Chen Wugui, Ling Guangfeng, Lin Chengshou, Chen Hengmei, Chen Kongning, Chen Shijie, Ye Yuchao, Liu Chengzhao
Department of Orthopedics, Fujian Medical University Affiliated With Mindong Hospital, Fujian, China.
Sci Rep. 2025 Jun 6;15(1):19976. doi: 10.1038/s41598-025-01340-4.
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.
评估内镜下含水环境对富含β-磷酸三钙（β-TCP）的自体骨髓间充质干细胞（MSC）活力和分化能力的影响。使用筛选-富集-组合循环系统（SECCS）从患者体内制备MSC/β-TCP。建立了不同冲洗时间（0-60分钟）的模拟脊柱内镜手术含水冲洗环境。采用扫描电子显微镜（SEM）确定MSC/β-TCP的黏附情况和状态。使用CCK-8检测贴壁MSC的活力。通过茜素红、碱性磷酸酶、油红O和甲苯胺蓝染色评估贴壁MSC的成骨、成脂和成软骨分化潜能。利用皮下植入小鼠模型，通过显微CT、苏木精-伊红染色和冯科萨染色评估不同含水冲洗条件下MSC/β-TCP的成骨能力。SEM显示，在所有冲洗持续时间（0-60分钟）内，MSC在β-TCP上持续黏附，黏附密度无差异。然而，30分钟后细胞活力显著下降（P<0.05）。分化试验显示出时间依赖性损伤：30分钟时成骨和成脂潜能显著降低（P<0.05），15分钟后软骨形成形态紊乱，而碱性磷酸酶活性仅在60分钟时下降（P<0.05）。在体内，冲洗时间>15分钟显著减少骨形成（P<0.05），矿化基质也相应减少（P<0.05）。总之，脊柱内镜含水环境中长时间冲洗（>15分钟）会损害MSC/β-TCP的黏附和成骨能力。优化手术效率并严格控制冲洗时间和压力对于保留移植物性能以实现成功骨融合至关重要。
