兔胫骨骨膜去细胞生物支架制备方案的优化

Optimization of the periosteal deceUularized bioscaffold

目的探讨制备兔胫骨骨膜去细胞生物支架的最佳方案。方法取健康新西兰大白兔70只，游离双侧胫骨近端内侧骨膜，共获取骨膜标本140个。用以下三种方案处理骨膜：方案1，物理冻融（-80℃，24h），置入含体积分数2％Triton-X100和3．5×10^-3mol／LPMSF的脱细胞混合液中振荡18h，10g／LSDS溶液中振荡12h，酶消化（DNA酶、RNA酶）；方案2，SDS仅振荡6h，其他同方案1；方案3，改10g／LSDS溶液中振荡12h为0．5mol／LNaCl溶液中振荡12h，其他同方案1。三种方案制备的支架及未处理的新鲜骨膜通过HE、DAPI和基因组DNA定量分析测定细胞结构及DNA含量是否小于评价去细胞的标准浓度50ng／mg；以番红“O”染色、Masson染色和羟脯氨酸测定法定性、定量检测骨膜细胞外基质主要成分（胶原、糖胺聚糖）的保留情况；以扫描电镜观察骨膜去细胞生物支架的表面微结构；以异体皮下包埋实验观察支架的炎细胞浸润等免疫排斥反应及组织重构现象。结果经方案1、2和3处理后，HE染色切片光镜下去细胞支架均无残留细胞，DAPI荧光染色未发现细胞核及核碎片存在，DNA定量检测组织去细胞率均达95％以上且含量均小于50ng／mg。方案3与方案1和2相比，骨膜去细胞支架中主要成分[胶原定量（36．94±0．70）μg／mg]保留更加完整，电镜下观察支架的胶原连续无断裂，胶原纤维排列规整，完整性好；异体皮下包埋后炎细胞浸润较少，免疫排斥反应不明显，并出现支架降解及重构现象。结论应用物理冻融（-80℃，24h）、含体积分数2％Triton-X100和3．5×10^-5mol／LPMSF的脱细胞混合液中振荡18h、0．5mol／LNaCl溶液中振荡12h及酶消化（DNA酶、RNA酶）处理后的骨膜生物支架去细胞更彻底，细胞外基质结构和成分保留更完整，且植入体内后免疫排斥反应小，生物相容性好。

Objective To explore an optimal protocol for the fabrication of a decellularized tibia periosteum biological scaffold. Methods Seventy healthy New Zealand white rabbits were selected, and both proximal tibia periosteums, namely 140 specimens, were collected. Specimens were divided into three groups and they were processed using the following three methods respectively. Method 1, specimens were freeze-thawed （-80 ℃, 24 h）, 2% Triton-X 100 mixed with 3.5×10^-5 mol/L PMSF aeellular solution for 18 h, 10 g/L SDS for 12 h and finally digested with enzyme （DNase and RNase）. Method 2, it＇s similar with method 1, but the processing time of SDS was 6 h. Method 3, it＇s similar with method 1, but the process of 10 g/L SDS for 12 h is replaced by the process of 0.5 mol/L NaCl for 12 h. Scaffolds fabricated using the three methods above, as well as the normal tibia periosteum, were characterized by HE staining, DAPI staining and quantitative analysis of genomic DNA to observe cellular structure and mea- sure DNA content, which would be compared with standard concentration 50 ng/mg in decellularization; characterized using Safra- nin O staining, Masson staining and the hydroxyproline measurement to measure the retain of the main components （collagen and glycosaminoglycan） in the ECM of periosteum qualitatively and quantitatively; characterized using scanning electron microscope to observe the micro structure on the surface of the scaffolds; characterized histologically using subcutaneous embedding test to ob- serve immunological rejection like the infiltration of inflammatory cells, and histological remodeling caused by scaffolds. Re-suits After the treatment by method 1, 2 and 3, HE staining showed the complete removal of ceils in the scaffolds. DAPI staining showed the absence of nucleus or nucleic debris. Quantitative analysis of DNA indicated that all the acellular ratios reached 95% and the contents were less than 50 ng/mg. Compared with method 1 and 2, the reserve of the main components （collagen 36.94± 0.70 μg/mg and glycosaminoglycan） in acellular scaffolds processed with method 3 were more complete, and the collagen fibers were in good condition without breakage under the SEM. Furthermore, the infiltration of inflammatory cells after the allogenic em- bedding test was less, and the immunological rejection was not obvious with preferable degradation of scaffolds and histological re- modeling. Conclusion An acellular tibia periosteum scaffold fabricated using method 3 showed more complete reservation of the structure and components of ECM, with minor immunological rejection and favorable biocompatibility.