Chitosan coatings for titanium implants: applications and strengths

doi:10.3969/j.issn.2095-4344.0975

Abstract

Abstract:

BACKGROUND: Titanium and its alloys as implants are extensively used in orthodontics, implant dentistry and oral and maxillofacial surgery due to their excellent physicochemical properties and biocompatibility. Titanium based implants cannot integrate with surrounding bone tissues due to their inherent surface bio-inertness. Meanwhile, bacteria tend to congregate on the implant surfaces. These two factors are mainly responsible for implant failure. To prevent such infections and increase low bone-implant contact, surface modification is feasible. Chitosan has excellent biocompatibility, antimicrobial properties and film-forming properties. It can be used as a coating to improve the biological properties of titanium.

OBJECTIVE: To summarize the research progress of chitosan modified titanium, and to discuss the effects and advantages of chitosan modification for titanium based implants.

METHODS: PubMed was retrieved for relevant articles published from January 2010 to May 2018. The key words were “chitosan; titanium; osseointegration; antibacterial" in English. After removal of repetitive and unrelated articles, 53 eligible articles were finally included in accordance with the inclusion criteria.

RESULTS AND CONCLUSION: Chitosan itself has certain antimicrobial and osteogenic properties. It can be combined with a variety of organic and inorganic materials due to its film forming ability and metal binding capacity to further improve its antimicrobial and osteogenic properties. In addition, chitosan has excellent metal binding capacity, which can improve the antimicrobial ability of metal and reduce its toxicity. Chitosan also can be used as drug carrier and control drug release by reinforcing the coating stability, and thus reduce bacterial adhesion and proliferation, enhance bone binding capability, and reduce the incidence of implant failure. The combination of chitosan and biological growth factors can promote angiogenesis and bone formation abilities of the coating. And multi-layer chitosan can control growth factor release and maintain regional growth factor concentration. If the chitosan modified titanium is applied to clinics, the physical properties of the coating, such as molecular weight, composition, hydrophilicity and porosity, need to be systematically studied, and animal trials and clinical trials are needed as well.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Chitosan, Titanium, Tissue Engineering

CLC Number:

R318

Fan Huayang, Yin Yijia, Wang Zheng, Pei Xuan, Han Xianglong. Chitosan coatings for titanium implants: applications and strengths[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(34): 5553-5558.

Figures/Tables 1

2.1 壳聚糖壳聚糖可以通过增加微生物细胞壁及细胞膜通透性、与微生物DNA结合等机制抑制微生物，使钛基涂层具有一定的抗菌性[15]。壳聚糖也具有诱导成骨的能力。Ghimire等[16]通过盐酸多巴胺修饰钛基表面，再将壳聚糖与多巴胺共价接枝，制备的涂层可以阻止细菌对成骨样细胞的入侵，显著增加细菌对抗生素的易感性，减少植入物相关感染和骨整合不良的发生。但壳聚糖的成骨性能尚存在争议[17]。Gilabert-Chirivella等[18]在钛表面上蚀刻了平行的微凹槽，使壳聚糖在16 μm深的凹槽底部以薄涂层的形式沉积，但未观察到MC3T3-E1前成骨细胞黏附在壳聚糖涂层上，证实其不具有成骨功能。 2.2 壳聚糖与其他载体物质壳聚糖还可以与其他载体物质结合构建复合涂层，如羟基磷灰石、二氧化钛、聚吡咯、磷酸钙等，取长补短，壳聚糖在这些涂层中发挥一定抗菌能力或作为载体起到稳定涂层、加载释放药物或降低另一载体的细胞毒性等的作用。 2.2.1 羟基磷灰石羟基磷灰石与人体骨组织的无机组成成分相似。壳聚糖可与羟基磷灰石涂层结合，发挥抗菌效果。Lei等[19]通过液体前驱体等离子喷涂在钛基表面制备羟基磷灰石涂层，再将水溶性壳聚糖加载到羟基磷灰石涂层中，负载壳聚糖的液体前驱体等离子喷涂多孔涂层表现出优良的抗菌效果。Chen等[20]通过逐层自装技术在钛种植体表面制备了壳聚糖-凝胶-羟基磷灰石复合涂层，该涂层能促进局部组织的修复及防止细菌黏附增殖，及促进骨生成及血管生成，凝胶可进一步提高壳聚糖生物学功能。 2.2.2 二氧化钛二氧化钛具有良好的生物效应和耐蚀性，二氧化钛纳米粒子膜可防止细菌黏附及增殖，并能作为药物输送和提高血凝块黏附的生长支撑平台，并可加速伤口愈合，壳聚糖-二氧化钛纳米颗粒-羟基磷灰石复合膜中壳聚糖起到稳定涂层，增强涂层耐腐蚀性和吸附作用，增加钛合金表面对二氧化钛及羟基磷灰石纳米颗粒吸附，并赋予涂层以良好抗菌性[21-22]。 2.2.3 聚吡咯聚吡咯具有良好生物相容性且成本低廉，可以加载及释放药物。Rikhari等[23]通过电化学沉积法技术在钛表面制备了聚吡咯-壳聚糖涂层，壳聚糖赋予复合涂层优良的黏附性及稳定性。 2.2.4 磷酸钙 Park等[24]和Zhou等[25]均制备了磷酸钙-壳聚糖涂层，壳聚糖使该复合涂层相比单纯磷酸钙涂层具更好生物活性并更有利于药物加载和释放。 2.2.5 多壁碳纳米管是由单层或多层石墨片卷曲而成的新型高强度碳纤维材料，具有优良的物理、化学性质，广泛的抗菌活性和促进成骨细胞分化作用。Zhu等[26]通过原子层沉积将壳聚糖沉积于多壁碳纳米管表面，该方法能精确控制壳聚糖膜沉积厚度，再通过EPD法将碳纳米管-壳聚糖沉积于碱热处理钛表面，证实壳聚糖可提高多壁碳纳米管的细胞相容性、抗菌效能并削弱其细胞毒性。 2.3 壳聚糖衍生物为解决壳聚糖抗菌性不足的问题，许多学者研究壳聚糖衍生物，如羟丙基三甲基氯化铵壳聚糖和羧甲基壳聚糖。Xu等[27]通过硅烷锚定将取代度为18%的羟丙基三甲基氯化铵壳聚糖连接在钛基表面，其涂层较壳聚糖具更强的溶解性及抗菌性，尤其是对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌和表皮葡萄球菌效果更佳。 2.4 壳聚糖与金属壳聚糖具有较高的金属结合效力，如银，镓等，尤其是与银，并起到缓释金属离子及降低金属离子毒性作用及一定抗菌性。 2.4.1 银纳米银粒子指直径在1-100 nm之间的银粒子[28]。纳米银粒子释放的Ag+和产生活性氧使其具有杀菌活性，还能防止多种细菌定植及生物膜的生长，并提高碱性磷酸酶活性，上调成骨向分化基因刺激人骨肉瘤细胞成骨向分化[29-31]。壳聚糖具有利于银的加载和缓释的能力，还可以降低银的毒性，可能是壳聚糖的添加导致涂层中纳米银粒子的均匀分布，阻止了大量Ag簇的形成，从而降低了Ag+的毒性。 Singh等[32]将纳米银粒子与壳聚糖混合溶解后可通过电泳沉积法沉积在钛片表面。壳聚糖可增强复合物与钛的结合强度，并能缓释Ag+。Li等[33]采取逐层自组装技术在钛基表面制备了巯基壳聚糖凝胶-银涂层，壳聚糖作为银离子的纳米容器，与银离子发生化学性的连接，提高银的粘接量。Mishra等[34]用硅烷锚定在钛基表面形成壳聚糖-聚乙烯醇-银涂层，壳聚糖作为缓释载体，使Ag+长期持续释放。Travan等[35]利用双酚A-甲基丙烯酸缩水甘油酯及三乙二醇三甲基丙烯酸酯，通过物理吸附的方式将银-壳聚糖混合液沉积在钛基表面，体内体外试验证实该涂层能稳定释放银及抗生物膜形成。Xie等[36]通过电化学沉积法方法制备了银-壳聚糖-羟基磷灰石涂层，后将骨形态发生蛋白2通过静电吸附于该涂层，通过Alamar Blue试验证实壳聚糖有效降低银离子毒性。 2.4.2 镓镓与Fe3+化学结构相似，故可以干扰各种细菌的铁代谢，增强乳铁蛋白的抗微生物作用，具有一定抑菌能力，并能通过降低破骨细胞活性来阻止骨吸收[37]。Bonifacio等[38]将镓置入壳聚糖与聚丙烯酸的双层膜中形成镓改性的壳聚糖-聚丙烯酸涂层，通过电化学沉积法方法将涂层沉积于钛基表面，聚丙烯酸稳定无毒且有促进涂层黏附作用，壳聚糖发挥抗菌性且作为加载金属抗菌剂的载体。 2.5 壳聚糖与抗生素口腔种植体周围炎，是发生在口腔种植体周围软组织的可逆炎症。其根本病因是种植体上细菌微生物的定植。庆大霉素、米诺环素、四环素、万古霉素通常用于治疗口腔常见感染。种植体手术后也需要患者配合使用抗生素以预防术后感染。壳聚糖作为多种抗生素载体，可有效抑制种植体周围相关细菌，确保局部药物浓度，保证疗效，减少全身抗生素应用导致的毒副作用，提高种植手术成功率。 2.5.1 氨基糖苷类抗生素硫酸庆大霉素为氨基糖苷类抗生素[39]。Kumeria等[40]通过电化学沉积法技术将壳聚糖沉积在二氧化钛纳米管表面，该涂层可装载和释放硫酸庆大霉素，壳聚糖可延长药物分子的释放时间至数周，硫酸庆大霉素与本身具有一定抗菌性能的壳聚糖一起发挥功效，阻止细菌扩散，并促进成骨细胞增殖和黏附。氨基糖苷类的妥布霉素可治疗革兰阴性菌引起的感染。Zhou等[41]通过逐层自组装技术在钛表面制备了妥布霉素-壳聚糖-肝素复合涂层，该涂层能够有效抑制细菌的黏附及生物膜的形成，并在酸性条件下呈现出长期抗菌的特点，其良好的成骨性能也得到了验证。 2.5.2 糖肽类抗生素糖肽类万古霉素可通过干扰细菌细胞中磷脂和多肽的生成，从而抑制细菌的生长和繁殖来杀死细菌。Ordikhani等[42]通过电泳沉积法将万古霉素-壳聚糖复合物沉积在钛合金上，壳聚糖发挥稳定涂层及缓释功能药物缓释总持续时间达30 d以上[43]。Ordikhani等[44]的另一项研究中运用EPD方法构建壳聚糖-生物活性玻璃-万古霉素涂层，生物活性玻璃可以稳定涂层结构，并延缓壳聚糖的降解从而延续药物释放功能。Kong等[45]通过电泳沉积法方法制备了壳聚糖纳米球矿化胶原蛋白涂层，壳聚糖纳米球作为药物载体加载盐酸万古霉素，具有良好加载释放作用。 2.5.3 四环素类抗生素四环素是一种广谱抗生素，对革兰阳性菌、革兰阴性菌及螺旋体均有抑制作用，为口腔常用药。Cai等[46]通过电泳沉积法方法在钛基种植体上制备了四环素-壳聚糖明胶纳米球涂层。米诺环素由天然四环素类抗生素半合成制得，是一种广谱四环素类抗生素[47]。Lü等[48]通过LBL方法在钛表面制备藻酸盐-壳聚糖-米诺环素复合涂层。在上述涂层中，壳聚糖作为复合涂层载体，加载并缓释四环素或米诺环素，提高涂层的抗菌能力、生物降解性和生物相容性。 2.6 壳聚糖与生长因子种植体稳定性的关键是其表面的血管及骨形成。壳聚糖帮助多种生长因子吸附于材料表面，提升其成骨及成血管能力。 2.6.1 骨形态发生蛋白骨形态发生蛋白能够诱导动物或人体间充质细胞分化为骨、软骨、韧带、肌腱和神经组织。Chung等[49]在钛棒表面制备了壳聚糖/γ-聚乙醇酸聚电解质多层涂层，壳聚糖控制释放骨形态发生蛋白2，并可促进间充质干细胞成骨向分化。Jung等[50]开发出1 μm厚，高度整齐的多孔骨形态发生蛋白2/二氧化硅/壳聚糖杂化物涂覆的钛支架。该支架能有效释放骨形态发生蛋白2长达26 d，在4周内释放的骨形态发生蛋白2的累积量是大约4 g，且有更好的骨组织再生能力。Poth等[51]将壳聚糖纳米颗粒涂覆在钛上并用于释放骨形态发生蛋白2使骨组织再生。Zheng等[52]将壳聚糖衍生物羧甲基壳聚糖联合骨形态发生蛋白2，通过硅烷锚定将其锚定在钛基表面，羧甲基壳聚糖同样具有稳定涂层的能力。 2.6.2 血管生长内皮因子血管内皮生长因子是一种刺激血管生成的信号蛋白。Hu等[53]共价接枝多巴胺于钛基表面，再接枝羧甲基壳聚糖使钛基的表面官能化，最后将血管内皮生长因子缀合至涂层表面。羧甲基壳聚糖增强了涂层中血管内皮生长因子的促成骨和血管生成功能，同时有效减少了细菌黏附。"

References

[1] Kulkarni M, Mazare A, Schmuki P, et al. Biomaterial surface modification of titanium and titanium alloys for medical applicationsnanomedicine. Int J Nanomedicine. 2017;12: 8711-8723.

[2] Neoh KG, Hu X, Zheng D, et al. Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces. Biomaterials. 2012;33(10):2813-2822.

[3] Liu X, Li M, Zhu Y, et al. The modulation of stem cell behaviors by functionalized nanoceramic coatings on Ti-based implants. Bioact Mater. 2016;1(1):65-76.

[4] Tobin EJ. Recent coating developments for combination devices in orthopedic and dental applications: a literature review. Adv Drug Deliv Rev. 2017;112:88-100.

[5] Liu Z, Zhu Y, Liu X, et al. Construction of poly (vinyl alcohol)/poly (lactide−glycolide acid)/vancomycin nanoparticles on titanium for enhancing the surface self-antibacterial activity and cytocompatibility. Colloids Surf B Biointerfaces.2016; 151:165-177.

[6] Arciola C R, Campoccia D, Speziale P, et al. Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials. Biomaterials. 2012;33(26):5967-5982.

[7] Wu S, Liu X, Yeung KWK, et al. Biomimetic porous scaffolds for bone tissue engineering. Mater Sci Eng R Rep. 2014;80(1):1-36.

[8] Ding F, Nie Z, Deng H, et al. Antibacterial hydrogel coating by electrophoretic co-deposition of chitosan/alkynyl chitosan. Carbohydr Polym. 2013;98(2):1547-1552.

[9] Venkatesan J, Anil S, Kim SK, et al. Chitosan as a vehicle for growth factor delivery: Various preparations and their applications in bone tissue regeneration. Int J Biol Macromol. 2017;104(Pt B):1383-1397.

[10] Husain S, Alsamadani KH, Najeeb S, et al. Chitosan Biomaterials for Current and Potential Dental Applications. Materials (Basel). 2017;10(6). pii: E602.

[11] Raphel J, Holodniy M, Goodman SB, et al. Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants. Biomaterials. 2016;84:301-314.

[12] Seuss S, Boccaccini AR. Electrophoretic deposition of biological macromolecules, drugs, and cells. Biomacromolecules. 2013;14(10):3355.

[13] Low CTJ, Wills RGA, Walsh FC. Electrodeposition of composite coatings containing nanoparticles in a metal deposit. Surf Coat Technol. 2006;201(1):371-383.

[14] Zhang B, Pan Y, Chen H, et al. Stabilization of starch-based microgel-lysozyme complexes using a layer-by-layer assembly technique. Food Chem. 2017;214:213-217.

[15] Pelgrift RY, Friedman AJ. Nanotechnology as a therapeutic tool to combat microbial resistance. Adv Drug Deliv Rev. 2013;65(13-14):1803-1815.

[16] Ghimire N, Foss BL, Sun Y, et al. Interactions among osteoblastic cells, Staphylococcus aureus and chitosan-immobilized titanium implants in a post-operative co-culture system: An in vitro study. J Biomed Mater Res A. 2016;104(3):586-594.

[17] Bumgardner JD, Wiser R, Gerard PD, et al. Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants. J Biomater Sci Polym Ed. 2003; 14(5):423-438.

[18] Gilabert-Chirivella E, Pérez-Feito R, Ribeiro C, et al. Chitosan patterning on titanium implants. Prog Org Coat. 2017;111: 23-28.

[19] Lei S, Xiao Y F, Lu G, et al. The effect of antibacterial ingredients and coating microstructure on the antibacterial properties of plasma sprayed hydroxyapatite coatings. Surf Coat Technol. 2012;206(11-12):2986-2990.

[20] Chen W, Xu K, Tao B, et al. Multilayered coating of titanium implants promotes coupled osteogenesis and angiogenesis in vitro and in vivo. Acta Biomater. 2018;74:489-504..

[21] Grassian VH, O'Shaughnessy PT, Adamcakova-Dodd A, et al. Inhalation exposure study of titanium dioxide nanoparticles with a primary particle size of 2 to 5 nm. Environ Health Perspect. 2007;115(3):397-402.

[22] Fekry AM. Electrochemical behavior of a novel nano-composite coat on Ti alloy in phosphate buffer solution for biomedical applications. RSC Adv. 2016;6(24):20276-20285.

[23] Rikhari B, Mani SP, Rajendran N. Electrochemical behavior of Polypyrrole/Chitosan composite coating on Ti metal for biomedical applications. Carbohydr Polym. 2018;189:126-137.

[24] Park KH, Kim SJ, Jeong YH, et al. Fabrication and biological properties of calcium phosphate/chitosan composite coating on titanium in modified SBF. Mater Sci Eng C Mater Biol Appl. 2018;90:113-118.

[25] Zhou J, Cai X, Cheng K, et al. Release behaviors of drug loaded chitosan/calcium phosphate coatings on titanium. Thin Solid Films. 2011;519(15):4658-4662.

[26] Zhu Y, Liu X, Yeung KWK, et al. Biofunctionalization of carbon nanotubes/chitosan hybrids on Ti implants by atom layer deposited ZnO nanostructures. Appl Surf Sci. 2016; 400: 14-23.

[27] Xu X, Wang L, Guo S, et al. Surface chemical study on the covalent attachment of hydroxypropyltrimethyl ammonium chloride chitosan to titanium surfaces. Appl Surf Sci. 2011; 257(24):10520-10528.

[28] Patra JK, Baek KH. Antibacterial activity and synergistic antibacterial potential of biosynthesized silver nanoparticles against foodborne pathogenic bacteria along with its anticandidal and antioxidant effects. Front Microbiol. 2017; 8(54):167.

[29] Wu M, Ma B, Pan T, et al. Silver-nanoparticle-colored cotton fabrics with tunable colors and durable antibacterial and self-healing superhydrophobic properties. Adv Funct Mater. 2016;26(4):569-576.

[30] Peetsch A, Greulich C, Braun D, et al. Silver-doped calcium phosphate nanoparticles: synthesis, characterization, and toxic effects toward mammalian and prokaryotic cells. Colloids Surf B Biointerfaces. 2013;102(2):724-729.

[31] Zhou W, Jia Z, Xiong P, et al. Bioinspired and biomimetic AgNPs/gentamicin-embedded silk fibroin coatings for robust antibacterial and osteogenetic applications. ACS Appl Mater Interfaces. 2017;9(31):166-173.

[32] Singh RK, Awasthi S, Dhayalan A, et al. Deposition, structure, physical and invitro characteristics of Ag-doped β-Ca3(PO4)2/chitosan hybrid composite coatings on titanium metal. Mater Sci Eng C Mater Biol Appl.2016;62:692-701.

[33] Li W, Xu D, Hu Y, et al. Surface modification of titanium substrates with silver nanoparticles embedded sulfhydrylated chitosan/gelatin polyelectrolyte multilayer films for antibacterial application.J Mater Sci Mater Med. 2014;25(6): 1435.

[34] Mishra SK, Ferreira JM, Kannan S. Mechanically stable antimicrobial chitosan-PVA-silver nanocomposite coatings deposited on titanium implants.Carbohydr Polym. 2015;121:37-48.

[35] Marsich E, Travan A, Donati I, et al. Biological responses of silver-coated thermosets: An in vitro and in vivo study. Acta Biomater. 2013;9(2):5088-5099.

[36] Xie CM, Lu X, Wang KF, et al. Silver nanoparticles and growth factors incorporated hydroxyapatite coatings on metallic implant surfaces for enhancement of osteoinductivity and antibacterial properties. Acs Appl Mater Interfaces. 2014; 6(11):8580-8589.

[37] Kelson AB, Carnevali M, Truongle V. Gallium-based anti-infectives: targeting microbial iron-uptake mechanisms. Curr Opin Pharmacol. 2013;13(5):707-716.

[38] Bonifacio MA, Cometa S, Dicarlo M, et al. Gallium-modified chitosan/poly(acrylic acid) bilayer coatings for improved titanium implant performances. Carbohydr Polym. 2017; 166:348-357.

[39] Ferrer M, Méndezgarcía C, Rojo D, et al. Antibiotic use and microbiome function. Biochem Pharmacol. 2016;134:114.

[40] Kumeria T, Mon H, Aw MS, et al. Advanced biopolymer-coated drug-releasing titania nanotubes (TNTs) implants with simultaneously enhanced osteoblast adhesion and antibacterial properties. Colloids Surf B Biointerfaces. 2015;130:255-263.

[41] Zhou W, Jia Z, Xiong P, et al. Novel pH-responsive tobramycin-embedded micelles in nanostructured multilayer-coatings of chitosan/heparin with efficient and sustained antibacterial properties. Mater Sci Eng C Mater Biol Appl. 2018;90:693-705.

[42] Ordikhani F, Tamjid E, Simchi A. Characterization and antibacterial performance of electrodeposited chitosan-vancomycin composite coatings for prevention of implant-associated infections. Mater Sci Eng C Mater Biol Appl. 2014;41(41):240-248.

[43] Yang CC, Lin CC, Liao JW, et al. Vancomycin-chitosan composite deposited on post porous hydroxyapatite coated Ti6Al4V implant for drug controlled release. Mater Sci Eng C Mater Biol Appl. 2013;33(4):2203-2212.

[44] Ordikhani F, Simchi A. Long-term antibiotic delivery by chitosan-based composite coatings with bone regenerative potential. Appl Surf Sci. 2014;317:56-66.

[45] Kong Z, Yu M, Cheng K, et al. Incorporation of chitosan nanospheres into thin mineralized collagen coatings for improving the antibacterial effect. Colloids Surf B Biointerfaces. 2013;111(111C):536-541.

[46] Cai X, Ma K, Zhou Y, et al. Surface functionalization of titanium with tetracycline loaded chitosan-gelatin nanosphere coatings via EPD: fabrication, characterization and mechanism. RSC Adv. 2016;6(9):7674-7682.

[47] Baym M, Stone L K, Kishony R. Multidrug evolutionary strategies to reverse antibiotic resistance. Science. 2016; 351(6268):aad3292.

[48] Lü H, Chen Z, Yang X, et al. Layer-by-layer self-assembly of minocycline-loaded chitosan/alginate multilayer on titanium substrates to inhibit biofilm formation. J Dent. 2014;42(11): 1464-1472.

[49] Chung RJ, Ou KL, Tseng WK, et al. Controlled release of BMP-2 by chitosan/γ-PGA polyelectrolyte multilayers coating on titanium alloy promotes osteogenic differentiation in rat bone-marrow mesenchymal stem cells. Surf Coat Technol. 2016;303:283-288.

[50] Jung HD, Yook SW, Han CM, et al. Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone regeneration. J Biomed Mater Res B Appl Biomater. 2014;102(5):913-921.

[51] Poth N, Seiffart V, Gross G, et al. Biodegradable chitosan nanoparticle coatings on titanium for the delivery of BMP-2. Biomolecules. 2015;5(1):3-19.

[52] Zheng D, Neoh K G, Shi Z, et al. Assessment of stability of surface anchors for antibacterial coatings and immobilized growth factors on titanium. J Colloid Interface Sci. 2013; 406(18):238-246.

[53] Hu X, Neoh KG, Shi Z, et al. An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion. Biomaterials. 2010; 31(34): 8854-8863.