摘要
目的利用牛膝关节透明软骨进行脱细胞处理,制备新型可塑形生物材料,探讨脱细胞软骨基质作为组织工程载体材料的可行性。方法采集新鲜牛膝关节,切取关节表面透明软骨,冻干后低温粉碎为软骨微粒,胰酶、TritonX-100及低张Tris-HCl溶液联合作用进行脱细胞处理,冷冻干燥塑形,紫外线交联后制备脱细胞软骨基质材料。采用组织学、免疫组织化学、扫描电镜、孔隙率测定及生物力学检测等对材料的理化性状进行观察分析。取4只成年新西兰白兔骨髓制备BMSCs,传至第3代进行实验。观察浓度分别为100%、10%及1%的材料浸提液培养BMSCs0、24、48及72h后相对乳酸脱氢酶(lactate dehydrogenase,LDH)释放率,以含5%FBS的DMEM培养基作为阴性对照,观察细胞毒性作用;并将浓度为1×107个/mL的BMSCs单细胞悬液与材料复合培养,观察细胞黏附情况。结果制备的脱细胞软骨基质材料呈白色多孔状结构。HE染色示材料由纤维状的软骨微粒形成网状结构,基质内无细胞成分残留;阿尔新蓝染色示微颗粒呈蓝色。材料Ⅱ型胶原免疫组织化学染色呈阳性。扫描电镜材料为多孔状海绵结构,孔径30～150μm。压汞法测定材料平均孔隙率为89.37%,平均孔径为90.8μm。力学分析示脱细胞软骨基质材料的压缩模量为(17.91±0.98)MPa,未经脱细胞处理的软骨微粒材料压缩模量为(15.12±0.77)MPa,差异无统计学意义(P>0.05);与正常牛关节软骨的(26.30±1.98)MPa比较差异均有统计学意义(P<0.05)。细胞毒性实验显示,培养0、24、48及72h,100%、10%、1%浓度材料浸提液条件培养基和阴性对照DMEM培养基相对LDH释放率差异无统计学意义(P>0.05)。细胞黏附实验显示细胞可黏附于材料上,生长状态良好。结论牛关节软骨经脱细胞处理后,制成的多孔状脱细胞软骨基质材料,既保持了软骨基质中的主要成分,又具有良好的理化性质和生物相容性,可作为组织工程研究的一种新型载体材料。
Objective To produce a decellularized cartilage matrix （DCM） and investigate its possibility to be used as a scaffold for tissue engineering. Methods Fresh bovine articular cartilage from knee joints was sliced, freeze-dried and freeze-ground into fine powder, and then was treated sequentially with Trypsin, Triton X-100 and hypotonic solution for decellularization. The decellularized matrix was freeze-dried for shaping and cross-linked by UV radiation. Histological, immunohistological, SEM, porosity assays and biomechanical assays were used to characterize the DCM. BMSCs were isolated from rabbit bone marrow aspirate and cultured in DCM extraction medium of different concentration （100%, 10% and 1%） for 0, 24, 48 and 72 hours, respectively, to detect the release rate of the lactate dehydrogenase （LDH）. The DMEM medium （5% FBS） served as the control. Biocompatibility was evaluated using BMSCs （1 × 10^7/mL） cultured with DCM. Results The DCM showed white spongy appearances, and histological analysis showed that the material was constructed by cartilage particles without any cells or cell fragments left in the matrix. Immunohistology staining and alcian blue staining showed that DCM retained the collagen and glycosaminoglycan components of cartilaginous matrix. SEM scanning showed that DCM had a porous spongy-1 ike structure with the aperture ranging 30-150 tam .The porosity assay showed that the average porosity was 89.37% and the average aperture was 90.8μm. The mechanical assay showed that there was no difference for the compress module before and after the decellularization process, which was （17.91 ± 0.98） MPa and （15.12 ± 0.77） MPa, respectively （P 〉 0.05）, but were both statistical different from normal articular cartilage [（26.30 ± 1.98） MPa, P 〈 0.05]. The LDH release rate in the DCM extraction medium of different concentration was not significantly different from that in the normal DMEM medium （P 〉 0.05）. I ne ceil adhesion test showed BMSCs grew well on DCM without any signs of growth inhibition. Conclusion Articular cartilage can be decellularized and fabricated into a scaffold which retains the major components of cartilaginous matrix. DCM has good biochemical, biophysical characteristics and good biocompatibility with cultured BMSCs and may be used as a novel scaffold for tissue engineering studies.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2008年第10期1232-1237,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30371440)
山西省国际合作重点资助项目(2007081037)~~
关键词
组织工程
载体材料
脱细胞软骨基质
ECM
生物相容性
Tissue engineering Carrier material Decellularized cartilage matrix ECM Biocompatibility
