Tissue Engineering of axially vascularized skeletal muscle tissue using functional nanoscaffolds in the rat animal model

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The creation of functional skeletal muscle is still a major challenge in the field of tissue engineering. Besides finding the optimal cell source, developing a suitable matrix for parallel and 3D alignment of the seeded cells is crucial step. Furthermore, growth factors play an important role during myogenesis. Growth differentiation factor-11 (GDF-11) as well as IGF-binding proteins 4, 5, and 6 (IGFBP 4, 5, 6) may influence myogenic differentiation via different actions. Since primary myoblasts lose their differentiation capacities after a few passages, the exclusive application of these cells is not suitable for the creation of large muscle tissues. Thus, mesenchymal stem cells are attractive candidates due to their proliferative and differentiation capacities. Bone-marrow derived stem cells (BMSC) in co-culture with primary myoblasts have shown their ability to differentiate into the myogenic line. An even more exciting cell source for regenerative applications are adipose-derived stem cells (ADSC), which can be harvested by minimally invasive procedures in a clinical setting.Electrospun nanoscaffolds with aligned fibers are a promising matrix as they reproduce the mechanical anisotropy and can be combined with ECM polymers, proteins and polysaccharides. An important aspect is the spinning process itself: toxic components should be avoided in light of a later bench-to-bedside application.In the current study the 3D co-culture of primary myoblasts with BMSC on electrospun parallel aligned PCL-collagen I-nanoscaffolds have already been established. Upcoming experiments would be focusing on replacing BMSC by clinically even more attractive ADSC. Most importantly the newly developed spinning process is now being based on the non-toxic solvent acetic acid instead of toxic solvents like HFIP. Development of this new process, including standardized assessment of batch-to-batch inconsistencies, has been a major effort, but now large quantities of nanoscaffolds can be spun for experiments aimed for in this proposal.Since the myogenic impact of differentiation factors investigated so far (in particular HFG) has only been weak, for the currently scheduled and hereby applied part of the proposal, the influence of GDF-11, IGFBP4, 5, and 6 on myogenic differentiation is analyzed as well as the influence of different culture conditions, static vs. dynamic via a bioreactor. Currently, nanoscaffolds are functionalized by integration of fibers containing growth factor aiming at myogenic differentiation. In addition, sacrificial fibers, containing polyethylenoxide, should facilitate the vascularisation of the scaffolds for in vivo application. Finally, the seeded scaffolds will be implanted into the rat EPI-loop-model, established recently by the applicant, containing an arteriovenous loop as well as a motor nerve. This model enables future vascularisation and neurotisation of the engineered neo-muscle tissue.