In situ electrospinning at the operating table to immobilise mesenchymal stem cells on the bony wall for the regeneration of osteoporotic bone defects.

Stem cell seeding is an important approach for the treatment of osteoporotic bone defects. However, existing seeding methods rely on carriers such as scaffolds to retain cells within the defect, making it difficult for cells to adhere directly to the complex surface of the bony wall. In this study, mesenchymal stem cells (MSCs) were delivered into in vivo bone defects with a handheld electrospinning device as an improved cell seeding strategy. First, flow cytometry and related assays confirmed that electrospinning had minimal effects on MSCs viability. MSCs were then immobilised by electrospinning in the calvarial defects of osteoporotic rats. Single-cell sequencing indicated that, compared with intraosseous injection, this approach effectively increased MSCs retention. The ElectroSpinning Group (ESG) also exhibited elevated expression of osteogenic marker proteins and enhanced bone repair. To elucidate the mechanism by which ESG promotes osteogenesis, this study first compared differences in bone repair between osteoporotic and normal rats. Tissue mRNA sequencing and related analyses verified that osteoporotic defects were characterised by reduced levels of the homing factor c-c motif chemokine ligand 2 (CCL2) and disruption of the osteogenic microenvironment. mRNA sequencing and related assays revealed that ESG upregulated CCL2 expression in response to these features of the osteoporotic microenvironment, thereby promoting the homing of endogenous stem cells. This study then employed electrospinning to seed green fluorescent protein (GFP)-labelled MSCs. Upon detection of green fluorescence in tissue sections, flow cytometry confirmed the retention of MSCs and was applied to quantify the recruitment of endogenous stem cells. With regard to microenvironmental modulation, this study focused on tumour necrosis factor-stimulated gene 6 (TSG6). The results indicated that, in ESG, MSCs increased regulatory T-cell infiltration and inhibited M1 macrophage polarisation via TSG6, thereby correcting the local microenvironmental imbalance. When TSG6 was blocked, these improvements in immunomodulation were attenuated. In summary, this study presents a novel stem cell delivery strategy that enables direct immobilization of MSCs at bone defect sites. Compared with non-electrospun MSCs, electrospun MSCs enhance autologous stem cell homing and modulate immunity to improve the osteogenic microenvironment, thereby promoting bone repair.