运用组织工程原理结合纳米技术构建骨组织的实验研究

RECONSTRUCTION OF BONE USING TISSUE ENGINEERING AND NANOSCALE TECHNOLOGY

目的 探索表面有纳米级沟槽的冻干脱钙骨基质（freeze-dried demineralized bone matrix with nanoscale topography，nFDBM）的制备方法，并探讨其构建组织工程骨的可行性。方法取犬趾皮质骨按改良Urist法制备FDBM，经掺钕钇铝石榴石（Nd：YAG）激光处理后，与犬自体骨髓间充质干细胞诱导分化来的成骨细胞体外构建nFDBM-细胞复合物，原子力显微镜及扫描电镜观察材料表面不同形态特征对细胞行为的影响;同时将片状nFDBM复合物植入右侧犬背深筋膜袋内（实验组A），短管状nFDBM植入右侧趾骨节段性缺损处（实验组C），以片状FDBM-成骨细胞复合物（对照组B）及短管状FDBM（对照组D）植入同一犬体内左侧相对应位置作为对照，术后第4、8、12周行X线片、组织学及扫描电镜观察。结果FDBM经Nd：YAG激光处理后表面形成了规则的纳米级沟槽状三维结构（深150nm，宽600-800nm）。成骨细胞在nFDBM上的黏附密度及分泌基质量均高于FDBM。实验组复合物植入体内12周时，HE染色、扫描电镜发现材料纳米沟槽一侧、材料内有新生骨组织，X线片检查呈部分钙化，而对照组几无成骨表现，其中X线片表现评分差异有统计学意义（P〈0．05）。结论Nd：YAG激光可在FDBM表面形成纳米沟槽结构，该结构有利于成骨细胞的黏附、生长和基质分泌。以组织工程方法结合纳米技术构建的nFDBM，成骨细胞复合物植入动物模型体内表现出一定的成骨能力。

Objective To explore the method of fabricating freeze-dried demineralized bone matrix with nanoseale topography （nFDBM） and to investigate the feasibility of reconstruction of tlssue-englneered bone with the novel scaffold. Methods Allogenie dogs＇ phalangeal cortical bone was fabricated into freeze-drled 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 mieroseope（AFM） and scanning electron microscope （SEM）. The osteoblasts were induced from autologous mesenehymal stem cells （MSCs） and mixed with nFDBM and FDBM in vitro. The effects of the different topography on cell-behavlor was identified by SEM. The complex of nFDBM and osteoblasts were implanted into faseial bags on dogs＇ back （experimental group A） and dogs＇ phalangeal defects on right （experimental group C）, while FDBM-osteoblast complex （control group B） and unique FDBM （control group D） were implanted into the corresponding sites on left as control groups. The osteogenie status was assessed by X-ray, HE and SEM at 4, 8 and 12 weeks after surgery. Results The surface of FDBM subjected to Nd ： YAG laser irradiation resulted in well-defined three-dlmensional nanoseale grooves （150 nm in depth and 600 to 800 nm in width）. When the osteoblasts were implanted on the scaffold, the cells adhering to nFDBM were more than those to FDBM and secreted more extraeellular matrix. Either new bone-llke thin layer on the nanoseale surface or a lot of new bone-formation inner the experimental complex was observed by HE after 12 weeks of surgery and the experimental complexes were partially calcified at the same time, while the control groups almost had no osteogenle phenomena. Conclusion Nd ： YAG laser could produce nanoseale grooves on the FDBM surface. The nanosoale grooves are conductive to adherence, proliferation and matrix secretion of osteoblasts. Complexes by tissue engineering and nanoseale technology have some osteogenie abilities in vivo after implanted the animal model.