新型生物陶瓷对体外培养成骨细胞影响的初步实验研究
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摘要
目的：通过研究ZrO_2-CBC对体外培养SD胎鼠成骨细胞生长的影响，初步探讨ZrO_2-CBC对成骨细胞的毒性作用。
材料与方法：(1)以SD胎鼠颅盖骨组织为材料，体外培养原代成骨细胞，并对细胞进行鉴定、纯化、传代、扩增；(2)将ZrO_2-CBC和纯Ti制成厚度1.0mm、直径15.0mm的圆形薄片，清洁、消毒备用；(3)ZrO_2-CBC为实验组，纯Ti为对照组，将两组材料与成骨细胞在体外复合培养15d，分别在1d、5d、10d和15d评价两组材料对成骨细胞的影响。
结果：(1)培养出的细胞经形态学观察、ALP定性定量分析及OC检测，确定为高纯度、高活性的成骨细胞；(2)与纯Ti相比，实验早期ZrO_2-CBC对成骨细胞增殖分化无抑制作用(P＞0.05)，后期轻度抑制成骨细胞的增殖，并抑制成骨细胞ALP的分泌(P＜0.05)；(3)电镜结果显示复合培养1d，成骨细胞就粘附在ZrO_2-CBC表面并开始生长。随着培养时间的延长，细胞数量不断增多，并有细胞突起伸入到ZrO_2-CBC内部，起到固定作用。
结论：本实验体外证明ZrO_2-CBC对SD胎鼠颅盖骨源性成骨细胞无明显毒性作用，是一种有前途的牙科种植体材料。
Objective: To evaluate the cell compatibility of ZrO2-CBC by studying the influence of ZrO2-CBC on the growth of osteoblasts derived from SD fetal rats in vitro.
Materials and Methods: Primary osteoblasts were cultured from SD fetal rats' calvaria and then were identified, purified and passaged. ZrO2-CBC (as experiment group) and pure titanium (as control group) were prepared to be round thin slice (thickness 1.0mm, diameter 15.0mm) and then cleaned and sterilized for spare use. After that, both materials and osteoblasts were co-cultured in vitro for 15 days to evaluate the effect of the materials on cells in Id, 5d, 10d and 15d, respectively. Results: Cells derived from SD fetal rats' calvaria were determined to be osteoblasts with higher purity and activity. It was found that ZrO2-CBC did not prevent the growth of osteoblasts at the early
phase (P>0.05 ) , but it restrained the cells' proliferation and ALP secretions at
the later phase (P<0.05) , compared with control group. Osteoblasts had
adhered to the surface of the material and begun to grow since the 1st day of
co-culture. The cells on the surface continued to increase and stretched into the
deep part of ZrO2-CBC.
Conclusion: It was proved in vitro that ZrO2-CBC did no visible harm to
osteoblasts derived from SD fetal rats' calvaria. It is a promising dental implant
material.
材料与方法：(1)以SD胎鼠颅盖骨组织为材料，体外培养原代成骨细胞，并对细胞进行鉴定、纯化、传代、扩增；(2)将ZrO_2-CBC和纯Ti制成厚度1.0mm、直径15.0mm的圆形薄片，清洁、消毒备用；(3)ZrO_2-CBC为实验组，纯Ti为对照组，将两组材料与成骨细胞在体外复合培养15d，分别在1d、5d、10d和15d评价两组材料对成骨细胞的影响。
结果：(1)培养出的细胞经形态学观察、ALP定性定量分析及OC检测，确定为高纯度、高活性的成骨细胞；(2)与纯Ti相比，实验早期ZrO_2-CBC对成骨细胞增殖分化无抑制作用(P＞0.05)，后期轻度抑制成骨细胞的增殖，并抑制成骨细胞ALP的分泌(P＜0.05)；(3)电镜结果显示复合培养1d，成骨细胞就粘附在ZrO_2-CBC表面并开始生长。随着培养时间的延长，细胞数量不断增多，并有细胞突起伸入到ZrO_2-CBC内部，起到固定作用。
结论：本实验体外证明ZrO_2-CBC对SD胎鼠颅盖骨源性成骨细胞无明显毒性作用，是一种有前途的牙科种植体材料。
Objective: To evaluate the cell compatibility of ZrO2-CBC by studying the influence of ZrO2-CBC on the growth of osteoblasts derived from SD fetal rats in vitro.
Materials and Methods: Primary osteoblasts were cultured from SD fetal rats' calvaria and then were identified, purified and passaged. ZrO2-CBC (as experiment group) and pure titanium (as control group) were prepared to be round thin slice (thickness 1.0mm, diameter 15.0mm) and then cleaned and sterilized for spare use. After that, both materials and osteoblasts were co-cultured in vitro for 15 days to evaluate the effect of the materials on cells in Id, 5d, 10d and 15d, respectively. Results: Cells derived from SD fetal rats' calvaria were determined to be osteoblasts with higher purity and activity. It was found that ZrO2-CBC did not prevent the growth of osteoblasts at the early
phase (P>0.05 ) , but it restrained the cells' proliferation and ALP secretions at
the later phase (P<0.05) , compared with control group. Osteoblasts had
adhered to the surface of the material and begun to grow since the 1st day of
co-culture. The cells on the surface continued to increase and stretched into the
deep part of ZrO2-CBC.
Conclusion: It was proved in vitro that ZrO2-CBC did no visible harm to
osteoblasts derived from SD fetal rats' calvaria. It is a promising dental implant
material.
引文
[1] Mayr Wohlfart U, et al. Proliferation and differentiation rates of a human osteoblast-like cell line (SaOS-2) in contact with different bone substitute materials. J Biomed Mater Res, 2001, 57(1): 132
[2] Webster TJ, et al. Enhanced functions of osteoblasts on nanophase ceramics. Biomaterials. 2000, 21 (17): 1803
[3] Mustafa K, et al. Determining optimal surface roughness of TiO_2 blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone. Clin Oral Implants Res, 2001, 12(5): 515
[4] Lehner CR, et al. All-ceramic crowns. Curr Opin Dent, 1992,2(6):45
[5] 施琥，等．复合生物陶瓷骨种植的扫描电镜研究．口腔材料器械杂志，1999，8(3)：131
[6] 刘娟，等．复合生物陶瓷修复髓室底的疗效观察．口腔材料器械志．1998，7(3)：139．157
[7] 李江，等．氧化锆添加剂对牙科用氧化铝玻璃复合体性能的影响．实用口腔医学杂志，2000，16(3)：171
[8] Letic Gavrilovic A, et al. Genetic potential of interfacial guided osteogenesis in implant devices. Dental Materials Journal, 2000, 19(2): 99
[9] 峰煜，等．纯钛表面牙周韧带细胞形成物骨钙素的表达．口腔颌面修复杂志，2002，3(3)：140
[10] Gough JE, Downes S. Osteoblast cell death on methacrylate polymers involves apoptosis. Journal of Biomedical Materials Research, 2001,57(2):291
[11] Mustafa K, et al. Determining optimal surface roughness of TiO_2 blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone. Clin Oral Implants Res. 2001,12(5): 515
[12] Schwartz Z, et al. Underlying mechanisms at the bone-surface interface during regeneration. J Periodontal Res. 1997,32(1 Pt 2): 166
[13] YangYunzhi, et al. Morphological behavior of osteoblast-like cells on surfacemodified titanium in vitro. Biomaterials. 2002, 23(5): 1383
[14] Torricelli P, et al. Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone. Biomaterials. 2001, 22(18): 2535
[15] 柴枫，等．烧结温度对氧化锆增韧纳米复合陶瓷基体性能的影响．口腔材料器械杂志．2002，11(2)：61
[16] Porter A E, et al. The ultrastructure of the plasma-sprayed hydroxyapatite-bone interface predisposing to bone bonding. Biomaterials. 2002, 23(3): 725
[17] Li D, et al. Bone interface of dental implants cytologically influenced by a modified sandblasted surface: a preliminary in vitro study. Implant Dent, 2001, 10(2): 132
[18] Nishio K, et al. The effect of alkali- and heat-treated titanium and apatiteformed titanium on osteoblastic differentiation of bone marrow cells. J Biomed Mater Res. 2000, 52(4): 652
[19] Ohgaki M, et al. Manipulation of selective cell adhesion and growth by surface charges of electrically polarized hydroxyapatite. J Biomed Mater Res, 2001, 57(3): 366
[20] Letic Gavrilovic A, et al. enetic potential of interfacial guided osteogenesis in implant devices. Dent Mater J, 2000, 19(2): 99
[21] 司徒镇强，吴军正．细胞培养．西安：世界图书出版西安公司，1996
[22] 薛庆善．体外培养的原理与技术．北京：科学出版社，2001
[23] 夏大弘．成骨细胞体外培养在种植体相关研究中的应用．国外医学口腔医学分册,2001，28(2)；73
[24] 李德华，刘宝林．人胚成骨细胞体外培养三维实验模型的建立．实用口腔医学杂志，2000，16(2)：99
[25] 唐昭，陈治清．大鼠成骨细胞体外培养的研究．华西口腔医学志，1997,15(1)：70
[26] Holzer Gerold, Einhorn Thomas A, Majeska Robert J. Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal
cells. Joumal of orthopaedic research, 2002,20 (2): 281
[27] Yeh Lee-Chuan C, Zavala Michelle C, Lee John C. Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation. Journal of cellular physiology, 2002, 190(3): 322
[28] Ho ML, Chang JK, et al. Characteristics of primary osteoblast culture derived from rat fetal calvaria. Kaohsiung J Med Sci, 1999, 15(5): 248
[29] Noriko yamamoto, et al. Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone formation. Biol Pharm Bull, 2002, 25(4): 509
[30] Nishiya Y, Kosaka N, et al. A potent 1,4-dihydropyridine L-type calcium channel blocker, benidipine, promotes osteoblast differentiation. Calcified tissue international, 2002, 70(1); 30
[31] Wlodarski KH. Properties and origin of osteoblasts. Clin orthop, 1990, 252(3):276
[32] Wada Y, et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone, 1998, 22(5): 479
[33] Rodan GA, et al. Diversity of the osteoblastic phenotype. Ciba Found Symp, 1988,136:78
[34] Duty P, et al. The osteoblast: A sophisticated fibroblast under central surveillance. Sci, 2000, 289:1501
[35] Safadi FF, et al. Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts. J Cell Biochem, 2001, 84(1): 12
[36] 陈治清．口腔材料学．第二版．北京：人民卫生出版社，2001．27
[37] ISO 7405：1997．牙科学-牙科医疗器械生物相容性临床前评价-牙科材料实验方法
[38] Vaes BL, et al. Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development. J Bone Miner Res, 2002, 17(12): 2106
[39] Ghannam A, et al. Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of
mineralized extracellular matrix. Biomaterials, 1997, 18(4): 295
[40] 王零森．二氧化锆陶瓷(一)(二)．陶瓷工程，1997，31(1)：41～44；1997，31(2)：43
[41] MV斯温．材料科学与技术从书(11卷)：陶瓷的结构和性能．北京：科学出版社，1998
[42] 王零森．特种陶瓷．长沙：中南工业大学出版社，1994．139～153
[43] Oliva A, et al. Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts. Biomaterials, 1996, 17(13): 1351
[44] Kue R, et al. Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs. Biomaterials, 1999, 20(13): 1195
[45] Wilke A, et al. Biocompatibility analysis of different biomaterials in human marrow cell cultures. J Biomed Mater Res, 1998, 40(2): 301
[46] Metzler V, et al. A novel method for quantifying shape deformation applied to biocompatibility testing. ASAIO J, 1999, 45(4): 264
[47] Granchi, et al. Endodontic cements induce alternations in the cell cycle of in vitro cultured osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radilo Endod, 1995, 79(3):359
[48] 傅远飞，等．锶磷灰石体外细胞毒性研究．上海口腔医学，2002，11(3)：229
[49] 黄培堂，等(译)，David L Spector,et al(著)．细胞实验指南．科学出版社，2001．1206
[2] Webster TJ, et al. Enhanced functions of osteoblasts on nanophase ceramics. Biomaterials. 2000, 21 (17): 1803
[3] Mustafa K, et al. Determining optimal surface roughness of TiO_2 blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone. Clin Oral Implants Res, 2001, 12(5): 515
[4] Lehner CR, et al. All-ceramic crowns. Curr Opin Dent, 1992,2(6):45
[5] 施琥，等．复合生物陶瓷骨种植的扫描电镜研究．口腔材料器械杂志，1999，8(3)：131
[6] 刘娟，等．复合生物陶瓷修复髓室底的疗效观察．口腔材料器械志．1998，7(3)：139．157
[7] 李江，等．氧化锆添加剂对牙科用氧化铝玻璃复合体性能的影响．实用口腔医学杂志，2000，16(3)：171
[8] Letic Gavrilovic A, et al. Genetic potential of interfacial guided osteogenesis in implant devices. Dental Materials Journal, 2000, 19(2): 99
[9] 峰煜，等．纯钛表面牙周韧带细胞形成物骨钙素的表达．口腔颌面修复杂志，2002，3(3)：140
[10] Gough JE, Downes S. Osteoblast cell death on methacrylate polymers involves apoptosis. Journal of Biomedical Materials Research, 2001,57(2):291
[11] Mustafa K, et al. Determining optimal surface roughness of TiO_2 blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone. Clin Oral Implants Res. 2001,12(5): 515
[12] Schwartz Z, et al. Underlying mechanisms at the bone-surface interface during regeneration. J Periodontal Res. 1997,32(1 Pt 2): 166
[13] YangYunzhi, et al. Morphological behavior of osteoblast-like cells on surfacemodified titanium in vitro. Biomaterials. 2002, 23(5): 1383
[14] Torricelli P, et al. Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone. Biomaterials. 2001, 22(18): 2535
[15] 柴枫，等．烧结温度对氧化锆增韧纳米复合陶瓷基体性能的影响．口腔材料器械杂志．2002，11(2)：61
[16] Porter A E, et al. The ultrastructure of the plasma-sprayed hydroxyapatite-bone interface predisposing to bone bonding. Biomaterials. 2002, 23(3): 725
[17] Li D, et al. Bone interface of dental implants cytologically influenced by a modified sandblasted surface: a preliminary in vitro study. Implant Dent, 2001, 10(2): 132
[18] Nishio K, et al. The effect of alkali- and heat-treated titanium and apatiteformed titanium on osteoblastic differentiation of bone marrow cells. J Biomed Mater Res. 2000, 52(4): 652
[19] Ohgaki M, et al. Manipulation of selective cell adhesion and growth by surface charges of electrically polarized hydroxyapatite. J Biomed Mater Res, 2001, 57(3): 366
[20] Letic Gavrilovic A, et al. enetic potential of interfacial guided osteogenesis in implant devices. Dent Mater J, 2000, 19(2): 99
[21] 司徒镇强，吴军正．细胞培养．西安：世界图书出版西安公司，1996
[22] 薛庆善．体外培养的原理与技术．北京：科学出版社，2001
[23] 夏大弘．成骨细胞体外培养在种植体相关研究中的应用．国外医学口腔医学分册,2001，28(2)；73
[24] 李德华，刘宝林．人胚成骨细胞体外培养三维实验模型的建立．实用口腔医学杂志，2000，16(2)：99
[25] 唐昭，陈治清．大鼠成骨细胞体外培养的研究．华西口腔医学志，1997,15(1)：70
[26] Holzer Gerold, Einhorn Thomas A, Majeska Robert J. Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal
cells. Joumal of orthopaedic research, 2002,20 (2): 281
[27] Yeh Lee-Chuan C, Zavala Michelle C, Lee John C. Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation. Journal of cellular physiology, 2002, 190(3): 322
[28] Ho ML, Chang JK, et al. Characteristics of primary osteoblast culture derived from rat fetal calvaria. Kaohsiung J Med Sci, 1999, 15(5): 248
[29] Noriko yamamoto, et al. Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone formation. Biol Pharm Bull, 2002, 25(4): 509
[30] Nishiya Y, Kosaka N, et al. A potent 1,4-dihydropyridine L-type calcium channel blocker, benidipine, promotes osteoblast differentiation. Calcified tissue international, 2002, 70(1); 30
[31] Wlodarski KH. Properties and origin of osteoblasts. Clin orthop, 1990, 252(3):276
[32] Wada Y, et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone, 1998, 22(5): 479
[33] Rodan GA, et al. Diversity of the osteoblastic phenotype. Ciba Found Symp, 1988,136:78
[34] Duty P, et al. The osteoblast: A sophisticated fibroblast under central surveillance. Sci, 2000, 289:1501
[35] Safadi FF, et al. Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts. J Cell Biochem, 2001, 84(1): 12
[36] 陈治清．口腔材料学．第二版．北京：人民卫生出版社，2001．27
[37] ISO 7405：1997．牙科学-牙科医疗器械生物相容性临床前评价-牙科材料实验方法
[38] Vaes BL, et al. Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development. J Bone Miner Res, 2002, 17(12): 2106
[39] Ghannam A, et al. Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of
mineralized extracellular matrix. Biomaterials, 1997, 18(4): 295
[40] 王零森．二氧化锆陶瓷(一)(二)．陶瓷工程，1997，31(1)：41～44；1997，31(2)：43
[41] MV斯温．材料科学与技术从书(11卷)：陶瓷的结构和性能．北京：科学出版社，1998
[42] 王零森．特种陶瓷．长沙：中南工业大学出版社，1994．139～153
[43] Oliva A, et al. Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts. Biomaterials, 1996, 17(13): 1351
[44] Kue R, et al. Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs. Biomaterials, 1999, 20(13): 1195
[45] Wilke A, et al. Biocompatibility analysis of different biomaterials in human marrow cell cultures. J Biomed Mater Res, 1998, 40(2): 301
[46] Metzler V, et al. A novel method for quantifying shape deformation applied to biocompatibility testing. ASAIO J, 1999, 45(4): 264
[47] Granchi, et al. Endodontic cements induce alternations in the cell cycle of in vitro cultured osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radilo Endod, 1995, 79(3):359
[48] 傅远飞，等．锶磷灰石体外细胞毒性研究．上海口腔医学，2002，11(3)：229
[49] 黄培堂，等(译)，David L Spector,et al(著)．细胞实验指南．科学出版社，2001．1206