保鲜异种骨的研制与骨移植免疫反应的实验研究
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摘要
目的 骨缺损的修复一直是骨科棘手的难题之一，寻找理想的骨修复材料是骨科领域长期以来研究的重要课题。异种骨在来源、韧性、功能适应症、理化性能、空间结构等方面优于其它植骨材料，但新鲜异种骨主要问题是生物相容性极差，有强烈的免疫排斥反应。通过建立大鼠肌袋实验检测受体免疫原性和测试保鲜异种骨抗压强度，判断胡杰教授发明“脱细胞保鲜异种骨”的生物相容性和理学强度以及与BCB的比较。
方法 制备新鲜骨、BCB、保鲜骨骨条，力学实验机分别测量各自的抗压强度，并进行统计学分析。以大鼠为动物模型，分别植入新鲜骨、BCB、以及北京生物组织工程科技有限公司制备的大块保鲜牛皮质骨、松质骨。建立鼠股后肌袋实验，于1、4、12周分别取材，采用体外淋巴细胞二次刺激增殖实验，酶联免疫吸附实验及组织学观察，研究移植后大鼠体内的细胞免疫、体液免疫及宿主对该处理骨移植物的局部病理反应。
结果 保鲜骨的制备过程对生物力学强度无显著性影响，BCB与新鲜松质骨与保鲜松质骨相比较，具有显著性差异。新鲜骨植入后1周，产生高浓度的特异性抗体，并持续到12周，特异性的IgG是实验组的3-7倍；细胞免疫SI值显著升高，4周时特异性T细胞转化率为13％-21％，12周时略有下降，但明显高于其他植入组。组织学检查，新鲜骨各个时间点都有大量的淋巴细胞浸润，具有严重的组织排斥反
军医进修学院硕士毕业论文
应。保鲜松质骨、皮质骨和BCB杭体呈现低滴度，虽时间延长滴度逐
渐降低。尤其松质骨和BCB淋巴细胞刺激指数接近1。组织学显示松
质骨和BCB植入体内后，大量的纤维组织长入骨小梁中，具有良好的
组织相容性。保鲜牛皮质骨、松质骨显著降低了异种骨的抗原性，其
中保鲜松质骨尤佳，与BCB相比较，没有统计学差异。
结论保鲜牛皮质骨、松质骨是一种来源广泛，免疫原性低、力
学强度高的植骨材料。与BCB相比较，保鲜骨有效去除引起免疫排斥
反应的血液成分和细胞成分，大大消弱移植排斥反应。处理过程相对
温和，不损伤胶原结构，矿物质损失很少，可以最大限度保持移植骨
的生物学和力学特性，有利于移植区域骨缺损的支撑强度。同时由于
保鲜骨创新的异种骨脱杭原方法，从根本改变了以往异种骨处理采用
的氯仿、甲醇、乙醚、双氧水等毒性物质，避免了毒性物质残留人体
应用时的局部和全身反应问题。
Objective Large skeletal repair is always a tough problem perplexing surgeons. The bone graft substitutes play an important role in repairing bone defects, so the search for ideal bone graft substitute to repair bone defects is always an important subject in orthopaedics. Xenogeneic bone graftion is an altermative to autogeneic bone grafting, but the intense immune rejection makes its clinical practicelimited. the author implanted Preserved bovine cancellous bone, Preserved bovine cortical bone, Fresh bovine cancellous and Antigen-extracted bovine cancellous bone carrier in rat muscle pouch to investigate how about the immune rejection of Preserved bovine bone.at the same time.the author studied the compressive strength of all kinds of bone abovely.
methods We performed lymphocytes proliferation assay, enzume-linked immunosorbent assay, and histological observation to evaluate the levels of cellular and humoral immunity, and the tissue reaction to the grafting of Wister rat receiving frech boving cancellous bone(FCB), Antigen-extracted bovine cancellous bone carrier(BCB), Preserved bovine cancellous bone(PBCCB) and Preserved bovine cortical bone(PBCTB), while nothing implantion was kept untreated as a control. After implantation ,we harvested at 1, 4, 12weeks. The biomechanics properties of all kinds of bone were evaluated by biomechanics testing instrument. Results Biomechanics test result showed that there was significant differemce in
both BCB and FCB and PBCB. Lymphocyte proliferation was increased in an early phase of the grafting and persisted for a long period in FCB group. Meanwhine higher levels of specific antibody production were not ascended and no appreciable histologic signs of immune or foreign body reaction both in BCB and PBCB groups. The results suggested that BCB and PBCCB were characterized less antigenicity and can be used as osteoconductive material of bone growth factors. Conclusion All the results suggested that PBCCB might be an ideal replacement material for bone transplantation, and might be used effectively as a kind of carrier of BMP. however ,its biomechanics intensity and how to combined with an approperiate dose of the BMPs needed further improving.
方法 制备新鲜骨、BCB、保鲜骨骨条，力学实验机分别测量各自的抗压强度，并进行统计学分析。以大鼠为动物模型，分别植入新鲜骨、BCB、以及北京生物组织工程科技有限公司制备的大块保鲜牛皮质骨、松质骨。建立鼠股后肌袋实验，于1、4、12周分别取材，采用体外淋巴细胞二次刺激增殖实验，酶联免疫吸附实验及组织学观察，研究移植后大鼠体内的细胞免疫、体液免疫及宿主对该处理骨移植物的局部病理反应。
结果 保鲜骨的制备过程对生物力学强度无显著性影响，BCB与新鲜松质骨与保鲜松质骨相比较，具有显著性差异。新鲜骨植入后1周，产生高浓度的特异性抗体，并持续到12周，特异性的IgG是实验组的3-7倍；细胞免疫SI值显著升高，4周时特异性T细胞转化率为13％-21％，12周时略有下降，但明显高于其他植入组。组织学检查，新鲜骨各个时间点都有大量的淋巴细胞浸润，具有严重的组织排斥反
军医进修学院硕士毕业论文
应。保鲜松质骨、皮质骨和BCB杭体呈现低滴度，虽时间延长滴度逐
渐降低。尤其松质骨和BCB淋巴细胞刺激指数接近1。组织学显示松
质骨和BCB植入体内后，大量的纤维组织长入骨小梁中，具有良好的
组织相容性。保鲜牛皮质骨、松质骨显著降低了异种骨的抗原性，其
中保鲜松质骨尤佳，与BCB相比较，没有统计学差异。
结论保鲜牛皮质骨、松质骨是一种来源广泛，免疫原性低、力
学强度高的植骨材料。与BCB相比较，保鲜骨有效去除引起免疫排斥
反应的血液成分和细胞成分，大大消弱移植排斥反应。处理过程相对
温和，不损伤胶原结构，矿物质损失很少，可以最大限度保持移植骨
的生物学和力学特性，有利于移植区域骨缺损的支撑强度。同时由于
保鲜骨创新的异种骨脱杭原方法，从根本改变了以往异种骨处理采用
的氯仿、甲醇、乙醚、双氧水等毒性物质，避免了毒性物质残留人体
应用时的局部和全身反应问题。
Objective Large skeletal repair is always a tough problem perplexing surgeons. The bone graft substitutes play an important role in repairing bone defects, so the search for ideal bone graft substitute to repair bone defects is always an important subject in orthopaedics. Xenogeneic bone graftion is an altermative to autogeneic bone grafting, but the intense immune rejection makes its clinical practicelimited. the author implanted Preserved bovine cancellous bone, Preserved bovine cortical bone, Fresh bovine cancellous and Antigen-extracted bovine cancellous bone carrier in rat muscle pouch to investigate how about the immune rejection of Preserved bovine bone.at the same time.the author studied the compressive strength of all kinds of bone abovely.
methods We performed lymphocytes proliferation assay, enzume-linked immunosorbent assay, and histological observation to evaluate the levels of cellular and humoral immunity, and the tissue reaction to the grafting of Wister rat receiving frech boving cancellous bone(FCB), Antigen-extracted bovine cancellous bone carrier(BCB), Preserved bovine cancellous bone(PBCCB) and Preserved bovine cortical bone(PBCTB), while nothing implantion was kept untreated as a control. After implantation ,we harvested at 1, 4, 12weeks. The biomechanics properties of all kinds of bone were evaluated by biomechanics testing instrument. Results Biomechanics test result showed that there was significant differemce in
both BCB and FCB and PBCB. Lymphocyte proliferation was increased in an early phase of the grafting and persisted for a long period in FCB group. Meanwhine higher levels of specific antibody production were not ascended and no appreciable histologic signs of immune or foreign body reaction both in BCB and PBCB groups. The results suggested that BCB and PBCCB were characterized less antigenicity and can be used as osteoconductive material of bone growth factors. Conclusion All the results suggested that PBCCB might be an ideal replacement material for bone transplantation, and might be used effectively as a kind of carrier of BMP. however ,its biomechanics intensity and how to combined with an approperiate dose of the BMPs needed further improving.
引文
1．刘玮，陆裕朴，胡蕴玉，等．异种骨移植抗原的免疫组织化学实验研究．中华骨科杂志．1989：9 (1) 53．
2．胡蕴玉，陆裕朴，刘玮，等．重组合异种骨的实验研究和临床应用．中华外科杂志．1993 31 (12) 709-713．
3．段海平，郑永唐，达天武，等．脱抗原牛角胎骨移植家兔后的特异性免疫反应．免疫学杂志．2002 18 (3) 204-206．
4．罗卓荆，胡蕴玉，王茜，等．去抗原异种松质骨移植的免疫反应研究．中华外科杂志．1997 35 (11) 690-693．
5．胡蕴玉，陆裕朴，刘玮，等．异种骨移植修复骨缺损的实验研究．中华骨科杂志．1990 10 (1) 33．
6．胡蕴玉，陆裕朴，刘玮，等．不同复合异种骨修复长骨缺损的实验研究．中华骨科杂志．1990 5：74
7．刘玮，胡蕴玉，陆裕朴．异种骨基质修复兔绕骨干缺损．第四军医大学学报．1991：12(4)：245
8．李亚非，胡蕴玉，宋海燕．重组合异种骨在小鼠体内诱导成骨活性的剂量依赖性研究．中华骨科杂志．1995：15(7)：447．
9. Skoglund A,Hising P, Young C. A clinical and histologic examination in humans of the osseous response to implanted natural bone mineral. Int Joral Maxillofac Implants, 1997; 12(2): 194.
10．桑宏勋，胡蕴玉，李雪冰，等．异种骨移植对T淋巴细胞总数及亚群的变化．中华外科杂志．1996 34 (8) 453．
11．罗卓荊，胡蕴玉．牛松质骨力学强度与去抗原处理时限的相关性实验研究．第四军医大学报．1996 17 (6) 434．
12. .Miyamoto S,Takaoka K,Okada T.Evaluation of lactic acid homopolymer as carries for bone morphogenetic protein.Clin Orthop,1992,278:274.
13．袁志，胡蕴玉，马平，等．新型植骨材料——复合rhBMP2的异种骨的
实验研究，现代康复，2000 4 (10) 1468．
14．徐虎，胡蕴．重组合异种骨移植局部IL-1α mRNA和蛋白质的表达．中华创伤杂志．2001：17(3)：175．
15．杨柳，胡蕴玉，吕荣，等．重组合异种骨的活性成分的分离与纯化，第四军医大学报．2001 22 (10) 922．
16．黄传书，金柏泉，杨连甲，等．骨形成蛋白抑制多克隆刺激剂诱导T淋巴细胞增殖及其机理的研究．中华微生物和免疫杂志，1991，11：373．
17. Sogal A, Tofe Aj. Risk assessment of bovine spongiform encephalopathy tranmission throuth bone graft material derived from bovine bone used for dental applications. J Periodontal, 1999; 70(9): 1053.
18. Young C, Sandstedt P, Skoglund AA,Comparative study of anorganic xenogenic bone regeneration on rabbits. Int Joral Maxillofac Implants, 1999; 14(1): 72.
19．刘玮，胡蕴玉，陆裕朴．重组合异种骨的研制及其生物活性分析．中华医学杂志．1991：71(7)：378．
20. Riley EA,Lane JM,Urist MR,et al.Bone morphogenetic pro-tein-2:Biologyandapplication. Clinic Orthop,1996; 324: 39～46.
21. Andriano KP, Chandrashekar B,McEnery K,et al.Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix.J Biomed Mater Res,2000;53(1):36～43.
22. Urist MR,et al.A bovine low molecular weight bone morpho-genetic protein(BMP) fraction.Clin Orthop, 1982; 162:219～227.
23. Urist MR,Kovacs S,Yates KA.Regeneration of an enchondroma defect under the influence of an implant of human bone morpho-genetic protein.J Hand Surg(Am), 1986;11:417～427.
24. Andriano KP, Chandrashekar B,McEnery K,et al.Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix.J Biomed Mater Res,2000;53(1):36～43.
25 罗卓荆，胡蕴玉，吕荣，等．块型重组合异种骨的诱导成骨活性实验研究，第四军医大学报．1996 17 (6) 468．
26. Isobe M,Yamazaki Y, Mori M,et al,Bone regeneration produced in rat femur defects by polymer capsules containing recom-binant human bone morphogenetic protein-2.J Oral Maxillofac Surg, 1999;57(6):695～698.
27. Teixeira JO,Urist MR.Bone morphogenetic protein induced repair of compartmentalized segmental diaphyseal defects.Arch Orthop Trauma Surg, 1998;117(1-2):27～34.
28. Meraw SJ,Reeve CM,Lohse CM,et al:Treatment of peri-implant defects with combination growth factor cement.J Periodontol,2000;71(1):8～13.
29. Chow LC.Development of self-setting calcium phosphate cements.J Ceram Soci Jap, 1991;99(10):954～960.
30. Costantino PD,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty.Plast Reconstr Surg, 1991,90(2): 174～185.
31. Otsuka M,Matsuda Y, Suwa Y, et al.Effect of particle size of metastable calcium phosphates on mechanical strength of a novel self-setting bioactive calcium phosphate cement.J Biomed Mater Res, 1995;29(1):25～32.
32. Ishikawa K,Asaoka K.Estimation of ideal mechanical str-ength and critical porosity of calcium phosphate cement.J Biomed Mater Res, 1995;29(12):1537～1543.
33. Fujikawa K,Sugawara A,Murai S,et al.Histopathological reaction of calcium phosphate cement in periodontal bone defect.Dent Mater J, 1995; 14(1): 45～57.
34. Bohner M,Lemaitre J,Landuyt PV,et al.Gentamicin-loaded hydraulic calcium phosphate bone cement as antibiotic delivery syetem[J].Journal of Pharmaceutical Sciences, 1995,84(5):565～272.
35. Otsuka M,Matsuda Y, Suwa Y, et al.A novel skeletal drug delivery system using self-setting calcium phosphate cement.2.Physicochemical properties and drug release rate of the cement-containing indomethacin。Journal of
Pharmaceutical Sciences, 1994,83(5):611～615.
36. Otsuka M,Matsuda Y, Suwa Y, et al.A novel skeletal drug delivery system using self-setting calcium phosphate cement.3.Physicochemical properties and drug release rate of bovine insulin and bovine albumin[J].Journal of Pharmaceutical Sciences, 1994,83(2):255～258.
37. Guicheux J,Grimandi G, Trecant M,et al.Apatite as carrieer for growth hormone:in vitro characterization of loading and release[J].J Biomed Mater Res, 1997,34(2): 165～170.
2．胡蕴玉，陆裕朴，刘玮，等．重组合异种骨的实验研究和临床应用．中华外科杂志．1993 31 (12) 709-713．
3．段海平，郑永唐，达天武，等．脱抗原牛角胎骨移植家兔后的特异性免疫反应．免疫学杂志．2002 18 (3) 204-206．
4．罗卓荆，胡蕴玉，王茜，等．去抗原异种松质骨移植的免疫反应研究．中华外科杂志．1997 35 (11) 690-693．
5．胡蕴玉，陆裕朴，刘玮，等．异种骨移植修复骨缺损的实验研究．中华骨科杂志．1990 10 (1) 33．
6．胡蕴玉，陆裕朴，刘玮，等．不同复合异种骨修复长骨缺损的实验研究．中华骨科杂志．1990 5：74
7．刘玮，胡蕴玉，陆裕朴．异种骨基质修复兔绕骨干缺损．第四军医大学学报．1991：12(4)：245
8．李亚非，胡蕴玉，宋海燕．重组合异种骨在小鼠体内诱导成骨活性的剂量依赖性研究．中华骨科杂志．1995：15(7)：447．
9. Skoglund A,Hising P, Young C. A clinical and histologic examination in humans of the osseous response to implanted natural bone mineral. Int Joral Maxillofac Implants, 1997; 12(2): 194.
10．桑宏勋，胡蕴玉，李雪冰，等．异种骨移植对T淋巴细胞总数及亚群的变化．中华外科杂志．1996 34 (8) 453．
11．罗卓荊，胡蕴玉．牛松质骨力学强度与去抗原处理时限的相关性实验研究．第四军医大学报．1996 17 (6) 434．
12. .Miyamoto S,Takaoka K,Okada T.Evaluation of lactic acid homopolymer as carries for bone morphogenetic protein.Clin Orthop,1992,278:274.
13．袁志，胡蕴玉，马平，等．新型植骨材料——复合rhBMP2的异种骨的
实验研究，现代康复，2000 4 (10) 1468．
14．徐虎，胡蕴．重组合异种骨移植局部IL-1α mRNA和蛋白质的表达．中华创伤杂志．2001：17(3)：175．
15．杨柳，胡蕴玉，吕荣，等．重组合异种骨的活性成分的分离与纯化，第四军医大学报．2001 22 (10) 922．
16．黄传书，金柏泉，杨连甲，等．骨形成蛋白抑制多克隆刺激剂诱导T淋巴细胞增殖及其机理的研究．中华微生物和免疫杂志，1991，11：373．
17. Sogal A, Tofe Aj. Risk assessment of bovine spongiform encephalopathy tranmission throuth bone graft material derived from bovine bone used for dental applications. J Periodontal, 1999; 70(9): 1053.
18. Young C, Sandstedt P, Skoglund AA,Comparative study of anorganic xenogenic bone regeneration on rabbits. Int Joral Maxillofac Implants, 1999; 14(1): 72.
19．刘玮，胡蕴玉，陆裕朴．重组合异种骨的研制及其生物活性分析．中华医学杂志．1991：71(7)：378．
20. Riley EA,Lane JM,Urist MR,et al.Bone morphogenetic pro-tein-2:Biologyandapplication. Clinic Orthop,1996; 324: 39～46.
21. Andriano KP, Chandrashekar B,McEnery K,et al.Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix.J Biomed Mater Res,2000;53(1):36～43.
22. Urist MR,et al.A bovine low molecular weight bone morpho-genetic protein(BMP) fraction.Clin Orthop, 1982; 162:219～227.
23. Urist MR,Kovacs S,Yates KA.Regeneration of an enchondroma defect under the influence of an implant of human bone morpho-genetic protein.J Hand Surg(Am), 1986;11:417～427.
24. Andriano KP, Chandrashekar B,McEnery K,et al.Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix.J Biomed Mater Res,2000;53(1):36～43.
25 罗卓荆，胡蕴玉，吕荣，等．块型重组合异种骨的诱导成骨活性实验研究，第四军医大学报．1996 17 (6) 468．
26. Isobe M,Yamazaki Y, Mori M,et al,Bone regeneration produced in rat femur defects by polymer capsules containing recom-binant human bone morphogenetic protein-2.J Oral Maxillofac Surg, 1999;57(6):695～698.
27. Teixeira JO,Urist MR.Bone morphogenetic protein induced repair of compartmentalized segmental diaphyseal defects.Arch Orthop Trauma Surg, 1998;117(1-2):27～34.
28. Meraw SJ,Reeve CM,Lohse CM,et al:Treatment of peri-implant defects with combination growth factor cement.J Periodontol,2000;71(1):8～13.
29. Chow LC.Development of self-setting calcium phosphate cements.J Ceram Soci Jap, 1991;99(10):954～960.
30. Costantino PD,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty.Plast Reconstr Surg, 1991,90(2): 174～185.
31. Otsuka M,Matsuda Y, Suwa Y, et al.Effect of particle size of metastable calcium phosphates on mechanical strength of a novel self-setting bioactive calcium phosphate cement.J Biomed Mater Res, 1995;29(1):25～32.
32. Ishikawa K,Asaoka K.Estimation of ideal mechanical str-ength and critical porosity of calcium phosphate cement.J Biomed Mater Res, 1995;29(12):1537～1543.
33. Fujikawa K,Sugawara A,Murai S,et al.Histopathological reaction of calcium phosphate cement in periodontal bone defect.Dent Mater J, 1995; 14(1): 45～57.
34. Bohner M,Lemaitre J,Landuyt PV,et al.Gentamicin-loaded hydraulic calcium phosphate bone cement as antibiotic delivery syetem[J].Journal of Pharmaceutical Sciences, 1995,84(5):565～272.
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