Self-assembling multilayer MSC-sheet promotes wound healing increasing M2 macrophage polarization.

Mesenchymal stem cell (MSC) therapy holds promise for treating chronic wounds. However, low cell density in the target tissue and short retention time limits its efficacy. This study investigated the therapeutic effects of the self-assembling multilayer MSC-sheet in a murine wound model, focusing on wound healing and macrophage infiltration. The MSC-sheet was prepared from human subcutaneous adipose tissue and characterized by histology and RNA sequencing. In vivo, the α-MEM (control), MSC-suspension, and MSC-sheet were applied to full-thickness skin defects in C57BL/6 J Jcl mice. On days 7, 14, and 21, the remaining wound area, neoepithelialization, granulation tissue, cell retention time, angiogenesis, and macrophage infiltration were evaluated using hematoxylin and eosin, Azan, anti-human nucleoli (HN), anti-CD31, anti-CD68, and anti-CD163 staining. Macrophage polarization was evaluated by RT-PCR on days 3, 7, and 14. The MSC-sheet formed a multilayer structure (6-7 layers) containing extracellular matrix (ECM). RNA sequencing identified 756 differentially expressed genes compared with MSC-suspension, highlighting pathways related to wound healing, anti-inflammation, angiogenesis, and macrophage polarization. In vivo, both the MSC-sheet and MSC-suspension significantly reduced the remaining wound area (days 7 and 14), increased neoepithelialization (day 14), granulation tissue formation (day 7), angiogenesis (day 7) and macrophage infiltration (days 7 and 14) compared to the control group. The MSC-sheet maintained superior MSC retention and greater M2 macrophage induction than suspension, confirmed by RT-PCR. The MSC-sheet and MSC-suspension accelerated wound healing and promoted M2 macrophage polarization. The MSC-sheet, with its multilayer structure and ECM, outperformed suspension in sustaining MSCs and enhancing M2 infiltration. These findings position the self-assembling multilayer MSC-sheet as a promising, scaffold-free cell delivery platform for chronic wounds, with potential scalability and clinical applicability for future translational use.