羟基磷灰石颗粒大小对成牙本质细胞MDPC-23生长的影响
摘要
目的：
观察不同大小的羟基磷灰石对成牙本质细胞(MDPC-23)的生长的影响。
方法：
将成牙本质细胞(MDPC-23)分别培养于不同颗粒大小的羟基磷灰石铺面的玻片,即20纳米大小的羟基磷灰石颗粒(nHAP)组和寻常大小羟基磷灰石颗粒(cHAP)组,不含羟基磷灰石的空白玻片作为对照组。Olympus倒置显微镜观察细胞生长情况,四唑盐比色实验法(MTT)测量吸光度OD值,计算细胞生长情况并绘制细胞生长曲线,观察不同颗粒大小的羟基磷灰石对成牙本质细胞生长的影响。
结果：
nHAP组MTT比值在第三天显著低于cHAP和对照组(P<0.05),但第五,七天MTT比值组间无显著性差异。
结论：
20nm羟基磷灰石颗粒nHAP对成牙本质细胞(MDPC-23)的生长无抑制作用,cHAP在MDPC-23细胞培养早期对细胞生长有促进作用。
Objective:
To observe the size-dependent effect of hydroxyapatite on the growth of odontoblast (MDPC-23).
Methods:
Three groups were designed according to the experimental materials used:1. nHAP group with 20nm Nano-hydroxyapatite,2. cHAP group with conventional particle size of the hydroxyapatite and 3. glass as control. Odontoblast MDPC-23 as the experimental cell was cultured in three groups. Morphology of cell were observed using Olympus inverted microscope, the growth of the cell in different groups were measured using Thiazolyl blue tetrazolium bromide test (MTT), and the growth curves of cell were drawn.
Results:
The MTT ratio of 20nm nHAP group was significant smaller than those in the cHAP group and the control (P<0.05); however, there was no significant difference after five-and seven-day's culture.
Conclusion:
20nm nHAP has no inhibitive effect on the growth of odontoblast MDPC-23, no different effect of particle size of hydroxyapatite on MDPC-23 growth was found.
观察不同大小的羟基磷灰石对成牙本质细胞(MDPC-23)的生长的影响。
方法：
将成牙本质细胞(MDPC-23)分别培养于不同颗粒大小的羟基磷灰石铺面的玻片,即20纳米大小的羟基磷灰石颗粒(nHAP)组和寻常大小羟基磷灰石颗粒(cHAP)组,不含羟基磷灰石的空白玻片作为对照组。Olympus倒置显微镜观察细胞生长情况,四唑盐比色实验法(MTT)测量吸光度OD值,计算细胞生长情况并绘制细胞生长曲线,观察不同颗粒大小的羟基磷灰石对成牙本质细胞生长的影响。
结果：
nHAP组MTT比值在第三天显著低于cHAP和对照组(P<0.05),但第五,七天MTT比值组间无显著性差异。
结论：
20nm羟基磷灰石颗粒nHAP对成牙本质细胞(MDPC-23)的生长无抑制作用,cHAP在MDPC-23细胞培养早期对细胞生长有促进作用。
Objective:
To observe the size-dependent effect of hydroxyapatite on the growth of odontoblast (MDPC-23).
Methods:
Three groups were designed according to the experimental materials used:1. nHAP group with 20nm Nano-hydroxyapatite,2. cHAP group with conventional particle size of the hydroxyapatite and 3. glass as control. Odontoblast MDPC-23 as the experimental cell was cultured in three groups. Morphology of cell were observed using Olympus inverted microscope, the growth of the cell in different groups were measured using Thiazolyl blue tetrazolium bromide test (MTT), and the growth curves of cell were drawn.
Results:
The MTT ratio of 20nm nHAP group was significant smaller than those in the cHAP group and the control (P<0.05); however, there was no significant difference after five-and seven-day's culture.
Conclusion:
20nm nHAP has no inhibitive effect on the growth of odontoblast MDPC-23, no different effect of particle size of hydroxyapatite on MDPC-23 growth was found.
引文
1 Liu, Y. et al. In vitro effects of nanophase hydroxyapatite particles on proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. J Biomed Mater Res A 90,1083-1091, doi:10.1002/jbm.a.32192 (2009).
2 Tang, R. et al. Dissolution at the nanoscale:self-preservation of biominerals. Angew Chem Int Ed Engl 43,2697-2701 (2004).
3 Shi, Z., Huang, X., Cai, Y., Tang, R.& Yang, D. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomater 5,338-345, doi:S1742-7061(08)00216-X [pii]10.1016/j.actbio.2008.07.023 (2009).
4 Okada, S., Ito, H., Nagai, A., Komotori, J.& Imai, H. Adhesion of osteoblast-like cells on nanostructured hydroxyapatite. Acta Biomaterialia 6,591-597 (2010).
5 Singh, S., Bhardwaj, P., Singh, V., Aggarwal, S.& Mandal, U. K. Synthesis of nanocrystalline calcium phosphate in microemulsion-effect of nature of surfactants. J Colloid Interf Sci 319,322-329, doi:DOI 10.1016/j.jcis.2007.09.059 (2008).
6 Stevens, M. M.& George, J. H. Exploring and engineering the cell surface interface. Science (New York, N.Y 310,1135-1138 (2005).
7 Arana-Chavez, V. E.& Massa, L. F. Odontoblasts:the cells forming and maintaining dentine. The international journal of biochemistry& cell biology 36, 1367-1373(2004).
8 Zhou, G. S. et al. Different effects of nanophase and conventional hydroxyapatite thin films on attachment, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells. Biomed Mater Eng 17,387-395 (2007).
9 Zhang, H. J., Zhu, X. D., Fan, H. S., Li, W.& Zhang, X. D. Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption. Acta Biomater, doi:S1742-7061(09)00467-X [pii]10.1016/j.actbio.2009.10.032 (2009).
10 Watari, F. et al. Material nanosizing effect on living organisms:non-specific, biointeractive, physical size effects. J R Soc Interface 6 Suppl 3, S371-388, doi:rsif.2008.0488.focus[pii]10.1098/rsif.2008.0488.focus(2009).
11 Yuan, Y, Liu, C., Qian, J., Wang, J.& Zhang, Y Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials, doi:S0142-9612(09)01056-4 [pii]10.1016/j.biomaterials.2009.09.088 (2009).
[1]. Slavkin H and Price P. Chemistry and biology of mineralized tissues. Excerpa Medica,1992:158～162.
[2]. 王夔主编.生物无机化学.北京：清华大学出版社,第四章,1998.
[3]. Young J R, Didymus J M, Bown P R, Prins B, Mann S. Crystal assemblv and phylogenetic evolution in heterococcoliths. Nature,1992,356:516～518.
[4]. Bundy K J. Determination of mineral—organic bonding effeetiveneSS in bone: theoretical considerations. Ann Biomed En9,1985,13:119～135.
[5]. Walsh W R, Labrador D P, Kim H K, et al. Ultrasonic properties of cortical bo,ne following fluoride ion treatment. Ann Biomed En9,1994,22:404～414.
[6]. Mann S. Molecular recognition in biomineralization. Nature,1988,332:119～ 124.
[7]. Vincent J, Structural Biomaterial. Princeton Univ. Press,1990.
[8]. 沈方宏.胶原蛋白调制碳酸钙晶体生长的研究.清华大学硕士论文,2002.
[9]. Simkiss K, Wilbur K M. Biomineralization:cell biology and mineral deposition. Academic Press, Inc.1998.
[10]. 张岑.合浦珠母贝贝壳形成相关单白及基因的研究.清华大学博士论文,2006.
[11]. Westbrock P, Martin F. Amarriage of bone and nacre. Nature 1998,392:861～ 862.
[12]. weiss I M, Gohring W, et al, A Haliotis laevigata(abalone)nacre protein is homologous to the insulin-like growth factor binding protein N—terminal module of vertebrates. Biochem Biophys Res Commun,2001,285:244～249.
[13]. Paine ML, White SN, LuoW, et al. Matrix Biol,2001,20:273～292.
[14].崔福斋,王秀梅,胡望.基因材料.北京：化学工业出版社,2004.
[15]. Boyde A. Electron microscopic observations relating to the nature and development of prism decussation in mammalian dental enamel. Bull Group Int RechSciStomatol,1969,12:151～207.
[16]. Boyde A. Microstructure of enamel. In:Dental enamel, Proceedings of ciba foundation symposium 205. Chichester, UK:John Wiley&Sons Ltd,1997, 18～31.
[17]. Veis A. A window on biomineralization[J]. Science,2005,307(5714): 1419-1420.
[18]. Kinney JH, Habelitz S, Marshall SJ, Marshall GW:The importance of intrafibrillar mineralization of collagen on the mechanical properties of dentin. J Dent Res 2003a;82:957-961.
[19]. Balooch M, Habelitz S, Kinney J, Marshall S, MarshallG:Mechanical properties of mineralizedcollagen fibrils as influenced by demineralization.J Struct Biol 2008; 162:404-410.
[20]. Kinney JH, Pople JA, Marshall GW, Marshall SJ. Collagen orientation and crystallite size in human dentin:a small angle X-ray scattering study. Calcif Tissue Int,2001,69:31-37.
[21]. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall Jr GW. In situ atomic force microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol,2002,138:227-236.
[22]. Cui F-Z, Li Y, Ge J. Self-assembly of mineralized collagen composites. Materials Science and Engineering:R:Reports 2007; 57:1-27.
[23]. Yang B, Cui, F.Z.. Molecular modeling and mechanics studies on the initial stage of the collagen-mineralization process. Current Applied Physics 2007; 7:Pages e2-e5
[24]. Milan AM, Sugars RV, Embery G, Waddington RJ. Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen. Eur J Oral Sci 2006; 114:223-31.(1)
[25]. Thakur AS, Robin G, Guncar G, et al. Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents. PLoS ONE 2007; 2:e1091.
[26]. Hoang QQ, Sicheri F, Howard AJ, Yang DS. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 2003; 425:977-80
[27]. Wang Y, Cui FZ, Hu K, Zhu XD, Fan DD. Bone regeneration by using scaffold based on mineralized recombinant collagen. J Biomed Mater Res B Appl Biomater 2008; 86:29-35.
[28]. Bertassoni LE, Habelitz S, Kinney JH, Marshall SJ, Marshall GW, Jr. Biomechanical Perspective on the Remineralization of Dentin. Caries Res 2009; 43:70-7.
[29]. Su X, Sun K, Cui FZ, Lindis WJ. Organization of apatite crystals in human woven bone[J]. Bone,2003,32:150-162.
[30]. Wassen MH, Lammens J, Tekoppele JM, Sakkers RJ, Liu Z, Verbout AJ Bank RA. Collagen structure regulates fibril mineralization in osteogenesis as revealed by cross-link patterns in calcifying callus. J Bone Miner Res,2000,15: 1776-1785.
[31]. Hartgerink J, Beniash E, Stupp S. Self-assembly and Mineralization of Peptide-amphiphile Nanofibers. Science,2001,294:1684-1688.
[32]. Tang RK, Wang LJ, Orme CA, Bonstein T, Bush PJ, Nancollas GH. Dissolution at the nanoscale:self-preservation of biominerals. Angew Chem Int Ed,2004,43:2697-2701.
[33]. Tao J, Pan H, Zeng Y, Xu X, Tang R. Roles of Amorphous Calcium Phosphate and Biological Additives in the Assembly of Hydroxyapatite Nanoparticles. J Phys Chem B,2007,111:13410-13418.
[34]. Li Li, Haihua Pan, Jinhui Tao, Xurong Xu, Caiyun Mao, Xinhua Gu and Ruikang Tang.Repair of enamel by using hydroxyapatite nanoparticles as the building blocks.J. Mater. Chem.,2008,18,4079-4084.
[35]. Beniash E, Traub W, Veis A, Weiner S. A transmission electron microscope study using vitrified ice sections of predentin:structural changes in the dentin collagenous matrix prior to mineralization. J Struct Biol,2000,132:212-225.
[36]. Liao SS, Cui FZ, Zhang W, Feng QL. Hierarchically biomimetic bone scaffold materials:nano-HA/collagen/PLA composite.J Biomed Mater Res Part B,2004, 69(2):158-165.
[37]. He G, Ramachandran A, Dahl T, George S, Schultz D, Cookson D, Veis A, George A. Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. J Biol Chem,2005,280(39):33109-33114.
[38]. George A, Hao J. Role of Phosphophoryn in Dentin Mineralization. Cells Tissues Organs,2005,181:232-240.
[39]. Xu L, Anderson AL, Lu Q, Wang J. Role of fibrillar structure of collagenous carrier in bone sialoprotein-mediated matrix mineralization and osteoblast differentiation. Biomaterials,2007,28:750-761.
[40]. He G and George A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem,2004,279(12): 11649-11656.
[41]. He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite formation in vitro[J]. Connect Tissue Res,2003,44:240-245.
[42]. Hao J, Zou B, Narayanan K, George A. Differential expression patterns of the dentin matrix proteins during mineralized tissue formation. Bone,2004, 34:921-932.
[43]. Luong LN, Hong SI, Patel RJ, Outslay ME, Kohn DH. Spatial control of protein within biomimetically nucleated mineral. Biomaterials,2006,27:1175-1186.
2 Tang, R. et al. Dissolution at the nanoscale:self-preservation of biominerals. Angew Chem Int Ed Engl 43,2697-2701 (2004).
3 Shi, Z., Huang, X., Cai, Y., Tang, R.& Yang, D. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomater 5,338-345, doi:S1742-7061(08)00216-X [pii]10.1016/j.actbio.2008.07.023 (2009).
4 Okada, S., Ito, H., Nagai, A., Komotori, J.& Imai, H. Adhesion of osteoblast-like cells on nanostructured hydroxyapatite. Acta Biomaterialia 6,591-597 (2010).
5 Singh, S., Bhardwaj, P., Singh, V., Aggarwal, S.& Mandal, U. K. Synthesis of nanocrystalline calcium phosphate in microemulsion-effect of nature of surfactants. J Colloid Interf Sci 319,322-329, doi:DOI 10.1016/j.jcis.2007.09.059 (2008).
6 Stevens, M. M.& George, J. H. Exploring and engineering the cell surface interface. Science (New York, N.Y 310,1135-1138 (2005).
7 Arana-Chavez, V. E.& Massa, L. F. Odontoblasts:the cells forming and maintaining dentine. The international journal of biochemistry& cell biology 36, 1367-1373(2004).
8 Zhou, G. S. et al. Different effects of nanophase and conventional hydroxyapatite thin films on attachment, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells. Biomed Mater Eng 17,387-395 (2007).
9 Zhang, H. J., Zhu, X. D., Fan, H. S., Li, W.& Zhang, X. D. Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption. Acta Biomater, doi:S1742-7061(09)00467-X [pii]10.1016/j.actbio.2009.10.032 (2009).
10 Watari, F. et al. Material nanosizing effect on living organisms:non-specific, biointeractive, physical size effects. J R Soc Interface 6 Suppl 3, S371-388, doi:rsif.2008.0488.focus[pii]10.1098/rsif.2008.0488.focus(2009).
11 Yuan, Y, Liu, C., Qian, J., Wang, J.& Zhang, Y Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials, doi:S0142-9612(09)01056-4 [pii]10.1016/j.biomaterials.2009.09.088 (2009).
[1]. Slavkin H and Price P. Chemistry and biology of mineralized tissues. Excerpa Medica,1992:158～162.
[2]. 王夔主编.生物无机化学.北京：清华大学出版社,第四章,1998.
[3]. Young J R, Didymus J M, Bown P R, Prins B, Mann S. Crystal assemblv and phylogenetic evolution in heterococcoliths. Nature,1992,356:516～518.
[4]. Bundy K J. Determination of mineral—organic bonding effeetiveneSS in bone: theoretical considerations. Ann Biomed En9,1985,13:119～135.
[5]. Walsh W R, Labrador D P, Kim H K, et al. Ultrasonic properties of cortical bo,ne following fluoride ion treatment. Ann Biomed En9,1994,22:404～414.
[6]. Mann S. Molecular recognition in biomineralization. Nature,1988,332:119～ 124.
[7]. Vincent J, Structural Biomaterial. Princeton Univ. Press,1990.
[8]. 沈方宏.胶原蛋白调制碳酸钙晶体生长的研究.清华大学硕士论文,2002.
[9]. Simkiss K, Wilbur K M. Biomineralization:cell biology and mineral deposition. Academic Press, Inc.1998.
[10]. 张岑.合浦珠母贝贝壳形成相关单白及基因的研究.清华大学博士论文,2006.
[11]. Westbrock P, Martin F. Amarriage of bone and nacre. Nature 1998,392:861～ 862.
[12]. weiss I M, Gohring W, et al, A Haliotis laevigata(abalone)nacre protein is homologous to the insulin-like growth factor binding protein N—terminal module of vertebrates. Biochem Biophys Res Commun,2001,285:244～249.
[13]. Paine ML, White SN, LuoW, et al. Matrix Biol,2001,20:273～292.
[14].崔福斋,王秀梅,胡望.基因材料.北京：化学工业出版社,2004.
[15]. Boyde A. Electron microscopic observations relating to the nature and development of prism decussation in mammalian dental enamel. Bull Group Int RechSciStomatol,1969,12:151～207.
[16]. Boyde A. Microstructure of enamel. In:Dental enamel, Proceedings of ciba foundation symposium 205. Chichester, UK:John Wiley&Sons Ltd,1997, 18～31.
[17]. Veis A. A window on biomineralization[J]. Science,2005,307(5714): 1419-1420.
[18]. Kinney JH, Habelitz S, Marshall SJ, Marshall GW:The importance of intrafibrillar mineralization of collagen on the mechanical properties of dentin. J Dent Res 2003a;82:957-961.
[19]. Balooch M, Habelitz S, Kinney J, Marshall S, MarshallG:Mechanical properties of mineralizedcollagen fibrils as influenced by demineralization.J Struct Biol 2008; 162:404-410.
[20]. Kinney JH, Pople JA, Marshall GW, Marshall SJ. Collagen orientation and crystallite size in human dentin:a small angle X-ray scattering study. Calcif Tissue Int,2001,69:31-37.
[21]. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall Jr GW. In situ atomic force microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol,2002,138:227-236.
[22]. Cui F-Z, Li Y, Ge J. Self-assembly of mineralized collagen composites. Materials Science and Engineering:R:Reports 2007; 57:1-27.
[23]. Yang B, Cui, F.Z.. Molecular modeling and mechanics studies on the initial stage of the collagen-mineralization process. Current Applied Physics 2007; 7:Pages e2-e5
[24]. Milan AM, Sugars RV, Embery G, Waddington RJ. Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen. Eur J Oral Sci 2006; 114:223-31.(1)
[25]. Thakur AS, Robin G, Guncar G, et al. Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents. PLoS ONE 2007; 2:e1091.
[26]. Hoang QQ, Sicheri F, Howard AJ, Yang DS. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 2003; 425:977-80
[27]. Wang Y, Cui FZ, Hu K, Zhu XD, Fan DD. Bone regeneration by using scaffold based on mineralized recombinant collagen. J Biomed Mater Res B Appl Biomater 2008; 86:29-35.
[28]. Bertassoni LE, Habelitz S, Kinney JH, Marshall SJ, Marshall GW, Jr. Biomechanical Perspective on the Remineralization of Dentin. Caries Res 2009; 43:70-7.
[29]. Su X, Sun K, Cui FZ, Lindis WJ. Organization of apatite crystals in human woven bone[J]. Bone,2003,32:150-162.
[30]. Wassen MH, Lammens J, Tekoppele JM, Sakkers RJ, Liu Z, Verbout AJ Bank RA. Collagen structure regulates fibril mineralization in osteogenesis as revealed by cross-link patterns in calcifying callus. J Bone Miner Res,2000,15: 1776-1785.
[31]. Hartgerink J, Beniash E, Stupp S. Self-assembly and Mineralization of Peptide-amphiphile Nanofibers. Science,2001,294:1684-1688.
[32]. Tang RK, Wang LJ, Orme CA, Bonstein T, Bush PJ, Nancollas GH. Dissolution at the nanoscale:self-preservation of biominerals. Angew Chem Int Ed,2004,43:2697-2701.
[33]. Tao J, Pan H, Zeng Y, Xu X, Tang R. Roles of Amorphous Calcium Phosphate and Biological Additives in the Assembly of Hydroxyapatite Nanoparticles. J Phys Chem B,2007,111:13410-13418.
[34]. Li Li, Haihua Pan, Jinhui Tao, Xurong Xu, Caiyun Mao, Xinhua Gu and Ruikang Tang.Repair of enamel by using hydroxyapatite nanoparticles as the building blocks.J. Mater. Chem.,2008,18,4079-4084.
[35]. Beniash E, Traub W, Veis A, Weiner S. A transmission electron microscope study using vitrified ice sections of predentin:structural changes in the dentin collagenous matrix prior to mineralization. J Struct Biol,2000,132:212-225.
[36]. Liao SS, Cui FZ, Zhang W, Feng QL. Hierarchically biomimetic bone scaffold materials:nano-HA/collagen/PLA composite.J Biomed Mater Res Part B,2004, 69(2):158-165.
[37]. He G, Ramachandran A, Dahl T, George S, Schultz D, Cookson D, Veis A, George A. Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. J Biol Chem,2005,280(39):33109-33114.
[38]. George A, Hao J. Role of Phosphophoryn in Dentin Mineralization. Cells Tissues Organs,2005,181:232-240.
[39]. Xu L, Anderson AL, Lu Q, Wang J. Role of fibrillar structure of collagenous carrier in bone sialoprotein-mediated matrix mineralization and osteoblast differentiation. Biomaterials,2007,28:750-761.
[40]. He G and George A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem,2004,279(12): 11649-11656.
[41]. He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite formation in vitro[J]. Connect Tissue Res,2003,44:240-245.
[42]. Hao J, Zou B, Narayanan K, George A. Differential expression patterns of the dentin matrix proteins during mineralized tissue formation. Bone,2004, 34:921-932.
[43]. Luong LN, Hong SI, Patel RJ, Outslay ME, Kohn DH. Spatial control of protein within biomimetically nucleated mineral. Biomaterials,2006,27:1175-1186.