CPC的制备、表征与荧光示踪研究
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摘要
磷酸钙骨水泥(calcium phosphate cement, CPC)由于其在化学组成上与骨组织的无机成分相同,同时具有优良的生物性能,即骨传导性和骨诱导性,得到了国内外广泛的关注和研究。
本文制备得到贝塔磷酸三钙(β-Ca3(PO4)2,β-TCP),将其作为骨水泥的固相成分,以壳聚糖、柠檬酸、聚乙烯醇混合溶液作为液相,采用正交试验,从凝固时间和抗压强度两方面确定了液相的最佳配比,制备可满足临床需要的磷酸钙骨水泥。
同时,本文设计制备了具有荧光效果的Eu3+掺杂β-TCP材料,利用铕的荧光特性,示踪β-TCP中钙离子在骨修复过程中的作用、迁移过程。
通过XRD分析表明,Eu3+掺杂β-TCP主晶相仍保持其原有的六方结构,通过计算得到其晶格常数发生变化,证明Eu3+进入β-TCP晶胞中；同时,在高分辨率透射电镜中也得到证实；在荧光显微镜下可看到明显的红色荧光,有利于其在骨诱导性的研究中进行示踪；同时,利用电感耦合等离子质谱跟踪检测了Eu离子,一定程度上示踪了Ca2+动向,从而为进一步研究骨诱导效应的具体过程和机理提供基础。
Nowadays, CPC is attractive in a broad range of special interest, due to its chemical component is similar to the mineral phase of hard tissue, and high biocompatibility--osteoinductivity and osteoconduction.
In this paper, self-madeβ-TCP powder used for solid phase of CPC. While the liquid phases were chitosan, citric acid and polyvinyl alcohol. The design orthogonal was tested to determine the best ratio of the system from the coagulation time and compressive strength of the CPC.
Then,β-TCP doped europium were prepared which have fluorescence effect, take advantage of fluorescence characteristics of europium to tracing the role and migration process of calcium in the bone repair.
XRD analysis represents thatβ-TCP containing europium have the same structure withβ-TCP. However, the changed lattice constants obtained by calculating shows that Eu3+ doping into the P-TCP unit cell which was also proved by HRTEM image. The fluoresence microscope demonstrate the complex show clear red luminescence, which are helpful to tracing studies in osteoinductive. ICP-MS analysis shows that, the contents of Eu3+ in the saline solution trace the Ca2+, it is beneficial in tracing study of osteoinductivity.
本文制备得到贝塔磷酸三钙(β-Ca3(PO4)2,β-TCP),将其作为骨水泥的固相成分,以壳聚糖、柠檬酸、聚乙烯醇混合溶液作为液相,采用正交试验,从凝固时间和抗压强度两方面确定了液相的最佳配比,制备可满足临床需要的磷酸钙骨水泥。
同时,本文设计制备了具有荧光效果的Eu3+掺杂β-TCP材料,利用铕的荧光特性,示踪β-TCP中钙离子在骨修复过程中的作用、迁移过程。
通过XRD分析表明,Eu3+掺杂β-TCP主晶相仍保持其原有的六方结构,通过计算得到其晶格常数发生变化,证明Eu3+进入β-TCP晶胞中；同时,在高分辨率透射电镜中也得到证实；在荧光显微镜下可看到明显的红色荧光,有利于其在骨诱导性的研究中进行示踪；同时,利用电感耦合等离子质谱跟踪检测了Eu离子,一定程度上示踪了Ca2+动向,从而为进一步研究骨诱导效应的具体过程和机理提供基础。
Nowadays, CPC is attractive in a broad range of special interest, due to its chemical component is similar to the mineral phase of hard tissue, and high biocompatibility--osteoinductivity and osteoconduction.
In this paper, self-madeβ-TCP powder used for solid phase of CPC. While the liquid phases were chitosan, citric acid and polyvinyl alcohol. The design orthogonal was tested to determine the best ratio of the system from the coagulation time and compressive strength of the CPC.
Then,β-TCP doped europium were prepared which have fluorescence effect, take advantage of fluorescence characteristics of europium to tracing the role and migration process of calcium in the bone repair.
XRD analysis represents thatβ-TCP containing europium have the same structure withβ-TCP. However, the changed lattice constants obtained by calculating shows that Eu3+ doping into the P-TCP unit cell which was also proved by HRTEM image. The fluoresence microscope demonstrate the complex show clear red luminescence, which are helpful to tracing studies in osteoinductive. ICP-MS analysis shows that, the contents of Eu3+ in the saline solution trace the Ca2+, it is beneficial in tracing study of osteoinductivity.
引文
[1]佘永华,肖洪文.系统解剖学.成都：四川大学出版社.2004,9-12.
[2]顾汉卿.《生物医用材料》.天津：天津大学出版社,1998,256-278.
[3]沈序辉,宋晨路,沈鸽等.有机一轻基磷灰石复合骨替代材料[J].材料与工程,1999(12)：85-90.
[4]邹仲之.组织学与胚胎学[M].北京：人民卫生出版社,2002.7：52-53.
[6]Brown WE, Chow LC. United States Patient,4518430.1985.
[7]Brown WE, Chow LC. United States Patient,4612053.1986.
[8]Gbureck U, Spatz K, Thull R. Improvement of mechanical properties of self setting calcium phosphate bone cement mixed with different metal oxides.Mat.-wiss.u.Werkstofftech.2003,34(12):1036-1040.
[9]Doi Y, Shimizu Y, Mouriwaki Y, Aga M,Iwanaga H, Shibutani T, Yamamoto K, Iwayama Y. Development of a new calcium phosphate cement that contains sodium calcium phosphate. Biomaterials,2001,22:847-854.
[10]Yokoyama A, Yamamoto S, Kawasaki T, Kohgo T, Nakasu M. Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials. Biomaterials,2002,23:1091-1101.
[11]Chow LC. Development of selfsetting calcium phosphate cements. Journal Ceramics Society Japan,1991,99(10):956-964.
[12]Constantz BR, Ison LC, Fulmer M T, et al. Skeletal repair by in situformation of the mineral phase of bone. Science,1995,267(5205):1796-1798.
[13]Friendman CD, Costantino PD, Chow LC, et al. Hydroxyapatite cement.Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surgery,1991,117(4):383-386.
[14]Costantino PD, Friendman CD, Jones K, et al. Experimental hydroxyapatite cement Cranioplasty. Plastic and Reconstructive sergery,1992,90(2):174-185.
[15]Karashinak K, Kurita H, Kotani A, et al. Experimental cranioplasty and skeletal augmentation using an alpha-tricalcium phosphate/dicalcium phosphate dibasic/tetracalcium phosphate monoxide cement:a preliminary short term experiment in rabbit. Biomaterials,1998,19(79):701-706.
[17]Ishikawa K, Miyamoto Y, Takechi M. Non-decay type fast-setting calcium phosphate cement:Hydroxyapatite putty containing an increased amount of sodium alginate. Journal of Biomedical Materials Research,
1997,36(3):393-399.
[18]陈发明,吴织芬,金岩等.智能凝胶药物控释系统在医学上的应用[J].人民军医,2005,48(4)：231-233.
[19]Jarcho M, Bolen C H. Hydroxyapatite synthesis and characterization in dense polycrystalline form[J]. J. Mater Sci,1976,11(2):227-235.
[20]Osaka A,Miura R,Taeuchi K, et al.Calcium apatite prepared from calcium hydroxide and orthophosphoric acid[J]. J Mater Sci Med,1991,2 (1):51-55.
[21]Hattori T, Iwadate Y. Hydrothermal preparation of calcium hydroxyapatite powders[J].J Am Ceram Soci,1990,73(6):1803-1805.
[22]武强,李祖兵.多孔β-TCP/BMG复合人工骨修复下颌骨缺损的实验研究[J].中国口腔种植学杂志,2001,6(4)：157-158.
[23]Costantino PD, Friedman CD, Lane A. Synthetic biomarerials in facial plastic and reconstructive surgery.Facial Plastic Surg.1993,9(1):1-7.
[24]Gerhart,Tobin N Miler,Richard L, et al. In vitro characterization and biomechanical opimization of a biodegrad-able particulate composite bone cement [J]. J Biomed Mater Res,1988,22:1071.
[25]Dai HL, Yan YH, Wang YF, et al. Evaluation of calcium phosphate cement as a root canal sealer filing material. Journal of Wuhan University of Technology:Mater Sci Ed,2001,16(4):14-18.
[26]郑承泽,苏国礼,王天兵,等.多孔磷酸三钙与自体红骨髓复合移植修复骨缺损的临床应用[J].中国修复重建外科杂志,1996,10(3)：164-167.
[27]Constantz BR, Ison LC, Fulmer MT, etal. Skeletal repair by in situ formation of the mineral phase of bone science,1995,267(5205):1796-1798.
[28]邸利芝,赵红.β-磷酸三钙的制备及骨科中的应用.天津理工学院学报.2003,19(2)：102-105.
[29]Driessens FCM, Boltong MG, de Maeyer EAP,et al. The Ca/P range of nanoapatite calcium phosphate cements.Biomaterials,2002;32(19):4011.
[30]Vande Vord PJ, Matthew HWT, DeSilva SP, Mayton L,Wu B, wooley PH. Evaluation of the Biocompatibility of a chitosan scaffold in mice.J Biomed Mater Res,2002,59:585-590.
[31]Senkoylu A, Simsek A, Sahin FI, Menevse S, Ozogul C,Denkbas EB, Piskin E. Interaction of cultured chondrocytes with chitosan scaffolf. Journal of Bioactive and Compatible Polymers,2001,16:136-144.
[32]Klokkevold PR, Vandemark L, Kenney EB, et al. Osteogenesis enhances by
chitosan (poly-N-acetyl glucosaminoglycan) in vitro, Periodotal,1996, 67(11):1170-1175.
[33]Friberg J, Fernandez E, Sarda S, et al. An experimental approach to the study of the rheology behaviour of synthetic bone calcium phosphate cements. Key Eng Mater,2001,192-195:777-780.
[34]Sarda S, Fernandez E, Nilsson M, et al. Kinetic study of citric acid influence on calcium phosphate bone cements as water-reducing agent. J Biomed Mater Res, 2002,61(4):653-659.
[35]杜嘉英等.聚乙烯醇在医疗中的应用进展[J].河北工业科技,2000,22(1)：52-54
[36]陈发明,吴织芬,金岩等.智能凝胶药物控释系统在医学上的应用[J].人民军医,2005,48(4)：231-233.
[37]王云普,袁昆,李全莲等.可生物降解的pH敏感水凝胶的合成及其溶胀性能研究[J].功能高分子学报,2004,17(12)：556-590.
[38]T.H.M.Abou-Aiada,KN.Abd-El-Nour,I.Khakima,M.Z.Elsabee.Dieleetric and interaction behavior of chitosan/polyvinyl alcohol and chitosan/polyvinyl pyrrolidone blends with some antimicrobial activities, Polymer,2006, (47):379-389.
[39]刘华骨诱导型可注射磷酸钙基骨修复材料的制备及性能研究[D]暨南大学博士论文2007.
[40]郭玉明,张秀英.高分子基质作用下碳酸钙的仿生合成.化学学报,2001,59(5)：755-762.
[41]Chow LC, Takagi S, Ishikawa K.Formation of hydroxyapatite in cement systems.In:Brown WE,Constanz E,editors. Hydroxyapatite and related materials.Boca Raion,FL:CRC Press,1994:127-137.
[42]A.Doat, F.pelle, N.Gardant,etal. Synthesis of luminescent bioapatite nanoparticles for utilization as a biologieal Probe, Jourual of Solid State Chemistry,2004,177:1179--1187.
[43]刘兵.磷酸钙骨水泥生物材料制备与性能研究[D]中南大学硕士学位论文,2005.
[44]Yamasaki H. Heterotopic bone formation around porous hydroxyapatite ceramics in the subcutis of dogs. J Oral Bio(Japan),1990;32:190.
[45]Zhang XD, Zou P, Wu C,et al. A study of porous block HA ceramics and its osteogeneses. In:Bioceramics and the Human Body. ED by Ravaglioli and Krajewski. Elsevier Applied Science,1991; 408.
[46]Ripamonti U. The morphogenesis of bone in replicas of poroushydroxyapatite obtained from conversion of calcium carbonate exoskeletons of coral. J Bone Joint Surg,1991;73:692.
[47]Z Yang, H Yuan, W Tong,et al. Osteogenesis inextraskeletally implaned porous phosphate ceramics:variabilityamong different kinds of animals. Biomaterials, 1996; 17:2131.
[48]Yuan HP, de Bruijin JD, Li YB,et al. Bone formatin inducedby calcium phosphate ceramics in soft tissue of dogs:a comparative study between porous alpha-TCP and beta-TCP. J Mater Sci, Mater Med,2001;12(1):7.
[49]Yuan H, Kurashina K, de Bruijin JD,et al. A preliminary study on osteoinduction of two kinds of calcin phosphateceramic, Biomaterials,1999;20:1799.
[50]H Yuan, Y Li, JD de Bruijin,et al. Tissue responses of calcium phosphate cement: a study in dogs. Biomaterials,2000; 21:1283.
[51]Xingdong Zhang, Huipin Yuan, K. de Groot. Calciumphosphate biomaterials with intrinsic osteoinductivity.Notebook of the 6th World Biomaterials Congress, Hawaii,May,2000.
[2]顾汉卿.《生物医用材料》.天津：天津大学出版社,1998,256-278.
[3]沈序辉,宋晨路,沈鸽等.有机一轻基磷灰石复合骨替代材料[J].材料与工程,1999(12)：85-90.
[4]邹仲之.组织学与胚胎学[M].北京：人民卫生出版社,2002.7：52-53.
[6]Brown WE, Chow LC. United States Patient,4518430.1985.
[7]Brown WE, Chow LC. United States Patient,4612053.1986.
[8]Gbureck U, Spatz K, Thull R. Improvement of mechanical properties of self setting calcium phosphate bone cement mixed with different metal oxides.Mat.-wiss.u.Werkstofftech.2003,34(12):1036-1040.
[9]Doi Y, Shimizu Y, Mouriwaki Y, Aga M,Iwanaga H, Shibutani T, Yamamoto K, Iwayama Y. Development of a new calcium phosphate cement that contains sodium calcium phosphate. Biomaterials,2001,22:847-854.
[10]Yokoyama A, Yamamoto S, Kawasaki T, Kohgo T, Nakasu M. Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials. Biomaterials,2002,23:1091-1101.
[11]Chow LC. Development of selfsetting calcium phosphate cements. Journal Ceramics Society Japan,1991,99(10):956-964.
[12]Constantz BR, Ison LC, Fulmer M T, et al. Skeletal repair by in situformation of the mineral phase of bone. Science,1995,267(5205):1796-1798.
[13]Friendman CD, Costantino PD, Chow LC, et al. Hydroxyapatite cement.Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surgery,1991,117(4):383-386.
[14]Costantino PD, Friendman CD, Jones K, et al. Experimental hydroxyapatite cement Cranioplasty. Plastic and Reconstructive sergery,1992,90(2):174-185.
[15]Karashinak K, Kurita H, Kotani A, et al. Experimental cranioplasty and skeletal augmentation using an alpha-tricalcium phosphate/dicalcium phosphate dibasic/tetracalcium phosphate monoxide cement:a preliminary short term experiment in rabbit. Biomaterials,1998,19(79):701-706.
[17]Ishikawa K, Miyamoto Y, Takechi M. Non-decay type fast-setting calcium phosphate cement:Hydroxyapatite putty containing an increased amount of sodium alginate. Journal of Biomedical Materials Research,
1997,36(3):393-399.
[18]陈发明,吴织芬,金岩等.智能凝胶药物控释系统在医学上的应用[J].人民军医,2005,48(4)：231-233.
[19]Jarcho M, Bolen C H. Hydroxyapatite synthesis and characterization in dense polycrystalline form[J]. J. Mater Sci,1976,11(2):227-235.
[20]Osaka A,Miura R,Taeuchi K, et al.Calcium apatite prepared from calcium hydroxide and orthophosphoric acid[J]. J Mater Sci Med,1991,2 (1):51-55.
[21]Hattori T, Iwadate Y. Hydrothermal preparation of calcium hydroxyapatite powders[J].J Am Ceram Soci,1990,73(6):1803-1805.
[22]武强,李祖兵.多孔β-TCP/BMG复合人工骨修复下颌骨缺损的实验研究[J].中国口腔种植学杂志,2001,6(4)：157-158.
[23]Costantino PD, Friedman CD, Lane A. Synthetic biomarerials in facial plastic and reconstructive surgery.Facial Plastic Surg.1993,9(1):1-7.
[24]Gerhart,Tobin N Miler,Richard L, et al. In vitro characterization and biomechanical opimization of a biodegrad-able particulate composite bone cement [J]. J Biomed Mater Res,1988,22:1071.
[25]Dai HL, Yan YH, Wang YF, et al. Evaluation of calcium phosphate cement as a root canal sealer filing material. Journal of Wuhan University of Technology:Mater Sci Ed,2001,16(4):14-18.
[26]郑承泽,苏国礼,王天兵,等.多孔磷酸三钙与自体红骨髓复合移植修复骨缺损的临床应用[J].中国修复重建外科杂志,1996,10(3)：164-167.
[27]Constantz BR, Ison LC, Fulmer MT, etal. Skeletal repair by in situ formation of the mineral phase of bone science,1995,267(5205):1796-1798.
[28]邸利芝,赵红.β-磷酸三钙的制备及骨科中的应用.天津理工学院学报.2003,19(2)：102-105.
[29]Driessens FCM, Boltong MG, de Maeyer EAP,et al. The Ca/P range of nanoapatite calcium phosphate cements.Biomaterials,2002;32(19):4011.
[30]Vande Vord PJ, Matthew HWT, DeSilva SP, Mayton L,Wu B, wooley PH. Evaluation of the Biocompatibility of a chitosan scaffold in mice.J Biomed Mater Res,2002,59:585-590.
[31]Senkoylu A, Simsek A, Sahin FI, Menevse S, Ozogul C,Denkbas EB, Piskin E. Interaction of cultured chondrocytes with chitosan scaffolf. Journal of Bioactive and Compatible Polymers,2001,16:136-144.
[32]Klokkevold PR, Vandemark L, Kenney EB, et al. Osteogenesis enhances by
chitosan (poly-N-acetyl glucosaminoglycan) in vitro, Periodotal,1996, 67(11):1170-1175.
[33]Friberg J, Fernandez E, Sarda S, et al. An experimental approach to the study of the rheology behaviour of synthetic bone calcium phosphate cements. Key Eng Mater,2001,192-195:777-780.
[34]Sarda S, Fernandez E, Nilsson M, et al. Kinetic study of citric acid influence on calcium phosphate bone cements as water-reducing agent. J Biomed Mater Res, 2002,61(4):653-659.
[35]杜嘉英等.聚乙烯醇在医疗中的应用进展[J].河北工业科技,2000,22(1)：52-54
[36]陈发明,吴织芬,金岩等.智能凝胶药物控释系统在医学上的应用[J].人民军医,2005,48(4)：231-233.
[37]王云普,袁昆,李全莲等.可生物降解的pH敏感水凝胶的合成及其溶胀性能研究[J].功能高分子学报,2004,17(12)：556-590.
[38]T.H.M.Abou-Aiada,KN.Abd-El-Nour,I.Khakima,M.Z.Elsabee.Dieleetric and interaction behavior of chitosan/polyvinyl alcohol and chitosan/polyvinyl pyrrolidone blends with some antimicrobial activities, Polymer,2006, (47):379-389.
[39]刘华骨诱导型可注射磷酸钙基骨修复材料的制备及性能研究[D]暨南大学博士论文2007.
[40]郭玉明,张秀英.高分子基质作用下碳酸钙的仿生合成.化学学报,2001,59(5)：755-762.
[41]Chow LC, Takagi S, Ishikawa K.Formation of hydroxyapatite in cement systems.In:Brown WE,Constanz E,editors. Hydroxyapatite and related materials.Boca Raion,FL:CRC Press,1994:127-137.
[42]A.Doat, F.pelle, N.Gardant,etal. Synthesis of luminescent bioapatite nanoparticles for utilization as a biologieal Probe, Jourual of Solid State Chemistry,2004,177:1179--1187.
[43]刘兵.磷酸钙骨水泥生物材料制备与性能研究[D]中南大学硕士学位论文,2005.
[44]Yamasaki H. Heterotopic bone formation around porous hydroxyapatite ceramics in the subcutis of dogs. J Oral Bio(Japan),1990;32:190.
[45]Zhang XD, Zou P, Wu C,et al. A study of porous block HA ceramics and its osteogeneses. In:Bioceramics and the Human Body. ED by Ravaglioli and Krajewski. Elsevier Applied Science,1991; 408.
[46]Ripamonti U. The morphogenesis of bone in replicas of poroushydroxyapatite obtained from conversion of calcium carbonate exoskeletons of coral. J Bone Joint Surg,1991;73:692.
[47]Z Yang, H Yuan, W Tong,et al. Osteogenesis inextraskeletally implaned porous phosphate ceramics:variabilityamong different kinds of animals. Biomaterials, 1996; 17:2131.
[48]Yuan HP, de Bruijin JD, Li YB,et al. Bone formatin inducedby calcium phosphate ceramics in soft tissue of dogs:a comparative study between porous alpha-TCP and beta-TCP. J Mater Sci, Mater Med,2001;12(1):7.
[49]Yuan H, Kurashina K, de Bruijin JD,et al. A preliminary study on osteoinduction of two kinds of calcin phosphateceramic, Biomaterials,1999;20:1799.
[50]H Yuan, Y Li, JD de Bruijin,et al. Tissue responses of calcium phosphate cement: a study in dogs. Biomaterials,2000; 21:1283.
[51]Xingdong Zhang, Huipin Yuan, K. de Groot. Calciumphosphate biomaterials with intrinsic osteoinductivity.Notebook of the 6th World Biomaterials Congress, Hawaii,May,2000.