新型多孔型磷酸钙骨水泥的生物相容性研究
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摘要
研究背景
骨缺损的修复重建是现代骨科治疗领域的热点之一，对骨移植材料的选择，除要具有来源广泛，易于携带，使用方便，成骨活性良好的特点外，还必须适应各种各样的骨缺损类型，具有可塑形和粘接功能，这点对于提高骨缺损的修复，显得尤为重要。目前，骨移植修复材料主要分两大类：生物材料和人工材料，具体材料多种多样，由于每种材料的性能和特点不同，实际应用中各有优缺点，因此联合采用两种以上的材料往往可以取长补短，达到理想效果。
磷酸钙骨水泥(Calcitun Phosphate cement，CPC)又称羟基磷灰石骨水泥(Hydroxyapatite cement，HA)，是一种具有生物学活性的新型非陶瓷型羟基磷灰石类人工骨材料，具有众多优点：具有自固化特点，可以任意塑形，有良好的生物相容性，有可降解性和骨传导性，这种骨水泥可以与骨组织紧密结合，CPC在固化过程中不产生热量，对周围组织不产生任何损伤。但是，CPC骨水泥的降解速度较慢、骨爬行替代时间较长。因此，改进CPC降解速度的研究是一个重要课题。CPC降解速度缓慢的原因与其结构有关，CPC虽然最大程度上模仿了天然骨的矿物相，但却结构致密，缺乏天然骨的孔隙结构，从而限制了新生骨向材料内部长入，因此，促进CPC降解与吸收也就成为当前CPC研究的热点之一。CPC植入体内会发生被动和主动两种方式的吸收。材料在体液条件下有一定的离散率，被动吸收程度依赖于水泥终成分，受材料的多孔性、离子置换、晶体特征以及水泥组织界面的PH值影响。主动吸收则与破骨细胞的细胞活性有关。研究显示吸收率和病人的性别、年龄、新陈代谢、社会习惯、材料植入部位、材料成分及几何学特征等许多因素有关。外科医生或者材料工程师很难控制病人因素，唯有通过改变材料的几何学特征和组成来促使其降解。Ooms等发现CPC植入后24周才吸收了20％。一般认为大孔隙有利于细胞渗入、骨内生和移植物的固定。因此增加孔隙尤其是增加大孔隙成为促CPC吸收的一个研究方向，许多实验表明磷酸钙的乳化技术，即添加适当大小的高溶性晶体：甘露醇、蔗糖等方法都可以制作大孔隙CPC。本实验研究材料——新型多孔型磷酸钙骨水泥(Porous Calcitun Phosphate cement，PCPC)是在普通磷酸钙骨水泥的基础上通过添加甘露醇配制而成，其孔隙率为44％，大孔率为18％，凝固时间为38min，抗压强度为8MPa。
目的
研究新型多孔磷酸钙骨水泥(PCPC)的生物相容性和安全性，探讨其降解和成骨性能，为其临床应用提供实验依据。
方法
通过采用浸提法制备PCPC浸提液；在小鼠腹腔内注射PCPC浸提液进行急性毒性试验，观察小白鼠一般状况(呼吸状态、进食及活动情况)和死亡情况；在豚鼠脊柱两旁作皮内注射PCPC浸提液进行致敏试验，观察注射点有无红斑、水肿、硬结及焦痂等形成情况；在兔血液中加入PCPC浸提液采用分光光度法进行溶血试验，观察PCPC是否具有溶血作用；自兔耳缘静脉缓慢注入PCPC混悬液(PCPC：0.9％氯化钠溶液=100 g／l)进行热原试验，观察PCPC是否具有致热性；通过新西兰兔股骨髁部骨内埋植试验观察PCPC在骨内的成骨活性和组织相容性。
结果
该材料原液对稀释兔血的溶血率＜5％，无溶血现象，材料原液未引起小鼠急性毒性反应、新西兰白兔热原反应及豚鼠过敏反应。骨内植入后无明显炎症反应，在不同时期的HE染色光镜下观察，在实验组(PCPC)可明显发现新生的编织骨不规则的分布于骨缺损中，成骨细胞蓝染，衬于新生骨小梁边缘，其间可见少量的巨噬细胞。骨小梁的长入分布与材料的孔隙空间一致，材料逐渐被新生骨小梁分割包绕，中央区开始出现小量的新生骨小梁，成骨活跃，边缘区逐渐可见到成熟骨组织，并出现骨髓腔结构，骨组织逐渐改建，趋于成熟。而对照组(CPC)，2周时骨—骨水泥材料界面整齐，无新骨生长。4，8，12，16周材料的边缘区有新骨生长，并随时间呈递增趋势，材料的中央区未见新骨组织。扫描电镜下观察：在低倍扫描电镜下发现，该新型PCPC的孔径在200-400μm之间分布，大部分孔隙与孔隙之间有90μm左右小孔道贯通，类似松质骨结构。在骨内植入4周时高倍扫描电镜下观察，在骨-骨水泥交接面可见新生骨小梁沿着PCPC的孔隙伸长入其内部，两者之间紧密结合，表明该PCPC具有较好的骨传导性。16周时，PCPC密度明显减低，说明其降解反应仍继续进行；其间可见网状结构的骨小梁形成，二者之间未见透亮线，表明已牢固结合，达成生物学铆定。随着骨水泥降解反应的逐步进行，新生骨组织的增多，其周围钙盐沉积、钙化，并进一步形成骨组织。
结论
通过以上实验，我们认为PCPC无毒性、无致敏、无致热和无溶血作用，植入后无炎症反应，可以与骨组织生物结合。上述结果均达到生物材料生物相容性和安全性检测标准，证明该新型PCPC具有良好的生物相容性、骨传导性和可降解性，多孔结构的存在明显加快了材料的降解，并为新骨组织的长入提供了有利条件，使材料早期获得稳定，具有广阔的临床应用前景。
Background
The reparation and reconstruction of bone defect is one of the hot spots of modem orthopedic research, and it's necessary to select the bone graft materials carefully to satisfy the needs. Recently, bone graft materials can be roughly grouped into two main categories: biomaterials and artificial materials. There are many kinds of materials to be selected and they all have their own advantages and disadvantages, so it is advisable for us to combine their advantages to come up with some satisfactory end-products. Ideal bone graft materials should have some important characteristics. They should be easily obtained, be convenient to apply and have good portability. They should have good osteogenesis, be suitable for different kinds of bone defects, and have good plasticity and cohesiveness, which are very important for promoting the reparation and reconstruction of bone defects.
The calcium phosphate bone cement (CPC), also called hydroxyapatite cement(HA), is a bioactive neotype non-ceramic hydroxyapatite artificial material. It has multiple merits: inclding self solidification and degradation, being modeled willfully, good biological compatibility and bone conduction. The bone cement may combine with the bone tissue tightly, and the calcium phosphate bone cement does not generate heat in the solidification process, and it can be organized into the surrounding tissues without any damages. However, the degradation of calcium phosphate bone cement is slow, and the bone crawling substitution time is long. Therefore, how to promote the degradation of calcium phosphate bone cement is an important research topic. The degradation of calcium phosphate bone cement is related to its structure. Although the calcium phosphate bone cement has imitated the natural bone mineral facies to the greatest extent, its structure is actually very compact and lacks the holes like those in the natural bone, which limits the newborn bone to enter the material. Promoting the CPC degradation and absorption is also one of the current CPC research hot spots. CPC implantation in vivo should be able to have the passive and the initiative absorptions. The material has a certain straggling rate under the body fluid condition, the degree of passive absorption relies on the cement end ingredient, material porosity, ion replacement, crystal characteristic, as well as the influence of cement organization contact surface PH value. The active absorption then is related to the osteoclast' cytoactivity. Some research demonstrated that the absorption rate was also related to the factors of patient's sex, the age, the metabolism, the social custom, the spot of the material implanted, the material ingredient, the geometry characteristic and so on. Surgeons, or material engineers, are very difficult to control the patient factor, they could only change material geometry characteristic and composition to promote degradation. Ooms discovered implanted CPC could only be absorbed by 20% after 24 weeks. Generally thought that big pore is advantageous in cell permeation, the bone survival and graft fixation. Therefore to increase pores, especially to increase big pores become a direction in the research of CPC. Some experiments indicated that the calcium phosphate emulsification technology and supplement of crystal with suitable size and high solubility, such as sucrose and mannitol, both could make the big pore CPC. Experimental material——neotype porous calcium phosphat bone cement (PCPC), were mainly composed of the ordinary CPC by adding mannito,the interval porosity is 44%, the macropole rate is 18%, the coagulation time is 38min, the crushing strength is 8MPa.
Objective
To provide experimental basis for clinical application through studying on the biocompatibility and security of pore configuration of calcium phosphate cement and discussing its degradation and osteogenous function.
Method
PCPC extracting solution was prepared by soaking the PCPC in normal sodium. The acute toxicity test was performed on the mice received intraperitoneal injection of PCPC extracting solution, in which the general condition (breath condition, foodintake state and active situation) and the death of the mice were observed. Sensitization study was performed on the guinea pig received intracutaneous injection of PCPC extracting solution on both sides of the spinal column. Whether there was a kind of red, dropsy, hardness and eschar response in the injected point or not was observed. Spectrophotometric method was used to examine the hemolytic response after adding PCPC extracting solution in the rabbit blood and the hemolytic action of PCPC was determined. Pyrogen test was produced when PCPC suspended solution (PCPC: 0.9% sodium chloride solution =100 g/l) was slowly injected into the vein of rabbit ear to determine whether the PCPC possess thermal property. PCPC was implanted in the condyles of femur of New Zealand rabbit. And it's ossific activity and the organization compatibility were observed.
Results
The hemolysis rate of the cement-extracted liquid in rabbits was less than 5 percent. The cement-extracted liquid didn't induced acute toxicity in mice, pyrogenic reaction in rabbits, or allergic reaction in guinea pigs. Implant test indicated that ambient tissue had no inflammatory reaction. When the HE dyeing tissue was observed with light microscope at different time point, the results showed that obvious newborn knittng bone irregularly distributed in the damaged area, the osteoblast was dyed blue lining in the edge of the newborn bone trabecula, and a few of macrophages were observed in the experimental group (PCPC). The entry and distribution of the bone trabecula were consistent with the material pore space. Gradually, the material was divided and circumvoluted by newborn bone trabecula with a few of newborn bone trabecula appearing in the central zone. The bone formation was active, and the mature bone tissue arrived in the fringe area. Then, the medullary cavity of bones structure formed, the bone tissue rebuilded gradually and tended to maturely. In the control group (CPC), the results showed that the bone - bone cement connection surface was neat without new bone growth at week 2. There was some new bone growing in the fringe area of the meterial after week 4 and there was an increasing tendency along with the time. However, no new bone tissue was observed in the central zone of the material. When we observed the tissue with SEM, the results showed that the aperture of this new PCPC was between 200 and 400μm with low power. There were about 90μm um-length passageways penetrating majority of every two holes, which was similar to the cancellated bone structure. When we observed with high power scanning electron microscope 4 weeks after implantation, the results showed that the newborn bone trabecula grew along the PCPC hole and reached the bone-bone cement connection suface, which made these two piece of bones bind tightly. It indicated that PCPC has a good conduction for the growth of the bone. When we observed at week 16, the results showed that the PCPC density decreases obviously, which suggests that its degradation reaction was going on. There was meshy bone trabecula forming between these two pieces of bones and there was no translucent line, which indicated that the bones had already combined reliably and achieved the biology riveting. With gradual degradation of the bone cement and increasing newborn bone tissue, the calcium salt deposited and calcificated around the bone cement, and further the bone tissue was developed.
Conclusion
In conclusion, the homemade PCPC have no toxicity, no allergic reaction, no pyrogenic reaction, no hemolytic reaction and no inflammatory reaction, but good tissue biocompatibility. All these results accord with the standards of biomaterials. It has been shown that the neotype pore configuration of calcium phosphate cement have good biocompatibility conductibility and degradation. The vesicular structure existence sped up the material degradation obviously, and deeply entered the new bone tissue has provided the advantage, caused the material stable early. It is a kind of promising material as bone substitute.
骨缺损的修复重建是现代骨科治疗领域的热点之一，对骨移植材料的选择，除要具有来源广泛，易于携带，使用方便，成骨活性良好的特点外，还必须适应各种各样的骨缺损类型，具有可塑形和粘接功能，这点对于提高骨缺损的修复，显得尤为重要。目前，骨移植修复材料主要分两大类：生物材料和人工材料，具体材料多种多样，由于每种材料的性能和特点不同，实际应用中各有优缺点，因此联合采用两种以上的材料往往可以取长补短，达到理想效果。
磷酸钙骨水泥(Calcitun Phosphate cement，CPC)又称羟基磷灰石骨水泥(Hydroxyapatite cement，HA)，是一种具有生物学活性的新型非陶瓷型羟基磷灰石类人工骨材料，具有众多优点：具有自固化特点，可以任意塑形，有良好的生物相容性，有可降解性和骨传导性，这种骨水泥可以与骨组织紧密结合，CPC在固化过程中不产生热量，对周围组织不产生任何损伤。但是，CPC骨水泥的降解速度较慢、骨爬行替代时间较长。因此，改进CPC降解速度的研究是一个重要课题。CPC降解速度缓慢的原因与其结构有关，CPC虽然最大程度上模仿了天然骨的矿物相，但却结构致密，缺乏天然骨的孔隙结构，从而限制了新生骨向材料内部长入，因此，促进CPC降解与吸收也就成为当前CPC研究的热点之一。CPC植入体内会发生被动和主动两种方式的吸收。材料在体液条件下有一定的离散率，被动吸收程度依赖于水泥终成分，受材料的多孔性、离子置换、晶体特征以及水泥组织界面的PH值影响。主动吸收则与破骨细胞的细胞活性有关。研究显示吸收率和病人的性别、年龄、新陈代谢、社会习惯、材料植入部位、材料成分及几何学特征等许多因素有关。外科医生或者材料工程师很难控制病人因素，唯有通过改变材料的几何学特征和组成来促使其降解。Ooms等发现CPC植入后24周才吸收了20％。一般认为大孔隙有利于细胞渗入、骨内生和移植物的固定。因此增加孔隙尤其是增加大孔隙成为促CPC吸收的一个研究方向，许多实验表明磷酸钙的乳化技术，即添加适当大小的高溶性晶体：甘露醇、蔗糖等方法都可以制作大孔隙CPC。本实验研究材料——新型多孔型磷酸钙骨水泥(Porous Calcitun Phosphate cement，PCPC)是在普通磷酸钙骨水泥的基础上通过添加甘露醇配制而成，其孔隙率为44％，大孔率为18％，凝固时间为38min，抗压强度为8MPa。
目的
研究新型多孔磷酸钙骨水泥(PCPC)的生物相容性和安全性，探讨其降解和成骨性能，为其临床应用提供实验依据。
方法
通过采用浸提法制备PCPC浸提液；在小鼠腹腔内注射PCPC浸提液进行急性毒性试验，观察小白鼠一般状况(呼吸状态、进食及活动情况)和死亡情况；在豚鼠脊柱两旁作皮内注射PCPC浸提液进行致敏试验，观察注射点有无红斑、水肿、硬结及焦痂等形成情况；在兔血液中加入PCPC浸提液采用分光光度法进行溶血试验，观察PCPC是否具有溶血作用；自兔耳缘静脉缓慢注入PCPC混悬液(PCPC：0.9％氯化钠溶液=100 g／l)进行热原试验，观察PCPC是否具有致热性；通过新西兰兔股骨髁部骨内埋植试验观察PCPC在骨内的成骨活性和组织相容性。
结果
该材料原液对稀释兔血的溶血率＜5％，无溶血现象，材料原液未引起小鼠急性毒性反应、新西兰白兔热原反应及豚鼠过敏反应。骨内植入后无明显炎症反应，在不同时期的HE染色光镜下观察，在实验组(PCPC)可明显发现新生的编织骨不规则的分布于骨缺损中，成骨细胞蓝染，衬于新生骨小梁边缘，其间可见少量的巨噬细胞。骨小梁的长入分布与材料的孔隙空间一致，材料逐渐被新生骨小梁分割包绕，中央区开始出现小量的新生骨小梁，成骨活跃，边缘区逐渐可见到成熟骨组织，并出现骨髓腔结构，骨组织逐渐改建，趋于成熟。而对照组(CPC)，2周时骨—骨水泥材料界面整齐，无新骨生长。4，8，12，16周材料的边缘区有新骨生长，并随时间呈递增趋势，材料的中央区未见新骨组织。扫描电镜下观察：在低倍扫描电镜下发现，该新型PCPC的孔径在200-400μm之间分布，大部分孔隙与孔隙之间有90μm左右小孔道贯通，类似松质骨结构。在骨内植入4周时高倍扫描电镜下观察，在骨-骨水泥交接面可见新生骨小梁沿着PCPC的孔隙伸长入其内部，两者之间紧密结合，表明该PCPC具有较好的骨传导性。16周时，PCPC密度明显减低，说明其降解反应仍继续进行；其间可见网状结构的骨小梁形成，二者之间未见透亮线，表明已牢固结合，达成生物学铆定。随着骨水泥降解反应的逐步进行，新生骨组织的增多，其周围钙盐沉积、钙化，并进一步形成骨组织。
结论
通过以上实验，我们认为PCPC无毒性、无致敏、无致热和无溶血作用，植入后无炎症反应，可以与骨组织生物结合。上述结果均达到生物材料生物相容性和安全性检测标准，证明该新型PCPC具有良好的生物相容性、骨传导性和可降解性，多孔结构的存在明显加快了材料的降解，并为新骨组织的长入提供了有利条件，使材料早期获得稳定，具有广阔的临床应用前景。
Background
The reparation and reconstruction of bone defect is one of the hot spots of modem orthopedic research, and it's necessary to select the bone graft materials carefully to satisfy the needs. Recently, bone graft materials can be roughly grouped into two main categories: biomaterials and artificial materials. There are many kinds of materials to be selected and they all have their own advantages and disadvantages, so it is advisable for us to combine their advantages to come up with some satisfactory end-products. Ideal bone graft materials should have some important characteristics. They should be easily obtained, be convenient to apply and have good portability. They should have good osteogenesis, be suitable for different kinds of bone defects, and have good plasticity and cohesiveness, which are very important for promoting the reparation and reconstruction of bone defects.
The calcium phosphate bone cement (CPC), also called hydroxyapatite cement(HA), is a bioactive neotype non-ceramic hydroxyapatite artificial material. It has multiple merits: inclding self solidification and degradation, being modeled willfully, good biological compatibility and bone conduction. The bone cement may combine with the bone tissue tightly, and the calcium phosphate bone cement does not generate heat in the solidification process, and it can be organized into the surrounding tissues without any damages. However, the degradation of calcium phosphate bone cement is slow, and the bone crawling substitution time is long. Therefore, how to promote the degradation of calcium phosphate bone cement is an important research topic. The degradation of calcium phosphate bone cement is related to its structure. Although the calcium phosphate bone cement has imitated the natural bone mineral facies to the greatest extent, its structure is actually very compact and lacks the holes like those in the natural bone, which limits the newborn bone to enter the material. Promoting the CPC degradation and absorption is also one of the current CPC research hot spots. CPC implantation in vivo should be able to have the passive and the initiative absorptions. The material has a certain straggling rate under the body fluid condition, the degree of passive absorption relies on the cement end ingredient, material porosity, ion replacement, crystal characteristic, as well as the influence of cement organization contact surface PH value. The active absorption then is related to the osteoclast' cytoactivity. Some research demonstrated that the absorption rate was also related to the factors of patient's sex, the age, the metabolism, the social custom, the spot of the material implanted, the material ingredient, the geometry characteristic and so on. Surgeons, or material engineers, are very difficult to control the patient factor, they could only change material geometry characteristic and composition to promote degradation. Ooms discovered implanted CPC could only be absorbed by 20% after 24 weeks. Generally thought that big pore is advantageous in cell permeation, the bone survival and graft fixation. Therefore to increase pores, especially to increase big pores become a direction in the research of CPC. Some experiments indicated that the calcium phosphate emulsification technology and supplement of crystal with suitable size and high solubility, such as sucrose and mannitol, both could make the big pore CPC. Experimental material——neotype porous calcium phosphat bone cement (PCPC), were mainly composed of the ordinary CPC by adding mannito,the interval porosity is 44%, the macropole rate is 18%, the coagulation time is 38min, the crushing strength is 8MPa.
Objective
To provide experimental basis for clinical application through studying on the biocompatibility and security of pore configuration of calcium phosphate cement and discussing its degradation and osteogenous function.
Method
PCPC extracting solution was prepared by soaking the PCPC in normal sodium. The acute toxicity test was performed on the mice received intraperitoneal injection of PCPC extracting solution, in which the general condition (breath condition, foodintake state and active situation) and the death of the mice were observed. Sensitization study was performed on the guinea pig received intracutaneous injection of PCPC extracting solution on both sides of the spinal column. Whether there was a kind of red, dropsy, hardness and eschar response in the injected point or not was observed. Spectrophotometric method was used to examine the hemolytic response after adding PCPC extracting solution in the rabbit blood and the hemolytic action of PCPC was determined. Pyrogen test was produced when PCPC suspended solution (PCPC: 0.9% sodium chloride solution =100 g/l) was slowly injected into the vein of rabbit ear to determine whether the PCPC possess thermal property. PCPC was implanted in the condyles of femur of New Zealand rabbit. And it's ossific activity and the organization compatibility were observed.
Results
The hemolysis rate of the cement-extracted liquid in rabbits was less than 5 percent. The cement-extracted liquid didn't induced acute toxicity in mice, pyrogenic reaction in rabbits, or allergic reaction in guinea pigs. Implant test indicated that ambient tissue had no inflammatory reaction. When the HE dyeing tissue was observed with light microscope at different time point, the results showed that obvious newborn knittng bone irregularly distributed in the damaged area, the osteoblast was dyed blue lining in the edge of the newborn bone trabecula, and a few of macrophages were observed in the experimental group (PCPC). The entry and distribution of the bone trabecula were consistent with the material pore space. Gradually, the material was divided and circumvoluted by newborn bone trabecula with a few of newborn bone trabecula appearing in the central zone. The bone formation was active, and the mature bone tissue arrived in the fringe area. Then, the medullary cavity of bones structure formed, the bone tissue rebuilded gradually and tended to maturely. In the control group (CPC), the results showed that the bone - bone cement connection surface was neat without new bone growth at week 2. There was some new bone growing in the fringe area of the meterial after week 4 and there was an increasing tendency along with the time. However, no new bone tissue was observed in the central zone of the material. When we observed the tissue with SEM, the results showed that the aperture of this new PCPC was between 200 and 400μm with low power. There were about 90μm um-length passageways penetrating majority of every two holes, which was similar to the cancellated bone structure. When we observed with high power scanning electron microscope 4 weeks after implantation, the results showed that the newborn bone trabecula grew along the PCPC hole and reached the bone-bone cement connection suface, which made these two piece of bones bind tightly. It indicated that PCPC has a good conduction for the growth of the bone. When we observed at week 16, the results showed that the PCPC density decreases obviously, which suggests that its degradation reaction was going on. There was meshy bone trabecula forming between these two pieces of bones and there was no translucent line, which indicated that the bones had already combined reliably and achieved the biology riveting. With gradual degradation of the bone cement and increasing newborn bone tissue, the calcium salt deposited and calcificated around the bone cement, and further the bone tissue was developed.
Conclusion
In conclusion, the homemade PCPC have no toxicity, no allergic reaction, no pyrogenic reaction, no hemolytic reaction and no inflammatory reaction, but good tissue biocompatibility. All these results accord with the standards of biomaterials. It has been shown that the neotype pore configuration of calcium phosphate cement have good biocompatibility conductibility and degradation. The vesicular structure existence sped up the material degradation obviously, and deeply entered the new bone tissue has provided the advantage, caused the material stable early. It is a kind of promising material as bone substitute.
引文
1. Williams DF.Definitions in biomaterials.New York:Elseriver Science Publ. 1987:66-72
2. Hench LL.Bioceramics and the origin life.J Biomed Mater Res,1989,23:685-703
3. Gruniger SE,Siew C,Chow L,et al.Evaluation of the biocompatibility of a new calcium phosphate setting cement.J Dent Res,1984; 63:200-206
4. Costantina PD,Friedman CD,Jones K, et al.Experimental hydroxyapatite cement cranioplasty.Plast Reconstr Surg, 1992, 90(Z): 174-185
5. 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.
6. (ISO).Biological Evaluation of Dental Materals TR 7405(E)1984
7．《CASTM》标准年鉴．医用装置标准；13(01)成都科技出版
8. Johnson HJ etal.Biocompatibility test procedures for materials evaluation in vitro.Ⅱ.Objective methods of toxicity assessment.J Biomed Mater Res 1985;19:489
9. Tsuchiya T. Study on the standardization of cytotoxicity tests and new standard reference materials useful for evaluating the safty of biomaterials.Biomater Appl. 1994;9(2):138
10. Oshima H,Nakamura M.A sdudy on reference standard for cytotoxicity assay of biomaterials.Biomed Mater, 1994; 4(4):327
11．孙胶，薛森等．遗传毒性试验中的接触法和浸提介质的探讨．生物医学工程学杂志，1994；11(1)：91-93
12. Eggli PS,Mtiller W, Schenk RK. Porous hydroxyapatite and tricalcium phosphate cylinders with two differentpore size ranges implanted in the cancellous bone of rabbits:A comparative histomorphometric and histologic study ofbony ingrowth and implant substitution. ClinOrthop,1988,232:127
13. Mandelkow HK,Hallfeldt KK,Kessler SB,etal.New boneformation following implantation of various hydroxyap-ante ceramics. Animal experiment with bore hole modelsof the sheep tibia.Unfallchirurg, 1990,93:376
14. Spektor M.Charakterisierung biokeramischer Kalzi-umphosphat-implantate.Hefte Unfallheilkd, 1991,216:11
15. Schlickewei W,Paul Ch.Experimentelle Untersuchungenzum knochenersatz mit bovinem Apatit.Hefte Unfall-heilkd,1991,216:59
16. Klein CPAT, Patka P, Den Hollander W, et al. Macroporous calcium phosphate ceremonies in dogfemora: a histological study of interface and biodegradation. Biomaterials, 1989, 10:59-62
17. Frayssinet P, Trouillet JL, Rouquent N, et al. Autefage A, Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition. Biomaterials, 1993, 14:432-29
18. Koerten HK, Vander Meulen J, Verhoeven MCH, et al. Degradation of caclium phosphate ceramics:a comparative study on three types of calcium phosphate. In:Ducheyme P, eds. Bone-Mboonding Biomaterinals. Leiderdorp:Reed Health Care Communications, 1992, 85:94
19. Renooij W, et al. Bioresorption of ceramic strontium-85 -- labelled calcium phosphate implants in dogs femora. Clin Orthorp 1985; 197:272
20. Kitsugi T et al J Biomed Mater Res_ 1987_ 21:1109
21. Kitsugi T et al Clin Orthop, 1988, 234:280
22. Kitsugi T et al. Clin Orthop, 1988, 45:90
23. 郑岳华,侯小妹,杨兆雄. 硅酸盐通报,1993, 3
24. Dear-mo Liu.Ceramics International, 1997,23:135-139
25. 黄学辉等. 武汉工业大学学报,1996,18(3):7
26. Goshima J,Goldberg VM,Caplan Al.The osteogenic potential culture expanded phosphate marrow mesenchymal cells assayed in intro Calcium ceramic blockes.Clin Orthop, 1991;262(1):298
27. Nade S,Armstrong L,Mccartney E et al.Osteogenesis after bone and bone marrow transplantation.Clin Orthop,1983;181(12): 255
28. Klawitter JJ,Hulbert SF.Application porous ceramicsfor the of attachmentof load bearing internal orthopedic ap-plications.J Biomed Mater Res,1971,2:161
29．陈力，Klaes W.Assenmacher S.五种骨骼移植替代材料成骨性能的比较研究(J)中华医学杂志，1996，76(7)：527-530
30. Hashimoto-Uoshima M,Ishikawa I,Kinoshita A,et al.Clinical and hitological observation of replacement of biphasic calcium phosphate by bone tissue in monkeys.Int J Periodonto Restor Dent, 1995,15:204-213
31. Damien CJ,Ricci JL,Christel P et al.Formation of a calcium phosphate-rich layer on absorbable calcium carbonate bone graft substitutes.Clacif Tissue Int, 1994,55(2): 151-158
32. Cornell CN,Lane JM.Current understanding of osteoconduction in bone regeneration.Clin Orthop, 1998,355s:s267-273
33. Pipamonti U,Mass,Vanden Heever B,et al. Plast Reconstr Surg,1992,90:382-393
34. Burchardt H.The biology of bone grafts repair.Clin Orthop, 1983,174:28
1 Brown W E,Chow L C.A new calsium phosphate,water setting cement.In: Brown PW E d.Cem ent Research Progress.Wasterville, Ohio: American Ceramic Society,1986: 352～379
2 Fukase Y, Eanes ED, Takagi S,et al. Setting reactions and compressive strengths ofcalciump hosphate cements. J DentRes,1990;69 (12):1852～1856
3 Ishikawa K ,Asaoka K.Estimation of idealmechanical strength and criticalporosity of calcium phosphate cement.J Biomed Mater Res,1995; 29 (12):11537～11543
4 Hamanishi C ,K itamoto K,Ohura K ,et al. Selfsetting,bioactive,and biodegradable T～T CPDCPD apatite cement. J Biomed Mater Res,1996; 32 (3):383～389
5 Ohura K ,Bohner M , Hardouin P ,et al.Resorption of and bone formation from ,new be- ta tricalcium phosphatem onocalcium phosphote cements: an in v ivo study. J Biomed Mater Res,1996; 30 (2): 193～200
6 Liu C Wang W, Shen W,et al .Evaluation of the biocompatibility of a nonceramic hydroxyapatite[J].J Endod,1997,23,(8):490-493
7 Costantino PD ,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty. Plast Reconstr Surg,1992,90:174-185.
8 Khairoun I,Boltong MG,Driessens FC,et al.Effect of calcium carbonate on clinical compliance of apatitic calcium phosphate bone cement.J Biomed Mater. 1997 Winter;38(4):356-60.
9 Yang QZ,Troczynski T,Dean-Mo Liu DM.Influence of apatite seeds on the synthesis of calcium phosphate cement [J].Biomaterials,2002,23:2751-2760.
10 Otsuka M,Matsuda Y,et al. Effect of particle size of metastable calcium phophates on mechanical strength of a novel self-setting bioactive calcium phophate cement [J]J Biomed Mater Res 1995, 29:25-32
11 Ginebra MP,Driessens FCM,Planell JA.ffect of particle size on the micro and nanostructural features of a calcium phophate cement :a kinetic analysis .[J] Biomatrials, 2004, 25:3453-3462
12 Ishikawat K, Takadi S, Chow LC, et al. Properties and mechanisms of fast-setting calacium phosphate cement.[J] Journal of Materials Science :Materials in Medicine,1995,6(9):528-533
13 Miyamoto Y, Ishikawa K,Takechi M,et al.Tissue response to fast-setting calacium phosphate cement in bone. Journal of Biomedical Materials Research [J],1997,37(4):457-464
14 Bohner M.New hydraulic cements based on α -tricalcium phosphate-calcium sulfate dihydrate mixtures .[J] Biomaterials,2004,25:741-749
15 Xu HHK,Quinn JB.Calcium phosphate cement containing resorbable fibles for short-rerm reinforcement and macroporoaty. [J]Biomaterials,2002,23(1):193
16 Bigi A ,Bracci B,Panzavolta S.Effect of added gelatin on the properties of calcium phosphate cement.[J] Biomaterials,2004,25:2893-2899
17 Wang XH,MaJB,et al.Structutal characterization of phosphorylated chitosan and their applications as effective additives of calcium phosphate cements. [J] Biomaterials ,2001,22:2247-2255
18 Gbureck U,Barralet JE,Spatz K,et al. Ionic modification of calcium phosphate cement viscosity.Part I:hypodermic injection and strength improvement of apatite cement [J]Biomaterials,2004,25:2187～2195
19 Dos Santos LA ,Carrodeguas RG, Dual-Setting Calcium Phosphate Cement Modified with Ammonium Polyacrylate .[J] Artifcial Organs, 1993,14:39～43
20 Leroux L,Hatim Z,Freche M,et al.Effects of various adjuvants (lactic acid,glycerol ,and chitosan ) on the injectability of a calium phosphate cement.[J] Bonen 1999n25(2):31～34
21 Takechi MnMiyamoto YnIshikawa K ,et al .Initial histological evaluation of anti-washout type fast-setting calcium phosphate cement following subcutaneous implantation.Biomaterials [J], 1998,19:2057～2063
22 Ishikawa K, Miyamoto Y, Kon M,et al. Non-decaytype fast-setting calcium phosphate cement :composite with sodium alginate .[J] Biomaterials,1995,16(7):527～532
23 Takechi M, Miyamoto Y ,et al.Effects of added antibiotics on the basic properties of anti-washout-type fast-setting calcium phosphate cement.[J] J Biomed Mater Res, 1998,39(2):308～316
24 Muzzarelli RRA,Zucchini C,Ilari P, et al.Osteoconduction exerted by methylpyrrolidinone chitosan used in dental surgery . [J] Biomaterials, 1993,14:39～43
25 纳米材料及其在生物医学工程中的应用。[J]国外医学·生物医学工程分册，1998，21(5)：262-265。
26 Wang XJ,Li YB. Development of biomimetic composite of nano hydroxyapatite and polyamide as a bone substi tute.[J] Chin J Biomed Engineer,2001,10 (2): 199-203.
27 俞兴，崔福斋．骨移植替代材料在脊柱外科中的应用。[J]生物骨科材料与临床研究，2004，1(4)：52-54。
28 Tahara Y ,Ishii Y .Apatite cement containing cis-diamminedichloroplatinum implanted in rabbit femur for sustained release of the anticancer drug and bone formation. [J] Orthop Sci,2001,6(6):556～565
29 Otsuka M Matsuda Y, Fox JL,et al. Anovel skeletal drug delivery system using self-setting calium phosphate cement. 9:Effects of the mixing solution volume on anticancer drug rlease from homogeneous drug-loaded cement。Jpharm Sci, 1995, 84:733～736.
30 张进军，李兵仓等．磷酸钙骨水泥承载头孢哌酮钠后对其理化特性的影响[J】生物医学工程学杂志，2000，17(4)：400～402
31 孙明林，胡蕴玉等。磷酸钙骨水泥/骨形态发生蛋白符合人工骨修复骨缺损的试验验究。现代康复，2001，5：32～33
32 BlomEJ,Klein-Nulend J,Klein CP, et al.Transforming growth factor betal incorporated during setting in calcium phosphate cement simulates bone cell differention in vitro.[J] J Biomed Mater Res,2000,50(1):67～74
33 Yetkinler DN,Ladd AL,Poser RD,et al.J Bone Joint Surg A m,1999;81(3):391～399
34 Zimmermann R,Gabl M,Lutz M,et al.Arch Orthop Trauma Surg,2003;123(1):22～27
35 Horstmann WG, Verbeyen OC,Leemans R,Injuey,2003 ;34(2): 141～144
36 Keating J, Hajducka C.The use of Norian SRS and minimal internal fixation in management of tibial plateau fractures. [J] Trans Orthop Trauma Assoc, 1999,15:411
37 Van Landuyt P, Peter B ,Beluze L ,rt al. Reinforcement of osteosynthesis screws with brushite cement. [J] Bone, 1999,25(2suppl):95s～98s
38 Mattsson P, Larsson S.Scand J Surg,2003;92(3):215～219
39 Yerby SA ,Toh E,Mclain RF. Revision of failed pedicle screws using hydroxyapatite cement: a biomechanial analysis. Spine, 1998,23:1657～1661
40 Ooms EM ,Wolk e J G, vande H euvel M T ,et al.Histo logical eva luation of the bone response to calciumphosphate cement implanted in cortical bone. Biomaterials,2003, 24 (6):989～1000。
41 Ooms EM, Egglezos EA ,Wolke JG, et al.Soft tissue response to injectable calcium phospha te cements.Biomateriais,2003, 24(5):749～757
42 Sasaki S ,Ishii Y.Apatite cement containing antibiotics: efficacy in treating experimental osteimyelitis. J Orthop Sci,1999,4:361-369
2. Hench LL.Bioceramics and the origin life.J Biomed Mater Res,1989,23:685-703
3. Gruniger SE,Siew C,Chow L,et al.Evaluation of the biocompatibility of a new calcium phosphate setting cement.J Dent Res,1984; 63:200-206
4. Costantina PD,Friedman CD,Jones K, et al.Experimental hydroxyapatite cement cranioplasty.Plast Reconstr Surg, 1992, 90(Z): 174-185
5. 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.
6. (ISO).Biological Evaluation of Dental Materals TR 7405(E)1984
7．《CASTM》标准年鉴．医用装置标准；13(01)成都科技出版
8. Johnson HJ etal.Biocompatibility test procedures for materials evaluation in vitro.Ⅱ.Objective methods of toxicity assessment.J Biomed Mater Res 1985;19:489
9. Tsuchiya T. Study on the standardization of cytotoxicity tests and new standard reference materials useful for evaluating the safty of biomaterials.Biomater Appl. 1994;9(2):138
10. Oshima H,Nakamura M.A sdudy on reference standard for cytotoxicity assay of biomaterials.Biomed Mater, 1994; 4(4):327
11．孙胶，薛森等．遗传毒性试验中的接触法和浸提介质的探讨．生物医学工程学杂志，1994；11(1)：91-93
12. Eggli PS,Mtiller W, Schenk RK. Porous hydroxyapatite and tricalcium phosphate cylinders with two differentpore size ranges implanted in the cancellous bone of rabbits:A comparative histomorphometric and histologic study ofbony ingrowth and implant substitution. ClinOrthop,1988,232:127
13. Mandelkow HK,Hallfeldt KK,Kessler SB,etal.New boneformation following implantation of various hydroxyap-ante ceramics. Animal experiment with bore hole modelsof the sheep tibia.Unfallchirurg, 1990,93:376
14. Spektor M.Charakterisierung biokeramischer Kalzi-umphosphat-implantate.Hefte Unfallheilkd, 1991,216:11
15. Schlickewei W,Paul Ch.Experimentelle Untersuchungenzum knochenersatz mit bovinem Apatit.Hefte Unfall-heilkd,1991,216:59
16. Klein CPAT, Patka P, Den Hollander W, et al. Macroporous calcium phosphate ceremonies in dogfemora: a histological study of interface and biodegradation. Biomaterials, 1989, 10:59-62
17. Frayssinet P, Trouillet JL, Rouquent N, et al. Autefage A, Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition. Biomaterials, 1993, 14:432-29
18. Koerten HK, Vander Meulen J, Verhoeven MCH, et al. Degradation of caclium phosphate ceramics:a comparative study on three types of calcium phosphate. In:Ducheyme P, eds. Bone-Mboonding Biomaterinals. Leiderdorp:Reed Health Care Communications, 1992, 85:94
19. Renooij W, et al. Bioresorption of ceramic strontium-85 -- labelled calcium phosphate implants in dogs femora. Clin Orthorp 1985; 197:272
20. Kitsugi T et al J Biomed Mater Res_ 1987_ 21:1109
21. Kitsugi T et al Clin Orthop, 1988, 234:280
22. Kitsugi T et al. Clin Orthop, 1988, 45:90
23. 郑岳华,侯小妹,杨兆雄. 硅酸盐通报,1993, 3
24. Dear-mo Liu.Ceramics International, 1997,23:135-139
25. 黄学辉等. 武汉工业大学学报,1996,18(3):7
26. Goshima J,Goldberg VM,Caplan Al.The osteogenic potential culture expanded phosphate marrow mesenchymal cells assayed in intro Calcium ceramic blockes.Clin Orthop, 1991;262(1):298
27. Nade S,Armstrong L,Mccartney E et al.Osteogenesis after bone and bone marrow transplantation.Clin Orthop,1983;181(12): 255
28. Klawitter JJ,Hulbert SF.Application porous ceramicsfor the of attachmentof load bearing internal orthopedic ap-plications.J Biomed Mater Res,1971,2:161
29．陈力，Klaes W.Assenmacher S.五种骨骼移植替代材料成骨性能的比较研究(J)中华医学杂志，1996，76(7)：527-530
30. Hashimoto-Uoshima M,Ishikawa I,Kinoshita A,et al.Clinical and hitological observation of replacement of biphasic calcium phosphate by bone tissue in monkeys.Int J Periodonto Restor Dent, 1995,15:204-213
31. Damien CJ,Ricci JL,Christel P et al.Formation of a calcium phosphate-rich layer on absorbable calcium carbonate bone graft substitutes.Clacif Tissue Int, 1994,55(2): 151-158
32. Cornell CN,Lane JM.Current understanding of osteoconduction in bone regeneration.Clin Orthop, 1998,355s:s267-273
33. Pipamonti U,Mass,Vanden Heever B,et al. Plast Reconstr Surg,1992,90:382-393
34. Burchardt H.The biology of bone grafts repair.Clin Orthop, 1983,174:28
1 Brown W E,Chow L C.A new calsium phosphate,water setting cement.In: Brown PW E d.Cem ent Research Progress.Wasterville, Ohio: American Ceramic Society,1986: 352～379
2 Fukase Y, Eanes ED, Takagi S,et al. Setting reactions and compressive strengths ofcalciump hosphate cements. J DentRes,1990;69 (12):1852～1856
3 Ishikawa K ,Asaoka K.Estimation of idealmechanical strength and criticalporosity of calcium phosphate cement.J Biomed Mater Res,1995; 29 (12):11537～11543
4 Hamanishi C ,K itamoto K,Ohura K ,et al. Selfsetting,bioactive,and biodegradable T～T CPDCPD apatite cement. J Biomed Mater Res,1996; 32 (3):383～389
5 Ohura K ,Bohner M , Hardouin P ,et al.Resorption of and bone formation from ,new be- ta tricalcium phosphatem onocalcium phosphote cements: an in v ivo study. J Biomed Mater Res,1996; 30 (2): 193～200
6 Liu C Wang W, Shen W,et al .Evaluation of the biocompatibility of a nonceramic hydroxyapatite[J].J Endod,1997,23,(8):490-493
7 Costantino PD ,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty. Plast Reconstr Surg,1992,90:174-185.
8 Khairoun I,Boltong MG,Driessens FC,et al.Effect of calcium carbonate on clinical compliance of apatitic calcium phosphate bone cement.J Biomed Mater. 1997 Winter;38(4):356-60.
9 Yang QZ,Troczynski T,Dean-Mo Liu DM.Influence of apatite seeds on the synthesis of calcium phosphate cement [J].Biomaterials,2002,23:2751-2760.
10 Otsuka M,Matsuda Y,et al. Effect of particle size of metastable calcium phophates on mechanical strength of a novel self-setting bioactive calcium phophate cement [J]J Biomed Mater Res 1995, 29:25-32
11 Ginebra MP,Driessens FCM,Planell JA.ffect of particle size on the micro and nanostructural features of a calcium phophate cement :a kinetic analysis .[J] Biomatrials, 2004, 25:3453-3462
12 Ishikawat K, Takadi S, Chow LC, et al. Properties and mechanisms of fast-setting calacium phosphate cement.[J] Journal of Materials Science :Materials in Medicine,1995,6(9):528-533
13 Miyamoto Y, Ishikawa K,Takechi M,et al.Tissue response to fast-setting calacium phosphate cement in bone. Journal of Biomedical Materials Research [J],1997,37(4):457-464
14 Bohner M.New hydraulic cements based on α -tricalcium phosphate-calcium sulfate dihydrate mixtures .[J] Biomaterials,2004,25:741-749
15 Xu HHK,Quinn JB.Calcium phosphate cement containing resorbable fibles for short-rerm reinforcement and macroporoaty. [J]Biomaterials,2002,23(1):193
16 Bigi A ,Bracci B,Panzavolta S.Effect of added gelatin on the properties of calcium phosphate cement.[J] Biomaterials,2004,25:2893-2899
17 Wang XH,MaJB,et al.Structutal characterization of phosphorylated chitosan and their applications as effective additives of calcium phosphate cements. [J] Biomaterials ,2001,22:2247-2255
18 Gbureck U,Barralet JE,Spatz K,et al. Ionic modification of calcium phosphate cement viscosity.Part I:hypodermic injection and strength improvement of apatite cement [J]Biomaterials,2004,25:2187～2195
19 Dos Santos LA ,Carrodeguas RG, Dual-Setting Calcium Phosphate Cement Modified with Ammonium Polyacrylate .[J] Artifcial Organs, 1993,14:39～43
20 Leroux L,Hatim Z,Freche M,et al.Effects of various adjuvants (lactic acid,glycerol ,and chitosan ) on the injectability of a calium phosphate cement.[J] Bonen 1999n25(2):31～34
21 Takechi MnMiyamoto YnIshikawa K ,et al .Initial histological evaluation of anti-washout type fast-setting calcium phosphate cement following subcutaneous implantation.Biomaterials [J], 1998,19:2057～2063
22 Ishikawa K, Miyamoto Y, Kon M,et al. Non-decaytype fast-setting calcium phosphate cement :composite with sodium alginate .[J] Biomaterials,1995,16(7):527～532
23 Takechi M, Miyamoto Y ,et al.Effects of added antibiotics on the basic properties of anti-washout-type fast-setting calcium phosphate cement.[J] J Biomed Mater Res, 1998,39(2):308～316
24 Muzzarelli RRA,Zucchini C,Ilari P, et al.Osteoconduction exerted by methylpyrrolidinone chitosan used in dental surgery . [J] Biomaterials, 1993,14:39～43
25 纳米材料及其在生物医学工程中的应用。[J]国外医学·生物医学工程分册，1998，21(5)：262-265。
26 Wang XJ,Li YB. Development of biomimetic composite of nano hydroxyapatite and polyamide as a bone substi tute.[J] Chin J Biomed Engineer,2001,10 (2): 199-203.
27 俞兴，崔福斋．骨移植替代材料在脊柱外科中的应用。[J]生物骨科材料与临床研究，2004，1(4)：52-54。
28 Tahara Y ,Ishii Y .Apatite cement containing cis-diamminedichloroplatinum implanted in rabbit femur for sustained release of the anticancer drug and bone formation. [J] Orthop Sci,2001,6(6):556～565
29 Otsuka M Matsuda Y, Fox JL,et al. Anovel skeletal drug delivery system using self-setting calium phosphate cement. 9:Effects of the mixing solution volume on anticancer drug rlease from homogeneous drug-loaded cement。Jpharm Sci, 1995, 84:733～736.
30 张进军，李兵仓等．磷酸钙骨水泥承载头孢哌酮钠后对其理化特性的影响[J】生物医学工程学杂志，2000，17(4)：400～402
31 孙明林，胡蕴玉等。磷酸钙骨水泥/骨形态发生蛋白符合人工骨修复骨缺损的试验验究。现代康复，2001，5：32～33
32 BlomEJ,Klein-Nulend J,Klein CP, et al.Transforming growth factor betal incorporated during setting in calcium phosphate cement simulates bone cell differention in vitro.[J] J Biomed Mater Res,2000,50(1):67～74
33 Yetkinler DN,Ladd AL,Poser RD,et al.J Bone Joint Surg A m,1999;81(3):391～399
34 Zimmermann R,Gabl M,Lutz M,et al.Arch Orthop Trauma Surg,2003;123(1):22～27
35 Horstmann WG, Verbeyen OC,Leemans R,Injuey,2003 ;34(2): 141～144
36 Keating J, Hajducka C.The use of Norian SRS and minimal internal fixation in management of tibial plateau fractures. [J] Trans Orthop Trauma Assoc, 1999,15:411
37 Van Landuyt P, Peter B ,Beluze L ,rt al. Reinforcement of osteosynthesis screws with brushite cement. [J] Bone, 1999,25(2suppl):95s～98s
38 Mattsson P, Larsson S.Scand J Surg,2003;92(3):215～219
39 Yerby SA ,Toh E,Mclain RF. Revision of failed pedicle screws using hydroxyapatite cement: a biomechanial analysis. Spine, 1998,23:1657～1661
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