Program - Symposium PP: Manipulating Cellular Microenvironments

Biomaterials play a critical role as both tools to advance understanding of basic biological processes and as therapies in medical devices. Here, we will discuss the design of novel hydrogel materials that allow simple and independent control of mechanical properties and chemical or biological functionality for studying stem cells. These new tools have allowed in depth probing of the integrated role of cell adhesion and matrix mechanical properties in stem cell differentiation. We have also designed materials to support that implementation of synthetic biology and genetic circuits. Specifically, a tunable genetic switch was incorporated into cells encapsulated in materials designed to present the switch inducer in multiple forms. Ultimately, biomaterials, specifically designed to address the challenges and requirements of biological questions and implementation, will make a significant advance in fundamental knowledge that will be used to advance medical science along numerous avenues.

Stem cells hold great potential for tissue repair due to their capacity for self-renewal, their ability to differentiate along multiple lineages, and the potential for autologous transplants. Scaffolds for tissue regeneration should provide signals that promote the targeted cell differentiation and tissue development. The extra-cellular matrix (ECM) and the mechanical properties of the microenvironment are key components of the stem cell niche in vivo that can play a critical role in stem cell fate regulation. I will discuss novel three-dimensional 3D combinatorial hydrogels developed to help understand how the complex interplay of micro-environmental signals influence stem cell fate decision, and to rapidly optimize the stem cell niche using high-throughput strategies.

One of the most significant challenges in the fabrication of keratoprostheses is the development of synthetic materials that promote re-epithelialization and maintenance of a self-renewing fully differentiated corneal epithelium. Although several groups have incorporated biophysical and biochemical cues within their scaffold design, they have failed to take into consideration the in vivo quantitative measurements of the anterior corneal basement membrane. In our previous work, we have thoroughly characterized and quantitated the biophysical properties of the basement membrane features including both topography and compliance underlying the human corneal epithelium. We have utilized our data as a guide for substrate fabrication and demonstrated the influence of topographic cues on individual human corneal epithelial cell (HCEC) behaviors including orientation, adhesion, migration, and proliferation. Our current goal is the development of biomimetic, clinically relevant materials that incorporate both biophysical and biochemical cues from the native basement membrane to influence HCEC cell response. We have utilized two separate materials platforms including poly (ethylene glycol) diacrylate (PEGDA) as well as a layer-by-layer approach of coating NOA81 substrates with reactive poly (ethylene imine) and poly (2-vinyl-4, 4-dimethylazlactone) (PEI/PVDMA) thin films to incorporate and present HCEC’s with both topography and a single adhesion peptide motif Arg-Gly-Asp (RGD). HCEC contact guidance in two culture mediums was used as a measurable endpoint to demonstrate that HCEC phenotype is dependent on the combination of three cues: topography, surface chemical composition and soluble medium components. Specifically, when cell culture medium does not contain serum, HCEC response to topographic cues is different between surfaces that are modified with RGD and surfaces that promote protein adsorption. HCEC alignment in serum-free media exhibited perpendicular alignment to all topographic pitches on all unmodified substrates that allowed for non-specific protein adsorption. In contrast, on substrates that promote specific RGD-cell interactions, cells demonstrate parallel alignment to 400, 800 and 4000 nm pitches. The switch in contact guidance from perpendicular to parallel between cells on protein adsorbing surfaces and RGD-modified surfaces, respectively, indicate that the surface chemical composition is influencing the cellular response to topographic cues. Understanding how HCECs respond to changes in biophysical and biochemical cues can be utilized to advance the field of biomaterials design for improved in vitro cell culture, prosthetics and many other biotechnology applications.

Cell interactions with their extracellular matrix (ECM) microenvironments play a major role in directing cellular processes that can drive wound healing and tissue regeneration but, if uncontrolled, lead to pathological progression. Epithelial to mesenchymal transition, or EMT, is one such process that if finely controlled could have significant potential in regenerative medicine approaches. Despite recent findings that highlight the influence of biochemical and mechanical properties of the ECM on EMT, it is still unclear how these two orthogonal cues act synergistically to control epithelial cell phenotype. Here, we cultured lung epithelial cells on combinations of different mutants of fibronectin’s cell binding domain that preferentially engage specific integrins and substrates of varying stiffness. Our results suggest that while stiff substrates induce spontaneous EMT, this response can be overcome by with fragments of fibronectin that support α3 and α5 integrin engagement. Furthermore, we found that substrate-induced EMT correlates with TGFβ activation by resident epithelial cells and is dependent on Rho/ROCK signaling. Suppressing cell-contractility was sufficient to maintain an epithelial phenotype. Our results suggest that integrin-specific engagement of fibronectin adhesive domains and the mechanics of the ECM act synergistically to direct EMT.

Tumor metastasis involves the migration of tumor cells from the original tumor mass, through the ECM and into neighboring regions such as other tissues or vasculature. While a lot is known regarding the various factors influencing individual cell migration through the ECM, very little quantitative information is available on collective migration of tumor cells in 3D environments. To better understand the factors that control the collective migration of cells, we have developed a multi-scale, integrated mechanistic and biomechanical model describing tumor cell motion in 3D. Using the visco-elastic characteristics of the matrix, the stiffness of single cells and tumors, the biochemical reaction rates for oxygen and nutrient transport and cell-matrix adhesions, we show how synergy between matrix mechanics, cell adhesion and nutrients regulate cell speed and persistence in collective motion and EMT. We show that based on the interplay between biochemical factors and physical parameters, using the model equations we are able to characterize in vitro, in vivo and clinically observed EMT and collective cell motion. Our experimental results validate our computational predictions and allow us to fine tune our model to breast cancer progression and metastasis. Good comparison with experiments suggests that our model opens new avenues of investigation for experimental studies as well as provides a theoretical tool which can be developed upon to be able to make accurate predictions regarding single and collective tumor cell migration during cancer metastasis.

Dendritic cells (DCs) migrate from sites of inflammation to secondary lymphoid organs where they initiate the adaptive immune response. While motility is essential to DC function, the mechanisms by which they migrate are not fully understood. We incorporated micropost array detectors into a microfluidic gradient generator to develop a novel method for probing low magnitude traction forces during directional migration. We found migration of primary murine DCs is driven by short-lived traction stresses at the leading edge or filopodia. The traction forces generated by DCs are smaller in magnitude than found in neutrophils, and of similar magnitude during chemotaxis and chemokinesis, at 18 +/- 1.4 and 16 +/- 1.3 nN/cell, respectively. The characteristic duration of local DC traction forces was 3 minutes. The maximum principal stress in the cell occurred in the plane perpendicular to the axis of motion, forward of the centroid. We illustrate that the spatiotemporal pattern of traction stresses can be used to predict the direction of future DC motion. Overall, DCs show a mode of migration distinct from both mesenchymal cells and neutrophils, characterized by rapid turnover of traction forces in leading filopodia. The methods described in this paper will be widely applicable to force measurements of cells of the immune system.

We report the use of a thiol-ene photopolymerization to synthetically engineer a peptide functionalized poly(ethylene glycol) (PEG) hydrogel for 3D cell culture. Specifically, we used norbornene functionalized, 8 arm, 40kDa PEG in conjunction with a cysteine flanked matrix metalloproteinase degradable peptide linker and a cysteine terminated RGDS sequence to form our hydrogel networks. Because of the click nature of the chemistry, the PEG molecular weight and functionality can be used to control the elastic modulus, while cysteine-containing peptides can be used to introduce biological ligands, rendering independent control of the mechanical properties versus biochemical functionality. For example, by varying cross-linking density, we obtained moduli spanning an order of magnitude (50 – 400 Pa, confirmed via shear rheometry). Further with a given cross-linking density, the RGD ligand was varied while maintaining a constant modulus. We validated the benefits of our hydrogel platform by testing the locomotion dependence of a tumor cell line, HT-1080s, on adhesion and stiffness. We systematically varied the RGD concentration within three gel formulations (50 Pa – soft, 110 Pa – intermediate, and 260 Pa – stiff) and quantified the percent of HT-1080s migrating and their persistence. Migrations statistics including velocity and distance to origin were also strongly influenced by adhesiveness, but only up to a threshold RGD concentration of 1mM upon where a transition to modulus dependence was observed. HT1080 morphologies ranged from amoeboid-like in soft hydrogels and RGD concentrations to mesenchymal-like in stiff hydrogels and RGD values, with mixed modes at intermediate concentrations. Similar findings were observed for the WM239a cells, a metastatic melanoma tumor cell line, suggesting that morphological qualities are not an inherent characteristic of a particular cell type, but rather a consequence of the specific microenvironment. Collectively, our results indicate a complex relationship in which parameter perturbations induce cellular morphology and migration changes. While we performed our cell experiments in hydrogels with moduli of less than 0.5 kPa, we demonstrate moduli of in excess of 30 kPa. By altering polymer length or functionality, we believe that this system is capable of reaching even higher values, which may be useful in other cellular studies, such as osteogenesis of mesenchymal stem cells. Our platform allows incorporation of any thiol containing ligands or proteins, is based on a facile synthesis protocol, is highly tunable, provides precise control, and is amenable to a wide range of biological assays commonly employed by molecular biologists.

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by diffuse infiltration of single cells into the brain parenchyma, which is a process that relies in part on aberrant biochemical and biophysical interactions between tumor cells and the brain extracellular matrix (ECM). A major obstacle to understanding ECM regulation of GBM invasion is the absence of model matrix systems that recapitulate the distinct composition and physical structure of brain ECM while allowing independent control of adhesive ligand density, mechanics, and microstructure. To address this need, we have synthesized brain-mimetic ECMs based on hyaluronic acid (HA) with a range of stiffnesses that encompasses normal and tumorigenic brain tissue and functionalized these materials with short Arg-Gly-Asp (RGD) peptides to facilitate cell adhesion. Scanning electron micrographs of the hydrogels revealed a dense, sheet-like microstructure with nanoscale porosity similar to brain extracellular space. On flat hydrogel substrates, glioma cell spreading area and actin stress fiber assembly increased strongly with increasing density of RGD peptide. Increasing HA stiffness under constant RGD density produced similar trends and increased the speed of random motility. In a three-dimensional (3D) spheroid paradigm, glioma cells invaded HA hydrogels with morphological patterns distinct from those observed on flat surfaces or in 3D collagen-based ECMs but highly reminiscent of those seen in brain slices. This result suggests that the distinctive motility exhibited by glioma cells invading brain tissue is strongly dependent on the unique physical structure of brain matrix, necessitating the use of similarly structured ECM-mimetic materials to interrogate the underlying mechanisms. We will present our work on delineating these mechanisms and thereby elucidating ECM mechanobiological regulation of brain tumor progression.

Introduction: Multiple features of the cellular microenvironment can influence cell function, including soluble factors, mechanics, and the extracellular matrix (ECM). Different cell types deposit their own characteristic ECM, modify the existing ECM, and interact with this dynamic ECM environment in different ways. Defects in the ECM during in vivo tissue development are known to cause significant tissue dysfunction, and a disorganized ECM structure is a hallmark feature of several types of diseases. Thus, from the perspective of tissue engineering, manipulating the ECM environment can be a powerful tool in directing cell fate and function. In this presentation, we focus on the role of ECM composition in two areas of cardiovascular biology: 1) the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes, and 2) the initiation of calcific disease in aortic valve cells and leaflets, and apply this information to the design of engineered environments to further study these phenomena. Methods: For hESC culture, embryoid bodies were generated from H9 hESCs according to established protocols (NSCB Cardiac differentiation – EB method, SOP-CH-203c) and cultured on uncoated TCPS, as well as on TCPS plates coated with collagen type I, laminin, or fibronectin. ECM remodelling, gene expression, and protein expression were quantified via ELISA, qRT-PCR (for 14 different genes), and flow cytometry, respectively, at Days 4, 8, and 12 post-seeding. For heart valve studies, both “bottom-up” and “top-down” approaches were employed, wherein valvular interstitial cells (VICs) were cultured on various defined ECM environments and analyzed for expression of disease markers, while the top-down approach consisted of modifying discrete ECM components within the native valve leaflet and analyzing for disease following in vitro organ culture in a bioreactor. Results: For both hESCs and VICs, the type of substrate upon which the cells were plated on Day 0 influenced the composition and remodelling of the ECM at all later time points. In the case of hESCs, each type of ECM coating yielded a different steady-state ECM composition profile, and each of these ECM composition/deposition profiles corresponded to differences in the differentiation lineage of these cells. In the case of VICs, it was found that certain ECM components corresponded to emergence of a disease-like cell phenotype, while others were able to maintain a quiescent VIC population. Manipulation of the 3D ECM in native valve leaflets revealed that certain cell-ECM interactions, specifically those between VICs and hyaluronic acid, are crucial in maintaining healthy valve function. Conclusions: Understanding the role of ECM composition in tissue development and disease can enable the construction of appropriate environments for applications such as guiding stem cell fate for tissue regeneration or elucidating disease etiology to identify potential treatment targets.

Statement of Purpose: Rational design of scaffolds to elicit desired mesenchymal stem cell (MSC) lineage progression has proven to be challenging due, in part, to our incomplete understanding of MSC responses to extracellular matrix (ECM)-mediated stimuli, including biochemical cue identity. In the present work, we begin to address this challenge by probing MSC responses to collagen-based biochemical motifs using novel hybrid hydrogels. These hydrogels were generated by covalently crosslinking diacrylate-derivatized poly(ethylene glycol) (PEGDA) and a novel collagen-mimetic protein, termed Scl2-1. Scl2-1 is unique in that it contains the GXY repeats and stable triple helical structure of native collagen but lacks collagen’s array of cell adhesion, cytokine binding, and enzymatic degradation sites. Thus, Scl2-1 provides a “blank-slate” into which desired collagen adhesion sequences can be programmed by site-directed mutagenesis while maintaining the triple helical context natively associated with these motifs. The current work explores the impact of a1b1 and a2b1 integrin binding and associated signaling on human MSCs (hMSCs) using modified Scl2-1 proteins. Specifically, the influence of Scl2-2 versus Scl2-3 (Scl2 based proteins containing a1b1/a2b1 and a1b1 binding motifs, respectively) on hMSC lineage progression was examined. Methods: Scl2 proteins were then reacted with acrylate-PEG-hydroxysuccinimide (Ac-PEG-NHS, MW 3400) at a 1:2 molar ratio inbicarbonate buffer. PEGDA-Scl2 gels were fabricated by combining 10 wt% PEGDA (10 kDa) with photoinitiator (Irgacure 2959), 1 mg/mL of Ac-PEG-Scl2, and 1x106 hMSCs (Lonza) per mL. The solutions were then crosslinked via exposure to 365 nm UV light. Results and Discussion: hMSCs were encapsulated in PEGDA-Scl2 hydrogels at a weight ratio of PEGDA to Scl2 of 100:1. This weight ratio was selected so that the elastic modulus, mesh size, and degradation rate of each hydrogel network would be dominated by PEGDA. Gels were cultured in media containing 10% FBS for 7 days. Over this time period, no alterations in gel volume or modulus were observed. Combined, these conditions ensured that Scl2 identity was the primary design variable across gels. Based on ELISA analyses, no differences in the levels of myoD (myogenic) or runx2 (osteogenic) were noted with varying integrin adhesion motif. However, significant differences in pparg (adipogenic) and sox9 (chondrogenic) levels were observed. Specifically, pparg expression was elevated by a2b1 signaling (Scl2-2). In contrast, sox9 levels were increased by a1b1 signaling (Scl2-3), but not in the presence of simultaneous a2b1 binding (Scl2-2). Given that a2b1 binding activates p38 whereas a1b1 does not, the p38 MAPK signaling cascade may play an important role in hMSC adipogenic differentiation. Future studies will incorporate gene silencing methods to elucidate causative cell signaling.

Synthetic matrices, such as poly(ethylene glycol) or PEG-based hydrogels, have been used to study cell morphogenesis in three-dimension (3D). PEG hydrogels crosslinked with defined peptide sequences are particularly suitable for identifying key matrix properties responsible for guiding cell morphogenesis. Although currently available PEG hydrogel systems have been used to answer many important questions related to cell fate processes in 3D, little is known about the utility of PEG hydrogels on regulating the proliferation and differentiation of pancreatic ductal epithelial cells (PANC-1). PANC-1 cells are immortalized epithelial cells that have been widely used to study pancreatic cancer cell metastasis. More recently, studies have shown that PANC-1 cells, when given appropriate soluble cues, can be differentiated into insulin secreting cell clusters that may provide an alternative cell source for cell transplantation to cure type 1 diabetes. While this process may be triggered by suspension culture in serum-free condition, no prior attempt has been made to establish a tunable 3D matrix for controlled differentiation of PANC-1 cells. PANC-1 cells exhibit spindle-shaped and fibroblastic morphology when cultured on 2D surfaces. We found that, when cultured in PEG hydrogels, PANC-1 cells form clusters with varying degrees of aggregation, depending on matrix composition, degradability, and media condition. Encapsulated PANC-1 cells cultured in serum-free medium containing standard ITS supplements had high level of MMP (matrix metalloproteinase) expression that accelerated both cell aggregation and matrix degradation. When cultured in non-MMP cleavable PEG hydrogels, encapsulated PANC-1 cells exhibited lower viability and cluster formation. These fundamental studies have established PEG-peptide hydrogels as a suitable platform for studying pancreatic cancer cell morphogenesis in 3D as well as for generating insulin-secreting cell clusters for the treatment of type 1 diabetes.

Macroporosity in scaffolds is crucial for successful tissue engineering (TE) process to provide space for blood vessel in-growth, cell proliferation, and cellular interactions. Current techniques in fabricating porous scaffold often involve processes that are harsh for cells and are thus only amenable for making scaffolds with preformed pores before cell seeding, and do not allow the tunability of scaffold porosity during cell-culture process. Scaffolds with dynamically tunable pores by using biocompatible porogens are highly desirable to overcome aforementioned limitations. Stimuli-responsive pore formation allows the control of cellular processes at desired time points, and biodegradable porogens can be used to serve as delivery vehicles for dispatching cells or biological cues to promote desired cellular processes. In this study, we aim to develop a novel method for fabricating TE scaffolds with dynamically-formed porosity. We developed three types of stimuli-responsive micro-spherical porogens from gelatin (GelMA), alginate (Alg), and glycidyl-methacrylated hyaluronan (GMHA), which dissolves upon the exposures to body temperature (BT), small-molecule chelating, and enzymatic digestion, respectively. To study the effect of stimuli-responsive pore-formation using the porogens, the three porogens were imbedded in a methacrylated gelatin (Gel-MA) scaffold, and the scaffold was exposed to different stimuli at sequential time points: BT on day 0, EDTA on day 2, and hyaluronidase (HAdase) on day 5. The subsequent porosity formation was evaluated using scanning electron-microscopy (SEM). To explore the feasibility of applying the dynamic pore-formation to cell delivery, human adipose derived stem cells (hADSCs) were encapsulated in the Alg porogen and the same experimental procedure was repeated. Cell viability and proliferation during the sequential stimuli treatments were evaluated using fluorescent microscopy (FLM), cell tittering assay, as well as SEM. SEM imaging confirmed sequential stimuli treatments led to gradual increases of the macropores' volume (MPV) in the 3D scaffold. Increasing temperature to BT led to about 15% MPV in the scaffold, the EDTA chelating led to about 30% MPV and enhanced pore interconnectivity, and at last the HAdase digesting increased the MPV to about 95% and led to highly interconnected pores in the scaffold. FLM and SEM imaging showed that hADSCs start to spread and migrate inside the scaffold 2 day after the treatment by EDTA, the cell population continued to grow after the HAdase treatment, and the cells distributed uniformly in the scaffold by day 14. The number of hADSC increased by more than 5 folds in the scaffold with dynamically formed pores. We have demonstrated a novel method to introduce dynamically formed macropores in TE scaffold by using cell-friendly porogens, which lead to an interconnecting porous network and support cell releasing, growth, spreading, and migration on designable time points.

Hydrogels, highly hydrated cross-linked polymer networks, have emerged as powerful synthetic analogs of extracellular matrices for basic cell studies as well as promising biomaterials for regenerative medicine applications. A critical advantage of these artificial matrices over natural networks is that bioactive functionalities, such as cell adhesive sequences and growth factors, can be incorporated in precise densities while the substrate mechanical properties are independently controlled. Polyethylene glycol (PEG) hydrogels represent the ‘gold standard’ due to their intrinsic low-protein adsorption properties, minimal inflammatory profile and history of safe in vivo use, ease in incorporating various functionalities, and commercial availability of reagents. We have explored the use of an alternative reactive cross-linking moiety for PEG hydrogels, the maleimide functional group. We demonstrate several advantages over other cross-linking chemistries, namely stoichiometric hydrogels with improved cross-linking efficiency, bioligand incorporation and reaction time scales appropriate for clinical use for in situ gelation. The maleimide reactive group is extensively used in peptide bioconjugate chemistry because of its fast reaction kinetics and high specificity for thiols at physiological pH. We have engineered PEG-maleimide hydrogels to incorporate VEGF as supportive matrices to improve pancreatic islet engraftment and vascularization. Precursor solutions of 4-arm PEG-maleimide were functionalized with RGD adhesion peptide and VEGF and cross-linked into a hydrogel by addition of cysteine-flanked collagenase-degradable peptides. Degradation studies demonstrated collagenase-dependent controlled release of VEGF from these hydrogels. These hydrogels supported in vitro islet survival, insulin production, and intra-islet endothelial cell sprouting. Furthermore, pancreatic islets delivered within hydrogels to the small bowel mesentery exhibited enhanced vascular reperfusion in vivo compared to other groups. Importantly, islets delivered within these functionalized hydrogels exhibited improved engraftment, vascularization and insulin production compared to islets delivered within other hydrogels and without a hydrogel carrier. In another application, we functionalized hydrogels with the integrin-specific, collagen-mimetic triple helical peptide GFOGER to promote osteogenic differentiation and bone repair. Human mesenchymal stem cells adhered well and maintained viability on both RGD and GFOGER hydrogels. However, alkaline phosphatase activity and mineralization was higher on GFOGER-hydrogels than on RGD-hydrogels. GFOGER-functionalized hydrogels significantly enhanced bone volume and mass in critically-sized, segmental bone defects in murine radii compared to other hydrogels and empty defects. These studies establish these maleimide-cross-linked hydrogels as promising biomaterial carriers for cell delivery, engraftment and enhanced tissue repair.

Endometriosis is a chronic condition affecting 5-10% of women in which endometrial tissue is found outside the uterus, resulting in pain and infertility. Despite its prevalence, relatively little is known about how endometriosis develops and progresses, leading to a lack of treatment options. The study of endometriosis is hampered by a lack of appropriate model systems and the overwhelming complexity that results from the interaction of multiple cell types that are responding to a milieu of inflammatory stimuli. To model the endometriotic microenvironment, we are developing an in vitro culture system in which epithelial cell, stromal cell, and macrophage interactions can be examined. Epithelial cells and/or stromal cells are cultured on basement membrane, resulting in organization into multi-cellular structures that are not observed on other substrates. In these multi-cellular structures, cells are viable and proliferate for several days. Using this system, we have examined the impact of soluble factors produced by macrophages on epithelial cells. In contrast to cells cultured on tissue culture plastic, epithelial cells cultured on basement membrane respond to macrophage-conditioned media, with significant increases in proliferation. Future work will examine the impact of the three cell types together, resulting in the first in vitro model of the endometriotic microenvironment.

Current synthetic materials in regenerative medicine applications are able to provide a scaffold to promote new tissue formation and act as a template to organise the resulting extracellular matrix (ECM). It remains a challenge to recreate the nanoscale structure, chemistry, or topography of native ECM in a single construct.[1] Incorporating biomolecules such as glycosaminoglycans (GAGs) in scaffolds is known to improve their biological function yet still requires placement and appropriate presentation for materials that mimic the exceptional properties and functions of native tissues. The next challenge in biomaterials is to create hierarchically organised scaffolds that mimic the spatial distribution of components found in native tissues. We have designed and synthesised novel polymer-peptide conjugates with a traditional biodegradable polymer poly(caprolactone) (PCL) and amino acid sequences to bind and distribute specific GAGs within a fibrous scaffold. Field-driven functionalisation during electrospinning[2] of these amphiphilic conjugates with unmodified high MW PCL forced peptides to the surface of the fibre while covalently-linked PCL anchored it within the fibre bulk. Combining electrospinning techniques developed in our laboratory[3] and previously[4], various concentration gradients of different GAG-binding peptides can be created through the fibrous 3D scaffold to mimic the GAG composition of complex tissues such as articular cartilage. GAGs are known to contribute to the unique mechanical properties and biological functions of articular cartilage, and these scaffolds provide a platform to study how gradients in GAG composition affect mechanical properties as well as cell behaviour. Peptides were synthesised manually using standard solid phase Fmoc synthesis techniques and purified by HPLC. Low MW PCL was modified with maleimide isocyanate using reported procedures[5] then coupled with the peptide via a cysteine to the maleimide. The conjugation steps were confirmed by 1H-NMR. Short synthetic peptides allow for functionalisation with biomolecules without risk of denaturing during electrospinning. The PCL-peptide conjugates offer the additional advantage of combining naturally-occurring amino acids with a biocompatible, biodegradable polymer. By simply changing the peptide sequence, this versatile strategy can be applied to a wide range of regenerative medicine applications. [1] (a) Stevens MM; George JH. Science 2005, 38, 1135-1138. (b) Place ES; George JH; Williams CK; Stevens MM. Chem Soc Rev 2009, 38, 1139-1151. [2] (a) Sun XY; Shankar R; Borner HG; Ghosh TK; Spontak RJ. Adv Matl 2007,19(1), 87-91. (b) Gentsch R; Pippig F; Schmidt S; Cernoch P; Polleux J; Borner HG. Macromol 2011, 44, 453-461. [3] McCullen SD; Autefage H; Callanan A; Gentleman E; Stevens MM. submitted 2011. [4] Sundararaghavan HG; Burdick JA. Biomacromol 2011,12(6), 2344-2350. [5] Annunziato ME; Patel US; Ranade M; Palumbo PS. Bioconj Chem 1993, 4, 221-218.

In the body cells are surrounded by the extracellular matrix (ECM), which is a complex arrangement of proteins and biomolecules that are rich in growth factor and cell binding sites.(1) Attempts to recreate this environment in vitro have yielded broadly applicable strategies, such as including the RGD peptide for cell adhesion, but attempts to include integrin specific ligands for controlled cell signaling have been more difficult to achieve. In vivo, a single cell surface protein can be bound simultaneously by several epitopes, as is the case with the synergy sequence found on fibronectin. This consists of an RGD and a PHSRN epitope synergistically binding the α5β1 integrin, and has been shown to induce osteogenesis in human mesenchymal stromal cells (hMSCs).(2) Efforts to synthetically recreate this effect in vitro have met little success, likely because these epitopes require a spacing around 3.3 nm to effective bind the α5β1 integrin, as determined through computer simulations.(3) To create a system that would controllably space two epitopes, we first designed and synthesized a novel self-assembling peptide, referred to as the backbone, to form a stable hydrogel during cell culture. This backbone peptide utilizes a β–sheet-forming domain that induces self-assembly into one-dimensional nanostructures. Transmission electron microscopy (TEM) showed these peptides form twisted nanofibers with diameters around 5 nm and lengths on the micron scale. This geometry suggests these nanostructures are one peptide across and only a few β–sheets thick with the β–sheets aligned with the long axis of the fiber. To control the spacing of biological epitopes, we synthesized the peptide backbone with selective modifications to display RGDS and PHSRN side chains at desired spacings. Since β–sheets are rigid in solution, the distances between any two amino acids along the backbone can be determined a priori. The RGDS/PHSRN peptide can be co-assembled with the backbone peptide to create a hydrogel with bioactivity from the synergistically displayed epitopes. Preliminary in vitro experiments confirm that these hydrogels support hMSC survival and growth, and ongoing experiments are looking at the effects of epitope spacing on α5β1 integrin activation and hMSC differentiation. 1. Stevens MM, George JH. Exploring and engineering the cell surface interface. Science. 2005 Nov. 18;310(5751):1135–1138. 2. Hamidouche Z, Fromigué O, Ringe J, Haeupl T, Vaudin P, Pagès J-C, et al. Priming integrin alpha 5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis. Proc Natl Acad Sci USA. 2009;106(44):18587–18591. 3. Krammer A, Craig D, Thomas WE, Schulten K, Vogel V. A structural model for force regulated integrin binding to fibronectin's RGD-synergy site. Matrix Biol. 2002 Mar.;21(2):139–147.

Self-assembled peptides that undergo hydrogelation via β-sheet fibrillization are a well-described platform for creating synthetic mimics of the extracellular matrix (ECM). However, to date these materials have been significantly limited by their being composed primarily of short peptides, lacking the precise biofunctionality afforded by folded proteins. Here we describe the development of a novel fusion protein strategy enabling the co-assembly of well-folded protein domains within fibrillar peptide assemblies. Because these materials exhibit minimal compositional drift upon assembly, hydrogels can be constructed that integrate precisely controlled combinations of multiple different proteins. The approach is based on a self-assembling fusion domain we term a “β-tail”, which allows for β-tail fusion proteins to be expressed and purified from microbial expression systems without undergoing premature self-assembly. Subsequently, co-assembly of β-tail fusion proteins with rapidly fibrillizing synthetic peptides leads to integration of the expressed proteins within β-sheet fibrillar hydrogels. Several different β-tail fusion proteins were constructed from green, red, and blue fluorescent proteins. Upon co-assembly of these differently colored proteins with the self-assembling peptide Q11, hydrogels were formed in which the fluorescence intensity indicated a near quantitative incorporation of each β-tail protein provided. Integration of the proteins into the peptide fibers was assessed with immunogold labeling and transmission electron microscopy, which determined that β-tail-GFP was specifically co-localized to the peptide nanofibers. Further, circular dichroism indicated that the β-tail peptide possessed a weak α-helical structure in isolation, which was abolished upon mixing with Q11 in favor of β-sheet structure, also suggesting integration within the peptide fiber. Exploiting the lack of compositional drift in this system, the color of the materials could be finely tuned across a wide spectrum of colors by mixing red, green, and blue fluorescent β-tail proteins, demonstrating that the β-tail proteins could be co-assembled both with compositional precision and while retaining proper protein folding. This approach is likely to enable the development of protein-presenting self-assembled hydrogels for diverse biomedical and biotechnological applications ranging from 3D cell culture to immunotherapies.