. Tissue Eng. 11, 5/6, 2005.7.Mohan N, Nair P D. Polyvinyl alcohol- polycaprolactone semi IPN scaffold with implication for cartilage tissue engineering. J Biomed Mater Res 84B, 584-594, 2008.
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Development and Evaluation of Conjugated Polymer Based Scaffolds for Stem Cell Differentiation and Tumor Suppression.
Anna Herland 1 , Vanessa Lundin 1 , Kristin Persson 2 , Maria Bolin 2 , Edwin Jager 2 , Magnus Berggren 2 , Ana Teixeira 1
1 Department of Neurology, Karolinska Institute, Stockholm Sweden, 2 Department of Science and Technology, Linköping University, Norrköping Sweden
Show AbstractStem cells (SC) are potentially the fundament of a whole new field of regenerative medicine from futuristic views of whole organ reconstruction to closer goals of CNS treatments. Successful SC therapy in the CNS requires proper integration of differentiated cells with surrounding tissues demanding good cell survival and differentiation in vivo. However, it is unlikely that an injured, or healthy, post-natal brain or spinal cord contains the instructive cues necessary to direct these events. We report on the development of a 3D hybrid polymer scaffold for spatial and temporal control of the SC microenvironment. The scaffold is a multi-component system consisting of (A) a conjugated polymer (CP), (B) a biodegradable support polymer and (C) soluble extrinsic factors directing SC differentiation or suppress tumor formation, available for controlled release. CPs such as PPy and PEDOT have been used as biomaterials for electrical and mechanical stimulation, as well as active release of compounds. (1) The component B, being co-polymers of PLLA and PGA in porous/fibrous scaffolds, have by us in vitro and by others in vivo, been demonstrated to give trophic support promoting cell survival, while not suppressing differentiation.Our primary target is a Parkinson’s therapy approach where the hybrid scaffold is adjusted to a mouse embryonic stem cell line (mESC) NesE-Lmx1a. In vitro NesE-Lmx1a cells can be successfully differentiated to cell type that is lost in Parkinson’s disease; 75-95% of the derived neurons express characteristics of mesencephalic dopamine neurons. (2) Upon transplantation these cells can integrate and innervate the striatum of 6-hydroxy-dopamine lesioned neonatal rats. However, both in vitro and in vivo overgrowth (tumor formation) of proliferating cells occurs and the degree of cell survival following transplantation is low.Soluble extrinsic factors that steer SC differentiation and proliferation include small molecules and growth factors (GFs). Incorporation and release of small charged molecules, which act as counter ions, in CPs have been shown. Retinoic acid and Notch inhibitors will via this strategy be used to actively suppress aberrant proliferation of NesE-Lmx1a. Active control of the presence of GFs necessary for differentiation, fibroblast growth factor (FGF8) and sonic hedgehog (Shh) for NesE-Lmx1a, demands a more delicate approach to maintain the biological activity. GFs can keep the differentiation ability for neural SCs, when associated to a hydrogel matrix. (3) For control of the presence of FGF8 and Shh, we are utilizing their high affinity for extracellular matrix components (ECMs). We have successfully incorporated and released ECMs as counter ions to PEDOT, a system where the doping state of the CP controls the presentation of GFs. 1 Bolin M , et al. Sensors and actuators B, 2009. accepted.2 Friling S, et al. PNAS, 2009, 106(18): 7613–183 Ilkhanizadeh S, Teixeira AI, Hermansson O. Biomat. 2007; 28(27):3936-43
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Enzymatic Synthesis of Amorphous Calcium Phosphate-Chitosan Nanocomposites and its Processing into Hierarchical Structures.
Francisco del Monte 1 , Maria C. Gutierrez 1 , Maria L. Ferrer 1 , Matias Jobbagy 1
1 , ICMM-CSIC, Madrid Spain
Show AbstractBiomineralization offers the opportunity to produce highly organized nanocomposite structures, controlling specific architectures over extended length scales for a wide range of inorganic materials. Enzymatically assisted routes also offer the possibility to synthesize a number of materials with excellent control on the structural organization. In particular, HA precursors and different calcium carbonate precipitates could be obtained in solutions by enzyme-catalyzed decomposition of urea by urease. Furthermore, the gradual generation of base provided by urea hydrolysis has recently been used for the preparation of monolithic and homogeneous chitosan hydrogels. The homogeneous pH modulation besides the low temperature used for urea hydrolysis allow for the achievement of CHI hydrogels with a homogeneous 3D network structure with superior biotechnological performance than chitosan solutions gelled by neutralization with alkaline solutions, gaseous NH3 or dialysis. Here in, we applied the urease assisted hydrolysis of urea for the preparation of nanocomposites (of turbid appearance) based on calcium phosphate precipitates and CHI hydrogel (see Scheme I in downloaded file). The base generated by urea hydrolysis promoted both CHI gelation and calcium phosphate precipitation at biological temperatures (~37 C). Otherwise (e.g., urea hydrolysis by thermal decomposition at 90 C), CHI would undergo partial decomposition. Macroporous scaffolds (e.g.;, hierarchically organized) were obtained by a cryogenic process (named ISISA, ice segregation induced self-assembly) that simply consist on the unidirectional freezing (at -196 C) of the hydrogel nanocomposites. Upon freezing, the ice formation (hexagonal form) causes every solute originally dispersed in the hydrogel to be segregated from the ice phase. After freeze-drying, the resulting hierarchical structures consists on well aligned micrometer-sized pores in the freezing direction corresponding to the empty areas where ice crystals originally resided, being the macrostructure supported by the matter (e.g., calcium phosphate nanoparticles dispersed within CHI matrix) accumulated between adjacent ice crystals. The excellent control on ice crystals formation, ice segregation matter and matter self-assembly between adjacent ice crystals allows ISISA for unique tailoring of the final macrostructural features of the resulting scaffolds. Thus, figure 1 (see at downloaded file) shows the porous channels of up to 90 micrometers that can be simply obtained by using different freezing rates in the application of the ISISA process to the hybrid hydrogel. The calcium phosphate nanoparticles entrapped within the CHI scaffold are identified as amorphous calcium phosphate as revealed by TEM, XRD and NMR experiments
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(Bio)degradable Urethane-Elastomers for Electrospun Vascular Grafts.
Stefan Baudis 1 , Maria Schwarz 1 , Christian Grasl 2 , Helga Bergmeister 3 , Guenter Weigel 3 , Heinrich Schima 2 , Robert Liska 1
1 Institute of Applied Synthetic Chemistry (Division: Macromolecular Chemistry), Vienna University of Technology, Vienna Austria, 2 Center of Biomedical Engineering and Physics, Vienna Medical University, Vienna Austria, 3 Ludwig-Boltzmann Cluster for Cardiovascular Research, Vienna Medical University, Vienna Austria
Show AbstractCardiovascular disease remains one of the leading causes of death in industrial countries. Up to now autologous vessels have been the preferred graft materials for diseased vascular segments smaller than 6 mm in diameter. Unfortunately, the number of appropriate vessels is limited in many patients because of previous vessel harvest or poor quality. Therefore we aim for the development of artificial cardiovascular grafts. Our approach is electrospinning (ES) of (bio)degradable polyurethane-based thermoplastic elastomers (TPUs).ES is a very powerful method to create cellular scaffolds for regenerative medicine – especially for artificial vascular grafts [1]. Commercially available TPUs, like Pellethane™ (Dow plastics) are FDA approved and have already shown excellent biomechanical properties as electrospun vascular grafts [2]. In order to induce the growth of a neo-artery and hence increase the long-term patency of the graft the use of biodegradable TPUs is beneficial [3]. Therefore we aim for the development of degradable TPUs. In preliminary studies the mechanical properties of segmented TPUs were examined. By the modification of soft-block length and the ratio of chain extender moduli ranging from 9 to 89 MPa at tensile strengths between 6 and 41 MPa and ultimate elongations from 770 to 900% could be achieved. These tendencies were also found for the electrospun materials. We could also show that the substitution of the aromatic 4,4'-methylene diphenyl diisocyanate (MDI) with the aliphatic hexamethylene diisocyanate (HMDI) – to avoid toxic aromatic amines as potential degradation products - only causes minor loss of strength. To obtain degradable TPUs our concept is to incorporate cleavable bonds into the polymer chain. For this purpose, lactid- and ethylene glycol-based cleavable chain extenders were used. The expected degradation products showed no cytotoxicity up to a concentration of 1mM in in-vitro tests. Currently, degradation tests of polymer samples in phosphate buffered saline (PBS) at elevated temperatures are performed by monitoring the molar mass with GPC using medical-grade PLA as reference.[1] Sell, S. A.; Bowlin, G. L. J. Mater. Chem. 2008, 18, 260-263.[2] Grasl, C.; Bergmeister, H.; Stoiber, M.; Schima, H.; Weigel, G. J. Biomed. Mater. Res. A 2009, in press.[3] Gogolewski, S.; Galletti, G.; Ussia, G. Colloid Polym. Sci. 1987, 265, 774-778.
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Synthesis, Characterization and Microfabrication of Poly(Glycerol Sebacate) for Human Mesenchymal Response Studies.
Israd Jaafar 3 , Mohamed Ammar 1 , Raymond Pearson 1 4 5 , John Coulter 3 , Sabrina Jedlicka 1 2 4
3 Mechanical Engineering & Mechanics, Lehigh University, Bethlehem, Pennsylvania, United States, 1 Materials Science & Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 4 Center for Advanced Materials and Nanotechnology, Lehigh University, Bethlehem, Pennsylvania, United States, 5 Center for Polymer Science & Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractCell fate modification is a critical step in preparing cells and tissues for implantation therapeutics. Novel materials possessing physical, mechanical, and chemical properties similar to those found in vivo provide a potential platform to build artificial microenvironments for therapeutic applications and well-defined biointerface platforms for examining differentiation potential in stem cell biology. Poly(glycerol sebacate) (PGS), a novel biocompatible and biodegradable elastomer is one such material. With tunable mechanical properties in the range of common soft tissue, the material provides an invaluable alternative platform for use in cell-to-substrate interaction studies. This paper describes the tunability of PGS mechanical properties based on variations in curing temperatures (140, 150, and 165 °C). We characterized the material by examining properties that include Young’s modulus, glass transition temperature, density, degree of crosslinking, cure kinetics, protein conformational changes, and molecular bonding compositions. Successful culturing of human mesenchymal stem cells (hMSCs) on PGS demonstrates a unique capability of the modulated polymer mechanics to tune the biological response with regards to ‘stemness’ of the hMSCs, including protein expression, mRNA, growth rate and morphology. We also provide a detailed account of microfabrication, where PGS micro-pillars were successfully replica molded from an inversely featured 3-inch silicon (Si) wafer. Variations in PGS curing temperature and microfeatures provide differences in physical and topographical cues presented to the hMSCs, and work is underway to examine the cellular responses of these hMSCs to microstructured PGS. Ultimately, micro- and nanostructured PGS may be useful tools in the maintenance, differentiation, and control of signaling pathways in hMSCs.
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Biocompatible Phosphorus-based Monomers for Radical Polymerization.
Christian Heller 1 , Claudia Dworak 1 , Robert Liska 1 , Franz Varga 2
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna Austria, 2 Ludwig Boltzmann-Institute of Osteology, Hanusch Hospital, Vienna Austria
Show AbstractAs many of the most important biochemical molecules are organophosphates, including DNA and RNA it was the aim of our studies to synthesize and characterize a series of monomers based on phosphorus-containing vinylesters and vinylcarbamates. Reactivity of the mono-, di- and trifunctional monomers was evaluated by Photo-Differential Scanning Calorimetry and values for the theoretical heat of polymerization were determined from FTIR measurements in combination with peak deconvolution. With respect to their potential application in the biomedical field, studies on cytotoxicity, hydrolytic degradation behaviour and mechanical stability were performed. Cell viability and experiments on the development of ALP-activity of osteoblast cells revealed a low toxicity of our monomers.
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Nanotopology of Coated poly(ε-caprolactone)/poly(D,L-lactide) Blends Scaffolds for ex vivo Artificial Organ Modeling.
Jolanta Marszalek 1 , Melissa Swint 2 , Eugene Apostolov 2 , Carl Simon 3 , Alexei Basnakian 2 , Alamgir Karim 1 3
1 Polymer Engineering, University of Akron, Akron, Ohio, United States, 2 , University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States, 3 Polymers Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States
Show AbstractThe design and manufacturing of 3D polymeric scaffolds for testing of ex vivo mini-organ model is of significant potential interest and value. To this end, we are interested in ultimately developing an ex vivo mini-kidney model in its natural spheroid growth environment and study its response to various toxic elements as is encountered in the body. Our design includes micro- and nanoporous biodegradable 3-D polymer blend scaffolds to serve as the platform for formation of spheroid mini-kidney and its subsequent toxicity assessment. The polymeric scaffolds are designed with high concentration microporosity as pathways to diffuse in nutrients and toxins, growth factors, and atmosphere, as well as for removal of waste products. In the first part of this project, we evaluated 2D and 2.5D polymer blend scaffold constructs for growth of mouse proximal tubular epithelial (TKPTS) cells. The constructs were prepared from a blend of two biodegradable polymers, cast as thin films from poly(ε-caprolactone)/poly(D,L-lactide) blends that exhibited a strong phase separation behavior influenced by a range of factors. For instance, we observed distinct differences in the surface morphology when the polymer blend was cast from various solvents. In addition, as expected, annealing at various temperatures influenced the phase separation of the two chosen polymers and induced nanotopology on a scale of few hundred nanometers.Our experiments demonstrated that TKPTS cells do not show sufficient attachment to the untreated surface of the phase-separated polymer blend. This is in contrast for instance to our previous osteoblast cell attachment behavior studies that readily attached to the pristine phase separated blend surfaces. To improve cell attachment, several coatings were tested at varying concentrations, including gelatin, fibronectin, agarose and poly-L-lysine. These experiments showed that immersion in the 0.01% poly-L-lysine aqueous solution proved to be beneficial for the proximal tubular epithelial cells attachment and proliferation. Advantageously, a preliminary evaluation of the surface with the gel coating showed no change in the nanotopology of the coated versus the underlying phase separated blend surface.
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Hydrocortisone and Triiodothyronine Inhibit Hyaluronate Production in a Human Dermal Equivalent through Independent Pathways.
Tara Pouyani 1 , Madhura Deshpande 1
1 Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts, United States
Show AbstractWe have previously reported the production of an organized extracellular matrix in vitro under serum free conditions from neonatal human dermal fibroblasts cultured without using a three dimensional artificial scaffold. These constructs resemble the human dermis and produce large amounts of hyaluronic acid (hyaluronate, HA). According to previous studies documented in the literature, hyaluronate synthesis and regulation in human dermal fibroblasts is hormonally controlled. We have studied the effects of the hormones hydrocortisone and triiodothyronine (T3) on HA and extracellular matrix synthesis, as these hormones are present in the base culture media used to prepare these constructs. We used varying concentrations of hydrocortisone and T3 and found that they are capable of inhibiting hyaluronate synthesis through independent pathways. Lower concentrations of hydrocortisone enhance HA synthesis while higher concentrations inhibit HA synthesis. T3 specifically inhibits HA synthesis. We sought to further investigate the effect of combination of these two hormones together on inhibition of HA synthesis in order to investigate its possible function in wound healing and the regenerative process. We find that hydrocortisone and T3 in conjunction with each other show higher inhibition of HA synthesis in the Human Dermal Equivalent than they do independently.
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Size Effects in the Mechanical Properties of the Drosophila Hox Transcription Factor Ultrabithorax Self-assembled Protein Fibers.
Zhao Huang 2 , Yang Lu 1 , Jaimin Shah 2 , Kathleen Matthews 2 , Sarah Bondos 3 2 , Jun Lou 1
2 Biochemistry and Cell Biology, Rice University, Houston, Texas, United States, 1 Mechanical Engineering and Materials Science, Rice University, Houston, Texas, United States, 3 Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, United States
Show AbstractThe remarkable properties of natural protein-based materials have not previously been reproduced in materials generated in vitro from recombinant self-assembling proteins. By controlling the diameter of fibers self-assembled from the Drosophila melanogaster transcription factor Ultrabithorax, we reproducibly generated materials with mechanical properties comparable to natural elastin. Diameter exerts a striking influence on the mechanical properties of Ubx fibers. In the elastic deformation of narrow Ubx fibers (<10μm diameter), strain and final fiber diameter after fracture are independent of the initial diameter, but breaking strength and Young’s modulus decrease sharply with increasing diameter. In contrast, the strain and extent of plastic deformation are dependent on the diameter of wide fibers (>12μm diameter). Wide fibers contain an elastic core, causing the plastically deformed outer layers to wrinkle upon unloading and occasionally separate upon rupture. Controlling fiber size may therefore be a facile way to manipulate the mechanical characteristics of protein fibers, and to rationally design and build large scale protein-based materials and structures with desired properties.
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A Structural Deterministic Model for Electrospun Scaffolds.
Antonio D'Amore 1 3 , John Stella 1 , David Schmidt 1 , William Wagner 1 2 , Michael Sacks 1
1 Bioengineering Department and the McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 3 Departments of Mechanics, University of Palermo, Palermo Italy, 2 Departments of Chemical Engineering and Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Show AbstractSoft tissue regeneration through cell-seeded engineered scaffolds has increased interest in electrospun elastomeric constructs. Structural deterministic modeling can provide mechanistic understanding of the complex multi-scale relations between the material structure and mechanical response, cell deformation and extracellular matrix synthesis. We present a novel modeling strategy to (1) characterize the material fibers network micro-architecture, (2) identify an appropriate Representative Volume Element (RVE) size, (3) reproduce stochastically equivalent fiber network models, and (4) predict the mechanical response at meso-macro level.Sets of electron micrographs (SEM) of electrospun poly (ester urethane) urea (PEUU) scaffolds were analyzed. Mechanical anisotropy and fiber alignment were controlled using different collecting mandrel tangential velocities (1.5, 4.5, 9.0 m/s). A combination of thresholding and morphological procedures enabled fiber-overlaps to be identified, and a modified Delaunay network was created from the fiber-overlap coordinates. Fiber-overlap positions, connectivity, and fiber angle distributions were extracted from the generated network to fully describe the network topology. The procedure was validated performing the image analysis on grids of known characteristics. The RVE size was determined studying the stabilization of the material extracted architectural features over areas of increasing sizes. Stochastically equivalent networks were generated (250x250 µm) for the 3 scaffold groups minimizing the discrepancies between the real material and the simulated networks. The simulated networks were then imported into FEM models wherein the fibers were assumed to be linear elastic using beam elements with known axial and bending stiffnesses. Equi-biaxial stretch conditions were simulated, so that the single fiber mechanical stiffness was the only parameter required to fit the equi-biaxial experimental data.Results indicated that the number of fiber-overlaps decreased as mandrel velocity was increased, and the fiber angle distribution was consistent with our previous findings. At the macro level the non-linear mechanical response agreed well with the experimental biaxial mechanical data. Single fiber mechanical stiffness was estimated to be ~380 kPa. At meso level under applied deformation, the simulated fiber networks exhibited long fiber-like behavior that in-turn dictated the macro-scale material structural anisotropy. The proposed modeling strategy was able to elucidate how local morphology shaped the mechanical response across the scales, and offers a tool for engineering bio-inspired elastomeric electrospun scaffolds.
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Nano-Engineering Biocompatibility of Implant Surfaces for Enhanced Biointegration.
Fereydoon Namavar 1 , John Jackson 2 , Renat Sabirianov 3 , John Sharp 4 , Thomas Gustafson 1 , Roxanna Namavar 1 , Hani Haider 1 , Edward Fehringer 1 , Kevin Garvin 1
1 Department of Orthopaedic Surgery and Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, United States, 2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States, 3 Department of Physics, University of Nebraska , Omaha, Nebraska, United States, 4 Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States
Show AbstractThe knee, hip, and shoulder rank 1-2-3 in terms of the numbers of each joint replaced in the United States, respectively. In 2005, over 249,000 Americans underwent hip replacement and 488,000 had knee replacements. Shoulders, wrists, and even spinal discs are also being replaced more frequently than ever before. In the future, we will see a massive increase in joint replacements as the baby boomers age. Kurtz, et al, [AAOS Chicago, IL, 2006] predict that by 2010 hip and knee replacements will number over one million, and by 2030 we will see 4.5 million annually. Revision replacements are required when primary replacements experience loosening, mechanical failure, or infection. Loosening and movement of the implant occurs because there is no or insufficient osseointegration. Therefore, there is a critical need to develop methods to enhance or prevent cell proliferation as needed, to both improve health and prevent disease. Increasing biointegration of prosthetic surfaces not only will lead to faster tissue integration and vascularization, but also will result in faster patient recovery and the savings of a substantial amount of healthcare dollars.Cell attachment and spreading in vitro is generally mediated by adhesive proteins such as fibronectin and vitronectin. The primary interaction between cells and adhesive proteins occurs through integrin and an RGD amino acid sequence. The adsorption of adhesive proteins plays an important role in cell adhesion and bone formation to an implant surface. The ability of the implant surface to adsorb these proteins determines its aptitude to support cell adhesion and spreading and its biocompatibility [C. Wilson, et, al .Tissue Engineering. 2005, 11 p1]. By applying ion beam assisted deposition (IBAD), we have produced pure cubic ZrO2 (a diamond simulant) coating with the spatial dispersion (roughness) comparable to the protein size (3-20nm). This nanostructured ZrO2 coating is transparent, hard (16 GPa), and displays a total wettability to water and calf serum. It can be deposited at room temperature onto any material with excellent adherence and mechanical integrity. Comparing this engineered coating to the orthopaedics materials, including Ti and hydroxyapatite (HA), indicates that our engineered surfaces are superior (300-500% increase) in supporting the adhesion and proliferation of osteoblasts-like cells of a mouse bone marrow stromal cell line. We will present and discuss experimental results for adhesion and growth of a bone marrow stromal cell on engineered nanocrystalline cubic ZrO2, TiO2, Ti, and compare them with conventional orthopedics grade of Ti and CoCr as well as HA. We will also discuss the application of two mechanisms, i.e., electrostatic and steric complementarity effects that may explain the enhanced adhesion and growth of bone marrow stromal cell to our engineered nanostructured surface as compared to conventional smooth surfaces.
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Delivering Neurotrophins from Microelectrode Arrays to Control the Tissue Response After Implantation.
Sheryl Kane 1 , Fei Wang 1 , Julia Ehrlich 1 , Stuart Cogan 1
1 , EIC Laboratories, Inc., Norwood, Massachusetts, United States
Show AbstractImplanted electrodes have two functions, recording electrical activity and stimulating nearby tissue. Both of these functions rely on the transmission of current between the electrode and the tissue. However, in the central nervous system, it can be very difficult to maintain continuous recording capability due to encapsulation of the electrode. In addition, the implantation process damages the tissue surrounding the implant, which can be particularly problematic in degenerative diseases, where the tissue is already damaged and cannot recover.One way to improve recording and stimulation and minimize post-implantation tissue damage is to deliver a neurotrophin from the electrode surface. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) promote neurite growth and cell survival in certain populations. Neurite extension toward the electrodes could produce neurites that span the encapsulation layer, mitigating is detrimental effects on charge transfer. Also, closer neurites could reduce lower charge injection levels, since charge conduction decreases with distance. Ultimately, these developments could improve the brain-machine interface in implanted devices. In addition, neurotrophin elution may improve neuron survival around the implant, regenerating a population of functional neurons. This would be especially beneficial for the treatment of degenerative diseases.We have developed a poly(ethylene glycol) (PEG)/poly(lactic acid) (PLA) coating to deliver neurotrophins from the surface of implanted electrodes. Precursor solution containing neurotrophin is photopolymerized on the surface of arrays with iridium oxide microelectrodes. Two arrays have been developed: a set of 32 penetrating electrodes which will be loaded with NGF and implanted into cat cortexes to evaluate recording and stimulation capability, and a set of 15 electrodes which will be loaded with BDNF and implanted into the retinas of rats with retinitis pigmentosa to evaluate the effect of BDNF on retinal degeneration.In vitro, the PEG-PLA hydrogel elutes NGF over the course of two weeks, which coincides with the period during which the encapsulation layer forms around electrodes. Cyclic voltammetry indicated that adding the hydrogel to the tips of the penetrating electrodes in the 32-electrode array caused a small immediate reduction in the charge capacity of the microelectrodes. However, the cyclic voltammograms and the charge capacity of the hydrogel-coated electrodes then remained stable for three weeks at 37°C in buffer. These results suggest that hydrogel-coated implanted electrodes should have the capability to elute neurotrophin while recording or stimulating the surrounding tissue during the critical period when implant encapsulation occurs. Additional in vitro studies will elucidate the time courses of BDNF elution and PEG-PLA degradation prior to implantation of neurotrophin-loaded, hydrogel-coated arrays into cortex and retina models.
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A Device for in vitro, High Throughput Cell Tissue Contractility Assay.
Anna Grosberg 1 , Adam Feinberg 1 , JJosue Goss 1 , Kevin Kit Parker 1
1 SEAS, Harvard University, Cambridge, Massachusetts, United States
Show AbstractTo evaluate the viability of cardiac tissue and its response to drug agents, it is essential to measure the tissues contractile performance. Accurate contractility data can aid in development of drugs and evaluation of stem cell derived cardiomyocytes. These research directions would be greatly aided with an efficient in vitro contractility assay. The novel device developed in this work is based on the muscular thin film technology [1], in which a thin film is manufactured with a cardiomyocyte monolayer on one surface. In the new device the muscular thin films remain secured at their base to a glass surface. During the contraction cycle the films bend up from the glass coverslips, and the degree of bending is directly correlated to the contractility of the cardiomyocytes. To construct these, a temperature sensitive polymer, poly(N-Isopropylacrylamide), was deposited in a thin layer onto the center portion of a glass cover slip.A biopolymer, Polydimethylsiloxane (PDMS), was deposited in a thicker layer (~ 10-25 μm) on top of the whole cover slip. An extracellular matrix protein (ECM), fibronectin, was stamped in a pattern onto the PDMS and the cardiomyocytes were seeded onto the ECM. After 3-4 days inculture, the film with the cells was cut and the unwanted regions peeled off the glass cover slip.The temperature was lowered to dissolve the temperature sensitive polymer, and as a result eightrectangles of film remained attached to the glass cover slip at one edge only. The dynamics ofthese tissue constructs was recorded under a stereo microscope and analyzed using ImageJ andMatLab. These proof of concept experiments demonstrated that the assay provides consistentmeasurements of cardiomyocyte contractility in an efficient manner. Additionally, the cardiactissue was exposed to standard inotropic agents, and our assay shows significant changes incontractility as a result. This novel device is a great improvement over current methods as theymeasure the contractility of cardiomyocytes either in vivo or in situ [2], which is time consumingand expensive, or in vitro on a single cells [3], which does not provide information about thewhole tissue.1. Feinberg, A.W., et al., Muscular Thin Films for Building Actuators and Powering Devices. Science, 2007. 317(5843): p. 1366-1370.2. Fujita, A., et al., IMPROVEMENT OF DRUG-INDUCED CARDIAC-FAILURE BY NKH477, A NOVEL FORSKOLIN DERIVATIVE, IN THE DOG HEART-LUNG PREPARATION. Japanese Journal of Pharmacology, 1992. 58(4): p. 375-381.3. Miyake, R., et al., Characterization of positive inotropic effect of colforsin dapropate hydrochloride, a water-soluble forskolin derivative, in isolated adult rat cardiomyocytes. Canadian Journal of Physiology and Pharmacology, 1999. 77(4): p. 225-234.
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Migration of Stem Cells in Response to Sustained Release of Stromal-Derived Factor-1α.
Xuezhong He 1 , Esmaiel Jabbari 1
1 Chemical Engineering, University of South Carolina, Columbia, South Carolina, United States
Show AbstractRecent clinical trials demonstrate the involvement of SDF-1α in mobilization of immature human CD-34+ cells. Furthermore, it has been demonstrated that marrow-derived osteo-progenitor cells are mobilized after bone fracture and migrate to the in injury site by chemoattraction of SDF-1α to form bone in transplanted animals. Therefore, addition of SDF-1α to the implanted scaffold can potentially enhance mobilization, migration, and homing of osteo-progenitor cells to the regenerating region and accelerate bone formation. However, native SDF-1α has a relatively short half-life. The objective of this work was to determine the release characteristics of SDF-1α from novel biodegradable poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogels and its effect on migration of bone marrow stromal cells.The polymerizing mixture consisting of sterile PLEOF macromer, N,N'-methylene-bis-acrylamide crosslinker, and SDF-1α (250 ng/ml) was crosslinked with ammonium persulfate (APS) and tetramethylethylenediamine (TMEDA) to form a hydrogel. For release experiments, disk-shaped hydrogels were incubated in PBS at 37°C under mild agitation. At each time point, the active concentration of SDF-1α was measured by ELISA using a recombinant human CXCL12/SDF-1α Quantikine ELISA Kit. A transwell cell migration assay was used to measure cell migration in response to different SDF-1α concentrations. Bone marrow stromal (BMS) cells, isolated from rats, (labeled with calcein AM) were seeded in the upper transwell. The seeded cells were allowed to migrate from the upper chamber to the lower side of the membrane for 12 h in response to SDF-1α gradient. Next, the cells in the upper chamber were removed using a sterile coated swab and migrated cells were enzymatically lifted from the lower side of the membrane. The number of migrated cells was quantified by measuring the fluorescence with a plate reader.Encapsulation efficiency of SDF-1α in PLEOF hydrogel was 74±3%. The release SDF-1α from PLEOF hydrogel consisted of a burst release (approximately 20%) followed by a constant release rate. Approximately 90% of the encapsulated SDF-1α was released in teh fisrt two weeks. In the absence of SDF-1α, only 4.5±0.4% of the seeded cells migrated to the lower side of the membrane after 12 h. On the other hand, after 15 days incubation, 170 ng (90% with 75% encapsulation efficiency) SDF-1α was released from the hydrogel which increased the fraction of migrated cells to 22±1%. Therefore, 15 days release of SDF-1α from the hydrogel resulted in 4.8 fold (4.5 to 22%) in migration rate. Migration of BMS cells in response to SDF-1α released from PLEOF hydrogel increased by 4.8 fold, compared to hydrogel without encapsulated SDF-1α. Sustained delivery of SDF-1α in biodegradable implants can potentially enhance mobilization, migration, and homing of progenitor cells to the regenerating.
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Functionalization of Nanofibrous Spiral Structured Scaffolds for Bone Tissue Engineering.
Junping Wang 1 , Chandra Valmikinathan 1 , Xiaojun Yu 1
1 , Stevens Institute of Technology, Hoboken, New Jersey, United States
Show AbstractWe have developed a 3D nanofibrous spiral scaffold for bone tissue engineering which has shown enhanced cell attachment, proliferation and differentiation compared to traditional cylindrical scaffolds due to the spiral structures and the nanofiber incorporation. In order to further improve the performance of the established PCL nanofibrous spiral structure scaffolds, we functionalized the scaffolds with Hydroxyapatite (HAP) and bone morphogenetic protein 2 (BMP-2). Specifically, we blended HAP into PCL at the ratio of 1:1 (wt:wt) and fabricated PCL-HAP spiral structured nanofibrous scaffolds through the combination of solvent casting, salt leaching and electrospinning. In addition, BMP-2 was loaded on the scaffolds by absorption. Three experiment groups were prepared: PCL spiral nanofibrous scaffold; PCL-HAP spiral nanofibrous scaffold w/o BMP-2. Human osteoblastic cells are seeded on these scaffolds and cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds are evaluated. Our results have suggested that 76.58% ±2.56% of BMP-2(0.3µg) can be efficiently loaded on this scaffold (80mg). And the scaffold was able to release BMP-2 over a 3-week period in a sustained manner. Interestingly, with the addition of HAP, cell attachment significantly decreased due to the surface roughness of the PCL-Hap scaffolds. Cell numbers on PCL-HAP scaffolds (w/o) BMP2 at day 4 and day 8are significantly lower than the PCL spiral scaffolds. However, the difference is decreasing with the time goes on. At day 14, there is no significantly difference of cell numbers between the control group and the PCL-HAP-BMP group. The alkaline phosphatase (ALP) study of the cell differentiation has shown that PCL-HAP scaffolds has significantly higher ALP expression level at day 4, 8and 14. Alizarin red S calcium staining and xyolene orange staining have demonstrated that calcium deposition and the newly formed mineralization matrix on PCL-HAP-BMP are significantly higher than those of other groups. Taken together, the addition of HAP and BMP-2 significantly improve the bioactivity of the spiral structure scaffolds, thus demonstrating the great potential of the functionalized spiral structured nanofibrous scaffolds for bone tissue engineering applications (The work was supported by the Early Career Translational Research Award in Biomedical Engineering from Wallace H.Coulter Foundation).
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Squid Beak Inspired DOPA-histidine Hydrogels.
Dominic Fullenkamp 1 , Kyle Holmberg 1 , Phillip Messersmith 1 2 3
1 Biomedical Engineering, Northwestern University, Evanston, Illinois, United States, 2 Materials Science and Engineering, Northwestern University, Evanston, Illinois, United States, 3 Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States
Show AbstractThe Dosidicus gigas squid beak is a non-mineralized, high modulus tissue composed primarily chitin and protein. Interestingly, histidine (His) makes up ~10% of the beak protein, an overrepresentation compared with most proteins. In addition, catechol-induced tanning heavily pigments the beak. Recent work from Waite and coworkers has shown that cross-links between 3,4-dihydroxyphenyl-L-alanine (DOPA) and His to be a dominate species in the quinone-tanned part of the beak. Similar catechol-His cross-links have long been observed in insect cuticles. Building upon these observations, we have investigated the conditions required to form cross-links from DOPA and His-modified linear polyethylene glycol (PEG). GPC and MALDI-TOF MS were used to characterize cross-link formation. Sodium periodate-initiated oxidation of DOPA to its quinone form was found to induce DOPA-His cross-links. Optimized conditions derived from these model studies were used to form hydrogels from 4-armed DOPA and His-modified PEG polymers that were studied by standard rheological techniques. These hydrogels may be candidates for medical adhesives, with cohesive mechanical properties contributed by DOPA-His cross-links and wet adhesive properties due to DOPA-mediated adhesion to tissues.
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Biocompatibility Assessment of SiC Surfaces After Functionalization with Self Sssembled Organic Monolayers.
Alexandra Oliveros 1 , Sebastian Schoell 2 , Christopher Frewin 1 , Marco Hoeb 2 , Ian Sharp 2 , Stephen Saddow 1
1 Electrical Engineering, University of South Florida, Tampa, Florida, United States, 2 Walter Schottky Institut, Technische Universität München, Garching Germany
Show AbstractThe integration of biological and micro-engineering techniques have allowed development of new platforms for improving both biomedical devices and pharmaceutical delivery technologies. Wide-bandgap semiconductors have played an important role in this field because they possess superior sensing capabilities and good biocompatibility. The use of such materials for long-term implantable devices requires a high degree of biocompatibility that implies cell surface attachment and spreading as well as a specific surface growth morphology. SiC is known to be a chemically inert and biologically-permissive substrate, making it a suitable material for the fabrication of biosensors. In this study we show that functionalization of SiC surfaces using methyl- and amine-terminal organosilane and alkyl self-assembled molecular monolayers significantly increases the substrate biocompatibility. We used two cell lines, H4 human neuroglioma and PC12 rat pheochromocytoma, and in vitro techniques to demonstrate the enhanced biocompatibility of SiC substrates following molecular surface modification. MTT, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide, assays were performed to determine general cell viability on each substrate. Atomic force microscopy (AFM) was used to quantify the general cell morphology on substrate surfaces along with the substrate permissiveness to cellular filopodia and lamellipodia extensions. The identified cell morphology (i.e. elongated and membrane extension) and low angle of attachment (~ 5°-25°) demonstrate the permissiveness of the evaluated substrates and the MTT assays show an increase in cell viability of as much as two times for the PC12 cell line and three times for the H4 cell line with respect to the untreated surfaces. These results represent a dramatic improvement in cell viability using these functionalization strategies which may be utilized for development of future biosensor and medical devices.
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Engineering Local Cytokine Delivery Systems for Preventing Open Fracture Associated Infections.
Bingyun Li 1 2 3 , Bingbing Jiang 1 , Matthew Dietz 1 , Murali Rao 4
1 Orthopaedics, West Virginia University School of Medicine, Morgantown, West Virginia, United States, 2 WVNano Initiative, West Virginia University, Morgantown, West Virginia, United States, 3 Chemical Engineering, West Virginia University, Morgantown, West Virginia, United States, 4 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Center for Disease Control and Prevention, Morgantown, West Virginia, United States
Show AbstractInfection is a significant clinical complication associated with a variety of synthetic biomedical devices. The infection rate is still high especially in patients with traumatic open fractures; the rate of infection may exceed 30% (1,2). Systemic antibiotics have been the primary treatment for infection. However, antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (3) and Acinetobacter baumannii (4,5) have been making treatment more problematic and challenging for civilian and military patients. We have developed advanced biomaterials using a combination of bioengineering, in vitro, and in vivo approaches. Nanocoatings are prepared on orthopaedic implants using electrostatic layer-by-layer self-assembly nanotechnology (6). Various drugs including cytokines (e.g. monocyte chemoattractant protein-1, interleukin-12) and growth factors (e.g. bone morphogenetic protein-2) are incorporated in the nanocoatings. The loading and release of drugs can be finely tuned. In vivo studies have shown that our developed non-antibiotic therapies, which stimulate cell-mediated immune response, can substantially decrease open fracture associated infection (infection rate decreased from 90% to 20%).References:1. Zalavras CG, Patzakis MJ. (2003). Open fractures: evaluation and management. J Amer Acad Orthop Surg. 11(3):212-9. 2. Schmidmaier G, et al. (2006). Prophylaxis and treatment of implant-related infections by antibiotic-coated implants: a review. Injury. 37(S2):S105-12.3. Klevens RM, et al. (2007). Invasive Methicillin-Resistant Staphylococcus aureus Infections in the United States. JAMA. 298:1763-71.4. Scott PT, et al. (2004). Acinetobacter baumannii infections among patients at military medical facilities treating injured U.S. service members, 2002—2004. Morbidity and Mortality Weekly Report, CDC. 53:1063-6.5. Murray CK, et al. (2006). Bacteria recovered from patients admitted to a deployed US military hospital in Baghdad, Iraq. Mil Med. 171(9):821-5.6. Li B, Jiang B, et al. (2009). Multilayer polypeptide nanoscale coatings for the prevention of biomedical device associated infections, Biomaterials. 30:2552-8.
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Magnesium-based Biodegradable Nanostructured Substrates: Hybrid Materials for Cellular Engraftment.
Tobias Schoen 1 , Tobias Wolfram 2 , Joachim Spatz 1
1 New Materials and Biosystems, MPI for Metals Research, Stuttgart Germany, 2 Research Group for Biomedical Engineered Surfaces, MPI for Metals Research, Stuttgart Germany
Show AbstractMagnesium and its alloys are used as implants because of its biocompatibility and its high strength-to-weight ratio. Medical engineering applications are restricted by the corrosion behavior of magnesium-containing materials. In contrast to other commonly used implantable materials such as stainless steel and Co-Cr-alloys, which may release toxic metallic ions, magnesium belongs to the natural composition of the human body. Magnesium may have stimulatory effects on bone tissue growth, and cells on Mg-enriched substrates express enhanced levels of α5β1 integrin receptors and of collagen1 proteins.Our approach is to engineer a cellular carrier system based on biodegradable magnesium substrates or magnesium alloys, which are decorated with gold nanostructures to form Mg-Au hybrid materials. For that purpose and to facilitate initial cell adhesion of specific cells on magnesium substrates, diblock copolymer micelle nanolithography was employed to fabricate gold nanoparticles on magnesium oxide single crystals (100 orientation) and on AZ31-Mg alloy substrates. The Au nanoparticles were arranged in ordered arrays with interparticle distances of 25 to 300 nm. In addition, homogeneous Au films were sputter-coated on the substrates as a comparison. The Au films and the Au nanoparticles were further functionalized with RGD-thiol or NTA-thiol that can be used to immobilize tagged proteins. This method allows the deposition of a protein carpet on top of the surface. The corrosive behavior in water, PBS and cell culture medium was studied by weighing the substrate and by scanning electron microscopy. Corrosion rates of Mg substrates in PBS and cell culture medium were significantly higher than in water. C2C12 mouse myoblasts and human mesenchymal stem cells were cultured on MgO and AZ31-alloys and investigated with scanning electron microscopy, fluorescence and phase contrast microscopy. Live cell imaging analysis was performed over 8 hours to investigate spreading and adhesion of C2C12-cells with and without presence of Mg2+ ions.The spreading and survival of C2C12 myoblasts on the Mg-Au hybrid materials was different to that on plain Mg surfaces. In contrast to Mg-Au hybrid nanostructures, cells on plain MgO crystals showed reduced filopodia activity. Control experiments revealed that dissolved Mg2+ ions had no influence on cell behaviour, confirming that the observed effects derived from the substrate surfaces.Specifically biofunctionalized and engineered magnesium-based substrates can be used as carriers for different cellular systems. By modifying the surface of these devices, the interaction properties of the material with biological systems can be tuned. Our results indicate first steps towards an implantable device with a defined biological surface activity made from magnesium materials, functioning as a biodegradable cell carrier system for example in cell-based therapies for structural heart disease.
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In-situ Patterning Multiple Cells based on Surface Tension of Fluids in Multi-level Microfluidic Environment.
Sung Hoon Lee 1 , Sunghwan Shin 1 , Wook Park 1 , Sung-Eun Choi 1 , Sunghoon Kwon 1
1 , Seoul National University, Seoul Korea (the Republic of)
Show AbstractWe propose a method for in-situ patterning multiple cells using surface tension of fluids in multi-level PDMS microfluidic channel, which is deep and shallow, fabricated with two-step lithography. After conducting O2 plasma surface treatment on PDMS channel and substrate, liquid solutions can be easily filled through shallow channel because of capillary force and maintained stable with surface tension of fluids. Gas can be ventilated through deep channel, which enables cells to be in proper condition.Patterning cells and proteins in vivo microenvironment provides new opportunities for tissue engineering, development of drug discovery and cell biology. Various techniques for patterning cells have been reported so far. Laminar flow method is a simple process with low cost, but it is limited to the shape of the flow profile. On the other hand, microcontact printing can make various shape of pattern. However, the laborious steps are required for heterogeneous patterning. Moreover it is hard to maintain the protein pattern after O2 plasma treatment, which is essential step for creating microfluidic cell-culture environment.Using the method based on surface tension that we proposed, complex cell pattern can be conveniently achieved with small amount of pipetting. By designing the channel in variety, arbitrary pattern of multiple cells can be generated and cultured inside the microfluidic channel. Heterogeneous cells, which is NIH-3T3 and Hela are injected and cultured in one single chip. The width, height of the shallow channel is controlled to observe the effect of channel dimension on pattern formation when the solution is injected. Detailed modeling regarding surface tension, hydrophilicity of surface and geometry of channels are also taken into account. For patterned cell culture, medium flow and air ventilation for cell living needs to be considered when designing the channel. The medium flow is initiated by the volume difference between inlet and outlet of shallow channels. Holes are punched into the inlets and outlets of the deep channel, so that air can be supplied easily into the microfluidic channel. When cells are seeded to shallow channel, it selectively flows into the channel, which patterns specific shape. After attaching cells on the substrate, the inlets are filled up with the media. In this way, multiple cells, which is NIH 3T3 and Hela cells are independently introduced and co-cultured in the single chip. Viability test confirms that cells inside the chip are in proper condition to be cultured.Using surface-tension based pattering method, we demonstrated that in situ multiple types of cells are patterned in microenvironment, in situ. This technique enables convenient fabrication of various patterns inside a microfludic channel. We envision that our technique can be used as a novel method for heterogeneous biomaterial patterning, which would be applicable for drug-screening, cell-cell interaction and biosensors.
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The Effect of Surface Roughness on the Attachment and Proliferation of Osteoblast Cells on 45S Bioactive Glass.
Raina Jain 1 , Shaojie Wang 2 1 , Hassan Moawad 2 , Matthias Falk 1 , Himanshu Jain 2
1 Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Department of Materials Science and Engineering, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractThe classic 45S bioactive glass appears promising as a bone implant material with superior biocompatibility and bioactivity. Past research on titanium implants showed that its surface roughness significantly affects the adhesion of bone cells. Much less is known about the role of surface roughness of glass on its use as an implant. In this work we systematically investigated the effect of surface roughness on cell adhesion and proliferation on 45S glass.The 45S glass samples were made by melting SiO2, CaCO3, Na2CO3 and Ca5(OH)(PO4)3. Varying surface roughnesses, with Ra = 1.2 to 0.01 micrometers were produced by polishing the samples with silicon carbide paper or cerium oxide. To assess the effect of surface roughness on cell proliferation and initial adhesion, MG63 osteosarcoma cells or MC3T3 preosteoblast cells were harvested by trypsinization and subsequently seeded on the glass samples and incubated for different times. After 1-7 days postseeding, MG63 cells were stained and photographed using an inverted fluorescent microscope. Cell density was determined manually to obtain the proliferation rate. The morphology and focal adhesion sites of MC3T3 cells were observed after staining for the vinculin adhesion protein, the F-actin cytoskeleton, and the nucleus. To quantify adhesion, a novel, simple method was developed: the glass samples were shaken for 30 seconds 3, 6, 12, and 24 hours postseeding thereby dislodging the poorly adhered cells. The nuclei of the remaining well-adhered cells were stained and counted. The proliferation results reveal that after the initial stress from trypsinization, the cells recover and begin to proliferate. The cell proliferation increases with increasing surface smoothness. In the early stages of proliferation the cells tend to be rounded and clumped together on rough surfaces in contrast to being elongated and spread out on smoother glass surfaces. Overall the proliferation rate was the highest on smoothest surfaces, similar to a previous report, but in contrast to the observations on titanium surface. Interestingly, cell alignment was observed on glass for the first time, when the scratch width was comparable to the cell size. In respect to the initial adhesion of MC3T3 cells, there were initially a higher number of well adhered cells on the roughest surface. However, over time, the number of well adhered cells on the rougher surface decreased while simultaneously, the number of well adhered cells on the smoother surfaces increased. Additionally, cells tended to spread out and form long extensions on smoother samples, indicating better adhesion over time, while cells on rougher surfaces tend to be rounded. Also seen, was a lack of connection between the focal adhesion protein vinculin and the actin cytoskeleton on three out of four roughnesses, revealing the effect of surface roughness on intracellular structure. The authors thank the NSF for supporting this work (DMR-0409588 and DMR-0602975).
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Effect of Surface Topography and Chemistry on Osteoblast Biomineralization.
Kathryn Dorst 1 , Yizhi Meng 1
1 Materials Science and Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractBone cell differentiation depends on the mineralization of an extracellular matrix (ECM), which functions as a substrate for motility, a reservoir for growth factors, and a barrier against host immunocompetent cells. How cellular functions are influenced by ECM-surface interactions remains to be explored. A determination of the topographical and chemical parameters that govern the initial attachment of osteoblasts on engineered tissues can provide valuable insight into determining strategies for biomineralization in vitro and bone regeneration in vivo. In our work, we examined the osteogenic development of two subclones of osteoblast-like cells cultured on micro- and nano-patterned surfaces that were functionalized with ECM proteins. Initial cell attachment and migration were observed with time-lapse microscopy. The formation of a fibrillar ECM network was characterized by scanning probe microscopy and SEM. Osteoblast differentiation was measured by alkaline phosphatase activity, osteocalcin and osteopontin production, and immunofluorescence.
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Cellular Interactions with a Tissue-engineered ECM Mimic.
Zhi Pan 1 , Kaustabh Ghosh 2 , Yajie Liu 3 , Xiaozheng Shu 4 , Glenn Prestwich 4 , Richard Clark 2 , Miriam Rafailovich 1
1 Materials Science & Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 2 Biomedical Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 3 Mechanical Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 4 Medicinal Chemistry, University of Utah, Salt Lake City, Utah, United States
Show AbstractTissue-engineered hydrogels composed of intermolecularlly crosslinked hyaluronan (HA-DTPH) and fibronectin functional domains (FNfds) were applied as a physiologically relevant ECM mimic with controlled mechanical and biochemical properties. Since both HA and FN are important components of ECM for cell migration and tissue organization during tissue repair, this hydrogel is an excellent agent for in vivo applications. Meanwhile, it provides an ideal system for in vitro studies at cell level because the mechanical and the biochemical properties of this hydrogel can be independently controlled by varying the crosslinking ratio and ligand (FNfds) type/bulk density respectively. Here, cellular interactions with this tissue-engineered environment, especially physical interactions (cellular traction forces), were quantitatively measured by using the digital image speckle correlation (DISC) technique and finite element method (FEM). By correlating with other cell functions such as cell morphology, stiffness and migration, a comprehensive structure-function relationship between cells and their environments was identified. Furthermore, spatiotemporal redistribution of cellular traction stresses was time-lapse measured during cell migration to better understand the dynamics of cell mobility. The results suggest that the reinforcement of the traction stresses around the nucleus, as well as the relaxation of nuclear deformation, are critical steps during cell migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration.
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High-Throughput Polymer Microarrays Screening –Identification of Stem Cell Selective Materials for Tissue Engineering.
Ferdous Khan 1 , Rahul Tare 2 , Mark Bradley 1 , Richard Oreffo 2
1 School of Chemistry, The University of Edinburgh, Edinburgh United Kingdom, 2 Bone and Joint Research Group, University of Southampton, Southampton United Kingdom
Show AbstractIt is established that mesenchymal stem cells (MSCs) present in bone marrow can give rise to cells of the stromal (adipogenic, chondrogenic, osteoblastic, myoblastic and fibroblastic) lineages and can generate intermediate progenitors. An ideal biomaterial would influence this heterogeneous progenitor cell mix in vivo. Here we present a high-throughput microarray screening, testing and analysis of some 500 bioresorbable / biodegradable polymers for the identification and selection of cell compatible polymers, which allow manipulation of MSCs / osteoprogenitors from hBMMSC populations. The microarray format allows massive parallel cellular analysis while consuming minute amounts of materials. Polymer microarrays were fabricated either by contact (QArray) or inkjet printing (Microdrop). The analysis of the cells immobilized polymer arrays was performed using an automated high content microscope that allowed capture and analysis of the images for each polymer spot using the software Pathfinder, allowed to identify specific cell binding polymers. Crucially different polymers were found that were able to selectively immobilize MSCs / osteoprogenitors from the hBMMSC populations, in a highly specific manner. Different polymers were also found to specifically bind Fetal, MG63 or SaOS cells. Some of the identified materials formed hydrogels that offered a non-biological alternative to Matrigel, and / or resulted in a microporous scaffold which supported cell growth in a 3D sense useful for tissue re engineering or regeneration.This study demonstrated that the polymer microarray is a high-throughput versatile format allowing the generation and identification of innovative biocompatible materials with wide range of applications in regenerative medicine. Microarrays allow large libraries of polymers to be screened for various cellular applications, the identification of new polymers for the attachment of various cell types, including adult stem cells, which are of significant interest. This approach allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth and differentiation both in in vitro and in vivo, and demonstrated the potential application in tissue engineering. Also this approach can serve as an important tool in applications where cell-selective polymers can be used for stem cells manipulation and to support specific types of cell growth.
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Expediting the Wound Healing Process using an Improved Alginate Wound Dressing.
Joseph White 1 , Xuan Luu 2 , Peter Wu 2 , Patrick Lee 2 , Susan Roberts 1 , Surita Bhatia 1
1 Chemical Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States, 2 Department of Surgery, Baystate Medical Center, Springfield, Massachusetts, United States
Show AbstractThe recent increase in patients with type 2 diabetes (T2D) has led to greater interest in the management of diabetic complications and an improved quality of life for patients suffering from the disease. One of the major sequelae of T2D is lower extremity neuropathy and poor wound healing, which leads to ulceration of the lower leg and foot. To provide optimal healing conditions for wound healing, a wide variety of dressings are employed, however, without clear best practice recommendations.The ideal wound dressing should provide a moist environment, the ability to absorb blood and excess exudate, and permit sufficient gaseous exchange while resisting pathogen invasion. Many commercial wound care products use alginate hydrogels as the dressing scaffold due to its tunable mechanical properties and biocompatibility. Although high porosity enables hydrogel wound dressings to hold up to 90% of their weight in water, hypoxic conditions at the wound site often cannot be avoided due slower diffusion of oxygen when compared to diffusion rates in monolayer culture and vascularized tissue. In diabetic patients, the wound site is hypoxic in the interior and exterior due to the lack of oxygen in the blood stream and the low oxygen diffusion through the wound dressing. This study uses a modified alginate formulation to overcome this diffusion limitation and increase oxygen transport through wound dressings to expedite the healing of diabetic foot and leg ulcers. We quantify the effect of the improved formulation via in vitro and in vivo experiments using human keratinocytes and diabetic vs. non-diabetic rats, respectively. This study also investigates the mechanical integrity of the alginate hydrogels by means of rheology and swelling analysis.
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Improved Alginate Formulation for Microencapsulation of Islet Cells.
Whitney Stoppel 1 , Melissa Brown 2 , Kyuong-sik Chin 1 , Alan Schneyer 2 , Susan Roberts 1
1 Chemical Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States, 2 , Pioneer Valley Life Sciences Institute , Springfield, Massachusetts, United States
Show AbstractCell encapsulation is a powerful in vivo technique for isolating cells and other particles from the host immune system for implantation applications. Numerous materials and cell types have been investigated in the past decade, but cell encapsulation is not widely used in the clinic due to poor oxygen and nutrient transport through the capsule compared to vascularized tissue. It has been shown that autoimmune diseases such as type 1 diabetes (TID), respond positively to cellular microencapsulation therapies through investigations such as those described by the Edmonton Protocol. Our laboratory explores fundamental challenges in encapsulating pancreatic islets of Langerhans for the treatment of T1D, with the goal of improving current methods and formulations while maintaining and enhancing islet functionality.Diffusion limitations between the patient’s blood stream and the transplanted islets pose another challenge. An islet is a cluster of many cell types, averaging about 150μm in diameter. The insulin producing beta cells are located in the center of the islet, making them the most susceptible to hypoxia and lack of nutrients. Previous work by islet experts has shown that culturing explanted islets with perfluorodecalin, a perfluorocarbon (PFC), enhances islet viability and functionality in culture. To improve oxygen diffusion and islet function, a novel alginate/ PFC formulation has been developed. Both islet function and capsule stability were monitored over time, suggesting formulations for both promoting capsule structural integrity and appropriate insulin secretion.
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Synthetic Viruses for Neural Tissue Regenerating Materials.
Seung-Wuk Lee 1 2 , Woo Jae Chung 1 2 , Anna Merzlyak 1 2
1 Bioengineering , University of California, Berkeley, Berkeley, California, United States, 2 Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States
Show AbstractWe have developed radically novel synthetic viruses which can control and guide cell behavior for tissue engineering materials using M13 bacteriophage (viruses). M13 viruses were engineered to display various eptides that promote cell interaction on all 2700 copies of major coat proteins. These viruses were biocompatible to neuronal cells which was verified using viability assays. Neural progenitor cells could both proliferate and differentiate when grown on viral surfaces and that there was a preference of specific cell interaction with the RGD- and IKVAV-peptide engineered phages over wildtype phage. We have demonstrated that such engineered phage can self-assemble into directionally organized structures, which in turn dictate the alignment and direction of cell growth in two dimensional and three dimensional tissue engineering matrices. These smart and novel engineered virus-based materials can be used for neural cell growth in vivo and cure challenge disease such as spinal injury in the future.
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Generation of Endothelialized Vascular Networks in Biodegradable Silk Fibroin Scaffolds.
Katie Megley 1 , Kimberly Wall 1 , David Truong 2 , James Hsiao 1 , Sarah Tao 1 , David Kaplan 3 , Ira Herman 2 , Jeffrey Borenstein 1
1 , Charles Stark Draper Laboratory, Cambridge, Massachusetts, United States, 2 Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts, United States, 3 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Show AbstractOver the past few years, substantial progress has been made in the development of engineered tissues for a wide range of clinical applications including wound and burn repair. However, progress towards engineering more complex tissues and organs has historically been limited by the difficulty in creating artificial tissues and organs complete with an integrated and functional vasculature, a necessity for delivery of oxygen and nutrients to the engineered construct. Vascular networks must be located within 100-200 µm of highly metabolic organs such as the heart, lung, and liver. And, while it has been demonstrated that host blood vessels can invade the implanted tissue, therein enabling spontaneous blood vessel formation, the rate of neovascularization is very slow, such that millimeter size implants would take weeks or months to re-vascularize. As a result, insufficient vascularization can cause improper cell integration or cell death in engineered tissue constructs. The complexity of achieving a replacement tissue with a patent and sustainable microvasculature represents a rate limiting step in the creation of most replacement tissues or organ systems.By creating a three dimensional scaffold with an intrinsic microvascular network, the necessary proximity to organs and tissues can be achieved to transport the required nutrients to a specific location. Presented here is a device fabricated from an FDA approved material, silk fibroin (SF), derived from the Bombyx mori (B. mori) silk worm. The biodegradable SF is a non-inflammatory, non-immunogenic, high strength biomaterial. Patent microvascular chambers were produced using soft lithographic techniques to create a polydimethylsiloxane (PDMS) inverse mold, and then silk solution cast over them to form a replica mold. Fabricated microvascular devices were seeded with human dermal microvascular endothelial cells (HDMVECs) at a concentration of 1 X 106 cells/mL and incubated at 37°C and 5% CO2. Supplemented media was perfused at a rate of 1 µL/min after a static attachment period of 16 hours. Cells were observed as attached and proliferating perfusing 2 and 6 days post perfusion. Our devices show the potential for growth of a confluent layer of endothelial cells within the microvascular network. Ultimately this device can be integrated into a stacked co-culture system that will be tailored with organ-specific cell types on the interior and exterior of the scaffold. By specifying these cells types for a particular application, the device can be integrated into a range of tissues and organs for regenerative therapy.
9:00 PM - RR3.7
Fabrication of an Artificial 3-Dimensional Vascular Network Using Sacrificial Cotton Candy.
Leon Bellan 1 2 , Teresa Porri 2 , Harold Craighead 2
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States, 2 Nanobiotechnology Center, Cornell, Ithaca, New York, United States
Show AbstractUsing sacrificial sugar structures, we have formed a 3D fluidic vascular network in a polymeric matrix. Melt-spun sugar fibers (cotton candy) were used to form channels with diameters and densities similar to those of natural capillaries. The sacrificial microstructures were formed using an inexpensive commercially available cotton candy machine and store-bought granulated sugar. To interface to macroscopic fluidic systems, larger sacrificial sugar structures were used to form artificial inlet and outlet macrochannels confluent with the microchannel network. A matrix material was poured over the sacrificial sugar structures, allowed to solidify, and then the sugar structures were removed by dissolution to leave a fluidic network. To verify that the channel network supported flow, we used video fluorescence microscopy to image both 2 µm fluorescent polystyrene spheres in an aqueous solution and fluorescently labeled blood. The channel network geometry was characterized using brightfield and multiphoton optical microscopy as well as scanning electron microscopy. Due to the large extent of the sacrificial microstructures, the resulting microchannel network had significant extent (on the order of centimeters) in all three dimensions. The three dimensional microfluidic networks produced using this technique have many potential applications, including artificial vascular networks for engineered tissue, fluid mixing, and providing healing agents in self-healing polymer systems. This fabrication process may be applied to a wide range of polymeric materials and is rapid, inexpensive, and highly scalable.
9:00 PM - RR3.8
A novel Elastin based Biomaterial for Enhanced Wound Repair and Regeneration.
Piyush Koria 1 2 3 , Zaki Megeed 1 2 3 , Yaakov Nahmias 1 2 3 , Martin Yarmush 1 2 3
1 Surgery, Massachusetts General Hospital, Charlestown, Massachusetts, United States, 2 , Harvard Medical School, Boston, Massachusetts, United States, 3 , Shriners Hospital for Children, Boston, Massachusetts, United States
Show AbstractA staggering one million burn injuries occur in the United States every year and more than half of those require hospitalization. Many clinical studies have demonstrated beneficial effects of exogenous growth factors e.g. Keratinocyte Growth Factor (KGF) on the healing process. Exogenous KGF improves wound healing significantly, but its bioavailability in therapeutic applications is limited by diffusion, clearance and short half life in the harsh wound environment. Here, we describe a novel biomaterial comprising of a fusion protein of KGF and Elastin like peptides (ELPs). This fusion protein retains the characteristic inverse phase transitioning behavior of ELPs as well as the bioactivity of recombinant KGF. The inverse phase transitioning behavior of ELP promotes the formation of 500 nm diameter particles at temperatures greater than 30 0 C. We further show that this biomaterial improves growth as well as wound healing in primary keratinocytes similar to exogenous KGF. Interestingly, the fusion protein was readily internalized by cells and this internalization was mediated by the KGF receptor. Our results suggest that KGF-ELP fusion protein could be delivered as particles at the wound site; where they may act as “drug depots” ensuring the presence of the growth factor by preventing its loss due to diffusion. This may help in accelerating the rate of healing of burn injuries, secondary donor sites, or chronic skin wounds.
9:00 PM - RR3.9
Using Hydrogel Arrays to Characterize the Influence of Environmental Cues on Stem Cell Behavior.
William King 1 , Leenaporn Jongpaiboonkit 1 , William Murphy 1 2 3
1 Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States, 2 Materials Science, University of Wisconsin-Madison, Madison, Wisconsin, United States, 3 Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin, United States
Show AbstractDevelopment of hydrogel materials that present specific biochemical signals to cells in 3D matrices has been an emerging research area in materials science. The traditional method to characterize the influence of environmental signals on cells involves varying one signal per material construct. This traditional approach could limit one’s ability to characterize cell behavior in 3D, because cell behavior is regulated by a diverse combination of soluble and insoluble signals. Recently, we have formed and characterized poly(ethylene glycol)-based hydrogel arrays that contain 16 independent array spots. These array spots can be filled with a combination of cells, growth factors, cell adhesion peptides, non-degradable polymers, and degradable polymers to define the cell’s local environment. In this study, we examined the combinatorial effects of fibroblast growth factor-2 (FGF2), the cell adhesion peptides RGSP and IKVAV, and hydrogel degradability on human mesenchymal stem cell (hMSC) survival and spreading. FGF2 increased hMSC survival in a dose-dependent manner in both degrading and non-degrading hydrogels. Significantly, a small molecule inhibitor of FGF2 receptor autophosphorylation abrogated FGF2-promoted hMSC survival. FGF2 had a decreased effect on cell survival in non-degrading hydrogels when hMSCs were cultured in the presence of RGDSP or IKVAV. Significantly, array spots with FGF2, cell adhesion peptides, and degradable polymers allowed for hMSC spreading, and the degree of cell spreading was FGF2 concentration-dependent. These results indicate that poly(ethylene glycol)-based hydrogel arrays may provide a broadly adaptable platform to combinatorially screen for environmental signals that influence hMSC behavior.
Symposium Organizers
Sujata Bhatia Dupont Corporation
Stephanie Bryant University of Colorado
Jason A. Burdick University of Pennsylvania
Jeffrey M. Karp Harvard-MIT Division of Health Sciences and Technology
Katie Walline CeraPedics, Inc.
RR4: Advanced Scaffold Design
Session Chairs
Milica Radisic
Katie Walline
Tuesday AM, December 01, 2009
Back Bay C (Sheraton)
9:30 AM - **RR4.1
New Strategies in Biomaterials Design for Musculoskeletal Tissue Engineering.
Molly Stevens 1 2
1 Materials, Imperial College London, London United Kingdom, 2 Institute for Biomedical Engineering, Imperial College London, London United Kingdom
Show AbstractThis talk will present our latest developments towards biomaterials for musculoskeletal repair. Tissues within the body are hierarchically organised from the nanoscale fibres of the extracellular matrix to the macroscale. The influence of nanoscale chemical and topographical cues on cell attachment and differentiation will be discussed. Lack of vascularisation remains one of the biggest hurdles in translation of large-scale tissue engineered constructs to the clinic. Here some new strategies to pre-vascularise tissue engineered matrices and also to induce efficient vascularisation in vivo will be presented. Finally the importance of cell source in influencing the final material properties of tissue engineered bone will be discussed.E. Place, N. D. Evans, M. M. Stevens, Nature Materials, 2009.E. Gentleman, R. Swain, N. D. Evans , S. Boorungsiman , G. Jell , M. Ball , T. Shean , M. Oyen , A. Porter, M. M. Stevens, Nature Materials, 2009.
10:00 AM - RR4.2
From Soft Hydrogels to Hybrid Elastomers: Advanced Biomaterials by Design.
Xinqiao Jia 1
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States
Show AbstractWe have designed and synthesized two types of advanced biomaterials that closely mimic the structure and functions of natural extracellular matrix (ECM) components. The first type of biomaterial was synthesized using naturally occurring hyaluronic acid as the starting material. HA-based hydrogel particles (HGPs) with defined size and porosity were prepared via inverse emulsion polymerization. When conjugated with a heparan sulfate proteoglycan, perlecan domain I, these particles allow for spatio/temporal release of therapeutically active growth factors. Hierarchically structured, doubly cross-linked networks (DXNs) were engineered using HA HGPs as the building blocks and a water-soluble HA derivative as the secondary cross-linker. The resulting hydrogels are soft and elastic; their viscoelastic properties can be readily modulated by varying the particle size, surface functional group, inter-particle and intra-particle crosslinking. The second type of biomaterial is multiblock hybrid polymers that not only capture the elasticity of native elastin, but also provide flexibility and tunability in a range of morphological and mechanical properties. These hybrid polymers were synthesized by condensation polymerization via Orthogonal Click Chemistry using flexible synthetic polymers and alanine-rich, lysine-containing peptides as the building blocks. Subsequent covalent cross-linking of the pre-registered lysin residue led to the formation of an elastomeric matrix. These custom-designed biomaterials are being evaluated as artificial ECM for cancer drug screening and soft tissue regeneration.
10:15 AM - RR4.3
Modular Scaffolds Assembled from PEG Microspheres with Pore Formation in the Presence of Living Cells.
Donald Elbert 1 , Evan Scott 1 , Michael Nichols 1
1 Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, United States
Show AbstractIntroduction: We introduce a new method to produce modular scaffolds for tissue engineering. The technique addresses a major challenge in the field - the production of macroporous scaffolds in the presence of living cells. The scaffolds are produced by crosslinking polyethylene glycol (PEG) microspheres in the presence of cells. The microspheres themselves are pre-formed in buffered salt solutions above the cloud point of PEG, without the use of organic solvents or surfactants.Methods: PEG microspheres are made by reacting PEG-octa-vinylsulfone (PEG-OVS, 10 kDa) or PEG-octa-acrylate (PEG-OAc, 10 kDa) with PEG-octa-amine (PEG-OA, 10kDa) or bovine serum albumin (BSA). The PEG solutions are allowed to react for a certain period of time and then are heated above the LCST in the presence of 0.6 M sodium sulfate, causing phase separation. If the gel point is reached with 5-15 min after phase separation, 1-100 micron-diameter microspheres are formed. The formed microspheres are recovered by centrifugation and buffer exchanged into PBS or cell culture medium. Scaffolds are formed by centrifuging microspheres in the presence of cells at 1000 g for 10 min. PEG-OVS may be functionalized with cell adhesion peptides to promote cell adhesion. Results: PEG-OVS/PEG-OA microspheres were modified with cell adhesion peptides. PEG-OVS/BSA microspheres were loaded with the angiogenic factor sphingosine 1-phosphate (S1P). Degradable PEG-OAc/PEG-OA microspheres were produced as porogens. Hepatocyte-like cells (HepG2) were added to the microsphere suspension in cell culture medium. Followed centrifugation, the microspheres were crosslinked around the cells via reaction of residual vinylsulfone groups on the microspheres with serum proteins in the medium. HepG2 cell viability following scaffold formation was 93.64 ± 3.34%, with the cells evenly distributed through the scaffolds. The porogenic PEG microspheres degraded within 48 h in culture, forming highly porous scaffolds without affecting HepG2 cell viability (91.94 ± 1.87%). The pores were designed to allow ingrowth of blood vessels following implantation. To test this in vitro, endothelial cells were seeded outside the scaffolds. The endothelial cells rapidly invaded scaffolds that released S1P (5.4 ± 1.0 micron/h with S1P release versus 2.6 ± 0.8 micron/h without S1P release). Gradients in porosity were also introduced by producing porogenic microspheres that were less buoyant than the other microsphere types. Endothelial cells migrated rapidly through the more porous regions, but only if the scaffolds were derivitized with cell adhesion peptides.Conclusions: Scaffolds were formed in the presence of living cells by crosslinking PEG microspheres around the cells. The PEG microspheres were formed by a novel aqueous process. The introduction of a non-cytotoxic porogen in the form of PEG microspheres may be useful for promoting vascularization of hydrogel scaffolds.
10:30 AM - RR4.4
Hierarchically Structured Polymer and Hybrid Tissue Scaffolds by Freeze-casting.
Philipp Hunger 1 , Amalie Oroho 1 , Matthew Schecter 1 , Thao Vi Le 1 , Benjamin Riblett 1 , Jenell Smith 1 , Ulrike G. Wegst 1
1 , Drexel University, Philadelphia, Pennsylvania, United States
Show AbstractThe design requirements for tissue scaffolds appear deceptively simple: to provide a porous matrix with interconnecting porosity that promotes rapid tissue ingrowth and, at the same time, possesses sufficient strength and toughness to prevent crushing under physiological loads until full integration and healing is reached. Yet, the challenge persists to synthesize a material that mimics both the structure and the mechanical performance of the natural tissue. Despite extensive efforts and first encouraging results, current porous materials for tissue regeneration tend to suffer a common limitation: the inherent lack of strength associated with porosity. Engineered high-performance composites with effective properties that, like the natural tissue (e.g. bone), exceed by orders of magnitude the properties of their constituent are rare. The reason for this is that the multi-level hierarchical composite structure, which is thought to be the origin of the observed ‘mechanical property amplification,’ is difficult to emulate in synthetic materials. One process with which it is possible to manufacture complex materials is freeze-casting. It utilizes the intricate process of ice formation to create hybrid materials with complex lamellar or brick-and-mortar-like structures that can be controlled across several length-scales. First results show that both strength and toughness of freeze-cast ceramic-polymer and ceramic-metal hybrid materials increase in a non-additive manner that goes well beyond the simple composite ‘rule of mixture.’ To date, there is little known about how to predict the mechanical performance of such lamellar composites based on volume fractions of the different constituents, on lamellar geometry and surface roughness, and on interfacial bonding. We use quantitative X-ray microtomography to establish simple structure-property linkages, to identify mechanisms that control mechanical behavior over multiple length-scales, and to propose new design concepts to guide the synthesis of custom-made, hierarchically structured hybrid materials.
10:45 AM - RR4.5
Photo-crosslinked PEO-PDMSstar Hydrogels: Synthesis, Characterization, and Potential Application for Tissue Engineering Scaffolds.
Yaping Hou 1 , Cody Schoener 1 , Katherine Regan 1 , Dany Munoz-Pinto 2 , Mariah Hahn 2 , Melissa Grunlan 1
1 Biomedical Engineering, Texas A&M University, College Station, Texas, United States, 2 Chemical Engineering, Texas A&M Univeristy, College Station, Texas, United States
Show AbstractPolyethylene oxide (PEO, or PEG)-based hydrogels have emerged as a promising class of tissue engineering scaffolds. The photochemical cure of diacrylated PEO (PEO-DA) permits the encapsulation and subsequent culture of cells in a physiologically relevant 3-dimensional environment. Scaffolds whose physical (e.g. mechanical) as well as chemical properties may be systematically tuned would be a tremendously useful tool to probe the effect of scaffold properties on cell behavior and end-point engineered tissue properties. However, tailoring the physical properties of PEO-DA hydrogels is limited to altering molecular weight (MW) and weight % of PEO-DA in the aqueous hydrogel precursor solutions. Herein, we report the preparation of tunable inorganic-organic hydrogels by the photopolymerization of methacrylated star polydimethylsiloxane (PDMSstar-MA) and PEO-DA. PDMSstar-MA (2k, 5k, and 7k g/mol) were each combined with PEO-DA (3k and 6k g/mol) at various weight ratios (1:99, 10:90, and 20:80) at a constant 10 weight % precursor solution concentration. The morphology of these hydrogels consisted of spherical PDMS-enriched domains disperse throughout a PEO-enriched matrix. In addition, the swelling ratio, mechanical properties, non-specific protein adhesion, and cell viability were measured.
11:30 AM - **RR4.6
Semi-synthetic Extracellular Matrices for Use in Translational Therapies.
Sarah Atzet 1 , Terry Tandeski 1 , Thomas Zarembinski 1 , Glenn Prestwich 1
1 , Glycosan BioSystems, Salt Lake City, Utah, United States
Show AbstractGlycosan BioSystems has developed easy-to-use semi-synthetic extracellular matrix (sECM) products that recapitulate the functions of the natural ECM in orchestrating cell proliferation, migration, differentiation, and angiogenesis. These versatile products use a crosslinkable, chemically-modified hyaluronan (HA) derivative, and allow full experimental control of composition and compliance, permit controlled release of growth factors, and support cell encapsulation in 3-D for in vitro and in vivo studies. The HyStem and Extracel product lines have been used in tumor xenograft models, for the deliver and localization of mesenchymal stem cells, and for feeder-free cultures of stem cells. Current research projects are evaluating the potential to promote stable expansion of stem cells lines including MSC's, hPSC's, mESC's, and hESC's on top of and encapsulated in Glycosan's sECM hydrogel products. mESC's cultured on HyStem-C show similar growth rates, plating efficiencies, pluripotent morphologies and markers compared to mouse embryonic fibroblast feeder cells. Additionally HyStem-C reduced variability in screening assays and enabled a more sensitive proteomic analysis. However, these hydrogels contained animal derived gelatin which can be potentially replaced by a combination of human extracellular matrix proteins (hECM). Hydrogels have been modified by covalent and non-covalent incorporation of ECM proteins and then evaluated for modulus changes and effects on pluripotency of the cultured cells. First an optimal combination of covalently-linked human collagen, vitronectin, fibronectin, and laminin (CVFL) proteins, as well as the minimum amounts required, for effective self-renewal of H9 hESCs was evaluated. Cells were recovered and analyzed for stem cell markers by flow cytometry. Next, human fibroblast derived ECM was incorporated into the hydrogels which were again evaluated for their ability to promote long-term propagation of stable hPSCs in surface and 3-D encapsulation cultures. Cells that have been encapsulated and cultured in a three dimensional environment are easily recovered by the dissolution of hydrogels using a non-enzymatic pathways. This resulted in the release of a new product called PEGSSDA that contains internal disulfide linkages which in the presence of a reducing agent such as N-acteyl L-cysteine allows for reverse gelation of the hydrogel. This research works towards the idea of easing "translatability" by delivering a simple and safe material that permits seamless transition from pre-clinical experimental optimization to human clinical use.
12:00 PM - **RR4.7
Silk/Silica Biomaterials for Tissue Engineering.
David Kaplan 1 , Aneta Mieszawska 1 , Carole Perry 2
1 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States, 2 School of Science and Technology, Nottingham Trent University, Nottingham United Kingdom
Show AbstractSilk/silica chimeric proteins are studied as new biocompatible biomimetic nanocomposites for bone repairs and tissue engineering. The material design is based on a biomimetic approach using fusion proteins that combine a self-assembling domain (spider dragline silk of Nephila clavipes) and a functional domain (silaffin derived R5 peptide of Cylindrotheca fusiformis). Silk serves as an organic support material that can be processed into films (studied here), fibers or porous matrices depending on the application, and forms β-sheet secondary structures that improve material stability. Genetic control over silk-silica fusion protein designs coupled with materials processing resulted in remarkable control of silica morphology and distribution. This control is assessed for influence on material properties with respect to bone regeneration. In a related approach to evaluate the impact of silica on bone formation, a composite materials approach is used. In this case silk protein (Bombyx mori) is blended with different sizes of silica nanoparticles (SNPs) and assessed for bone formation. Both systems, the recombinant silk/silica fusion proteins and the silk fibroin protein/SNPs are assessed in vitro for osteogenic outcomes using human bone marrow derived mesenchymal stem cells (hMSC).
12:30 PM - RR4.8
Hydrogel-Based Nerve Guidance Channels for Peripheral Nerve Regeneration.
Shawn Lim 1 , Ruifa Mi 3 , Changqing Shi 3 , Ahmet Hoke 3 , Hai-Quan Mao 2
1 Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 3 Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 2 Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractAutografts are the current gold standard of treatment for nerve transection injuries. Shortcomings of this approach are donor site morbidity as well as a finite supply of implantable material. Synthetic nerve guidance conduits (NGCs) offer an attractive alternative because they can be produced in large quantities, and tailored to match the dimensions of the nerve defect. However, clinical success of commercially-available NGCs has been limited to short gap lengths, likely because they only provide gross mechanical guidance to the regenerating nerve stump. We hypothesize that repair over long defects requires the presence of additional cues in the form of chemoattractive molecules or physical contact guidance.We designed and tested the efficacy of a hydrogel-based NGC in a rat sciatic nerve model of peripheral nerve injury. Neurotrophic factors were encapsulated within a polymeric hydrogel with pore size tailored to permit controlled release of growth factor over a prolonged time following implantation. Physical guidance for axons was provided in the form of aligned polymer nanofibers within the lumen of the tube. Additionally, the possible synergistic effect of extracellular matrix molecules was tested by conjugating the surface of the fibers with either laminin or heparin. The success of regeneration within our NGCs over a 15-mm gap was compared with commercially-available conduits. All conduits were harvested after three months in vivo. Regeneration was evaluated by histomorphometry, retrograde transport of FluoroGold dye injected into the distal nerve stump, as well as electrophysiological recordings of the nerve.Regeneration in empty gel tubes was generally poor, with few animals containing retro-labeled neuronal cell bodies; this was associated with a lack of myelinated axons and lack of functional recovery. Both functional recovery and presence of myelinated axons within the regenerated nerve cables was observed in animals implanted with tubes containing GDNF, demonstrating that controlled release of growth factor plays a vital role in promoting regeneration. However, there was no significant enhancement in regeneration within tubes that contained the additional nanofiber guidance cues. It is likely that the ECM molecules provided an overly permissive microenvironment that trapped and hindered regenerating axons from extending beyond the distal end of the NGC.Our results demonstrate that growth factors encapsulated within our NGCs maintain bioactivity and promote regeneration when released in a controlled fashion into a nerve gap. Studies are currently under way to optimize ECM coatings onto nanofiber cues to best complement the effect of the chemoattractive cues. The eventual goal of the study is to develop a novel NGC containing the ideal combination of cues capable of promoting regeneration over long distances.
12:45 PM - RR4.9
New 3D-Biophotopolymers with Selective Surface-cell Interactions for Regenerative Medicine.
Christian Heller 1 2 , Martin Schwentenwein 1 , Robert Liska 1 , Juergen Stampfl 2 , Franz Varga 3 , Maja Porodec 4 , Michaela Schulz-Sigmund 4 , Guenter Russmueller 5
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna Austria, 2 Institute of Materials Science and Technology, Vienna University of Technology, Vienna Austria, 3 Ludwig Boltzmann-Institute of Osteology, Hanusch Hospital, Vienna Austria, 4 Department of Cranio-, Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna Austria, 5 Institute of Pharmacy, University of Leipzig, Leipzig Germany
Show AbstractThe design of a 3D scaffold with defined pore sizes offering good cell adhesion is still an important topic in tissue engineering and regenerative medicine.[1] The use of (meth)acrylate based photopolymers as biomaterials has recently gained increasing interest because of their easy access, tailorable mechanical properties and their ability of being structured by the means of Additive Manufacturing Technology (AMT). However, methacrylates suffer from poor photoreactivity and acrylates show high affinity to side-reactions with amines like proteins causing adverse effects in the human body, making them less suitable for regenerative medicine applications and providing the need for other polymerizable groups. Therefore, new monomers based on vinyl esters, vinyl carbonates and vinyl carbamates, giving water-soluble and biocompatible poly(vinyl alcohol) upon hydrolytic degradation, were synthesized.[2]The new monomers were up to two orders of magnitude less toxic compared to acrylates and their photoreactivity was between the ones of acrylates and methacrylates. Furthermore, similar mechanical properties compared to their (meth)acrylate references but also PLA was found. Furthermore,, in-vitro degradation behaviour and biocompatibility, measured by their cytotoxicity towards osteoblast-like cells and cell culture tests on polymer specimens, were tested. In order to provide the ability of a selective interaction between the polymer and human cells, an immobilization of proteins like collagen on the polymer surface is desirable. Therefore, monomers containing functional groups being readily reactive towards nucleophiles like epoxides and cyclic carbonates have been copolymerized successfully with the new monomers. To study surface modification reactions, alkaline phosphatase, was used as model protein.Cellular 3D structures were printed by AMT techniques such as microstereolithography, digital light processing and two-photon induced photopolymerization, showing the suitability as starting materials for several regenerative medicine applications. Finally, 3D cellular structures of selected composition were tested in vivo in rabbits[1] Agrawal, C. M. et al.; Journal of ASTM International 2006, 3[2] R. Liska , S. Baudis, M. Schuster, C. Heller , F. Varga, J. Stampfl, H. Bergmeister, G. Weigel: Patent application: AT A19032007
RR5: Nanotechnology
Session Chairs
Tuesday PM, December 01, 2009
Back Bay C (Sheraton)
2:30 PM - **RR5.1
Multi-functional and Dynamic Fibrous Scaffolds for Tissue Engineering and Controlled Release.
Robert Mauck 1 , Brendon Baker 1 , Lara Ionescu 1 , Nandan Nerurkar 1 , Jason Burdick 1
1 Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractDense connective tissues of the musculoskeletal system are typified by a hierarchical collagen organization consisting of fibers arranged in across multiple length scales. This ordered ensemble provides such tissues as tendon, ligament, the annulus fibrosus of the intervertebral disc, and the knee meniscus with mechanical properties that are tuned to enable operation in dynamic mechanical loading conditions and over a lifetime of use. Specifically, these tissues all possess high tensile moduli that are highest in the primary load bearing direction. Damage and degeneration of these structures results in a significant loss in load bearing capacity, and endogenous healing is limited by the dense and relatively acellular and avascular nature of the native tissue. To overcome the natural sequelae of damage to such tissues, namely scar formation, we have developed an electrospinning system for the production of dynamic fibrous scaffolds for tissue repair. These scaffolds can be formed in such a way as to recapitulate key architectural features and mechanical anisotropies critical to native tissue function. Moreover, with modification of the electrospinning system, dynamic multi-polymer assemblies can be generated. Such composite scaffolds, with their multiple fiber families, show time dependent evolution of both pore size and mechanical properties based on the identity and amount of different component polymer fractions, and their behavior can be predicted through theoretic al modeling of the mixture. These scaffolds can likewise be tuned to promote enhanced cellular infiltration and matrix deposition via the removal of rapidly degrading (sacrificial) fiber populations with time in culture. Additional functionality and cellular interactions can be imparted via the inclusion of both synthetic (biodegradable polyester) and biologic (natural ECM) fiber populations within these composite networks. Moreover, these scaffolds can be further functionalized to enable controlled release of growth factors and other agents. We have recently shown, for example, that the mechanical properties and micro-patterning aspects of the scaffold can be decoupled from drug or biofactor release via the inclusion of microspheres in the fast eroding or sacrificial fiber populations of these nanofibrous composites. In such a way, multiple factors can be released in differing amounts and rates by tuning microsphere composition and density within the fibrous assembly. Further, matrix formation can be modulated by mechanical stimulation of these cell-seeded networks using custom mechanical bioreactor systems that mimic in vivo loading conditions. These advances in scaffold formation and maturation hold great promise for the regeneration and replacement of dense connective tissues.
3:00 PM - RR5.2
Bioactive Nanomaterials: Directing Cell Morphology and Patterning with Arrays of Nanopillars.
Michael Bucaro 1 , Benjamin Hatton 1 , Joanna Aizenberg 1
1 , harvard university, Cambridge, Massachusetts, United States
Show AbstractMedical implant surfaces can be engineered to direct cell fate by providing tissue-specific chemical, mechanical and spatial cues. The aim of our research is to create multifunctional biomaterial surfaces based on nanoscale topography to direct and monitor cell behavior. Herein, we demonstrate the use of nanopillar arrays to manipulate cell morphology and behavior. Silicon surfaces were lithographically patterned with vertical arrays of high aspect ratio nanopillars with varied geometry, spacing and chemical functionality. Murine fibroblasts were induced to form polarized cell bodies with elongated axon-like extensions aligned with the symmetry of the nanopillar array. The results demonstrate a platform to create tunable bioactive nanostructures capable of eliciting a spectrum of morphological characteristics and patterned growth in pluripotent cells. This approach is compatible with integrated circuit fabrication and can be applied to create bioactive surfaces that probe and direct cell behavior. Applications include the formation of cellular networks on neural chips, stem cell lineage specification and manipulation of cell activity for a variety of biomedical applications.
3:15 PM - RR5.3
Effects of particulate and cellular Integration and Mandrel Size on the Structure and Mechanical Anisotropy of Electrospun Constructs.
Nicholas Amoroso 1 , William Wagner 1 , Michael Sacks 1
1 Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Show AbstractConcurrent electrospinning of polymer fibers while electrospraying living cells has been recently introduced to provide the ability to rapidly fabricate highly cellularized engineered tissue constructs. [1] To date, this technique has not been closely studied to evaluate its effects on the mechanical properties and structure of these constructs. Here we present a detailed analysis of the effect of cell and particulate micro-integration on the construct microstructure and mechanical anisotropy. Electrospun constructs were fabricated using an experimental setup similar to that described previously [1]. Briefly, poly(ester urethane)urea (PEUU) was electrospun onto one of two steel cylindrical mandrels rotated at similar tangential velocities. Polystyrene microspheres were utilized as rigid bodies in order to elucidate an upper limit in stiffness for the effect of particulate inclusion. Concurrently, known concentrations of polystyrene microspheres, vascular smooth muscle cells, or sterile media was electrostatically sprayed onto the mandrel in a perpendicular orientation to the polymer. Constructs were then sectioned and mechanically tested using a biaxial testing device described previously [2]. Cross-sections of a microsphere integrated construct shows the microstructure surrounding the microspheres and cells. Interestingly, we observed that decreasing mandrel diameter produced reversed mechanical anisotropy at the same tangential velocity. This effect is maintained with the inclusion of particulates. We have demonstrated that a change in the mandrel geometry can induce high levels of mechanical anisotropy in a scaffold at very low tangential velocities that would not normally induce these behaviors [2]. Presumably, this effect results from changes in polymer fiber architecture due to bending to conform to a smaller diameter mandrel, and has the greatest effect on the preferred fiber direction. Tissue culture media only specimens, used a controls for the cell integration, produce scaffolds with pronounced fiber loops that contributed to enhanced longitudinal compliance. Graduations of cell integration concentration produced ranges of mechanical anisotropy, but only in the longitudinal direction. Moreover, the levels of achieved anisotropy are comparable to those induced by high mandrel rotational velocities [2], and may allow for the development of mechanically anisotropic cellular infiltrated elastomeric scaffolds without rapid mandrel speeds.References:1. Stankus J.J. et. al. Biomaterials 27, 735, 2006., 2. T. Courtney, M.S. Sacks, J. Stankus, J. Guan, and W.R. Wagner, Biomaterials, Vol. 27, pp. 3631-3638, 2006.Acknowledgements NIH grant HL68816.
3:30 PM - RR5.4
The Use of Electrospun Polycaprolactone as a Dermal Scaffold for Skin Tissue Engineering.
Ming Chen 2 , Manisha Chopra 2 , Sankha Bhowmick 1 2
2 Biomedical Engineering and Biotechnology, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts, United States, 1 Mechanical Engineering, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts, United States
Show AbstractRapid healing of acute and chronic skin defects is an important objective. Skin tissue engineering using controlled biodegradable synthetic matrices provide a prospective source of advanced therapies for treatment of acute and chronic skin wounds. In the present work, we report on the design and feasibility of a co-culture system for fibroblasts and keratinocytes by using electrospun polycaprolactone (PCL) scaffolds. We first seeded fibroblasts inside the scaffolds by controlled vacuum seeding and then, the keratinocytes were seeded on top of the scaffolds. Finally, the keratinocyte layer was lifted to the air/liquid interface to differentiate. We studied the effect of scaffold fiber diameter on keratinocyte attachment, proliferation and differentiation along with effect of vacuum seeding and fiber diameter on collagen secretion. 400 nm and 1000 nm electrospun scaffolds were prepared using 15wt% and 18 wt% PCL solutions with certain other fixed parameters. For cell culture, Clonetics® NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes and NIH 3T3 mouse fibroblast cells were used. The CellTiter 96TM Aqueous One Solution Cell Proliferation Assay (MTS assay) was used to measure cell attachment and proliferation. The collagen secretion analysis was done by SircolTM Assay. Our results show that after 12 hours, the normalized MTS value of keratinocytes on 400 nm scaffolds is significantly higher than on 1000 nm scaffolds at all time points. It was found that fibroblasts secreted more collagen in 400 nm scaffold than 1000nm scaffolds in both 1 day and 3 day periods. For the same scaffold, when fibroblasts were co-cultured with keratinocytes, they significantly secreted more collagen than fibroblasts in single culture. Also, fibroblasts secreted significantly more collagen when they were separated from keratinocytes in different layers of scaffold than when they were just mixed with keratinocytes on the top of scaffolds. These results suggest that collagen secretion can be controlled by either fiber diameter or using a co-culture method. The antihuman keratinocyte transglutaminase was used for localization of keratinocyte differentiation marker transglutaminase. For the keratinocyte differentiation in co-culture model, the results show that keratinocytes differentiation depends on culture time and scaffolds and they fully differentiate in 7 days. Cells differentiate more on 400 nm scaffolds than on 1000 nm scaffolds. This result also gives us an option to control keratinocyte differentiation by varying scaffold fiber diameter. In our case, the keratinocyte differentiation expression is mainly dependent on cell number as the differentiation conditions were same. We propose that keratinocyte differentiation and fibroblast collagen secretion are controllable through varying scaffold fiber diameter and the approach to skin modeling reported here may find application both in tissue engineering and screening of new pharmaceuticals.
3:45 PM - RR5.5
Marrow Stromal Cell Reponse to Fiber-Reinforced Laminated Nanocomposites.
Junyu Ma 1 , Weijie Xu 1 , Esmaiel Jabbari 1
1 Chemical Engineering, University of South Carolina, Columbia, South Carolina, United States
Show AbstractThere have been numerous efforts to develop synthetic and/or natural tissue engineering scaffolds that are suitable for bone regeneration applications to replace autograft and allograft bones. Current biomaterials as a scaffold for bone regeneration are limited by the extent of degradation concurrent with bone formation, mechanical strength, and the extent of osteogenic differentiation of marrow stromal cells migrating from the surrounding tissues. In this project, a novel laminated nanocomposite scaffold is fabricated, consisting of poly (L-lactide ethylene oxide fumarate) (PLEOF) hydrogel reinforced with poly (L-lactide acid) (PLLA) electrospun nanofibers and hydroxyapatite (HA) nanoparticles. PLEOF is a novel in situ crosslinkable macromer synthesized from biocompatible building units which can be functionalized with bioactive peptides like the cell-adhesive Arg–Gly–Asp (RGD) amino acid sequence. The hydrophilicity and degradation rate of the macromer can be tailored to a particular application by controlling the ratio of PEG to PLA blocks in the macromer and the unsaturated fumarate units can be used for in-situ crosslinking. The PLLA nanofibers were electrospun from high molecular weight PLLA. The laminated nanocomposites were fabricated by dry-hand lay up technique followed by compression molding and thermal crosslinking. The laminated nanocomposites were evaluated with respect to degradation, water uptake, mechanical strength, and the extent of osteogenic differentiation of bone marrow stromal (BMS) cells. Laminates with or without HA nanoparticles showed modulus values much higher than that of trabecular bone (50-100 MPa). The effect of laminated nanocomposites on osteogenic differentiation of BMS cells was determined in terms of cell number, ALPase activity and calcium content. Our results demonstrate that grafting RGD peptide and HA nanoparticles to a PLEOF hydrogel reinforced with PLLA nanofibers synergistically enhance osteogenic differentiation of BMS cells. In conclusion, the laminated nanocomposite with controllable degradation characteristics and robust mechanical properties is attractive as a synthetic bone-mimetic matrix for skeletal tissue regeneration.
4:30 PM - **RR5.6
Nanotechnology for Treating Damaged Tissues: Hype or Reality?
Thomas Webster 1
1 Biomedical Engineering, Brown University, Providence, Rhode Island, United States
Show AbstractNanotechnology is being used to mimic structural components of tissues in synthetic materials intended for various implant applications. Recent studies have highlighted that when compared to flat or micron rough surfaces, surfaces with nanofeatures promote optimal initial protein interactions necessary to mediate cell adhesion and subsequent tissue regrowth. This has been demonstrated for a wide range of implant chemistries (from ceramics to metals to polymers) and for a wide range of tissues (including bone, vascular, cartilage, bladder, skin, and the central and peripheral nervous system). Importantly, these results have been seen at the in vitro and in vivo level. This talk will cover some of the more significant advancements in creating better vascular, cardiovascular, and orthopedic implants through nanotechnology efforts. It will also cover recent in vitro and in vivo studies which highlight better tissue regeneration. This talk will also address recent concerns of nanoparticle toxicity.
5:00 PM - RR5.7
Modification of Vertically Aligned Carbon Nanofiber Array with Conductive Polymer Coating as a Novel Neural Electric Interface for Neural Stimulation.
Jun Li 1 2 , Edward de Asis 2 3 , T. D. Barbara Nguyen-Vu 2 , Prabhu Arumugam 2 , Hua Chen 2 , Alan Cassell 2 , Russell Andrews 2 , Meyya Meyyappan 2
1 Chemistry, Kansas State University, Manhattan, Kansas, United States, 2 Center for Nanotechnology, NASA Ames Research Center, Moffett Field, California, United States, 3 , Santa Clara University, Santa Clara, California, United States
Show AbstractElectrical stimulation has been demonstrated as an effective regenerative medical technology for neurological diseases and neural injuries. Long-term neuroprosthes and functional electrical stimulation require chronic implanted electrodes with a stable neural electrical interface which can efficiently stimulate neural tissues without toxic effects. An optimum neural electrical interface requires the device surface to be compatible with neural cells and tissues in molecular, topographical, mechanical, and electrical properties. Here, we report on the investigation of a nanostructured materials, i.e. vertically aligned carbon nanofiber arrays (VACNFs), as a novel neural electrical interface. After proper modification with electrical conductive polymer coatings, such novel nanostructured materials can serve multiple functions toward a reliable neural electrical interface as demonstrated with neuron-like PC12 cell culture and electrical stimulation of rat hippocampal slices. VACNF arrays are brush-like nanostructures consisting of freestanding bamboo-like multiwalled carbon nanotubes (i.e. carbon nanofibers, CNFs) grown by plasma enhanced chemical vapor deposition. The individual carbon nanofibers can be controlled at ~50-150 nm in diameter and ~2-10 microns in length. They are fully separated from each other with an average spacing of ~300-400 nm. VACNF arrays can be grown on lithographically patterned electric circuits as microbrushes. By electrodeposition, conductive polymers such as PPy or PEDOT can be conformally coated around each CNFs. The thin polymer coating dramatically enhances the mechanical properties of the VACNFs and also provides a large pseudocapacitance which serves as an ideal medium for conversion of electronic currents to ionic currents. PC12 cells were found to proliferate and form extensive neural network on the polymer coated VACNF surface with neurites intermingled with individual carbon nanofibers. Electrical stimulation of rat hippocampal slices with such polymer-coated VACNF microbrushes have been systematically studied in comparison with tungsten microwires and planar Pt microelectrode arrays. The results indicate that the intimate neural electrical interface between neurites and CNFs provides much higher stimulation efficacy. A unique fast neural excitation wave with much narrower peak width can be excited with much lower stimulation current with polymer coated VACNF microbrushes. The excitation voltage is below 1.0 V, which can completely avoid electrolyzing water. These results highlight the unique properties of PPy-coated VACNF electrodes in lower electrical impedance, ability to stimulate tissue through a biocompatible chloride flux, and stable vertical alignment in liquid which ensures the access to spatially confined regions of electrically active cells. A stable neural electrical interface may be developed for chronic neuromodulations with such new electrode materials.
5:15 PM - RR5.8
Redox Activity of Cerium Oxide Nanoparticles for Scavenging Radicals – Activity and Regeneration.
Ajay Karakoti 1 , Jessica King 1 , Sanjay Singh 3 , Talgat Inerbaev 2 , Artem Masunov 2 , William Self 3 , Sudipta Seal 1 2
1 Advanced Materials Processing and Characterization, University of Central Florida, Orlando, Florida, United States, 3 Microbiology and Molecular Biology, University of Central Florida, Orlando, Florida, United States, 2 Nanoscience and Technology Center, University of Central Florida, Orlando, Florida, United States
Show AbstractCerium oxide nanoparticles (CNPs), a versatile catalyst, have found a new niche in biomedical applications by showing tremendous potential in radical scavenging antioxidant properties. It was shown recently that nanoceria can scavenge radicals at a rate faster than some of the biologically existing enzymes. Recently CNPs have been demonstrated to protect biological tissues against; radiation induced damage, scavenging of superoxide anions, prevention of laser induced retinal damage, reduction of spinal injury in a tissue culture model, prevention of cardiovascular myopathy, pH dependent antioxidant properties, as a tool for immunoassays and other inflammatory diseases. One of the important characteristics of CNPs is the retention and regeneration of active Ce3+ oxidation state which is more potent in scavenging reactive oxygen species (ROS). Despite several studies, the precise mechanism of action of CNPs in scavenging radicals is not yet fully understood. Likewise we are unable to determine the mechanism of the regenerative nature of this unique catalyst. In the present investigation we outline some of the key concepts in the radical scavenging action and regeneration of CNPs through simple experiments using hydrogen peroxide as a source of peroxide radicals. The observations were further supported by theoretical investigations using DFT. As CNPs can act as an oxygen buffer, the role of dissolved oxygen (DO) in ROS scavenging activity of CNps and interaction of CNPs directly with DO content will be outlined. The tailor made 3-5nm CNPs show predominant Ce3+ oxidation state and are unstable at alkaline pH. CNPs were coated with polyethylene glycol (PEG) and dextran to increase the stability in neutral to alkaline medium and the effect of such biocompatible coatings on the redox properties on nanoceria will be discussed. It will be shown how PEG coating on CNPs can tune the redox properties of CNPs and accelerate the regeneration of Ce3+ valence state. The role of increasing PEG concentration as well as molecular weight in tuning the size, shape and redox properties of CNPs will be discussed. The biological activity was tested using classical superoxide dismutase (SOD) mimic model - competition with ferricytochrome C for reduction by superoxide. Initial results showed no decrease in activity of CNPs with increasing concentration of PEG.
5:30 PM - RR5.9
Nanoparticle-mediated Polymer Network Enabling Outstanding Shape Memory Properties at Physiological Temperatures.
Jianwen Xu 1 , Jie Song 1 2
1 Orthopaedic, Umass Medical School, Worcester, Massachusetts, United States, 2 Cell Biology, Umass Medical School, Worcester, Massachusetts, United States
Show AbstractA major roadblock in translating scaffold-based regenerative medicine into clinical practice is the lack of materials combining tissue-like mechanical and biochemical properties with clinically relevant deployability for safe delivery and integration with target tissues. Thermal-responsive shape memory polymers (SMPs), fixable at a temporary shape below transition temperature, and recoverable to their original shapes upon thermal triggering, hold great promise as minimally invasive self-fitting tissue scaffolds. Conventional SMP networks contain either un-tethered polymer chains resulting in plastic deformations and broad transitions, or excessive chain-chain interactions requiring extra energy to overcome. Consequently, they require harsh temperatures to fix temporary shape (<0 °C) or trigger shape recovery (>70 °C) that is often slow and incomplete. Moreover, they often lack desired bioactivities and adequate mechanical strength at body temperature. Thus, practical clinical use of existing SMPs has been hampered by their inefficient shape memory properties and inadequate mechanical strengths around physiological temperatures. Inspired by nanoparticle’s capability in modulating chain motion, we designed bulky nanoparticle-derived multi-armed polyester macromer building blocks and crosslinked them to form homogenous SMP network. The resulting materials showed high-modulus (>2.0 GPa) at body temperature. It exhibited stable shape fixing at room temperature (> 1 year) and rapid (~ 1 sec) and complete shape recovery at 50 °C. In vitro degradation assay indicated that the 50% weight loss could be achieved from 3 to 7 months depending on the polyester arm length. Using “click” chemistry, bioactive motifs was incorporated with the SMP without compromising its outstanding shape memory performance and mechanical strength. This is the first time that a well-defined nanoparticle is used, in the form of nanoparticle-derived macromer building block, to control the spatial distribution and interactions of polymer chains in a SMP network, enabling maximal participation of polymer chains in the elastic deformation and recovery process. This nanostructured design platform provides a unique opportunity to integrate physical, biochemical, and mechanical signals for complex regenerative medicine applications.
5:45 PM - RR5.10
Nanoporous Biodegradable Polymers for Ocular Drug Delivery.
Daniel Bernards 1 , Mark Steedman 1 , Paula Wynn 2 , On-Tat Lee 2 , Robert Bhisitkul 2 , Tejal Desai 1
1 Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, United States, 2 Ophthalmology, University of California, San Francisco, San Francisco, California, United States
Show AbstractIn clinical ophthalmology, there is significant demand for viable intraocular sustained-release therapeutic devices to improve contemporary therapies. Topical eye drops and intraocular injections are the current standard of care for delivery of most ocular medications. For instance, intraocular treatment of age-related macular degeneration (AMD) with Lucentis involves monthly injections over the course of two years. A drug delivery device capable of sustained release over several months can significantly improve this therapy by maintaining effective therapeutic concentrations while reducing the number of injections. For this application, a flexible device utilizing a biodegradable polymer would allow for injection and eliminate the need for surgical excision. Poly(caprolactone) (PCL) is an ideal biodegradable material for this purpose as it does not lose its structural integrity during the majority of the degradation time course: this allows structural degradation to follow the effective therapeutic lifetime of the device. In order to achieve constant release rates, nanoscale porosity is a useful approach. Nanostructures have been demonstrated as an approach to achieve non-Fickian diffusion when therapeutic size is comparable to pore size. In this work, we characterize the delivery of AMD-relevant therapeutics from nanoporous PCL thin films over extended periods. In addition, intraocular biocompatibility and structural integrity of micro- and nano-strucutured PCL films implanted or injected into rabbit eyes over 6 months will be discussed.