犬股骨髁脱蛋白骨关节的理化特性和生物安全性

Physicochemical characteristics and biological safety of allogeneic deproteinized osteo-articulation

摘要:

目的：探讨用过氧化氢-乙醚法制备同种异体脱蛋白骨关节（deproteinized osteo-articulation，DPOA），讨论其理化特性和生物安全性。方法：采用过氧化氢-乙醚法将成年杂交犬股骨下端半关节松质骨脱脂、脱蛋白制作成生物衍生骨关节材料DPOA。体视学显微镜、扫描电子显微镜观察DPOA材料外表面、孔隙内表面结构、孔径大小和孔间交通情况。HE染色观察 DPOA材料的细胞成分清除情况；pH计检测仪检测 DPOA材料的培养液 pH值；微量凯氏定氮法检测DPOA材料中蛋白质含量；生物力学测试DPOA应力负荷；细胞毒性实验及溶血实验测试体外生物安全性。结果：体视学显微镜、扫描电子显微镜观察结果显示，DPOA材料具有原骨组织的三维多孔-网架结构系统。计算机图像分析系统结果显示，孔隙率为（84.76±4.63）%，孔径最大为（318.64±16.35） μm，最小为（112.13±15.42） μm。HE染色显示DPOA材料细胞成分得到了彻底清除。浸泡DPOA材料组培养液pH为7.382±0.009。DPOA蛋白质含量为（0.017±0.003）%。生物力学测试做最大应力负荷为（226.00±7.65） N，接近正常骨关节力学载荷。细胞毒性实验测试细胞毒性0级，溶血试验< 5%。结论：用过氧化氢-乙醚法制作的同种异体天然衍生DPOA，保持了原骨组织密集的孔隙网状结构，并具有原有的解剖结构外形，其理化特性和生物安全性符合要求，可作为一种组织工程支架或犬股骨半关节移植材料。

Abstract:

Objective：To explore a method to prepare deproteinized osteo-articulation（DPOA） with hydrogen peroxide and ether as well as to discuss its physicochemical characteristics and biological safety. Methods：Distal femur semi-joint cancellous bones of adult hybrid canine were made into bio-derived osteo-articulation materials with hydrogen peroxide and ether by degreasing and deproteinizing；outsaide surface，pore texture of interal surface，pore size and traffic situation among pores of DPOA were observed by stereology microscope and scanning electron microscope；eliminated cell composition of DPOA was observed by HE staining；culture solution pH values of DPOA were detected by instrument；protein contents of DPOA were detect by micro Kjeldahl method；stress weight was detect by biomechanics test；biological safety in vitro was tested by cytotoxicity test and hemolysis test. Results：Results of stereology microscope and scanning electron microscope reveal that DPOA had three-dimensional and porus-grid structure system of original bone tissue. Results of computer picture analytical system reveal that porosity was （84.76±4.63）%，maximum aperture was （318.64±16.35） μm and minimum aperture was （112.13±15.42） μm. HE staining revealed that cell composition of DPOA was eliminated entirely. pH value of DPOA culture solution was 7.382±0.009. Protein content of DPOA was （0.017±0.003）%. Maximum stress weight of biomechanics test was （226.00±7.65） N，approaching mechanics loading of normal osteo-articulation.Cytotoxicity was 0 grade. Result of hemolysis test was < 5%. Conclusions：Allogeneic natural derived DPOA made by hydrogen peroxide and ether method retains pore net structure of original bone tissue and possesses the intrinsic anatomical configuration. Its physicochemical characteristics and biological safety are qualified and can be used as an osteo-articulation grafting trestle material.