Highly regulated signals in the stem cell microenvironment, such as growth factor presentation and concentration, matrix stiffness, and ligand adhesion density have been implicated in modulating stem cell proliferation and fate decisions. To address the relative and combined effects of these parameters on stem cell function, it is desirable to have independent control over their presentation to a cell. Accordingly, we have developed different synthetic platforms using either semi-interpenetrating polymer networks (sIPN) or variable moduli interpenetrating polymer networks (vmIPNs) to assess the effects of soluble signals, adhesion ligand presentation, and material moduli on stem cell function. Employing these networks, we have demonstrated that the mechanical and biochemical properties of a stem cell’s microenvironment can be tuned to regulate self-renewal and differentiation.

Electrospinning (ES) receives a lot of attention as novel approach to produce sub-500 nm fibrous scaffolds for tissue engineering in order to mimic topographically the ECM structures of natural tissues. ES is a fast and low-cost technology to produce polymer scaffolds with large surface area-to-volume ratio and high porosity, exploiting electrical forces to produce fibres with nm-scale diameters from natural or synthetic polymers. Tailoring polymer nanofibers is made possible by means of specific biological functions modifying the external surface of nanofibers by linking biomolecules, such as cell adhesive proteins and peptides, or directly incorporating biomolecules in the electrospinning solution. Here, several approaches are investigated in order to support the biochemical and/or topological requirements of several cell lines in vitro. A deep study of surface modification has lead to identify the best chemical/physical route to link an adhesive ECM protein to prototypal polymer nanofibers, improving their biological properties for human renal cells. The development of composite nanofibers, made of a synthetic polymer organic phase and biomimetic inorganic phases, allows to enhance the differentiation of human adult stem cells towards the osteogenic lineage. Finally, we discuss the cellular response to different topographies induced by random and aligned nanofibrous scaffolds.References:Polini et al. Soft Matter 6, 1668 (2010).Polini et al. submitted (2010).

Corneal disease is a major cause of blindness affecting more than 10 million people worldwide with corneal transplants being the only currently available treatment. Shortcomings of the procedure include immune rejections, infections and donor shortages. This has motivated attempts to develop tissue engineered corneal replacements. However, a clinically viable implant has still not been produced largely due to the difficulty in recreating the nanostructure of the cornea. The cornea has three main layers – epithelium, endothelium and stroma. The stroma consists of cells called corneal fibroblasts (characterized by crystallin proteins such as Transketolase) that exist within 30 nm parallel collagen fibrils, further stacked into orthogonal layers called lamellae. This nanoscale architecture is integral for providing mechanical strength and transparency to the cornea. Upon injury, corneal fibroblasts transform into repair cells called myofibroblasts, characterized by the expression of α–smooth muscle actin and increased matrix production and scarring. However, during freeze injuries which leave the matrix intact, corneal cells regenerate without transforming into the repair phenotype, thus indicating the importance of matrix for regeneration.Our objective is to evaluate whether environmental cues presented through substrate stiffness and matrix topography regulate corneal fibroblast phenotype and matrix production in vitro. To this end, cell response was measured by culturing cells on the following substrates (1) PDMS substrates of varying stiffness: 1800 kPa, 260 kPa (to mimic stiffness in vivo) and 50 kPa, or (2) Collagen fibers of varying nanotopography: 30 nm parallel fibers (to mimic fiber arrangement in vivo), 30 nm random fibers (effect of alignment) and 200 nm parallel fibers (effect of diameter). Collagen fibers were fabricated using a proprietary matrix weaving technology from Fibralign Corp. Purified monomeric collagen solution (bovine type I) in a liquid crystal state was deposited on a glass slide using a patented liquid film applicator assembly. Fiber diameter and alignment were varied by changing pH, collagen concentration, humidity and other factors. Our studies show that mechanical cues such as substrate stiffness and matrix topography down-regulate the repair phenotype and promote formation of the regenerative phenotype. Interestingly, these cues do not seem to influence matrix synthesis. Phenotype transformation and matrix synthesis are regulated by different pathways in corneal fibroblasts. Our results suggest that mechanical cues act via pathways that promote phenotype transformation and that this effect could be uncoupled from matrix synthesis. These results have significant implications for both the design of corneal replacements and for promoting regenerative healing of the cornea after disease or injury.

INTRODUCTION: A critical need exists for long-term replacements of articular cartilage that has been damaged by trauma or degenerative diseases such as osteoarthritis. Current treatments are limited by the amount of donor material and injury at the donor site or result in production of fibrocartilage that is mechanically inferior to native tissue. To address the need for a long-term, functional cartilage replacement, tissue engineering strategies combine biomaterials, cells, and bioactive molecules. Although cartilage cells have the desired phenotype, their use is constrained by low cell numbers and loss of phenotype in cell culture. Due to the ease of harvesting and high proliferation capacity, adult stem cells are a promising cell type for use in tissue-engineered constructs. Challenges that need to be addressed before stem cells can be used in clinical applications include 1) the undesirable use of super-physiological concentrations of growth factors in current differentiation methods and 2) directing differentiation to the desired cell type and maintaining the correct phenotype in vivo. In addition, MSCs do not secrete as much matrix as cartilage cells, which results in tissue-engineered constructs with reduced mechanical properties.RESULTS: To fully harness the potential of MSCs and overcome their associated challenges, we are developing an artificial protein matrix that includes peptide cues for influencing and maintaining the cartilage cell fate. MSCs are typically differentiated to cartilage through the use of soluble factors such as transforming growth factor-beta 3 (TGF-β3) and bone morphogenetic protein 2 (BMP-2). A major challenge of this method is to maintain the articular cartilage phenotype and prevent chondrocyte hypertrophy. The first part of our work examines the ability of agonist peptides derived from chondrogenic growth factors to influence MSCs to undergo cartilage differentiation. The most promising of these peptides is derived from the knuckle epitope of BMP-2. In our study, the BMP-2 peptide was added to the medium over a range of concentrations. The normalized GAG production of cells cultured with >100 μg/mL of the peptide was 74% of the positive control (cells cultured with 200 ng/mL BMP-2 growth factor). The effect of the peptide (100 μg/mL) on normalized GAG and collagen production and AP activity were studied over time in the presence and absence of TGF-β signaling. The second part of our work is to develop an artificial protein matrix that includes the promising bioactive peptides, mechanical domains, and crosslinking sites. We have completed the cloning of this recombinant protein and are currently characterizing its effect on cell behavior.

Schemes to mimic tissue development and engineer functional tissues are likely to benefit from control over the cell’s local signaling environment. This concept is particularly important in stem cell-based applications, in which local signaling can dictate self renewal and differentiation. Recent studies have demonstrated that the characteristics of the local microenvironment – including substrate mechanics and cell adhesion – can significantly impact stem cell fate. Collectively, these studies suggest that engineered biomaterials may be useful as platforms to actively regulate the microenvironment and, in turn, stem cell behavior. We are interested in assembling biomaterials that actively control the presence and activity of growth factors in the local stem cell microenvironment. Specifically, we have used tailored growth factor-material affinity to build biomaterials that regulate stem cell behavior. The affinity and specificity of these interactions can be engineered to regulate local growth factor signaling and mimic the natural extracellular matrix. This presentation will detail an approach in which modular growth factors are engineered to include an ECM binding moiety and a receptor-binding “active” site. Specific examples will include: i) a mineral-binding version of bone morphogenetic protein-2 (BMP-2), which promotes osteogenic differentiation of human mesenchymal stem cells (hMSCs) and promotes bone tissue formation in vivo; ii) a mineral-binding version of VEGF that promotes endothelial cell proliferation, migration, and angiogenesis in vivo; and iii) proteoglycan-binding growth factors that promote hMSC proliferation and osteogenic differentiation. In view of the large number of possible ECM-binding motifs and “active” motifs, the potential for general applicability of these approaches will be highlighted in the presentation.

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering. Transforming growth factor β-1 (TGF-β) can signal MSCs to differentiate into both a smooth muscle cell (SMC) lineage as well as a chondrogenic lineage. Here we showed that matrix stiffness modulated these differential responses. MSCs had more stress fibers and α-actin/calponin-1 assembly on stiff substrates but less spreading, fewer stress fibers and a lower proliferation rate on soft substrates. MSCs had higher expression of SMCs markers on stiff substrates but had a drastic increase in collagen-II on soft substrates; these trends were further increased by TGF-β. The differential gene expression on stiff and soft substrates was reversible in the absence of TGF-β, suggesting matrix stiffness alone was not sufficient to drive terminal differentiation of a specific lineage. Our data also indicated that soft matrix induced the expression of adipogenic marker, suggesting that the specificity of differentiation required additional differentiation factor(s). The mechanotransduction induced by matrix stiffness required actin stress fibers, and to a lesser extent, contractility, as demonstrated by the treatment of Rho kinase inhibitor and non-muscle myosin inhibitor. However, overexpression of constitutively activated Rho GTPase induced SMC markers on stiff substrates and chondrogenic marker on soft surface, suggesting Rho is not the molucular switch for the differential effects of matrix stiffness. MSCs on soft matrices had weaker cell adhesion, which decreased the formation of stress fibers and differentially regulated the expression of SMC, chondrogenic and adipogenic markers. The results advance our understanding on the mechanism of mechanotransduction and the crosstalk of TGF-β and matrix stiffness, and will provide a rational basis for the design and selection of appropriate scaffolds for tissue regeneration.

Stem cell behavior is closely linked to the surrounding microenvironment. As a result, control over stem cell microenvironments can potentially be used to regulate stem cell fate and produce specific cell types for a range of therapeutic applications. A main thrust of this current work was to generate substrates with arrays of chemically well-defined microenvironments to screen for the affects of peptides on human mesenchymal stem cell (hMSC) activity. These substrates were created using mixed self-assembled monolayers (SAMs) of oligo(ethylene glycol) bearing alkanethiolates on gold, which included multiple, orthogonal reactive functionalities for covalent immobilization of specific biomolecules. Array fabrication was achieved by selectively removing regions of hydroxy-terminated oligo(ethylene glycol) followed by local reformation of SAMs with mixtures of hydroxyl and carboxylic acid-terminated alkanethiolates. Localized SAM removal was performed using aqueous NaBH4 or H2 plasma to locally destroy regions of SAM and expose bare gold for subsequent SAM reformation. Conjugation of peptides via carbodiimide condensation yielded a patterned substrate containing features with varied biomolecule density, surrounded by a homogeneous, protein-resistant background. Screening of hMSC behavior on arrays presenting varied densities of the cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) indicated that cells attach to patterned features presenting a broad range of cell adhesion peptide densities, and that peptide density strongly influences cell attachment and focal adhesion formation. Furthermore, immobilization of both RGDSP and a heparin-binding peptide led to a localized increase in hMSC proliferation within patterned regions containing the heparin-binding peptide. Taken together, these results demonstrate that hMSC behavior can be regulated via controlled cell adhesion ligand density as well as presentation of peptides that locally bind heparin at the cell culture surface.

Slow vascularization of functional blood limits the transplantation of tissue constructs and the recovery of ischemic and wounded tissues. Despite the widespread investigation of polysaccharide-based hydrogel scaffolds for their therapeutic applications, blood vessel ingrowth into these hydrogel scaffolds remains a challenge.To promote neovascularization or angiogenesis, we have developed tunable dextran-based hydrogel by incorporating different functional groups, including allyl isocyanate (AI), ethylamine (AE), chloroacetic acid (AC) and maleic anhydride (AM). The modified dextran macromers were then incorporated with polyethylene glycol diacrylate (PEGDA) to prepare a novel biodegradable dextran-based hydrogel. Physical and biological properties - such as swelling, degradation rate, in vitro and in vivo biocompatibility, and vascular endothelial growth factor release were assessed. To expedite angiogenesis, we then remodeled the hydrogel structure by decreasing crosslinking density via reduced degree of substitution of crosslinking groups, which further improved the capacity of hydrogels to promote angiogenesis. We demonstrate that rapid, efficient, and functional neovascularization can be achieved via precise manipulation of hydrogel scaffold properties. To further evaluate their potential as wound healing scaffolds, we tested it in a burn injury model. To mimic current clinical practice, burn wound excisions were performed after 48 hours. These wounds were covered with either dextran hydrogels or Integra, and non-treatment as control followed coverage with Duoderm dressings. The burn site and surrounding tissue were collected at different time intervals and immunohistochemical stains for wound responses and skin regeneration were performed. We demonstrate that the dextran hydrogel not only promoted abundant tissue in growth and angiogenesis, but also promoted regeneration of hair follicles, epithelial reticulation, and differentiated dermal structure within 21 days. Our results indicate that dextran hydrogel can be tailored to promote vascularization and wound healing.

Tissue cells constantly probe their surroundings. They lack eyes to see and ears to hear but sense their microenvironment through adhesion and cell-generated deformation. To address how deeply cells feel into the depths of a matrix - a question of relevance to myoblasts on basal lamina, chondrocytes surrounded by pericellular matrix, and osteoblasts on osteoid - we cultured mesenchymal stem cells, as prototypical but particularly sensitive adherent cells (1), on collagen-impregnated hydrogels of controlled elasticity (E) and micro-thickness (h). By 12-36 hrs in culture, cell morphology was distinct on thick and soft matrices compared to either thin or stiff films, correlating well with nuclear morphology. A morphological transition at ~5 microns gel thickness defines a tactile length scale for mechanosensitivity. Matrix-dependent cytoskeletal organization exhibits thickness-dependent nematic (2) and smectic-like assembly, and some nuclear structure components widely implicated in cell differentiation (3), are also found to change for thin films relative to thick gels of the same E. To improve sensitivity and accuracy of transcriptional microarrays we developed a novel algorithm that shows gene expression of many nuclear genes, including histones, also depend on h. The changes suggest direct physical links to regulation of gene expression by matrix.REFERNCES: (1) Engler et al, CELL (2006). (2) Zemel et al, Nature Physics (2010). (3) Pajerowski et al, PNAS (2007).