纳米双相陶瓷人工骨的制备和实验研究
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摘要
目的
为获得有良好降解性能及成骨性能的人工骨，采用化学沉淀法制备纳米羟基磷灰石／磷酸三钙双相陶瓷人工骨并对人工骨的结构、成骨性能以及降解性能进行研究，将人工骨植入骨缺损，进行人工骨促进骨折愈合方面的临床研究，同时进行恒古速效骨伤愈的辅助促进骨折愈合的研究。研究。研究内容包括：(1)探索纳米双相陶瓷人工骨的制备方法并进行表征；(2)研究纳米双相陶瓷人工骨的生物相容性并进行评价；(3)研究纳米双相陶瓷人工骨在生物体内的降解性能；(4)进行纳米双相陶瓷人工骨的修复兔桡骨骨缺损的实验研究；(5)应用纳米双相陶瓷人工骨于临床及恒古速效骨伤愈辅助促进骨折愈合的研究。
方法
1、纳米双相陶瓷人工骨的制备
采用磷酸钠和氯化钙并用氢氧化钠控制溶液pH值的情况下制备人工骨支架材料。进行支架材料的X线晶体衍射(XRD)，形貌分析(SEM)，复合材料孔径和孔隙率的测定以及生物力学测试。
2、纳米双相陶瓷人工骨的生物相容性
按照国家的(GB／T)16886系列标准对人工骨进行溶血试验、皮内刺激试验、致敏试验和急性全身毒性试验。
3、纳米双相陶瓷人工骨的降解实验
将纳米双相陶瓷人工骨植入新西兰兔肌袋内通过大体和组织学观察人工骨的降解情况。
4、将纳米双相陶瓷人工骨植入兔桡骨骨缺损内，通过大体观察、成骨情况、组织学观察以及电镜观察，反应人工骨的降解情况。
5、将纳米双相陶瓷人工骨植入患者骨缺损部位；患者采用恒古骨伤愈合剂术后第二天开始服用，成人每次25 ml每次0.5 ml／kg，摇匀服用，隔日服药1次，12天为1个疗程，共2个疗程。观察患者的疼痛、肿胀缓解和骨折愈合情况。
结果
1、所得XRD图谱表明，粉体由羟基磷灰石和磷酸三钙两相组成，该粉体的钙磷摩尔比约为1.67。经计算可知：该粉体中羟基磷灰石与磷酸三钙的质量百分比为70／30；电镜扫描材料为蜂窝状多孔结构，颗粒约为100nm左右。分析结果表明，纳米双相陶瓷人工骨孔隙率为81.5％左右；测得其平均抗压强度为5.5MPa。
2、生物相容性实验
2．1、人工骨试验材料的溶血率为0.72％，远低于5％的规定值，该材料不会引起急性溶血。
2．2、通过计算得出试验材料浸提液的原发刺激指数为0.167，属于无刺激反应类型。
2．3、出现过敏反应的动物数为0／10，表明材料不引起致敏反应。
2．4、材料浸提液腹腔注射后，经24h、48h和72h连续观察，试验组情况良好，活动、食欲正常，呼吸平稳，无惊厥、瘫痪和死亡现象，试验动物未见毒性表现。
3、体内降解实验
4W时纳米双相陶瓷人工骨降解率约为10％。8W时，纳米双相陶瓷人工骨降解约25％，体积较4W时减小，内部材料失去规整的外观，形状不规则，表面仍被软组织包围。12W时纳米双相陶瓷人工骨降解约35％，材料形状、体积明显减小。
4、人工骨植入兔桡骨骨缺损实验
实验组术后4周材料周围可见一层结缔组织膜包绕，术后8周可见材料明显降解，材料有新骨形成，12周可见材料部分被骨松质所取代。X线表现4周时材料有少许降解，未见明显新生骨生成，8周时植入材料有明显降解，可见有新生骨形成，12周时可见材料大部分降解，连续性骨痂通过骨缺损部位。X线骨形成及塑形定量分析结果统计学分析显示：纳米双相陶瓷人工骨内：4周＜8周＜12周，差异有显著性意义。组织学检查实验组：植入4周后材料孔隙内有成骨细胞，成软骨细胞及成纤维母细胞，材料孔隙内纤维结缔组织增生，8周后有大量骨细胞，成骨细胞和软骨细胞，以成骨细胞和软骨细胞为主伸向材料孔隙中，孔隙内以纤维增生，材料部分降解，12周后以骨细胞和成骨细胞为主，有少量软骨细胞，出现散乱的骨松质，材料大部分降解。成骨情况组织学定量分析统计学分析结果显示：实验组内各个时期在期间比较，4周＜8周＜12周，差异有显著性意义。扫描电镜观察实验组：4周可见支架孔隙完整，材料内有纤维组织填充。8周材料部分降解，有新生骨组织及纤维组织填充，12周后材料部分降解，骨缺损部分被新生骨松质取代。
5、治疗组一周内缓解疼痛效果90.9％，缓解肿胀效果90.9％，治疗组临床愈合时间为95.7天。
结论
1、采用化学沉淀方法所制备的人工骨达到纳米水平的双相(包括羟基磷灰石和磷酸三钙)陶瓷人工骨，具有三维多孔结构以及和松质骨相似的抗压强度。
2、纳米双相羟基磷灰石具有良好的生物相容性，植入体内后安全无毒，无致敏性，是一种比较安全的生物材料。
3、纳米双相羟基磷灰石具有一定的降解率，植入体内后进行性降解。
4、新型纳米双相陶瓷人工骨通过植入兔桡骨后显示，该支架有良好的生物相容性，植入体内安全无毒，未见明显炎症反应，骨组织再生后，支架材料逐渐降解，最终被新生的骨组织所取代，材料采用的高孔隙率结构，可有效引导骨再生。
5、临床上显示纳米双相陶瓷人工骨能够有效引导骨再生，恒古骨伤愈合剂能够较快地缓解骨折后肿胀，疼痛，促进骨折愈合。
Objects
For the purpose of obtaining a good degradation and osteogenicproperties of artificial bone,Prepare biphasic nano-ceramic artificial bone bynano-technology and study it, carry out simultaneously in promoting fracturehealing medicine: (1) Study preparation methods of biphasic nano-ceramicartificial bone and characterization; (2) Research biphasic nano-ceramicartificial bone biocompatibility; (3) Research biphasic nano-ceramic artificialbone degradation; (4) Research biphasic nano-ceramic artificial bone to repairbone defects in rabbits experimental study; (5) Application of nanotechnologyin biphasic ceramic bone in clinical and heng gu su xiao gu shang yu onfracture healing studies.
Methods
1、Nano-biphasic ceramic bone preparation: the use of sodium andcalcium chloride in the PH value controlled by the sodium hydroxide.Detection of the X-ray diffraction, Analysis of the IR, Dtermination of thecomposite materials aperture and porosity, biomechanical testing.
2、Biocompatibility of the biphasic ceramic bone: According tonational standards, test of the hemolysis, stimulation and sensitization, acutesystemic toxicity.
3、Implant the biphasic nano-ceramic artificial bone in muscle bag and studythe degradation of artificial bone by general observation and histologicalobservation.
4、Artificial bone was implanted in bone defects and study the degradationof artificial bone by general observation, osteoblasts, histologicalobservation,and electron microscopy.
5、The biphasic nano-ceramic artificial bone was implanted inbone defect site; Hncient bone healing agent after beginning the second day oftaking it 25 ml per adult per 0.5 ml / kg) shake taking it one day, medicationtimes 12 days for a course of treatment, taking two courses. Observation ofpatients with pain, swelling and fracture healing to alleviate the situation.
Results
1、XRD patterns showed that the powder compose hydroxyapatite andtricalcium phosphate, the powder is 1.67 molar ratio of calcium andphosphorus. After calculations, It can be seen: the quality of the percentageof 70/30; Scanning electron microscopy of porous honeycomb structure,Nano-particles is about 100 nanometer;Analysis results showed that nanoceramic artificial bone's porosity is about 81.5%; The average compressivestrength is about 5.5MPa.
2.1、Test Materials hemolysis rate was 0.72 percent, well below the 5% of thevalue, the material will not cause acute hemolysis.
2.2、The stimulation index by calculating the Materials extraction is 0.167,and belong to no stimulus-response type.
2.3、Emergenge of allergic reactions of animals is 0 / 10, show that thematerial does not cause allergic reactions.
2.4、After intraperitoneal injection and 24h, 48h and 72h continuousobservation, the experimental group in good condition, activity, appetite normal, smooth breathing, no convulsions, paralysis and death situation, Theexperimental animals demonstrated no toxicity.
3、In vivo degradation experiments, the nano-ceramic artificial bonedegradation is 10% in 4W, , the nano-biphasic ceramic bone degradation byabout 25% in 8W, The volume decreases, the internal material lose regularityof appearance and irregular shape, the surface still being surrounded by softtissue; The artificial bone degradation by about 35% in 12W, material shapeand size significantly reduced.
4、After 4 weeks,the material enveloped by membrane in experimental group;After 8 weeks, Materials can be seen clearly degradation, there is new boneformation Materials, Materials shows that was replaced by cancellous boneafter 12 weeks. X-ray showed there is some degradation of the material after 4weeks, There is obvious degradation of implant after 8 weeks;We can seethere is new bone formation after 12 weeksThe results of statistical analysis of the X-ray bone formation and remodelingquantitative showed: Nano-ceramic artificial bone: 4 weeks＜8 weeks＜12weeks, the difference was significant. Histological examination on theexperimental group: After 4 weeks the bone porosity were jncludingOsteoblasts and chondrocytes and fibroblast cells, There were materialsdesmoplastic porosity with the fiber, the bone cells, osteoblasts andchondrocytes came into porous materials, the fibrous is hyperplasia in porosity,and material are part of degradation, After 12 weeks, There are a smallnumber of cartilage cells, appear scattered cancellous bone.The bonehistology quantitative analysis of statistical analysis show: The experimentalgroup at various times during the period compared to 4 weeks＜8 weeks＜12weeks, the difference was significant. Scanning electron microscope testgroup: 4 weeks can be seen stent porosity integrity, material has fibrous tissuefiller. Degradation of Materials Part 8 weeks, there is new bone tissue and fibrous tissue filling, 12 weeks later material degradation, bone defect site wasreplaced by cancellous bone.
5、The pain relief effect is 90.9% in the treatment group within 1 week, Theswelling of the effect is 90.9% in the treatment group, Clinical healing time is95.7 days.
Conclusions:
1、Using chemical precipitation method achieve nano-level duplex(including hydroxyapatite and tricalcium phosphate), with three-dimensionalporous structure,The artificial bone is similar to the compressive strength ofthe cancellous bone.
2、Nano artificial bone has good biocompatibility, It is a relatively safebiological materials with non-toxic, non-allergenic in vivo.
3、Nano artificial bone is a certain degree of degradation rate, and degradeafter implantation in vivo.
4、New nano-biphasic ceramic bone implants in rabbits reveal that the stenthas good biocompatibility, implants with non-toxic in vivo and no obviousinflammatory response, After bone regeneration, the scaffold materialgradually degraded eventually ,New bone tissue was replaced by thematerial. The material has high porosity that can effectively guided boneregeneration.
5、Clinical display nano biphasic ceramic artificial bone can be effectivelyguided bone regeneration, Henggusuxiaogushangyu can quickly ease thepost-fracture swelling, pain, and promote fracture healing.
为获得有良好降解性能及成骨性能的人工骨，采用化学沉淀法制备纳米羟基磷灰石／磷酸三钙双相陶瓷人工骨并对人工骨的结构、成骨性能以及降解性能进行研究，将人工骨植入骨缺损，进行人工骨促进骨折愈合方面的临床研究，同时进行恒古速效骨伤愈的辅助促进骨折愈合的研究。研究。研究内容包括：(1)探索纳米双相陶瓷人工骨的制备方法并进行表征；(2)研究纳米双相陶瓷人工骨的生物相容性并进行评价；(3)研究纳米双相陶瓷人工骨在生物体内的降解性能；(4)进行纳米双相陶瓷人工骨的修复兔桡骨骨缺损的实验研究；(5)应用纳米双相陶瓷人工骨于临床及恒古速效骨伤愈辅助促进骨折愈合的研究。
方法
1、纳米双相陶瓷人工骨的制备
采用磷酸钠和氯化钙并用氢氧化钠控制溶液pH值的情况下制备人工骨支架材料。进行支架材料的X线晶体衍射(XRD)，形貌分析(SEM)，复合材料孔径和孔隙率的测定以及生物力学测试。
2、纳米双相陶瓷人工骨的生物相容性
按照国家的(GB／T)16886系列标准对人工骨进行溶血试验、皮内刺激试验、致敏试验和急性全身毒性试验。
3、纳米双相陶瓷人工骨的降解实验
将纳米双相陶瓷人工骨植入新西兰兔肌袋内通过大体和组织学观察人工骨的降解情况。
4、将纳米双相陶瓷人工骨植入兔桡骨骨缺损内，通过大体观察、成骨情况、组织学观察以及电镜观察，反应人工骨的降解情况。
5、将纳米双相陶瓷人工骨植入患者骨缺损部位；患者采用恒古骨伤愈合剂术后第二天开始服用，成人每次25 ml每次0.5 ml／kg，摇匀服用，隔日服药1次，12天为1个疗程，共2个疗程。观察患者的疼痛、肿胀缓解和骨折愈合情况。
结果
1、所得XRD图谱表明，粉体由羟基磷灰石和磷酸三钙两相组成，该粉体的钙磷摩尔比约为1.67。经计算可知：该粉体中羟基磷灰石与磷酸三钙的质量百分比为70／30；电镜扫描材料为蜂窝状多孔结构，颗粒约为100nm左右。分析结果表明，纳米双相陶瓷人工骨孔隙率为81.5％左右；测得其平均抗压强度为5.5MPa。
2、生物相容性实验
2．1、人工骨试验材料的溶血率为0.72％，远低于5％的规定值，该材料不会引起急性溶血。
2．2、通过计算得出试验材料浸提液的原发刺激指数为0.167，属于无刺激反应类型。
2．3、出现过敏反应的动物数为0／10，表明材料不引起致敏反应。
2．4、材料浸提液腹腔注射后，经24h、48h和72h连续观察，试验组情况良好，活动、食欲正常，呼吸平稳，无惊厥、瘫痪和死亡现象，试验动物未见毒性表现。
3、体内降解实验
4W时纳米双相陶瓷人工骨降解率约为10％。8W时，纳米双相陶瓷人工骨降解约25％，体积较4W时减小，内部材料失去规整的外观，形状不规则，表面仍被软组织包围。12W时纳米双相陶瓷人工骨降解约35％，材料形状、体积明显减小。
4、人工骨植入兔桡骨骨缺损实验
实验组术后4周材料周围可见一层结缔组织膜包绕，术后8周可见材料明显降解，材料有新骨形成，12周可见材料部分被骨松质所取代。X线表现4周时材料有少许降解，未见明显新生骨生成，8周时植入材料有明显降解，可见有新生骨形成，12周时可见材料大部分降解，连续性骨痂通过骨缺损部位。X线骨形成及塑形定量分析结果统计学分析显示：纳米双相陶瓷人工骨内：4周＜8周＜12周，差异有显著性意义。组织学检查实验组：植入4周后材料孔隙内有成骨细胞，成软骨细胞及成纤维母细胞，材料孔隙内纤维结缔组织增生，8周后有大量骨细胞，成骨细胞和软骨细胞，以成骨细胞和软骨细胞为主伸向材料孔隙中，孔隙内以纤维增生，材料部分降解，12周后以骨细胞和成骨细胞为主，有少量软骨细胞，出现散乱的骨松质，材料大部分降解。成骨情况组织学定量分析统计学分析结果显示：实验组内各个时期在期间比较，4周＜8周＜12周，差异有显著性意义。扫描电镜观察实验组：4周可见支架孔隙完整，材料内有纤维组织填充。8周材料部分降解，有新生骨组织及纤维组织填充，12周后材料部分降解，骨缺损部分被新生骨松质取代。
5、治疗组一周内缓解疼痛效果90.9％，缓解肿胀效果90.9％，治疗组临床愈合时间为95.7天。
结论
1、采用化学沉淀方法所制备的人工骨达到纳米水平的双相(包括羟基磷灰石和磷酸三钙)陶瓷人工骨，具有三维多孔结构以及和松质骨相似的抗压强度。
2、纳米双相羟基磷灰石具有良好的生物相容性，植入体内后安全无毒，无致敏性，是一种比较安全的生物材料。
3、纳米双相羟基磷灰石具有一定的降解率，植入体内后进行性降解。
4、新型纳米双相陶瓷人工骨通过植入兔桡骨后显示，该支架有良好的生物相容性，植入体内安全无毒，未见明显炎症反应，骨组织再生后，支架材料逐渐降解，最终被新生的骨组织所取代，材料采用的高孔隙率结构，可有效引导骨再生。
5、临床上显示纳米双相陶瓷人工骨能够有效引导骨再生，恒古骨伤愈合剂能够较快地缓解骨折后肿胀，疼痛，促进骨折愈合。
Objects
For the purpose of obtaining a good degradation and osteogenicproperties of artificial bone,Prepare biphasic nano-ceramic artificial bone bynano-technology and study it, carry out simultaneously in promoting fracturehealing medicine: (1) Study preparation methods of biphasic nano-ceramicartificial bone and characterization; (2) Research biphasic nano-ceramicartificial bone biocompatibility; (3) Research biphasic nano-ceramic artificialbone degradation; (4) Research biphasic nano-ceramic artificial bone to repairbone defects in rabbits experimental study; (5) Application of nanotechnologyin biphasic ceramic bone in clinical and heng gu su xiao gu shang yu onfracture healing studies.
Methods
1、Nano-biphasic ceramic bone preparation: the use of sodium andcalcium chloride in the PH value controlled by the sodium hydroxide.Detection of the X-ray diffraction, Analysis of the IR, Dtermination of thecomposite materials aperture and porosity, biomechanical testing.
2、Biocompatibility of the biphasic ceramic bone: According tonational standards, test of the hemolysis, stimulation and sensitization, acutesystemic toxicity.
3、Implant the biphasic nano-ceramic artificial bone in muscle bag and studythe degradation of artificial bone by general observation and histologicalobservation.
4、Artificial bone was implanted in bone defects and study the degradationof artificial bone by general observation, osteoblasts, histologicalobservation,and electron microscopy.
5、The biphasic nano-ceramic artificial bone was implanted inbone defect site; Hncient bone healing agent after beginning the second day oftaking it 25 ml per adult per 0.5 ml / kg) shake taking it one day, medicationtimes 12 days for a course of treatment, taking two courses. Observation ofpatients with pain, swelling and fracture healing to alleviate the situation.
Results
1、XRD patterns showed that the powder compose hydroxyapatite andtricalcium phosphate, the powder is 1.67 molar ratio of calcium andphosphorus. After calculations, It can be seen: the quality of the percentageof 70/30; Scanning electron microscopy of porous honeycomb structure,Nano-particles is about 100 nanometer;Analysis results showed that nanoceramic artificial bone's porosity is about 81.5%; The average compressivestrength is about 5.5MPa.
2.1、Test Materials hemolysis rate was 0.72 percent, well below the 5% of thevalue, the material will not cause acute hemolysis.
2.2、The stimulation index by calculating the Materials extraction is 0.167,and belong to no stimulus-response type.
2.3、Emergenge of allergic reactions of animals is 0 / 10, show that thematerial does not cause allergic reactions.
2.4、After intraperitoneal injection and 24h, 48h and 72h continuousobservation, the experimental group in good condition, activity, appetite normal, smooth breathing, no convulsions, paralysis and death situation, Theexperimental animals demonstrated no toxicity.
3、In vivo degradation experiments, the nano-ceramic artificial bonedegradation is 10% in 4W, , the nano-biphasic ceramic bone degradation byabout 25% in 8W, The volume decreases, the internal material lose regularityof appearance and irregular shape, the surface still being surrounded by softtissue; The artificial bone degradation by about 35% in 12W, material shapeand size significantly reduced.
4、After 4 weeks,the material enveloped by membrane in experimental group;After 8 weeks, Materials can be seen clearly degradation, there is new boneformation Materials, Materials shows that was replaced by cancellous boneafter 12 weeks. X-ray showed there is some degradation of the material after 4weeks, There is obvious degradation of implant after 8 weeks;We can seethere is new bone formation after 12 weeksThe results of statistical analysis of the X-ray bone formation and remodelingquantitative showed: Nano-ceramic artificial bone: 4 weeks＜8 weeks＜12weeks, the difference was significant. Histological examination on theexperimental group: After 4 weeks the bone porosity were jncludingOsteoblasts and chondrocytes and fibroblast cells, There were materialsdesmoplastic porosity with the fiber, the bone cells, osteoblasts andchondrocytes came into porous materials, the fibrous is hyperplasia in porosity,and material are part of degradation, After 12 weeks, There are a smallnumber of cartilage cells, appear scattered cancellous bone.The bonehistology quantitative analysis of statistical analysis show: The experimentalgroup at various times during the period compared to 4 weeks＜8 weeks＜12weeks, the difference was significant. Scanning electron microscope testgroup: 4 weeks can be seen stent porosity integrity, material has fibrous tissuefiller. Degradation of Materials Part 8 weeks, there is new bone tissue and fibrous tissue filling, 12 weeks later material degradation, bone defect site wasreplaced by cancellous bone.
5、The pain relief effect is 90.9% in the treatment group within 1 week, Theswelling of the effect is 90.9% in the treatment group, Clinical healing time is95.7 days.
Conclusions:
1、Using chemical precipitation method achieve nano-level duplex(including hydroxyapatite and tricalcium phosphate), with three-dimensionalporous structure,The artificial bone is similar to the compressive strength ofthe cancellous bone.
2、Nano artificial bone has good biocompatibility, It is a relatively safebiological materials with non-toxic, non-allergenic in vivo.
3、Nano artificial bone is a certain degree of degradation rate, and degradeafter implantation in vivo.
4、New nano-biphasic ceramic bone implants in rabbits reveal that the stenthas good biocompatibility, implants with non-toxic in vivo and no obviousinflammatory response, After bone regeneration, the scaffold materialgradually degraded eventually ,New bone tissue was replaced by thematerial. The material has high porosity that can effectively guided boneregeneration.
5、Clinical display nano biphasic ceramic artificial bone can be effectivelyguided bone regeneration, Henggusuxiaogushangyu can quickly ease thepost-fracture swelling, pain, and promote fracture healing.
引文
[1] Langer R., Vacanti J. P. Tissue engineering. Science, 93; 260: 920-926.
[2] Rose F. R. A. J., Oreffo R. O. C. Breakthroughs and views bone tissue engineering:hopevshype. Biochemical and Biophysical Research. Communications, 2002; 292:1-7.
[3] Proussaefs P, Lozada J, Valencia q Rohrer MD. Histologicevaluation of a hydroxyapatite on lay bone graft retrieved after 9 years: a clinical report. J Prosthet Dent, 2002;87: 481-484.
[4] Lewandrowski K, Gresser JD, Wise DL, Trantolo DJ. Bioresorbable bone graft substitutes of different osteoconductivities: an istologic evaluation of osteointegration of poly (propylene glycol-co-fumaric acid) based cement implants in rats. Biomaterials, 2000; 21(8):757-764.
[5] Suchanek W., Yoshimura M. processing, properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J. Mater. Res., 1998;13:94-117.
[6] 孙雪，奚廷斐．第三代生物医学材料与再生医学．中国临床康复，2005；9(26)：109-110．
[7] Lin AS, Barrows TH, CartmellSH etal. Microarchitectural and mechan, characterization of oriented Porous Polymer seaffolds. Biomaterials, 2003; 24: 481-489.
[8] LI S, DeWijnJR, Li J etal. Macro Porous biPhasieealeium PhosPhateseaflbld high Permeability/Porosityratio. Tissue Eng., 2003; 90: 535-548.
[9] 李家顺，吕宏．纳米工程多孔隙高强度磷酸钙人工骨细胞爬行支架对 传统骨移植替代物理念的彻底变革．中国药物与临床，2003；3(3)：174-175．
[10] G. Gollera, H. Demirkirana, F. N. Oktarb, et al. Processing and characterization of bioglass reinforced hydroxyapatite composites. Ceramics Int, 2003; 29: 721 724.
[11] Klein CPAT., de Blieck-Hogervorst JMA., Wolke JGC. A Study of solubility and surface features of different calcium phosphate coatings in vitro and in vivo: In: Ceramics in Substitutive and Reconstructive Surgery. Ed. By Vincenzini P, Elsevier Science Publishers. 1991; 363-373.
[12] Hasegawa S, Tamura J, Neo M, et al. In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for use as a bone substitute. J Biomed Mater Res A, 2005;75(3):567-579.
[13] Du C, Cui FZ, Feng QL, et al. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity. J Biomed Mater Res, 1998;42(4):540-548.
[14] Liao SS, Cui FZ, Zhang W, et al. Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite. J Biomed Mater Res B Appl Biomater, 2004;69(2):158-165.
[15] Wang J S. Basic fibroblast growth factor for stimulation of bone formation in osteoinduction or conductive implants. Acta Orthop Scand, 1996; 67: 1-32.
[16] Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res, 2000;55:15-35.
[17] Pilliar RM, Filiaggi MJ, Wells JD. Porous calcium polyphosphate scaffolds for bone substitute applications in vitro characterization. Biomaterials, 2001;22(9): 963-972.
[18] 肖建德主编．实用骨移植学．北京：科学出版社，2006，497-498．
[19] 何伟，肖建德．复合型纳米羟基磷灰石人骨的研究进展，国际骨科学杂志，2007；4：222-223．
[20] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite:Effect of pore size studied in rabit, Acta. Orthop Scand, 1994; 65: 246-252.
[21] de Groot K. Effect of porosity and physicochemical properties on the stability, resorption, and strength of calcium phosphate ceramics. In E3ioceramics: Material characteristics versus in vivo behavior. Ducheyne P&Lemons J, eds. Annals of NY Acad. Sci.,]988; 523: 268-272.
[22] Legeros R Z. Biodegradation and bioresorption of calcium phosphate ceramics. Clinical Materials, 1993; 14: 65-88.
[23] 俞耀庭，张兴栋．生物医用材料．天津：天津大学出版社，2002，12．
[24] Michael Hadjiargyrou, Frank Lombardo, Shanchuan Zhao, et al. Transcriptional Profiling of Bone Regeneration. J. Biol. Chem., 2002; 277: 30177-3018 2.
[25] J. P. Bilezikian, L. G. Raisz, G. A. Rodan. Principles of Bone Biology. California: Academic Press, 2002;471-494.
[26] Hollinger JO, Brekke J, Gruskin, et al. Role of bone substitutes. Clin. Orthop. 1996; 324: 55-65.
[27] 刘柏龄主编．中医骨伤科学．北京：人民卫生出版社，2003，116．
[1] GUY Daculsi, OLIVIER Laboux, OLIVIR Malard, at al. Current state of the art of biphasic calcium phosphate bioceramics. J Mater Sci: Mater Med, 2003; 14: 195-200.
[2] DACULSI G, PASSUTIN, MARTIN S, et al. Macroporous calcium phosphate ceramic for long hone surgery in humans and dogs: Clinical and histological study. J Biomed Mater lies, 1990; 24(3): 379-396.
[3] LOGEROS R Z, LIN S, ROHANIZADEH R, et al. Biphasic calcium phosphate hioceramics: preparation, properties and applications. J Mater Sci: Mater M ed, 2003; 14: 201-209.
[4] 肖建德主编．实用骨移植学．北京：科学出版社，2006，414-415．
[5] Daiwon Choi, Prashant N. Kumta. An Alternative Chemical Route for the Synthesis and Thermal Stability of Chemically Enriched Hydroxyapatite:The American Ceramic Society, 2006;89(2):444-449.
[6] Loty C, Sautier JM, Boulekbache H, et al. In vitro bone formation on a bone-like apatite layer prepared by a biomimetic process on a bioactive glass-ceramic. J Biomed Mater Res, 2000; 49(4): 423-434.
[7] Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials, 2004; 25(17): 3829-3835.
[8] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite:Effect of pore size studied in rabit, Acta. Orthop Scand, 1994;65:246-252.
[1] ISO 10993: Biological evaluation of medical devices.
[2] 李玉宝主编．生物医学材料．北京：化学工业出版社，2003．
[3] Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin/poly (carbonate)-urethane as a substrate for cell growth: in vitro interactions with human cells. Biomaterials, 2003; 24: 789-799.
[4] Kneser U, Voogd A, Ohnolz J, et al. Fibrin gel-immobilized primary osteoblasts in calcium phos phate bone cement: in vivo evaluation with regard to application as injectable biologics 1 bone substitute. Cells Tissues Organs, 2005; 179(4): 158-169.
[5] 郝和平主编：生物医学材料生物学评价标准实施指南，北京，中国标准出版社，2000．
[1] Pistner H, Gutwald R, Ordung R, et al. Poly(L-lactide): a long-term degradation study in vivo. I. Biological results. Biomaterials. 1993; 14: 671-677。
[2]. Uchida A, Araki N, Shinto Y, et al: The use of calcium hydroxyapatite ceramic in bone tumour surgery. J Bone Joint Surg. 1990; 72B: 298-302。
[3]．郑启新，杜靖远，朱通伯等．多孔磷酸三钙陶瓷人工骨生物降解的实验观察．同济医科大学学报．1996，25(1)：37-40
[4].Sharpe T, adsorption Sammons RL, Marquis PM. Effect of PH on protein to hydroxyapatite and tricalcium phosphateceramics. Biomaterial, 1997;18(6):471-476.
[1] 程顺巧，苟立，季金苟等．双相HA／β-TCP陶瓷的多孔结构对类骨磷灰石形成的影响．现代技术陶瓷，2004；1：14-17
[2] 何伟，肖建德．复合型纳米羟基磷灰石人工骨的研究进展．国际骨科学杂志．2007；28(4)：222-223．
[3] Gutwein LG, Webster TJ. Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles. Biomaterials, 2004; 25(18): 4175-4183.
[4] 张力德．纳米材料，北京，化学工业出版社，2000：39-41．
[5] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite: Effect of pore size studied in rabbit. Acta Orthop Scand, 1994; 65(3): 246-252.
[1] 卢世壁 主译，坎贝尔骨科手术学，济南，山东科学技术出版社，2002，3：2278．
[2] 刘柏龄主编，中医骨伤科学，北京，人民卫生出版社，1998，116．
[3] 刘强，陈君长．重组转化因子TGF-β1的表达及修复骨缺损的研究中国骨伤，2000；13(12)：715-717．
[1] G.Gollera,H.Demirkirana,F.N.Oktarb,et al. Processing and characterization of bioglass reinforced hydroxyapatite composites. Ceramics Int,2003 ; 29: 721 724.
[2] Huang.M,Feng.J.Q,Wang.J.X,et al. Synthesis and characterization of nano-HA/PA66 composite. Journal of materials science: materials in science, 2003; 14: 655-660.
[3] Wei.J, Li.Y.B,Chun.W.Q,et al.A study on nano-composite of hydroxyapatite and polyamide. Journal of materials science,2003; 38:3303-3306
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[7] Murugan R,Rarnakrishna S.Bioresorbable composite bone paste using polysaccharide based nano hydroxypatite (J). Biomaterials, 2004; 25: 3829-3835.
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[14] 卢志华，孙康宁，赵中帆．原位合成制备碳纳米管／羟基磷灰石复合粉体．硅酸盐学报，2007；35(2)：212-221．
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[17] Wang JS. Basic fibroblast growth factor for stimulation of bone formation in osteoinduction or conductive implants, Acta Orthop Scand, 1996; 67: 1-32.
[18] Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res, 2000;55:15-35.
[19] 程顺巧，苟立，季金苟等．双相HA／β-TCP陶瓷的多孔结构对类骨磷灰石形成的影响．现代技术陶瓷，2004；1：14-17．
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[21] Chida A, Nade SML, Mecartney ER, et al. The use of ceramics for bone replacement. Bone jiont. 1984; 66B: 269-275.
[22] Kuhne J,, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite:Effect of pore size studied in rabit, Acta. Orthop Scand, 1994; 65: 246-252.
[23] H. Yuan, JD de Bruijin Y. Li, K. De Groot,,et al. Bone formation induced by calcium phosphate ceramics in soft tissue of dogs:a comparative study between porous alpha-TCP and Beta TCP. J. Materials Science Materials Medicine, 2001;12(1):7-13.
[2] Rose F. R. A. J., Oreffo R. O. C. Breakthroughs and views bone tissue engineering:hopevshype. Biochemical and Biophysical Research. Communications, 2002; 292:1-7.
[3] Proussaefs P, Lozada J, Valencia q Rohrer MD. Histologicevaluation of a hydroxyapatite on lay bone graft retrieved after 9 years: a clinical report. J Prosthet Dent, 2002;87: 481-484.
[4] Lewandrowski K, Gresser JD, Wise DL, Trantolo DJ. Bioresorbable bone graft substitutes of different osteoconductivities: an istologic evaluation of osteointegration of poly (propylene glycol-co-fumaric acid) based cement implants in rats. Biomaterials, 2000; 21(8):757-764.
[5] Suchanek W., Yoshimura M. processing, properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J. Mater. Res., 1998;13:94-117.
[6] 孙雪，奚廷斐．第三代生物医学材料与再生医学．中国临床康复，2005；9(26)：109-110．
[7] Lin AS, Barrows TH, CartmellSH etal. Microarchitectural and mechan, characterization of oriented Porous Polymer seaffolds. Biomaterials, 2003; 24: 481-489.
[8] LI S, DeWijnJR, Li J etal. Macro Porous biPhasieealeium PhosPhateseaflbld high Permeability/Porosityratio. Tissue Eng., 2003; 90: 535-548.
[9] 李家顺，吕宏．纳米工程多孔隙高强度磷酸钙人工骨细胞爬行支架对 传统骨移植替代物理念的彻底变革．中国药物与临床，2003；3(3)：174-175．
[10] G. Gollera, H. Demirkirana, F. N. Oktarb, et al. Processing and characterization of bioglass reinforced hydroxyapatite composites. Ceramics Int, 2003; 29: 721 724.
[11] Klein CPAT., de Blieck-Hogervorst JMA., Wolke JGC. A Study of solubility and surface features of different calcium phosphate coatings in vitro and in vivo: In: Ceramics in Substitutive and Reconstructive Surgery. Ed. By Vincenzini P, Elsevier Science Publishers. 1991; 363-373.
[12] Hasegawa S, Tamura J, Neo M, et al. In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for use as a bone substitute. J Biomed Mater Res A, 2005;75(3):567-579.
[13] Du C, Cui FZ, Feng QL, et al. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity. J Biomed Mater Res, 1998;42(4):540-548.
[14] Liao SS, Cui FZ, Zhang W, et al. Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite. J Biomed Mater Res B Appl Biomater, 2004;69(2):158-165.
[15] Wang J S. Basic fibroblast growth factor for stimulation of bone formation in osteoinduction or conductive implants. Acta Orthop Scand, 1996; 67: 1-32.
[16] Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res, 2000;55:15-35.
[17] Pilliar RM, Filiaggi MJ, Wells JD. Porous calcium polyphosphate scaffolds for bone substitute applications in vitro characterization. Biomaterials, 2001;22(9): 963-972.
[18] 肖建德主编．实用骨移植学．北京：科学出版社，2006，497-498．
[19] 何伟，肖建德．复合型纳米羟基磷灰石人骨的研究进展，国际骨科学杂志，2007；4：222-223．
[20] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite:Effect of pore size studied in rabit, Acta. Orthop Scand, 1994; 65: 246-252.
[21] de Groot K. Effect of porosity and physicochemical properties on the stability, resorption, and strength of calcium phosphate ceramics. In E3ioceramics: Material characteristics versus in vivo behavior. Ducheyne P&Lemons J, eds. Annals of NY Acad. Sci.,]988; 523: 268-272.
[22] Legeros R Z. Biodegradation and bioresorption of calcium phosphate ceramics. Clinical Materials, 1993; 14: 65-88.
[23] 俞耀庭，张兴栋．生物医用材料．天津：天津大学出版社，2002，12．
[24] Michael Hadjiargyrou, Frank Lombardo, Shanchuan Zhao, et al. Transcriptional Profiling of Bone Regeneration. J. Biol. Chem., 2002; 277: 30177-3018 2.
[25] J. P. Bilezikian, L. G. Raisz, G. A. Rodan. Principles of Bone Biology. California: Academic Press, 2002;471-494.
[26] Hollinger JO, Brekke J, Gruskin, et al. Role of bone substitutes. Clin. Orthop. 1996; 324: 55-65.
[27] 刘柏龄主编．中医骨伤科学．北京：人民卫生出版社，2003，116．
[1] GUY Daculsi, OLIVIER Laboux, OLIVIR Malard, at al. Current state of the art of biphasic calcium phosphate bioceramics. J Mater Sci: Mater Med, 2003; 14: 195-200.
[2] DACULSI G, PASSUTIN, MARTIN S, et al. Macroporous calcium phosphate ceramic for long hone surgery in humans and dogs: Clinical and histological study. J Biomed Mater lies, 1990; 24(3): 379-396.
[3] LOGEROS R Z, LIN S, ROHANIZADEH R, et al. Biphasic calcium phosphate hioceramics: preparation, properties and applications. J Mater Sci: Mater M ed, 2003; 14: 201-209.
[4] 肖建德主编．实用骨移植学．北京：科学出版社，2006，414-415．
[5] Daiwon Choi, Prashant N. Kumta. An Alternative Chemical Route for the Synthesis and Thermal Stability of Chemically Enriched Hydroxyapatite:The American Ceramic Society, 2006;89(2):444-449.
[6] Loty C, Sautier JM, Boulekbache H, et al. In vitro bone formation on a bone-like apatite layer prepared by a biomimetic process on a bioactive glass-ceramic. J Biomed Mater Res, 2000; 49(4): 423-434.
[7] Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials, 2004; 25(17): 3829-3835.
[8] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite:Effect of pore size studied in rabit, Acta. Orthop Scand, 1994;65:246-252.
[1] ISO 10993: Biological evaluation of medical devices.
[2] 李玉宝主编．生物医学材料．北京：化学工业出版社，2003．
[3] Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin/poly (carbonate)-urethane as a substrate for cell growth: in vitro interactions with human cells. Biomaterials, 2003; 24: 789-799.
[4] Kneser U, Voogd A, Ohnolz J, et al. Fibrin gel-immobilized primary osteoblasts in calcium phos phate bone cement: in vivo evaluation with regard to application as injectable biologics 1 bone substitute. Cells Tissues Organs, 2005; 179(4): 158-169.
[5] 郝和平主编：生物医学材料生物学评价标准实施指南，北京，中国标准出版社，2000．
[1] Pistner H, Gutwald R, Ordung R, et al. Poly(L-lactide): a long-term degradation study in vivo. I. Biological results. Biomaterials. 1993; 14: 671-677。
[2]. Uchida A, Araki N, Shinto Y, et al: The use of calcium hydroxyapatite ceramic in bone tumour surgery. J Bone Joint Surg. 1990; 72B: 298-302。
[3]．郑启新，杜靖远，朱通伯等．多孔磷酸三钙陶瓷人工骨生物降解的实验观察．同济医科大学学报．1996，25(1)：37-40
[4].Sharpe T, adsorption Sammons RL, Marquis PM. Effect of PH on protein to hydroxyapatite and tricalcium phosphateceramics. Biomaterial, 1997;18(6):471-476.
[1] 程顺巧，苟立，季金苟等．双相HA／β-TCP陶瓷的多孔结构对类骨磷灰石形成的影响．现代技术陶瓷，2004；1：14-17
[2] 何伟，肖建德．复合型纳米羟基磷灰石人工骨的研究进展．国际骨科学杂志．2007；28(4)：222-223．
[3] Gutwein LG, Webster TJ. Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles. Biomaterials, 2004; 25(18): 4175-4183.
[4] 张力德．纳米材料，北京，化学工业出版社，2000：39-41．
[5] Kuhne J, Bartl R, Frish B, et al. Bone formation in coralline hydroxyapatite: Effect of pore size studied in rabbit. Acta Orthop Scand, 1994; 65(3): 246-252.
[1] 卢世壁 主译，坎贝尔骨科手术学，济南，山东科学技术出版社，2002，3：2278．
[2] 刘柏龄主编，中医骨伤科学，北京，人民卫生出版社，1998，116．
[3] 刘强，陈君长．重组转化因子TGF-β1的表达及修复骨缺损的研究中国骨伤，2000；13(12)：715-717．
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