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 PM, 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
11:00 AM - RR1: Bio
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 pat