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

功能性骨骼肌的构建仍然是组织工程领域的一大挑战。除了找到最佳的细胞来源外，开发合适的矩阵用于种子细胞的平行和三维比对是至关重要的一步。此外，生长因子在肌肉发生过程中起着重要作用。生长分化因子-11(GDF-11)以及IGF结合蛋白4、5和6(IGFBP4、5、6)可能通过不同的作用影响成肌细胞的分化。由于原代成肌细胞在传代几代后失去分化能力，因此单独应用这些细胞不适合于建立大的肌肉组织。因此，间充质干细胞因其增殖和分化能力而成为有吸引力的候选细胞。骨髓来源干细胞(BMSC)在与原代成肌细胞共培养中显示出分化为肌源性细胞的能力。更令人兴奋的再生细胞来源是脂肪干细胞(ADSC)，它可以在临床环境中通过微创程序获得。具有排列的纤维的电纺纳米支架是一种很有前途的基质，因为它们再现了机械各向异性，并可以与ECM聚合物、蛋白质和多糖结合。一个重要的方面是纺丝过程本身：考虑到后来的床边应用，应该避免有毒成分。在目前的研究中，已经建立了原代成肌细胞与骨髓基质细胞在电纺平行排列的PCL-胶原I-纳米支架上的3D共培养。即将到来的实验将集中在用临床上更有吸引力的ADSC取代BMSC。最重要的是，新开发的纺丝工艺现在基于无毒的溶剂醋酸，而不是像HFIP这样的有毒溶剂。这一新过程的开发，包括批次间不一致性的标准化评估，一直是主要的努力，但现在可以旋转大量的纳米支架用于本提案中的实验。由于迄今为止研究的分化因子(尤其是HFG)对肌源性细胞的影响很弱，对于目前预定和据此应用的部分提案，通过生物反应器分析了GDF-11、IGFBP4、5和6对肌源性分化的影响，以及不同培养条件(静态和动态)的影响。目前，纳米支架的功能化是通过整合含有生长因子的纤维来实现的，目的是为了向肌细胞分化。此外，含有聚环氧乙烷的牺牲纤维应该有助于支架的血管化，以便在体内应用。最后，种植的支架将被植入到申请者最近建立的大鼠外周静脉环模型中，该模型包含动静脉环和运动神经。这个模型使未来的工程化新肌肉组织的血管化和神经化成为可能。

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.