猪异种骨移植靶抗原分布及除抗原处理的研究
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摘要
目的：(1)探讨猪骨组织靶抗原分布特点。(2)观察脱脂部分脱蛋白、超深低温冷冻、超深低温冷冻＋酶消化对猪骨组织抗原表达的影响，并研究其性状和生物力学特性改变。(3)研究不同方法处理的猪骨材料与兔成骨细胞组织相容性的变化。(4)不同方法处理的猪骨材料植入兔体内，观察宿主免疫排斥反应和移植骨的转归。
方法：(1)采用免疫组化的方法观察α-Gal抗原、MHC-Ⅰ抗原和MHC-Ⅱ抗原在猪骨中的分布情况。(2)观察不同方法处理的猪骨材料的异种抗原及性状改变：实验分为A、B、C、D组。A组：脱脂部分脱蛋白组；B组：超深低温冷冻组；C组：超深低温冷冻＋酶消化组；D组：新鲜猪骨移植组。免疫组化方法观察各组骨材料异种抗原的变化情况，大体观察不同组骨材料的形貌特征结构，扫描电镜观察各组材料的细微结构变化，比较各组骨材料的孔径大小和孔隙率；生物力学实验测定各组骨材料的力学性能变化。(3)不同方法处理的异种骨材料的细胞相容性：将A、B、C组骨材料体外分别与兔成骨细胞联合培养，每块材料上加入浓度为1×10~7╱ml的细胞悬液20μl，连续培养7天。分别于1、3、5、7天采用MTT法，检测兔成骨细胞在各组骨材料上的增殖情况，倒置显微镜和扫描电镜观察细胞在骨材料上的粘附生长，流式细胞仪分析细胞周期的变化。(4)不同方法处理的异种骨材料体内移植免疫研究：实验分为A、B、C、D、E、F组，A组：脱脂部分脱蛋白组；B组：超深低温冷冻组；C组：超深低温冷冻＋酶消化组；D组：新鲜异种骨移植组；E组：自体骨移植组；F组：对照组(假手术组)。将各组骨材料分别植入新西兰大白兔双侧骶棘肌内，分别于术后1、2、4周取兔耳缘静脉血，流式细胞仪检测各组外周血中CD4~+、CD8~+T淋巴细胞亚群和CD4~+／CD8~+比值的变化；ELISA定量检测各组术后1、2、4、6周血清中IL-2和IL-4水平的变化；组织病理学观察移植骨材料周围炎性细胞浸润情况。(5)不同方法处理的异种骨材料修复兔桡骨缺损的研究：实验分组同(4)，将各组骨材料分别植入兔桡骨缺损处，分别于术后6、12、24周取材，X线片检查，根据Lane-Sandhux的X线评分标准，定量观察各组骨缺损的修复情况；组织切片行HE和Masson染色，显微镜下观察各组骨愈合和新骨形成情况。
结果：
(1)免疫组化研究发现正常猪骨中，α-Gal、MHC-Ⅰ抗原广泛表达在骨髓细胞和骨细胞、成骨细胞胞膜及哈佛氏管周围，MHC-Ⅱ抗原主要表达于骨髓细胞。
(2)不同方法处理的猪骨材料的异种抗原及性状改变：①各组抗原表达的比较，A组无骨细胞存留，无抗原阳性表达；B组骨陷窝和Harversian管周围仍有骨细胞存留，骨细胞表面可见少量α-Gal抗原、MHC-Ⅰ和MHC-Ⅱ抗原的阳性表达；C组可见骨陷窝内残存蓝染细胞核，无异种抗原的阳性表达；D组骨髓及骨细胞表面大量α-Gal抗原、MHC-Ⅰ和MHC-Ⅱ抗原的阳性表达。②扫描电镜观察，各组骨材料孔径大小比较A组＞C组＞B组＞D组，B、D组间比较无统计学差异(P＞0．05)，其余各组间比较均有差异(P＜0．05)；材料孔隙率比较A组＞C组＞B组＞D组，其中A组与C组、B组与D组之间比较无明显差别(P＞0．05)，其余各组间差别明显(P＜0．05)。③轴向压缩实验，最大载荷比较D组＞C组＞B组＞A组，其中A组明显小于其他各组(P＜0．05)，而B、C、D组间无明显差别(P＞0．05)。
(3)不同方法处理的异种骨材料的细胞相容性：兔成骨细胞在各组猪骨材料表面上均有生长，并且在第5天后成骨细胞增殖达到高峰，扫描电镜观察和MTT检测结果显示，各组生物相容性能C组＞A组＞B组。流式细胞术检测A、C组骨材料表面成骨细胞的细胞周期之间无明显差异，这两组细胞的S期＋G_2／M期明显高于B组(P＜0．05)。
(4)不同方法处理的异种骨材料体内移植免疫研究：各组术后1-2周CD4~+、CD8~+T淋巴细胞均逐渐升高，CD4~+／CD8~+比值在各组术后1周最高，以后逐渐下降，2周时B、D组较高，A、C、E组比较无明显差别。ELISA法检测外周血清IL-2、IL-4水平，术后D组外周血IL-2、IL-4水平最高，术后1周时达到最高峰，以后逐渐下降，F组水平最低。A、B、C组间比较结果显示，术后不同时间点B组血清中IL-2、IL-4水平均明显高于A组和C组(P＜0．05)。术后6周，各组组织病理学研究发现，D组移植骨材料周围大量淋巴细胞和巨噬细胞聚集；其次为B组，植骨材料周围中等量的淋巴细胞浸润；A组、C组、E组骨材料周围少量淋巴细胞浸润。
(5)不同方法处理的异种骨材料修复兔桡骨缺损的研究：不同方法处理的骨材料修复兔桡骨缺损，经放射学和组织学评估，术后6周、12周和24周各组成骨能力依次为：E组＞C组＞A组＞B组＞D组＞F组。
结论：
(1)α-Gal、MHC-Ⅰ抗原广泛表达在猪骨骨髓细胞和骨细胞、成骨细胞膜及哈佛氏管周围，MHC-Ⅱ抗原主要表达于骨髓细胞。
(2)脱脂部分脱蛋白和超深低温冷冻＋酶处理方法可以有效去除异种主要抗原，单纯超深低温冷冻可以明显降低猪骨的异种抗原表达，但是不能完全有效去除异种主要抗原。
(3)各组骨材料孔径大小比较，三种处理方法获得的异种骨材料的孔径大小均可满足成骨细胞生长所需要的孔径要求。但是超深低温冷冻组骨材料孔隙率最小，可能会影响成骨细胞的长入。脱脂部分脱蛋白组生物力学性能最差，超深低温冷冻和超深低温冷冻＋酶消化法对猪骨生物力学性能无明显影响，接近正常新鲜猪骨移植组。
(4)各组骨材料体外与兔成骨细胞复合培养，超深低温冷冻＋酶消化联合处理后的骨材料的生物相容性优于脱蛋白处理和单纯超深低温冷冻处理。
(5)各组骨材料植入兔体内，异种骨移植免疫排斥反应主要发生在术后1-2周，超深低温冷冻＋酶消化组和脱脂部分脱蛋白组骨材料免疫排斥反应较轻，单纯超深低温冷冻组免疫排斥较重。
(6)超深低温冷冻＋酶消化组和脱脂部分脱蛋白组骨缺损修复能力无明显差别，两组骨缺损修复能力均明显优于超深低温冷冻组。
(7)超深低温冷冻＋酶处理可以有效去除异种骨抗原，同脱脂部分脱蛋白组相比，具有良好的生物力学性能；具备合适的孔径大小和孔隙率，适合成骨细胞生长，具有良好的细胞生物相容性能；体内移植免疫排斥反应较轻微，骨缺损修复能力强，有望将来作为临床异种骨移植处理的有效方法之一。
Objective: (1) To investigate the distribution of xenogenous targeted antigen on porcine bone tissue. (2) To study the change of xenogenous antigen、biomechanical and physical properties, the porcine bone tissues were processed with three different methods. (3) To investigate the cellular compatibility of xenogenous bone scaffold materials. (4) To observe the immunological rejection and the turnover of the bone graft, the porcine bone tissues with different methods was implanted into the rabbits.
Methods: (1) Distribution ofα-Gal、MHC-Ⅰand MHC-Ⅱantigen on porcine bone tissue were observed by immunohistochemistry. (2) Physical properties and heteroantigens of porcine bone were detected: The experiment was divided into group A、B、C、D according to different materials. Group A: porcine bone was processed with defatting and partial deproteinization; Group B: porcine bone was preserved in liqiud nitrogen;Group C: porcine bone was preserved in liqiud nitrogen and was digested by enzyme; Group D: fresh porcine bone. The change of expression of heteroantigen was detected by immunohistochemistry, and the microstructure and biomechanical properties of different groups were observed by Electronic Scanning and INSTRON 8874. (3) Cellular compatibility of xenogenous bone scaffold materials: Osteoblasts of rabbit were cultured with the bone scaffold materials of group A, group B, group C,respectively. Osteoblasts in 1×10~7/ml density were cultured with the three materials for 7 days. And the adhesion and growth of osteoblasts were observed with phase microscope and electronic scanning microscope, the cell cycle was analyzed with flow cytometer. The growth of osteoblasts were observed on 1、3、5、7 day were detected by MTT assay. (4) The immune reaction of bone xenotransplantaion: The experiment was divided into group A、B、C、D、E、F according to different materials. Group A:porcine bone was processed with defatting and partial deproteinization;Group B: porcine bone was preserved in liqiud nitrogen; Group C: porcine bone was preserved in liqiud nitrogen and was digested with enzyme;Group D: fresh porcine bone; Group E: bone atuograft; Group F: control group. The bone scaffold materials were implanted into the bilaterial spinal muscles of rabbits. At 1、2、4 weeks postoperatively, the examination of CD4~+、CD8~+、CD4~+/CD8~+ in blood was detected by flow cytometer, and the levels of IL-2 and IL-4 in blood were analyzed by quantitative ELISA assay.The infiltration of lymphocytes and macrophages surrounding soft tissue of bone graft were also observed in order to evaluation biocompatibility of bone graft. (5) The effect of repairing bone defect with different processed porcine bone scaffold materials: Sixty New Zealand white rabbits were chosen, and the radial critical bone defects were made for 15mm long. The experiment was randomly divided into group A、B、C、D、E、F according to different materials, and each group details was equal to part (4). The bone defects were grafted by xenogenous bone materials derived from different groups. Groups of rabbits were sacrificed and the specimens were procured at 6、12、24 weeks after surgery for examination X-rays photography and histology evaluation. The ability of repaii'ing bone defect of each group was evaluated by Lane-Sandhux radiographic scoring. To study the bone defect repairing and reconstruction, new bone formation and substitute, the 5μm paraffin slices were obtained and were stained by hematoxylin-eosin and Masson method.
Results:
(1)α-Gal、MHC-Ⅰxenogenous antigens were extensively observed on the surface of bone marrow cells、osteocytes, osteoblasts and Harversian canals, and the MHC-Ⅱantigen mainly expressed on the bone marrow cells.
(2) Change of physical property and xenogenous antigens: No positive antigen expression in group A and group C, a few positive xenogenous antigens expression were detected in group B, while many positive xenogenous antigens expression were detected in group D. The results of pore size showed that group A＞group C＞group B＞group D, there was no significant difference between group B and group D (P＞0.05), and there was significant difference among the other groups (P＜0.05); The results of porosity showed that group A＞group C＞group B＞group D, there was no significant difference between group A and group C, group B and group D (P＞0.05). Biomechanical analysis demonstrated that the maximum load in axial compression test, group D＞group C＞group B＞group A, the minimum load was observed in group A and it was significantly inferior than the other groups (P＜0.05), there was no significantly difference among group B、group C and group D (P＞0.05).
(3) The results of cellular compatibility of xenogenous bone scaffold materials demonstrated that rabbit osteoblasts were survival in all groups, and the cell proliferation reached the peak at the fifth day. The cellular compatibility was detected by electronic scanning microscope and MTT assay, the results showed that group C＞group A＞group B. Compared to group B, the xenogenous bone materials in group A and group C had little influence on the G_0/G_1 stage and S stage of the rabbit osteoblasts.
(4) CD4~+, CD8~+ T lymphocytes gradually increased in 2 weeks after operation. The CD4~+/CD8~+ ratio reached the peak at 1 week after operation, then decreased gradually. The detection of CD4~+/CD8~+ ratio at 2 weeks postoperatively, there was no significant difference among group A、group C and group E, while the CD4~+/CD8~+ ratio reached higher level. Evaluation of IL-2 and IL-4 in serum by ELISA assay, the highest level" of IL-2 and IL-4 was detected in group D at 1 week after operation, the lowest level was in group F. Compared to group A and group C, the level of IL-2 and IL-4 were increased obviously in group B at different intervals after operation (P＜0.05). There was medium dose of lymphocytes infiltration surrounding the soft tissue of bone graft in group B, and there was few lymphocytes infiltration in group A and group C.
(5) According to the Lane-Sandhux radiographic scoring and the histological evaluation, the ability of reconstruction of radial defect was followed, group E＞group C＞group A＞group B＞group D＞group F.
Conclusions:
(1)α-Gal、MHC-Ⅰxenogenous antigens were extensively observed on the surface of bone marrow cells、osteocytes、osteoblasts and Harversian canals, and the MHC-Ⅱantigen mainly expressed on the surface of bone marrow cells.
(2) The expression of xenogenous antigen was decreased obviously in group B, but there was still some of positive expression of antigen on the porcine bone tissues. The xenogenous antigen was effectively deleted in group A and group C.
(3) The results of pore size showed that group A＞group C＞group B, the pore size of porcine bone materials derived these groups could meet the requirement of osteoblasts ingrowth, but porosity of group B was lowest, which maybe hinder the ingrowth of osteoblast. The xenogenous bone materials had the poor biomechanical performance in group A. Compared to the normal fresh porcine bone tissue, there was no significantly influence on the biomechanical performance in group B and group C.
(4) The cellular biocompatibility showed that group C＞group A＞group B.
(5) Immunological rejection of bone xenograft occurred during 2 weeks after operation, the host immunological response was lightly in group A and group C, and there was obviously immunological rejection in group B.
(6) The ability of reconstruction of radial bone defect demonstrated that group C＞group A＞group B.
方法：(1)采用免疫组化的方法观察α-Gal抗原、MHC-Ⅰ抗原和MHC-Ⅱ抗原在猪骨中的分布情况。(2)观察不同方法处理的猪骨材料的异种抗原及性状改变：实验分为A、B、C、D组。A组：脱脂部分脱蛋白组；B组：超深低温冷冻组；C组：超深低温冷冻＋酶消化组；D组：新鲜猪骨移植组。免疫组化方法观察各组骨材料异种抗原的变化情况，大体观察不同组骨材料的形貌特征结构，扫描电镜观察各组材料的细微结构变化，比较各组骨材料的孔径大小和孔隙率；生物力学实验测定各组骨材料的力学性能变化。(3)不同方法处理的异种骨材料的细胞相容性：将A、B、C组骨材料体外分别与兔成骨细胞联合培养，每块材料上加入浓度为1×10~7╱ml的细胞悬液20μl，连续培养7天。分别于1、3、5、7天采用MTT法，检测兔成骨细胞在各组骨材料上的增殖情况，倒置显微镜和扫描电镜观察细胞在骨材料上的粘附生长，流式细胞仪分析细胞周期的变化。(4)不同方法处理的异种骨材料体内移植免疫研究：实验分为A、B、C、D、E、F组，A组：脱脂部分脱蛋白组；B组：超深低温冷冻组；C组：超深低温冷冻＋酶消化组；D组：新鲜异种骨移植组；E组：自体骨移植组；F组：对照组(假手术组)。将各组骨材料分别植入新西兰大白兔双侧骶棘肌内，分别于术后1、2、4周取兔耳缘静脉血，流式细胞仪检测各组外周血中CD4~+、CD8~+T淋巴细胞亚群和CD4~+／CD8~+比值的变化；ELISA定量检测各组术后1、2、4、6周血清中IL-2和IL-4水平的变化；组织病理学观察移植骨材料周围炎性细胞浸润情况。(5)不同方法处理的异种骨材料修复兔桡骨缺损的研究：实验分组同(4)，将各组骨材料分别植入兔桡骨缺损处，分别于术后6、12、24周取材，X线片检查，根据Lane-Sandhux的X线评分标准，定量观察各组骨缺损的修复情况；组织切片行HE和Masson染色，显微镜下观察各组骨愈合和新骨形成情况。
结果：
(1)免疫组化研究发现正常猪骨中，α-Gal、MHC-Ⅰ抗原广泛表达在骨髓细胞和骨细胞、成骨细胞胞膜及哈佛氏管周围，MHC-Ⅱ抗原主要表达于骨髓细胞。
(2)不同方法处理的猪骨材料的异种抗原及性状改变：①各组抗原表达的比较，A组无骨细胞存留，无抗原阳性表达；B组骨陷窝和Harversian管周围仍有骨细胞存留，骨细胞表面可见少量α-Gal抗原、MHC-Ⅰ和MHC-Ⅱ抗原的阳性表达；C组可见骨陷窝内残存蓝染细胞核，无异种抗原的阳性表达；D组骨髓及骨细胞表面大量α-Gal抗原、MHC-Ⅰ和MHC-Ⅱ抗原的阳性表达。②扫描电镜观察，各组骨材料孔径大小比较A组＞C组＞B组＞D组，B、D组间比较无统计学差异(P＞0．05)，其余各组间比较均有差异(P＜0．05)；材料孔隙率比较A组＞C组＞B组＞D组，其中A组与C组、B组与D组之间比较无明显差别(P＞0．05)，其余各组间差别明显(P＜0．05)。③轴向压缩实验，最大载荷比较D组＞C组＞B组＞A组，其中A组明显小于其他各组(P＜0．05)，而B、C、D组间无明显差别(P＞0．05)。
(3)不同方法处理的异种骨材料的细胞相容性：兔成骨细胞在各组猪骨材料表面上均有生长，并且在第5天后成骨细胞增殖达到高峰，扫描电镜观察和MTT检测结果显示，各组生物相容性能C组＞A组＞B组。流式细胞术检测A、C组骨材料表面成骨细胞的细胞周期之间无明显差异，这两组细胞的S期＋G_2／M期明显高于B组(P＜0．05)。
(4)不同方法处理的异种骨材料体内移植免疫研究：各组术后1-2周CD4~+、CD8~+T淋巴细胞均逐渐升高，CD4~+／CD8~+比值在各组术后1周最高，以后逐渐下降，2周时B、D组较高，A、C、E组比较无明显差别。ELISA法检测外周血清IL-2、IL-4水平，术后D组外周血IL-2、IL-4水平最高，术后1周时达到最高峰，以后逐渐下降，F组水平最低。A、B、C组间比较结果显示，术后不同时间点B组血清中IL-2、IL-4水平均明显高于A组和C组(P＜0．05)。术后6周，各组组织病理学研究发现，D组移植骨材料周围大量淋巴细胞和巨噬细胞聚集；其次为B组，植骨材料周围中等量的淋巴细胞浸润；A组、C组、E组骨材料周围少量淋巴细胞浸润。
(5)不同方法处理的异种骨材料修复兔桡骨缺损的研究：不同方法处理的骨材料修复兔桡骨缺损，经放射学和组织学评估，术后6周、12周和24周各组成骨能力依次为：E组＞C组＞A组＞B组＞D组＞F组。
结论：
(1)α-Gal、MHC-Ⅰ抗原广泛表达在猪骨骨髓细胞和骨细胞、成骨细胞膜及哈佛氏管周围，MHC-Ⅱ抗原主要表达于骨髓细胞。
(2)脱脂部分脱蛋白和超深低温冷冻＋酶处理方法可以有效去除异种主要抗原，单纯超深低温冷冻可以明显降低猪骨的异种抗原表达，但是不能完全有效去除异种主要抗原。
(3)各组骨材料孔径大小比较，三种处理方法获得的异种骨材料的孔径大小均可满足成骨细胞生长所需要的孔径要求。但是超深低温冷冻组骨材料孔隙率最小，可能会影响成骨细胞的长入。脱脂部分脱蛋白组生物力学性能最差，超深低温冷冻和超深低温冷冻＋酶消化法对猪骨生物力学性能无明显影响，接近正常新鲜猪骨移植组。
(4)各组骨材料体外与兔成骨细胞复合培养，超深低温冷冻＋酶消化联合处理后的骨材料的生物相容性优于脱蛋白处理和单纯超深低温冷冻处理。
(5)各组骨材料植入兔体内，异种骨移植免疫排斥反应主要发生在术后1-2周，超深低温冷冻＋酶消化组和脱脂部分脱蛋白组骨材料免疫排斥反应较轻，单纯超深低温冷冻组免疫排斥较重。
(6)超深低温冷冻＋酶消化组和脱脂部分脱蛋白组骨缺损修复能力无明显差别，两组骨缺损修复能力均明显优于超深低温冷冻组。
(7)超深低温冷冻＋酶处理可以有效去除异种骨抗原，同脱脂部分脱蛋白组相比，具有良好的生物力学性能；具备合适的孔径大小和孔隙率，适合成骨细胞生长，具有良好的细胞生物相容性能；体内移植免疫排斥反应较轻微，骨缺损修复能力强，有望将来作为临床异种骨移植处理的有效方法之一。
Objective: (1) To investigate the distribution of xenogenous targeted antigen on porcine bone tissue. (2) To study the change of xenogenous antigen、biomechanical and physical properties, the porcine bone tissues were processed with three different methods. (3) To investigate the cellular compatibility of xenogenous bone scaffold materials. (4) To observe the immunological rejection and the turnover of the bone graft, the porcine bone tissues with different methods was implanted into the rabbits.
Methods: (1) Distribution ofα-Gal、MHC-Ⅰand MHC-Ⅱantigen on porcine bone tissue were observed by immunohistochemistry. (2) Physical properties and heteroantigens of porcine bone were detected: The experiment was divided into group A、B、C、D according to different materials. Group A: porcine bone was processed with defatting and partial deproteinization; Group B: porcine bone was preserved in liqiud nitrogen;Group C: porcine bone was preserved in liqiud nitrogen and was digested by enzyme; Group D: fresh porcine bone. The change of expression of heteroantigen was detected by immunohistochemistry, and the microstructure and biomechanical properties of different groups were observed by Electronic Scanning and INSTRON 8874. (3) Cellular compatibility of xenogenous bone scaffold materials: Osteoblasts of rabbit were cultured with the bone scaffold materials of group A, group B, group C,respectively. Osteoblasts in 1×10~7/ml density were cultured with the three materials for 7 days. And the adhesion and growth of osteoblasts were observed with phase microscope and electronic scanning microscope, the cell cycle was analyzed with flow cytometer. The growth of osteoblasts were observed on 1、3、5、7 day were detected by MTT assay. (4) The immune reaction of bone xenotransplantaion: The experiment was divided into group A、B、C、D、E、F according to different materials. Group A:porcine bone was processed with defatting and partial deproteinization;Group B: porcine bone was preserved in liqiud nitrogen; Group C: porcine bone was preserved in liqiud nitrogen and was digested with enzyme;Group D: fresh porcine bone; Group E: bone atuograft; Group F: control group. The bone scaffold materials were implanted into the bilaterial spinal muscles of rabbits. At 1、2、4 weeks postoperatively, the examination of CD4~+、CD8~+、CD4~+/CD8~+ in blood was detected by flow cytometer, and the levels of IL-2 and IL-4 in blood were analyzed by quantitative ELISA assay.The infiltration of lymphocytes and macrophages surrounding soft tissue of bone graft were also observed in order to evaluation biocompatibility of bone graft. (5) The effect of repairing bone defect with different processed porcine bone scaffold materials: Sixty New Zealand white rabbits were chosen, and the radial critical bone defects were made for 15mm long. The experiment was randomly divided into group A、B、C、D、E、F according to different materials, and each group details was equal to part (4). The bone defects were grafted by xenogenous bone materials derived from different groups. Groups of rabbits were sacrificed and the specimens were procured at 6、12、24 weeks after surgery for examination X-rays photography and histology evaluation. The ability of repaii'ing bone defect of each group was evaluated by Lane-Sandhux radiographic scoring. To study the bone defect repairing and reconstruction, new bone formation and substitute, the 5μm paraffin slices were obtained and were stained by hematoxylin-eosin and Masson method.
Results:
(1)α-Gal、MHC-Ⅰxenogenous antigens were extensively observed on the surface of bone marrow cells、osteocytes, osteoblasts and Harversian canals, and the MHC-Ⅱantigen mainly expressed on the bone marrow cells.
(2) Change of physical property and xenogenous antigens: No positive antigen expression in group A and group C, a few positive xenogenous antigens expression were detected in group B, while many positive xenogenous antigens expression were detected in group D. The results of pore size showed that group A＞group C＞group B＞group D, there was no significant difference between group B and group D (P＞0.05), and there was significant difference among the other groups (P＜0.05); The results of porosity showed that group A＞group C＞group B＞group D, there was no significant difference between group A and group C, group B and group D (P＞0.05). Biomechanical analysis demonstrated that the maximum load in axial compression test, group D＞group C＞group B＞group A, the minimum load was observed in group A and it was significantly inferior than the other groups (P＜0.05), there was no significantly difference among group B、group C and group D (P＞0.05).
(3) The results of cellular compatibility of xenogenous bone scaffold materials demonstrated that rabbit osteoblasts were survival in all groups, and the cell proliferation reached the peak at the fifth day. The cellular compatibility was detected by electronic scanning microscope and MTT assay, the results showed that group C＞group A＞group B. Compared to group B, the xenogenous bone materials in group A and group C had little influence on the G_0/G_1 stage and S stage of the rabbit osteoblasts.
(4) CD4~+, CD8~+ T lymphocytes gradually increased in 2 weeks after operation. The CD4~+/CD8~+ ratio reached the peak at 1 week after operation, then decreased gradually. The detection of CD4~+/CD8~+ ratio at 2 weeks postoperatively, there was no significant difference among group A、group C and group E, while the CD4~+/CD8~+ ratio reached higher level. Evaluation of IL-2 and IL-4 in serum by ELISA assay, the highest level" of IL-2 and IL-4 was detected in group D at 1 week after operation, the lowest level was in group F. Compared to group A and group C, the level of IL-2 and IL-4 were increased obviously in group B at different intervals after operation (P＜0.05). There was medium dose of lymphocytes infiltration surrounding the soft tissue of bone graft in group B, and there was few lymphocytes infiltration in group A and group C.
(5) According to the Lane-Sandhux radiographic scoring and the histological evaluation, the ability of reconstruction of radial defect was followed, group E＞group C＞group A＞group B＞group D＞group F.
Conclusions:
(1)α-Gal、MHC-Ⅰxenogenous antigens were extensively observed on the surface of bone marrow cells、osteocytes、osteoblasts and Harversian canals, and the MHC-Ⅱantigen mainly expressed on the surface of bone marrow cells.
(2) The expression of xenogenous antigen was decreased obviously in group B, but there was still some of positive expression of antigen on the porcine bone tissues. The xenogenous antigen was effectively deleted in group A and group C.
(3) The results of pore size showed that group A＞group C＞group B, the pore size of porcine bone materials derived these groups could meet the requirement of osteoblasts ingrowth, but porosity of group B was lowest, which maybe hinder the ingrowth of osteoblast. The xenogenous bone materials had the poor biomechanical performance in group A. Compared to the normal fresh porcine bone tissue, there was no significantly influence on the biomechanical performance in group B and group C.
(4) The cellular biocompatibility showed that group C＞group A＞group B.
(5) Immunological rejection of bone xenograft occurred during 2 weeks after operation, the host immunological response was lightly in group A and group C, and there was obviously immunological rejection in group B.
(6) The ability of reconstruction of radial bone defect demonstrated that group C＞group A＞group B.
引文
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