冻干脱钙骨表面纳米结构对细胞行为的影响

Effection of nano-structure on the surface of freeze-dried demineralized bone matrix to osteoblast-behavior

目的探讨表面有纳米结构的冻干脱钙骨基质(nFDBM)的制备方法及该结构对成骨细胞生物学行为的影响。方法取新鲜狗趾皮质骨按改良Urist法制备冻干脱钙骨基质(FDBM),经NdYAG激光表面处理后,用原子力显微镜、扫描电镜观察其表面形态结构学变化。nFDBM、FDBM分别与狗自体骨髓基质细胞诱导分化来的成骨细胞体外构建材料——细胞复合物并植入该狗脊柱两侧深筋膜袋内,于植入前及术后第4、8周取材行扫描电镜及组织学观察。结果FDBM经NdYAG激光处理后表面形成了规则的纳米级沟槽状三维结构。体外构建材料——细胞复合物后扫描电镜观察nFDBM表面细胞黏附密度及分泌基质量均高于FDBM。术后HE染色、扫描电镜观察见左侧植入物基本纤维化,表面未见明显成骨细胞及基质;右侧植入物原纳米沟槽一侧形成薄层骨样组织,表面为成骨细胞分泌的大量基质所覆盖。结论NdYAG激光可在FDBM表面形成纳米级沟槽状三维结构;该结构有利于成骨细胞的黏附、生长和基质分泌。

Objective: To investigate the method of how the freeze-dried demineralized bone matrix with nanoscale topography (nFDBM) was fabricated and effects of the nano-structure on osteoblast-behavior.Method:Allogenic dogs' phalangeal cortical bone was fabricated into freeze-dried demineralized bone (FDBM) with modified Urist's method.FDBM was subjected to Nd:YAG laser irradiation under special conditions.The surface topography was identified by atomic force microscope and scanning electron microscope (SEM).The osteoblasts were induced from autologous bone marrow stromal cells (BMSCs) and mixed with nFDBM and FDBM in vitro.Cell-material constructs were implanted into fascial bags on dogs' back.The results were assessed by histomorphologic and SEM preoperatively and in 4th,8th weeks postoperatively surgery.Result:The surface of FDBM resulted in well-defined three-dimensional nanoscale grooves.The osteoblasts from BMSCs adhered on nFDBM were more than those on FDBM and secreted more extracellular matrix after implanted on the scaffold.The constructs on the left of dogs' spine became fibrosis in 4th and 8th weeks postoperatively,while new bone-like thin layers were observed on the nanoscale surface of the right constructs by histological examination.Abundant osteoblasts and matrix were observed by SEM on the surface of the constructs on the right and no obvious cells and matrix on the irregular surface of the left constructs in 8th weeks postoperatively.Conclusion:Nd:YAG laser could produce the well-defined three-dimensional nanoscale grooves on the FDBM surface.The nanoscale grooves have advantage to the adherence,proliferation and matrix secretion of osteoblasts.