Engineering complex 3-dimensional tissues presents great promises to improve treatment of tissue defects and to provide better understanding of emergent behavior of normal and pathologic cells. Hence, there is an immediate need for systems that can recapitulate and further manipulate the tissues of interest, in a high throughput and automated manner while maximizing the cell and tissue functionality. Such approaches can also be used to develop cellular systems and to study the emergent behavior of integrated cellular systems. In this talk, we will present an overview of our work using 3-D stereolithography to develop polymeric structures embedded with live cells for a range of applications. Firstly, we show that cell-encapsulated hydrogels with complex three-dimensional (3D) structures can be fabricated from photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) using modified â?~top-downâ?T and â?~bottoms-upâ?T versions of a commercially available stereolithography apparatus (SLA). Long-term viability of encapsulated NIH/3T3 cells was quantitatively evaluated using an MTS assay and shown to improve over 14 days by increasing the Mw of the hydrogels. Secondly, we show that we can spatially organize primary hippocampus neurons (HNs) and skeletal muscle myoblast cells (MCs) in a 3D hydrogel matrix with tunable mechanical and degradation properties. The spatial organization of these multiple cell types revealed that the presence of MCs resulted in increased cholinergic functionality of the HNs, as quantified by their choline acetyltransferase activity. And finally, we show that a â?~livingâ?T microvascular stamp can be fabricated which releases multiple angiogenic factors and subsequently creates neovessels with the same pattern as that engraved in the stamp. The microvascular stamp developed in this study would serve to direct the emergent cellular behavior towards vascularization, improve the quality of revascularization therapies, and allow the vascularization of biological machines in-vitro.

Reconstruction of phylogenetic trees based on 16S rRNA gene sequencing reveals that so far only a tiny fraction of microbial diversity has been cultured in the laboratory. One major reason behind this â?ounculturabilityâ? is that many microbes function in symbiosis, frequently exchanging metabolites to sustain their own metabolism, while key exchanged metabolites have hardly been identified. To advance the culturability of diverse microbes we propose a method to engineer a microfluidic co-culture platform that mimics natural conditions for bacterial growth. The key to innovation is to physically isolate bacteria while allowing chemical communication through metabolite diffusion. In our method, we use a porous material, poly(2-hydroxyethyl methacrylateco-ethylene dimethacrylate) (HEMA-EDMA), to fabricate a microwell array with 105 individual culture chambers. Pore size of HEMA-EDMA was confirmed by ESEM imaging to be less than 200 nm, adequate for isolating all identified bacteria. We have video-recorded fluorescence labeled Escherichia coli swimming in confined of HEMA-EDMA wells and observe that E. coli is unable to go travel between culture chambers. In our culturing process, we stochastically seed fluorescently-labeled E. coli into each 1 nanoliter sized well with prescribed cell solution, a 500 times dilution of the E. coli culture solution in stationary phase. Our statistical results of 300 wells showed that on average 0.92 cells were seeded in each well with a standard deviation of 0.79. In order to prevent evaporation of growth media, we packaged the device with a PDMS cap and immersed the device in water. After about 36 hours of cell culture at 30°C without refreshment of growth media, 53.7% of the seeded wells were observed to have exponential bacterial growth. In this case only one well with observed growth was not initially seeded. Suggesting that cross-contamination by way of â?oswimmingâ? from one well to neighboring wells was minimal. In this presentation we will explore co-culture of bacterial species which rely upon a syntrophic relationship in our novel device. This will be demonstrated via symbiotic strains of E. coli and Salmonella genetically engineered to exchange lactate to ensure survival. In the future we will utilize this device to search for previously uncultivable microbes that rely upon syntrophic relationships.

Cell migration is a dynamic process strongly regulated by biophysical interactions between cells and the extracellular matrix (ECM). These mechanosensitive interactions are driven by sub-cellular mechanisms including protrusions at the leading edge of the migrating cell, adhesions with the ECM, and actomyosin contractility, all of which depend on both the stiffness and geometry of the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility have been extensively studied in two-dimensional ECMs, extension of this concept to three-dimensional ECMs that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change matrix porosity. This convolution of ECM biophysical properties is particularly problematic given that pore size has been independently demonstrated to regulate migration speed. Here we investigate this problem using a novel microscale culture platform and mathematical modeling. First, we introduce a microchannel-based matrix platform that allows orthogonal variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also causes migration speed to increase monotonically with ECM stiffness, in contrast with the biphasic relationship observed on unconfined ECMs. We attribute this effect to the fact that channel confinement forces polarization of traction force, which in turn enhances fast, persistent migration. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. To integrate all of these hypotheses into a coherent, quantitative framework, we develop a predictive multiscale model of a cell migrating in an ECM channel of defined width and stiffness, which we show recapitulates key experimental trends. These studies introduce a new paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement modulates the relationship between cell migration speed and ECM stiffness.

Photochemical patterning is a powerful tool for manipulating the biochemical composition of extracellular matrix mimetic hydrogels. The general approach has been to swell in a biomolecule such as a peptide, and then conjugate it to the hydrogel matrix via a photo-initiated reaction. Excellent 2D patterning fidelity can be achieved with this method by using conventional photomasking technology, and complex 3D patterns can be created by two-photon irradiation. It is even possible to create complex microenvironments with multiple biomolecules by sequential patterning. Nevertheless, the ability of the soluble biomolecule to interact with cells during the swelling process is a potential limitation. To address this problem, we propose here the use of a biomolecule caging strategy. In short, biomolecule caging is the general approach of modifying a biomolecule with a photo-labile moiety to temporarily render it biologically inactive, but photo-activatible. Thus, caging is also a photochemical approach, and the same techniques for hydrogel patterning described above can be utilized. However, because the molecule must be irradiated and â?oswitched onâ? to become bioactive, this approach circumvents the potential for unwanted interactions with cells outside of the patterned region of the hydrogel. Importantly, caging has shown exceptional utility in biochemistry studies because it allows for user-controlled signal activation with spatio-temporal resolution. Numerous caged biomolecules have been described in the literature including morpholinos, oligonucleotides, peptides, and even proteins. Nevertheless, biomolecule caging has only been used minimally in biomaterials and tissue engineering. To show proof of concept, we have designed caged RGD peptides that can either be incorporated into the hydrogel matrix during initial gel formation, or patterned in at a later time point with an orthogonal photo-initiated reaction. RGD caging with nitrobenzyl and amino coumarinyl moieties, which have the potential for orthogonal photo-cleavage, was explored. Solution studies of uncaging showed that the proper irradiation conditions resulted in efficient uncaging to produce the bioactive RGD peptide without side reactions. Our efforts to dynamically regulate cell-matrix interactions using these caged RGD peptides will be presented.

There is a growing interest in the development of novel biomaterial platforms that can be tuned both dynamically and in real time to probe the effects of the surrounding materials on cell function. Taking lead from the recently popularized â?oclickâ? philosophy, where chemical reactions proceed efficiently and with high specificity, orthogonal reactions can be exploited to control different physical and chemical aspects of cell-laden polymer scaffolds. This contribution will detail one such synthetic platform to engineer multifunctional networks based on three orthogonal reactions that enable user-defined biochemical and biomechanical cues to be introduced in the presence of cells at any point in time and space. Gels are first formed via a copper-free click reaction between azide and cyclooctyne moieties, enabling the encapsulation of cells in idealized, initially-uniform networks. Subsequently, a thiol-ene photocoupling reaction is introduced that enables patterning of biological functionalities within the gel, allowing one to tailor the chemical properties of the cell culture niche in situ. Finally, a photodegradable nitrobenzyl ether is incorporated into the hydrogel backbone that allows real-time manipulation of mechanical properties of the system. As both the photocoupling and photodegradation reactions are initiated with different wavelengths of light (365 and >500 nm), they can be performed orthogonally, as verified with NMR, and with full spatial and temporal control illustrated in 3D. This presentation will detail our efforts towards network characterization of chemical functionalization and degradation with 3D confocal microscopy, profilometery, and rheometry. Further, results will be presented to highlight the versatile nature of the chemistry to create programmable niches to study and direct human mesenchymcal stem cell function and mouse embryonic stem cell pluripotency by modifying the local hydrogel environment.

Introduction: Scaffolds with properties that mimic the natural extracellular matrix is highly desirable for regulating stem cell fate and promoting tissue regeneration. Conventional 3D biomaterials are unable to vary biochemical and mechanical cues independently. For example, increasing collagen increases cell adhesion density but also changes the mechanical stiffness of the resulting network. To address these limitations, the goal of this study is to develop an interpenetrating network which allows independent tuning of the biochemical and mechanical properties of the hydrogel. To construct two polymer networks simultaneously and independently, two different crosslinking mechanisms were utilized, namely radical polymerization of methacrylate and coupling of amine with N-Hydroxysuccinimide ester (NHS). Both approaches can be carried out at physiological conditions and are cell-friendly. Materials and Methods: We chose poly(ethylene-glycol) (PEG) as the backbone structure due to its â?oblank slateâ? structure and amenability to chemical modification. To introduce pendent modifiable sites, we propose to perform condensation of PEG, then introduce methacrylate end group to make it photocrosslinkable, followed by pendent NHS introduction and peptides incorporation. Peptides derived from both insoluble ECM or soluble growth factors were conjugated. The mechanical network was constructed from linear amine terminated PEG (molecular weight variable) coupling with 4-arm NHS terminated PEG (Mw~10k). The linear PEG component was synthesized by coupling carboxylic terminated PEG with MMP-1 sensitive peptide sequence GPQGâ?"IWGQK to make the network enzymatically degradable, or by coupling carboxylic terminated PEG with PEG diol to introduce ester linkage into the backbone and make it hydrolytically degradable. The degradation rate can be tailored by changing the concentration and type of the degradable linkages. Results and Discussion: Using a modular design approach, we have developed an interpenetrating network with two distinct crosslinking mechanisms for the biochemical and mechanical â?obuilding blocksâ?. We have successfully synthesized the precursor polymers with final yield higher than 70% and the structures of newly synthesized polymers were verified by NMR. The biochemical precursor with pendent carboxylic groups formed hydrogel upon light exposure (365nm, 5mW cm-2) using I2959 as initiator. The mechanical precursor polymer with amine terminated groups formed hydrogel with 4-arm NHS terminated PEG, and the mechanical strength can be varied across a broad range by changing the number-average molecular weight between crosslinks (Mc) and the concentration of the polymer. The technology platform is very versatile and can be adapted to optimize niche cues for directed differentiation of any type of stem cells towards any lineages.

An ongoing effort in tissue engineering is to design hydrogels with tunable degradability for controlled release and cell encapsulation studies. Recently, hydrogels prepared from step-growth thiol-ene photochemistry have been used to encapsulate a variety of cell types for tissue regeneration, including pancreatic beta cells and human mesenchymal stem cells (hMSCs). In this gelation scheme, norbornene acid functionalized 4-arm poly(ethylene glycol) (PEG) was crosslinked, via a thiol-ene photo-click reaction, with bis-cysteine terminated peptides to form PEG-peptide hydrogels. Due to the presence of an ester bond between the cyclic olefin and PEG, these hydrogels degrade hydrolytically via ester hydrolysis. Furthermore, these thiol-ene hydrogels may be degraded enzymatically with the use of enzyme cleavable peptides as hydrogel crosslinkers. We have proven that the degradation characteristic can be â?~switchedâ?T from surface erosion to bulk degradation through modulating peptide crosslinkers with different enzyme sensitivities. We also synthesized a series of thiol-ene hydrogels with different peptide crosslinker sequences and functionalities, from which to tune the time required for complete gel degradation (from weeks to months in vitro). In addition, we utilized a statistical-co-kinetic model to predict the degradation profiles of these thiol-ene hydrogels. Specifically, this model takes into account the variations in hydrogel crosslinking efficiency, hydrolysis kinetics, as well as polymer structural information (including molecular weight and functionality of the macromer and crosslinker). While the degradation of ester-linked thiol-ene hydrogels is base-catalyzed, our experimental results indicated a slower than predicted degradation rate at basic condition (pH8), indicating the existence of another base-catalyzed mechanism within the hydrogel network that retards gel degradation. We hypothesized that this phenomenon was due to the oxidation of thioether bonds between PEG macromer and thiol-terminated crosslinker. The benefits of highly tunable and predictable thiol-ene hydrogel degradation will be realized via controlled cell morphogenesis in 3D.

The cellular microenvironment plays an integral role in cell adhesion, proliferation, and gene expression and has been shown to guide stem cell fate. Stem cells have a host of applications in tissue engineering and regenerative medicine including skeletal muscle regeneration and cardiac repair. In spite of the large therapeutic impact of stem cells, their use is hindered in part by the use of undefined substrate culture conditions which use animal-derived products leading to issues with xenogenic contamination and batch to batch variation. Some of these concerns could be eliminated through the use of synthetic substrates. Our lab has demonstrated long term maintenance of human embryonic stem cells (hESCs) on a synthetic zwitterionic polymer, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), generated using a â?ografting toâ? approach.1 However, the influence of substrate properties on cell morphology and function is not clearly understood. In order to elucidate the material properties that influence hESC behavior on PMEDSAH, a more controlled polymerization process that utilizes a â?ografting fromâ? approach, atom transfer radical polymerization (ATRP), is employed to fabricate PMEDSAH brushes. The physical properties of this polymer are modulated by brush thickness and in this work the influence of polymer chemistry on cell-substrate interactions is decoupled from the physical properties of the polymer by generating PMEDSAH brushes of varying thickness. In particular, the wettability, surface roughness, and antigen binding capabilities of the PMEDSAH brush as a function of thickness is assessed. Furthermore, the interaction of human mesenchymal stem cells (hMSCs) and hESCs on PMEDSAH coatings of varying thickness is undertaken. This work elucidates the influence of PMEDSAH material properties on hMSC and hESC adhesion, morphology, and differentiation. Results from this work may be applied to material design for stem cell culture systems. 1. Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat Biotechnol 28, 581-583 (2010).

Variation of multiple signals within a cellular microenvironment can lead to a change in cell response that is not easily predictable from investigations of single signals. Cells can respond to several types of environmental signal, including both biological signals (such as adhesion proteins, growth factors and small peptide analogs) and physical features such as topography and mechanical properties. A platform is required where both physical and biochemical signals can be varied independently of each other within a three dimensional environment. Our work has studied electrospun nanofibres produced from poly(styrene-co-vinylbenzyl chloride). The VBC monomer subunits allow for the subsequent grafting of polymer brushes onto the fibres via atom transfer radical polymerisation (ATRP) without requiring further chemical activation. This process creates a bottlebrush-like core-shell structure. Polymer brush layers containing poly(ethylene glycol) can provide a low protein fouling background, and can be co-grafted with other functional monomers. This has allowed the creation of low-fouling electrospun fibres that contain reactive groups, such as alkynes, for highly specific covalent attachment of bioactive peptides via click-chemistry. At the same time, the physical properties of the material can be altered easily through optimisation of the electrospinning and grafting conditions, independently of changes in peptide attachment. This type of material can provide a highly controlled 3D nanofibre substrate within which interfacial interactions may in the future be investigated and optimised.

Conducting polymers (CPs) are organic compounds with special chemical characteristics that endow them with electrical conductivity on the same order as inorganic semiconductors or metals. In addition, CPs exhibit the traditional advantages of polymeric materials: they are light, easy and inexpensive to synthesize, amenable to blending and copolymerization with other materials, and may be doped with a variety of compounds that can be adsorbed and released under desired conditions. As a result, CPs have been investigated for use in a wide range of applications such as implantable biosensors, organic solar cells, water-purification membranes, artificial muscles, advanced textiles, flexible electronics, and clinical devices for delivering biologically active compounds. The fact that a variety of cell types respond to electrical stimuli also renders CPs especially useful for tissue engineering scaffolds. However, the brittleness and poor long-term stability of CPs have greatly impeded their widespread application. To reduce the inherent brittleness of CPs, we have synthesized blends with polyurethane (PU). The improved mechanical properties of this material, compared to pure CPs, allow it to be processed into a range of morphologies (films, foams, fibers) with widely tunable mechanical and electrical properties. Once synthesized, the material may be dissolved in a variety of solvents and electrospun to produce elastic fibers with dimensions as small as 100 nanometers. The resistance of these fibers ranges from 1 â?" 20 Mohm, depending on the chemical formulation and electrospinning parameter setpoints. Although other research groups, including ours, have previously generated conductive fibers by chemically coating non-conductive nanofibers with CPs, these materials have not demonstrated appreciable elasticity. Our elastomeric fibers combine electrical conductivity, biocompatibility, and significant mechanical resilience to create a three-dimensional (3D) cell-culture microenvironment that mimics the native extracellular matrix. This hybrid scaffold thereby enables electrically-stimulated cellular growth and differentiation in a mechanically stressful environment, e.g. for peripheral nerve and cardiac tissue regeneration.

Islet transplantation is currently the only option to cure insulin-dependent (Type 1) diabetes. Unfortunately, the clinical prevalence of islet transplantation is greatly hindered by the shortage of donor islets. Thus, biomaterials capable of promoting the proliferation of insulin-secreting cells in 3D have the potential to provide large quantity of insulin secreting cell spheroids for the treatment of Type 1 diabetes. Furthermore, a strategy that allows for facile recovery of the expanded cell spheroids is beneficial for biological analysis of the expanded cell constructs. Towards achieving these goals, we utilized poly(ethylene glycol) (PEG) hydrogels formed by thiol-ene photopolymerization as a platform for 3D culture of pancreatic beta-cells (MIN6) and explored the influence of gel matrix degradation on cell proliferation and spheroid formation. We found that high macromer concentrations resulted in decreased β-cell survival and proliferation, which may be attributed to lower cell survival following photo-encapsulation and slower hydrolytic gel degradation over time. Macromer concentration, however, had minimum impact on the size of cell spheroids but resulted in decreased number of spheroids formed. Furthermore, we studied the influence of hydrogel degradation on the formation of cell spheroids and found that β-cell proliferated faster when encapsulated in gels capable of undergoing hydrolytic degradation. Using a chymotryspin-sensitive peptide linker, we further examined hydrogel erosion kinetics and its effect on recovery of naturally formed cell spheroid within the hydrogel matrix. In summary, thiol-ene hydrogels provide a cytocompatible environment for promoting the proliferation of beta-cell in 3D, and the formation and recovery of beta-cell spheroids depend largely on hydrogel properties. This hydrogel system may serve as a cell culture platform for generation and recovery of other tissue-engineered cell constructs for regenerative medicine applications.

The therapeutic potential of stem cells is imperative to the future of personalized medicine. This includes areas such as cell therapy, diagnostics, and drug discovery. However, in order to make use of stem cells for these purposes, it is of vital importance that their differentiation be directed into specific cell types. One component that contributes greatly to the differentiation of stem cells is their microenvironment or niche. In vivo, the microenvironment of cells is three-dimensional so it is necessary to fabricate a microenvironment of similar dimension in vitro. Another aspect of the microenvironment that must be taken into account during fabrication is its fluidity. A prominent example of a material that can take a three-dimensional shape and maintain fluidity is the hydrogel. It is proposed here that a hydrogel-microstrand microenvironment with a liquefied core will facilitate the differentiation of mouse embryonic stem cells (mESCs) into adipocytes. These microstrands were fabricated using a simple approach, which involved the dispensing of an alginate-cell solution into calcium chloride. The parameters to control the size and the swelling rate of the hydrogel microstrands were investigated. The self-assembly behaviors and growth of mESCs in alginate microstrands were studied. The cells in the microstrands were then subjected to directed differentiation into adipocytes over the course of two to three weeks. After careful execution, adipocyte differentiation within the microstrands was confirmed by oil red O staining and immunocytochemistry for adipocyte-specific markers. Samples were examined using the Nikon Eclipse 80i fluorescence microscope and Leica SP5 confocal microscope. Further characterization of these microstrands will include elasticity measurements and functional analysis of adipocytes. In summary, we fabricated three-dimensional alginate-microstrand constructs for ESC self-assembly and preferentially differentiated the mESCs into adipocytes with high efficiency. This shows significant promise in fabrication of a novel system that can direct stem cell differentiation and growth for future clinical applications.

The function of a living cell relies on the balance of supramolecular forces that govern the self -assembly of molecules such as proteins and lipids. Understanding the role of these forces in a synthetic context is important for the development of extracellular mimetic materials and to elucidate events at the cell-material interface. In our approach, we utilize a platform of self-assembling peptide amphiphile (PA) molecules as a building block to form high aspect ratio nanofibers. PAs are comprised of a peptide sequence covalently linked to an alkyl tail and are modular in design, affording control over hydrophobic collapse and intermolecular hydrogen bonding, the two dominant supramolecular forces that drive their assembly. To first probe the interaction between the cell membrane and PA, we cultured our materials with MC3T3-E1 preosteoblast cells and found that assemblies with less stable hydrogen bonding induced catastrophic rupture of the cell membrane within minutes, while the nanofibers that were reinforced with ordered beta sheet hydrogen bonding or a stable hydrophobic core supported cell survival. Next, we investigated the interface between integrins and PA, and observed differences in cell adhesion and morphology by simply tailoring the supramolecular forces within the assemblies. Specifically, cells on stiffer nanofibers exhibited a flat morphology, supported by strong focal adhesion complexes and a stable actin cytoskeletal framework. In contrast, cells on softer nanofibers retained a rounded morphology with weaker focal adhesions and less organized actin filaments. Finally, we probed cell function and found that assemblies with increased hydrogen bonding promoted increased proliferation rates and alkaline phosphatase activity as compared to their weaker counterparts. Overall, our results emphasize the significance of supramolecular interactions when using artificial scaffolds in the extracellular milieu, and can provide further insight for materials design to guide biological response in a variety of applications from cancer therapy to regenerative medicine.

Natural polymers such as silk and chitin/chitosan have been shown to have good biocompatibility and low immune response. Unlike synthetic polymers the degraded products of these natural polymer scaffolds are not likely to damage the tissues during regeneration. We prepared scaffolds from silk and chitin/chitosan and their blends with different blend composition to test their ability to support adult mesenchymal stem cells (MSC) growth and differentiation. These scaffolds were analysed for levels of viable cell adhesion, and production of various differentiation and transcription markers using both Real time and Quantative polymerase chain reaction. Adult MSCs were grown on the pure and blended scaffolds and viability assay was done to find out surface attachment of cells followed by quantitative assay for cell attachment. Real time and quantitative PCR was conducted to determine expression for Adipogenic and Chondrogenic markers. All membranes showed good viability and proliferation of cells. No expression for adipogenic markers was observed and an up regulation of chondrogenic markers was observed. The findings suggest that these natural polymers and their blends hold a great potential to be used for MSC growth and differentiation for cartilage tissue engineering.

Recent studies suggest that bioactive scaffolds can be used to regenerate cartilage by local cell homing. However, the specific cell types participating in regeneration are not fully understood. In this study, we identified a novel type of neural crest like synovial stem cell (NCL-SSC) residing within the synovial membrane, which has several important characteristics distinct from previously identified stem cells. Protein marker screening revealed that NCL-SSCs homogeneously expressed neural crest stem cell markers such as Sox1, Sox10, Snail and Vimentin. Furthermore, differentiation assays NCL-SSCs can be induced into not only into mesenchymal lineages including osteoblasts, adipocytes, and chondrocytes, but can also differentiate into peripheral neurons and Schwann cells. In addition, we found that the combination of chick embryo extract and basic fibroblast growth factor can maintain the multipotency and maker expression of NCL-SSCs. Cloning assay showed that the average plating efficiency of NCL-SSCs is about 10% and the cloned colonies retained the marker expression and multipotency. NCL-SSCs can form neurosphere like aggregates when cultured in ultra-low attachment culture dishes. In summary, our study indentified a new type of local cells in synovial knee joint, which could be an important cell source for in situ tissue engineering.

We have developed a novel printing technique of colloidal inks, based on electrohydrodynamically assisted micro- and nanodroplet ejection from a microscopic nozzle. The application of an electrical potential between the ink-loaded pipette and the substrate results in the formation of a liquid meniscus and highly periodic ejection of ink droplets from its apex. The frequency of droplet formation is between 1-10 kHz and can be controlled with the voltage, while the pulse length defines the total number of deposited droplets. By careful matching of the solvent evaporation and ejection frequency, the nanoparticles in the colloidal ink can combine to form three-dimensional out-of-plane structures via self-assembly. Direct printing of dots, lines and pillars with widths well below 100 nm have been demonstrated (Galliker et al., under review, 2011). With the abovementioned technique, larger structures are also accessible simply by adopting wider nozzles and ink solvents with higher vapor pressures. By combining sequential (i.e. dotwise) and continuous printing, freeform wires and complex arc structures with diameters around 1 µm have been fabricated. These structures provide a controlled and precisely engineered microenvironment for the study of the migration of cells within three dimensional tissues. It has been shown that highly malignant tumor cells can plastically adapt their migration behavior to the surrounding microenvironment in order to bypass obstacles or penetrate pores (Wolf et al., J. Cell Biol. 160, 2003). The reconstituted collagen matrices mainly used for these studies on interstitial cell migration closely simulate the physiological environment, yet have limited optical accessibility and contain wide pore size variability. New platforms produced by microfabrication methods like 3D lithography (Klein et al., Adv. Mater. 23, 2011) or two-photon laser ablation (Ilina et al., Phys. Biol. 8, 2011) have recently been proposed. Until now, though, a flexible and economic method for the fabrication of large scale, mixed pore size microenvironments totally accessible by high-resolution microscopy techniques was still not available. Optically accessible cyclo-olefin-copolymer substrates were pre-structured by thermal nanoimprint lithography (NIL) to form a basal scaffold for cell contact guidance (Ferrari et al., Nano Lett. 11, 2011). Arrays of freestanding arcs with varying cross sections between 10-100 μm2 were then fabricated by 3D printing of a colloidal gold nanoparticle ink. Having precise control over the pore shape, size and stiffness, the influence of these parameters on the ability of the cell to undergo interstitial pore penetration can be decoupled. The visualization of cell migration, particularly upon penetration of the pore structures is explored with cancer cells by wide-field, confocal and total internal reflection fluorescence (TIRF) microscopy.

In order to study the evolution of drug resistance in cancer, it is important to mimic the tumor microenvironment, in which cells are exposed to not uniform concentrations but rather gradients of drugs, nutrients, and other factors. However, it is hard to control the temporal and spatial profile of gradients using traditional in-vitro techniques. In this paper, we report the generation of stable gradients of doxorubicin, using a microfluidic gradient generating device for the human metastatic breast cancer cell line MDA-MB-231, and the successful long-term culture of such cells in the chip. Exposing cells in a microfluidic environment to constant fluid flow has a negative effect cell viability [1] (T. M. Keenan et al, Lab on a Chip, 2007). We found that flow rates as small as 100 microns/sec does change morphology of the cells after two days. We then fabricated a structure in which DC fluid flows outside the culture region are used to establish drug concentration boundary conditions, and arrays of micro-posts serve as perfusion barriers to allow the drug to diffuse across the culture chamber to establish , but isolate it from DC fluid flows. The culture of mammalian cells in microfluidic environments is much more demanding than that of bacteria such as E-Coli. MDA-MB-231 cells were introduced into the gradient zone via an extra port, and their successful on-chip culture required several factors. The chip surface was coated in-situ with appropriate extracellular matrix. A micro-incubator with controlled humidity, temperature, oxygen, and carbon dioxide concentrations and a PDMS chip sealing method was used to allow the ambient to diffuse into the culture zone were used. The input fluid for the gradient culture chip contains DMEM growth media (with 10% fetal bovine serum), and was also preconditioned with oxygen and carbon dioxide. After seeding, the breast cancer cells grow exponentially for 5 days, and then enter a stationary phase, in which the cells are still healthy with stable population after 16 days. Finally, we report the adaptation of the breast cancer cells under the stress of doxorubicin gradients (0-200nM/mm). The cell population increases quickly in the low drug region, but also increases (slower) in the high drug region. Further work is ongoing to determine whether resistance is emerging or if cells are migrating from the low to high drug concentration regions.

Cellulose nanowhiskers (CNWs) are high aspect-ratio nanoparticles with diameters of a few nanometres which are prepared by partial hydrolysis of native cellulose. Although normally found in plant cell walls, cellulose is also produced by some marine invertebrates. Such animal-derived cellulose is highly crystalline and allows the production of particularly high aspect-ratio CNWs with lengths on the order of a micrometre. We have shown that such CNWs are non-cytotoxic to skeletal muscle myoblasts and that their unique combination of physical, chemical and biological properties makes them a highly suitable material for engineering the cell microenvironment for applications in tissue engineering and regenerative medicine. Highly oriented surfaces of deposited CNWs were prepared using spin coating. The degree of CNW deposition and relative orientation was effectively controlled and modulated and the surfaces were characterized by atomic force microscopy and image analysis. The mean feature height was only 5.5 nm and yet the topographical cues provided by the deposited CNWs induced contact guidance in C2C12 skeletal muscle myoblasts that were seeded to the surfaces. The myoblasts adopted highly oriented morphologies and upon differentiation fused to form highly oriented arrays of myotubes. Furthermore, the degree of terminal differentiation and myoblast fusion was upregulated on the oriented CNW surfaces and the myoblasts deposited highly oriented fibrils of extracellular fibronectin. The CNW surfaces were also shown to support the adhesion of human mesenchymal stem cells (hMSCs) and to direct their morphology. Using a co-culture system the hMSCs were induced to fuse with C2C12 myotubes demonstrating that CNWs are a potentially valuable tool for engineering human skeletal muscle tissue with physiologically relevant structure across several length scales. As extracellular matrix mimics the CNWs are some of the smallest features ever demonstrated to induce contact guidance in mammalian cells.

As the cost and duration of clinical trials continue to increase, a rising emphasis is being placed on improving preclinical testing methods. The current standard preclinical absorption model utilizes human colon cancer-derived cellular monolayers grown on collagen-coated membranes. Despite its prevalence of use, this model suffers from multiple limitations, including inaccuracies in predicting paracellular transport as well as poor reproducibility. The goal of this work is to exploit the molecular-level precision of protein-engineered scaffold materials to create an improved in vitro mimic of intestinal tissue, the primary point of absorption for most orally administered drugs. We have developed a modular elastin-like protein (ELP) in which we can independently tune cell adhesive and mechanical properties. Through selectively incorporating RGD ligand domains, we can obtain up to 9300 cell adhesive ligands per square micron of ELP gel surface. Further, by altering the concentration of chemical crosslinker, we have generated thin films with elastic moduli ranging from 20 to 100 kPa. It is hypothesized that both scaffold biomechanics and biochemistry will affect cytoskeletal organization and focal adhesion formation, cell processes known to correlate with tight junction formation and therefore paracellular transport. Independent tuning of these individual properties is thus hypothesized to facilitate the use of scaffold property optimization to regulate paracellular permeability. We demonstrate that our ELP is a suitable extracellular matrix protein mimic for use as an adsorbed coating in the culture of epithelial cell lines, resulting in comparable metabolic health and cell density of confluent monolayers relative to adsorbed collagen, fibronectin, and laminin. Our results also demonstrate that this material is a suitable matrix for culturing primary intestinal organoids in 3D, enabling the maintenance of mature primary intestinal epithelial layers for up to 6 weeks in culture. We further demonstrate that replacement of the standard collagen coating with our ELP material provides a novel absorption model that performs comparably to the collagen standard, rendering statistically equivalent fluxes of a transport marker in both the apical-to-basal and basal-to-apical directions. Further scaffold property optimization will enable the generation of a new model for intestinal absorption that is not only more consistent and reliable but also facilitates the tailoring of monolayer permeability to match various conditions in which intestinal barrier function is altered.

Human embryonic stem cells (hESCs) have applications in numerous different areas of biological engineering, but there are still difficulties with efficiently proliferating and directing differentiation of these cells in vitro. Studying the effect of colony size and spacing on hESCs may allow for better control of their proliferation and differentiation. I have developed a soft lithography technique using PDMS stencils to micropattern an interpenetrating polymer network composed of poly(acrylamide-co-ethylene glycol). The IPN is a superior surface for blocking protein and cell adhesion because it is very robust and has been shown to reduce fibrinogen adsorption by more than 96% compared to TCPS. During patterning by oxygen plasma etching, the areas blocked by the stencil will remain non-adhesive, while the areas exposed to plasma will be etched to the base TCPS substrate. The success of this patterning has been shown by patterning 3T3 fibroblasts on this surface for up to a week. Currently, the most common surface for in vitro culture of hESCs is Matrigel, which is not well defined, contains animal products, and suffers from batch-to-batch variability. Therefore, it has been a focus of research in our lab to develop a completely defined, synthetic surface for the culture of hESCs. Work in our group has developed a low cost, synthetic polymer surface composed of aminopropylmethacrylamide (APAAm) that has been used to successfully culture hESCs for over 20 passages in mTeSR media. Immunostaining and quantitative RT-PCR studies demonstrated that cells cultured on the APAAm surface have similar proliferation and pluripotency markers to cells cultured on Matrigel. The incorporation of this material into my micro-patterned culture surface will allow for defined in vitro culture of hESCs and allow for the study of colony size and spacing on cell growth, proliferation, and differentiation.

Here we present the development of an array of electrical nano-biosensorsembedded in a microfluidic channel, which we hereby refer to as Nanoneedle Biosensors, capable of label-free electronic detection of protein biomarkers. The ultimate goal of the nanoneedle biosensor is to insert the needle tip into a cell membrane and make direct in-vivo measurements of protein binding inside the cell without disturbing the cell itself. In this paper, we will present the proof of concept study for protein detection. The nanoneedle biosensor is a platform, capable of electrical detection, both label free and in real-time. Our sensor, which is capable of high sensitivity detection, operates by measuring the impedance modulation across a nanoneedle tip, due to the binding of biomolecules such as proteins or nucleic acids. We show that the sensors are capable of protein detection. We functionalized the Nanoneedle Biosensors with the receptors specific to a target protein via physical adsorption for immobilization. We have used biotinylated bovine serum albumin as the receptor and different concentrations of Sterpavidin as the target analyte. The detection of Streptavidin binding to the receptor protein is presented. We also present measurements for the sensitivity of the sensor for a range of target protein concentrations.

Creating micropatterned coculture of different cell types is useful for the investigation of cell behaviors and communications, or engineering tissues composed of multiple cell types. Molecules with the cell adhesive property switchable by external stimuli would provide additional capability to manipulate different cell types with more complexity. Here, we have developed a new photoresponsive molecule that can switch the cell adhesive property on the surface using a photocleavable moiety which cleaves upon exposure to long wavelength UV. We demonstrate the principle to investigate the intercellular communication between hepatocytes /endothelial cells and endothelial cells/pericytes. This method provides a tool for studying cell-cell interactions and mimicking the complex tissue structure in vitro.

Although most orthopaedic fractures heal, the management of critical sized (>3mm) bone defects continues to present major challenges clinically. During normal bone healing, mesenchymal stem cells (MSCs) are recruited, proliferate, and produce extracellular matrix that subsequently becomes mineralized to form bone in a highly-orchestrated process. Therefore, a means to deliver and control MSC proliferation represents a promising regenerative strategy in bone tissue engineering. Thus, we are designing poly(ethylene glycol) (PEG) hydrogel microenvironments to exploit the action of the soluble small molecule GSK3β (glycogen synthase kinase 3β) inhibitor, 6-bromoindirubin-3â?T-oxime (BIO). Previously, we demonstrated dose dependent MSC proliferation in vitro utilizing transient (24 hr) BIO treatment. We observed a 4.5-fold and 5.5-fold increase in cell number under 2 μM and 5 μM BIO dosing conditions, respectively, as compared to untreated controls. In the current work, we developed a PEG-based degradable hydrogel network to provide reproducible BIO delivery and exploit BIO-mediated MSC proliferation. Various ratios of hydrolytically degradable PEG-PLA-DM [poly(lactide)-b-PEG-b-poly(lactide) dimethacrylate] and PEG-CAP-DM [poly(caprolactone)-b-PEG-b-poly(caprolactone) dimethacrylate] tri-block copolymers were tuned to degrade completely over 35 days, as required by typical bone healing. Furthermore, we utilized previously-developed controlled release strategies to tether and release BIO with tunable delivery kinetics. Total BIO dose was controlled through overall BIO conjugate incorporation, and the delivery kinetics were controlled by the number of degradable linkages incorporated into the BIO conjugate. When encapsulated in these hydrogels, MSCs exhibited high viability and similarly-enhanced proliferation as observed previously in our two-dimensional studies. We are currently exploiting these degradable, BIO-releasing hydrogel microenvironments to promote fracture healing in mouse models.

Two-Photon-Polymerisation (2PP) is a rapidly emerging platform technology for the microfabrication of three-dimensional biocompatible scaffolds for tissue engineering at the nanolevel of resolution. The required near-infrared laser emits light of minimally damaging wavelength for biological tissue. This makes it potentially very attractive to apply 2PP for developing new and advanced functional materials to be structured in direct contact with cells or other living organisms. It is crucial to design a polymerisable formulation to be minimally toxic. Furthermore toxicity assays need to be developed allowing for high throughput screening using life organisms. This paper reports the biofabrication of three-dimensional scaffolds using two-photon polymerisable hydrogels. Caenorhabditis elegans has been used as a test living organism for toxicity studies. The structuring was performed with a pulsed near-infrared laser with a wavelength of 810 nm and adjustable power up to 400 mW. Using a two-photon active, water soluble initiator (WSPI) it was possible to polymerise 3D structures in resins having M9 buffer medium contents of up to 80%; the highest aqueous content reported for the use with 2PP. High writing speeds of 10 mm/s allowed very short fabrication times. The little damaging wavelength of a near-infrared laser and a novel photo-sensitive material system with a high aqueous content made the rapid biofabrication of 3D scaffold with an embedded organism possible for the first time. These data demonstrate the feasibility and possible potential of 2PP and advanced photo-sensitive biomaterials to biofabricate 3D tissue constructs directly in the context of a living organism.

Biomaterial properties including stiffness, degradation rate, and presentation of cell adhesion ligands are known to affect a variety of cell behaviors including spreading, proliferation, and differentiation. To engineer complex tissues, it may be important to spatially pattern these properties to create local microenvironments presenting desired signals. The capacity to achieve this in 3D would permit potential mimicry of the in vivo mechanical and chemical signaling microenvironments present during developmental or healing processes for application in tissue regeneration approaches. Here, alginate, a naturally derived polysaccharide, is used to create hydrogels with regions of different crosslinking and subsequently different material properties. Alginate can be crosslinked ionically with divalent cations, chemically with compounds such as gluteraldehyde, and in the presence of UV light and a photoinitiator if the material is appropriately modified. Methacrylated alginate, which is UV-crosslinkable, retains its ability to crosslink ionically. Because hydrogel exposure to UV light can be controlled spatially with a photomask, combining these two crosslinking mechanisms permits creation of a material that is crosslinked ionically through its bulk, and additionally crosslinked in selected areas with UV light. Alginates with 5-11% methacrylation (MA) of carboxyl groups were prepared, and for cell studies the cell adhesion peptide GGGGRGDSP was subsequently covalently attached to the methacrylated alginate. Alginates were dissolved in media containing Irgacure D-2959 photoinitiator. Calcium-only gels were crosslinked with solutions of 100 mM calcium chloride in deionized water. Dual crosslinked gels were first crosslinked with 320-500 nm light and then calcium crosslinked. In all groups, dual crosslinked hydrogels had higher shear moduli and exhibited less swelling after 48 hours than the same material crosslinked with calcium alone. These differences were statistically significant for both 7% and 11% MA hydrogels (p<0.05). Shear storage moduli ranged from 10 kPa (calcium only, 7% MA) to 25 kPa (dual crosslinking, 7% MA). Swelling ratios ranged from 40 (dual crosslinking, 11% MA) to 100 (calcium crosslinking, 7% MA). Live/dead staining demonstrated that the dual crosslinking process was not toxic to encapsulated human mesenchymal stem cells, which remained viable after one week of culture. When UV light was shone through a photomask with a 100 um checkerboard pattern onto a calcium crosslinked hydrogel, visual inspection demonstrated that this pattern was imparted to the hydrogel. Such a system will be useful for studying effects of 3D hydrogel material properties on encapsulated cells, and for patterning these properties in a scaffold for tissue regeneration applications.

Neural stem cells (NSCs) are multipotent and differentiate into neurons and glial cells, which can provide essential sources of engraftable neural cells for devastating diseases such as Alzheimerâ?Ts disease, Parkinsonâ?Ts disease and spinal cord injury. One of the major challenges involved in the differentiation of NSCs is to identify and optimize factors which result in an increased proportion of NSCs differentiating into neurons as opposed to glial cells. The research toward studying the function of the two microenvironmental cues - cell-cell interactions and insoluble cues - during the neuro-differentiation of NSCs is limited, mainly due to the lack of availability of methods for the investigation. Herein, we demonstrate the effect of extracellular matrix (ECM) protein patterns and nanotopography, as insoluble cues, on the differentiation of NSCs. First, bio-surface chemistry combined with soft lithography was used to generate patterns with varying geometries and dimensions of the ECM protein, laminin, to study the influence of surface patterns and cell-cell interactions on the differentiation of NSCs. Second, we used varying nanotopographies, generated by films of different sizes of nanoparticles coated with laminin, to deliver plasmid DNA and siRNA against a specific gene to enhance the neuronal differentiation of NSCs. Our nanotopography-based gene delivery platform allows for the uptake of only the siRNA/DNA and not the nanoparticles, which is extremely advantageous. Our results confirmed that ECM protein patterns with variant geometries and dimensions guided cell-cell communications and cell-ECM interactions in a controlled manner, which ultimately led to a pattern geometry-dependent and dimension-dependent neuronal differentiation. In addition, by controlling gene expression levels using nanotopography-based delivery of siRNA, we observed a remarkably high number of NSCs undergoing neuronal differentiation. Importantly, all the experiments were carried out in the absence of exogenous factors promoting neuronal differentiation. Differentiation of NSCs into neurons was confirmed using RT-PCR and immunostaining. The down-regulation of the NSC marker (Nestin), the up-regulation of the early neuronal marker (TuJ1) and mature neuronal marker (MAP2) were monitored. Furthermore, the extent of glial differentiation of NSCs was assessed by monitoring the expression of the protein GFAP, a specific glial marker. In addition, neurite outgrowths were observed by using an inverted phase contrast microscope. Our results could be significant for tissue engineering for brain and spinal cord injuries, where NSCs can be transplanted into the damaged regions with scaffolds having specific geometries or nanotopographies for delivery of genetic material into stem cells. For example, scaffolds having nanotopographies and patterns promoting cell-cell interactions in a controlled manner could potentially lead to increased neuronal differentiation in vivo.

Incorporating bioactive molecules in biomaterials is increasingly preferred to instruct the cells and to obtain a specific and desired biological response. Surface tethered growth factors offer a better control of their spatial and temporal availability in the extracellular environment in contrast to soluble proteins. Due to challenges in simultaneous patterning of multiple biomolecules, only few studies have investigated the effect of co-patterning multiple different molecules to (stem-) cells. Such co-patterns would be very instrumental in studying the synergistic- or antagonistic activity of different bioactive molecules such as growth factors. Although inkjet printing allows arraying of multiple biomolecules, no control is available over the shape and size (>100 µm). Parallel patterning with micrometer level resolution and with desired shapes can be provided using microcontact printing and combined with its low fabrication costs, simplicity and reproducibility, however more wide spread implementation is challenged by the necessity of patterning multiple biomolecules with a single stamp. Here, we present hydrogel-filled silicon stamps having individually addressable ink reservoirs with which multiplexicity can be achieved while avoiding the need for re-inking. We used these stamps to micropattern and covalently immobilize multiple different bioactive molecules on surfaces of biocompatible polymers for investigating the interaction of stem cells with multiple bioactive molecules. We fabricated silicon microstructures having separate wells (320Ã-320Ã-380 µm) and each well having a 25 µm thick membrane (144 microchannels measuring 5 µm in diameter) on the printing side. The reservoirs and microchannels of the silicon microstructures were filled with macroporous poly(2-hydroxyethyl methacrylate-co-ethylene glycol dimethacrylate) hydrogels that were covalently bound to the surface of the silicon. After filling the separate reservoirs of the stamps with different growth factors (VEGF, EGF, bFGF, TGF-β, and BMP-6) and fibronectin simultaneously a multi-content micropatterns were generated on epoxy-functionalized poly(dimethylsiloxane) (PDMS) or poly(trimethylene carbonate) substrates. Moreover, up to twenty times pattern replication was possible without re-fill. The integrity of the multi-content micropatterns was verified with immunofluorescence stainings and currently the bioactivity of these surfaces for the differentiation of mesenchymal stem cells towards different lineages is studied. These engineered bioactive surfaces may become very instructive tools in designing future biomaterials for regenerative medicine.

Tissue-specific stem cells with potent regenerative properties are present in many adult tissues including blood and muscle, but their â?~stemnessâ?T is rapidly lost upon culture in traditional plastic dishes. We hypothesized that a bioengineered substrate which recapitulated key biophysical and biochemical niche features could overcome this limitation. Using a novel hydrogel culture substrate in conjunction with timelapse microscopy and a highly automated data analysis algorithm, we tracked the behavior of clones derived from single muscle stem cells (MuSC) in culture, and then subjected them to a stringent assay of function: transplantation into mouse muscles followed by a quantitative assessment of regeneration by noninvasive bioluminescence imaging. We found that MuSCs cultured on a substrate with the elastic modulus of muscle tissue and tethered with a niche extracellular matrix protein, proliferated without loss of regenerative capacity illustrating the power of biomaterials to direct stem cell fate and overcome roadblocks to stem cell therapeutic utility. This platform provides a novel basis for screening for drugs that enhance stem cell function and expansion and for elucidating the signalling mechanisms by which cells â?~senseâ?T the rigidity of the substrate/tissue with which they are in contact. Such studies are of fundamental interest and will aid in the treatment of muscle wasting disorders.

Endothelial cell sprouting morphogenesis is a critical early step in angiogenesis, the formation of new blood vessels from existing conduits. We have designed a microfluidic chemotactic generator that enables formation of stable, soluble gradients and real-time visualization of collective cell migration within 3D biomaterials. These microfluidic platforms are being used to study the biomechanical and biochemical factors that regulate endothelial cell movement during sprouting morphogenesis. Using this platform, we have identified that the G-protein coupled receptor 124 (GPR124) is a previously unknown regulator of blood vessel development in the brain. Furthermore, we have used these devices to screen various biomaterial formulations for their ability to induce stable endothelial sprouting upon exposure to vascular endothelial growth factor (VEGF) gradients. Intriguingly, our experiments find that endothelial sprouts alter their sensitivity to VEGF depending on the matrix density, suggesting a complex interplay between biochemical and biomechanical factors. As matrix density increases, steeper VEGF gradients and higher VEGF absolute concentrations are required to induce directional sprouting. In lower density matrices, endothelial sprouts were frequently observed to change their direction of growth by turning to reorient parallel to the VEGF gradient, a behavior reminiscent of the path-finding behavior of neuronal axons. In contrast, in higher density matrices this turning phenomenon was only rarely observed. These results demonstrate that identical soluble gradient profiles can result in dramatically different cell responses depending on the 3D biomaterial environment. These findings encourage the further development of 3D culture models as screening tools for new anti-angiogenic strategies for potential cancer treatment as well as pro-angiogenic strategies for regenerative medicine.

The clinical application of stem cells calls for large amount of cells with well-controlled phenotypes, which is currently impossible to achieve using conventional static cell culture systems. Here, we combine microcarriers-based cell culture technology with bioreactors for the efficient large-scale culture and manipulation of stem cells. Droplet-based microfluidics was employed for the reliable high-throughput generation of poly(ethylene glycol) (PEG)-based hydrogel microcarriers. The developed microfluidic platform allows (i) to strictly control the distribution of the microcarrier size, (ii) to tune their physicochemical properties by simply changing the flow rates, and (iii) their biofunctionalization with desired proteins or peptides to generate cell type-specific microcarriers. As a first application of this platform, mouse embryonic stem cells (ESC) were cultured on gelatin-functionalized PEG-hydrogel microcarriers in suspension in a rotating bioreactor. ESCs were efficiently expanded on the microcarriers, with a 35-fold increase in Oct4-positive cells after 4 days in culture as was shown by flow cytometric analysis.

The mammary gland is a dynamic tissue in which cells are continuously interacting with each other and with cells and molecules in the surrounding microenvironment. When the microenvironment receives signals from cells in the mammary epithelium, it sends back cues that regulate tumor growth and its progression to malignancy. While the importance of the microenvironment is clear, current screening platforms are generally limited to traditional two-dimensional (2D) cell culture, and often exclude the influence of microenvironmental components, such as stromal cells and extracellular matrix (ECM) molecules, in modulating cellular behavior. Therefore, there is a need for more in vivo-like screening platforms that allow a screening approach to elucidating the regulatory role of the ECM and stromal components. In this work, we present an automated microfluidic platform that cultures breast carcinoma cells (T47D) and human mammary fibroblasts (HMF) in 3D microenvironments of defined composition, and screens for cellular and ECM compositions that support T47D cell growth. Seven different combinations of three different ECM molecules (collagen type-I, fibronectin, laminin) were used to culture T47D cells in monocultures and in co-cultures with HMF cells. First, a size-based screening identified significant differences in T47D cluster size in microenvironments composed of T47D and HMF cells cultured with collagen type-I mixed with 0, 100μg/mL of fibronectin (FN), or 100μg/mL of laminin (LN). Second, a total cell number-based screening revealed that small additions of LN (e.g., 10μg/mL and 50μg/mL) affected paracrine signals between HMF and T47D cells. The platform presented in this work shows for the first time a screening platform capable of screening for breast carcinoma cell growth under the influence of different ECM compositions and stromal cells. Applying the concepts presented in this work to higher throughput screening platforms promises to be useful for future cell screenings that will facilitate the study of cell-ECM interactions in breast cancer.

Interfaces play an important role in a wide spectrum of cellular processes ranging from cell adhesion to tissue morphogenesis. Designing functional interfaces integrating multiple components and structures plays a pivotal role in successful outcome of regenerative medicine approaches. In this talk, I will discuss the engineering of the cell-matrix and cell-cell interfaces to control stem cell fate. In particular, I will talk about our recent efforts in the development of biomaterials with defined physico-chemical properties for controlling various cellular processes, with an emphasis on ex vivo expansion of human pluripotent stem cells (hPSCs) and tissue specific differentiation of stem cells. These synthetic biomaterials serve as excellent platforms for studying molecular mechanisms that regulate stem cell proliferation and differentiation. Moreover, these cost-effective and scalable biomaterials that recapitulate various attributes of the native extracellular matrix could accelerate the translational potential of hPSCs.

We are interested in developing and using microfluidics for high-throughput studies in developmental biology, cancer and immunology. These engineered chips allow us to manipulate cells, particles, and embryos at the right length scale with precise controls and dexterity unavailable with conventional tools. I will show a microfluidic system for arraying, aligning/orienting, and imaging embryos to study the signaling in embryonic development. We use a similar principle to manipulate cells and perform long-term imaging of cells under a variety of conditions to study. The advantage of this approach is that we have robust particle loading (very high loading occupancy and high single cell/particle/embryo loading efficiency) in a short amount of time just using flow passively. In combinations with other microfluidic modules and functionalities such as gradient generator and pulse generator, these devices allow us to study signal transductions and complex behaviors of cells, tissues, and whole embryos.

Multiple biochemical and physical signals are orchestrated between several cell types to sculpt the functional architectures of tissues and organs during morphogenesis. Traditional three-dimensional (3D) culture models aimed at recapitulating and dissecting morphogenetic mechanisms ex vivo are not fully quantitative and suffer from lack of spatial and temporal control. We have used lithography-based microfabrication approaches to generate epithelial tissues of precisely controlled size and geometry, embedded within an engineered stroma containing native extracellular matrix, with or without mesenchymal cells. This method produces hundreds of tissues of identical size and shape, which enables rigorous quantitative analysis of the extent and spatiotemporal pattern of morphogenesis. We have used this platform to define the molecular and biophysical regulators of branching morphogenesis, the process whereby the treelike structures of the lung, kidney and mammary gland are formed. We discovered that autocrine inhibitory morphogens cooperate with endogenous mechanical gradients to specify the final pattern of branching. In particular, branches initiated from sites experiencing high tensile forces and low concentrations of TGFβ. Branch sites were defined by activation of focal adhesion kinase and neo-expression of mesenchymal markers, including the transcription factors Snail, Slug and E47. Incorporating adipocytes in the matrix to mimic the fatty stroma of the mammary gland revealed that the extent of mammary epithelial branching is controlled in part by the surrounding adipose tissue through paracrine signals including hepatocyte growth factor. These engineered organotypic models may thus shed light on the integration of morphogenetic programs and how they are circumvented and co-opted during disease.

Invasion is the first step of metastasis and represents a potential target for preventing the spread of cancer to distant organs. Studying the dependence of invasion on specific influences of the microenvironment is limited by control over standard culture platforms such as tissue culture polystyrene (TCP), collagen, and Matrigel. Here, we present a systematic approach for studying the influence of the microenvironment on invasion by taking advantage of the precision of engineered materials to complement well-established 2D and 3D culture platforms. In order to study tumor cell function in well-defined 3D culture with quantitative control over matrix properties, we developed a platform that takes advantage of photoinitiated reactions between alkenes and thiol groups ("thiol-ene" reaction) to form poly(ethylene glycol) (PEG) hydrogels. Our strategy allows us to couple cysteine containing peptides for functionality such as protease degradation and adhesion while maintaining strict control over cell-materials interactions. We compared our 3D engineered platform to 2D self-assembled monolayers with similar functionality, as well as TCP and collagen, in order to deconstruct the influence of the microenvironment on tumor cell function in a systematic fashion. Differences between primary and transformed cell migration mechanisms will be illustrated, with an emphasis on how the engineered matrix provided additional insight that would be difficult to achieve with standard platforms. Finally, examples of how strictly controlled 3D culture platforms can be useful for studying tumor cluster growth and the influence of the microenvironment on invasion will be discussed.

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 HCECâ?Ts 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 fibronectinâ?Ts 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 â?" 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 â?" soft, 110 Pa â?" intermediate, and 260 Pa â?" 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.

Fibronectin (FN) is an abundant extracellular matrix protein that cells secrete, bind to and forcefully unfold to assemble into a fibrillar matrix. Increases in both FN production and tissue matrix stiffness are associated with normal development and with defects that occur in tumor malignancy, tissue fibrosis, and other disease states. Little is known about the specific effects of rigidity on the process of cell-mediated FN matrix assembly. To address this question, polyacrylamide gels of varying stiffnesses are being used to mimic the range of rigidities observed in normal and diseased tissues. Using immunofluorescence and quantitation of detergent insoluble matrix to measure cell-mediated assembly of FN, we have found that assembly is significantly affected by substrate stiffness. Assembly requires changes in cytoskeletal contractility, modulation of integrin receptor activity and integrin-FN bond strength, as well as changes in FN conformation. We are investigating how these processes respond to changes in rigidity. This information will provide mechanistic insights into regulation of FN assembly by tissue compliance and will aid in designing materials that promote or suppress matrix assembly.

Focal adhesions are important transducers of signals from the extracellular matrix (ECM) to the cell and vice versa. Most biomaterials are coated with an ECM ligand uniformly to promote adhesion, but that does not match heterogeneous adhesive site distribution in native ECM. Mixed copolymer vesicles undergo interface-confined phase separation; by using diblock copolymer mixtures of non-adhesive and adhesive components, i.e. polyethylene oxide (PEO)-polystyrene (PS) and polyacrylic acid (PAA)-PS, respectively, we determined if foam structures made in a high internal phase emulsion would have surfaces composed of adhesive and non-adhesive domains just as with ECM. Bulk copolymer incorporation scaled with input composition but neither foam morphology nor surface roughness dramatically changed as a result. However, surface phase separation did occur between PAA-PS and PEO-PS; 0.1 µm2 nano-domains spaced ~0.5 µm apart were found when either copolymer fraction was â?¤25%. That size and spacing is similar to the heterogeneous adhesivity of native ECM. When incubated with an ECM ligand, protein attached to PAA-PS and produced nano-domains with a distribution that also mimicked native ECM. Two human mesenchymal stem cell sources cultured on the foams were adherent on and express the most robust vinculin-containing adhesions on 25% PAA foams. qPCR microarray data indicates that these two mesenchymal stem cell sources undergo both nano-domain-dependent and â?"independent differentiation depending on the lineage to which the cell commits. Thus interface-confined phase separation in copolymer foam mixtures can create adhesive nano-domains mimicking native ECM, induce stem cells to differentiate, and should be considered in future regenerative strategies.

Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins, and these forces are presumed to activate integrin â?oswitchesâ?. The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechano-transduction events within the cellular microenvironment.

The extracellular matrix (ECM) contains components with remarkable mechanical properties, including fibronectin (Fn) fibers with extensibilities of greater than 700% strain. We utilized a novel technique to quantify the extent of molecular unfolding that contributes to Fn fiber extension, and we compared this behavior with stochastic models of Fn â?ofibersâ? with different molecular arrangements. In vitro unfolding as a function of strain was measured by fluorescently labeling cysteines in modules FnIII7 and III15 in artificial Fn fibers. A novel calibration technique made it possible to demonstrate that 44% of cysteines in these modules were exposed in Fn fibers strained to 421% extension, up from 8% exposure without strain. In silico unfolding was measured by applying a constant strain rate to a fiber represented by a network of worm like chain springs, each representing an individual Fn molecule. Unfolding rates were calculated with a tension dependent stochastic model applied to FnIII modules in each molecule. A comparison of these approaches revealed that only a molecular arrangement permitting unequal mechanical loading of Fn molecules recapitulates in vitro unfolding. These data have implications for Fn-dependent mechanotransduction and give insight into how the molecular architecture of natural materials permits such remarkable extensibility.

Fibronectin (Fn), an extracellular matrix (ECM) dimeric glycoprotein, has been implicated as a possible critical mechanotransducer that regulates cell signaling and behavior, wound healing, and embryogenesis (Smith et al. PLoS biology 2007). There is a need to understand the mechanism(s) by which the ECM in pathogens such as breast cancer, dysregulate normal cell signaling and behavior to better develop diagnostics and therapies to target local diseased tissue. We utilize intramolecular fluorescence resonance energy transfer (FRET) to elucidate how Fn molecular conformation and strain are affected by tumor soluble factors as it is known that Fn is up-regulated in tumors (Chander et al. Physical Biology 2011). Murine 3T3-L1 pre-adipocyte cells were pre-conditioned for 3 days with Control (Ctrl) and Tumor Conditioned Media (TCM). Preconditioned cells were seeded into Lab-Tek^TM wells at a density of 20,000 cells with reduced serum (1% fetal bovine serum) and exogenous Fn (10% FRET-labeled Fn, 90% unlabeled Fn). After 24 hours in culture, cells were: (i) fixed with formaldehyde or (ii) unfixed. In each case, the culture systems were either left with cells or decellularized with Triton X-100, and imaged through confocal microscopy. Matlab analyses of z-stack images of fluorescent intensity ratios were used to assess FRET in the ECM, and to discriminate stretched and partially unfolded fibers (low FRET) from relaxed and folded fibers (high FRET). Our results demonstrate both a larger amount of deposited Fn fibers and a lower FRET in ECM treated with TCM, indicating overall highly stretched and unfolded Fn fibers. We also show that cross-linking causes the ECM to contract and increase strain, leading to higher unfolding of fibers as indicated by lower FRET. This suggests that the commonly used fixation protocol causes Fn fibers to stretch and to exhibit more unfolded conformations. Additionally, decellularizing the ECM leads to higher FRET intensity, indicating that Fn fibers adopt a more relaxed conformation as it is no longer bearing the tension from adherent cells. Our conformational study suggests that tumor soluble factors are at the origin of dysregulated polymerization of Fn. Cells deposit an altered ECM made of highly stretched Fn fibers likely exposing cryptic sites that further disrupt biochemical signaling (i) between the cell and its ECM, and (ii) between ECM components. Finally, as it is also known that tumors are stiffer than their surrounding tissue, the mechanical properties of the above-mentioned ECMs were also investigated in a separate study. Overall, conformational and mechanical signals from the ECM may up-regulate certain proliferative signaling pathways, creating a dysregulated positive feedback mechanism that may be implicated in tumor progression and eventual metastases.

Increased stiffness represents a hallmark of breast cancer that is mediated by physicochemical alterations of the extracellular matrix (ECM). However, the cellular and molecular mechanisms underlying tumor-mediated ECM stiffening and the resulting effects on tumor angiogenesis, and hence growth, are poorly understood. We have investigated whether paracrine signaling by breast cancer cells regulates ECM assembly by adipose-derived stem cells (ASCs) located in the breast stroma and if these changes, in turn, stimulate tumor vascularization. Specifically, we have evaluated the effect of tumor-derived soluble factors on the proliferation and differentiation of 3T3-L1 preadipocytes and primary, human ASCs using conditioned media collected from various breast cancer cell lines and non-malignant MCF-10A breast epithelial cells as a negative control. Our results indicate that tumors induce adipose progenitor cells to differentiate into myofibroblasts and that transforming growth factor-beta (TGF-beta) plays a role in this process. These changes not only promote tissue-stiffening by enhancing ECM deposition and contraction, but also angiogenesis by increasing endothelial cell proliferation, migration, and tube formation through elevated concentrations of pro-angiogenic molecules (e.g., vascular endothelial growth factor [VEGF]). Using Foerster Resonance Energy Transfer (FRET) imaging and the Surface Forces Apparatus (SFA) we have quantified the conformation and rigidity of fibronectin-based ECMs deposited at the fiber and tissue level, respectively, and confirmed that varied fibronectin assembly contributes to ECM stiffening by tumor-associated ASCs. Furthermore, we have applied biomaterials approaches to determine whether increased stiffness reconstitutes a positive feedback mechanism that further regulates myofibroblast differentiation and pro-angiogenic signaling in vitro and have determined the relevance of our findings in vivo. Collectively, these results suggest that ASCs play a critical role in the stiffening of the tumor-associated stroma. Furthermore, they indicate that these changes promote tumor vascularization and growth by up-regulating the pro-angiogenic capability of both ASCs and endothelial cells. In summary, these studies suggest that ASCs stimulate tumor vascularization in a stiffness-dependant manner and represent a promising target for improved anti-angiogenic therapies.

Cancer is a disease characterized by uncontrolled growth, which invades surrounding tissues and migrates to other parts of the body. Invasion is one of the hallmarks of cancer cell. Cancer cell has the capacity to secrete the matrix degrading enzymes (MDE) and invade the extracellular matrix (ECM), which is driven by haptotaxis. Chemotherapy is one of the principal models of treatment for cancer patients. Chemotherapeutic drug aims to kill these abnormal or cancer cells, however normal cells and healthy tissues are also adversely affected. Hence, the sophisticate plan of chemotherapy for specific patients is needed to be prepared based on reliable information, which induces the recent requests of the advanced computational simulation technology. Here, we present three-dimensional models to simulate the invasion process of cancer cells and the chemotherapeutic process. The model incorporates multiple mechanisms and various components of system. This study provides a reliable framework for the comprehensive simulation technology of cancer cells.

Recent studies suggest that the molecular conformation of fibronectin (FN) dictates the mechanical properties of the extracellular matrix (ECM), which then regulates cell signaling and behavior at the microscopic (cellular) scale. We introduce the use of the surface forces apparatus (SFA) as a mechanical tool to directly measure the compressive elastic moduli, E, of ECMs assembled by 3T3-L1 pre-adipocytes. Using the SFA, we tested both (i) decellularized and (ii) cell-containing ECMs assembled in absence (Control) or presence (TCM) of tumor soluble factors to determine how these biochemical cues affect the mechanical behavior of the ECM at the cellular level. Atomically smooth mica sheets were glued onto cylindrical silica disks, and murine pre-adipocyte 3T3-L1s were directly seeded on top of one of these disks (25,000 cells/disk) for 24hrs, while the other bare disk was used for indentation measurements. Normalized force curves were generated as a function of ECM thickness, and, E was extracted using Hertz contact mechanics modeling and fitting of the following force-indentation expression: F/R=Ï?E(δ^2/D) where F, is the applied load; R, the radius of curvature of the discs; D, the thickness of the relaxed ECM; δ, the indentation, and E, the effective compressive modulus. Our data indicate that ECM assembled by 3T3-L1s after pre-conditioning with tumor factors (TCM) is more rigid (E=0.8kPa) than ECM assembled without tumor factors (Control) (E=0.4kPa). This correlates well with previous FRET studies indicating more unfolding in tumor FN fibers and suggesting simultaneous FN stiffening. (Chandler et al., Phys Biol, 2011). Overall, this study demonstrates that the SFA is a robust tool for the direct assessment of mechanical properties of ultra compliant ECM layers, at the cellular/tissue level. In such a context, it complements well the conformational properties gained by techniques such as FRET, covering the molecular level of FN; the combination of both tools would definitely help gaining insight into the ultimate structure-functionality relationship of ECMs.

Obesity represents a risk factor of breast cancer and is characterized by excess adipose tissue with increased interstitial fibrosis. Adipose tissue represents a primary stromal component of the breast and its tumor-promoting capabilities are typically attributed to various endocrine functions. However, it remains unclear if obesity-mediated alterations of mammary fat can also contribute to pre-tumorigenic stiffening of the breast and thereby promote the development and progression of mammary tumors. Here, we have investigated the hypothesis that obesity-associated adipose stromal cells (OA-ASC) vary with regards to their ability to deposit and contract extracellular matrix components (ECM) relative to ASCs from lean individuals and that these changes generate a desmoplastic predisposition of the breast microenvironment that may ultimately promote tumorigenesis and malignancy. To address this hypothesis, we first examined desmoplasia in histological cross-sections from lean and obese breast cancer patients. Additionally, we compared adipose tissue from lean and obese mouse models (both diet-induced and genetic models) with regards to cellular and molecular features that are typically associated with tumor desmoplasia; i.e., the content of contractile myofibroblasts as well as conformational changes of the fibrillar ECM component fibronectin (Fn), which plays a critical role in collagen matrix assembly. Our results suggest that tumor samples from obese patients exhibit a higher degree of desmoplasia and greater levels of Fn as compared to those of lean patients. Moreover, adipose tissues from obese mice contained more Fn and greater levels of contractile α-SMA and vimentin positive myofibroblastic cells than those from lean mice. Accordingly, OA-ASCs isolated from obese mice exhibited higher levels of α-SMA cells and Fn matrix assembly as compared to ASCs from lean mice. FRET (Foerster Resonance Energy Transfer) and SFA (Surface Forces Apparatus) analysis additionally indicated that OA-ASCs assembled Fn matrices that were characterized by increased Fn unfolding, stiffening, and overall matrix thickness relative to those deposited by ASCs from lean mice. Furthermore, histological analysis of tumor-free mammary tissues revealed that mammary fat from obese mice exhibited enhanced desmoplastic traits compared to mammary fat from lean mice. This trend was enhanced in mammary tissue from genetically obese mice as compared to those from diet-induced obese mice. Decellularized matrices from lean and OA-ASCs were utilized to assess the response of breast cancer cells to these changes in matrix assembly. Collectively, OA-ASCs may generate a physicochemical microenvironment, which may favor breast tumorigenesis by increased stroma stiffening due to altered deposition and contraction of ECM components.

We theorize that function based adaptations alter elastic gradients within load bearing tissues and biological interfaces, developing a positive feedback leading to impaired joint function. Proposed key parameter as a metric for loss of function will be â?oshifts in elastic gradientsâ? i.e. â?ofrom naturally graded to an abrupt transition or discontinuity" between dissimilar materials (Hurng et al. 2011, Biomaterials). This study will highlight elastic discontinuities within human alveolar bone (AB) and in a seemingly functional bone-tooth fibrous joint. METHODS: X-ray attenuation and elemental spatial variations on 1-2 µm sections from AB were identified using X-ray transmission microscopy (TXM, 5.4 keV) and microprobe X-ray fluorescence imaging (µ-XRF, 12 keV). Proteoglycans with fibrogenic (fibromodulin; FMOD) and osteogenic (biglycan; BGN) potential were identified using immunohistochemistry. Mechanical resistance of interfaces, specific regions in AB, and cementum was investigated using nanoindentation technique. RESULTS: Within the narrowed PDL-space, two types of bones marked by differences in X-ray attenuation, calcium (Ca), phosphorus (P), and zinc (Zn) were identified in AB. Bone with radial fibers (RFB) closer to cementum was identified as a higher X-ray attenuating material and µ-XRF measurements yielded higher counts of Ca, P, and Zn. Higher X-ray attenuating bands were observed in interlamellae regions of lamellar bone (LB) with a presence of FMOD, and PDL-cementum and PDL-bone attachment sites demonstrated presence of BGN. RFB also demonstrated FMOD expression. AFM wet scans illustrated hygroscopic lamellae relative to interlamellae regions. Interestingly, radial PDL-inserts changed their orientation into circumferential in LB within a junction of 10-30µm. Hygroscopicity, higher X-ray attenuation, and an elastic discontinuity were observed at this junction. As a result of narrowing PDL-space due to growth of RFB, steep modulus gradients representing discontinuities (analogous to stress concentrations sites) were generated between RFB and cementum. Heterogeneity was associated with variations in elastic modulus values with 23.9 ± 2.8 GPa for lamellae and 33.2 ± 0.4 GPa for interlamellae, resulting in significant elastic modulus differences as confirmed via Student's t-test (P<0.05); and 2-8 GPa for RFB. CONCLUSIONS: Despite hygroscopic in nature, higher collagen packing density, not necessarily mineral changes, within RFB, LB, and the junction could contribute to higher X-ray attenuation. From a tissue mechanics perspective the discontinuity in modulus profile between RFB and LB could compromise the functional quality of AB. The change in bone quality due to elastic discontinuities would further affect mechanobiology at the attachment sites and trigger overall adaptation of the fibrous joint, which was observed as a narrowed PDL-space. SUPPORT: NIH-NIDCR R00DE018212-03 (SPH), NIH-NIBIB R01EB004321 (PP), and DOE-BES.

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 â?" 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 â?obottom-upâ? and â?otop-downâ? 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 collagenâ?Ts array of cell adhesion, cytokine binding, and enzymatic degradation sites. Thus, Scl2-1 provides a â?oblank-slateâ? 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 â?~gold standardâ?T 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 βâ?"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 βâ?"sheets thick with the βâ?"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 βâ?"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):1135â?"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):18587â?"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):139â?"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 â?oβ-tailâ?, 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.

Faculty entrepreneurism is a scholarly activity. First, I will describe how we have implemented policies at the University of Utah that encourage faculty and student entrepreneurial activities through the Entrepreneurial Faculty Scholars and the Lassonde Entrepreneur Center student programs. Then, I will describe a case study for commercialization of a biomaterials technology for regenerative medicine. Injectable and biocompatible vehicles for delivery, retention, growth, and differentiation of progenitor cells are needed for cell therapy. We created a synthetic extracellular matrix (sECM) from hyaluronic acid (HA) that affords highly reproducible, manufacturable, approvable, and affordable biomaterials. The in situ crosslinkable sECM hydrogels has been customized for use with progenitor and mature cell populations obtained many tissues, including skin, fat, liver, heart, brain, muscle, bone, and cartilage. In addition, sECMs have been developed for rapid expansion and recovery of cells in 3-D, and for the bioprinting of engineered tissue constructs. The technology is being commercialized in three fields of use: human medical devices, cell therapy and research tools for 3-D cell culture, and veterinary wound care and adhesion prevention. 1. Prestwich, G.D. Evaluating drug toxicity and efficacy in three dimensions: using synthetic extracellular matrices in drug discovery, Acc. Chem. Res., 41, 139-148 (2008). 2. Prestwich, G.D. Engineering a clinically-useful matrix for cell therapy. Organogenesis 4, 42-47 (2008). 3. J. Burdick, G. D. Prestwich, Hyaluronic acid hydrogels for biomedical applications, Adv. Mater. 23, H41-H56 (2011) 4. G. D. Prestwich, Clinical biomaterials derived from hyaluronic acid for use in cell and molecule delivery in regenerative medicine,� J. Controlled Release, 155, 193-199 (2011).

The extracellular matrix (ECM) is natureâ?Ts scaffold, and in recent years, researchers have isolated these scaffolds for tissue engineering applications by removing all of the cellular components, a process called decellularization. These scaffolds are known to promote cell influx, regeneration, and healing in a variety of tissues, and their degradation products have angiogenic, chemoattractant, and antimicrobial properties, as well as promote cell migration and proliferation. By removal of the cellular antigens, these scaffold are considered biocompatible, and xenogeneic sources can be used. While these scaffolds retain the native ECM structure, they are not amenable to minimally invasive, injectable procedures. We have recently developed a variety of injectable ECM derived materials that self-assemble to form porous, nanofibrous scaffolds once injected in vivo or brought to physiological temperature. These scaffolds are showing promise in a variety of tissues including the myocardium, skeletal muscle, and adipose tissue. These materials can also be utilized as coatings for in vitro cell culture and have been shown to increase differentiation and maturation of a variety of cell types.

Hydrogel materials are finding use for the encapsulation and delivery of mammalian cells for tissue regenerative therapy and cytomedical applications. We have designed a class of small peptides that undergo a triggered intramolecular folding event to form an amphiphilic beta-hairpin conformation that is prone to self-assembly. Self-assembly leads to the formation of a physically crosslinked network of beta-sheet rich fibrils that constitute a mechanically rigid hydrogel. When peptide folding and self-assembly is triggered in the presence of cells, they are directly encapsulated in a uniform and cytocompatible manner. Peptide hydrogels, with or without cells, are shear thinning but have the capacity to quickly re-heal after the cessation of shear. This attribute allows hydrogel loaded with cells to be easily delivered from a syringe/catheter. Primary chondrocyte encapsulation and delivery is demonstrated and the influence of peptide structure on cell response studied, highlighting our efforts towards cartilage engineering.

Injectable stem cell therapies are a promising strategy for the treatment of many injuries and degenerative diseases. Unfortunately, poor and unpredictable cell transplantation to, retention at, and integration with the injury site currently constitute major roadblocks in the development of these therapies. One potential solution to these problems is the use of a hydrogel cell-carrier to protect and localize the cells, facilitating their contribution to the healing process. Currently the majority of commercially available hydrogels are not ideal for these therapies as they require potentially cell-damaging, non-physiological triggers for gelation. To overcome this limitation, we have developed a novel family of protein hydrogels: Mixture-Induced Two-Component Hydrogels (MITCH) in which the two components are recombinantly engineered block-co-poly-peptides containing repeats of WW and proline-rich peptide domains interspersed with random coil segments. These polymers are designed to hetero-assemble into a network at constant physiological conditions. Cells are encapsulated in MITCH by mixing with the two components, resulting in a hydrogel with cells evenly distributed throughout. Shear storage moduli can be tuned over a narrow range (10 to 50 Pa), dependent on the choice of binding partners. Rheology confirms that the hydrogels undergo shear thinning and re-gel upon force removal, as expected due to the transient physical crosslinks that form the network. These thixotropic properties enable hand injection of a pre-formed gel through a syringe needle and rapid gel recovery. MITCH have proven to support three-dimensional culture of multiple clinically-relevant cell types, including human and mouse adipose-derived stem cells (hASCs, mASCs), human umbilical vein endothelial cells, rat mesenchymal stem cells, and rat neural progenitor cells. Recent in vivo studies have shown transplantation in MITCH to improve cell retention and localization to the injection site. mASCs harvested from luciferase-positive transgenic mice were encapsulated in MITCH, alginate, collagen, or saline and injected subcutaneously into the dorsa of athymic mice. The retention of viable cells, compared to day one, was monitored through bioluminescence imaging (BLI). At day three, retention in MITCH was significantly higher than all other carriers, with a 3.4-fold improvement over alginate. Through day 14, retention in MITCH continued to show a significant, greater than 2-fold improvement over collagen and saline. MITCH also achieved successful host-integration without chronic inflammation. Transplanted cell retention to an injury site is critical to the efficacy of an injectable therapy, as functional recovery has been directly correlated to the number of viable cells present. These results, coupled with the ability to further tailor MITCH using the protein-engineered synthesis strategy, make MITCH promising for a myriad of stem cell clinical therapies.

As stem cells mature within the body during development, tissue healing, and normal homeostasis, their microenvironment is highly regulated, both spatially and temporally. While the tissue engineering field has aspired to engineer mimics of the developmental microenvironment, the complexity required to introduce a wide variety of diverse cues at the right time, the right place, and at the right concentrations is an overwhelming challenge. Emerging evidence suggests that microRNAs (miRNA, endogenously expressed siRNAs) play essential roles in initiation of stem cell differentiation and tissue patterning during development, essentially acting as â?~master switchesâ?T responsible for signal integration and subsequent phenotypic regulation of cells. While siRNA therapeutics promise the ultimate level of simplicity, specificity, and reproducibility to control stem cell differentiation and tissue evolution, delivery remains a major challenge. In this work, we successfully developed a novel delivery system for siRNA treatment of mesenchymal stem cells (MSCs) to control differentiation patterns. This delivery system was specially designed to protect siRNA, enhance its uptake, and promote efficient cytosolic delivery into MSCs. Specifically, when we delivered fluorescently-labeled siRNA with our delivery system, we observed diffuse intracellular accumulation of fluorescent siRNA, indicating successful cytosolic siRNA delivery. Using a glyceraldehyde 3 phosphate dehydrogenase (GAPDH)-specific siRNA, we observed efficient gene reduction (18%) while maintaining high cell viability. Importantly, this delivery system resulted in >90% siRNA positive cells, as indicated using flow cytometry of FAM-labeled siRNA. We then treated cells with siRNAs that mimic known miRNAs upregulated in osteogenesis and chondrogenesis. Specifically, we delivered siRNA mimicking either miRNA-24 or miR-199a and observed marked upregulation of core binding factor alpha 1 (CBFA1, 3-fold) or SRY-box 9 (sox9, 8-fold), markers for osteogenesis and chondrogenesis, respectively. Moreover, the specific action by the siRNA, rather than the delivery vehicle, became evident when cells that were treated with control siRNA showed no altered MSC phenotype on surface marker expression (CD44+/CD45-/CD90+/CD105+) over a 15-day period. Our current pursuits are focused on controlling siRNA delivery to MSCs in space and time to regenerate tissues of the musculoskeletal system.

Hydrogel scaffolds are used in biomedicine to study cell differentiation and tissue evolution, where it is critical to be able to deliver chemical cues (therapeutics and nutrients) in a spatially and temporally defined manner. High molecular weight therapeutics such as large proteins can by physically entrapped in hydrogels; because their diffusion is retarded by the hydrogel, they are slowly released from the hydrogel in a predictable and sustained manner. It is more difficult to control the release of small and moderate molecular weight therapeutics physically entrapped in hydrogels because highly swollen nature of the hydrogel allows for rapid diffusion of species. The release of therapeutic agents from hydrogels can be better controlled if the agent is covalently tethered into the network through a degradable linkage instead of physically entrapped. Hydrolyzable and, to a lesser extent, enzymatically degradable linkages have been reported in controlled release systems. While hydrolysis and enzymolysis are both effective methods for sustained release of therapeutic agents, the rate of release cannot be adjusted or arrested after the hydrogel is fabricated, and release is not spatially controlled. We are therefore interested in using photochemistry as an alternative. In contrast to both hydrolysis and enzymolysis, photodegradation is externally controlled, allowing user-dictated on-demand release. We synthesized and characterized a model photoreleasable therapeutic agent using an ortho-nitrobenzylether (o-NB) linker as the photosensitive moiety and fluorescein as the model drug. This photoreleasable fluorescein-conjugate was copolymerized into hydrogel depots of photoreleasable fluorescein. The release of fluorescein as a function of light exposure at multiple wavelengths (365-436 nm), intensities (5-20 mW/cm2) and durations (0-20 minutes) was quantified. We also prepared o-NBs with different functionalities (alcohol, amine, carboxylic acid, activated disulfide) at the benzylic position to allow direct conjugation of nearly any type of therapeutic agent to the linker, and its subsequent controlled release from a hydrogel depot. We have developed a predictive model of photodegradation for materials containing multiple photodegradable groups and demonstrate the predictable release of biologically relevant molecules, including cell-adhesive peptides, enzymes, proteins and growth factors. Each of these agents retains full biological activity after it is released from the hydrogel.

Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. In this study we were interested in determining if the manner in which VEGF is presented from a scaffold to endothelial cells influences the phosphorylation of VEGFR-2 and the architecture of the blood vessels formed. We covalently bound VEGF to either self-assembled monolayers on gold or to polymeric nanoparticles to modulate the presentation of VEGF for both molecular analyses and cellular assays. We found that covalently bound VEGF (Vc) is able to phosphorylate VEGFR-2 (VR-2) to the same extent as soluble VEGF (Vs) in endothelial cells (EC), but that the mode of VEGF presentation alters the tyrosine residues that are phosphorylated, the time course of phosphorylation, and the resulting downstream signaling. Further, we found that Vc need not be internalized along with the receptor for receptor phosphorylation and downstream signaling to occur. Using the nanoparticle VEGF clusters and an EC branching assay or CAM assay we found that VEGF clusters led to an increase in EC sprout branching, thickness, and total vessel network length compared to soluble VEGF or less clustered VEGF. We believe the presentation of growth factors within biomaterial is an important parameter to be consider when designing scaffolds for tissue regeneration and stem cell culture.

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 Ã- 10,000 cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigenâ?"specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigenâ?"specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

Improving stem cell differentiation methods to generate pure populations of target cells may enhance the development of clinically relevant cell therapies and physiologically relevant models. The convention for driving differentiation of human embryonic stem cells involves temporal addition of dissolved bioactive factors to suspended cell culture supernatant. However, differentiating cells spontaneously aggregate to form embryoid bodies (EBs) and undergo epithelialization. Densely packed, highly spread, epithelial-like cells form tight cell junctions and encapsulate EBs in an extra-embryonic shell of basement membrane proteins, creating a barrier to diffusion and inner gradients of bioactive cues. Subsequently, EBs experience internal cavitation and programed cell death in response to elevated local H2O2 within the shell. Together, these phenomena lead to non-uniform instructional cues and potential target cell loss. We hypothesized that modulation of local H2O2 concentrations, coupled with inside-out exogenous bioactive factor delivery, mediated via triggered microcapsule release, will enhance efficiency of target human stem cell differentiation. Our lab has optimized a suspension culture system to support evenly dispersed microcapsule scaffolds in human stem cell derived EBs. We also previously reported novel H2O2 responsive biocompatible nanocapsules that exploit thioether oxidation to induce their solubility in the presence of H2O2. We demonstrated triggered release of model proteins (ovalbumin) and model hydrophobic small molecules (Nile red) separately in buffer and intracellularly in a monocyte/macrophage cell line. We have combined these technologies to deliver, via bioresponsive microcapsule scaffolds, bioactive factors that modulate intercellular H2O2, thereby attenuating cavitation in human embryonic stem cell-derived EBs. Using this scheme we are separately screening co-delivery of catalase, an enzyme that decomposes H2O2 into water and oxygen, with established Wnt-signaling promotors of cardiomyogenesis or small molecule modulators of hypoxia inducible factor.