For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active, differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in statically cultured cardiac constructs, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability. Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in static dishes. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct. Oxygen concentration and cell viability decreased linearly and the live cell density decreased exponentially with the distance from the construct surface. Physiological density of live cells was present only within the first 128 microm of the construct thickness. Medium flow significantly increased oxygen concentration within the construct, correlating with the improved tissue properties observed for constructs cultured in convectively mixed bioreactors.

Native myocardium consists of several cell types, of which approximately one-third are myocytes and most of the nonmyocytes are fibroblasts. By analogy with monolayer culture in which fibroblasts were removed to prevent overgrowth, early attempts to engineer myocardium utilized cell populations enriched for cardiac myocytes (CMs; ~80-90% of total cells). We hypothesized that the pre-treatment of synthetic elastomeric scaffolds with cardiac fibroblasts (CFs) will enhance the functional assembly of the engineered cardiac constructs by creating an environment supportive of cardiomyocyte attachment and function. Cells isolated from neonatal rat ventricles were prepared to form three distinct populations: rapidly plating cells identified as CFs, slowly plating cells identified as CMs, and unseparated initial population of cells (US). The cell fractions (3 × 10 6 cells total) were seeded into poly(glycerol sebacate) scaffolds (highly porous discs, 5 mm in diameter × 2-mm thick) using Matrigel ™ , either separately (CM or CF), concurrently (US), or sequentially (CF pre-treatment followed by CM culture, CF + CM), and cultured in spinner flasks. The CF + CM group had the highest amplitude of contraction and the lowest excitation threshold, superior DNA content, and higher glucose consumption rate. The CF + CM group exhibited compact 100-to 200-μm thick layers of elongated myocytes aligned in parallel over layers of collagen-producing fibroblasts, while US and CM groups exhibited scattered and poorly elongated myocytes. The sequential co-culture of CF and CM on a synthetic elastomer scaffold thus created an environment supportive of cardiomyocyte attachment, differentiation, and contractile function, presumably due to scaffold conditioning by cultured fibroblasts. When implanted over the infarcted myocardium in a nude rat model, cell-free poly(glycerol sebacate) remained at the ventricular wall after 2 weeks of in vivo, and was vascularized.

Conventional treatment options for myocardial infarction are limited by the inability of mature myocardium to regenerate after injury. Although functional improvements after injection of cells and growth factors have been demonstrated, the clinical utility of this procedure has been hampered by poor cell localization, low survival, and rapid clearance of injected growth factors. The main objective of this study was to evaluate the applicability of a hydrogel, based on photocrosslinkable chitosan and acryloyl-poly(ethylene glycol)-RGDS (Az-chitosan/Acr-PEG-RGD) for myocyte cell culture and myocardial injection. Chitosan was modified with photoreactive azidobenzoic acid and Acr-PEG-RGD was synthesized by reacting YRGDS with an equimolar amount of acryloyl-PEG-N-hydroxysuccinimide. For injection and encapsulation each polymer was dissolved in Di-H(2)O (pH 6.4), the solutions were mixed and crosslinked by UV application (4 mW/cm(2)). C2C12 myoblasts proliferated and differentiated on hydrogels containing 5 mM RGD but not on the pure photocrosslinked chitosan. In vitro, the crosslinked hydrogels retained 80% of encapsulated VEGF for 24 days. Live/dead staining of neonatal rat cardiomyocytes encapsulated into Az-chitosan/Acr-PEG-RGD hydrogels indicated high cell viability upon UV crosslinking. Ex vivo, we localized the hydrogel on the surface and in the ventricle wall of an adult rat heart by brief (2 min) UV light application.

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