去抗原羊椎松质骨支架微观空间结构的分析

The microstructure of antigen-extracted heterologous bone

背景:实验证实不同去除免疫原性方法对去抗原异种骨材料微观空间结构有着不同的影响。目的:比较不同方法处理条件下去抗原异种松质骨的微观空间结构。方法:取成年绵羊新鲜椎骨松质部分制备成特定形状的圆柱体,其长轴与所取松质骨骨小梁排列方向一致,行振动冲洗、不同频率超声冲洗后随机分为3组:物理煅烧组采用甲醇/氯仿、过氧化氢等化学制剂序列脱脂脱细胞脱蛋白后,与焦磷酸钠溶液水浴并直接在煅烧炉内1 000℃煅烧3 h;化学处理组采用甲醇/氯仿、过氧化氢等化学制剂序列脱脂脱细胞脱蛋白;对照组直接置于室温自然晾干。通过孔隙率测定、扫描电镜观察、X射线衍射分析、X射线原子能谱元素分析比较各组样品的微观空间结构。结果与结论:与对照组相比,物理煅烧组、化学处理组均不同程度保持了原骨组织的天然网状孔隙结构,大孔孔径为50-600μm,微孔孔径为2μm左右,孔隙率55%-70%;物理煅烧组的主要成分为羟基磷灰石和一定量的β-磷酸三钙,化学处理组与对照组的主要成分为羟基磷灰石;经化学或物理煅烧处理的松质骨立体空间结构并无很大破坏,保留了孔径不等、交通性良好、表面开放的孔隙和高度的孔隙间连接;并且经物理煅烧步骤可更彻底地去除异种松质骨的抗原成分,制成的去抗原异种骨支架材料基本符合组织工程骨支架材料的要求。

BACKGROUND：Different methods to remove immunogenicity have different effects on the spatial microstructure of antigen-extracted heterologous bone. OBJECTIVE：To compare the microstructure of the antigen-extracted heterologous bone via different methods to provide experimental data for tissue engineering industrialization. METHODS：Fresh cancellous bones extracted from adult sheep vertebrae were prepared into cylinders. Their long axis direction was the same with orientation of the trabeculae. After vibration washing and different-frequency ultrasound rinsing, the cylinder samples were randomly divided into three groups：in physical calcined group, the samples were defatted, decellularized and deproteinized sequential y using methanol/chloroform and hydrogen peroxide, then bathed in sodium pyrophosphate and directly calcined at 1 000 ℃ for 3 hours;in chemical group, the samples were defatted, decellularized and deproteinized sequential y using methanol/chloroform and hydrogen peroxide;in control group, the samples were dried natural y at room temperature. Microstructure of the samples in each group was analyzed and compared through determination of porosity, scanning electron microscopy observation, X-ray diffraction analysis, X-ray atomic spectroscopy elemental analysis microscopic spatial structure. RESULTS AND CONCLUSION：The physical calcined and chemical groups maintained natural network pore system to different extents. The size of the large pore was 50-600μm and that of the smal one was about 2μm. The porosity was 55%to 70%. Hydroxyapatite was the main component of the physical calcined group which was determined by X-ray diffraction, and a smal amount of theβ-Tricalcium phosphate was also determined. In the chemical group, the main component was only hydroxyapatite. The three-dimensional spatial structures of the deproteinized cancellous bones were not damaged greatly, and they had a natural pore network system. Antigen component of xenogeneic cancellous bone can be more thoroughly removed by physical calcination step. The scaffold material made by antigen-extracted heterologous bone may satisfy the demands for bone tissue-engineering scaffolds.