骨胶原基质的制备、理化性能及其生物相容性研究
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摘要
寻找合适的骨修复材料一直是骨科和生物材料领域面临的的一个艰巨挑战。
目前使用的修复材料都存在不同的缺陷，难以完全满足临床需要。骨组织工程学
为大范围骨缺损的修复开辟了新的渠道，细胞外基质材料的开发是其中的关键。
天然异种骨具有来源丰富、价格低廉等优点，是骨组织工程细胞外基质材料和骨
缺损移植材料的潜在来源之一。
本文主要研究一种新型骨胶原基质(bone collagen matrix,BCM)的制备方法，
采用优化的脱脂、部分脱钙、盐酸胍浸泡、胰蛋白酶消化等一系列生物和化学方
法对牛骨进行脱抗原处理，对所得材料的各项理化性能进行测试，并进行体外细
胞相容性和体内的组织相容性实验研究。主要研究内容和结论如下：
(1) 屠宰场获得新鲜牛骨，经适当前处理后，采用如下的方法制备BCM：
加工好的骨块→50℃热水反复冲洗→室温稀碱液浸泡→脱脂剂脱脂→部分脱钙→
脱非胶原蛋白→胰蛋白酶消化→50℃真空干燥→消毒→密封包装，备用。
通过上述方法最终制备出两种BCM材料，即松质骨胶原基质(Trabecular Bone
Collagen Matrix，TBCM)和皮质骨胶原基质(Cortical Bone Collagen Matrix，
CBCM)，TBCM与PDLLA复合成TBCM-PDLLA复合材料。采用色氨酸含量作
为非胶原蛋白去除效果的评价指标，以羟脯氨酸的含量来衡量胶原的破坏程度，
并以XRD结果来定性分析部分脱钙对骨矿结构的影响。结果表明经HC1胍处理
24h后两种骨块中的色氨酸浓度接近2mg／g，胰蛋白酶消化后降低到1mg／g左右。
胰蛋白酶消化能够切除胶原蛋白具有免疫原性的端肽部分，其三螺旋结构得以保
留，脱蛋白后胶原溶出的比例低于1％。XRD结果表明经过盐酸部分脱钙，骨组
织的主要的晶相成分保持不变，骨矿的晶体结构也没有发生显著变化。
(2) 付立叶红外光谱(FTIR)、x-射线衍射光谱(XRD)、能量散射X-射
线荧光光谱(EDXRF)和差热扫描分析(DSC)对材料进行成分和物相分析；力
学测试仪测试材料的力学性能；扫描电镜(SEM)观察材料的微观结构；并测试
了材料中各种成分的含量和孔隙率。结果如下：
① 光谱学分析表明三种材料中的骨矿成分主要是碳酸盐羟基磷灰石，Ca/P摩
尔比为1． 95-2． 03。骨的有机成分主要是胶原，脂肪和细胞成分被完全脱除。
② 力学性能测试表明CBCM的拉伸杨氏模量为5956±1259 Mpa，其压缩模量
达到12． 47±7． 64Gpa，TBCM和TBCM-PDLLA的压缩模量达到90-100 Mpa，
与原骨的力学性能相近。表明整个加工没有对材料的力学性能造成太大的影响，
重庆大学博士学位论文
PDLLA的加入有助于提高松质骨材料的力学强度。
③扫描电镜观察表明，CBCM的孔径为10一40阿，TBcM和TBcM一PDLLA
的孔径为200~500脚，且孔壁光滑，孔隙连通较好。液体置换法测得TBCM和
TBCM一PDLLA的孔隙率为50%以上，而CBCM的孔隙率低于10%。
(3)将材料与大鼠成骨细胞在体外复合培养，形态学和组织学观察发现成骨
细胞能够在材料上粘附并保持其原有形态。MTT检测结果表明成骨细胞能够在材
料上正常伸展和增殖，证明材料无明显细胞毒性。碱性磷酸酶(ALP)和成骨细胞
分泌钙质的测定结果显示复合培养的成骨细胞能够正常分化和分泌细胞外基质，
反映材料具有较好的生物相容性，不损害细胞的生物功能;各项指标均显示松质
骨材料明显优于皮质骨材料，而TBCM一PDLLA材料略优于TBCM材料。
(4)设计兔双侧挠骨骨干缺损模型，将材料植入体内双侧挠骨和背部骸棘肌
下，通过大体观察、组织学观察、ELISA和局部细胞免疫测定等方法，研究了材
料在体内的毒性反应和免疫排斥反应。结果表明材料均无细胞毒性，但具有较低
的免疫原性，在兔体内引起免疫反应的大小为TBCM>TBCM一PDLLA>CBCM，
由于免疫反应持续时间较短，未超出动物的耐受能力，不会对机体的生长造成大
的伤害。
(5)以兔双侧挠骨骨干缺损模型对各种材料的体内成骨性能进行研究，通过
X线片和组织学分析研究材料的成骨能力。结果显示三种材料的成骨能力大小依次
为:TBCM一PDLLA、TBCM)CBCM，统计学分析表明TBCM一PDLLA与TBCM
的成骨能力无显著性差异。在术后12周:TBCM一PDLLA与TBCM组骨缺损区
被新骨替代而修复;CBCM组有新生骨组织生成，骨缺损区部分被修复;空白对
照组缺损未被修复。各组材料主要以传导成骨方式实现骨的“爬行替代”，材料的表
面结构和多孔结构对成骨能力有重要影响。TBCM一PDLLA与TBCM具有天然多
孔的结构特点，能够为成骨细胞提供良好的微环境，有利于细胞粘附生长和营养
物质进入，适宜新骨的长入，可以作为骨移植材料和骨组织工程细胞外基质材料
使用。CBCM材料的力学强度使之有可能用于节段性骨缺损的修复，但其孔隙率
和降解速度大大影响了材料的成骨能力。
Developing of a suitable bone graft materials to replace the lost region of bone has been a formidable challenge in orthopaedics and biomaterials research. The materials currently used for bone defect repair, including autografts and allografts as well as synthetic bone substitutes, each has its own shortcomings and difficult to completely meet the need of clinical applications. Bone tissue engineering is a new research area, it provides solutions for generating a new bone tissue with good functional on a large scale, the extracellular matrix (ECM) plays a pivotal role on current bone tissue engineering. Xenogenic bone is easy to obtain and has lower cost than autogeneic or allogeneic bone graft. Therefor, it can be a potential alternatives for bone grafting materials and ECM materials for bone tissue engineering.
In this work, a novel processing technique of bone collagen matrix (BCM) has been developed. A series of biological and chemical treatment procedure including defatting, deproteination, partial decalcification, extraction with guanidine-HCl, digestion with trypsin were applied to remove the strongly antigenic proteins and the cellular elements of bovine bone. A wide range of analytical methods was used to investigate the properties of these prepared materials, the cytocompatibility in vitro and the histocompatibility in vivo were studied. The main works and conclusions are included as follows:
(1) Bovine bones were obtained from a local slaughterhouse and used in fresh. The bones were stripped of muscle and fat, cleaned of periosteum, demarrowed by pressure with cold water, and stored at -20 C, Bone Collagen Matrix (BCM) was prepared as follows. The precut frozen bone cubes were first thawed in water at 50C, then soaked in dilute NaOH at ambient temperature, followed by a thorough rinse in the running water. They were then refluxed in a mixture of defatting agent, subsequently, the cubes were demineralized by 0.5 mol/1 HC1, and extracted to release noncollagenous proteins with guanidine-HCl, followed by a digestion with trypsin. Finally, the samples were dried at 50 C in vacuo, packed and sterilized by gamma irradiation.
Two types BCM involving TBCM and CBCM were prepared as described above, and TBCM-PDLLA composite material was prepared by mergeing PDLLA into TBCM. The tryptophan content of the matrices is used as an indicator of noncollagenous protein, the hydroxyproline content dissolved in the solution is used as an marker of the
distroyed degree of bone collagens, and the analysis of X-ray diffraction (XRD) is used to evaluate the effect on structure of the bone mineral. It is showed that the tryptophan content of two kinds bone cubes near to 2mg/g after extracted with guanidine-HCl for 24h, and less than lmg/g after digested with trypsin. Non-helical telopeptide regions of collagen which are thought to be responsible for the immunogenicity can be removed by digestion with trypsin, and the rigid triple-helical structure keep intact, the mass content of collagen dissolved is less than 1%. XRD analysis of demineralized bone show that both the main component of materials and the crystals of HA had no significant change.
(2) The bone specimens were characterized by various of analytical techniques involving infared spectroscopy (FTIR), XRD, differential scanning calorimetry (DSC), energy dispersive X-ray fluorescence (EDXRF), mechanical tests and scanning electron microscopy (SEM), and the composition and the porosity were estimated. The results as follows:
(1) The spectroscopy indicated that the major component of the bone blocks was carbonated hydroxyapatite, and the ratio of Ca/P was 1.95~2.03, whereas the major organic component was collagen, the fatty and cellular components were completely eliminated.
(2) The results of mechanical tests showed that the tensile Young's modulus value of CBCM was 5956+1259 Mpa, and the compressive Young's modulus value was 12.47+7.64Gpa. Whereas that of TBCM or TBCM-PDLLA was about 90~100 Mpa. These close to that of original native bovine bone respectively. Th
目前使用的修复材料都存在不同的缺陷，难以完全满足临床需要。骨组织工程学
为大范围骨缺损的修复开辟了新的渠道，细胞外基质材料的开发是其中的关键。
天然异种骨具有来源丰富、价格低廉等优点，是骨组织工程细胞外基质材料和骨
缺损移植材料的潜在来源之一。
本文主要研究一种新型骨胶原基质(bone collagen matrix,BCM)的制备方法，
采用优化的脱脂、部分脱钙、盐酸胍浸泡、胰蛋白酶消化等一系列生物和化学方
法对牛骨进行脱抗原处理，对所得材料的各项理化性能进行测试，并进行体外细
胞相容性和体内的组织相容性实验研究。主要研究内容和结论如下：
(1) 屠宰场获得新鲜牛骨，经适当前处理后，采用如下的方法制备BCM：
加工好的骨块→50℃热水反复冲洗→室温稀碱液浸泡→脱脂剂脱脂→部分脱钙→
脱非胶原蛋白→胰蛋白酶消化→50℃真空干燥→消毒→密封包装，备用。
通过上述方法最终制备出两种BCM材料，即松质骨胶原基质(Trabecular Bone
Collagen Matrix，TBCM)和皮质骨胶原基质(Cortical Bone Collagen Matrix，
CBCM)，TBCM与PDLLA复合成TBCM-PDLLA复合材料。采用色氨酸含量作
为非胶原蛋白去除效果的评价指标，以羟脯氨酸的含量来衡量胶原的破坏程度，
并以XRD结果来定性分析部分脱钙对骨矿结构的影响。结果表明经HC1胍处理
24h后两种骨块中的色氨酸浓度接近2mg／g，胰蛋白酶消化后降低到1mg／g左右。
胰蛋白酶消化能够切除胶原蛋白具有免疫原性的端肽部分，其三螺旋结构得以保
留，脱蛋白后胶原溶出的比例低于1％。XRD结果表明经过盐酸部分脱钙，骨组
织的主要的晶相成分保持不变，骨矿的晶体结构也没有发生显著变化。
(2) 付立叶红外光谱(FTIR)、x-射线衍射光谱(XRD)、能量散射X-射
线荧光光谱(EDXRF)和差热扫描分析(DSC)对材料进行成分和物相分析；力
学测试仪测试材料的力学性能；扫描电镜(SEM)观察材料的微观结构；并测试
了材料中各种成分的含量和孔隙率。结果如下：
① 光谱学分析表明三种材料中的骨矿成分主要是碳酸盐羟基磷灰石，Ca/P摩
尔比为1． 95-2． 03。骨的有机成分主要是胶原，脂肪和细胞成分被完全脱除。
② 力学性能测试表明CBCM的拉伸杨氏模量为5956±1259 Mpa，其压缩模量
达到12． 47±7． 64Gpa，TBCM和TBCM-PDLLA的压缩模量达到90-100 Mpa，
与原骨的力学性能相近。表明整个加工没有对材料的力学性能造成太大的影响，
重庆大学博士学位论文
PDLLA的加入有助于提高松质骨材料的力学强度。
③扫描电镜观察表明，CBCM的孔径为10一40阿，TBcM和TBcM一PDLLA
的孔径为200~500脚，且孔壁光滑，孔隙连通较好。液体置换法测得TBCM和
TBCM一PDLLA的孔隙率为50%以上，而CBCM的孔隙率低于10%。
(3)将材料与大鼠成骨细胞在体外复合培养，形态学和组织学观察发现成骨
细胞能够在材料上粘附并保持其原有形态。MTT检测结果表明成骨细胞能够在材
料上正常伸展和增殖，证明材料无明显细胞毒性。碱性磷酸酶(ALP)和成骨细胞
分泌钙质的测定结果显示复合培养的成骨细胞能够正常分化和分泌细胞外基质，
反映材料具有较好的生物相容性，不损害细胞的生物功能;各项指标均显示松质
骨材料明显优于皮质骨材料，而TBCM一PDLLA材料略优于TBCM材料。
(4)设计兔双侧挠骨骨干缺损模型，将材料植入体内双侧挠骨和背部骸棘肌
下，通过大体观察、组织学观察、ELISA和局部细胞免疫测定等方法，研究了材
料在体内的毒性反应和免疫排斥反应。结果表明材料均无细胞毒性，但具有较低
的免疫原性，在兔体内引起免疫反应的大小为TBCM>TBCM一PDLLA>CBCM，
由于免疫反应持续时间较短，未超出动物的耐受能力，不会对机体的生长造成大
的伤害。
(5)以兔双侧挠骨骨干缺损模型对各种材料的体内成骨性能进行研究，通过
X线片和组织学分析研究材料的成骨能力。结果显示三种材料的成骨能力大小依次
为:TBCM一PDLLA、TBCM)CBCM，统计学分析表明TBCM一PDLLA与TBCM
的成骨能力无显著性差异。在术后12周:TBCM一PDLLA与TBCM组骨缺损区
被新骨替代而修复;CBCM组有新生骨组织生成，骨缺损区部分被修复;空白对
照组缺损未被修复。各组材料主要以传导成骨方式实现骨的“爬行替代”，材料的表
面结构和多孔结构对成骨能力有重要影响。TBCM一PDLLA与TBCM具有天然多
孔的结构特点，能够为成骨细胞提供良好的微环境，有利于细胞粘附生长和营养
物质进入，适宜新骨的长入，可以作为骨移植材料和骨组织工程细胞外基质材料
使用。CBCM材料的力学强度使之有可能用于节段性骨缺损的修复，但其孔隙率
和降解速度大大影响了材料的成骨能力。
Developing of a suitable bone graft materials to replace the lost region of bone has been a formidable challenge in orthopaedics and biomaterials research. The materials currently used for bone defect repair, including autografts and allografts as well as synthetic bone substitutes, each has its own shortcomings and difficult to completely meet the need of clinical applications. Bone tissue engineering is a new research area, it provides solutions for generating a new bone tissue with good functional on a large scale, the extracellular matrix (ECM) plays a pivotal role on current bone tissue engineering. Xenogenic bone is easy to obtain and has lower cost than autogeneic or allogeneic bone graft. Therefor, it can be a potential alternatives for bone grafting materials and ECM materials for bone tissue engineering.
In this work, a novel processing technique of bone collagen matrix (BCM) has been developed. A series of biological and chemical treatment procedure including defatting, deproteination, partial decalcification, extraction with guanidine-HCl, digestion with trypsin were applied to remove the strongly antigenic proteins and the cellular elements of bovine bone. A wide range of analytical methods was used to investigate the properties of these prepared materials, the cytocompatibility in vitro and the histocompatibility in vivo were studied. The main works and conclusions are included as follows:
(1) Bovine bones were obtained from a local slaughterhouse and used in fresh. The bones were stripped of muscle and fat, cleaned of periosteum, demarrowed by pressure with cold water, and stored at -20 C, Bone Collagen Matrix (BCM) was prepared as follows. The precut frozen bone cubes were first thawed in water at 50C, then soaked in dilute NaOH at ambient temperature, followed by a thorough rinse in the running water. They were then refluxed in a mixture of defatting agent, subsequently, the cubes were demineralized by 0.5 mol/1 HC1, and extracted to release noncollagenous proteins with guanidine-HCl, followed by a digestion with trypsin. Finally, the samples were dried at 50 C in vacuo, packed and sterilized by gamma irradiation.
Two types BCM involving TBCM and CBCM were prepared as described above, and TBCM-PDLLA composite material was prepared by mergeing PDLLA into TBCM. The tryptophan content of the matrices is used as an indicator of noncollagenous protein, the hydroxyproline content dissolved in the solution is used as an marker of the
distroyed degree of bone collagens, and the analysis of X-ray diffraction (XRD) is used to evaluate the effect on structure of the bone mineral. It is showed that the tryptophan content of two kinds bone cubes near to 2mg/g after extracted with guanidine-HCl for 24h, and less than lmg/g after digested with trypsin. Non-helical telopeptide regions of collagen which are thought to be responsible for the immunogenicity can be removed by digestion with trypsin, and the rigid triple-helical structure keep intact, the mass content of collagen dissolved is less than 1%. XRD analysis of demineralized bone show that both the main component of materials and the crystals of HA had no significant change.
(2) The bone specimens were characterized by various of analytical techniques involving infared spectroscopy (FTIR), XRD, differential scanning calorimetry (DSC), energy dispersive X-ray fluorescence (EDXRF), mechanical tests and scanning electron microscopy (SEM), and the composition and the porosity were estimated. The results as follows:
(1) The spectroscopy indicated that the major component of the bone blocks was carbonated hydroxyapatite, and the ratio of Ca/P was 1.95~2.03, whereas the major organic component was collagen, the fatty and cellular components were completely eliminated.
(2) The results of mechanical tests showed that the tensile Young's modulus value of CBCM was 5956+1259 Mpa, and the compressive Young's modulus value was 12.47+7.64Gpa. Whereas that of TBCM or TBCM-PDLLA was about 90~100 Mpa. These close to that of original native bovine bone respectively. Th
引文
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[30] 李涛，李幼平，杨志明．猪到人异种移植抗原与移植免疫研究．中国修复重建外科杂志. 1998，12(1) ：42-47
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[35] 夏烈文．异种(猪)骨酶法处理及用作骨修复材料的研究(重庆大学硕士学位论文)．重庆
市：重庆大学，2002：13-19
[36] 吴多能，林坚平，陈剑飞，等．生物性骨载体的制备．生物医学工程学杂志，2000，17(3) ： 346-347
[37] 夏烈文，王远亮，何创龙等．酶处理制备力学性能优异的骨修复材料．中国临床康复． 2003，7(26) ：3537-3539
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[41] 罗卓荆，胡蕴玉．新型块形脱脂去抗原异种骨移植材料及其制备方法．中国专利：ZL 97108538． 2，2001
[42] Urist M R, Strates B S. Bone formation in implants of partially and wholly demineralized bone matrix. Clin Orthop, 1970, 71: 271-272
[43] Zhang Q, Couru O, Delloye C H. Ethylene oxide dose not extinguish the osteoinductive capacity of demineralized bone. Acta Orthop Scand. 1997,68: 104-108
[44] 何去华，赵小瑾．流动注射化学发光法测定L-色氨酸．汉中师范学院学报．2000，18(2) ： 49-52
[45] 张奇，徐巧娣，沈冬莲．应用HPLC进行流动荧光注射测定饲料中的色氨酸．氨基酸与生 物资源．1997，19(2) ：52-55
[46] 雷绍荣，胡谟彪，罗玲．饲料中色氨酸含量的测定．化验与检测1997，18(12) ：31-32
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[49] Sikavitsas V, Temenoff J S, Mikos A G Biomaterials and bone mechanotransduction. Biomaterials, 2001, 22: 2581-2593
[50] 杨志明．组织工程．北京：化学工业出版社．2002：290-300
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[52] Stark M, Rauterberg J, Kuhn K. Evidence for a non-helical region at the carboxyl terminus of
the collagen molecule. FEBSLett. 1971, 13: 101-104
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