新型脱细胞骨基质材料的组织学结构及其细胞相容性观察  

HISTOLOGICAL STRUCTURE AND CYTOCOMPATIBILITY OF NOVEL ACELLULAR BONE MATRIX SCAFFOLD

作　　者：赵艳红[1,2] 杨强[3] 彭江[1] 郭全义[1] 夏群[3] 马信龙[3] 徐宝山[3] 赵斌[1] 张莉[1] 伍耀宏[3] 刘越[1] 许文静[1] 卢世璧[1] 

机构地区：[1]解放军总医院骨科研究所北京,100853 [2]天津医科大学口腔医院正畸科 [3]天津医院脊柱外科

出　　处：《中国修复重建外科杂志》2013年第7期781-785,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31000432、30330570);中国博士后科学基金资助项目(2011M500530、2012T50235);天津市卫生局科技基金资助项目(2012KY24)~~

摘　　要：目的对新型脱细胞骨基质材料(acellular bone matrix,ACBM)的组织学结构及细胞相容性进行观察,探讨其作为组织工程骨支架的可行性。方法取健康18～24月龄雄性杂种犬股骨头负重区骨柱,高压水枪冲洗、脱脂,Trixon X-100、脱氧胆酸钠等进行脱细胞等理化处理,制备新型ACBM支架;对其行扫描电镜观察,HE染色、天狼星红染色及Hoechst 33258染色,评估其脱细胞程度及结构。采用密度梯度离心法分离培养犬BMSCs,取第3代BMSCs种植至ACBM支架上,MTT法分析支架浸提液毒性;复合培养3 d行倒置显微镜、扫描电镜、活/死细胞荧光染色、组织学等观察细胞在支架的生长、分化情况。结果 HE染色示ACBM支架去细胞彻底,无细胞碎片残留;天狼星红染色示支架呈深红及黄红相间染色;Hoechst 33258染色未见细胞残留。扫描电镜观察示ACBM支架内孔洞相互贯通,具有天然骨的孔径和孔隙率。MTT法检测示,培养1～6 d不同浓度(25%、50%、100%)支架浸提液与对照H-DMEM培养液吸光度(A)值比较差异均无统计学意义(P>0.05)。倒置显微镜、扫描电镜、组织学观察结果均显示细胞在支架上黏附良好,增殖显著,细胞基质分泌增加;活/死细胞染色示ACBM支架内细胞均呈绿色。结论 ACBM支架去细胞彻底,具备良好的孔径和孔隙率,无毒,细胞相容性良好,可作为良好的支架载体用于组织工程骨的构建。Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix （ACBM） and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope （SEM）; HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells （BMSCs） from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance （A） values between different extracts （25%, 50%, and 100%） and H-DMEM culture media （P 〉 0.05）, indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone t

关 键 词：组织工程骨 脱细胞骨基质材料 细胞相容性 犬 

分 类 号：R318.08[医药卫生—生物医学工程]