We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 x 10(8) cells/cm(3)) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric alpha-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter x 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-microm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.

Hybrid cardiac constructs with mechanical properties suitable for in vitro loading studies and in vivo implantation were constructed from neonatal rat heart cells, fibrin (Fn), and biodegradable knitted fabric (Knit). Initial (2-h) constructs were compared with native heart tissue, studied in vitro with respect to mechanical function (stiffness, ultimate tensile strength [UTS], failure strain epsilon(f), strain energy density E) and compositional remodeling (collagen, DNA), and implanted in vivo. For 2-h constructs, stiffness was determined mainly by the Fn and was half as high as that of native heart, whereas UTS, epsilon(f), and E were determined by the Knit and were, respectively, 8-, 7-, and 30-fold higher than native heart. Over 1 week of static in vitro culture, cell-mediated, serum-dependent remodeling was demonstrated by a 5-fold increase in construct collagen content and maintenance of stiffness not observed in cell-free constructs. Cyclic stretch further increased construct collagen content in a manner dependent on loading regimen. The presence of cardiac cells in cultured constructs was demonstrated by immunohistochemistry (troponin I) and Western blot (connexin 43). However, in vitro culture reduced Knit mechanical properties, decreasing UTS, epsilon(f), and E of both constructs and cell-free constructs and motivating in vivo study of the 2-h constructs. Constructs implanted subcutaneously in nude rats for 3 weeks exhibited the continued presence of cardiomyocytes and blood vessel ingrowth by immunostaining for troponin I, connexin 43, and CD-31. Together, the data showed that hybrid cardiac constructs initially exhibited supraphysiologic UTS, epsilon(f), and E, and remodeled in response to serum and stretch in vitro and in an ectopic in vivo model.

Using a clinically well-established collagen matrix, contractile myocardial tissue can be engineered in vitro successfully. Mechanical and biologic properties of artificial myocardial tissue resemble native cardiac tissue. Use of artificial myocardial tissues might be a promising approach to reconstitute degenerated or failing cardiac tissue in many disease states and therefore provide a reasonable alternative to whole organ transplantation.