BACKGROUND The creation of functional skeletal muscle via tissue engineering holds great promise without sacrificing healthy donor tissue. Different cell types have been investigated regarding their myogenic differentiation potential under the influence of various media supplemented with growth factors. Yet, most cell cultures include the use of animal sera, which raises safety concerns and might lead to variances in results. Electrospun nanoscaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining both biocompatibility and stability. We therefore aimed to develop a serum-free myogenic differentiation medium for the co-culture of primary myoblasts (Mb) and mesenchymal stromal cells derived from the bone marrow (BMSC) and adipose tissue (ADSC) on electrospun poly-ε-caprolacton (PCL)-collagen I-nanofibers. RESULTS Rat Mb were co-cultured with rat BMSC (BMSC/Mb) or ADSC (ADSC/Mb) two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-nanofibers. Differentiation media contained either AIM V, AIM V and Ultroser® G, DMEM/Ham's F12 and Ultroser® G, or donor horse serum (DHS) as a conventional differentiation medium. In 2D co-culture groups, highest upregulation of myogenic markers could be induced by serum-free medium containing DMEM/Ham's F12 and Ultroser® G (group 3) after 7 days. Alpha actinin skeletal muscle 2 (ACTN2) was upregulated 3.3-fold for ADSC/Mb and 1.7-fold for BMSC/Mb after myogenic induction by group 3 serum-free medium when compared to stimulation with DHS. Myogenin (MYOG) was upregulated 5.2-fold in ADSC/Mb and 2.1-fold in BMSC/Mb. On PCL-collagen I-nanoscaffolds, ADSC showed a higher cell viability compared to BMSC in co-culture with Mb. Myosin heavy chain 2, ACTN2, and MYOG as late myogenic markers, showed higher gene expression after long term stimulation with DHS compared to serum-free stimulation, especially in BMSC/Mb co-cultures. Immunocytochemical staining with myosin heavy chain verified the presence of a contractile apparatus under both serum free and standard differentiation conditions. CONCLUSIONS In this study, we were able to myogenically differentiate mesenchymal stromal cells with myoblasts on PCL-collagen I-nanoscaffolds in a serum-free medium. Our results show that this setting can be used for skeletal muscle tissue engineering, applicable to future clinical applications since no xenogenous substances were used.

中文翻译：

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在无血清条件下，在PCL-胶原I-纳米管上，原代成肌细胞和间充质基质细胞的成肌分化。

背景技术在不牺牲健康的供体组织的情况下，通过组织工程创建功能性骨骼肌具有广阔的前景。在补充了生长因子的各种培养基的影响下，已经研究了不同细胞类型的成肌分化潜能。但是，大多数细胞培养都包括使用动物血清，这引起了安全隐患，并可能导致结果差异。电纺纳米支架代表了生物相容性和稳定性相结合的适用于骨骼肌组织工程的基质。因此，我们旨在开发一种无血清的成肌分化培养基，用于在静电纺丝聚ε-己内酰胺（PCL）上共培养原代成肌细胞（Mb）和源自骨髓（BMSC）和脂肪组织（ADSC）的间充质基质细胞）胶原蛋白I-纳米纤维。结果大鼠Mb与大鼠BMSC（BMSC / Mb）或ADSC（ADSC / Mb）在对齐的PCL-胶原I-纳米纤维上以二维（2D）单层或三维（3D）共培养。分化培养基包含AIM V，AIM V和Ultroser®G，DMEM / Ham's F12和Ultroser®G或供体马血清（D​​HS）作为常规分化培养基。在2D共培养组中，7天后，含DMEM / Ham's F12和Ultroser®G的无血清培养基可以诱导成肌标志物的最高上调。与DHS刺激相比，第3组无血清培养基成肌诱导后，α肌动蛋白骨骼肌2（ACTN2）的ADSC / Mb升高3.3倍，BMSC / Mb升高1.7倍。Myogenin（MYOG）在ADSC / Mb中上调了5.2倍，在BMSC / Mb中上调了2.1倍。在PCL胶原蛋白I纳米脚手架上，在与Mb共培养中，ADSC与BMSC相比显示出更高的细胞活力。与无血清刺激相比，DHS长期刺激后，肌球蛋白重链2，ACTN2和MYOG作为晚期成肌标记，显示出更高的基因表达，尤其是在BMSC / Mb共培养中。用肌球蛋白重链进行的免疫细胞化学染色验证了在无血清和标准分化条件下均存在收缩装置。结论在这项研究中，我们能够在无血清培养基中用PCL-胶原I-纳米管的成肌细胞对间充质基质细胞进行成肌分化。我们的结果表明，此设置可用于骨骼肌组织工程，由于未使用异种物质，因此可用于将来的临床应用。与无血清刺激相比，DHS和作为长期成肌标记的MYOG在长期刺激后显示出更高的基因表达，尤其是在BMSC / Mb共培养中。用肌球蛋白重链进行的免疫细胞化学染色验证了在无血清和标准分化条件下均存在收缩装置。结论在这项研究中，我们能够在无血清培养基中，用PCL-胶原I-纳米管的成肌细胞对间充质基质细胞进行成肌分化。我们的结果表明，该设置可用于骨骼肌组织工程，由于未使用异种物质，因此可用于将来的临床应用。与无血清刺激相比，长期使用DHS刺激的MYOG和MYOG表现出更高的基因表达，特别是在BMSC / Mb共培养中。用肌球蛋白重链进行的免疫细胞化学染色验证了在无血清和标准分化条件下均存在收缩装置。结论在这项研究中，我们能够在无血清培养基中，用PCL-胶原I-纳米管的成肌细胞对间充质基质细胞进行成肌分化。我们的结果表明，该设置可用于骨骼肌组织工程，由于未使用异种物质，因此可用于将来的临床应用。用肌球蛋白重链进行的免疫细胞化学染色验证了在无血清和标准分化条件下均存在收缩装置。结论在这项研究中，我们能够在无血清培养基中，用PCL-胶原I-纳米管的成肌细胞对间充质基质细胞进行成肌分化。我们的结果表明，该设置可用于骨骼肌组织工程，由于未使用异种物质，因此可用于将来的临床应用。用肌球蛋白重链进行的免疫细胞化学染色验证了在无血清和标准分化条件下均存在收缩装置。结论在这项研究中，我们能够在无血清培养基中，用PCL-胶原I-纳米管的成肌细胞对间充质基质细胞进行成肌分化。我们的结果表明，该设置可用于骨骼肌组织工程，由于未使用异种物质，因此可用于将来的临床应用。