不同钙磷摩尔比/多重孔隙磷酸钙骨水泥的研究
|
摘要
由于磷酸钙骨水泥(Calcium Phosphate Cement, CPC)具有良好的生物相容性和骨传导性,室温下可自固化塑型,固化时不放热等优点,已经成功地被应用于临床硬组织修复和替代。但是,大部分磷酸钙骨水泥的固相成分都是难溶的磷酸钙盐并且具有较低的钙磷摩尔比,这将会减缓磷酸钙骨水泥的溶解以及向羟基磷灰石的转变,影响磷酸钙骨水泥的生物相容性和降解速率。其中Biocement D由于其优良的性能被广泛研究,但是它的固相成分均为难溶性钙盐,而且它的钙磷摩尔比只有1.50。另外,传统磷酸钙骨水泥缺少适合细胞生长的微孔和骨组织长入的大孔,生物降解较缓慢,虽有研究制备具有多孔结构的磷酸钙骨水泥。然而,过大的孔径和过快的成孔速率将影响磷酸钙骨水泥植入人体前期的力学性能,因此,本论文的目的通过添加易溶钙盐和可降解的壳聚糖,制备不同钙磷摩尔比的磷酸钙骨水泥,利用易溶钙盐的溶解和壳聚糖的降解在体内不同时间形成不同尺寸的多重孔隙结构的磷酸钙骨水泥。
本论文采用磷酸钙骨水泥Biocement D的配方,添加具有一定粒径(20-37.5μm)的可溶性钙盐氯化钙,制备钙磷摩尔比分别为1.60、1.67和1.80的磷酸钙骨水泥。在此基础上,分别在磷酸钙骨水泥(Biocement D)的固相配方中添加不同量的粒径为20-37.5μm的氯化钙颗粒和粒径为150-300 gm的壳聚糖作为成孔剂制备具有多重孔隙结构的磷酸钙骨水泥。测试不同钙磷摩尔比磷酸钙骨水泥和多重孔隙磷酸钙骨水泥的凝结时间,所制备磷酸钙骨水泥进行体外模拟浸泡研究,通过X-射线衍射(XRD)、力学性能实验机和扫描电镜(SEM)等研究磷酸钙骨水泥相成分、抗压强度和断面微观形貌,并且通过细胞实验评价所制备磷酸钙骨水泥的生物学性能。
结果表明：在添加氯化钙的磷酸钙骨水泥中,随着钙磷摩尔比的增加,磷酸钙骨水泥的初、终凝时间都有显著性增加,初凝时间在7-10 min,终凝时间在12-14 min,除1.80-CPC外,凝固时间能满足临床应用的需要。体外模拟浸泡3 d和7 d后,添加了氯化钙的磷酸钙骨水泥的抗压强度都有了显著性提高,但是和对照组相比没有显著性差异。加入CaCl2可提供较多的Ca2+,在水化和体外浸泡过程中促进CPC的起始原料向终相HA生成,1.67-CPC和1.80-CPC体外模拟浸泡后,形成类骨弱结晶磷灰石结构的终产物,具有快速降解和优异生物相容性的特点。水化24小时候后,随着钙磷摩尔比的提高,磷酸钙骨水泥的孔隙率有了显著性提高,钙磷摩尔比为1.80的磷酸钙水泥的孔隙率最大,为34.2±0.3%。
经过研究发现：添加氯化钙和壳聚糖的CPC的凝结时间显著性减小,四组磷酸钙骨水泥的初凝时间为5-7 min,终凝时间为10-14 min。水化24小时后,多重孔隙磷酸钙骨水泥的抗压强度最大为4.78±0.17 MPa,体外模拟浸泡7天后,抗压强度最大为17.8±0.601 MPa,与对照组磷酸钙骨水泥显著性降低。多重孔隙磷酸钙骨水泥的孔隙率显著性提高,水化24小时后最大为40.5±2.2%,体外模拟浸泡7天后,最大为36.2±0.4%。多重孔隙磷酸钙骨水泥经过体外模拟浸泡之后,形成孔径为数百微米的多孔疏松结构,以及弱结晶类骨磷灰石的终产物。
体外细胞培养结果显示,经过7天的细胞培养,钙磷摩尔比为1.60、1.67和1.80三组磷酸钙骨水泥较对照组磷酸钙骨水泥的细胞数量有了显著性提高。增加钙磷摩尔比的CPC实验组细胞的碱性磷酸酶(alkaline phosphatase, ALP)活性显著性提高,说明提高钙磷摩尔比可以提高磷酸钙骨水泥的生物相容性。钙磷摩尔比为1.67和1.80的两组磷酸钙骨水泥的内部清晰地看到成骨细胞沿着孔壁粘附增殖,细胞形态完整,说明较高钙磷摩尔比所产生的多孔疏松结构有利于细胞在磷酸钙骨水泥内部粘附增殖。不同钙磷摩尔比磷酸钙骨水泥和多重孔隙磷酸钙骨水泥的对比细胞实验中,经过7d的细胞培养,钙磷摩尔比为1.60和1.67的多重孔隙磷酸钙骨水泥的细胞数量都有了显著性增加；钙磷摩尔比为1.50和1.67的多重孔隙磷酸钙骨水泥的细胞活性也有了显著性提高。
添加氯化钙的磷酸钙骨水泥具有较高的孔隙率和合适的抗压强度,相成分为类骨弱结晶羟基磷灰石,通过细胞实验发现添加了氯化钙的磷酸钙骨水泥具有更加优良的生物学性能。添加了氯化钙和壳聚糖的磷酸钙骨水泥的孔隙率更高并且具有数百微米的孔隙以及类骨弱结晶羟基磷灰石的相成分,通过细胞实验发现,它的生物学性能较添加氯化钙的磷酸钙骨水泥更加优良。
The calcium phosphate cement (CPC) have many advantages, such as, the excellent biocompatibility, self-setting under ambient conditions, and almost without heat release. Therefore it has attained more attentions and been applied in clinic to repair and replace the bone defects.
However, the solid components of most CPC were insoluble calcium phosphate and the CPC had a lower molar ratio, which would slow down the dissolution CPC as well as changed to the hydroxyapatite(HA)and afected the biocompatibility and the degradation rate of CPC. Biocement D has been studied extensively because of its excellent performance, but it's solid components were insoluble calcium salt, and it's molar ratio of Ca/P was only 1.50. Meanwhile, the traditional CPC was lack of microporous for cell growth and macroporous for tissue ingrowth, and the biodegradable of CPC was slow. Therefore, many studies solved the problem by preparing porous CPC. However, the large size of the pore and fast rate of generating pores affect the mechanical properties of CPC implanted to human in the early. To preparing suitable pore size and rate of generating pores in CPC, the purpose of this study is preparation CPC with different molar ratio of Ca/P by adding easily dissolved calcium chloride particles and biodegradable chitosan particles to CPC, using dissolution of soluble calcium salt and degradation of chitosan formation multiple porous structure in CPC in the body at different times and different size.
This study prepared CPC with 1.60、1.67 and 1.80 molar ratio of Ca/P by adding certain size (20-37.5μm) of soluble calcium salt (calcium chloride) to the solid of Biocement D. On this basis, multiple porous CPC was prepared by adding calcium chloride particles with size of 20-37.5μm and chloride particles with size of 150-300μm to CPC. The initial setting time (IT) and final setting time (FT) of CPC with different molar ratio of Ca/P and multiple porous CPC were studied. X-ray diffraction (XRD), mechanical testing, and scanning electron microscope (SEM) were used to characterize the phase composition, compressive strength and the morphology of the fracture surface of CPC with different Ca/P molar ratios and multiple porous CPC after soaking in phosphate buffer solution (PBS). Biological properties of CPC prepared in this study were evaluated by Cell experiment.
The results show that:with the increase of molar ratio of Ca/P, initial and final setting time of CPC added calcium chloride increased significantly, initial setting time was 7-10 min, final setting time was 12-14 min, the setting time meet the needs of clinical applications except 1.80-CPC. After soaking in PBS for 3 and 7 days, the compressive strength of CPC added calcium chloride increased significantly, but no significant different compared with the control group. Provide more Ca2+ by adding calcium chloride promote the starting material of CPC to the final phase-HA in hydration and soaking in PBS,1.67-CPC and 1.80-CPC formed the poor crystalline apatite similar to those of the inorganic composition of bone after soaking in PBS, which have degradation and excellent biocompatibility. With the increase of molar ratio of Ca/P, the porosity of CPC has improved significantly after hydration for 24 hours, the largest porosity of CPC was 34.2±0.3% with Ca/P molar ratio of 1.80.
The research indicates:the setting time of CPC added calcium chloride and chitosan was significantly reduced which initial setting time was 5-7 min, final setting time was 10-14 min. The maximum compressive strength of multiple porous CPC was 4.78±0.17 MPa after hydration for 24 hours. The maximum compressive strength of multiple porous CPC was 17.8±0.601 MPa after soaking in PBS for 7 days. The compressive strength decreased significantly compared with the control group. The porosity of multiple porous CPC improved significantly, the maximum porosity was 40.5±2.2% after hydration for 24 hours and 36.2±0.4% after soaking in PBS for 7 days. The multiple porous CPC formed the poor crystalline apatite with porous of hundreds of microns in diameter similar to those of the inorganic composition of bone
The results of cell culture in vitro showed that:after 7 days of cell culture, the cell numbers of CPC with Ca/P molar ratio of 1.60,1.67 and 1.80 were improved significantly than CPC with Ca/P molar ratio of 1.50. The alkaline phosphatase (ALP) activity of cells in the experimental group of CPC increased molar ratio of Ca/P improved significantly, it illustrated that promote the molar ratio of Ca/P would improve the biocompatibility of CPC. The osteoblasts in CPC with Ca/P molar ratio of 1.60,1.67 and 1.80 adhered and proliferationto the pore. From the cell experiment between CPC with different molar ratio of Ca/P and multiple porous CPC, the cell numbers of multiple porous CPC with Ca/P molar ratio of 1.60 and 1.67 were increased significantly after 7 days of cell culture; the ALP activity of multiple porous CPC with Ca/P molar ratio of 1.50 and 1.67 had improved significantly. It illustrated that the multiple porous CPC had better performance of biology than CPC with same molar ratio of Ca/P.
The CPC added calcium chloride had high porosity and suitable strength, poor crystalline apatite similar to those of the inorganic composition of bone. The cell experiments showed more excellent biological properties of CPC added calcium chloride. The CPC added calcium chloride and chitosan had higher porosity and pores with hundreds of microns and poor crystalline apatite similar to those of the inorganic composition of bone. From the cell experiment between CPC added calcium chloride to CPC added calcium chloride and chitosan, it showed that CPC added calcium chloride and chitosan had more excellent biological properties.
本论文采用磷酸钙骨水泥Biocement D的配方,添加具有一定粒径(20-37.5μm)的可溶性钙盐氯化钙,制备钙磷摩尔比分别为1.60、1.67和1.80的磷酸钙骨水泥。在此基础上,分别在磷酸钙骨水泥(Biocement D)的固相配方中添加不同量的粒径为20-37.5μm的氯化钙颗粒和粒径为150-300 gm的壳聚糖作为成孔剂制备具有多重孔隙结构的磷酸钙骨水泥。测试不同钙磷摩尔比磷酸钙骨水泥和多重孔隙磷酸钙骨水泥的凝结时间,所制备磷酸钙骨水泥进行体外模拟浸泡研究,通过X-射线衍射(XRD)、力学性能实验机和扫描电镜(SEM)等研究磷酸钙骨水泥相成分、抗压强度和断面微观形貌,并且通过细胞实验评价所制备磷酸钙骨水泥的生物学性能。
结果表明：在添加氯化钙的磷酸钙骨水泥中,随着钙磷摩尔比的增加,磷酸钙骨水泥的初、终凝时间都有显著性增加,初凝时间在7-10 min,终凝时间在12-14 min,除1.80-CPC外,凝固时间能满足临床应用的需要。体外模拟浸泡3 d和7 d后,添加了氯化钙的磷酸钙骨水泥的抗压强度都有了显著性提高,但是和对照组相比没有显著性差异。加入CaCl2可提供较多的Ca2+,在水化和体外浸泡过程中促进CPC的起始原料向终相HA生成,1.67-CPC和1.80-CPC体外模拟浸泡后,形成类骨弱结晶磷灰石结构的终产物,具有快速降解和优异生物相容性的特点。水化24小时候后,随着钙磷摩尔比的提高,磷酸钙骨水泥的孔隙率有了显著性提高,钙磷摩尔比为1.80的磷酸钙水泥的孔隙率最大,为34.2±0.3%。
经过研究发现：添加氯化钙和壳聚糖的CPC的凝结时间显著性减小,四组磷酸钙骨水泥的初凝时间为5-7 min,终凝时间为10-14 min。水化24小时后,多重孔隙磷酸钙骨水泥的抗压强度最大为4.78±0.17 MPa,体外模拟浸泡7天后,抗压强度最大为17.8±0.601 MPa,与对照组磷酸钙骨水泥显著性降低。多重孔隙磷酸钙骨水泥的孔隙率显著性提高,水化24小时后最大为40.5±2.2%,体外模拟浸泡7天后,最大为36.2±0.4%。多重孔隙磷酸钙骨水泥经过体外模拟浸泡之后,形成孔径为数百微米的多孔疏松结构,以及弱结晶类骨磷灰石的终产物。
体外细胞培养结果显示,经过7天的细胞培养,钙磷摩尔比为1.60、1.67和1.80三组磷酸钙骨水泥较对照组磷酸钙骨水泥的细胞数量有了显著性提高。增加钙磷摩尔比的CPC实验组细胞的碱性磷酸酶(alkaline phosphatase, ALP)活性显著性提高,说明提高钙磷摩尔比可以提高磷酸钙骨水泥的生物相容性。钙磷摩尔比为1.67和1.80的两组磷酸钙骨水泥的内部清晰地看到成骨细胞沿着孔壁粘附增殖,细胞形态完整,说明较高钙磷摩尔比所产生的多孔疏松结构有利于细胞在磷酸钙骨水泥内部粘附增殖。不同钙磷摩尔比磷酸钙骨水泥和多重孔隙磷酸钙骨水泥的对比细胞实验中,经过7d的细胞培养,钙磷摩尔比为1.60和1.67的多重孔隙磷酸钙骨水泥的细胞数量都有了显著性增加；钙磷摩尔比为1.50和1.67的多重孔隙磷酸钙骨水泥的细胞活性也有了显著性提高。
添加氯化钙的磷酸钙骨水泥具有较高的孔隙率和合适的抗压强度,相成分为类骨弱结晶羟基磷灰石,通过细胞实验发现添加了氯化钙的磷酸钙骨水泥具有更加优良的生物学性能。添加了氯化钙和壳聚糖的磷酸钙骨水泥的孔隙率更高并且具有数百微米的孔隙以及类骨弱结晶羟基磷灰石的相成分,通过细胞实验发现,它的生物学性能较添加氯化钙的磷酸钙骨水泥更加优良。
The calcium phosphate cement (CPC) have many advantages, such as, the excellent biocompatibility, self-setting under ambient conditions, and almost without heat release. Therefore it has attained more attentions and been applied in clinic to repair and replace the bone defects.
However, the solid components of most CPC were insoluble calcium phosphate and the CPC had a lower molar ratio, which would slow down the dissolution CPC as well as changed to the hydroxyapatite(HA)and afected the biocompatibility and the degradation rate of CPC. Biocement D has been studied extensively because of its excellent performance, but it's solid components were insoluble calcium salt, and it's molar ratio of Ca/P was only 1.50. Meanwhile, the traditional CPC was lack of microporous for cell growth and macroporous for tissue ingrowth, and the biodegradable of CPC was slow. Therefore, many studies solved the problem by preparing porous CPC. However, the large size of the pore and fast rate of generating pores affect the mechanical properties of CPC implanted to human in the early. To preparing suitable pore size and rate of generating pores in CPC, the purpose of this study is preparation CPC with different molar ratio of Ca/P by adding easily dissolved calcium chloride particles and biodegradable chitosan particles to CPC, using dissolution of soluble calcium salt and degradation of chitosan formation multiple porous structure in CPC in the body at different times and different size.
This study prepared CPC with 1.60、1.67 and 1.80 molar ratio of Ca/P by adding certain size (20-37.5μm) of soluble calcium salt (calcium chloride) to the solid of Biocement D. On this basis, multiple porous CPC was prepared by adding calcium chloride particles with size of 20-37.5μm and chloride particles with size of 150-300μm to CPC. The initial setting time (IT) and final setting time (FT) of CPC with different molar ratio of Ca/P and multiple porous CPC were studied. X-ray diffraction (XRD), mechanical testing, and scanning electron microscope (SEM) were used to characterize the phase composition, compressive strength and the morphology of the fracture surface of CPC with different Ca/P molar ratios and multiple porous CPC after soaking in phosphate buffer solution (PBS). Biological properties of CPC prepared in this study were evaluated by Cell experiment.
The results show that:with the increase of molar ratio of Ca/P, initial and final setting time of CPC added calcium chloride increased significantly, initial setting time was 7-10 min, final setting time was 12-14 min, the setting time meet the needs of clinical applications except 1.80-CPC. After soaking in PBS for 3 and 7 days, the compressive strength of CPC added calcium chloride increased significantly, but no significant different compared with the control group. Provide more Ca2+ by adding calcium chloride promote the starting material of CPC to the final phase-HA in hydration and soaking in PBS,1.67-CPC and 1.80-CPC formed the poor crystalline apatite similar to those of the inorganic composition of bone after soaking in PBS, which have degradation and excellent biocompatibility. With the increase of molar ratio of Ca/P, the porosity of CPC has improved significantly after hydration for 24 hours, the largest porosity of CPC was 34.2±0.3% with Ca/P molar ratio of 1.80.
The research indicates:the setting time of CPC added calcium chloride and chitosan was significantly reduced which initial setting time was 5-7 min, final setting time was 10-14 min. The maximum compressive strength of multiple porous CPC was 4.78±0.17 MPa after hydration for 24 hours. The maximum compressive strength of multiple porous CPC was 17.8±0.601 MPa after soaking in PBS for 7 days. The compressive strength decreased significantly compared with the control group. The porosity of multiple porous CPC improved significantly, the maximum porosity was 40.5±2.2% after hydration for 24 hours and 36.2±0.4% after soaking in PBS for 7 days. The multiple porous CPC formed the poor crystalline apatite with porous of hundreds of microns in diameter similar to those of the inorganic composition of bone
The results of cell culture in vitro showed that:after 7 days of cell culture, the cell numbers of CPC with Ca/P molar ratio of 1.60,1.67 and 1.80 were improved significantly than CPC with Ca/P molar ratio of 1.50. The alkaline phosphatase (ALP) activity of cells in the experimental group of CPC increased molar ratio of Ca/P improved significantly, it illustrated that promote the molar ratio of Ca/P would improve the biocompatibility of CPC. The osteoblasts in CPC with Ca/P molar ratio of 1.60,1.67 and 1.80 adhered and proliferationto the pore. From the cell experiment between CPC with different molar ratio of Ca/P and multiple porous CPC, the cell numbers of multiple porous CPC with Ca/P molar ratio of 1.60 and 1.67 were increased significantly after 7 days of cell culture; the ALP activity of multiple porous CPC with Ca/P molar ratio of 1.50 and 1.67 had improved significantly. It illustrated that the multiple porous CPC had better performance of biology than CPC with same molar ratio of Ca/P.
The CPC added calcium chloride had high porosity and suitable strength, poor crystalline apatite similar to those of the inorganic composition of bone. The cell experiments showed more excellent biological properties of CPC added calcium chloride. The CPC added calcium chloride and chitosan had higher porosity and pores with hundreds of microns and poor crystalline apatite similar to those of the inorganic composition of bone. From the cell experiment between CPC added calcium chloride to CPC added calcium chloride and chitosan, it showed that CPC added calcium chloride and chitosan had more excellent biological properties.
引文
[1]LC. Chow. Development of self-setting calcium phosphate cements. J Ceram Soci Jap.1991,99 (10):954-964.
[2]BR. Constantz, IC. Ison, MT. Fulmer, et al. Skeletal repair by in situ formation of the mineral Phase of bone. Science.1995,267 (5205):1796-1799.
[3]PD. Costantino, CD. Friedman, LC. Chow, et al. Hydroxyapatite cement: Basic chemistry and histology properties. Arch Otolaryngol Head Neek Surg. 1991,117 (4):379-384.
[4]PD. Constantino, CD. Friendman, LC. Chow, et al. Experimental Hydroxyapatite cement. eranioPlasty. Plast Re Constr Surg.1992,90 (2): 174-185.
[5]PD. Constantino, CD. Friedman, A. Lane. Synthetic biomaterials in facial plastic and reconstructive surgery. Facial Plastic Surg.1993,9 (1):1-15.
[6]CD. Friedman, PD. Costantino, LC. Chow, et al. Hydroxyapatite cement. Ⅱ. Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surg.1991,117 (4):385-389.
[7]朱静,潘可风.磷酸钙骨水泥的研究和临床进展.上海生物医学工程,2001；22：37.
[8]EM. Oomsa, EA. Egglezosb, JGC. Wolke. Soft-tissue response to injectable calcium phosphate cements. Biomaterials.2003, (24):749-757.
[9]郑昌琼,冉军国,新型无机材料.北京：科学出版社,2003.
[10]I. Khairoun, MG. Boltong, FC. Driessens. Effect of calcium carbonate on clinical compliance of apatitic calcium phosphate bone cement. J Biomed Mater Res.1997,38 (4):356-60.
[11]B. Bai. The Use of an Injectable, Biodegradable Calcium Phosphate Bone Subsitute for the Prophylactic Augmentation of. Osteroporotic Vertebral Compression. Spine,1999.24 (8):1521-1526.
[12]JL. Lacout, E. Mejdoub, M. Hamad. Crystallization mechanisms of calcium phosphate cement for biological uses. J Mater Sei.1996,7:371-374.
[13]M.Otsuka, Y.Nakahigashi, Y.Matsuda, JL. Fox, WI. Higuchi, Y.Sugiyama. A novel skeletal drug delivery system using self-setting calcium phosphate cement Vlll:The relationship between invitro and invivo drug release from indomethacin containing cement. J.Controlled Release.1997,43:115-122.
[14]A. Bigi, B. Bracci, S. Panzavolta. Effect of added gelatin on the properties of calcium phosphate cement. Biomaterials.2004,25:2893-2899.
[15]K. Ishikawa. Self-setting barrier membrane for guided tissue regeneration method:Initial evaluation of alginate membrane made with sodium alginate and calcium chloride aqueous solutions. J Biomed Mater Res.1999,47 (2):111-115.
[16]EM. Oomsa, N. Verdonschotb, JGC. Wolke. Enhancement of initial stability of press-fit femoral stems using injectable calcium phosphate cement:an in vitro study in dog bones. Biomaterials.2004, (25):3887-3894.
[17]P. Dennis, VD. Juliette. Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles. Biomaterials,2006 (27):4941-4947.
[18]SV. Dorozhkin. Calcium Orthophosphates in Nature, Biology and Medicine. Materials.2009,2:399-498.
[19]M. Driessens, G. Boltong, O. Bermudez, et al. Formulation and setting times of some calcium orthophosphate cenments:a pilot study. Mater Sci Mater Med, 1994,5:164-170.
[20]W. Habraken, J. Wolke, J. Jansen. Ceramic composites as matrices and scaffolds for drug delivery in tissue engineering. Advanced Drug Delivery Reviews.2007,59:234-248.
[21]FCM. Driessens. Formation and stability of calcium phosphate inrelation to the phase composition of the mineral in calcied tissues. Bioceramics of calcium phosphates. Boca, Raton, FL:CRC Press; 1983:1-32.
[22]A. Tofighi, K. Schaffer, R. Palazzolo. Calcium phosphate cement (CPC): acritical development path. Key Eng Mater.2008,361-363:303-306.
[23]M. Pi, P. Faber, G. Ekema, PD. Jackson, A. Ting, N. Wang, et al. Identication of a novel extracellular cation-sensing G-protein-coupled receptor. J Biol Chem 2005,280 (40):201-209.
[24]XP. Wang, JD. Ye, Y Wang. Influence of calcium to phosphate molar ratio on performance of a novel calcium puosphate cement. Journal of the Chinese Ceramic Society.2007,35 (12):1582-1586.
[25]张爱娟,王卫伟.磷酸钙骨水泥的制备及性能.材料科学与工程学报.2008,26(5)：779-782.
[26]H. Guo, J. Su, J. Wei, H. Kong, CS. Liu. Biocompatibility and osteogenicity of degradable Ca-decient hydroxyapatite scaffolds from calcium phosphate cement for bone tissue engineering. Acta Biomaterialia.2009,5:268-278.
[27]JX. Lu, B. Flautre, K. Anselme. et al. Role of interconnections in porous biocermics on bone recoloniztion in vitro and in vivo. J Mater Sci:Mater Med. 1999,10:111-118.
[28]顾汉卿,徐国风.生物医学材料学.天津,天津科技翻译出版社.1993,335-351.
[29]L L. Hench. Bioceramics. Am Ceram Soc.1998,81:1705-1728.
[30]唐佩福,姚琦,黄鹏,王继芳.碳酸化羟基磷灰石骨水泥的开发与多孔化研究.中国矫形外科杂志.2008,216：124-126.
[31]董浩,叶建东,王秀鹏,杨娟娟.磷酸钙骨水泥大孔径多孔组织工程支架的制备及其纤维增强.无机材料学报.2007,5(22)：1007-1010.
[32]董浩,叶建东,王秀鹏.磷酸钙骨水泥明胶复合组织工程支架材料的制备及其结构与性能.功能材料.2006,37(11)：1805-1807.
[33]WJ. Habraken, Z. Zhang, JG. Wolke, DW. Grijpma, AG. Mikos, J. Feijen, JA. Jansen. Introduction of enzymatically degradable poly (trimethylene carbonate) microspheres into an injectable calcium phosphate cement. Biomaterials.2008,29: 2464-2476.
[34]PQ. Ruhe, OC. Boerman, FGM. Russel, PHM. Spauwen, AG. Mikos, JA. Jansen. Controlled release of rhBMP-2 loaded poly (DL-lactic-co-glycolicacid)/calcium phosphate cement composites in vivo. Journal of Controlled Release.2005,106:162-171.
[35]朱广楠,孙康宁.明胶对多孔磷酸钙骨水泥结构及性能的影响.硅酸盐学报.2008,3(36)：383-386.
[36]姚子昂,吴海歌,邢福有.壳聚糖在组织工程中应用的研究进展.中国生物工程杂志,2003,23(10)：32-36.
[37]吕彩霞,姚子华.羟基磷灰石／壳聚糖复合材料研究进展.化工进展.2006,25(7)：755-759.
[38]PJ. Vord, HWL. Matthew, SP. Desilva, et al. Evaluation of the biocompatibility of a chitosan scaffold in mice. J. Biomed. Mater. Res.2002,59 (3):585-590.
[39]F. Driessens, MG. Boltong, O. Bermudez. Formulation and setting times of some calcium orthophosphate cements:a pilot study. Mater Sci-Mater M.1991,4: 503-508
[40]HHK. Xu, S. Takagi, JB. Quinn. Fast-setting calcium phosphate scaffolds with tailored macropore formation rates for bone regeneration. J Biomed Mater Res.2004,68:725-734.
[41]A Cherng, S. Takagi, LC. Chow. Effects of hydroxypropyl methylcellulose and other gelling agents on the handling properties of calcium phosphate cement. J Biomed Mater Res.1997,35:273-277.
[42]华中师范大学.分析化学[M](第二版).北京,高等教育出版社.1996：436-438.
[43]吴飞雁,刘兴炎,陈克明,等.大孔径速降解磷酸钙骨水泥的制备.中国矫形外科杂志.2008,16(15)：1181-1184.
[44]沈卫,刘昌胜,顾燕芳.磷酸钙骨水泥的水化反应、凝结时间及抗压强度.酸盐学报.1998,26(2)：129-135.
[45]I. Khairoun, MG. Boltong, FCM. Driessens. et al. Effect of calcium carbonate on the compliance of an apatite calcium phosphate bone cement. Biomaterials. 1997,18:1535-1539.
[46]K. Tadashi, T. Hiroaki. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials.2006,27:2907-2915.
[47]罗方聪,叶建东.层状结构磷酸钙骨水泥组织工程支架材料的制备与表征.硅酸盐通报.2009,28(1)：27-30,37.
[48]P. Real, G. Wolke, M. Vallet, et al. A new method to produce macropores in calcium phosphate cements. Biomaterials,2002,23:3673-3680.
[49]何莉萍,吴振军,陈宗璋,米耀荣.纳米晶状磷酸钙盐生物复合材料的制备与结构表征.无机化学学报.2004,20(3)：273-277.
[50]M. Bohner. Calcium orthophosphates in medicine:from ceramics to calcium phosphate cements. Injury. Int. J. Care Injured.2000,31:37-47.
[51]T. Sadao, LC. Chow, I. Kiichi. Formation of hydroxyapatite in new calcium phosphate cements. Biomaterials.1998,19:1593-1599.
[52]叶建东,王秀鹏,白波,徐谦.一种可注射可降解磷酸钙骨水泥的结构与性能.功能材料.2008,2(39)：271-274.
[53]陈芳萍,王凯,刘昌胜,李伟翔.聚磷酸钙骨支架材料的可控降解性和 细胞毒性研究.无机化学学报.2008,24(1)：88-92.
[54]钟吉品,刘宣勇,常江.激活基因的玻璃.无机材料学报.2002,17(5)：897-907.
[55]叶建东,王秀鹏,白波,等.一种可注射可降解磷酸钙骨水泥的结构与性能.功能材料.2008,2(39)：271-274.
[56]韩媛媛,李红,容建华,周长忍.羧甲基壳聚糖复合磷酸钙骨水泥的实验.暨南大学学报(自然科学版.2007,28(3)：288-291.
[57]LX. Xu, XT. Shi, YP.Wang, ZL. Shi. Performance of calcium phosphate bone cement using chitosan and gelatin as well as citric acid as harding liquid. Journal of clinical rehabilitative tissue engineering research.2008,12 (32): 6381-6384.
[58]I. Sopyan, M. Mel. S. Ramesh, et al. Porous hydroxyapatite for articial bone applications. Science and Technology of Advanced Materials.2007,8:116-123.
[59]T. Kokubo, Apatite formation on organic polymers by a biomimetic process. Eur J Solid State Inorg Chem.1995,32:819-827.
[60]JG. Li, HH. Liao, M. Sjosrom. Characterization of calcium phosphate precipitated from simulated body fluid of different buffering capacities. Biomaterials.1997.18:743-747.
[61]D. Klee, H. Hoecker. Polymers for biomedical applications:improvement of the interface compatibility. Adv Polym Sci.1999,149:1-57.
[62]A. Lucke, J. Tessmar, E. Schnell. Biodegradable poly (D, L-lacticacid)-poly (ethylene glycol)-monomethyl ether diblock copolymers:structures and surface properties relevant to their use as biomaterials. Biomaterials.2000,21: 2361-2370.
[2]BR. Constantz, IC. Ison, MT. Fulmer, et al. Skeletal repair by in situ formation of the mineral Phase of bone. Science.1995,267 (5205):1796-1799.
[3]PD. Costantino, CD. Friedman, LC. Chow, et al. Hydroxyapatite cement: Basic chemistry and histology properties. Arch Otolaryngol Head Neek Surg. 1991,117 (4):379-384.
[4]PD. Constantino, CD. Friendman, LC. Chow, et al. Experimental Hydroxyapatite cement. eranioPlasty. Plast Re Constr Surg.1992,90 (2): 174-185.
[5]PD. Constantino, CD. Friedman, A. Lane. Synthetic biomaterials in facial plastic and reconstructive surgery. Facial Plastic Surg.1993,9 (1):1-15.
[6]CD. Friedman, PD. Costantino, LC. Chow, et al. Hydroxyapatite cement. Ⅱ. Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surg.1991,117 (4):385-389.
[7]朱静,潘可风.磷酸钙骨水泥的研究和临床进展.上海生物医学工程,2001；22：37.
[8]EM. Oomsa, EA. Egglezosb, JGC. Wolke. Soft-tissue response to injectable calcium phosphate cements. Biomaterials.2003, (24):749-757.
[9]郑昌琼,冉军国,新型无机材料.北京：科学出版社,2003.
[10]I. Khairoun, MG. Boltong, FC. Driessens. Effect of calcium carbonate on clinical compliance of apatitic calcium phosphate bone cement. J Biomed Mater Res.1997,38 (4):356-60.
[11]B. Bai. The Use of an Injectable, Biodegradable Calcium Phosphate Bone Subsitute for the Prophylactic Augmentation of. Osteroporotic Vertebral Compression. Spine,1999.24 (8):1521-1526.
[12]JL. Lacout, E. Mejdoub, M. Hamad. Crystallization mechanisms of calcium phosphate cement for biological uses. J Mater Sei.1996,7:371-374.
[13]M.Otsuka, Y.Nakahigashi, Y.Matsuda, JL. Fox, WI. Higuchi, Y.Sugiyama. A novel skeletal drug delivery system using self-setting calcium phosphate cement Vlll:The relationship between invitro and invivo drug release from indomethacin containing cement. J.Controlled Release.1997,43:115-122.
[14]A. Bigi, B. Bracci, S. Panzavolta. Effect of added gelatin on the properties of calcium phosphate cement. Biomaterials.2004,25:2893-2899.
[15]K. Ishikawa. Self-setting barrier membrane for guided tissue regeneration method:Initial evaluation of alginate membrane made with sodium alginate and calcium chloride aqueous solutions. J Biomed Mater Res.1999,47 (2):111-115.
[16]EM. Oomsa, N. Verdonschotb, JGC. Wolke. Enhancement of initial stability of press-fit femoral stems using injectable calcium phosphate cement:an in vitro study in dog bones. Biomaterials.2004, (25):3887-3894.
[17]P. Dennis, VD. Juliette. Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles. Biomaterials,2006 (27):4941-4947.
[18]SV. Dorozhkin. Calcium Orthophosphates in Nature, Biology and Medicine. Materials.2009,2:399-498.
[19]M. Driessens, G. Boltong, O. Bermudez, et al. Formulation and setting times of some calcium orthophosphate cenments:a pilot study. Mater Sci Mater Med, 1994,5:164-170.
[20]W. Habraken, J. Wolke, J. Jansen. Ceramic composites as matrices and scaffolds for drug delivery in tissue engineering. Advanced Drug Delivery Reviews.2007,59:234-248.
[21]FCM. Driessens. Formation and stability of calcium phosphate inrelation to the phase composition of the mineral in calcied tissues. Bioceramics of calcium phosphates. Boca, Raton, FL:CRC Press; 1983:1-32.
[22]A. Tofighi, K. Schaffer, R. Palazzolo. Calcium phosphate cement (CPC): acritical development path. Key Eng Mater.2008,361-363:303-306.
[23]M. Pi, P. Faber, G. Ekema, PD. Jackson, A. Ting, N. Wang, et al. Identication of a novel extracellular cation-sensing G-protein-coupled receptor. J Biol Chem 2005,280 (40):201-209.
[24]XP. Wang, JD. Ye, Y Wang. Influence of calcium to phosphate molar ratio on performance of a novel calcium puosphate cement. Journal of the Chinese Ceramic Society.2007,35 (12):1582-1586.
[25]张爱娟,王卫伟.磷酸钙骨水泥的制备及性能.材料科学与工程学报.2008,26(5)：779-782.
[26]H. Guo, J. Su, J. Wei, H. Kong, CS. Liu. Biocompatibility and osteogenicity of degradable Ca-decient hydroxyapatite scaffolds from calcium phosphate cement for bone tissue engineering. Acta Biomaterialia.2009,5:268-278.
[27]JX. Lu, B. Flautre, K. Anselme. et al. Role of interconnections in porous biocermics on bone recoloniztion in vitro and in vivo. J Mater Sci:Mater Med. 1999,10:111-118.
[28]顾汉卿,徐国风.生物医学材料学.天津,天津科技翻译出版社.1993,335-351.
[29]L L. Hench. Bioceramics. Am Ceram Soc.1998,81:1705-1728.
[30]唐佩福,姚琦,黄鹏,王继芳.碳酸化羟基磷灰石骨水泥的开发与多孔化研究.中国矫形外科杂志.2008,216：124-126.
[31]董浩,叶建东,王秀鹏,杨娟娟.磷酸钙骨水泥大孔径多孔组织工程支架的制备及其纤维增强.无机材料学报.2007,5(22)：1007-1010.
[32]董浩,叶建东,王秀鹏.磷酸钙骨水泥明胶复合组织工程支架材料的制备及其结构与性能.功能材料.2006,37(11)：1805-1807.
[33]WJ. Habraken, Z. Zhang, JG. Wolke, DW. Grijpma, AG. Mikos, J. Feijen, JA. Jansen. Introduction of enzymatically degradable poly (trimethylene carbonate) microspheres into an injectable calcium phosphate cement. Biomaterials.2008,29: 2464-2476.
[34]PQ. Ruhe, OC. Boerman, FGM. Russel, PHM. Spauwen, AG. Mikos, JA. Jansen. Controlled release of rhBMP-2 loaded poly (DL-lactic-co-glycolicacid)/calcium phosphate cement composites in vivo. Journal of Controlled Release.2005,106:162-171.
[35]朱广楠,孙康宁.明胶对多孔磷酸钙骨水泥结构及性能的影响.硅酸盐学报.2008,3(36)：383-386.
[36]姚子昂,吴海歌,邢福有.壳聚糖在组织工程中应用的研究进展.中国生物工程杂志,2003,23(10)：32-36.
[37]吕彩霞,姚子华.羟基磷灰石／壳聚糖复合材料研究进展.化工进展.2006,25(7)：755-759.
[38]PJ. Vord, HWL. Matthew, SP. Desilva, et al. Evaluation of the biocompatibility of a chitosan scaffold in mice. J. Biomed. Mater. Res.2002,59 (3):585-590.
[39]F. Driessens, MG. Boltong, O. Bermudez. Formulation and setting times of some calcium orthophosphate cements:a pilot study. Mater Sci-Mater M.1991,4: 503-508
[40]HHK. Xu, S. Takagi, JB. Quinn. Fast-setting calcium phosphate scaffolds with tailored macropore formation rates for bone regeneration. J Biomed Mater Res.2004,68:725-734.
[41]A Cherng, S. Takagi, LC. Chow. Effects of hydroxypropyl methylcellulose and other gelling agents on the handling properties of calcium phosphate cement. J Biomed Mater Res.1997,35:273-277.
[42]华中师范大学.分析化学[M](第二版).北京,高等教育出版社.1996：436-438.
[43]吴飞雁,刘兴炎,陈克明,等.大孔径速降解磷酸钙骨水泥的制备.中国矫形外科杂志.2008,16(15)：1181-1184.
[44]沈卫,刘昌胜,顾燕芳.磷酸钙骨水泥的水化反应、凝结时间及抗压强度.酸盐学报.1998,26(2)：129-135.
[45]I. Khairoun, MG. Boltong, FCM. Driessens. et al. Effect of calcium carbonate on the compliance of an apatite calcium phosphate bone cement. Biomaterials. 1997,18:1535-1539.
[46]K. Tadashi, T. Hiroaki. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials.2006,27:2907-2915.
[47]罗方聪,叶建东.层状结构磷酸钙骨水泥组织工程支架材料的制备与表征.硅酸盐通报.2009,28(1)：27-30,37.
[48]P. Real, G. Wolke, M. Vallet, et al. A new method to produce macropores in calcium phosphate cements. Biomaterials,2002,23:3673-3680.
[49]何莉萍,吴振军,陈宗璋,米耀荣.纳米晶状磷酸钙盐生物复合材料的制备与结构表征.无机化学学报.2004,20(3)：273-277.
[50]M. Bohner. Calcium orthophosphates in medicine:from ceramics to calcium phosphate cements. Injury. Int. J. Care Injured.2000,31:37-47.
[51]T. Sadao, LC. Chow, I. Kiichi. Formation of hydroxyapatite in new calcium phosphate cements. Biomaterials.1998,19:1593-1599.
[52]叶建东,王秀鹏,白波,徐谦.一种可注射可降解磷酸钙骨水泥的结构与性能.功能材料.2008,2(39)：271-274.
[53]陈芳萍,王凯,刘昌胜,李伟翔.聚磷酸钙骨支架材料的可控降解性和 细胞毒性研究.无机化学学报.2008,24(1)：88-92.
[54]钟吉品,刘宣勇,常江.激活基因的玻璃.无机材料学报.2002,17(5)：897-907.
[55]叶建东,王秀鹏,白波,等.一种可注射可降解磷酸钙骨水泥的结构与性能.功能材料.2008,2(39)：271-274.
[56]韩媛媛,李红,容建华,周长忍.羧甲基壳聚糖复合磷酸钙骨水泥的实验.暨南大学学报(自然科学版.2007,28(3)：288-291.
[57]LX. Xu, XT. Shi, YP.Wang, ZL. Shi. Performance of calcium phosphate bone cement using chitosan and gelatin as well as citric acid as harding liquid. Journal of clinical rehabilitative tissue engineering research.2008,12 (32): 6381-6384.
[58]I. Sopyan, M. Mel. S. Ramesh, et al. Porous hydroxyapatite for articial bone applications. Science and Technology of Advanced Materials.2007,8:116-123.
[59]T. Kokubo, Apatite formation on organic polymers by a biomimetic process. Eur J Solid State Inorg Chem.1995,32:819-827.
[60]JG. Li, HH. Liao, M. Sjosrom. Characterization of calcium phosphate precipitated from simulated body fluid of different buffering capacities. Biomaterials.1997.18:743-747.
[61]D. Klee, H. Hoecker. Polymers for biomedical applications:improvement of the interface compatibility. Adv Polym Sci.1999,149:1-57.
[62]A. Lucke, J. Tessmar, E. Schnell. Biodegradable poly (D, L-lacticacid)-poly (ethylene glycol)-monomethyl ether diblock copolymers:structures and surface properties relevant to their use as biomaterials. Biomaterials.2000,21: 2361-2370.