Abstract: Objective: To determine the adhesion behavior of bone marrow mesenchymal stem cells (MSCs) on a titanium surface with a nanostructured strontium-doped hydroxyapatite (Sr-HA) coating. Methods: Sr-HA coatings were applied on roughened titanium surfaces using an electrochemical deposition method. Primary cultured rat MSCs were seeded onto Sr-HA-, HA-coated titanium, and roughened titanium surfaces, and they were then cultured for 1, 6, and 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the metabolic condition of the cells. Scanning electron microscopy (SEM) was used to observe the cell morphology. The cytoskeletal structure was analyzed using fluorescence actin staining to characterize cell adherence. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the gene expression levels of FAK (focal adhesion kinase), vinculin, integrin β1, and integrin β3 after culturing for 24, 48, and 72 h. Results: MSCs cultured on the Sr-HA surface showed better cell proliferation and viability. Improvement of cell adhesion and structural rearrangement of the cytoskeleton were observed on the Sr-HA surface. The gene expression of FAK, vinculin, integrin β1, and integrin β3 was also elevated on the Sr-HA surface. Conclusions: Cell viability, adhesion, cell morphology, and the cytoskeletal structure were all upregulated considerably by the titanium surface modified with a Sr-HA coating.

多孔纯钛表面掺锶羟基磷灰石涂层对骨髓间充质干细胞粘附的影响

[1]Abert, J., Bergmann, C., Fischer, H., 2014. Wet chemical synthesis of strontium-substituted hydroxyapatite and its influence on the mechanical and biological properties. Ceram. Int., 40(7):9195-9203.

[2]Aina, V., Bergandi, L., Lusvardi, G., et al., 2013. Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells. Mater. Sci. Eng. C Mater. Biol. Appl., 33(3):1132-1142.

[3]Anselme, K., 2000. Osteoblast adhesion on biomaterials. Biomaterials, 21(7):667-681.

[4]Brammer, K.S., Choi, C., Frandsen, C.J., et al., 2011. Hydrophobic nanopillars initiate mesenchymal stem cell aggregation and osteo-differentiation. Acta Biomater., 7(2):683-690.

[5]Branemark, P.I., 1983. Osseointegration and its experimental background. J. Prosth. Dent., 50(3):399-410.

[6]Braux, J., Velard, F., Guillaume, C., et al., 2011. A new insight into the dissociating effect of strontium on bone resorption and formation. Acta Biomater., 7(6):2593-2603.

[7]Cox, S.C., Jamshidi, P., Grover, L.M., et al., 2014. Preparation and characterisation of nanophase Sr, Mg, and Zn substituted hydroxyapatite by aqueous precipitation. Mater. Sci. Eng. C Mater. Biol. Appl., 35:106-114.

[8]Dalby, M.J., Gadegaard, N., Oreffo, R.O., 2014. Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Nat. Mater., 13(6):558-569.

[9]Fage, S.W., Muris, J., Jakobsen, S.S., et al., 2016. Titanium: a review on exposure, release, penetration, allergy, epidemiology, and clinical reactivity. Contact Dermat., 74(6):323-345.

[10]Fu, D.L., Jiang, Q.H., He, F.M., et al., 2012. Fluorescence microscopic analysis of bone osseointegration of strontium-substituted hydroxyapatite implants. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 13(5):364-371.

[11]Gao, J., Wang, M., Shi, C., et al., 2016. Synthesis of trace element Si and Sr codoping hydroxyapatite with non-cytotoxicity and enhanced cell proliferation and differentiation. Biol. Trace Element Res., 174(1):208-217.

[12]Guo, D.G., Hao, Y.Z., Li, H.Y., et al., 2013. Influences of Sr dose on the crystal structure parameters and Sr distributions of Sr-incorporated hydroxyapatite. J. Biomed. Mater. Res. Part B Appl. Biomater., 101(7):1275-1283.

[13]Hurtel-Lemaire, A.S., Mentaverri, R., Caudrillier, A., et al., 2009. The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways. J. Biol. Chem., 284(1):575-584.

[14]Jiang, Q.H., Gong, X., Wang, X.X., et al., 2015. Osteogenesis of rat mesenchymal stem cells and osteoblastic cells on strontium-doped nanohydroxyapatite-coated titanium surfaces. Int. J. Oral Maxillofac. Implants, 30(2):461-471.

[15]Jiao, M.J., Wang, X.X., 2009. Electrolytic deposition of magnesium-substituted hydroxyapatite crystals on titanium substrate. Mater. Lett., 63(27):2286-2289.

[16]Kaneko, K., Ito, M., Naoe, Y., et al., 2014. Integrin αv in the mechanical response of osteoblast lineage cells. Biochem. Biophys. Res. Commun., 447(2):352-357.

[17]Kaygili, O., Keser, S., Kom, M., et al., 2015. Strontium substituted hydroxyapatites: synthesis and determination of their structural properties, in vitro and in vivo performance. Mater. Sci. Eng. C Mater. Biol. Appl., 55: 538-546.

[18]Khang, D., Choi, J., Im, Y.M., et al., 2012. Role of subnano-, nano- and submicron-surface features on osteoblast differentiation of bone marrow mesenchymal stem cells. Biomaterials, 33(26):5997-6007.

[19]Krause, A., Cowles, E.A., Gronowicz, G., 2000. Integrin-mediated signaling in osteoblasts on titanium implant materials. J. Biomed. Mater. Res., 52(4):738-747.

[20]Krishna, O.D., Jha, A.K., Jia, X., et al., 2011. Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide. Biomaterials, 32(27):6412-6424.

[21]Kumar, A., Nune, K.C., Basu, B., et al., 2016. Mechanistic contribution of electroconductive hydroxyapatite-titanium disilicide composite on the alignment and proliferation of cells. J. Biomater. Appl., 30(10):1505-1516.

[22]Kuo, S.W., Lin, H.I., Ho, J.H., et al., 2012. Regulation of the fate of human mesenchymal stem cells by mechanical and stereo-topographical cues provided by silicon nanowires. Biomaterials, 33(20):5013-5022.

[23]Lee, J., Chu, B.H., Chen, K.H., et al., 2009. Randomly oriented, upright SiO₂ coated nanorods for reduced adhesion of mammalian cells. Biomaterials, 30(27):4488-4493.

[24]Lim, J.Y., Dreiss, A.D., Zhou, Z., et al., 2007. The regulation of integrin-mediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography. Biomaterials, 28(10):1787-1797.

[25]Lindahl, C., Pujari-Palmer, S., Hoess, A., et al., 2015. The influence of Sr content in calcium phosphate coatings. Mater. Sci. Eng. C Mater. Biol. Appl., 53:322-330.

[26]Liu, J., Wang, X.D., Jin, Q.M., et al., 2012. The stimulation of adipose-derived stem cell differentiation and mineralization by ordered rod-like fluorapatite coatings. Biomaterials, 33(20):5036-5046.

[27]Liu, L., Song, L.N., Yang, G.L., et al., 2011. Fabrication, characterization, and biological assessment of multilayer DNA coatings on sandblasted-dual acid etched titanium surface. J. Biomed. Mater. Res. Part A, 97A(3):300-310.

[28]Loya, M.C., Brammer, K.S., Choi, C., et al., 2010. Plasma-induced nanopillars on bare metal coronary stent surface for enhanced endothelialization. Acta Biomater., 6(12):4589-4595.

[29]Madamanchi, A., Santoro, S.A., Zutter, M.M., 2014. α2β1 Integrin. Adv. Exp. Med. Biol., 819:41-60.

[30]Matschegewski, C., Staehlke, S., Loeffler, R., et al., 2010. Cell architecture-cell function dependencies on titanium arrays with regular geometry. Biomaterials, 31(22):5729-5740.

[31]Ni, G.X., Yao, Z.P., Huang, G.T., et al., 2011. The effect of strontium incorporation in hydroxyapatite on osteoblasts in vitro. J. Mater. Sci. Mater. Med., 22(4):961-967.

[32]Omar, S., Repp, F., Desimone, P.M., et al., 2015. Sol–gel hybrid coatings with strontium-doped 45S5 glass particles for enhancing the performance of stainless steel implants: electrochemical, bioactive and in vivo response. J. Non-Crystal. Solids, 425:1-10.

[33]Park, J.H., Wasilewski, C.E., Almodovar, N., et al., 2012. The responses to surface wettability gradients induced by chitosan nanofilms on microtextured titanium mediated by specific integrin receptors. Biomaterials, 33(30):7386-7393.

[34]Park, J.K., Kim, Y.J., Yeom, J., et al., 2010. The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration. Adv. Mater., 22(43):4857-4861.

[35]Peng, S., Zhou, G., Luk, K.D., et al., 2009. Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway. Cell. Physiol. Biochem., 23(1-3):165-174.

[36]Seo, C.H., Jeong, H., Furukawa, K.S., et al., 2013. The switching of focal adhesion maturation sites and actin filament activation for MSCs by topography of well-defined micropatterned surfaces. Biomaterials, 34(7):1764-1771.

[37]Shi, J., Dong, L.L., He, F., et al., 2013. Osteoblast responses to thin nanohydroxyapatite coated on roughened titanium surfaces deposited by an electrochemical process. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 116(5):e311-e316.

[38]Sirin, H.T., Vargel, I., Kutsal, T., et al., 2015. Ti implants with nanostructured and HA-coated surfaces for improved osseointegration. Artif. Cells Nanomed. Biotechnol., 44(3):1023-1030.

[39]Sisti, K.E., de Andres, M.C., Johnston, D., et al., 2015. Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification. Biochem. Biophys. Res. Commun., 473(3):719-725.

[40]Takaoka, S., Yamaguchi, T., Yano, S., et al., 2010. The calcium-sensing receptor (CAR) is involved in strontium ranelate-induced osteoblast differentiation and mineralization. Horm. Metab. Res., 42(9):627-631.

[41]Tao, Z.S., Bai, B.L., He, X.W., et al., 2016a. A comparative study of strontium-substituted hydroxyapatite coating on implant’s osseointegration for osteopenic rats. Med. Biol. Eng. Comput., 54(12):1959-1968.

[42]Tao, Z.S., Zhou, W.S., He, X.W., et al., 2016b. A comparative study of zinc, magnesium, strontium-incorporated hydroxyapatite-coated titanium implants for osseointegration of osteopenic rats. Mater. Sci. Eng. C Mater. Biol. Appl., 62:226-232.

[43]Uccelli, A., Moretta, L., Pistoia, V., 2008. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol., 8(9):726-736.

[44]Wang, W., Zhao, L., Wu, K., et al., 2013. The role of integrin-linked kinase/β-catenin pathway in the enhanced MG63 differentiation by micro/nano-textured topography. Biomaterials, 34(3):631-640.

[45]Wong, K.L., Wong, C.T., Liu, W.C., et al., 2009. Mechanical properties and in vitro response of strontium-containing hydroxyapatite/polyetheretherketone composites. Biomaterials, 30(23-24):3810-3817.

[46]Xu, J., Yang, Y., Wan, R., et al., 2014. Hydrothermal preparation and characterization of ultralong strontium-substituted hydroxyapatite whiskers using acetamide as homogeneous precipitation reagent. Sci. World J., 2014: 863137.

[47]Yang, F., Yang, D., Tu, J., et al., 2011. Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/Catenin signaling. Stem Cells, 29(6):981-991.

[48]Yang, H.W., Lin, M.H., Shang, G.W., et al., 2015. Osteogenesis of bone marrow mesenchymal stem cells on strontium-substituted nano-hydroxyapatite coated roughened titanium surfaces. Int. J. Clin. Exp. Med., 8(1):257-264.

[49]Yim, E.K., Darling, E.M., Kulangara, K., et al., 2010. Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells. Biomaterials, 31(6):1299-1306.

[50]Zhao, L., Liu, L., Wu, Z., et al., 2012. Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation. Biomaterials, 33(9):2629-2641.

[51]Zhao, L., Wang, H., Huo, K., et al., 2013. The osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates. Biomaterials, 34(1):19-29.

[52]Zhao, S.F., Dong, W.J., Jiang, Q.H., et al., 2013. Effects of zinc-substituted nano-hydroxyapatite coatings on bone integration with implant surfaces. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 14(6):518-525.

[53]Zhao, S.F., Shi, J., He, F.M., et al., 2014. Design and in vitro evaluation of simvastatin-hydroxyapatite coatings by an electrochemical process on titanium surfaces. J. Biomed. Nanotechnol., 10(7):1313-1319.

[54]Zhou, J., Li, B., Lu, S., et al., 2013. Regulation of osteoblast proliferation and differentiation by interrod spacing of Sr-HA nanorods on microporous titania coatings. ACS Appl. Mater. Interf., 5(11):5358-5365.

[55]Zhou, J., Han, Y., Lu, S., 2014. Direct role of interrod spacing in mediating cell adhesion on Sr-HA nanorod-patterned coatings. Int. J. Nanomed., 9:1243-1260.

[56]Zhuang, X.M., Zhou, B., Ouyang, J.L., et al., 2014. Enhanced MC3T3-E1 preosteoblast response and bone formation on the addition of nano-needle and nano-porous features to microtopographical titanium surfaces. Biomed. Mater., 9(4):045001.