rhBMP-2/GMS/CPC复合材料促进骨缺损愈合的实验研究
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摘要
一、研究背景
骨缺损的修复是骨科临床常见和多发的棘手难题之一，传统的自体及异体骨移植一直仍为首选，但均存在各自的缺点。磷酸钙骨水泥(calcium phosphatecements，CPCs)作为新型的骨修复材料，具有可注射、塑形好、骨传导性强等优点，是一种新型自固化人工骨替代材料，但较慢的吸收降解率阻碍了其广泛应用；目前人们在骨组织工程的研究中，期望rhBMP-2的参与能获得比较理想的骨组织再生。但由于将BMP单纯植入机体内扩散太快，并且BMP易被蛋白酶水解，故不能在有效的时间内作用于更多靶细胞，生物学效能难以充分发挥，故BMP的临床应用需要一定的缓释载体。虽然通过将CPC直接负载骨生长因子治疗骨缺损效果良好，但CPC的因子释放能力有限。微球用于药物载体的研究始于20世纪70年代中期，自此以后的研究发展十分迅速。目前生长因子及可降解的微球载体材料生物学的最新成就己经成功运用于软骨、骨以及其他相关组织的组织工程研究中。可降解微球携载因子后复合磷酸钙骨水泥，在微球降解成孔的同时使因子释放率大大增加，明显加速复合材料的降解和诱导成骨。目前主要以PLGA材料制备微球，但它仍存在较为缓慢的降解率及降解产物呈酸性易损伤周围组织细胞的缺点，而明胶微球生物相容性好，降解后形成胶原，无毒副作用，有望代替PLGA作为磷酸钙骨水泥的良好改性材料。
二、研究目的
采用无毒、可生物降解、生物相容性好的明胶作为载体材料，优化的双相乳化冷凝聚合法制备rhBMP-2/GMS，对其形态、粒径、包封率、载药率进行检测，测定其体外因子缓释曲线，进一步将其负载于磷酸钙骨水泥，制备多孔载因子CPC，在骨缺损模型体内，采用“生物力学特性-X线观察-组织形态”的综合手段，评估新型复合人工骨材料对骨缺损愈合的作用。
三、研究内容和方法
1基因重组人骨形态发生蛋白-2/明胶微球(rhBMP-2/GMs)的制备及性质检测根据天然明胶材料经京尼平处理后可交联成球及可固化的特性，采用改进的双相乳化冷凝聚合法制备rhBMP-2/GMs ;检测rhBMP-2/GMs载药率及包封率，扫描电镜观察rhBMP-2/GMs形态及粒径。
rhBMP-2/GMs体外释药实验研究：以10ml无菌PBS作为释药介质，加入适当浓度的rhBMP-2/GMS溶液1ml，混合均匀后，放置于37℃恒温水浴振荡器中，记录释药起始时间。分别于第1-10天吸取释药介质5ml，ELISA法测试药物含量，并同时补充等量5ml介质，测量每个时间段所释放的药物总量，进行百分比统计，描绘药物累积释药曲线。
根据rhBMP-2/GMs载药率和包封率计算出rhBMP-2含量后，以相同比例与CPC混合，分别将rhBMP-2/GMs/CPC组及rhBMP-2/CPC组各以5ml生理盐水作为释药介质，分别于1、4、7、14、21、28天收集全部浸提液，ELISA法测定rhBMP-2的浓度，从而计算出各个时间点的累积释药量，描绘累计释药曲线。
2.rhBMP-2/GMs/CPC体内降解、诱导成骨实验
制备复合rhBMP-2/GMS/CPC(A组)、GMS/CPC (B组)和rhBMP-2/CPC (C组)，分别植入兔桡骨骨缺损模型中，术后6、12周行大体、X线检查、骨密度检查，术后12周处死动物，收集标本，分别行生物力学测定，脱钙切片、HE染色，不脱钙切片进行荧光显微镜下观察双标间距，计算平均矿化率。
3.统计学处理
实验结果以x±s表示，组间差异比较分别采用方差分析和q检验，P<0.05为差异显著，P<0.01为差异特别显著。
四、结果
1 rhBMP-2/GMs性质检测结果.
形态及粒径：经图像分析仪检测，圆形规则，直径为78±20μm，平均直径为87.8μm。扫描电镜观察固化后的微球表面均匀、圆整，微球之间粘连少，分散性好。
载药率及包封率：微球内药物的包封率80.5%，微球载药率为9.8%，即每毫克微球中含98μg。
稳定性观察：明胶微球在放置于4℃的恒温冰柜6个月以后，倒置显微镜下观察其外观、形态明显无变化，无明显粘连，再分散性良好，置于生理盐水中后成乳状液，散布均匀，无沉淀。
体外释药实验: rhBMP-2/GMs微球释药结果表明微球的体外释药符合双相动力学释药规律，即初相为快速释药相，后相为平稳缓释相。体外释药曲线可见约70%的rhBMP-2在10天左右释放完成。两种复合材料对比释药结果表明：rhBMP-2/GMs/CPC组的24小时体外释放率为8.3±1.5%，随后28天内逐渐缓慢释放，不同时间点的释放率均高于rhBMP-2/CPC组。
2 .rhBMP-2/GMs/CPC体内降解、诱导成骨实验
X线检查：术后第12周，A组骨缺损修复明显快于B组、C组，且材料降解较B组快。骨密度检测显示术后6周三组未见明显差异，但12周后A组明显高于B、C组。组织病理学提示：术后12周材料周围、邻近区域A组的平均骨矿化率均明显高于B组及C组。HE染色见术后12周A组复合材料大部分降解，内部可见大量新生骨组织生成，新生骨组织的形成量明显高于B组、C组，不脱钙切片观察：A组材料骨界面见双条状新骨荧光着色带且距离较宽，新骨生成较多；B组见骨界面材料荧光着色极少，距离最窄；而C组片状着色且距离相对较窄，说明材料周围新骨生成相对不稳定。
五、结论
1．探索出:rhBMP-2/GMs的最佳制备方法，所得方法制备工艺稳定，重复性好。
2．rhBMP-2/GMs及rhBMP-2/GMs/CPC体外释药实验均证明其具有良好的缓释效果。
3. rhBMP-2/GMs复合于磷酸钙骨水泥上的诱导成骨及降解效果分别优于GMS/CPC和rhBMP-2/CPC。
4．复合rhBMP-2明胶微球缓释系统在骨组织工程研究中具有广阔的应用前景。
Background
The management of bone defects has always been a challenge in clinicalperiodontics. The traditional autogenous and allograft always are first selections,but there are many limitations with the materials used in the operation. calciumphosphate cements，(CPCs) is a new bone substitute material with manyadvangtanges，but it’s widely use was set back by slow degradation. rhBMP-2 caninduce the regeneration of bone，Therefore，the application of rhBMP-2 is expectedto obtain more realistic bone regeneration.When BMP is transplanted into the body，too rapid diffusion and hydrolyzation restricts its osteoinduction and its applicationon the target cells. Therefore，the clinical application of BMP must have sustainedrelease carrier. Normal bone defects can heal fast with rhBMP-2/CPC，but therelease of the CPC factor capacity is limited. The study of microspheres as drugcarriers begun in the middle term of 1970s，after that，microspheres as drug delivery system have draw much attention in pharmaceutical field and have beensuccessfully applied in some clinical cases. When degradable micropheres loadedwith growth factors are mixed with CPC to construct a macroporous composite，therelease ability and degradation rate of the composite can be enhanced. At presentpeople mainly use PLGA material as microspheres,but it has some limitations: slowdecomposition rate and degradation products can damage the surrounding tissue andcells.GMs are biocompatible and controlled-degradable.GMs may replace PLGAmicrospheres as good modified materials.
Objective
The carrier material for preparation of microspheres is gelatin，which holdssuch characteristic as excellent biodegradability，low or no toxicity andbiodegradability. On the basis of gelatin's self-crosslinking and its solidification byaldehyde,rhBMP-2/GMs were prepared by improved emulsificationchemical-crosslinking method according to orthogonal factorization optimizationmethod. Many other methods were used to evaluate morphologic property，encapsulation rate，ectopia osteogenesis，drug release property of rhBMP-2/GMs.To evaluate the healing effects of the new artificial composite bone in bone defectmodels, using the "biomechanical characteristics - X-ray observations - theorganization form" comprehensive evaluation methods.
Methods
1 Preparation and quality examination of rhBMP-2/GMs
1.1 On the basis of gelatin's self-crosslinking and its solidification by aldehyde，rhBMP-2/GMs were prepared by improved emulsification chemica crosslinkingmethod according to orthogonal factorization optimization method.
1.2 Evaluate the drug content，encapsulation rate，morphologic property and microsphere-size with a scanning electron microscope.
1.3 Investigation of rhBMP-2-releaseing characters in vitro. Phosphate buffer salinesolution was regard as rhBMP-2-releaseing medium,and numerical data wererecorded at different time.The curve of accumulative rhBMP-2-releaseing ofrhBMP-2-GMS in vitro was describeed.The leaching liquor of rhBMP-2/GMs/CPCwas collected after the composites had been soaked in the physiological saline for1、4、7、14、21 and 28 days，and the release of rhBMP-2 was measured by ELISAmethod.
2 Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vivio.
Macroporous CPC was developed using GMs with 5 wt% weight ratios. ThreeCPC loaded rhBMP-2/GMs，GMS，rhBMP-2 were fabricated and implanted intothe bone defect of rabbits radius. After 6，12 weeks，the defects were detected byX-ray and dual energy X-rayabsorption meter. After 12weeks，the rabbits werekilled. rhBMP-2/GMs/CPC，GMS/CPC and rhBMP-2/CPC were obtained andhistopathological method was used to research the bone ingrowth and the materialdegradation.Osteo inductive ability and degradation rate of three scaffolds wasevaluated histologically.
3 Statistical methods: Measurement data were presented as mean x±s standarddeviation and analyzed by q test and ANOVA，respectively. Significance differencedefined，as P value is less than 0.05
Results
1.Quality examination of rhBMP-2/GMs
1.1 The microspheres were spherical in shape and easily dispersible in character，with a diameter of 78±20μm，(average diameter 87.2μm).
1.2 The drug content and encapsulation rate were 9.8% and 80.5%，respectively.
1.3 The stability and redispersity of GMs were fairly excellent.
1.4 rhBMP-2-releasing kinetics could be divided into two stages: the fast-releasestage and the sustained-release stage. 70% of total loaded rhBMP-2 was released inthe first ten days. The release of different time points of rhBMP-2/GMs/CPCwashigher than rhBMP - 2 / CPC group.
2.Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vitro.
X-ray analysis showed that bone defect healing of group A was the fastest，andremiain-volume of group A was smaller than these of group B and C.Both CPCloaded rhBMP-2 and rhBMP-2/GMs could induce bone formation. Significantlymore bone was found in rhBMP-2/GMs/CPC group comparing with GMS/CPCgroup and rhBMP-2/CPC group.Histomorphologically，the amount of new bonewas observed deeper into the rhBMP-2/GMs/CPC composite，but little resorption ofrhBMP-2/CPC could be detected.Mineralization rate and bone density showed nodifference between three groups after 6 weeks，but after 12 weeks the values werehightest in the group A. New bones grew into the cores of the rhBMP-2/GMs/CPCwhich resulted from GMs degradation and rhBMP-2 bone induction
Conclusion
1 The best method of preparation of rhBMP-2/GMs was worked out，and thisstudy indicated that the technology of preparing rhBMP-2/GMs was stable andrepeatable.
2 The experiments of rhBMP-2-releasing kinetics indicated that rhBMP-2/GMs andrhBMP-2/GMs/CPC possessed excellent sustained-release character.
3 The degradation performance and bone induction abilities of CPC loadedrhBMP-2/GMs were stronger than those of application of rhBMP-2/CPC and GMs/CPC.
4 Microspheres that of CPC loaded rhBMP-2. sustained-release system will bepossible to apply in bone tissue engineering in the future.
骨缺损的修复是骨科临床常见和多发的棘手难题之一，传统的自体及异体骨移植一直仍为首选，但均存在各自的缺点。磷酸钙骨水泥(calcium phosphatecements，CPCs)作为新型的骨修复材料，具有可注射、塑形好、骨传导性强等优点，是一种新型自固化人工骨替代材料，但较慢的吸收降解率阻碍了其广泛应用；目前人们在骨组织工程的研究中，期望rhBMP-2的参与能获得比较理想的骨组织再生。但由于将BMP单纯植入机体内扩散太快，并且BMP易被蛋白酶水解，故不能在有效的时间内作用于更多靶细胞，生物学效能难以充分发挥，故BMP的临床应用需要一定的缓释载体。虽然通过将CPC直接负载骨生长因子治疗骨缺损效果良好，但CPC的因子释放能力有限。微球用于药物载体的研究始于20世纪70年代中期，自此以后的研究发展十分迅速。目前生长因子及可降解的微球载体材料生物学的最新成就己经成功运用于软骨、骨以及其他相关组织的组织工程研究中。可降解微球携载因子后复合磷酸钙骨水泥，在微球降解成孔的同时使因子释放率大大增加，明显加速复合材料的降解和诱导成骨。目前主要以PLGA材料制备微球，但它仍存在较为缓慢的降解率及降解产物呈酸性易损伤周围组织细胞的缺点，而明胶微球生物相容性好，降解后形成胶原，无毒副作用，有望代替PLGA作为磷酸钙骨水泥的良好改性材料。
二、研究目的
采用无毒、可生物降解、生物相容性好的明胶作为载体材料，优化的双相乳化冷凝聚合法制备rhBMP-2/GMS，对其形态、粒径、包封率、载药率进行检测，测定其体外因子缓释曲线，进一步将其负载于磷酸钙骨水泥，制备多孔载因子CPC，在骨缺损模型体内，采用“生物力学特性-X线观察-组织形态”的综合手段，评估新型复合人工骨材料对骨缺损愈合的作用。
三、研究内容和方法
1基因重组人骨形态发生蛋白-2/明胶微球(rhBMP-2/GMs)的制备及性质检测根据天然明胶材料经京尼平处理后可交联成球及可固化的特性，采用改进的双相乳化冷凝聚合法制备rhBMP-2/GMs ;检测rhBMP-2/GMs载药率及包封率，扫描电镜观察rhBMP-2/GMs形态及粒径。
rhBMP-2/GMs体外释药实验研究：以10ml无菌PBS作为释药介质，加入适当浓度的rhBMP-2/GMS溶液1ml，混合均匀后，放置于37℃恒温水浴振荡器中，记录释药起始时间。分别于第1-10天吸取释药介质5ml，ELISA法测试药物含量，并同时补充等量5ml介质，测量每个时间段所释放的药物总量，进行百分比统计，描绘药物累积释药曲线。
根据rhBMP-2/GMs载药率和包封率计算出rhBMP-2含量后，以相同比例与CPC混合，分别将rhBMP-2/GMs/CPC组及rhBMP-2/CPC组各以5ml生理盐水作为释药介质，分别于1、4、7、14、21、28天收集全部浸提液，ELISA法测定rhBMP-2的浓度，从而计算出各个时间点的累积释药量，描绘累计释药曲线。
2.rhBMP-2/GMs/CPC体内降解、诱导成骨实验
制备复合rhBMP-2/GMS/CPC(A组)、GMS/CPC (B组)和rhBMP-2/CPC (C组)，分别植入兔桡骨骨缺损模型中，术后6、12周行大体、X线检查、骨密度检查，术后12周处死动物，收集标本，分别行生物力学测定，脱钙切片、HE染色，不脱钙切片进行荧光显微镜下观察双标间距，计算平均矿化率。
3.统计学处理
实验结果以x±s表示，组间差异比较分别采用方差分析和q检验，P<0.05为差异显著，P<0.01为差异特别显著。
四、结果
1 rhBMP-2/GMs性质检测结果.
形态及粒径：经图像分析仪检测，圆形规则，直径为78±20μm，平均直径为87.8μm。扫描电镜观察固化后的微球表面均匀、圆整，微球之间粘连少，分散性好。
载药率及包封率：微球内药物的包封率80.5%，微球载药率为9.8%，即每毫克微球中含98μg。
稳定性观察：明胶微球在放置于4℃的恒温冰柜6个月以后，倒置显微镜下观察其外观、形态明显无变化，无明显粘连，再分散性良好，置于生理盐水中后成乳状液，散布均匀，无沉淀。
体外释药实验: rhBMP-2/GMs微球释药结果表明微球的体外释药符合双相动力学释药规律，即初相为快速释药相，后相为平稳缓释相。体外释药曲线可见约70%的rhBMP-2在10天左右释放完成。两种复合材料对比释药结果表明：rhBMP-2/GMs/CPC组的24小时体外释放率为8.3±1.5%，随后28天内逐渐缓慢释放，不同时间点的释放率均高于rhBMP-2/CPC组。
2 .rhBMP-2/GMs/CPC体内降解、诱导成骨实验
X线检查：术后第12周，A组骨缺损修复明显快于B组、C组，且材料降解较B组快。骨密度检测显示术后6周三组未见明显差异，但12周后A组明显高于B、C组。组织病理学提示：术后12周材料周围、邻近区域A组的平均骨矿化率均明显高于B组及C组。HE染色见术后12周A组复合材料大部分降解，内部可见大量新生骨组织生成，新生骨组织的形成量明显高于B组、C组，不脱钙切片观察：A组材料骨界面见双条状新骨荧光着色带且距离较宽，新骨生成较多；B组见骨界面材料荧光着色极少，距离最窄；而C组片状着色且距离相对较窄，说明材料周围新骨生成相对不稳定。
五、结论
1．探索出:rhBMP-2/GMs的最佳制备方法，所得方法制备工艺稳定，重复性好。
2．rhBMP-2/GMs及rhBMP-2/GMs/CPC体外释药实验均证明其具有良好的缓释效果。
3. rhBMP-2/GMs复合于磷酸钙骨水泥上的诱导成骨及降解效果分别优于GMS/CPC和rhBMP-2/CPC。
4．复合rhBMP-2明胶微球缓释系统在骨组织工程研究中具有广阔的应用前景。
Background
The management of bone defects has always been a challenge in clinicalperiodontics. The traditional autogenous and allograft always are first selections,but there are many limitations with the materials used in the operation. calciumphosphate cements，(CPCs) is a new bone substitute material with manyadvangtanges，but it’s widely use was set back by slow degradation. rhBMP-2 caninduce the regeneration of bone，Therefore，the application of rhBMP-2 is expectedto obtain more realistic bone regeneration.When BMP is transplanted into the body，too rapid diffusion and hydrolyzation restricts its osteoinduction and its applicationon the target cells. Therefore，the clinical application of BMP must have sustainedrelease carrier. Normal bone defects can heal fast with rhBMP-2/CPC，but therelease of the CPC factor capacity is limited. The study of microspheres as drugcarriers begun in the middle term of 1970s，after that，microspheres as drug delivery system have draw much attention in pharmaceutical field and have beensuccessfully applied in some clinical cases. When degradable micropheres loadedwith growth factors are mixed with CPC to construct a macroporous composite，therelease ability and degradation rate of the composite can be enhanced. At presentpeople mainly use PLGA material as microspheres,but it has some limitations: slowdecomposition rate and degradation products can damage the surrounding tissue andcells.GMs are biocompatible and controlled-degradable.GMs may replace PLGAmicrospheres as good modified materials.
Objective
The carrier material for preparation of microspheres is gelatin，which holdssuch characteristic as excellent biodegradability，low or no toxicity andbiodegradability. On the basis of gelatin's self-crosslinking and its solidification byaldehyde,rhBMP-2/GMs were prepared by improved emulsificationchemical-crosslinking method according to orthogonal factorization optimizationmethod. Many other methods were used to evaluate morphologic property，encapsulation rate，ectopia osteogenesis，drug release property of rhBMP-2/GMs.To evaluate the healing effects of the new artificial composite bone in bone defectmodels, using the "biomechanical characteristics - X-ray observations - theorganization form" comprehensive evaluation methods.
Methods
1 Preparation and quality examination of rhBMP-2/GMs
1.1 On the basis of gelatin's self-crosslinking and its solidification by aldehyde，rhBMP-2/GMs were prepared by improved emulsification chemica crosslinkingmethod according to orthogonal factorization optimization method.
1.2 Evaluate the drug content，encapsulation rate，morphologic property and microsphere-size with a scanning electron microscope.
1.3 Investigation of rhBMP-2-releaseing characters in vitro. Phosphate buffer salinesolution was regard as rhBMP-2-releaseing medium,and numerical data wererecorded at different time.The curve of accumulative rhBMP-2-releaseing ofrhBMP-2-GMS in vitro was describeed.The leaching liquor of rhBMP-2/GMs/CPCwas collected after the composites had been soaked in the physiological saline for1、4、7、14、21 and 28 days，and the release of rhBMP-2 was measured by ELISAmethod.
2 Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vivio.
Macroporous CPC was developed using GMs with 5 wt% weight ratios. ThreeCPC loaded rhBMP-2/GMs，GMS，rhBMP-2 were fabricated and implanted intothe bone defect of rabbits radius. After 6，12 weeks，the defects were detected byX-ray and dual energy X-rayabsorption meter. After 12weeks，the rabbits werekilled. rhBMP-2/GMs/CPC，GMS/CPC and rhBMP-2/CPC were obtained andhistopathological method was used to research the bone ingrowth and the materialdegradation.Osteo inductive ability and degradation rate of three scaffolds wasevaluated histologically.
3 Statistical methods: Measurement data were presented as mean x±s standarddeviation and analyzed by q test and ANOVA，respectively. Significance differencedefined，as P value is less than 0.05
Results
1.Quality examination of rhBMP-2/GMs
1.1 The microspheres were spherical in shape and easily dispersible in character，with a diameter of 78±20μm，(average diameter 87.2μm).
1.2 The drug content and encapsulation rate were 9.8% and 80.5%，respectively.
1.3 The stability and redispersity of GMs were fairly excellent.
1.4 rhBMP-2-releasing kinetics could be divided into two stages: the fast-releasestage and the sustained-release stage. 70% of total loaded rhBMP-2 was released inthe first ten days. The release of different time points of rhBMP-2/GMs/CPCwashigher than rhBMP - 2 / CPC group.
2.Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vitro.
X-ray analysis showed that bone defect healing of group A was the fastest，andremiain-volume of group A was smaller than these of group B and C.Both CPCloaded rhBMP-2 and rhBMP-2/GMs could induce bone formation. Significantlymore bone was found in rhBMP-2/GMs/CPC group comparing with GMS/CPCgroup and rhBMP-2/CPC group.Histomorphologically，the amount of new bonewas observed deeper into the rhBMP-2/GMs/CPC composite，but little resorption ofrhBMP-2/CPC could be detected.Mineralization rate and bone density showed nodifference between three groups after 6 weeks，but after 12 weeks the values werehightest in the group A. New bones grew into the cores of the rhBMP-2/GMs/CPCwhich resulted from GMs degradation and rhBMP-2 bone induction
Conclusion
1 The best method of preparation of rhBMP-2/GMs was worked out，and thisstudy indicated that the technology of preparing rhBMP-2/GMs was stable andrepeatable.
2 The experiments of rhBMP-2-releasing kinetics indicated that rhBMP-2/GMs andrhBMP-2/GMs/CPC possessed excellent sustained-release character.
3 The degradation performance and bone induction abilities of CPC loadedrhBMP-2/GMs were stronger than those of application of rhBMP-2/CPC and GMs/CPC.
4 Microspheres that of CPC loaded rhBMP-2. sustained-release system will bepossible to apply in bone tissue engineering in the future.
引文
1.Brown WE,Chow LC.A new calcium phosphate,water-setting cement.In:Brown PW Ed.Cement Research Progress.ohio:American Ceramic Society.1986:325-329
2.周馨,郑昌琼,王方瑚,等.骨水泥及磷酸钙生物活性骨水泥.硅酸盐通报.1998;5:33-37
3.Lacout JL,Mejdoub E,Hamadm.Crystallization mechanisms of calcium phosphate cement for biological uses.J T Mater Sci 1996;7:371
4. Friedman CD, Costantino P D, Jones K, et al.Hydroxyapitite Cement. J Arch Otolaryngol Head NeckSurg 1991;117~385.
5.Li Qing,Ouyang Jia-hu,Lei Ting-quan.Setting reactionsand compressive strengths of calcium phosphat cement.J Material Science and Technology 1996; 4(4):22~36
6. DelRealRP , Wolke JG , Vallet-RegiM , et al . A new method to producemacropores in calcium phosphate cements.J Biomaterials 2002;23(7): 3673-3680
7.BohnerM. Calcium orthophosphates in medicine: From ceramics to calcium phosphate cements. J Injury 2000;31 ( Suppl 4 ):SD37-SD47.
8.Guo F, Li B.Recent developments of researches on calcium phosphate cements.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.2009;26(1):202-205
9.Fernandez E,Vald MD,Gel MM,et al.Modulation of porosity in apatitic cements by the use of alpha-tricalcium phosphate-calcium sulphate dihy drate mixtures. Biomaterials 2005;26(17):3395-3404
10.Theiss F,Apelt D,Brand B,et al.Biocompatibility and resorption of a brushite calcium phosphate cement.Biomaterials 2005;26(21):4383-4394
11.Lu J,Descamps M,Dejon J,et al.The biodegradation mechanism of calciumphosphate biomaterials in bone. J Biomed Mater Res 2002;63(4):408-412
12.Ooms EM, Wolke JG, van de Heuvel MT,et al.Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone.Biomaterials 2003;24:989-1000
13.Liu C,Wang W,ShenW,et al.Evaluation of the biocompatibility of a nonceramic hydroxyapatite.J Endod 1997;23:490-493
14.del Real RP,Ooms E,Wolke JG,et al.In vivo bone response to porous calcium phosphate cement.J Biomed Master Res A 2003;65(1):30-36
15.Fernandez E,Vald M D,Gel M M,et al.Modulation of porosity in apatitic cement by the use of the alpha-tricalcium phosphate-calcium sulphate dihydrate mixtures.Biomaterials 2005;26(17):3395-3404
16.Ito A, Kawamura H, Otsuka M, et al.Mater Sci Eng C 2002; 22(1),21-25
17.Ishikaw K,Miyamoto Y, Yuasa T, et al.Biomaterials 2002; 23(2):423-428
18.Kim HW, Koh YH, Kong YM, et al.Strontium substituted calcium phosphate biphasic ceramics obtained by a powder precipitation method. J Mater Sci Mater Med 2004;15:1129-1134
19.Lacout JL,Leroux L.Mater Res 2001; 16(1);171-178
20.陈德敏.傅飞远,顾国珍,等.中国生物医学工程学报2001; 20(3),27-28
21.Kroese-Deutman HC,Ruhe PQ,Spanwen PH,et al.Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site inrabbis.J Biomaterials 2005;10:1131-1138.
22.孙明林,胡蕴玉,张瑞萍,等.磷酸钙骨水泥/骨形态发生蛋白复合人工骨修复骨缺损的实验研究.现代康复2001;5(8):32-33.
23.Seeherman HJ,Azari K,Bidic S,et al.rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radius.JBone Joint Surg Am 2006;7:1553-1565.
24 .Jansen JA,Vehof JW,Ruhe PQ,et al.Growth factor-loaded scaffolds for bone engineering.J Control Release 2005;1(3):127-136.
25. Blom EJ,Klein-Nulend J,Wolke JG,et al.Transforming growth factor-betal incorporation in an alpha-tricalcium phosphate J .Biomaterials 2002;4:1261-1268.
26.Xu HH,Quinn JB.Calcium phosphate cement containing resorable fibers for short-term reinforcement and macroporosity.Biomaterials 2002;23(1):193-202
27.Meraw SJ, Reeve CM, Lohse CM. et al.Treatment of perimplant defects with combination growth factor cement.J Periodontol 2000;71:8-13.
28.Habraken WJ,Wolke JG,Mikos AG,et al.Injectable PLGA microsphere/calcium phosphate cements:physical properties and degradation characteristics.J Biomater Sci Polym Ed 2006;17(9):1057-74
29.Ruhe PQ,Hedberg-Dirk et al.Padron NT,et al.Porous poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composite for reconstuction of bone defects.Tissure Eng 2006;12(4):789-800
30.蔡舒,王彦伟,姚康德.骨水泥自固化法制备磷酸钙多孔材料.硅酸盐学报2004;32:1174-1177
31.潘朝晖,范清宇,蔡和平,等.明胶对自固化骨水泥性能影响.医学研究生学报2005;18(8):696-699
32. Liang HC,Chang WH,Lin KJ,et al.Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration:in vivo and in vivo studiea.J Biomed Mater Rres A 2003;65(2):271-282
33.厉孟,刘旭东,刘兴炎.京尼平与戊二醛交联明胶微球的性能比较.中国修复重建外科杂志2009;23(1):87-92
34. Simon CG Jr,Khatri CA,Wight SA,et al.Preliminary report on thebiocompatibility of a moldable，resorbable. composite bone graft consisting ofcalcium phosphate cement and poly（lactide-co-glycol-ide) microspheres J OrthopRes. 2002;20(3):473~482
35.Fei Z,Hu Y,Wu D,et al．Preparation and property of a novel bone graft compositeconsisiting of rhBMP-2 loaded PLGA microspheres and calcium phosphatecement．J Mater Sci:mater Med 2008;19(3):1109-16
36.Link DP,Van den Dolder J,van den Beucken JJ,et al．Bone response andmechanical strenth of a rabbit femoral defects filled with injectable CaP cementscontaining TGF-β1 loaded gelatin microparticles．Biomaterials 2008;29(6):675-82
37董方言．现代实用中医药新剂型新技术.北京：人民卫生出版社，2001;341-343
38费正奇，胡蕴玉，吴道澄．携载rhBMP-2微球的新型可注射自凝固复合人工骨的制备及特性研究.中国矫形外科杂志2006；14(2):124-138
39.Ruhe PQ，Boerman OC，Russel OC，et al．Controlled release of rhBMP-2 loadedpoly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo．JControl Release 2005;106(1-2):162-171
40.费正奇，胡蕴玉，张德志．携载rhBMP-2微球的新型可注射自凝固复合人工骨的释药及成骨活性研究．中华实验外科杂志2006；23(2):151-153
41.厉孟，刘旭东，刘兴炎,等．京尼平与戊二醛交联明胶微球的性能比较．中国修复重建外科杂志2009;23(1):87-91
42.厉孟，刘旭东，刘兴炎等．多孔明胶微球/磷酸钙骨水泥的制备及性能研究．中国矫形外科杂志2009;7:526-529
43.侯锐．生长因子及其不同缓释系统在骨组织工程中的应用．第四军医大学学位论文（博士），2004
44.陆扬．明胶微球的研究进展(1)．明胶科学与技术2006;26(2)：57-68
45. Liang HC，Chang WH,Lin KJ,et al．Genipin-crosslingked gelatin microspheresas a drug carrier for intramuscular administration:in vitro and in vivo studies．JBiomed Master Res A 2003;65(2):271-282
46.Clokie CM, Moghadam H, Jackson MT, et al．Closure of sized defects withallogenic and alloplastic bone substitutes. J fac Surg, 2002;13(1):111
47.MoghadamHG, Sandor GK, HolmesHH, et al．Histomorphevaluation of boneregeneration using allogeneic and alloplast substitutes. J Oral Maxillofac Surg,2004;62(2): 202
48.仲荣洲，费琴明，张键．载rhBMP-2人工骨促进家兔脊柱融合实验研究．中国修复重建外科杂志2009;23(2)：231-234
49.刘兴炎，厉孟，刘旭东，等．rhBMP-2/CPC对骨质疏松骨缺损的治疗作用的研究．中国骨质疏松杂志2009;15(1)：16-19
50.孙明林，胡蕴玉．磷酸钙骨水泥的研究和应用进展．中华骨科杂志2002;22(1):49-52
51.白波，吴显培，徐谦．新型可注射磷酸钙骨水泥对成骨作用影响的实验研究．中华创伤骨科杂志2006;8(6)：557-561
52.孙明林，胡蕴玉．磷酸钙骨水泥作为骨形成蛋白修复阶段性骨缺损及相关研究．中华骨科杂志2003;23(2):114-119
53.LU L,Stamatas GN,Mikos AG．Controlled release of transforming growth factorβ1 from biodegradable polymer microparticles．J Biomed Mater Res2000;50:440-451
54.XU HHK,Janet B．Calcium phosphate cement containing resorbable fibers forshort-term reinforcement and macroporosity．J Biomaterials 2002;23:193-202.
55.Woo BH，Fink BF，Page R．Enhancement of bone growth by sustaied deliveryof recombinant human bone morphogenetic protein-2 in a polymeric matrix．J
2.周馨,郑昌琼,王方瑚,等.骨水泥及磷酸钙生物活性骨水泥.硅酸盐通报.1998;5:33-37
3.Lacout JL,Mejdoub E,Hamadm.Crystallization mechanisms of calcium phosphate cement for biological uses.J T Mater Sci 1996;7:371
4. Friedman CD, Costantino P D, Jones K, et al.Hydroxyapitite Cement. J Arch Otolaryngol Head NeckSurg 1991;117~385.
5.Li Qing,Ouyang Jia-hu,Lei Ting-quan.Setting reactionsand compressive strengths of calcium phosphat cement.J Material Science and Technology 1996; 4(4):22~36
6. DelRealRP , Wolke JG , Vallet-RegiM , et al . A new method to producemacropores in calcium phosphate cements.J Biomaterials 2002;23(7): 3673-3680
7.BohnerM. Calcium orthophosphates in medicine: From ceramics to calcium phosphate cements. J Injury 2000;31 ( Suppl 4 ):SD37-SD47.
8.Guo F, Li B.Recent developments of researches on calcium phosphate cements.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.2009;26(1):202-205
9.Fernandez E,Vald MD,Gel MM,et al.Modulation of porosity in apatitic cements by the use of alpha-tricalcium phosphate-calcium sulphate dihy drate mixtures. Biomaterials 2005;26(17):3395-3404
10.Theiss F,Apelt D,Brand B,et al.Biocompatibility and resorption of a brushite calcium phosphate cement.Biomaterials 2005;26(21):4383-4394
11.Lu J,Descamps M,Dejon J,et al.The biodegradation mechanism of calciumphosphate biomaterials in bone. J Biomed Mater Res 2002;63(4):408-412
12.Ooms EM, Wolke JG, van de Heuvel MT,et al.Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone.Biomaterials 2003;24:989-1000
13.Liu C,Wang W,ShenW,et al.Evaluation of the biocompatibility of a nonceramic hydroxyapatite.J Endod 1997;23:490-493
14.del Real RP,Ooms E,Wolke JG,et al.In vivo bone response to porous calcium phosphate cement.J Biomed Master Res A 2003;65(1):30-36
15.Fernandez E,Vald M D,Gel M M,et al.Modulation of porosity in apatitic cement by the use of the alpha-tricalcium phosphate-calcium sulphate dihydrate mixtures.Biomaterials 2005;26(17):3395-3404
16.Ito A, Kawamura H, Otsuka M, et al.Mater Sci Eng C 2002; 22(1),21-25
17.Ishikaw K,Miyamoto Y, Yuasa T, et al.Biomaterials 2002; 23(2):423-428
18.Kim HW, Koh YH, Kong YM, et al.Strontium substituted calcium phosphate biphasic ceramics obtained by a powder precipitation method. J Mater Sci Mater Med 2004;15:1129-1134
19.Lacout JL,Leroux L.Mater Res 2001; 16(1);171-178
20.陈德敏.傅飞远,顾国珍,等.中国生物医学工程学报2001; 20(3),27-28
21.Kroese-Deutman HC,Ruhe PQ,Spanwen PH,et al.Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site inrabbis.J Biomaterials 2005;10:1131-1138.
22.孙明林,胡蕴玉,张瑞萍,等.磷酸钙骨水泥/骨形态发生蛋白复合人工骨修复骨缺损的实验研究.现代康复2001;5(8):32-33.
23.Seeherman HJ,Azari K,Bidic S,et al.rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radius.JBone Joint Surg Am 2006;7:1553-1565.
24 .Jansen JA,Vehof JW,Ruhe PQ,et al.Growth factor-loaded scaffolds for bone engineering.J Control Release 2005;1(3):127-136.
25. Blom EJ,Klein-Nulend J,Wolke JG,et al.Transforming growth factor-betal incorporation in an alpha-tricalcium phosphate J .Biomaterials 2002;4:1261-1268.
26.Xu HH,Quinn JB.Calcium phosphate cement containing resorable fibers for short-term reinforcement and macroporosity.Biomaterials 2002;23(1):193-202
27.Meraw SJ, Reeve CM, Lohse CM. et al.Treatment of perimplant defects with combination growth factor cement.J Periodontol 2000;71:8-13.
28.Habraken WJ,Wolke JG,Mikos AG,et al.Injectable PLGA microsphere/calcium phosphate cements:physical properties and degradation characteristics.J Biomater Sci Polym Ed 2006;17(9):1057-74
29.Ruhe PQ,Hedberg-Dirk et al.Padron NT,et al.Porous poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composite for reconstuction of bone defects.Tissure Eng 2006;12(4):789-800
30.蔡舒,王彦伟,姚康德.骨水泥自固化法制备磷酸钙多孔材料.硅酸盐学报2004;32:1174-1177
31.潘朝晖,范清宇,蔡和平,等.明胶对自固化骨水泥性能影响.医学研究生学报2005;18(8):696-699
32. Liang HC,Chang WH,Lin KJ,et al.Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration:in vivo and in vivo studiea.J Biomed Mater Rres A 2003;65(2):271-282
33.厉孟,刘旭东,刘兴炎.京尼平与戊二醛交联明胶微球的性能比较.中国修复重建外科杂志2009;23(1):87-92
34. Simon CG Jr,Khatri CA,Wight SA,et al.Preliminary report on thebiocompatibility of a moldable，resorbable. composite bone graft consisting ofcalcium phosphate cement and poly（lactide-co-glycol-ide) microspheres J OrthopRes. 2002;20(3):473~482
35.Fei Z,Hu Y,Wu D,et al．Preparation and property of a novel bone graft compositeconsisiting of rhBMP-2 loaded PLGA microspheres and calcium phosphatecement．J Mater Sci:mater Med 2008;19(3):1109-16
36.Link DP,Van den Dolder J,van den Beucken JJ,et al．Bone response andmechanical strenth of a rabbit femoral defects filled with injectable CaP cementscontaining TGF-β1 loaded gelatin microparticles．Biomaterials 2008;29(6):675-82
37董方言．现代实用中医药新剂型新技术.北京：人民卫生出版社，2001;341-343
38费正奇，胡蕴玉，吴道澄．携载rhBMP-2微球的新型可注射自凝固复合人工骨的制备及特性研究.中国矫形外科杂志2006；14(2):124-138
39.Ruhe PQ，Boerman OC，Russel OC，et al．Controlled release of rhBMP-2 loadedpoly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo．JControl Release 2005;106(1-2):162-171
40.费正奇，胡蕴玉，张德志．携载rhBMP-2微球的新型可注射自凝固复合人工骨的释药及成骨活性研究．中华实验外科杂志2006；23(2):151-153
41.厉孟，刘旭东，刘兴炎,等．京尼平与戊二醛交联明胶微球的性能比较．中国修复重建外科杂志2009;23(1):87-91
42.厉孟，刘旭东，刘兴炎等．多孔明胶微球/磷酸钙骨水泥的制备及性能研究．中国矫形外科杂志2009;7:526-529
43.侯锐．生长因子及其不同缓释系统在骨组织工程中的应用．第四军医大学学位论文（博士），2004
44.陆扬．明胶微球的研究进展(1)．明胶科学与技术2006;26(2)：57-68
45. Liang HC，Chang WH,Lin KJ,et al．Genipin-crosslingked gelatin microspheresas a drug carrier for intramuscular administration:in vitro and in vivo studies．JBiomed Master Res A 2003;65(2):271-282
46.Clokie CM, Moghadam H, Jackson MT, et al．Closure of sized defects withallogenic and alloplastic bone substitutes. J fac Surg, 2002;13(1):111
47.MoghadamHG, Sandor GK, HolmesHH, et al．Histomorphevaluation of boneregeneration using allogeneic and alloplast substitutes. J Oral Maxillofac Surg,2004;62(2): 202
48.仲荣洲，费琴明，张键．载rhBMP-2人工骨促进家兔脊柱融合实验研究．中国修复重建外科杂志2009;23(2)：231-234
49.刘兴炎，厉孟，刘旭东，等．rhBMP-2/CPC对骨质疏松骨缺损的治疗作用的研究．中国骨质疏松杂志2009;15(1)：16-19
50.孙明林，胡蕴玉．磷酸钙骨水泥的研究和应用进展．中华骨科杂志2002;22(1):49-52
51.白波，吴显培，徐谦．新型可注射磷酸钙骨水泥对成骨作用影响的实验研究．中华创伤骨科杂志2006;8(6)：557-561
52.孙明林，胡蕴玉．磷酸钙骨水泥作为骨形成蛋白修复阶段性骨缺损及相关研究．中华骨科杂志2003;23(2):114-119
53.LU L,Stamatas GN,Mikos AG．Controlled release of transforming growth factorβ1 from biodegradable polymer microparticles．J Biomed Mater Res2000;50:440-451
54.XU HHK,Janet B．Calcium phosphate cement containing resorbable fibers forshort-term reinforcement and macroporosity．J Biomaterials 2002;23:193-202.
55.Woo BH，Fink BF，Page R．Enhancement of bone growth by sustaied deliveryof recombinant human bone morphogenetic protein-2 in a polymeric matrix．J