Methacrylated human recombinant collagen peptide as a hydrogel for manipulating and monitoring stiffness-related cardiac cell behavior.

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Ice-templating of anisotropic structures with high permeability.

Nutrient diffusion and cellular infiltration are important factors for tissue engineering scaffolds. Maximizing both, by optimizing permeability and scaffold architecture, is important to achieve functional recovery. The relationship between scaffold permeability and structure was explored in anisotropic scaffolds from a human collagen I based recombinant peptide (RCP). Using ice-templating, scaffold pore size was controlled (80-600µm) via the freezing protocol and solution composition. The transverse pore size, at each location in the scaffold, was related to the freezing front velocity, via a power law, independent of the freezing protocol. Additives which interact with ice growth, in this case 1wt% ethanol, altered ice crystallization and increased the pore size. Variations in composition which did not affect the freezing, such as 40wt% hydroxyapatite (HA), did not change the scaffold structure, demonstrating the versatility of the technique. By controlling the pore size, scaffold permeability could be tuned from 0.17×10-8 to 7.1×10-8m2, parallel to the aligned pores; this is several orders of magnitude greater than literature values for isotropic scaffolds: 10-9-10-12m2. In addition, permeability was shown to affect the migration of osteoblast-like cells, suggesting that by making permeability a design parameter, tissue engineering scaffolds can promote better tissue integration.

Osteogenesis and mineralization of mesenchymal stem cells in collagen type I-based recombinant peptide scaffolds.

Recombinant peptides have the power to harness the inherent biocompatibility of natural macromolecules, while maintaining a defined chemistry for use in tissue engineering. Creating scaffolds from peptides requires stabilization via crosslinking, a process known to alter both mechanics and density of adhesion ligands. The chemistry and mechanics of linear scaffolds from a recombinant peptide based on human collagen type I (RCP) was investigated after crosslinking. Three treatments were compared: dehydrothermal treatment (DHT), hexamethylene diisocyanate (HMDIC), and genipin. With crosslinking, mechanical properties were not significantly altered, ranging from 1.9 to 2.7 kPa. However, the chemistry of the scaffolds was changed, affecting properties such as water uptake, and initial adhesion of human mesenchymal stem cells (hMSCs). Genipin crosslinking supported the lowest adhesion, especially during osteoblastic differentiation. While significantly altered, RCP scaffold chemistry did not affect osteoblastic differentiation of hMSCs. After four weeks in vitro, all scaffolds showed excellent cellular infiltration, with up-regulated osteogenic markers (RUNX2, Osteocalcin, Collagen type I) and mineralization, regardless of the crosslinker. Thus, it appears that, without significant changes to mechanical properties, crosslinking chemistry did not regulate hMSC differentiation on scaffolds from recombinant peptides, a growing class of materials with the ability to expand the horizons of regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1856-1866, 2017.