Perfusable adipose decellularized extracellular matrix biological scaffoldco-recellularizedwith adipose-derived stem cellsand L6 promotes functional skeletal muscle regeneration following volumetric muscle loss

In this study, we used a recellularization method of perfusing cells into biological scaffolds through vascular pedicles. ASCs and L6 cells were co-seeded into adipose decellularized extracellular matrix (adECM) biological scaffolds. On one hand, this strategy ensures uniform distribution of seeded cells in the scaffold and avoids the formation of non-functional "muscle islands" later. On the other hand, co-seeding of L6 cells and ASCs addresses the issue of low myogenic differentiation potential of ASCs. Subsequently, the recellularized biological scaffold treatment of VML animal experiments showed that the combined recellularization strategy had significantly better muscle regeneration and angiogenesis than the single ASCs recellularization strategy, and the former had better functional recovery of tibialis anterior muscle (TA). Further single cell sequencing analysis found that compared to singly seeded ASCs in biological scaffolds, L6 cells in the combined recellularization induced ASCs to transform into a new subpopulation of cells highly expressing Mki67, CD34 and CDK1 genes, which had stronger ability of oriented myogenic differentiation and formed more mature and larger muscle fibers, significantly restoring TA muscle function.本研究采用通过血管蒂向生物支架内灌注细胞的细胞再殖化方案。将脂肪来源干细胞（Adipose-derived Stem Cells, ASCs）与L6细胞共接种至脂肪脱细胞外基质（adipose decellularized extracellular matrix, adECM）生物支架中。一方面，该策略可保障接种细胞在支架内均匀分布，避免后续形成无功能的“肌岛”结构；另一方面，L6细胞与ASCs共接种可解决ASCs成肌分化潜能偏低的问题。后续针对体积性肌肉缺损（Volumetric Muscle Loss, VML）动物开展的再殖化生物支架治疗实验表明，联合细胞再殖化策略的肌肉再生与血管生成效果显著优于单纯ASCs细胞再殖化策略，且前者可更有效地恢复胫前肌（Tibialis Anterior, TA）的功能。进一步的单细胞测序分析显示，相较于单独接种于生物支架的ASCs，联合再殖体系中的L6细胞可诱导ASCs转化为一个高表达Mki67、CD34与CDK1基因的全新细胞亚群，该亚群具备更强的定向成肌分化能力，可形成更成熟、体积更大的肌纤维，显著恢复了胫前肌的功能。