Prolonged irrigation time in endoscopic aqueous medium impairs MSC/β-TCP adhesion and osteogenic potential

To evaluate the effects of an endoscopic aqueous environment on the viability and differentiation capacity of autologous bone marrow mesenchymal stem cells (MSCs) enriched with β-tricalcium phosphate (β-TCP). A screening-enrichment-combination circulating system (SECCS) was used to prepare MSCs/β-TCP from a patient. A simulated aqueous flushing environment for spinal endoscopic surgery was established with different flushing times (0-60 min). Scanning electron microscopy (SEM) was employed to determine the adhesion and condition of MSCs/β-TCP. CCK-8 was used to determine the viability of adherent MSCs. The osteogenic, adipogenic, and chondrogenic differentiation potentials of adherent MSCs were assessed with alizarin red, alkaline phosphatase, Oil Red O, and toluidine blue staining. A subcutaneous implantation mouse model was utilized to evaluate the osteogenic capacity of MSCs/β-TCP under different aqueous flushing conditions using micro-CT, haematoxylin-eosin staining, and Von Kossa staining. SEM revealed sustained MSC adhesion on β-TCP across all irrigation durations (0-60 min), with no differences in adhesion density. However, cell viability declined significantly after 30 min(P < 0.05). Differentiation assays demonstrated time-dependent impairments: osteogenic and adipogenic potentials decreased significantly at 30 min (P < 0.05), chondrogenic morphology disorganized after 15 min, while ALP activity declined only at 60 min (P < 0.05). In vivo, irrigation > 15 min markedly reduced bone formation (P < 0.05) and parallel reductions in mineralized matrix(P < 0.05). In summary, prolonged irrigation (> 15 min) in the spinal endoscopic aqueous environment compromises MSC/β-TCP adhesion and osteogenic capacity. Optimizing surgical efficiency and strictly controlling the irrigation time and pressure are critical in preserving graft performance for successful bone fusion.