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
Monday AM, November 30, 2009
Back Bay C (Sheraton)
9:30 AM - **RR1.1
Biomaterial Regulation of Molecular Signaling in Engineered Tissues.
John Fisher 1 Show Abstract
1 Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States
Soluble 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 Show Abstract
1 Biomedical Department, McGill University, Montreal, Quebec, Canada, 2 Department of Surgery, McGill University, Montreal, Quebec, Canada
Pancreatic 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 Show Abstract
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 Biological Sciences, University of Delaware, Newark, Delaware, United States
We 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 Show Abstract
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
Understanding 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 Show Abstract
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
The 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 , and a collagen mimetic domain that can physically attach to natural collagen through triple helical interaction as previously reported by our group . 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.  D’Andrea, L. et al. PNAS. 2005, 102, 14215-14220.  Wang, A. et al. Biomacromolecules. 2008, 9, 1755-1763
11:30 AM - **RR1.6
Materials To Program Cells In Situ.
David Mooney 1 2 Show Abstract
1 , Harvard, Cambridge, Massachusetts, United States, 2 , Wyss Institute for Biologically Inspired Engineering, Cambridge, Massachusetts, United States
There 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 Show Abstract
1 Biomedical Engineering, Technion - Israel Institute of Technology, Haifa Israel
The 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 Show Abstract
1 Chemical & Biological Engineering, University of Colorado, Boulder, Colorado, United States, 2 , Howard Hughes Medical Institute, Boulder, Colorado, United States
Since 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 Show Abstract
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
By 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
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 Show Abstract
1 , University of Wisconsin, Madison, Wisconsin, United States
Schemes 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 Show Abstract
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
Stem 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 Show Abstract
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)
This 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 Show Abstract
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
Stem 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 Show Abstract
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
The 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 Show Abstract
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
It 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 Show Abstract
1 , University of Toronto, Toronto, Ontario, Canada
Vascularization 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 Show Abstract
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 2 , McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario, Canada
Human 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 Show Abstract
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
This 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 Show Abstract
1 Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Materials Science and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Stem 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
Monday PM, November 30, 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 Show Abstract
1 Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States, 2 Mechanical Engineering, Stony Brook University, Stony Brook, New York, United States
Marrow 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 Show Abstract
1 Bioengineering, University of Washington, Seattle, Washington, United States, 2 , University of Maryland, College Park, Maryland, United States
The 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 Show Abstract
1 School of Enigneering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
The 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 Show Abstract
1 Department of Biological Sciences, Korea Advaced Institute of Science and Techonology, Daejeon Korea (the Republic of)
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 Show Abstract
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
Introduction: 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 . 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 Show Abstract
1 Bioengineering, Clemson University, Clemson, South Carolina, United States
Cardiomyocyte 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 Show Abstract
1 School of Materials, University of Manchester, Manchester United Kingdom, 2 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester United Kingdom
Inkjet 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 Show Abstract
1 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Silk 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 Show Abstract
1 Material Sciences Center, Philipps University, Marburg Germany, 2 Department of Biological and Chemical Engineering, Gunma University, Kiryu Japan
Silk 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 Show Abstract
1 Materials Science and Engineering, Univ. of Washinginton, Seattle, Washington, United States, 2 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey
Uncontrolled 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 Show Abstract
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
Implant-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 Show Abstract
1 Institute of Materials Science, University of Connecticut, Storrs, Connecticut, United States, 2 Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States
Shape 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 Divi