mussel inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications

HOSTED BY Available online at www.sciencedirect.com Biosurface and Biotribology ] (]]]]) ]]]–]]] www.elsevier.com/locate/bsbt Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications Zhenming Wanga,1, Pengfei Lia,1, Yanan Jianga, Zhanrong Jiaa, Pengfei Tanga, Xiong Lua,b,n, Fuzen Renc, Kefeng Wangb, Huiping Yuand a Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China b National Engineering Research Center for Biomaterials, Genome Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, China c Department of Materials Science and Engineering, South University of Science and Technology, Shenzhen, Guangdong 518055, China d College of Physical Science and Technology, Sichuan University, Chengdu, Sichuan 610064, China Received September 2016; received in revised form 13 January 2017; accepted 25 January 2017 Abstract Producing hierarchical nanostructured coatings with a biomimetic composition is an effective surface modification strategy to improve the bioactivity of biomaterials In this study, mussel-inspired polydopamine nanoparticles (PDA-NPs) and hydroxyapatite (HA) nanorods were used to modify Ti surfaces Firstly, the PDA-NPs were prepared via oxidative self-polymerization of dopamine Secondly, the HA nanorods were decorated with a PDA nanolayer in order to improve the adhesion of the HA nanorods Thirdly, the PDA-NPs and PDA-decorated HA nanorods were alternately assembled to form a porous and hierarchical micro/nanostructured {PDA/HA} composite coating on the Ti surfaces Finally, Bone morphogenetic protein-2 (BMP-2) was immobilized on the {PDA/HA} composite coating using the functional groups of PDA The BMP-2-loaded {PDA/HA} composite coating exhibited excellent biocompatibility and promoted the adhesion, proliferation, and differentiation of bone marrow stromal cells The animal implantation tests indicated that the BMP-2-loaded {PDA/HA} composite coating promoted the formation of new bone tissue & 2017 Southwest Jiaotong University Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Polydopamine nanoparticle; Hydroxyapatite; BMP; Self-assembly; Hierarchical structure; Biocompatibility Introduction Nanostructured coatings with a biomimetic composition are an effective surface modification method to improve the bioactivity of biomaterials Previous studies indicate that substrate-decorated polydopamine (PDA) coatings can significantly improve cell affinity and promote cell behavior compared with bare substrates [1,2] Most of the reported PDA coatings are dense and consist of solid films that completely cover the substrates and not have n Corresponding author at: Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China Tel.: ỵ 86 28 87634023; fax: ỵ86 28 87601371 E-mail address: luxiong_2004@163.com (X Lu) These two authors contributed equally to this work Peer review under responsibility of Southwest Jiaotong University micro/nanostructures to facilitate cell adhesion and tissue ingrowth Recently, PDA nanoparticles (PDA-NPs) have been studied in the energy [3], environmental [4], and biomedical fields [5] Some studies have revealed that PDA-NPs-decorated substrates promote cell behavior and tissue ingrowth due to the micro/nanostructures and cell affinity of the PDA-NPs Wang et al [6] used PDA-NPs to decorate a β-tricalcium phosphate (TCP) scaffold, and the results demonstrated that the PDA-NPs provided multiple bioactive sites for the adsorption of proteins and peptides, while improving the adhesion of bone marrow stromal cells (BMSCs) on the TCP scaffold However, pristine PDAdecorated substrates lack the necessary osteoinductivity to be used for bone reparation Several studies indicate that hydroxyapatite (HA) is the main inorganic component of the bone matrix and has excellent biocompatibility [7,8] Nevertheless, compared with http://dx.doi.org/10.1016/j.bsbt.2017.01.001 2405-4518/& 2017 Southwest Jiaotong University Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] traditional HA, nanoscaled HA has a higher biocompatibility and bioactivity, and is similar to the inorganic components of natural bone tissue [9–11] Recently, Tsai et al [12] used a deposition method based on dopamine polymerization to prepare nanostructured PDA/HA coatings on Ti implants, and their results indicated that the PDA/HA coatings enhanced the proliferation and osteodifferentiation of human BMSCs compared with pristine PDA coatings Chien et al [13] prepared nanostructured PDA/HA coatings on Ti implants using the adhesive properties of PDA, and their results demonstrated that the incorporation of nanoscaled HA enhanced the osteoinductivity of the PDA coatings and promoted new bone formation In summary, the incorporation of nanoscaled HA on PDA coatings can improve their bioactivity and induce bone ingrowth In addition, previous studies also demonstrated that porous and hierarchical micro/nanostructured composite coatings have high bioactivity and cell affinity Wang et al [14] prepared porous micro/nanostructured PDA microcapsules/chitosan composite coatings on Ti surfaces using a layer-by-layer self-assembly technique; their results indicated that the porous micro/nanostructures of the composite coatings promoted both the adhesion and proliferation of BMSCs Wu et al [15] fabricated porous nanostructured poly(L-lactide)/HA honeycomb films using a self-assembly technique, and their results showed that the porous nanostructures of the honeycomb films improved the affinity of a MC3T3-E1 cell line Based on previous studies, the development of a nanoscaled selfassembly technique for preparing porous and hierarchical micro/nanostructured functional coatings is of great interest In this study, bioactive coatings with porous and hierarchical micro/nanostructures containing HA nanorods and PDA-NPs were self-assembled using the unique adhesive properties of PDA Firstly, the HA nanorods were synthesized using a chemical precipitation method To facilitate the self-assembly, the HA nanorods were decorated with a PDA nanolayer Secondly, the PDA-NPs were prepared via oxidative selfpolymerization of dopamine Thirdly, the PDA-NPs and PDAdecorated HA nanorods were alternately assembled on the Ti surface to form a {PDA/HA} composite coating Finally, BMP-2 was immobilized on the {PDA/HA} composite coating to improve the osteoinductivity of the Ti substrates The physicochemical properties of the composite coatings were analyzed, and the biological properties were evaluated using in vitro cell cultures and in vivo animal implantation The experimental procedure is illustrated in Fig Fig Schematic of the {PDA/HA} composite coating self-assembly and characterization of the biological properties on the Ti surface (a) Preparation of PDANPs; (b) preparation of PDA-decorated HA nanorods; (c) self-assembly of the PDA-NPs and PDA-decorated HA nanorods; (d) immobilization of BMP-2 on the {PDA/HA} composite coating; (e) cytocompatibility and osteoinductivity of the coating were investigated via in vitro cell culture and in vivo implantation, respectively Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] Material and methods 2.4 Characterization of the HA nanorods, PDA-decorated HA nanorods, PDA-NPs, and various coatings 2.1 Preparation of the PDA-decorated HA nanorods The HA nanorods were synthesized using a previously reported chemical precipitation method [16] Briefly, Ca (NO3)2 Á 4H2O and (NH4)2HPO4 were first dissolved in distilled water to prepare a 0.5 mol L À Ca(NO3)2 solution and a 0.3 mol L À (NH4)2HPO4 solution, respectively Then, the (NH4)2HPO4 solution (100 mL) was added dropwise to the Ca (NO3)2 solution (100 mL) at a rate of mL À under vigorous stirring Ammonium hydroxide was immediately added to adjust the pH of the mixed solution to 11 After vigorous stirring for 12 h, the suspension was centrifuged (8800 g, 15 min) three times The HA suspension was cooled at À 20 1C in a freezer for 24 h, and then subsequently freezedried at À 40 1C for 36 h using a freeze dryer The dopamine hydrochloride (DA, 0.05 g) was dissolved in a Tris–HCl solution (100 mL, pH ¼ 8.5) The HA nanorods (0.4 g) were then dispersed in the DA solution (100 mL) After vigorous stirring for 12 h in the dark, the PDA-decorated HA nanorods were obtained via centrifugation (8800 g, 15 min) and freezedrying ( À 40 1C, 36 h) 2.2 Preparation of the PDA-NPs PDA-NPs were synthesized according to a modified procedure previously reported [17,18] Briefly, an ammonia aqueous solution (0.8 mL, NH4OH, 28–30%) was mixed with deionized water (90 mL) and ethanol (40 mL) under mild stirring at room temperature for 10 DA (0.5 g) was dissolved in deionized water (10 mL) Then, the DA solution was injected on the previously mixed solution and the reaction proceeded for 48 h under vigorous stirring The PDA-NPs were obtained by centrifugation (13,000g , 15 min) and washed with deionized water three times Finally, the PDA-NPs were dried under vacuum at 60 1C 2.3 Coating of the Ti surfaces Various coatings were prepared on Ti surfaces using a selfassembly technique，including bare Ti, {PDA} coating, {HA} coating and {PDA/HA} composite coating Briefly, the PDANPs and PDA-decorated HA nanorods were separately dissolved in distilled water to prepare mg mL–1 suspensions of PDA-NPs and PDA-decorated HA nanorods The Ti plates (Ф 10 Â mm) were then immersed on the PDA-NP suspension for 10 at room temperature, and the PDA-NPs assembled on the Ti surface after washing for three times Finally, the PDA-NPs-coated Ti substrates were immersed in the PDA-decorated HA nanorod suspension These steps were repeated five times to obtain a {PDA/HA} composite coating on the Ti substrates For the preparation of the {PDA} and {HA} coating, the Ti substrates were immersed on the corresponding PDA-NPs and PDA-decorated HA nanorod suspensions ten times, as previously described The particle size distribution and zeta potential of the HA nanorods, PDA-decorated HA nanorods, and PDA-NPs were evaluated using a laser particle analyzer (ZETA-AIZER, Malvern Instruments Ltd., UK) The crystal phase of the PDA-decorated HA nanorods was determined by X-ray diffraction (XRD, X’pert PRO, Philips, The Netherlands) over the 2θ range of 15–751, with a rate of 0.1 s À The morphology of the HA nanorods, PDA-decorated HA nanorods, PDA-NPs, {PDA} coating, {HA} coating, and {PDA/HA} composite coating was investigated using scanning electron microscopy (SEM, JSM 6390, JEOL, Japan) and transmission electron microscopy (TEM, JEOL, Japan) 2.5 BMP-2 adsorption and release BMP-2 was first dissolved in distilled water to prepare a BMP-2 solution (10 μg mL–1) Then, the various composite coatings (bare Ti, {PDA}, {HA} and {PDA/HA}) were immersed in the BMP-2 solution (200 μL) to absorb BMP-2 for 24 h at room temperature The adsorbed BMP-2 was obtained by subtracting the amount of BMP-2 left in the distilled solution from the initial amount The BMP-2 loaded bare Ti and {PDA/HA} composite coating are denoted as Ti/BMP-2 and {PDA/HA}/BMP-2, respectively To determine the BMP-2 release kinetics from the Ti/BMP-2 and {PDA/HA}/BMP-2, the BMP-2-loaded samples were placed in phosphate buffer saline (PBS, mL) and agitated at 37 1C The BMP-2 concentration in the solution was analyzed at different times (1, 5, 8, and 12 days) using the human BMP-2 ELISA kits 2.6 In vitro cell experiments BMSCs were cultured on various coatings, including bare Ti plates (Ф 10 Â mm) and {PDA}, {HA}, {PDA/HA}, and {PDA/HA}/BMP-2 coated Ti plates, to evaluate their cytocompatibility The detailed process for the cell adhesion, proliferation, and differentiation of BMSCs on the various coatings was described in Supporting Information, as reported in Ref [19] 2.7 In vivo animal experiments Five groups of implant materials, including bare Ti bars (Ф Â 20 mm) and {PDA}, {HA}, {PDA/HA}, and {PDA/ Table Particle size and zeta potential of different particles Samples Particle size (nm) Zeta potential (mV) HA nanorods PDA-decorated HA nanorods PDA-NPs 18378 18676 46279 4.517 0.27 À 18.537 0.44 À 25.417 0.95 Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] HA}/BMP-2 coated Ti bars, were implanted in the femoral bone marrow cavity of SD rats to evaluate their osteoinductivity in vivo Each group contained four parallel samples The detailed process for the intramedullary implantation, histological staining (toluidine blue and magenta), and new bone area ratio (NBAR) around the implant materials was described in Supporting Information, as reported in Ref [20] Results 3.1 Characterization of the HA nanorods, PDA-decorated HA nanorods, and PDA-NPs The particle size and zeta potential of different particles (HA nanorods, PDA-decorated HA nanorods, and PDA-NPs,) are shown in Table The average particle size of the PDA-NPs is 462 nm, and their zeta potential is À 25.41 0.95 mV The average particle size of HA nanorods is 183 nm and the average particle size of PDA-decorated HA nanorods is 186 nm, which indicates that the size of HA nanorods is not significantly change after HA nanorods were decorated by PDA However, the zeta potential of the HA nanorods is 4.51 0.27 mV After decoration with PDA, the zeta potential of the HA nanorods is changed to À 18.53 0.44 mV These results show that PDA was successfully grafted on the surface of the HA nanorods The morphologies of the PDA-NPs and PDA-decorated HA nanorods are shown in Fig 2(a)–(c) The SEM and TEM images reveal that the PDA-NPs are spherical with a rough surface, and possess a diameter of $ 450 nm (Fig 2(a) and (b)) The TEM micrographs reveal that the HA nanorods have a rod-like structure, with a length and width of $ 100 and $ 10 nm, respectively (Fig 2(c)) The selected area electron diffraction (SAED) patterns of the PDA-decorated HA nanorods show distinct diffraction rings, representing the (002), (211), (310), and (213) crystallographic planes (Fig 2(c)) The XRD patterns of the PDA-decorated HA nanorods show intense diffraction peaks at 25.81, 32.11, 39.81, 46.81, and 49.51, which can be assigned to the (0 2), (2 1), (3 0), (2 2), and (2 3) lattice planes of HA, respectively 3.2 Characterization of various coatings The SEM images shown in Fig reveal the morphology of the {PDA} coating, {HA} coating, and {PDA/HA} composite coating on the Ti surface If only PDA-NPs are assembled, the distribution of the PDA-NPs on the {PDA} coating is uneven and the Ti substrate can be observed (Fig 3(a)) If only PDAdecorated HA nanorods are assembled, the {HA} coating is compact and dense (Fig 3(b)) However, if the PDA-NPs and PDA-decorated HA nanorods are alternately assembled, the spherical PDA-NPs and rod-like HA nanorods result in porous Fig (a) SEM and (b) TEM images of the PDA-NPs; (c) TEM image of the PDA-decorated HA nanorods (the inset shows the SAED patterns of the PDAdecorated HA nanorods); and (d) XRD pattern of the PDA-decorated HA nanorods Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] Fig SEM images of various coatings (a) {PDA} coating, (b) {HA} coating, and (c) {PDA/HA} composite coating (the inset shows a magnified image of the {PDA/HA} composite coating morphology); and (d) cross-section view of the {PDA/HA} composite coating Table The BMP-2 loading ability of various composite coatings Samples Ti {PDA} {HA} {PDA/HA} The amount of adsorption (ng cm À 2) 897 11 1967 17 172 713 214712 PDA-NPs A cross-section image of the {PDA/HA} composite coating shows a coating thickness of $ μm after five assembly cycles (Fig 3(d)), and a uniform distribution of both PDA-NPs and PDA-decorated HA nanorods in the coating In summary, the biomimetic {PDA/HA} composite coating with a porous and hierarchical micro/nanostructure can be prepared by alternately assembling PDA-NPs and PDA-decorated HA nanorods on the Ti surface 3.3 BMP-2 adsorption and release Fig Cumulative BMP-2 release from Ti/BMP-2 and {PDA/HA}/BMP-2 and hierarchical micro/nanostructures (Fig 3(c)) The PDAdecorated HA nanorods are attached to the surface of the PDANPs, as shown in the high magnification SEM image (inset of Fig 3(c)), which is ascribed to the intrinsic adhesion of the The Table showed the BMP-2 loading ability of various coatings after soaking in the BMP-2 solution (10 μg mL À 1, 200 μL) for 24 h The BMP-2 loading ability of {PDA} and {HA} coatings was significantly higher than that of bare Ti, which indicated that the PDA increased the BMP-2 loading ability of the coatings Moreover, the BMP-2 loading ability of {PDA/HA} was the highest among all coatings, which revealed that adhesive PDA and the microporous structures synergistically enhanced the BMP-2 loading ability of the functional coatings The cumulative BMP-2 release performance from Ti/BMP-2 and {PDA/HA}/BMP-2 was investigated by immersing the BMP-2-containing samples in PBS (Fig 4) Ti/BMP-2 shows a burst release, with more than 80% of BMP-2 being released during the first day In contrast, {PDA/HA}/BMP-2 showed sustained release, and only 35% of BMP-2 was released during the first day This result demonstrates that the {PDA/HA} coating Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] Fig SEM micrographs of BMSCs after days of culture on various coatings (a) Bare Ti; (b) {PDA} coating; (c) {HA} coating; (d) {PDA/HA} coating; (e) {PDA/HA}/BMP-2 coating; and (f) high-magnification micrographs of BMSCs on the {PDA/HA}/BMP-2 coating is excellent BMP-2 release carrier The main reason is attributed to the {PDA/HA} coating that possesses porous micro/nanostructures, providing multiple BMP-2 adsorption sites 3.4 Cell adhesion, proliferation, and differentiation The morphology and adhesion of BMSCs after days of culture on various coatings were examined using SEM (Fig 5) BMSCs on bare Ti surfaces (Fig 5(a)) showed scarce filopodia formation However, BMSC adhesion was better on the {PDA} and {HA} coatings (Fig 5(b) and (c)) Specifically, BMSCs on the {PDA/HA} composite coating with a porous and hierarchical micro/nanostructure produced various filopodia to attach on these coating surfaces (Fig 5(d)–(f)) In summary, porous and hierarchical micro/nanostructures enhanced the adhesion of BMSCs Alamar blue assays indicated that the {PDA/HA} composite coating favored the proliferation of BMSCs (Fig 6(a)) The number of BMSCs on various coatings increased after days, which reveals that all coatings promoted the proliferation of BMSCs The number of BMSCs on the {PDA} and {HA} coatings was significantly higher than that on bare Ti surfaces Furthermore, the number of BMSCs on the {PDA/HA} composite coating was higher than that on the {PDA} and Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] {HA} (Fig 7(e) and (f)) and {PDA/HA} composite coatings (Fig 7(g) and (h)) After BMP-2 was loaded, the new bone tissue formation increased significantly (Fig 7(i) and (j)) A quantitative analysis (Fig 8) indicated that the NBAR around the bare Ti and {PDA} coating was 0% However, the NBAR for the {HA} and {PDA/HA} composite coatings was $ 15% This result indicates that the osteoinductivity was significantly improved due to the addition of HA nanorods to the coating After the BMP-2 loading, the NBAR of the {PDA/ HA}/BMP-2 coating increased to 30% In summary, these results show that the incorporation of both HA and BMP-2 to the coatings synergistically enhanced the bone formation Discussion Fig (a) Proliferation of BMSCs after and days of culture on various coatings (b) ALP activity of BMSCs after culturing for 14 days * Indicates the significant difference (p o 0.05) {HA} coatings After the BMP-2 was loaded, {PDA/HA} showed the highest number of BMSCs These results demonstrate that the PDA-NPs and HA nanorods can synergistically enhance the proliferation of BMSCs The ALP (alkaline phosphatase) activity test indicated that the {PDA/HA} composite coating induced the BMSC differentiation (Fig 6) The ALP activity of BMSCs on the {HA} and {PDA/HA} coatings was significantly higher than that on the surface of bare Ti and {PDA}; this is because HA in the {PDA/HA} composite coating had a good osteoinductivity Moreover, the ALP activity of BMSCs on the {PDA/HA}/BMP-2 composite coating was even higher because BMP-2 improved the osteoinductivity of the coatings In summary, the {PDA/HA} composite coating with a porous and hierarchical micro/nanostructure enhanced BMSC proliferation and differentiation 3.5 In vivo evaluation Intramedullary tests showed that the {PDA/HA} composite coating could induce bone regeneration in the bone marrow cavity of SD rats after 12 weeks of implantation Fig shows the histological section stained with toluidine blue and magenta There is almost no new bone tissue formation around the bare Ti (Fig 7(a) and (b)) and {PDA} coating (Fig 7(c) and (d)), which indicates that the bare Ti and {PDA} coating could not induce bone regeneration However, new bone tissue formed around the The self-assembled {PDA/HA} composite coating can enhance the behavior of BMSCs and bone regeneration, which could be ascribed to three reasons The first reason is the intrinsic cell affinity of PDA Previous studies have reported that PDA coatings can promote cell adhesion Cho et al [21] reported that PDA coatings of the surfaces of polyethylene glycol adipate and polystyrene substrates could promote the proliferation and spreading of human neural stem cells She et al [22] reported that PDA coatings of polylactic acid scaffold surfaces increased the adhesion, proliferation, and differentiation of human adiposederived stem cells PDA can adsorb extracellular (ECM) proteins, such as fibronectin and collagen, providing a favorable environment for cell proliferation and spreading [12,23] The second reason is the porous and hierarchical micro/ nanostructure produced by self-assembly of PDA-NPs and PDA-decorated HA nanorods It should be noted that most of the previous studies focused on dense PDA films [24,25] In this study, the {PDA/HA} composite coating containing PDANPs and PDA-decorated HA nanorods, has a porous and hierarchical micro/nanostructure that facilitates cell adhesion It is commonly accepted that porous and hierarchical micro/ nanostructures can improve the adsorption of ECM biomolecules, which can enhance the osteoinductivity of the coatings [26,27] Thus, the self-assembled PDA-NP-based porous and hierarchical micro/nanostructure reported in this study can promote cell activity The third reason for the good bioactivity of the {PDA/HA} composite coating is attributed to the presence of PDA-decorated HA nanorods It has been reported that nanoscaled HA has good bioactivity [9,10] PDA-decorated nanoscaled HA can significantly enhance the differentiation and mineralization of osteoblasts [28] Note that PDA decoration had no effect on Ca ion release from on HA nanorods, as demonstrated by the cumulative ion release experiments (Fig S1, Supporting information) In this study, we used PDA-decorated HA nanorods to assemble composite coatings Compared with pure PDA coatings, the in vitro study demonstrated that the HA nanorods promoted cell differentiation, and the in vivo study indicated that the HA nanorods accelerated the bone tissue regeneration The PDAdecorated HA nanorods reported in this study have the following advantages: (1) the preparation process of the PDA-decorated HA nanorods is simple and effective, and does not require complex Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] Fig Histological section stained with toluidine blue and magenta of different implant materials after they were implanted in the bone marrow cavity of SD rats for 12 weeks (a) and (b) bare Ti; (c) and (d) {PDA} coating; (e) and (f) {HA} coating; (g) and (h) {PDA/HA} composite coating; and (i) and (j) {PDA/HA}/BMP-2 coating The right column shows magnified images of the left column S represents the implanted samples, and NB represents the new bone around the implanted samples Please cite this article as: Z Wang, et al., Mussel-inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 Z Wang et al / Biosurface and Biotribology ] (]]]]) ]]]–]]] Fig NBAR around the implant materials * Indicates the significant difference (po0.05) factor affinity, bioactive HA nanorods, and hierarchical micro/ nanostructures Firstly, the PDA-NPs improved both cell adhesion and proliferation; secondly, the PDA-decorated HA nanorods promoted the osteoinductivity of the coatings; and thirdly, the self-assembled porous and hierarchical micro/ nanostructure provided a favorable microenvironment to recruit and host new cells Furthermore, the PDA-NPs, HA nanorods, and porous micro/nanostructures are beneficial for the BMP-2 immobilization [26,32] The incorporation of BMP-2 on the {PDA/HA} composite coating further enhanced the cell activity and bone regeneration [33,34] Hence, the {PDA/HA} composite coating with a porous and hierarchical micro/nanostructure closely mimics ECM microenvironments, promoting cell growth and tissue regeneration Conclusions and expensive equipment; (2) HA nanorods modified by PDA can improve the bonding strength of the composite coatings; (3) the assembly process is conducted under a mild aqueous environment and does not use organic solvents, avoiding chemical toxicity of cells and tissues; and (4) the PDAdecorated HA nanorods, along with the PDA-NPs, impart the composite coatings with nanoporous structures, enhancing the biomolecule adsorption ability of the coatings In conclusion, the PDA-decorated HA nanorods have excellent biocompatibility and osteogenic activity, and are excellent building blocks for preparing nanostructured materials with improved bioactivities The {PDA/HA} composite coating had a high growth factor loading ability and provided a sustained release of BMP-2, which was attributed to two reasons First, the PDA-NPs in the coating provided multiple bioactive sites for the BMP-2 immobilization Pan et al [29] immobilized BMP-2 on a PDA-coated poly(lactic-coglycolic acid) polymer scaffold, and their results showed that PDA provided multiple bioactive sites for BMP-2 immobilization, and that BMP-2 improved the osteoinductivity of the scaffold Shin et al [30] immobilized BMP-2 on the surface of PDA-decorated polylactic acid nanofibers, and their results indicated that the incorporation of BMP-2 increased significantly the ALP activity and calcification of human mesenchymal stem cells In this study, the PDA-NPs were more favorable for BMP-2 immobilization because of the amounts of functional groups exposed on the surface Second, the porous and hierarchical structures of the composite coating facilitate the BMP-2 adsorption Wang et al [14] reported that PDA capsules assembled on Ti surfaces had large specific surface areas, effectively immobilizing a high dose of BMP-2 Li et al [31] prepared a nanoporous structured Mg/Zn/Si xerogel for BMP-2 immobilization using a sol-gel method, and their results indicated that the nanoporous structure could provide a sustained release of BMP-2 and promote the proliferation and differentiation of osteoblasts, compared with a xerogel without a nanoporous structure In summary, both PDA and microporous structures impart a growth factor immobilization ability to the composite coatings In summary, the {PDA/HA} composite coating showed good osteoinduction and accelerated the in vivo bone tissue formation, which consisted of a synergistic effect of three coating features, including PDA-NP cell affinity and growth In this study, a {PDA/HA} composite coating with a porous and hierarchical micro/nanostructure was prepared on the Ti surface by self-assembly of PDA-NPs and PDA-decorated HA nanorods The composite coating provided both a high loading and a sustained release of BMP-2 due to the porous micro/nanostructure and PDA growth factor affinity The BMP-2-loaded {PDA/HA} composite coating possessed an excellent cytocompatibility and it could significantly promote the adhesion, proliferation, and differentiation of BMSCs In addition, the BMP-2-loaded {PDA/HA} composite coating was implanted in vivo, and exhibited excellent osteoinductivity due to the synergistic effect of the porous micro/nanostructure, HA-nanorods, and BMP-2 Acknowledgements The work was financially supported by the National Key Research and Development Program of China (2016YFB0700802), the 863 Program (2015AA034202), NSFC (81671824), Open fund of Key Lab of Advanced Technologies of Materials (MOE), Fundamental Research Funds for the Central Universities (2682016CX075) Appendix A Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bsbt.2017.01.001 References [1] C Wu, W Fan, J Chang, Y Xiao, Mussel-inspired porous SiO2 scaffolds with improved mineralization and cytocompatibility for drug delivery and bone tissue engineering, J Mater Chem 21 (2011) 18300–18307 [2] S.H Ku, C.B Park, Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering, Biomaterials 31 (2010) 9431–9437 [3] X Yu, H Fan, Y Liu, Z Shi, Z Jin, Characterization of carbonized polydopamine nanoparticles suggests ordered supramolecular structure of polydopamine, Langmuir 30 (2014) 5497–5505 [4] J.H Jiang, L.P Zhu, H.T Zhang, B.K Zhu, Y.Y Xu, 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using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017), http://dx.doi.org/10.1016/j.bsbt.2017.01.001 ... as: Z Wang, et al., Mussel- inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017),... as: Z Wang, et al., Mussel- inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications, Biosurface and Biotribology (2017),... the {PDA} and Please cite this article as: Z Wang, et al., Mussel- inspired nanostructured coatings assembled using polydopamine nanoparticles and hydroxyapatite nanorods for biomedical applications,