The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.

Despite extensive use of type I collagen for research and medical applications, its fibril-forming or polymerization potential has yet to be fully defined and exploited. Here we describe a type I collagen formulation that is acid solubilized from porcine skin (PSC), quality controlled based upon polymerization potential, and well suited as a platform polymer for preparing three-dimensional (3D) culture systems and injectable/implantable in-vivo cellular microenvironments in which both relevant biochemical and biophysical parameters can be precision-controlled. PSC is compared with 3 commercial collagens in terms of composition and purity as well as polymerization potential, which is described by kinetic parameters and fibril microstructure and mechanical properties of formed matrices. When subjected to identical polymerization conditions, PSC showed significantly decreased polymerization times compared to the other collagens and yielded matrices with the greatest mechanical integrity and broadest range of mechanical properties as characterized in oscillatory shear, uniaxial extension, and unconfined compression. Compositional and intrinsic viscosity analyses suggest that the enhanced polymerization potential of PSC may be attributed to its unique oligomer composition. Collectively, this work demonstrates the importance of standardizing next generation collagen formulations based upon polymerization potential and provides preliminary insight into the contribution of oligomers to collagen polymerization properties.

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >108 ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([3H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGF beta. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGF beta-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling.