Collagen scaffold arrays for combinatorial screening of biophysical and biochemical regulators of cell behavior.

Arrays of 3D macroporous collagen scaffolds with orthogonal gradations of structural and biomolecular cues are described. Gradient maker technology is applied to create linear biomolecular gradients within microstructurally distinct sections of a single CG scaffold array. The array set up is used to explore cell behaviors including proliferation and regulation of stem cell fate.

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials.

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue engineering.

The promotion of HL-1 cardiomyocyte beating using anisotropic collagen-GAG scaffolds.

Biomaterials for myocardial tissue engineering must balance structural, mechanical and bioactivity concerns. This work describes the interaction between HL-1 cardiomyocytes and a series of geometrically anisotropic collagen-GAG (CG) scaffolds with aligned tracks of ellipsoidal pores designed to mimic elements of the native geometric anisotropy of cardiac tissue. Here we report the role scaffold geometric anisotropy and pore size plays in directing cardiomyocyte bioactivity. Notably, HL-1 cardiomyocytes showed good proliferation and metabolic activity in all variants out to 14 days in culture. Critically, HL-1s exhibited significantly elevated 3D alignment and earlier spontaneous beating within anisotropic CG scaffolds relative to isotropic scaffold controls. This spontaneous beating occurred at significantly higher instances for larger pore size anisotropic variants. Gene expression and immunohistochemical analyses for key cardiac marker (α-myosin heavy chain, connexin-43) suggest that the isotropic and anisotropic scaffolds support expression of key transcriptomic markers of cardiomyocyte phenotype as well as the formation of gap junctions and elongated, aligned cell morphologies. Collectively, these results suggest that a geometrically anisotropic scaffold with sufficiently large pore size (>150 µm) provides a suitable microenvironment to induce cardiomyocyte alignment, beating, and bioactivity for cardiac tissue engineering applications.