体外磷灰石晶体矿化模型构建及应用的研究
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摘要
生物矿化是生物体内矿物形成过程的统称,是一个受到众多因素精密调控的生理过程。其研究涉及生物学、化学、结晶学、材料学、矿物学和医学等学科,是一门多学科共同推进的交叉前沿学科。生物体内矿化过程调控机制的研究在很大程度上依赖于体外矿化模型的构建和应用。目前应用较多的单分子膜模型和凝胶矿化模型由于自身的缺陷难以对生物体内硬组织矿化过程进行有效模拟。本研究拟利用具有离子选择透过性的阳离子膜和透析膜构建双膜体外矿化系统,并应用此模型对牙体硬组织生物矿化相关的无机因素、有机因素及无机/有机因素的协同作用进行研究,以期构建一个有效的生物矿化模型,对牙体组织中生物磷灰石晶体的矿化调控机制进行探讨。所取得的研究结果如下:
1体外矿化双膜系统的构建及检测
利用阳离子膜和透析膜构建双膜系统,对系统的稳定性、可靠性进行了检测。结果表明,双膜系统在溶液环境中反应3日即可获得有效的矿化物晶体沉积,随着反应时间的延长,系统的失败率增加,但晶体成份及形态无显著变化。在系统内加入1ppm氟离子后,在阳离子膜表面形成了大量杆状氟磷灰石晶体;而加入含10%胎牛血清的培养基后,阳离子膜表面则形成了大量片层状晶体,晶体成份为磷酸八钙。通过对双膜系统矿化模型的改建,初步构建了稳定的矿化反应系统,并通过氟离子和含10%胎牛血清的培养基初步证实了双膜体外矿化模型的可靠性及有效性,为进一步的研究打下了基础。
2无机因素在体外对磷灰石晶体矿化作用的研究
利用先前实验构建的双膜体外矿化模型对氟离子、镁离子及碳酸氢根离子在体外对磷灰石晶体矿化的作用进行研究。实验结果显示,不同浓度氟离子的加入对晶体的成份及形态均产生了影响。低浓度的氟即可对晶体形态产生显著作用,晶体由不规则的蠕虫状转变为具有一定排列方向的杆状晶体,成份也由磷酸八钙转化为磷酸八钙和氟磷灰石的混合物。氟离子浓度达到或超过5ppm后,大量杆状的氟磷灰石晶体优先在磷酸盐溶液中形成,而阳离子膜表面无明显晶体沉积。加入镁离子后,阳离子膜表面晶体沉积受到抑制,晶体结晶度降低,但晶体成份仍为磷酸八钙;碳酸氢根离子的加入则促进了磷酸八钙晶体的矿化,并且直接参与了晶体的构成。
以上结果表明,低浓度的氟离子即可在磷酸八钙向羟基磷灰石的转化过程中发挥作用,并导致晶体形态的变化。镁离子则可能通过和钙离子的竞争作用抑制磷酸八钙晶体的生长,碳酸氢根离子促进磷酸八钙晶体的矿化,取代磷酸氢根离子,参与晶体的构成。其对局部矿化微环境pH值的影响作用也是其促进磷酸八钙晶体矿化的可能因素之一。无机因素在磷灰石晶体的成核、生长过程中发挥了重要的调控作用。
3有机因素在体外对磷灰石晶体矿化作用的研究
利用双膜系统对单一组份有机大分子物质(牛血清白蛋白,提取的猪釉原蛋白片段及重组小鼠细胞外基质磷酸化糖蛋白),多组份有机混合物(大鼠牙髓细胞矿化诱导培养上清)在体外对磷灰石晶体矿化的影响进行研究。实验结果显示,在有机物的参与下,阳离子膜表面沉积的晶体主要成份均为磷酸八钙。在矿化相关蛋白组(釉原蛋白和细胞外基质磷酸化糖蛋白)中,形成了呈球状聚集的晶体团,而牛血清白蛋白组中则未发现这种现象。有机大分子的加入对晶体生长均产生了抑制作用。大鼠牙髓细胞矿化诱导后培养上清对磷酸八钙晶体形态作用明显,相对于对照组,形成了形态和排列更规则的片层状晶体。
上述结果说明,相对于晶体的成份,有机大分子对晶体形态的影响作用更强。矿化相关蛋白可能直接参与生物磷灰石晶体的成核,并通过与晶体特定晶面的相互作用调控生物晶体的取向性生长。
4无机/有机因素共同作用对磷灰石晶体体外矿化的影响
利用双膜系统对无机/有机因素共同作用对磷灰石晶体体外矿化作用的影响进行研究。分别检测了牛血清白蛋白和氟离子,及釉原蛋白和氟离子的共同作用对晶体矿化的影响。实验结果显示,无机/有机因素的协同作用对磷灰石晶体的形态及成份均产生了作用,当牛血清白蛋白与氟离子共同作用时,磷酸八钙晶体向另一种羟基磷灰石的前体物质磷酸氢钙转化,而当釉原蛋白和氟离子共同作用时,晶体则向磷灰石转化。这说明矿化相关的釉原蛋白可能通过参与成核及与特定晶面的相互作用,对特定晶体的生长有特异性的作用。
综上所述,无机离子和有机大分子在磷灰石晶体的生物矿化过程中发挥着不同的调控作用。无机离子通过取代羟基磷灰石前体物质中的羟基(氟离子)、与钙离子竞争磷灰石晶体表面生长点(镁离子)等作用,改变晶体的热力学稳定性,从而影响矿化物的成份。而有机大分子则通过参与成核及其功能基团与生长中晶体特定晶面的结合,调控晶体的形态。此外,釉原蛋白和细胞外基质磷酸化糖蛋白有可能直接参与了晶体的成核。有机/无机因素的协同作用则是生物矿化物成份和形态的决定因素。
Biomineralization is a mineral forming process in organism, it is a physiological process that regulated precisely by numerous factors. Its research involves disciplines like biology, chemistry, crystallography, material science, mineralogy medicine and so on, is a multi-disciplines field overlapping several front disciplines. Mechanisms researches of the regaulation process are largely depend on the establishment and application of the mineralization system in vitro. Monolayer system and gel system are the most widely used models in this field, but for their own flaw, they are not the best choice for the mechanism research of dental tissue. In this paper, we use Cation-selective membrane(CMV) and dialysis membrane to construct a dual-membrane mineralization system, by using this model, the inorganic and organic factors involved in biomineralization process of dental tissue were investigated to reveal the regaluation mechanism of the mineralization process.
Part 1 Establishment and test of the dual-membrane mineralization system
The model was constructed by using the cation-selective membrane and dialysis membrane. The reliability and stability of the system was tested. Results showed that, after 3 days’reaction, OCP crystal can deposite effectively on the CMV, extended reaction time caused an increased failure rate of the in vitro system without any significent changes of crystal components. When 1ppm fluoride was added into the reaction space, large number of short, rod-like crystal deposited on the CMV, the product was a mixture of OCP and apatite; When cell culture medium supplemented with 10%FBS was added, plate-like OCP was the main component deposited on the CMV. After some improvements, the in vitro mineralization model was established.
Part 2 Effects of inorganic factors on apatite mineralization in vitro
Using the dual-membrane system, effects of F-, Mg2+ and HCO3- on apatite mineralization were investigated. Results showed that, flouride can change the crystal shape and component effectively, even at very low concentration(1ppm). Compared with the worm-like crystal derived from DDW(double-distilled water) group, when flouride was added, oriented rod-like crystal was the main component of the product on CMV. When flouride concentration rised to or exceeded 5ppm, large amount of rod-like flouroapatite crystal deposited in the phosphate solution beside the CMV surface. When Mg2+ was added to the system, deposition of OCP on CMV was inhibited accompany with the decreased crystallinity. HCO3- ions can accelerate the mineralizaton process of OCP, and take part in the crystal lattice directly.
These results revealed that, floride can accelerate the transform process of OCP to apatite and cause the change of the crystal shape. Mg’s inhibit effects of OCP mineralization can be attributed to the competition of Mg with Ca, for their similar ion diameter. HCO3- can replace the phosphate in the OCP lattice and modify the local pH value of the mineralization environment, that promoted the mineralizaton process of OCP. The inorganic factors play important rule during the nucleation and growth process of the apatite crystal.
Part 3 Effects of organic factors on apatite mineralization in vitro
The effects of single-component organic molecule(BSA, P-Amel and MEPE), multi-component (the culture medium of rat DPC after mineralization induction), were inverstigated using the dual-membrane system. When mineralization related proteins(Amel and MEPE) were applied, OCP crystals aggregated to form crystal sphere, which could not be found in BSA group. All the organic molecules tested can inhibit the growth of OCP crystal, Amel and BSA had stronger effect. The induced culture medium of DPC showed impressive effects on OCP crystal’s shape when compared with control group, large amount of well oriented, plate-like crystals were deposited on CMV surface.
All the evidences indicated that, organic molecules’effects on crystal mainly focus on the crystal morphology. They may concentrate Ca ions and act as the crystal nuclui directly. Also, the interaction of their functional groups and specific crystal surface may play critical rules of the oriented growth of the apatite crystal.
Part 4 Co-effects of organic/inorganic factors on apatite crystal mineralization in vitro
The co-effects of BSA and fluoride, Amel and fluoride on apatite crystal mineralization were tested. when BSA was applied accompany with fluoride, morphology of the deposites changed, interestingly, at the same time, XRD results revealed that, the crystal component changed from OCP to another HAP precursor, DCPD. While in the Amel and fluoride group, the crystal component changed from OCP to HAP. That means the Amel may regulated the HAP nuclearation and growth through some specific effects that BSA doesn’t have.
In summary, both organic and inorganic factors play critical rules when regulating the biomineralization process. The inorganic ions mainly affect the component of the apatite through chemistry reactions, while the organic molecules affect the crystal nuclearation and morphology processes through their functional groups. The co-regulation of the organic and inorganic factors is the key element of biomineralization.
1体外矿化双膜系统的构建及检测
利用阳离子膜和透析膜构建双膜系统,对系统的稳定性、可靠性进行了检测。结果表明,双膜系统在溶液环境中反应3日即可获得有效的矿化物晶体沉积,随着反应时间的延长,系统的失败率增加,但晶体成份及形态无显著变化。在系统内加入1ppm氟离子后,在阳离子膜表面形成了大量杆状氟磷灰石晶体;而加入含10%胎牛血清的培养基后,阳离子膜表面则形成了大量片层状晶体,晶体成份为磷酸八钙。通过对双膜系统矿化模型的改建,初步构建了稳定的矿化反应系统,并通过氟离子和含10%胎牛血清的培养基初步证实了双膜体外矿化模型的可靠性及有效性,为进一步的研究打下了基础。
2无机因素在体外对磷灰石晶体矿化作用的研究
利用先前实验构建的双膜体外矿化模型对氟离子、镁离子及碳酸氢根离子在体外对磷灰石晶体矿化的作用进行研究。实验结果显示,不同浓度氟离子的加入对晶体的成份及形态均产生了影响。低浓度的氟即可对晶体形态产生显著作用,晶体由不规则的蠕虫状转变为具有一定排列方向的杆状晶体,成份也由磷酸八钙转化为磷酸八钙和氟磷灰石的混合物。氟离子浓度达到或超过5ppm后,大量杆状的氟磷灰石晶体优先在磷酸盐溶液中形成,而阳离子膜表面无明显晶体沉积。加入镁离子后,阳离子膜表面晶体沉积受到抑制,晶体结晶度降低,但晶体成份仍为磷酸八钙;碳酸氢根离子的加入则促进了磷酸八钙晶体的矿化,并且直接参与了晶体的构成。
以上结果表明,低浓度的氟离子即可在磷酸八钙向羟基磷灰石的转化过程中发挥作用,并导致晶体形态的变化。镁离子则可能通过和钙离子的竞争作用抑制磷酸八钙晶体的生长,碳酸氢根离子促进磷酸八钙晶体的矿化,取代磷酸氢根离子,参与晶体的构成。其对局部矿化微环境pH值的影响作用也是其促进磷酸八钙晶体矿化的可能因素之一。无机因素在磷灰石晶体的成核、生长过程中发挥了重要的调控作用。
3有机因素在体外对磷灰石晶体矿化作用的研究
利用双膜系统对单一组份有机大分子物质(牛血清白蛋白,提取的猪釉原蛋白片段及重组小鼠细胞外基质磷酸化糖蛋白),多组份有机混合物(大鼠牙髓细胞矿化诱导培养上清)在体外对磷灰石晶体矿化的影响进行研究。实验结果显示,在有机物的参与下,阳离子膜表面沉积的晶体主要成份均为磷酸八钙。在矿化相关蛋白组(釉原蛋白和细胞外基质磷酸化糖蛋白)中,形成了呈球状聚集的晶体团,而牛血清白蛋白组中则未发现这种现象。有机大分子的加入对晶体生长均产生了抑制作用。大鼠牙髓细胞矿化诱导后培养上清对磷酸八钙晶体形态作用明显,相对于对照组,形成了形态和排列更规则的片层状晶体。
上述结果说明,相对于晶体的成份,有机大分子对晶体形态的影响作用更强。矿化相关蛋白可能直接参与生物磷灰石晶体的成核,并通过与晶体特定晶面的相互作用调控生物晶体的取向性生长。
4无机/有机因素共同作用对磷灰石晶体体外矿化的影响
利用双膜系统对无机/有机因素共同作用对磷灰石晶体体外矿化作用的影响进行研究。分别检测了牛血清白蛋白和氟离子,及釉原蛋白和氟离子的共同作用对晶体矿化的影响。实验结果显示,无机/有机因素的协同作用对磷灰石晶体的形态及成份均产生了作用,当牛血清白蛋白与氟离子共同作用时,磷酸八钙晶体向另一种羟基磷灰石的前体物质磷酸氢钙转化,而当釉原蛋白和氟离子共同作用时,晶体则向磷灰石转化。这说明矿化相关的釉原蛋白可能通过参与成核及与特定晶面的相互作用,对特定晶体的生长有特异性的作用。
综上所述,无机离子和有机大分子在磷灰石晶体的生物矿化过程中发挥着不同的调控作用。无机离子通过取代羟基磷灰石前体物质中的羟基(氟离子)、与钙离子竞争磷灰石晶体表面生长点(镁离子)等作用,改变晶体的热力学稳定性,从而影响矿化物的成份。而有机大分子则通过参与成核及其功能基团与生长中晶体特定晶面的结合,调控晶体的形态。此外,釉原蛋白和细胞外基质磷酸化糖蛋白有可能直接参与了晶体的成核。有机/无机因素的协同作用则是生物矿化物成份和形态的决定因素。
Biomineralization is a mineral forming process in organism, it is a physiological process that regulated precisely by numerous factors. Its research involves disciplines like biology, chemistry, crystallography, material science, mineralogy medicine and so on, is a multi-disciplines field overlapping several front disciplines. Mechanisms researches of the regaulation process are largely depend on the establishment and application of the mineralization system in vitro. Monolayer system and gel system are the most widely used models in this field, but for their own flaw, they are not the best choice for the mechanism research of dental tissue. In this paper, we use Cation-selective membrane(CMV) and dialysis membrane to construct a dual-membrane mineralization system, by using this model, the inorganic and organic factors involved in biomineralization process of dental tissue were investigated to reveal the regaluation mechanism of the mineralization process.
Part 1 Establishment and test of the dual-membrane mineralization system
The model was constructed by using the cation-selective membrane and dialysis membrane. The reliability and stability of the system was tested. Results showed that, after 3 days’reaction, OCP crystal can deposite effectively on the CMV, extended reaction time caused an increased failure rate of the in vitro system without any significent changes of crystal components. When 1ppm fluoride was added into the reaction space, large number of short, rod-like crystal deposited on the CMV, the product was a mixture of OCP and apatite; When cell culture medium supplemented with 10%FBS was added, plate-like OCP was the main component deposited on the CMV. After some improvements, the in vitro mineralization model was established.
Part 2 Effects of inorganic factors on apatite mineralization in vitro
Using the dual-membrane system, effects of F-, Mg2+ and HCO3- on apatite mineralization were investigated. Results showed that, flouride can change the crystal shape and component effectively, even at very low concentration(1ppm). Compared with the worm-like crystal derived from DDW(double-distilled water) group, when flouride was added, oriented rod-like crystal was the main component of the product on CMV. When flouride concentration rised to or exceeded 5ppm, large amount of rod-like flouroapatite crystal deposited in the phosphate solution beside the CMV surface. When Mg2+ was added to the system, deposition of OCP on CMV was inhibited accompany with the decreased crystallinity. HCO3- ions can accelerate the mineralizaton process of OCP, and take part in the crystal lattice directly.
These results revealed that, floride can accelerate the transform process of OCP to apatite and cause the change of the crystal shape. Mg’s inhibit effects of OCP mineralization can be attributed to the competition of Mg with Ca, for their similar ion diameter. HCO3- can replace the phosphate in the OCP lattice and modify the local pH value of the mineralization environment, that promoted the mineralizaton process of OCP. The inorganic factors play important rule during the nucleation and growth process of the apatite crystal.
Part 3 Effects of organic factors on apatite mineralization in vitro
The effects of single-component organic molecule(BSA, P-Amel and MEPE), multi-component (the culture medium of rat DPC after mineralization induction), were inverstigated using the dual-membrane system. When mineralization related proteins(Amel and MEPE) were applied, OCP crystals aggregated to form crystal sphere, which could not be found in BSA group. All the organic molecules tested can inhibit the growth of OCP crystal, Amel and BSA had stronger effect. The induced culture medium of DPC showed impressive effects on OCP crystal’s shape when compared with control group, large amount of well oriented, plate-like crystals were deposited on CMV surface.
All the evidences indicated that, organic molecules’effects on crystal mainly focus on the crystal morphology. They may concentrate Ca ions and act as the crystal nuclui directly. Also, the interaction of their functional groups and specific crystal surface may play critical rules of the oriented growth of the apatite crystal.
Part 4 Co-effects of organic/inorganic factors on apatite crystal mineralization in vitro
The co-effects of BSA and fluoride, Amel and fluoride on apatite crystal mineralization were tested. when BSA was applied accompany with fluoride, morphology of the deposites changed, interestingly, at the same time, XRD results revealed that, the crystal component changed from OCP to another HAP precursor, DCPD. While in the Amel and fluoride group, the crystal component changed from OCP to HAP. That means the Amel may regulated the HAP nuclearation and growth through some specific effects that BSA doesn’t have.
In summary, both organic and inorganic factors play critical rules when regulating the biomineralization process. The inorganic ions mainly affect the component of the apatite through chemistry reactions, while the organic molecules affect the crystal nuclearation and morphology processes through their functional groups. The co-regulation of the organic and inorganic factors is the key element of biomineralization.
引文
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[32] Igarashi M, Kamiya N, Takagi M, et al. In situ localization and in vitro expression of osteoblast/osteocyte factor 45 mRNA during bone cell differentiation. Histochem J. 2002; 34(5): 255-63.
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[37] MacDougall M, Simmons D, Gu TT, Dong J. MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21. Connect Tissue Res. 2002; 43(2-3): 320-30.
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[41]艾林人细胞外基质磷酸化糖蛋白的重组表达及在牙本质形成中的作用和机理研究博士研究生论文2008
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[43] George A, Gui J, Jenkins NA, et al . In situ localization and chromosomal mapping of the AG1 (Dmp1) gene . J Histochem Cytochem ,1994 ,42 (12) :1527-31
[44] Septier D, Torres - Quintana MA, Menashi S, et al . Inositol hexasulphate , a casein kinase inhibitor , alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs. Eur J Oral Sci ,2001 ,109 (3) :198-203
[45] He G, George A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem. 2004;279(12): 11649-56
[46] He G, Dahl T, Veis A, George A. Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1. Nat Mater. 2003;2(8):552-8
[47] Gajjeraman S, Narayanan K, George A, et al. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly ofhydroxyapatite. J Biol Chem. 2007;282(2):1193-204
[48] Robinson C, Connell S, Smith AM, et al. The effect of fluoride on the developing tooth.Caries Res. 2004, 38(3):268-76.
[49]生命科学中的微量元素王夔主编第二版北京中国计量出版社1996: 70-72
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