Tissue Engineered Skeletal Muscle

Muscle structural anomalies, whether they evolve from a congenital condition, accident, or from removal of a tumor, can affect physiological performance as well as the psychological health of a patient. Objective: Our long-term goal is to develop a striated muscle implant, derived from a patient's own skeletal muscle satellite cells, that mimics the architectural organization and physiological function of intact muscle. Methods: To this end, we have used an aligned collagen gel system to engineer a 3D prosthesis of skeletal muscle cells. In the aligned collagen gel system, cells are plated onto a polarized matrix of Type I collagen fibrils prepared on a solid substrate. Applying a solution of ice cold, neutral type I collagen to a culture dish or extruded collagen surface, at a concentration of 0.62ìg/mm2, produces this matrix and allowed to polymerize at 37 C for 1 hour. Satellite cells were isolated from neonatal (3-4day old) rat hind limbs by 1.25% protease digestion on a reciprocating rocker at 37C for 60 min. The released cells were harvested and plated on the aligned collagen gels. Results: Over time (7 days) the satellite cells are aligned in the direction of the painted collagen and fused into myotubes. At weekly intervals more satellite cells were added to the cultures resulting in multilayered tissue engineered constructs. The constructs were analyzed light and confocal microscopy, real time RT PCR, and the ability to generate force in an isolated muscle preparation. The myotubes were positive for MyoD and possessed parallel banded myofibrils. The constructs expressed mRNA for a number of markers that are present in differentiated skeletal muscle and were capable of developing force that approximates that seen for muscle of similar thickness. Conclusion: The TE constructs are similar to intact muscle in both appearance and function. NIH grant DE 014599.