骨膜去细胞生物支架的制备和鉴定

Preparation and identification of the periosteal decellularized bioscaffold

目的制备兔骨膜去细胞生物支架,为骨缺损、骨不愈的组织工程研究提供天然的生物支架材料。方法取健康新西兰大白兔,游离双侧胫骨近端内侧骨膜,通过物理冻融(-80℃,24h)、去污剂洗脱(triton-X 100、SDS)和酶消化(DNA酶、RNA酶)获取骨膜去细胞生物支架。通过HE染色、DAPI染色、琼脂糖电泳和基因组DNA定量分析(n=5)测定细胞结构及DNA成分残留;Masson染色和羟脯氨酸测定法(n=6)定性定量检测骨膜细胞外基质的主要成分(胶原)的保留情况;扫描电子显微镜下观察骨膜去细胞生物支架的表面微结构;CCK8法检测支架浸提液毒性;皮下包埋实验(n=4)观察该支架的免疫排斥反应。结果 HE染色和4’,6-二脒基-2-苯基吲哚(DAPI)染色表明去细胞支架无残留细胞;琼脂糖电泳未见明显DNA条带;DNA定量检测显示组织去细胞率达95%以上;Masson染色及羟脯氨酸测定表明去细胞支架胶原成分被保留;扫描电子显微镜下细胞外基质呈现三维网状疏松结构;不同体积分数的浸提液对骨膜细胞的增殖与对照组(普通培养基)比较无明显抑制作用(P>0.05);异体皮下包埋实验显示,该去细胞支架免疫排斥反应不明显。结论运用物理冻融、去污剂洗脱和酶消化等方法所获取的骨膜去细胞生物支架细胞去除彻底,细胞外基质的结构及主要成分保留完好,生物相容性良好。

Objective To prepare rabbit periosteal decellularized bioscaffold andprovide a natural bioscaffold for the treatment for bone defect or bone nonunion in bone tissue engineering. Methods Bilateral medial proximal tibia periosteum of healthy New Zealand rabbits was obtained. To prepare the scaffold,we used physical freeze thawing（- 80℃,24h）,eluted detergent（ triton-X 100,SDS） and enzyme digestion（ DNA enzymes,RNA enzymes）. After decellularization process, normal periosteum and decellularized periosteum were examined by HE staining,4 ＇,6-diamidino-phenylindole（ DAPI） staining,agarose gel electrophoresis and quantitative analysis of genomic DNA（ n = 5） to evaluate the residual cell components. The retention of main components of the extracellular matrix（ collagen） was examined by Masson staining and the hydroxyproline measurement（ n = 6）; Scanning electron microscopy（ SEM） was used to observe the microstructure of the scaffold; The toxicity of leaching liquor from scaffolds was tested by CCK8 assay; The immunological rejection of the scaffold was evaluated by subcutaneous implantation（ n = 4）. Results HE and DAPI staining showed that no cells remained in the scaffold. After separation,the visible DNA bands were not found in the agarose electrophoresis gel for the decellularized periosteum. The DNA quantitative analysis showed that more than 95% of periosteal cells were removed; Masson staining and hydroxyproline measurement revealed that the collagen of the extracellular matrix was preserved; SEM showed the loose three-dimensional network of the extracellular matrix. The CCK8 assays demonstrated that there was no significant difference of periosteal cells proliferation among different volume fractions of leaching liquor from scaffolds and the control group（ normal medium）（ P〉0. 05）. The subcutaneous implantation showed no obvious immune response to the decellularized periosteum. Conclusion The decellularized periosteum obtained by the use of physical freeze thawing,eluted detergent and enzyme digestion methods was found to remove periosteal cells completely,while the extracellular matrix structure and the main components were well-preserved and the biocompatibility was excellent.