自分泌胞外全基质修饰钛种植体表面的研究
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摘要
背景自骨整合(osseointegration)理论成为口腔种植学发展的主导理论,口腔种植学得到广泛的发展和应用。但是,目前种植修复也存在植入后愈合时间较长,在骨质、骨量不良的部位失败率较高等问题。
种植体-组织界面的反应被认为是决定牙种植体成功和失败的重要因素之一,所以学者们纷纷通过对种植材料表面进行有效的控制,促进骨整合。近年来在种植体表面粗糙化和表面氧化物活化的基础上,将生物活性分子复合在种植体表面的生化改性成为种植体设计研究中较为活跃和发展较快的领域。为了模拟胞外基质对生物行为的调控功能,通常采用基质中的活性成分对钛表面进行改性,并取得较为肯定的结果。但这些单一的活性成分与天然基质仍有差距。因此为了使钛种植体的仿生构建更接近天然情况,本次研究试想用生物自行分泌的胞外基质作为一种新型生物材料,进行初步探讨。
目的在体外研究成骨细胞自身分泌的胞外基质(ECM)修饰的钛表面对骨髓基质干细胞生物学行为的影响,并为进一步指导钛种植体表面的仿生构建提供依据。
方法取SD乳鼠,采用改良组织块法培养成骨细胞。取直径12 mm的钛棒,加工成厚1 mm纯钛片,消毒后放入24孔培养板中,用DMEM培养液将成骨细胞浓度调整为3×105/mL,取1 mL/孔接种于钛片上,经过反复冻融脱去细胞留下基质,即为基质化钛片(Ti/ECM)。取SD大鼠,采用全血贴壁法培养骨髓基质干细胞(BMSCs),取传至第3代的细胞,用DMEM培养液将其浓度调整为1×105/mL,取1 mL/孔分别接种于基质化钛片和纯钛片上,分为基质化钛片组(Ti/ECM/BMSCs)、纯钛片组(CpTi/BMSCs)。通过荧光免疫组化、扫描电镜的观察和MTT检测、ALP活性检测,观察基质化钛片的表面形貌变化、胞外基质形态和标识成分,并比较两组钛片上骨髓基质细胞的早期黏附、铺展、生长增殖、分化情况。
结果关于钛片的基质化构建,荧光显微镜显示去细胞后,钛表面存有胞外基质的生物活性成分,其呈现为不规则絮状,SEM显示基质化后钛片表面被一层似胶状物质覆盖;关于两组细胞行为比较,SEM显示骨髓基质细胞在基质化钛片表面黏附、铺展良好,在接种4h时,Ti/ECM/BMSCs细胞黏附率与CpTi/BMSCs组有统计学差异(P<0.05),接种1、3、5、7天后,Ti/ECM组的细胞增殖与CpTi组之间存在显著性差异(P<0.05);接种5天后,Ti/ECM表面的细胞分化与CpTi的之间存在显著性差异(P<0.05)。
结论经过初步基质化构建,钛片表面存有多种胞外基质的生物活性成分,基质化钛片更有利于骨髓基质细胞的早期黏附和进一步铺展、增殖,并可能具有诱导细胞向成骨细胞分化的作用。
Background Since osseointegration become the main theory of dental implantology by Branenmark, titanium implant devices are widely used. But many problems still exist ,such as a slow healing or a failure because of poor bone quality and insufficient quantity of bone.
Interfacial interactions at the bone-implant interface are recognised as the key to osseointegration, so a huge number of approaches to the surface modification of titanium, to improve further clinical results and extend the spectrum of indications, are carried out, as well. Based on surface roughness techniques and surface oxidic activitation, biochemical modification by immobilization of biomolecules to titanium surfaces is focused to the enhancement of bone regeneration at the interface with implant devices. Active component of matrix is frequently used to modify titanium surface in order to simulate regulatory function of extracellular matrix to biological behavior. Although positive results were showed in such experiments, those single biomolecule are still different from natural matrix. Thus, to simulate natural construct more, as a new biomaterial, in this study all components of extracellular matrix screted by living cells were coated onto titanium surface.
Objective to observe the early behavior of bone marrow stromal cells on the titanium coated by extracellular matrix from osteoblast, and supply experimental basis for improved biomimetic properties of titanium implants.
Methods Osteoblasts from neonate rats were cultured using improved tissue method. Titanium rod of 12 mm diameter was sheared into sections of 1 mm thickness which were put into 24-well culture plate after sterilization. Concentration of osteoblasts was adjusted to 3×108 L-1 in DMEM culture medium. Thereafter, the osteoblasts (1 mL/well) were inoculated on titanium section and freeze-thawed to obtain matrix, namely matrix-coated titanium sheet. Bone marrow stromal cells from adult SD rats were cultured using whole blood adherence method. The third-generation cells were assigned into matrix-coated titanium group and pure titanium group. The concentration of cells was adjusted to 1×108 L-1 in the DMEM culture medium, and then the cells were inoculated on matrix-coated titanium sheet and pure titanium sheet, respectively, 1 mL/well. The morphologic changes of modified titanium surface were observed by scanning electron microscopy. Besides, biologic behavior of bone marrow stromal cells in the two groups ,including early attachment ,spreading, growth and differentiation, were detected using fluorescence immunohistochemistry, scanning electron microscopy, MTT assay and ALP activity .
Results After the freeze/thaw cycles, biomoleculars secreted by osteoblasts were found under fluoroscope. At 4 hours, cell adhesion in the matrix-coated titanium group was significantly higher than pure titanium group (P < 0.05). At 4 hours after inoculation, bone marrow stromal cells attached to matrix-coated titanium to a certain degree; at 24 hours after inoculation, the cells were spread well and tightly attached to matrix-coated titanium, showing a pseudopodium process which was importance for intercommunication between every two cells; at 72 hours after inoculation, cells were sufficiently spread, and spreading area of soma was enlarged rapidly. BMSCs on Ti/ECM always grew better statistically than those on CpTi according to MMT assays(P<0.05).and statistic significance of BMSCs’differentiation was showed by alkaline phosphatase activity after 5 days(P<0.05).
Conclusions After biomimetic construct, many biomolecules of extracellular matrix were laid onto titanium surfaces. And titanium surfaces coated by ECM from osteoblasts are more helpful for BMSCs’early attachment and spreading growth and osseoblastic differentiation.
种植体-组织界面的反应被认为是决定牙种植体成功和失败的重要因素之一,所以学者们纷纷通过对种植材料表面进行有效的控制,促进骨整合。近年来在种植体表面粗糙化和表面氧化物活化的基础上,将生物活性分子复合在种植体表面的生化改性成为种植体设计研究中较为活跃和发展较快的领域。为了模拟胞外基质对生物行为的调控功能,通常采用基质中的活性成分对钛表面进行改性,并取得较为肯定的结果。但这些单一的活性成分与天然基质仍有差距。因此为了使钛种植体的仿生构建更接近天然情况,本次研究试想用生物自行分泌的胞外基质作为一种新型生物材料,进行初步探讨。
目的在体外研究成骨细胞自身分泌的胞外基质(ECM)修饰的钛表面对骨髓基质干细胞生物学行为的影响,并为进一步指导钛种植体表面的仿生构建提供依据。
方法取SD乳鼠,采用改良组织块法培养成骨细胞。取直径12 mm的钛棒,加工成厚1 mm纯钛片,消毒后放入24孔培养板中,用DMEM培养液将成骨细胞浓度调整为3×105/mL,取1 mL/孔接种于钛片上,经过反复冻融脱去细胞留下基质,即为基质化钛片(Ti/ECM)。取SD大鼠,采用全血贴壁法培养骨髓基质干细胞(BMSCs),取传至第3代的细胞,用DMEM培养液将其浓度调整为1×105/mL,取1 mL/孔分别接种于基质化钛片和纯钛片上,分为基质化钛片组(Ti/ECM/BMSCs)、纯钛片组(CpTi/BMSCs)。通过荧光免疫组化、扫描电镜的观察和MTT检测、ALP活性检测,观察基质化钛片的表面形貌变化、胞外基质形态和标识成分,并比较两组钛片上骨髓基质细胞的早期黏附、铺展、生长增殖、分化情况。
结果关于钛片的基质化构建,荧光显微镜显示去细胞后,钛表面存有胞外基质的生物活性成分,其呈现为不规则絮状,SEM显示基质化后钛片表面被一层似胶状物质覆盖;关于两组细胞行为比较,SEM显示骨髓基质细胞在基质化钛片表面黏附、铺展良好,在接种4h时,Ti/ECM/BMSCs细胞黏附率与CpTi/BMSCs组有统计学差异(P<0.05),接种1、3、5、7天后,Ti/ECM组的细胞增殖与CpTi组之间存在显著性差异(P<0.05);接种5天后,Ti/ECM表面的细胞分化与CpTi的之间存在显著性差异(P<0.05)。
结论经过初步基质化构建,钛片表面存有多种胞外基质的生物活性成分,基质化钛片更有利于骨髓基质细胞的早期黏附和进一步铺展、增殖,并可能具有诱导细胞向成骨细胞分化的作用。
Background Since osseointegration become the main theory of dental implantology by Branenmark, titanium implant devices are widely used. But many problems still exist ,such as a slow healing or a failure because of poor bone quality and insufficient quantity of bone.
Interfacial interactions at the bone-implant interface are recognised as the key to osseointegration, so a huge number of approaches to the surface modification of titanium, to improve further clinical results and extend the spectrum of indications, are carried out, as well. Based on surface roughness techniques and surface oxidic activitation, biochemical modification by immobilization of biomolecules to titanium surfaces is focused to the enhancement of bone regeneration at the interface with implant devices. Active component of matrix is frequently used to modify titanium surface in order to simulate regulatory function of extracellular matrix to biological behavior. Although positive results were showed in such experiments, those single biomolecule are still different from natural matrix. Thus, to simulate natural construct more, as a new biomaterial, in this study all components of extracellular matrix screted by living cells were coated onto titanium surface.
Objective to observe the early behavior of bone marrow stromal cells on the titanium coated by extracellular matrix from osteoblast, and supply experimental basis for improved biomimetic properties of titanium implants.
Methods Osteoblasts from neonate rats were cultured using improved tissue method. Titanium rod of 12 mm diameter was sheared into sections of 1 mm thickness which were put into 24-well culture plate after sterilization. Concentration of osteoblasts was adjusted to 3×108 L-1 in DMEM culture medium. Thereafter, the osteoblasts (1 mL/well) were inoculated on titanium section and freeze-thawed to obtain matrix, namely matrix-coated titanium sheet. Bone marrow stromal cells from adult SD rats were cultured using whole blood adherence method. The third-generation cells were assigned into matrix-coated titanium group and pure titanium group. The concentration of cells was adjusted to 1×108 L-1 in the DMEM culture medium, and then the cells were inoculated on matrix-coated titanium sheet and pure titanium sheet, respectively, 1 mL/well. The morphologic changes of modified titanium surface were observed by scanning electron microscopy. Besides, biologic behavior of bone marrow stromal cells in the two groups ,including early attachment ,spreading, growth and differentiation, were detected using fluorescence immunohistochemistry, scanning electron microscopy, MTT assay and ALP activity .
Results After the freeze/thaw cycles, biomoleculars secreted by osteoblasts were found under fluoroscope. At 4 hours, cell adhesion in the matrix-coated titanium group was significantly higher than pure titanium group (P < 0.05). At 4 hours after inoculation, bone marrow stromal cells attached to matrix-coated titanium to a certain degree; at 24 hours after inoculation, the cells were spread well and tightly attached to matrix-coated titanium, showing a pseudopodium process which was importance for intercommunication between every two cells; at 72 hours after inoculation, cells were sufficiently spread, and spreading area of soma was enlarged rapidly. BMSCs on Ti/ECM always grew better statistically than those on CpTi according to MMT assays(P<0.05).and statistic significance of BMSCs’differentiation was showed by alkaline phosphatase activity after 5 days(P<0.05).
Conclusions After biomimetic construct, many biomolecules of extracellular matrix were laid onto titanium surfaces. And titanium surfaces coated by ECM from osteoblasts are more helpful for BMSCs’early attachment and spreading growth and osseoblastic differentiation.
引文
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6.Stanford C M. Surface modification of implants . Oral Maxillofacial Surg Clin N Am, 2002,14: 39;
7.栗兴超,董福生种植体表面优化处理对其骨结合促进的研究进展现代口腔医学杂志2007, 21 (6):645-646
8.吴应龙,梁剑秋,刘族志纯钛金属种植体表面活性化的处理方法中国组织工程研究与临床康复2008,12(14):2693-2696
9.庄燕燕,胡仁,陈菲等钛植入体表面生物化学改性的研究进展生物医学工程学杂志,2005,22 (3)∶6 18-621
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2.Puleo DA, Nanci A. Understanding and controlling the bone-implant interface. Biomaterials, 1999; 20(23-24): 2311-2321
3. Puleo D A, Thoma MV . Implant surface, Dent Clin N Am 2006, 50: 323-328
4. Bengt Kasemo and Jukka Lausmaa Material-tissue Interfaces: The Role of Surface Properties and Processes Environmental Health Perspectives 1994,102(Suppl5): 41-45
5. Tirrell M, Kokkoli E, Biesalski M. The role of surface science in bioengineered materials. Surface Science 2002, 500: 61–83
6.Stanford C M. Surface modification of implants . Oral Maxillofacial Surg Clin N Am, 2002,14: 39;
7.栗兴超,董福生种植体表面优化处理对其骨结合促进的研究进展现代口腔医学杂志2007, 21 (6):645-646
8.吴应龙,梁剑秋,刘族志纯钛金属种植体表面活性化的处理方法中国组织工程研究与临床康复2008,12(14):2693-2696
9.庄燕燕,胡仁,陈菲等钛植入体表面生物化学改性的研究进展生物医学工程学杂志,2005,22 (3)∶6 18-621
10.Morra M Biochemical modification of titanium surfaces: peptide and ECM proteins. European Cells and Materials 2006 Jul 24; 12:1-15
11.Roehlecke C, Witt M, Kasper M Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells. Cells Tissues Organs. 2001;168(3):178-187
12.Schliephake H, Scharnweber D, Roesseler S,et al .Biomimetic calcium phosphate composite coating of dental implants Int J Oral Maxillofac Implants. 2006 ,21(5): 738-746
13.Sumner DR, Turner TM, Urban RM. et al Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model. J Bone Joint Surg Am, 2006, 88(4):806- 817
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