骨形成蛋白脱钙骨基质颗粒骨水泥复合材料的评价
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摘要
目的：骨缺损修复是骨科临床常见并涉及多方面理论和技术的难题，不论是先天性还是外伤、感染、肿瘤等原因造成的骨缺损，还是老年或其他原因所致的骨萎缩，都将引起严重的功能障碍和外观畸形，对此，唯一可行的治疗方法是对其进行修复，以恢复骨骼的正常功能和矫正骨骼畸形。最早用骨移植进行骨缺损修复的手术是在1668年，当时一位荷兰医生在战争时期用狗的颅盖骨修复了一名战士的颅盖骨缺损，虽然术后发现患者的颅盖骨缺损愈合，但并没有给人们以很大的启示。1674年，荷兰医生Leeuwenkoek开始对骨的结构进行观察和描述，以此为基础，Duhamel和Heyde发现和认识到了骨生成的现象及其重要性，其后法国人Ollier在狗和兔子身上做实验观察到，如果在机体内得到适宜的环境，游离的自体骨仍然具有生长能力。真正的第一例临床自体骨移植术是1820年由德国医生Walther完成的，而第一例临床同种异体骨移植术是苏格兰人William Macewen与1880年完成的。由此人们认识到对于一些较大的骨缺损可以用某些骨材料进行修复，以此来弥补骨组织自身有限的再生修复能力。用骨移植来修复骨缺损真正的广泛开展始于二十世纪初期。1923年Albee发表了他总结的3000例自体骨移植术，1924年Lexer又报道了1000例手术，其中还报道了大量有关同种异体骨移植修复骨缺损的手术。虽然这期间开展了大量的此类手术，但结果成败参半，而对于骨移植如何促进骨缺损修复的机理仍缺乏明确的认识。
早在1930年，Huggins报道了将膀胱粘膜植入肌肉中可以诱导生成新骨组织的结果，从而对异位成骨的概念有了初步的认识。真正对骨缺损修复和骨再生过程进行广泛深入研究的应当是美国的Marshall R．Urist教授。1952年，Urist将未分化的结缔组织细胞植入动物眼前房后诱导形成了新生骨组织，1965年，他又发现将动物的长骨皮质骨基质脱钙冻干后植入同种动物的肌肉内也可以诱导形成新生骨组织，从而建立了可靠的实验动物模型。这一模型的建立为揭示失去活性的脱钙骨基质在异位的宿主区诱导未分化间充质细胞分化并形成骨组织的本质提供了前提。为了进一步研究这一骨诱导现象，Lash等人参照Grobstein的方法将冻干脱钙骨基质装入扩散盒内，然后一起植入动物皮下组织或肌肉陷窝内，一定时间后发现在扩散盒的微孔滤膜外有新生骨组织沉积。扩散盒的引用排除了细胞间直接作用的可能，但仍不能解释这种骨诱导现象。1967年，Urist等人在成骨诱导广泛和深入研究的基础上，总结和回顾了以往所做的各种研究结果，提出了骨诱
第四军医大学博士学位论文
导物质（Bone Induction Pincghe，BIP）的新概念。
BIP被人们初步证实后，Uist等人又经过了大量的研究以后，于1971年首次提出了
骨形成蛋白（又称为骨形态发生蛋白，Bone Mdsenehc Prten，BMP）的概念，他认
为BIP实质上就是BMP。这一概念的提出使研究范围大大缩小，从而更加明确了这一领
．域的研究方向。在以后的十余年的时间里，Urist等人通过不懈的努力，她BMP进行了
更加广泛深人的研究，并对其进行了分离和提纯。近年来的研究证明，BMP是一种高效
的骨诱导物质，在促进骨缺损的修复过程中，其作用几乎与自体骨移植的效果相同，因
此，它在骨缺损修复的实验研究和临床应用过程中占有极其重要的地位，为充分发挥它
的诱导成骨的功能，目前研究的焦点是对其载体释放系统进行探索。
本实验的目的是制备骨形成蛋白（BMP）脱钙骨基质颗粒（DBM）骨水泥（BC）复合
材料，将DBM作为BWi的载体，用BC赋予材料可塑型性，对其进行综合评价，为临床应
用和批量化生产提供方法和依据。
方法：制备狗的异体脱钙骨基质颗粒（DBM），使其直径＜2．0。，消毒备用。提取牛
的骨形成蛋白（bBMP），消毒，将其埋人小白鼠后腿股部肌肉内行成骨诱导活性测定后备
用。将Mpp与mM按l：25的质量比用直接掺合法和吸附法复合后，再与骨水泥（BC）
按质量比0：10、4：6、5：5、6：4和7．5：2．5的比例复合，至眈呈面团期时制成诱
导成骨活性测定标本、生物相容性研究标本、生物力学测定标本和扫描电镜观察标本，
分别埋入小白鼠后腿股部肌肉内行成骨诱导活性测定、植入小白鼠腹腔内行生物相容性
研究、体外行生物力学强度和弹性模量测定以及扫描电镜下观察。应用自行研制的骨肿
瘤微波治疗仪，以1．skHZ频率、70W功率加热至5055oC，持续20分钟，建立成年狗股
骨微波灭活骨缺损的实验动物模型，将4：6、5：5、6：4和7．5：2．5的复合材料植入
右侧狗股骨骨缺损处，左侧单纯植入骨水妮做对照，术后不同时间进行X射线照相、
一TC－－MDP骨显像、大体标本观察、生物力学测定、组织学观察、土霉素荧光标记和印度
墨汁灌注观察。
结果：自行提取的bBWi具有很强的成骨诱?
OBJECTIVE It is common to be met in orthopedic clinic that the bone defects caused by bone tumors which have no ideal methods to repair at present However the clinical results that arrest people's attention have been got by the surgical treatment for bone tumors with microwave-induced hyperthermia and the allograft decalcified bone matrix (DBM) combined with bone cement (BC) had been used to try repairing the bone defects caused by microwave-induced hyperthermia in recent years. In order to further improve the bone formation inductive activity, we try to mix the bovine bone morphogenetic protein (bBMP) with this composite material and this experiment was designed to determine the biological procedure of the composite material of DBM, BC and bBMP when it was implanted in the canine femoral defect caused by microwave-induced hyperthermia. The aim of the present experiment is to prepare and evaluate the composite material of bovine bone morphogenetic protein (bBMP), decalcified bone matrix (DBM) and bone cement
(BC) for the clinical use and the batch process.
METHODS The allograft canine DBM which diameter was less than 2.0mm was prepared and sterilized to use. The bBMP was extracted from the fresh cortex of the four limbs of the young ox and its bone formation inductive activity was determined. First mix the bBMP and DBM together with the proportion of 1:25 with adsorption and directly, then add BC with different weight proportion and finally the composite material was examined by biomechanics, scan electron microscope, determinations of bone formation inductive activity and biocompatibility. The canine femoral defect model was caused by microwave-induced hyperthermia and the composite material was implanted into the canine right femoral defect and the left one was filled up with BC only as a contrast. The canine femurs were examined by X-ray, 99mTc-MDP bone scintigraphy, biomechanics, histology, flurescence labeling of terramycin and India ink staining.
RESULTS The bBMP had strong bone formation inductive activity. At the third day when it was buried in the muscles of the behind legs of the mouse, there were a large number of
6
mesenchymal cells around the bBMP and at the seventh day the mesenchymal cells were differentiated into the chondrocytes. At the fourteenth day the cartilage was formed and at the twenty-first day and the twenty-eighth day the new bone was formed. Though the bone inductive activity of the composite material was weaker and the amount of the new bone which it inducted to form was less than that of bBMP, it had very strong bone inductive activity. There was no affect for the bone formation inductive activity of the composite material when the bBMP was combined with DBM with different method. However the direct method was the most convenient. When the composite material was implanted into the abdominal cavity of the mouse, no animal died in the acute toxicity test and the body weight, red blood cells, white blood cells and hemoglobin of the mouse were not affected in the subacute test. The composite material did not affect the function of hemolysis and coagulation. The determinative results of biomechanics was that the biomechanical intensity of the composite material decreased when the proportion of DBM was from 0% to 75%. However the composite material had enough intensity to bear a load when the proportion of DBM was 50% and 60%. Under the scan electron microscope, there were irregular gaps in the composite material and most of them with the diameter was less than 100 u m and the porosity was less than 20% when the proportion of DBM was 40%. So it was unfavorable for the new bone to form and the new blood vessels to reconstruct. However there were many irregular gaps with diameter of 200 u m to 800 u m in the composite material with the proportion of DBM was 50% and 60% and the porosity was 32% to 55%. There were more irregular gaps with diameter of 200 u m to 800 u m in the composite material with the proportion of DBM was 75% and the porosity was more than 60%. However the DBM
早在1930年，Huggins报道了将膀胱粘膜植入肌肉中可以诱导生成新骨组织的结果，从而对异位成骨的概念有了初步的认识。真正对骨缺损修复和骨再生过程进行广泛深入研究的应当是美国的Marshall R．Urist教授。1952年，Urist将未分化的结缔组织细胞植入动物眼前房后诱导形成了新生骨组织，1965年，他又发现将动物的长骨皮质骨基质脱钙冻干后植入同种动物的肌肉内也可以诱导形成新生骨组织，从而建立了可靠的实验动物模型。这一模型的建立为揭示失去活性的脱钙骨基质在异位的宿主区诱导未分化间充质细胞分化并形成骨组织的本质提供了前提。为了进一步研究这一骨诱导现象，Lash等人参照Grobstein的方法将冻干脱钙骨基质装入扩散盒内，然后一起植入动物皮下组织或肌肉陷窝内，一定时间后发现在扩散盒的微孔滤膜外有新生骨组织沉积。扩散盒的引用排除了细胞间直接作用的可能，但仍不能解释这种骨诱导现象。1967年，Urist等人在成骨诱导广泛和深入研究的基础上，总结和回顾了以往所做的各种研究结果，提出了骨诱
第四军医大学博士学位论文
导物质（Bone Induction Pincghe，BIP）的新概念。
BIP被人们初步证实后，Uist等人又经过了大量的研究以后，于1971年首次提出了
骨形成蛋白（又称为骨形态发生蛋白，Bone Mdsenehc Prten，BMP）的概念，他认
为BIP实质上就是BMP。这一概念的提出使研究范围大大缩小，从而更加明确了这一领
．域的研究方向。在以后的十余年的时间里，Urist等人通过不懈的努力，她BMP进行了
更加广泛深人的研究，并对其进行了分离和提纯。近年来的研究证明，BMP是一种高效
的骨诱导物质，在促进骨缺损的修复过程中，其作用几乎与自体骨移植的效果相同，因
此，它在骨缺损修复的实验研究和临床应用过程中占有极其重要的地位，为充分发挥它
的诱导成骨的功能，目前研究的焦点是对其载体释放系统进行探索。
本实验的目的是制备骨形成蛋白（BMP）脱钙骨基质颗粒（DBM）骨水泥（BC）复合
材料，将DBM作为BWi的载体，用BC赋予材料可塑型性，对其进行综合评价，为临床应
用和批量化生产提供方法和依据。
方法：制备狗的异体脱钙骨基质颗粒（DBM），使其直径＜2．0。，消毒备用。提取牛
的骨形成蛋白（bBMP），消毒，将其埋人小白鼠后腿股部肌肉内行成骨诱导活性测定后备
用。将Mpp与mM按l：25的质量比用直接掺合法和吸附法复合后，再与骨水泥（BC）
按质量比0：10、4：6、5：5、6：4和7．5：2．5的比例复合，至眈呈面团期时制成诱
导成骨活性测定标本、生物相容性研究标本、生物力学测定标本和扫描电镜观察标本，
分别埋入小白鼠后腿股部肌肉内行成骨诱导活性测定、植入小白鼠腹腔内行生物相容性
研究、体外行生物力学强度和弹性模量测定以及扫描电镜下观察。应用自行研制的骨肿
瘤微波治疗仪，以1．skHZ频率、70W功率加热至5055oC，持续20分钟，建立成年狗股
骨微波灭活骨缺损的实验动物模型，将4：6、5：5、6：4和7．5：2．5的复合材料植入
右侧狗股骨骨缺损处，左侧单纯植入骨水妮做对照，术后不同时间进行X射线照相、
一TC－－MDP骨显像、大体标本观察、生物力学测定、组织学观察、土霉素荧光标记和印度
墨汁灌注观察。
结果：自行提取的bBWi具有很强的成骨诱?
OBJECTIVE It is common to be met in orthopedic clinic that the bone defects caused by bone tumors which have no ideal methods to repair at present However the clinical results that arrest people's attention have been got by the surgical treatment for bone tumors with microwave-induced hyperthermia and the allograft decalcified bone matrix (DBM) combined with bone cement (BC) had been used to try repairing the bone defects caused by microwave-induced hyperthermia in recent years. In order to further improve the bone formation inductive activity, we try to mix the bovine bone morphogenetic protein (bBMP) with this composite material and this experiment was designed to determine the biological procedure of the composite material of DBM, BC and bBMP when it was implanted in the canine femoral defect caused by microwave-induced hyperthermia. The aim of the present experiment is to prepare and evaluate the composite material of bovine bone morphogenetic protein (bBMP), decalcified bone matrix (DBM) and bone cement
(BC) for the clinical use and the batch process.
METHODS The allograft canine DBM which diameter was less than 2.0mm was prepared and sterilized to use. The bBMP was extracted from the fresh cortex of the four limbs of the young ox and its bone formation inductive activity was determined. First mix the bBMP and DBM together with the proportion of 1:25 with adsorption and directly, then add BC with different weight proportion and finally the composite material was examined by biomechanics, scan electron microscope, determinations of bone formation inductive activity and biocompatibility. The canine femoral defect model was caused by microwave-induced hyperthermia and the composite material was implanted into the canine right femoral defect and the left one was filled up with BC only as a contrast. The canine femurs were examined by X-ray, 99mTc-MDP bone scintigraphy, biomechanics, histology, flurescence labeling of terramycin and India ink staining.
RESULTS The bBMP had strong bone formation inductive activity. At the third day when it was buried in the muscles of the behind legs of the mouse, there were a large number of
6
mesenchymal cells around the bBMP and at the seventh day the mesenchymal cells were differentiated into the chondrocytes. At the fourteenth day the cartilage was formed and at the twenty-first day and the twenty-eighth day the new bone was formed. Though the bone inductive activity of the composite material was weaker and the amount of the new bone which it inducted to form was less than that of bBMP, it had very strong bone inductive activity. There was no affect for the bone formation inductive activity of the composite material when the bBMP was combined with DBM with different method. However the direct method was the most convenient. When the composite material was implanted into the abdominal cavity of the mouse, no animal died in the acute toxicity test and the body weight, red blood cells, white blood cells and hemoglobin of the mouse were not affected in the subacute test. The composite material did not affect the function of hemolysis and coagulation. The determinative results of biomechanics was that the biomechanical intensity of the composite material decreased when the proportion of DBM was from 0% to 75%. However the composite material had enough intensity to bear a load when the proportion of DBM was 50% and 60%. Under the scan electron microscope, there were irregular gaps in the composite material and most of them with the diameter was less than 100 u m and the porosity was less than 20% when the proportion of DBM was 40%. So it was unfavorable for the new bone to form and the new blood vessels to reconstruct. However there were many irregular gaps with diameter of 200 u m to 800 u m in the composite material with the proportion of DBM was 50% and 60% and the porosity was 32% to 55%. There were more irregular gaps with diameter of 200 u m to 800 u m in the composite material with the proportion of DBM was 75% and the porosity was more than 60%. However the DBM
引文
1 Urist MR and McLean FC. Osteogenic potency and new bone formation by induction in transplants to the anterior chamber of the eye. J Bone Joint Surg, 1952, 34-A: 443-470.
2 Urist MR. Bone: formation by autoinduction. Science, 1965, 150:893-899.
3 Urist MR, Silverman BF, Buring K, et al. The bone induction principle. Clin Orthop, 1967,53:243-283.
4 赵廷宝，范清宇，郭照江．骨形成蛋白在骨缺损修复中的矛盾与克服．医学与哲学，2000，21(8)：38～39．
5 金岩，杨连甲．生物活性材料修复骨缺损过程的基本内容与概念．见杨连甲，金岩，胡蕴玉主编：口腔和骨科的生物活性材料．1993，陕西科学技术出版社，7．
6 Bang, G., and Urist, M. R.. Bone induction in excavation chambers in matrix of decalcified dentin. Arch. Surg. 1967,94:781.
7 Dubuc, F. L., and Urist, M. R. The acceaaibility of the bone induction principle in surface-decalcified bone implants. Clin. Orthop. 1967,55:217.
8 Strates, B. S., and Urist, M. R.. Origin of the inductive signal in implants of normal and lathyritic bone matrix, clin. Orthop.1969,66:226.
9 Urist, M. R.. Mesenchymal cell reaction to inductive substrates for new bone formation, in Dunphy, J.E.,and van Winkle,W,Jr, et al, (eds). Repair and regeneration, New York:McGraw-Hill, Inc., 1969,229～259.
10 Urist, M.R,Dowe,T.A.,Hay, P.H. and Swales, B. S..Inductive substrates for bone formation. Clin. Orthop. 1968,59:59.
11 Urist, M. R.,Jurist,J.M., Dubuc, E L., and Strates, B. S..Quantitation of new bone formation in intramuscular implants of bone matrix in rabbits. Clin. Orthop. 1970,68:279.
12 Urist, M. R.,and Strates, B. S..Bone formation in implants of partially and wholly demineralized bone matrix. Clin. Orthop. 1970,71:271～278.
13 Urist, M. R.,and Strates, B. S..Bone morphgenetic protein. J Dent Res,50[Suppl.6]: 1971,1392.
14 Urist, M. R.,Iwata, H,Ceccotti,P,W.L.,et al. Bone morphgenesis in implants of insoluble bone gelatin. Proc Natl Acad Sci USA, 1973,70:3511.
15 Urist, M. R.,Mikulski,A.and Lietze,A..Solubilized and insolubilized bone morphgenetic protein. Proc Natl Acad Sci USA, 1979,76:1828.
16 Urist, M. R., Lietze,A., Mizutani, H.,et al. A bovine low molecular weight bone morphgenetic protein(BMP)fraction. Clin. Orthop. 1982,162:219.
17 Urist, M. R., Mizutani,H.,Conover, M.A.,et al. Dentin ,bone and osteosarcoma tissue bone morphgenetic proteins. In Factors and Mechanisms Influencing Bone Growth. New York, Allen R. Liss, 1983,61～81.
18 Urist, M. R.,DeLange, R J, and Finerman, G A M.. Bone cell differentiation and growth factors: Induced activity of chondro-osteogenetic DNH. Science, 1983,220:680.
19 Urist, M. R.,Huo,Y K, Brownell,A G,et al. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci USA,1984,81:371.
20 Urist, M. R.,Chang J J,Lietze,A, et al. Methods of preparation and bioassay of bone morphogenetic protein and polypeptide fragments. In Bames, D and Sirbaska, D A (eds.):Methods in Enzymology, vol. 146.New York,Academic Press, 1987,294～312.
21 吴祖尧．诱导成骨与骨形态发生蛋白．中华骨科杂志，1988，8(3)：231～234．
22 唐六一，吴祖尧．骨形态发生蛋白的载体及其释放系统．中华骨科杂志，1993，13(2)：140～142．
23 董玉峰，戴克戎．骨形态发生蛋白载体的研究进展．国外医学．创伤与外科基本问题分册，1999，20(1)：6～9．
24 梁戈，胡蕴玉，郑昌琼，等．多孔β-TCP／BMP复合人工骨的研制和动物体内的相关研究．中华骨科杂志，1998，18(2)：75～79．
25 Urist, M. R., Lietze,A, Dawson E. β-tricalcium phosphate delivery system for bone morphogenetic protein. Clin Orthop,1984,187:277～280.
26 Ono I, Ohura T, Murata M, et al. A study on bone induction in hydroxyapatite combined with bone morphogenetic protein. Plast Reconstr Surg, 1992,90:870～879.
27 Hollinger J O, Kleinschmidt J G The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg, 1990,1:60～68.
28 罗卓荆，胡蕴玉，王茜．去抗原牛松质骨块/bBMP复合材料修复兔长骨骨缺损．第四军医大学学报，2000，21(1)：48～51．
29 陈克明，李旭生，刘兴炎，等．骨形态发生蛋白复合纤维蛋白载体修复骨缺损的实验研究．中华骨科杂志，1998，18(2)：68～70．
30 胡晓波，夏筠，李群，等．骨诱导性载体复合骨形态发生蛋白的双重成骨作用．中华骨科杂志，1998，18(2)：80～83．
31 Kawamura M, Urist M R. Human fibrin is a physiological delivery system for bone morphogenetic protein. Clin Orthop,1988,235:302～310.
32 De Bore H, H. The history of bone graft. Clin Orthop, 1988,226:292.
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