载TGF-β_1微球涂层多孔钛对成骨细胞功能的影响
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摘要
目的：研究载不同浓度TGF-β1微球缓释药动力特性,评价载TGF-β1微球涂层多孔钛对成骨细胞影响。
方法：采用粉末注射成形(Metal Injection Molding, MIM)技术制备60%孔隙度具有连通孔结构多孔钛；用改良乳化冷凝聚合交联法制备明胶缓释微球；用渗涂法在多孔钛表层孔隙内涂覆载TGF-β1明胶微球涂层,用MTT法检测载TGF-β1微球涂层多孔钛的细胞毒性,ELISA法检测该涂层的药物动力特性；体外细胞实验评价载不同浓度的TGF-β1明胶微球对成骨细胞增殖和分化的影响并优化TGF-β1浓度,比较研究载TGF-β1微球涂层多孔钛组(A组),添加TGF-β1溶液多孔钛组(B组),不加TGF-β1多孔钛组(C组)对成骨细胞黏附增殖分化的影响。
结果：用MIM技术制备多孔钛的结构特点及力学性能为：孔隙度为60%,孔径范围为50-300μm的连通孔结构。载TGF-β1明胶微球平均粒径为(20.33+3.67)μm,用5wt%明胶溶液处理后再渗涂20mg/ml明胶微球发现孔隙内均匀分布微球且不阻塞孔隙；MTT法检测结果显示：100%、50%、10%浓度的明胶微球涂层多孔钛的浸提液对L929细胞均无细胞毒性;载不同TGF-β1微球涂层药动力特征为：首天释放率达约20-30%,随着时间的递增,速率逐渐减缓,持续约12天,累计释放达93%。TGF-β1的浓度影响成骨细胞增殖和分化,在0.025-2.5ng/mg范围内,成骨细胞增殖分化与TGF-β1呈剂量正效应,当浓度为2.5ng/mg浓度时促增殖分化作用最佳；低于0.025ng/mg对成骨细胞增殖分化无明显作用,高于2.5ng/mg时抑制MG63细胞的增殖,但有利于细胞分化。体外细胞实验3、7天检测MG63细胞增殖,A组(载TGF-β1微球涂层多孔钛组)>B组(添加TGF-β1溶液多孔钛组)>C组(不加TGF-β1多孔钛组),差异显著(P<0.05)；14天时,A组细胞数量大于B组和C组,差异显著(P<0.05),B组C组之间含量差异无显著性(P>0.05)；体外细胞培养7天后扫描电镜发现：三组材料表面黏附MG63细胞的数量、形貌及孔隙内生长的情况均有差异,A组试样表面黏附MG63细胞数多于B组和C组,A组试样MG63细胞形态不规则,伪足多,平铺于材料表面,细胞多附着于孔隙边缘,有的细胞已迁移至孔隙内；B组试样MG63细胞伸展良好,附着在孔隙边缘,垂直向孔隙内生长,形成向孔隙内迁移的趋势；C组试样MG63细胞形态呈梭形,平铺于多孔钛表面,伪足少,未见细胞向孔隙内迁移;14天时扫描电镜发现3组细胞无明显外观差异,均生长良好,细胞多呈不规则多边形,呈叠瓦状生长,叠层平铺于材料表面及孔隙内。A组细胞在孔隙内结成网状结构；B组细胞呈叠瓦状铺于多孔钛几乎看不到多孔钛表面结构；C组可见成骨细胞紧贴孔壁生长,长入孔底,但仍可看见多孔钛表面。
体外实验3天、7天时检测MG63细胞分化,A组B组试样碱性磷酸酶(alkaline phosphatase ALP)、骨钙素(boney-carboxyglutamic acid BGP)含量均高于C组,差异显著(P<0.05),A组B组之间含量差异无显著性(P>0.05)；培养14天ALP、BGP含量A组>B组>C组,差异显著(P<0.05)。
结论：1)载TGF-β1微球涂层具有缓释功能,释放时间能维持12天。
2)不同浓度TGF-β1影响成骨细胞增殖和分化,TGF-β1浓度在0.25-2.5ng/mg范围内,与成骨细胞增殖分化呈剂量正效应关系,并以2.5ng／mg浓度时促增殖分化作用最为显著；低于0.25ng/mg对成骨细胞增殖分化无明显作用,高于2.5ng/mg时抑制成骨细胞的增殖,但有利于细胞分化。
3)添加TGF-β1溶液多孔钛3、7天可促进MG63细胞增殖,14天时无明显促进增殖作用,但在3、7、14天均有利于MG63细胞分化,而载TGF-β1微球涂层多孔钛在三个时间段均能有效促进MG63细胞的增殖和分化。
Objective:This study investigated the characterization of porous titanium coated with different concentrations of TGF-β1 loaded gelatin microspheres, and estimate the effects on function of osteoblast cells after surface modificated with TGF-β1 loaded gelatin microspheres in vitro.
Materials and methods:Porous titanium with porosity of 60% and cross-connection pore structure were prepared by metal injection molding.Gelatin microspheres were prepared by improved emulsified cold condensation method. The porous titanium were coated with TGF-β1 loaded gelatin microspheres, MTT assay method was used to evaluate the cytotoxicity of coating to the rat fibroblast cells L929. The encapsulation rate, drug content were test with TGF-β1 ELISA kit.Assessed the influence of different concentrations of TGF-β1 loaded gelatin microspheres on proliferation and differentiation of osteoblast cells MG63 in vitro and select the best concentrations of TGF-β1 comparative studied influence of porous titanium coated with TGF-β1 loaded gelatin microspheres(group A), porous titanium with TGF-β1 (group B), porous titanium (group C)on adhesion, proliferation and differentiation of osteoblast cells MG63.
Result:Porous titanium implants were produced by MIM, which microstructure characters were 60% porosity with cross-connection pore structure and pore size range of 50-300μm. The size of gelatin microspheres were (20.33±3.67)μm in average.The MTT test showed the leaching liquor with 100%、50%、10% concentrations of porous titanium coated with gelatin microspheres was nontoxic to L929.The porous titanium implants were pretreated by 5wt% gelatin and coated with 20mg/ml TGF-β1 loaded gelatin microspheres were appropriate,the structure of pores were kept completely. In vitro,20-30% of TGF-β1 released from gelatin microspheres coating in 24h and releasing rat gradually decreased and became stable as time increased. Finally,93% of TGF-β1 were released during 12days. The concentrations of TGF-β1 effected proliferation and differentiation of osteoblast cells, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.After cultured 3、7 days it showed the amount of cells in A group>B group>C group, and the difference was remarkable. on 14th day, the amount of cells in A group> B group and C group,there was no significant difference between group B and group C (P>0.05). (P<0.05); Observing cell culture in vitro on 7th day by SEM, we comed to a conclusion that:The quantity, morphology and growing status in interspaces of MG63 A were different among 3 groups. Samples on group A own more cells than group B and C. MG63 in group A had irregular shapes and more pseudopod, they laid on the surface of materials, most of cells adhered to the border of pores while a few of them have migrated inside. MG63 in group B spreaded well, they adhered to the border and even grow into the pores vertically, trended to migrate inside. MG63 in group C is spindle shaped, laid on the surface of porous titanium and had few pseudopod, they got no trend of migrating inside. Observed by SEM in 14th day,3 groups of cells develop well, they had no differences in appearance,most of cells are in irregular polygon shaped.MG63 in group A formed an net work. MG63 in group B laminated lay on and inside pores, can barely show surface structure of porous titanium. Most of cells in group C grew vertically to the bottom of pores in reticular structure.After cultured 3、7days, no difference in ALP and BGP production was detected on group A and group B (P>0.05). But there was significant (P<0.05) between other groups. No difference in BGP production was detected on 5% and 30% group (P>0.05). But the difference was remarkable (P<0.05) on the other three groups,in 14th day,it showed the amount in A group>B group>C group, and the difference was remarkable.
Conclusion: 1) In vitro, porous titanium coated with TGF-β1 loaded gelatin microspheres could controlledly release, TGF-β1 was released for 12 days.
2) Proliferation and differentiation of osteoblasts were effected by the concentration of TGF-b1, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.
3) Porous titanium with TGF-β1 is propitious to osteoblast cell's proliferation on 3th and 7th has no significant effect on 14th,but it is propitious to proliferation. Porous titanium coated with TGF-β1 loaded gelatin microspheres is propitious to osteoblast cell's adhesion, proliferation and differentiation on the three time slot.
方法：采用粉末注射成形(Metal Injection Molding, MIM)技术制备60%孔隙度具有连通孔结构多孔钛；用改良乳化冷凝聚合交联法制备明胶缓释微球；用渗涂法在多孔钛表层孔隙内涂覆载TGF-β1明胶微球涂层,用MTT法检测载TGF-β1微球涂层多孔钛的细胞毒性,ELISA法检测该涂层的药物动力特性；体外细胞实验评价载不同浓度的TGF-β1明胶微球对成骨细胞增殖和分化的影响并优化TGF-β1浓度,比较研究载TGF-β1微球涂层多孔钛组(A组),添加TGF-β1溶液多孔钛组(B组),不加TGF-β1多孔钛组(C组)对成骨细胞黏附增殖分化的影响。
结果：用MIM技术制备多孔钛的结构特点及力学性能为：孔隙度为60%,孔径范围为50-300μm的连通孔结构。载TGF-β1明胶微球平均粒径为(20.33+3.67)μm,用5wt%明胶溶液处理后再渗涂20mg/ml明胶微球发现孔隙内均匀分布微球且不阻塞孔隙；MTT法检测结果显示：100%、50%、10%浓度的明胶微球涂层多孔钛的浸提液对L929细胞均无细胞毒性;载不同TGF-β1微球涂层药动力特征为：首天释放率达约20-30%,随着时间的递增,速率逐渐减缓,持续约12天,累计释放达93%。TGF-β1的浓度影响成骨细胞增殖和分化,在0.025-2.5ng/mg范围内,成骨细胞增殖分化与TGF-β1呈剂量正效应,当浓度为2.5ng/mg浓度时促增殖分化作用最佳；低于0.025ng/mg对成骨细胞增殖分化无明显作用,高于2.5ng/mg时抑制MG63细胞的增殖,但有利于细胞分化。体外细胞实验3、7天检测MG63细胞增殖,A组(载TGF-β1微球涂层多孔钛组)>B组(添加TGF-β1溶液多孔钛组)>C组(不加TGF-β1多孔钛组),差异显著(P<0.05)；14天时,A组细胞数量大于B组和C组,差异显著(P<0.05),B组C组之间含量差异无显著性(P>0.05)；体外细胞培养7天后扫描电镜发现：三组材料表面黏附MG63细胞的数量、形貌及孔隙内生长的情况均有差异,A组试样表面黏附MG63细胞数多于B组和C组,A组试样MG63细胞形态不规则,伪足多,平铺于材料表面,细胞多附着于孔隙边缘,有的细胞已迁移至孔隙内；B组试样MG63细胞伸展良好,附着在孔隙边缘,垂直向孔隙内生长,形成向孔隙内迁移的趋势；C组试样MG63细胞形态呈梭形,平铺于多孔钛表面,伪足少,未见细胞向孔隙内迁移;14天时扫描电镜发现3组细胞无明显外观差异,均生长良好,细胞多呈不规则多边形,呈叠瓦状生长,叠层平铺于材料表面及孔隙内。A组细胞在孔隙内结成网状结构；B组细胞呈叠瓦状铺于多孔钛几乎看不到多孔钛表面结构；C组可见成骨细胞紧贴孔壁生长,长入孔底,但仍可看见多孔钛表面。
体外实验3天、7天时检测MG63细胞分化,A组B组试样碱性磷酸酶(alkaline phosphatase ALP)、骨钙素(boney-carboxyglutamic acid BGP)含量均高于C组,差异显著(P<0.05),A组B组之间含量差异无显著性(P>0.05)；培养14天ALP、BGP含量A组>B组>C组,差异显著(P<0.05)。
结论：1)载TGF-β1微球涂层具有缓释功能,释放时间能维持12天。
2)不同浓度TGF-β1影响成骨细胞增殖和分化,TGF-β1浓度在0.25-2.5ng/mg范围内,与成骨细胞增殖分化呈剂量正效应关系,并以2.5ng／mg浓度时促增殖分化作用最为显著；低于0.25ng/mg对成骨细胞增殖分化无明显作用,高于2.5ng/mg时抑制成骨细胞的增殖,但有利于细胞分化。
3)添加TGF-β1溶液多孔钛3、7天可促进MG63细胞增殖,14天时无明显促进增殖作用,但在3、7、14天均有利于MG63细胞分化,而载TGF-β1微球涂层多孔钛在三个时间段均能有效促进MG63细胞的增殖和分化。
Objective:This study investigated the characterization of porous titanium coated with different concentrations of TGF-β1 loaded gelatin microspheres, and estimate the effects on function of osteoblast cells after surface modificated with TGF-β1 loaded gelatin microspheres in vitro.
Materials and methods:Porous titanium with porosity of 60% and cross-connection pore structure were prepared by metal injection molding.Gelatin microspheres were prepared by improved emulsified cold condensation method. The porous titanium were coated with TGF-β1 loaded gelatin microspheres, MTT assay method was used to evaluate the cytotoxicity of coating to the rat fibroblast cells L929. The encapsulation rate, drug content were test with TGF-β1 ELISA kit.Assessed the influence of different concentrations of TGF-β1 loaded gelatin microspheres on proliferation and differentiation of osteoblast cells MG63 in vitro and select the best concentrations of TGF-β1 comparative studied influence of porous titanium coated with TGF-β1 loaded gelatin microspheres(group A), porous titanium with TGF-β1 (group B), porous titanium (group C)on adhesion, proliferation and differentiation of osteoblast cells MG63.
Result:Porous titanium implants were produced by MIM, which microstructure characters were 60% porosity with cross-connection pore structure and pore size range of 50-300μm. The size of gelatin microspheres were (20.33±3.67)μm in average.The MTT test showed the leaching liquor with 100%、50%、10% concentrations of porous titanium coated with gelatin microspheres was nontoxic to L929.The porous titanium implants were pretreated by 5wt% gelatin and coated with 20mg/ml TGF-β1 loaded gelatin microspheres were appropriate,the structure of pores were kept completely. In vitro,20-30% of TGF-β1 released from gelatin microspheres coating in 24h and releasing rat gradually decreased and became stable as time increased. Finally,93% of TGF-β1 were released during 12days. The concentrations of TGF-β1 effected proliferation and differentiation of osteoblast cells, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.After cultured 3、7 days it showed the amount of cells in A group>B group>C group, and the difference was remarkable. on 14th day, the amount of cells in A group> B group and C group,there was no significant difference between group B and group C (P>0.05). (P<0.05); Observing cell culture in vitro on 7th day by SEM, we comed to a conclusion that:The quantity, morphology and growing status in interspaces of MG63 A were different among 3 groups. Samples on group A own more cells than group B and C. MG63 in group A had irregular shapes and more pseudopod, they laid on the surface of materials, most of cells adhered to the border of pores while a few of them have migrated inside. MG63 in group B spreaded well, they adhered to the border and even grow into the pores vertically, trended to migrate inside. MG63 in group C is spindle shaped, laid on the surface of porous titanium and had few pseudopod, they got no trend of migrating inside. Observed by SEM in 14th day,3 groups of cells develop well, they had no differences in appearance,most of cells are in irregular polygon shaped.MG63 in group A formed an net work. MG63 in group B laminated lay on and inside pores, can barely show surface structure of porous titanium. Most of cells in group C grew vertically to the bottom of pores in reticular structure.After cultured 3、7days, no difference in ALP and BGP production was detected on group A and group B (P>0.05). But there was significant (P<0.05) between other groups. No difference in BGP production was detected on 5% and 30% group (P>0.05). But the difference was remarkable (P<0.05) on the other three groups,in 14th day,it showed the amount in A group>B group>C group, and the difference was remarkable.
Conclusion: 1) In vitro, porous titanium coated with TGF-β1 loaded gelatin microspheres could controlledly release, TGF-β1 was released for 12 days.
2) Proliferation and differentiation of osteoblasts were effected by the concentration of TGF-b1, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.
3) Porous titanium with TGF-β1 is propitious to osteoblast cell's proliferation on 3th and 7th has no significant effect on 14th,but it is propitious to proliferation. Porous titanium coated with TGF-β1 loaded gelatin microspheres is propitious to osteoblast cell's adhesion, proliferation and differentiation on the three time slot.
引文
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[1]Pan J.Wang YL.Cao YB.et al. Study of adhesion property of Wistar rat osteoblast on polylactide and maleic anhydride modified polylactide [J]. Prog Biochem biochem biophys.2001,28(5):688
[2]Anselme K. Osteoblast adhesion on biomaterials [J]. Biomaterials,2000,21(7): 667-681
[3]Lim JY,Hansen JC, Siedleeki CA,et al.Osteoblast adhesion on poly(L-lactic acid) / polystyrene demixed thin film blends:effect of nanotopography. surface chemistry, and wettability[J]. Biomaterials,2005,6(6):3319-3327
[4]Gronthos S.Stewart K,Graves SE,et al.Integrin expression and function on human osteoblast-like cells.[J]. Bone Miner Res.1997,12(8):1189
[5]schuler M, Owen GR. Hamilton DW. et al.Biomimetic medification of titanium dental implant model surfaces using the RGD-SP-peptide sequence:a cell morphology study[J]. Biomaterials,2006,27 (21):4003-4015
[6]Bagno A, Piovan A. Dettin M et al. Human osteoblast-like cell adhesion on titanium substrates covalently funetiormlized with synthetic peptides[J]Bone, 2007,40(3)693-699
[7]Pallu S, Bourget C, Bareille R, et al. The effect Of cyclo—DfKRG pettide immobilization on titanium on the adhesion and diferentiatinn of human osteop-rogenitor cells [J]. Biomaterials,2005,26(34):6932—6940
[8]IrvineDJ. HueKA. Mayes AM. et al. Simulations of cell-surface integrin binding to nanoscale-clustered adhesion ligands [J]. Biophys,.2002,82(1):120—132
[9]Guo KT, ScharnweberD, SchwenzerB, et al.The effect electrochemical functionalization of Ti—alloy surface by aptamerbased capture molecules on cell adhesion[J]. Biomaterials,2007.28(3):468—474
[10]Cheng SL,Lecanda F,Davidson M.et al.Human osteoblasts express a repertoire of cadherins. [J]. Bone Miner Res.1998,13(4); 633
[11]Cheng SL, Shin CS,Towler DA et al. A dominant negative cadherin inhibits osteoblast differentiation and protein synthesis of human osteoblast-like cells(MG63). [J]. Bone Mater Res,1995,29(12):389
[12]Martin JY,Schwartz Z,Hummert TW,et al.Effect of titanium surface roughness
on proliferation,differentation and protein synthesis of human osteoblast- like cells(MG63). [J] Bone Mater Res,1995,29(3):389
[13]Langer R,Liithen F,Beck U,et al. Cell-extacellular matrix interaction and physicochemical characterristics of titanium surfaces depend on the roughness of the material [J]. Biomolecular Engineering.2002,19(2-6):255
[14]Keller JC,Schneider GB,Stanford CM,et al. Effects of implant microto- pography on osteoblast cell attachment[J] Implant Dent,2003,12(2):175-181
[15]Deligianni DD,Katasla N ,Ladas S,et al.Effect of surface roughness of the titanium alloy Ti-6A1-4V on human bone marrow cell response and on protein adsorption [J]. Biomaterials,2001,22(11):1241-1251
[16]Anselme K, Bigerene M, Noel B, et al. QIlalitative and quantitotive study of human osteoblast adhesion on materials with various surface roughness[J]. Biomed Mater Res 2000.49(2):155—166
[17]Bignrelle M, Anselme K, Dufresne E, et al, An unsealed parameter to measure the order of surface :a new surface elaboration to increase cells adhesion[J]. Biomol Eng,2002.19(2—6):79—83
[18]Anaelme K, BigerelIe M .Topography effects of pure titanium substrates on human osteoblast long-term adhesion[J]Acta Biomater.2005,1(2):211-222
[19]Bagno A, Genovese M,LucilnliA, Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coatad Ti and Ti6A14V disks[J].Biomaterials,2004,25(12):2437-2445
[20]Anita Kapanen,Anatoli Danilov,Petri Lenenkari,et al. Effect of metal alloy surface stresses on the viability of ROS-17/2.8 osteoblastiic cell.[J] Biomaterials,2002,23(17):3733
[21]Lim JY, Dreiss AD, Zhou Z, et al. The regulatlon Of integrinmediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography[J]. Biomalerials.2007,28(10):1787—1797
[22]Lim JY,Hansen JC, Siedlecki CA. et al. Human foetal osteoblastic cell response to polymer—demixed nanotopographic interfaces[J].R Soc Interface,2005,2(2): 97-108
[23]Tomas CH, McFland CD, Jenkins ML, et al. The role of vitronectin in the attachment and spatial distribution of bonederived cells on materials with patterned surfaces chemistry [J].Biomed Mater Res.1997,37(1):81
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