The successful engineering of a truly biomimetic model of skeletal muscle could have a significant impact on a number of biomedical disciplines. Although a variety of techniques are currently being developed, there is, as of yet, no widely available and easily reproducible culture system for the synthesis of 3D artificial muscle tissues. In attempting to generate such a model it is essential to optimise any protocol in order to generate a tissue that best represents the in vivo environment. Since the maturation of muscle derived cells in culture is critically dependent on density, a major factor to be addressed in the development of these models is the ideal concentration at which to seed cells in order to generate an optimal response. In studying the effect of cell density on the performance of cells in an established 3D collagen based model of skeletal muscle, we demonstrate that an optimum density does exist in terms of peak force generation and myogenic gene expression data. Greater densities however, lead to the formation of a more physiologically relevant tissue with a phenotype characteristic of slow, postural muscle.

In vitro skeletal muscle engineering involves the culture of isolated primary myogenic cells in an environment conducive to the formation of a three-dimensional (3D) tissue construct capable of generating force. Isolated human myogenic cells have been used to study cell-cell interactions, permitting identification of functions intrinsic to skeletal muscle in two dimensions (2D). However, the independent contribution of human myogenic and non-myogenic cell types that comprise skeletal muscle to myogenic cell differentiation, force generation and matrix remodelling has yet to be established in 3D. The objective of this study was to use isolated human myogenic and non-myogenic muscle-derived cells (MDC) seeded in 3D collagen constructs to engineer a biomimetic craniofacial skeletal construct. The aim was to purify the two subpopulations of myogenic and non-myogenic cells from human masseter muscle and quantitate myogenic cell differentiation, force generation and matrix remodelling of the 3D collagen construct. The results showed that both the heterogeneous mixture of cells and the purified myogenic cell population expressed myogenin, indicative of myogenic cell differentiation. Further, there was a synergistic effect as the heterogeneous co-culture of myogenic and non-myogenic cells generated the highest peak force and expressed the most MMP-2 mRNA compared to isolated individual cell populations.

Tissue-engineering strategies for the treatment of osteoarthritis would benefit from the ability to induce chondrogenesis in precursor cells. One such cell source is bone marrow-derived stromal cells (BMSCs). Here, we examined the effects of moderate-strength static magnetic fields (SMFs) on chondrogenic differentiation in human BMSCs in vitro. Cells were cultured in pellet form and exposed to several strengths of SMFs for various durations. mRNA transcript levels of the early chondrogenic transcription factor SOX9 and the late marker genes ACAN and COL2A1 were determined by reverse transcription-polymerase chain reaction, and production of the cartilage-specific macromolecules sGAG, collage type 2 (Col2), and proteoglycans was determined both biochemically and histologically. The role of the transforming growth factor (TGF)-β signaling pathway was also examined. Results showed that a 0.4 T magnetic field applied for 14 days elicited a strong chondrogenic differentiation response in cultured BMSCs, so long as TGF-β3 was also present, that is, a synergistic response of a SMF and TGF-β3 on BMSC chondrogenic differentiation was observed. Further, SMF alone caused TGF-β secretion in culture, and the effects of SMF could be abrogated by the TGF-β receptor blocker SB-431542. These data show that moderate-strength magnetic fields can induce chondrogenesis in BMSCs through a TGF-β-dependent pathway. This finding has potentially important applications in cartilage tissue-engineering strategies.