The use of a single well identified molecule to

The use of a single well-identified molecule to induce AChR clusters formation can simplify complicated cell manipulation procedures and may provide a more efficient means of developing biological substitutes for functional muscle tissue restoration in vivo. Pharmacological approaches dealing with bioactive molecules and drugs can be utilized to accelerate and enhance tissue maturation, innervations, and function in vitro and further promote accelerated growth of blood vessels and nerves into the engineered muscle tissue [36]. In this study we focused on the use of the postsynaptic regulator agrin for this purpose. Agrin is a large proteoglycan and its best-characterized role is in the development of the neuromuscular junction during embryogenesis as well as in later muscle development and healing. It exhibits the ability to express AChR and cluster on muscle cells, and this provides the foundation on which vascular and neural networks can be built. We hypothesized that pre-treatment of engineered muscle fibers with agrin would enhance AChR clusters formation and thereby accelerate the contacts with host nerves in vivo following implantation. Our results indicate that agrin treatment increased AChR clusters formation on C2C12 myotubes in both 2-D and 3-D muscle tissue constructs and that these pre-fabricated AChR clusters facilitated accelerated contacts with DRG nerve in vitro. Interestingly, engineered C2C12 muscle constructs treated with agrin also promoted innervations with host nerve in vivo. The implanted agrin-treated 3-D muscle constructs displayed a mature myofiber structure with MHC Lapatinib in the vicinity of the host nerve, indicating enhanced interaction with the host nerve [37], [38]. This is in agreement with the findings of Bian and Bursac [19] who showed that when neonatal rat skeletal muscle constructs were treated with miniagrin, there was a 1.7 fold increase in twitch-tetanus force amplitude and an enhanced ACh receptor cluster formation. We also present experimental evidence that agrin is involved in the maintenance of normal nerve structure and function. Thus, we observed an enhanced DRG neurite outgrowth on agrin-treated myotubes in vitro compared to untreated myotubes, and that the host nerve (CPN) appeared to have more intact filaments with continuous axon structure in the agrin-treated constructs (Fig. S4(e)). Moreover, our TEM study further supported the positive effect of agrin treatment on the maintenance of nerve by demonstrating that the CPN within the agrin-treated constructs contained more myelinated axons than the untreated construct (Fig. S7) [39]. This data is consistent with the previous finding that neural agrin contributes to the establishment of axonal pathways by modulating the function of neurite promoting molecules such as fibroblast growth factor-2 (FGF-2). From these results, it can be inferred that agrin is also an important factor in maintaining neural functions. We also observed that agrin treatment increased angiogenesis at the vicinity of the implant and host nerve (CPN) in vivo. Agrin treatment of the engineered muscle tissue significantly enhanced recruitment of a number of large blood vessels to the implants. Although full length agrin was reported to play a role in modulating the activities of heparin-binding angiogenic proteins such as FGF-2 [40], [41], in this study, miniagrin without heparin-binding sites was used. Further investigations are required to examine the pro-angiogenic effect of miniagrin. Taken together, our results combined with previously reported findings provide evidence that the activity of agrin is not restricted solely to the formation of AChR clusters but might be of broader significance than previously anticipated [42], [43], [44]. Thus, we suggest that agrin treatment of engineered muscle tissue has implications for the restoration of skeletal muscle function, and we hypothesize that agrin may play multiple roles in many processes in the neuromuscular system including myogenesis, neurogenesis, and angiogenesis through well-orchestrated biological pathways.