The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells

Accepted Manuscript Full length article The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells Cancan Zhao, Xiaoya Wang, Long Gao, Linguo Jing, Quan Zhou, Jiang Chang PII: DOI: Reference:

S1742-7061(18)30233-2 https://doi.org/10.1016/j.actbio.2018.04.030 ACTBIO 5430

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

20 December 2017 14 April 2018 16 April 2018

Please cite this article as: Zhao, C., Wang, X., Gao, L., Jing, L., Zhou, Q., Chang, J., The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells, Acta Biomaterialia (2018), doi: https://doi.org/10.1016/j.actbio.2018.04.030

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The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells Cancan Zhao a, b, Xiaoya Wang a, Long Gao a, b, Linguo Jing a, Quan Zhou a, Jiang Chang a, * a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences. Shanghai 200050, P.R.China b University of Chinese Academy of Sciences, Beijing 100049, P.R.China

* Corresponding Author Email: [email protected] (J. Chang) Tel: +86-21-52412804, Fax: +86-21-52413903

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Abstract The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect.

Keywords: micro/nano hybrid; osteogenic differentiation; synergy effect; integrins; BMP2

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signaling pathway; cell-cell communication. 1. Introduction It is known that the structural microenvironment of stem cells plays a vital role in regulating stem cell fate, and developing artificial microenvironments by designing surface topography on the biomaterials is a possible approach to regulate stem cell differentiation and tissue regeneration [1]. Some studies have shown that the micro- or nano-structure could enhance osseointegration and bone regeneration by accelerating osteogenic differentiation of mesenchymal stem cells even in the absence of growth factors [2-4]. The regulation of cell behaviors using nanostructure is inspired by the nanoscale fibrous structure of the natural extracellular matrix (ECM) [5,6]. Furthermore, some studies have shown that micropatterns could also regulate nucleolus morphology and reformation of the cytoskeleton, and promote bone apposition during early stages of bone regeneration and improve the fixation of the implant in vivo [6,7], indicating the important role of the multi-scale structure from nano- to micro-range in regulating cell behaviors. Our recent studies also demonstrated that the surface modification of bioceramics with both micropatterns and nano-structure showed activity to stimulate osteogenic gene expression and enhance osteogenesis, which suggests that the surface topographical design can be considered as an effective approach to develop bioactive biomaterials for enhanced bone regeneration [8,9]. However, a finding by Meirelles indicates that the nanostructure alone may not be the optimal structure to induce bone integration [10]. Considering the hierarchical features of natural bone in both micro- and nano-scales, it is reasonable to assume that the micro- and nano-structural characteristics may have different function on stimulating tissue regeneration. However, the mechanisms of the micro- and nano-structures regulating stem cell differentiation are still unknown, and the elucidation of the role of both micropatterns and nano-topography, and

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in particular the combinatorial effect of the micro- and nano-structures on the regulation of stem cell differentiation will provide a clue to guide the design of optimal biomaterials for bone regeneration. Previous studies indicated that cells could sense the surface structure first through adhesion upon seeding them on the biomaterials [11]. Integrins are known as transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion. When cells are seeded on biomaterials, integrins play a critical role in regulating cell adhesion and other cellular activities by transmitting signals via the cell membrane [12]. In addition, integrins can stimulate downstream signaling cascades by organizing the cytoskeleton [13]. It has been reported that the micro- or nano-structure could enhance integrin activation [14-16]. However, the effect of the hybrid structure on integrins during osteogenic differentiation has not been reported. The fundamental questions to be answered are whether the micropattern and nano-topography play similar role in integrin activation, and the combination of micro- and nano-structures have synergetic or additive effects on osteogenic differentiation of stem cells. Our hypothesis is that the integrin activation effect of micro- and nano-structures are different, and the combination of two different structural characteristics may result in an additive or even synergetic stimulatory effect on osteogenesis. Some results have indicated that the collaboration between integrins and bone morphogenetic protein 2 (BMP2) in BMP2 signaling pathway could govern osteogenic differentiation [13,17,18]. Still, the interrelation of integrins and BMP2 has not been reported in material structure induced osteogenic differentiation, though micro- or nano-structure could promote the activation and expression of integrins and BMP2 respectively. Previous research has suggested that the BMP2 signaling pathway is one of the most essential pathways for osteogenic

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differentiation and necessary for the Runt-related transcription factor 2 (Runx2)-dependent induction of osteogenic gene expression [13,19-21]. Moreover, it had been found that both micro- and nano-structures could enhance cell-biomaterial interactions and promote cell behaviors via activating BMP2 signaling pathways, respectively [22,23], although the mechanisms of the activation are still unclear. In addition, the differentiation of stem cells is also regulated via cell-cell interactions [24]. On the one hand, cell-biomaterial interactions and in particular the biomaterial-induced cell-cell communication via gap junction could be considered to play a crucial role in osteogenic differentiation [25-28]. Connexin 43 (Cx43), a component of gap junction proteins, is fundamental to stimulate ALP activity and regulate Runx2 function [2933]. On the other hand, integrins have been found to interact directly with Cx43 and activate the opening of the Cx43 hemichannel for the small molecule exchange [34]. These results imply that integrin might be an important aspect of the activation of BMP2 signaling pathway and cell-cell communication in response to micro- or nano-structure of biomaterials [12,16]. Furthermore, a previous study has shown that BMP2 could modulate chondrogenic and neuronal differentiation in part through up-regulation of Cx43 expression [35,36], indicating a close connection between BMP2 signaling pathway and cell-cell communication. However, it has been reported that BMP2 signaling effects on gap junction-mediated intercellular communication during osteogenic differentiation. We hypothesize that the activation of integrins, BMP2 signaling pathway and Cx43-mediated gap junctional communication of cells may be induced by both surface micropattern and nano-topography of biomaterials in a different way, so that the combination of micro- and nano-structures has a synergistic stimulatory effect on osteogenic differentiation as compared to the single-scale structural characteristic. Among bone regeneration biomaterials, hydroxyapatite (HA, Ca10(PO4)6(OH)2) bioceramics

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have been widely applied as bone grafts owing to the excellent osteoconductivity and the similar chemical component as the mineral phase of the human hard tissues [37-39]. However, one of the challenges is to fabricate the micropatterns and nano-structure, in particular, the micro/nano hybrid structure directly on HA bioceramics because of the brittleness of the materials. In this study, we utilized a technique to prepare HA bioceramics with the hierarchical hybrid surface structure combining micropatterns with nanorods, and investigated the surface structural activation of the expression of the BMP2 signaling pathway related genes, integrins and gap junction related Cx43 protein to elucidate the activation mechanisms of the micro/nano hybrid structure.

2. Materials and methods 2.1. Fabrication and characterization of HA bioceramics with different surfaces structure In this study, HA bioceramics with different surfaces structure were respectively fabricated by template method following hydrothermal treatment. First of all, HA powders were fabricated by wet chemical precipitation. In the process, Ca(NO3)2•4H2O and (NH4)2HPO4 were dissolved respectively in distilled water to obtain 0.5 M Ca(NO3)2 solutions and 0.3 M (NH4)2HPO4 solutions, and the pH of both solutions was adjusted to 10.8 by adding ammonia solution. Then, (NH4)2HPO4 solution was added into the Ca(NO3)2 solution dropwise, and the pH of the suspension was maintained at 10.8 using ammonia solution. The suspension was further stirred for 24 h, and then washed with distilled water and anhydrous ethanol for three times. Finally, the synthesized powders were dried at 120 oC for 24 h, and sintered at 850 oC for 3 h. The obtained HA powders were dispersed in alcohol and the particle size was measured using a Laser diffraction particle size analyzer (mastersizer 3000, Malvern, England), showing that the particle

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size (D50) of the powders was 14 µm. HA bioceramics discs with micropatterned surface were fabricated by employing 400 mesh nylon sieves as the templates. The nylon sieve templates with a suitable diameter that matches with the mold were first put on the surface of steel die before adding HA powders and the discs were obtained by pressing at 10 MPa pressure. The obtained discs were sintered in air at 1100 oC for 5 h at a heating rate of 2 oC min-1 to remove the nylon sieve templates for fabricating the micropattern structured HA bioceramics (S2) [9]. In addition, HA bioceramics with the flat surface (S0) were pressed without nylon sieve templates and sintered under same condition. Then, samples S0 and S2 were treated in an aqueous system containing 0.055 M ethylenediamine tetraacetic acid disodium calcium (EDTANa2Ca), and 0.125 M Na3PO4•12H2O, under 120 oC hydrothermal condition for 12 h to prepare HA bioceramics with nanorod structure (S1) and micropattern/nanorod hybrid structured HA (S3), respectively (Scheme 1). Field emission scanning electron microscopy (FESEM: JSM-6700F, JEOL, Japan) was used to characterize the surface morphologies of HA bioceramics with the different structures. In addition, X-ray diffraction (XRD, D/max2550 V, Rigaku Co., Japan) was applied to determine the phase of the samples S0-S3. Furthermore, the surface contact angle of the samples S0-S3 was measured by automatic contact angle meter (Kruss, Kruss GmbH Germany) to characterize materials wettability, and the average contact angle of each group was calculated from the values of three samples. Brunauer–Emmett–Teller surface area analyser (AsAp2010, Micromeritics, USA) was used to measure the surface area of the samples S0-S3. For protein adsorption study, bovine serum albumin (BSA) and fibronectin (FN) were purchased from Sigma. HA disks were incubated in BSA solution (1 mg ml-1) and FN solution (1

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mg ml-1), respectively at 37oC for 4 h. The solution was centrifuged at 10000 rpm for 10 min and the supernatant was collected after incubation. Subsequently, the concentration of unadsorbed proteins in the supernatant was determined using Pierces BCA Protein Assay Kit (Thermo ScientificTM, USA). The adsorption of the proteins by the samples were calculated by subtracting the residual protein concentration from the total protein concentration. All experiments were performed in triplicate. To study the degradability of the samples, HA disks were soaked in the buffer solution of tris-(hydroxymethyl)-aminomethane (Tris, (CH2OH)3CNH2) and hydrochloric acid (HCl) (pH = 7.40) at 37 °C for 1,3,5 and 7 days, and the ratio of disk surface area to solution volume of TrisHCl was 0.1 cm2 ml−1. After soaking, the disks were removed and the concentrations of calcium (Ca) and phosphorus (P) ions in the Tris-HCl buffer were determined quantitatively by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Perkin–Elmer, Optima 3000DV, USA). All experiments were performed in triplicate. 2.2. Culturing of human bone marrow stromal cells 2.2.1. Cells culture and seeding Human bone marrow stromal cells (hBMSCs) were cultured in the Basal Medium for human mesenchymal stem cells (Cyagen) with 10% fetal bovine serum (FBS). hBMSCs were grown in an incubator as mentioned in our previous paper [9]. The non-adherent cells were discarded by changing culture medium every two days. In order to characterizing cell responses to the samples (such as attachment, proliferation and osteogenic differentiation), 1ml cell suspension were seeded into each well of 6-well tissue culture plates containing samples S0-S3, before the cells were digested by pancreatic enzyme at 90% confluence and resuspended in fresh culture medium with the density of 3
104 cells per ml.

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2.2.2. Adhesion of the cells seeded on the samples S0-S3 The cells cultured on the samples S0-S3 for 24 h were treated for determining the morphology through two means. In the first method, the samples were primarily rinsed with phosphate-buffered saline (PBS) two times for removing the non-adherent cells, and then the adherent cells were fixed in 4% paraformaldehyde (PFA) for 40 min. Following this treatment, the samples were rinsed three times with PBS. After the actin cytoskeletons were labeled by incubating with Phalloidin- TRITC (Sigma, USA) for 30 min, PBS was employed to rinse three times. Then, the cell nuclei were contrast-labeled in blue by 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, USA) [40]. Finally, the actin cytoskeletons of cells were imaged via a confocal laser scanning microscope (CLSM, Leica, Germany). For the purpose of analyzing the cell spreading area, Image-Pro plus 6.0 (Media Cybernetics, USA) software was used. 2.2.3. Cells proliferation assay After culturing for 1, 3 and 7 days, hBMSCs seeded on HA bioceramics surfaces in 24-well plates were characterized to assay cell proliferation. After 300 µl Basal Medium for human mesenchymal stem cells with 30 µl cell counting kit-8 (cck-8, Beyotime, USA) solution were added to each well, the cells were incubated at 37 oC for 2 h in the incubator [41]. To obtain optical density (OD) values, ELX Ultra Microplate Reader (Bio-tek, USA) was utilized to measure at 450 nm. All experiments were performed in triplicate. 2.2.4. Alkaline phosphatase (ALP) activity To assay for ALP activity, hBMSCs were cultured on the samples S0-S3 for 14 days. On the one hand, ALP staining was performed by using operating fluid from Beyotime. On the other hand, according to the manufacturer’s protocol, ALP activity was further quantified by an Alkaline Phosphatase Assay Kit (Colorimetric, Abcam, UK). In brief, to remove the residual

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medium, the cells lysed in 200 µl lysis buffer (0.1% Triton
100) at -20 oC for 10 min, following three-time rinsing with phosphate buffered saline (PBS). The cell lysates were removed into an Eppendorf microcentrifuge tube and centrifuged at 10000 rpm for 10 min at 4 o

C. Nearly 100 µl of the supernatant was transferred to a 96-well plate and mixed with 200 µl p-

nitrophenyl-phosphate (pNPP: Sigma, St. Louis, USA) solution. After incubation in darkness for 30 min at room temperature. Finally, ALP activity was quantified by determining absorbance at 405 nm by a Microplate Reader (Bio-tek, USA). In addition, data were normalized to the total cell protein content as measured by using a Pierces BCA Protein Assay Kit (Thermo ScientificTM, USA). 2.2.5. Quantitative real-time PCR assay The cells were seeded on flat HA bioceramics (S0) and HA bioceramics with micropattern or nanorod (S1-S3) for 7 days. The total RNA was extracted from the cells cultured on the samples S0-S3 utilizing Trizol reagent (Invitrogen, USA). Each of samples was reversetranscribed to cDNA as the previous study [9]. PCR reaction was performed by Ex Taq DNA polymerase (TaKaRa, China) for the following genes: Collagen 1 (COL1), runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2), ALP, Cx43, and integrin subunits α5 (ITGA5), αv (ITGAV), β1 (ITGB1), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered as the house-keeping gene for normalization. All experiments were performed in triplicate. The sequences of the specific primers used are as follows: For ITGA5: F: 5’-GGCAGCTATGGCGTCCCACTGTGG-3’; R: 5’- GGCATCAGAGGTGGCTGGAGGCTT-3’.

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For ITGAV: F: 5’-GTTGCTACTGGCTGTTTTGG-3’; R: 5’-AAGTTCCCTGGGTGTCTG-3’. For ITGB1: F: 5’-ATTACTCAGATCCAACCAC-3’; R: 5’- CTGCTCCCTTTCTTGTTCTTC-3’. 2.2.6. BMP2-related signaling pathway of hBMSCs To investigate the activation of the BMP2 signaling pathway in hBMSCs, several genes or receptors were analyzed by RT-qPCR as mentioned in Section 2.2.5, including BMP2 signaling pathway related genes [Mothers against decapentaplegic homologue 1/4/5 (SMAD1, SMAD4, SMAD5)], bone morphogenetic protein receptor, type IA (BMPR1A) and type IB (BMPR1B) and bone morphogenetic protein receptor, type II (BMPR2). 2.2.7. Assessment of gap junction communication in hBMSCs Gap junction communication in hBMSCs was characterized by immunofluorescence staining when hBMSCs were grown on the samples S0-S3 after 10 days. The cells sticking on the samples were fixed for 30 min with 4% PFA and permeabilized with 0.3% Triton X-100 for 10 min. The samples were blocked with 2% bovine serum albumin (BSA) for 1 h. Immunofluorescence staining was performed using the antibody which purified mouse monoclonal specific for Cx43 (BD Biosciences, MD, USA; dilution 1:250). Prior to adding AlexaFluor secondary antibodies (Invitrogen) into the samples for 1 h at room temperature, the cells were incubated at 4 oC overnight. Filamentous actin and the nuclei were labeled as described in Section 2.2.2, then, the cells were visualized with a confocal laser scanning microscope (CLSM, Leica, Germany), and the staining intensity of Cx43 was analyzed using an

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Image-Pro plus 6.0 software. 2.2.8. Integrins and Cx43 analysis in the presence of BMP2-inhibitor noggin For the purpose of investigating the role of BMP2 signaling pathway in activation of integrins and Cx43-related cell-cell communication by micro/nano surface structure, the related integrin subunits and Cx43 protein were analyzed when BMP2 activity was blocked using inhibitor noggin (Gibco®, PHC1506). Cells were seeded in two 6-well plates as experimental group and control group respectively, and each 6-well plate contained four samples with different surface structure (S0, S1, S2 and S3). After 6 h, BMP2 blocking antibodies noggin were added to a final concentration of 1.5 µg/ml only in the experimental group. After four days culturing, the gene expression of integrin subunits and Cx43 was evaluated by RT-qPCR as described in Section 2.2.5. 2.2.9. Statistical Analysis Data were expressed as means ± standard deviation of the mean. Differences between two groups were analyzed by ANOVA (SPSS, v.17.5, USA). It was considered statistically significant when p < 0.05.

3. Results 3.1. Characterization of HA bioceramics From the SEM in Figure 1, the surface features of the fabricated samples were clearly revealed. As expected, the sample S0 with the flat and dense surface was obtained without using nylon sieve as a template, and ceramic particles were well sintered with the crystal size range from 2 µm to 5 µm. In contrast, it is clear to see that the sample S1 fabricated by hydrothermal treatment of S0 has nanorods grown on the ceramic surface with the rod diameter of 70-100 nm.

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The sample S2 has micro-patterned quadrate concave-convex surface structure, which was constructed using template method, and the width and the space between the convex were about 28 µm and 24 µm. The sample S3 was constructed with the hybrid surface structure of micropatterns and nanorods by hydrothermal treatment of the sample S2. It is worth to indicate that, in the surface structure of the sample S3, the three-dimensional size of micropatterns with the width of 28 µm and the space between the convex up to 24 µm was the closest dimension to the cell diameter, while the nanorods were similar to that of the sample S1. The XRD spectra confirmed that the samples with different surface structure could be identified as pure HA (JCPDS card no. 09-0432) phase (Figure S1), which suggests that there was no phase change after hydrothermal treatment at 120 oC for 12 h. Moreover, the ratio of different XRD peak intensities could serve as proof to demonstrate the specific orientation of the HA crystals on the ceramic disc surface. And the sharp and intensive peak of all the samples in (002) suggested that the HA crystals preferred to be aligned along the c-axis. The orientation degree of the samples S0-S3 was calculated by intensity (I) of diffraction peaks at 2θ= 25.8° for (002) and 31.8° for (211) according to a method described in the literature [42]. As shown in Table 1, the final orientation degrees for samples S0–S3 were 0.28, 0.34, 0.29 and 0.31, respectively. The hydrophilicity was directly relevant to the contact angle of the samples. The result revealed that the contact angle of the samples with surface structure (S1-S3) was smaller than that of the control sample (S0), and there was no significant difference between the average values of the sample S1 and that of the sample S2 (Figure S2). The study verified that the micropattern or nanorod could apparently improve the hydrophilicity of the HA bioceramics. In particular, the micro/nano hybrid structure of the sample S3 significantly improved the

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hydrophilicity of the surface as compared with the nanorod structure or micropattern structure only. The surface areas of the samples S0-S3 were 0.10, 0.40, 0.32 and 0.47 m2 g-1, respectively (Table 2). It is clear to see that all the micro- and nano- structures have higher the specific surface area than that of the flat surface, and the sample S3 with micro/nano hybrid structures showed the largest specific surface area among all the samples. Adsorption of BSA and FN proteins on samples S0-S3 was determined after 4 h incubation (Figure S3). Compared with the flat surface structure (S0), the micro- and nano- structures (S1S3) revealed higher adsorption of nonspecific protein BSA and specific protein FN, in which the sample S3 with the hybrid surface structure showed the highest adsorption, while the nanostructure showed higher capacity of protein adsorption than the micro-pattern structure. The release profiles of Ca, and P ions of the samples S0-S3 within 7 days are shown in Figure S4. It is clear to see that, although the concentration of ions gradually increased within a 7-day period, both the Ca and P ions maintained at low levels. The highest Ca and P concentrations are 0.6 (Figure S4a) and 0.37 ppm (Figure S4b), respectively, which are significantly lower than the Ca and P concentrations in the cell culture medium with Ca concentration of 70.03 ppm and P concentration of 35.87 ppm. 3.2. The adhesion and growth of the cells seeded on the different surface structure After culturing for 24 h, actin cytoskeletons were labeled to observe the cell morphology. In the Figure 2A, the cells morphology on the samples S1-S3 showed apparent difference when compared with the cells on the flat HA sample (S0). The cells seeded on the sample S0 with flat surface showed roundness with limited spreading and in the absence of filopodia. Instead, the apparent cytoplasmic extensions and much longer filopodia both were exhibited when the cells

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cultured on all samples with structured surfaces (S1-S3), especially for S3. It suggested that the micropattern and nanorod could enhance cell attachment. Furthermore, the quantification of cell spreading area also showed that the micro/nano hybrid structure appeared higher cell adhesion than a single-scale structure including micropattern and nanorod structure. 3.3. The proliferation of the cells seeded on ceramics the different surface structure As shown in the Figure 3, the amount of hBMSCs was similar in all the four groups at day 1, which indicated that the cells were seeded equably on the samples. Furthermore, after culture for 3 days, the OD values of hBMSCs showed a higher proliferation on the samples S1-S3 than that on flat sample S0, while the proliferation on S3 is significantly higher than S1 and S2. When hBMSCs were cultured up to 7 days, all the samples S1-S3 demonstrated significantly higher proliferation as compared with control sample S0. Obviously, the proliferation of S3 is still significantly higher than sample S1 and S2. 3.4. The effect of the different surface structure on ALP activity of hBMSCs Figure 4 showed the effect of the different surface structure on ALP activity of hBMSCs. ALP staining was performed after hBMSCs cultured on the samples for 14 days (Figure 4A). The results showed that the staining was the weakest when the cells were cultured on the sample S0. On the contrary, more intense and obvious staining was observed on the surface of the samples S1-S3, especially for S3. In addition, the quantitative result confirmed the optical observation of ALP staining, and revealed that the ALP activity for the cells cultured on the samples S1-S3 was significantly higher than that on flat sample S0. More interesting is that the ALP activity of cells on sample S3 with micro/nano hybrid structure is clearly higher than that of cells on samples S1S2 (Figure 4B) suggesting that the micro/nano hybrid structure may have an additive effect of micro- and nano-structures.

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3.5. The effect of the different surface structure on the expression of osteogenic genes and integrins The osteogenic differentiation of hBMSC on samples S0∼S3 was evaluated by the analysis of expression of osteogenic genes after culturing for 7 days (Figure 5). The result shows that the cells cultured on the samples S1-S3 displayed considerable increase in the expression of BMP2, Runx2, ALP, and COL1 as compared to that on the sample S0, suggesting that the surface micropattern and nanorod of bioceramics stimulated the osteogenic differentiation. Obviously, the sample S3 with micropattern/nanorod hybrid surface resulted in significantly higher expression of BMP2 as compared with samples S1-S2 with single-scale structure such as micropattern or nanorod. In addition, the cells seeded on S3 showed a significant increase of Runx2 expression as compared with that on S2, and a significant increase of COL1 as compared with that on S1, respectively. We also found that the cells cultured on S1 displayed a higher expression of OCN as compared with that on S2. In addition to the osteogenic genes, integrin expression was also affected by the surface structure. Having shown in Figure 6, the expression of integrin subunits α5, αv, β1 was significantly enhanced on the samples S1-S3 as compared to the sample S0. Furthermore, the sample S3 with micro/nano hybrid surface showed the highest integrin expression than the samples only with nano or micro surface structure (samples S1 and S2). In addition, the cells cultured on S2 showed a higher expression in integrin subunits α5 and β1 than that on S1. 3.6. BMP2 signaling pathway-related gene expression The gene expressions of the BMP2 signaling pathway were further examined to explore the mechanisms of the surface structure induced osteogenesis. As expected, the samples S1-S3 with micro- and nano-structures up-regulated the BMP2 signaling pathway related genes as compared

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with the sample S0. BMP2 has two possible receptors, type I (BMPR1A and BMPR1B), and the receptor, type II (BMPR2). It is interesting to see that BMPR1A and BMPR1B were up-regulated by S1 and S3, while BMPR2 was up-regulated by S2 and S3. Considering that S1 only has micropatterned surface structure and S2 only has nanorod surface structure, this result suggests that the micropattern and nanorod might promote osteogenesis by activating different BMP receptor route. Furthermore, the expression of SMAD1, SMAD5 and SMAD4 in hBMSCs cultured on samples S1-S3 are all significantly higher than those cultured on S0 (P < 0.05) (Figure 7). Then, a significant difference between groups S1 and S2 was observed in gene BMPR1A, BMPR1B and SMAD4, while cells seeded on S2 showed a significant increase of BMPR2 expression as compared with that on S1. In addition, S3 showed the higher expression of these genes as compared to S1 and S2, indicating an additive activation of these genes by the combination of micropatterns and nanorods. 3.7. The effect of the different surface structure on gap junction communication in hBMSCs The cell-cell communication via gap junction in hBMSCs plays a crucial role in osteogenesis. Cx43 is a major connexin in gap junction communication in order to coordinate the synthesis of new bone [43]. Figure 8A shows the immunostaining of Cx43 in hBMSCs, in which the cells appeared to have a close connection when cultured on S0-S3 for 10 days. We ascertained the distribution of Cx43 (Figure 8A and Figure 8B: green), hBMSCs cultured on S0 with flat surface had few gap junction. In contrast, the amount of Cx43 is higher when hBMSCs cultured on S1-S3, and it was significantly higher in cells cultured on S3 than on S1 and S2. The quantitative analysis of Cx43 staining also showed the same trend (Figure 8C), indicating that the micro/nano hybrid structure could stimulate communication between cells. The result (Figure 8) revealed that the expression of Cx43 gene in hBMSCs cultured on S1-

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S3 was much higher than in cells cultured on S0, especial for S3. In addition, the Cx43 gene expression in hBMSCs cultured on S1 with nano-structure and S3 with hybrid structure was significantly up-regulated as compared to that of cells grown on S2 with single-scale structure. 3.8. The role of BMP2 in activation of integrins and cell-cell communication by surface topography In order to elucidate the role of BMP2 in the activation of integrins and cell-cell communication, BMP2 activity was blocked with antibody noggin and the expression of integrins and Cx43 was analyzed. The results showed BMP2 was inhibited (about 50%) in the presence of noggin, indicating that noggin has blocked the activation of BMP2 signaling pathway. And our data showed no significant difference between the expression of integrin subunit αv for groups with and without BMP2 blocking. However, integrin subunit β1 in hBMSCs cultured on S1 and S3 was significantly inhibited in the presence of noggin, suggesting that integrins are partially regulated by BMP2, in particular when cells are cultured on a surface containing micro/nano hybrid topography (Figure 9). Furthermore, expression of gap junction-associated Cx43 was significantly decreased with the addition of noggin as compared with the control group, showing that the autocrine BMP2 protein is critical for the nano- and micro- surface topography activated cell-cell communication.

4. Discussion The micro/nano hybrid structure can be frequently observed in nature such as gecko feet and butterfly wings, which exhibit multifunctional properties [44]. Bone tissue also consists of hierarchical structure both in micro- and nanoscales. In some previous studies, we have demonstrated that microstructure and nanostructure of bioceramics have the activity to stimulate

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bone regeneration [8,40]. However, it is unknown if the micro- and nano-structures affect bone regeneration through same mechanisms, and it is important to elucidate the mechanisms for designing optimal bone repairing biomaterials. Considering the different functions of the biological structures in different scales, it is reasonable to assume that the micro- and nanosurface structures of bioceramics may affect bone regeneration through different pathways, and if that is the case, the combination of micro- and nano-structures may have a synergistic stimulatory effect on bone regeneration. To answer this question, one of the challenges is to fabricate micro/nano hybrid structure because of the hardness and brittleness of bioceramics. In the present study, to prove our hypothesis, micro/nano hybrid structures on HA bioceramics were obtained successfully through the combination of template masking and hydrothermal treatment processes. The fabricated HA bioceramics have a micro-patterned surface structure, which was covered by the nano-crystal structure. This structural combination allows us to compare the stimulatory effect of different surface characteristics of bioceramics on osteogenesis by comparing with bioceramics only with micropattern or nano-structure. In bone tissue engineering approaches, once cells are cultured on the surface of biomaterials with micro- or nano-structure, the integrin mediated cell adhesion is usually the first cellular event, which further regulates upcoming cellular behaviors, for instance, cell proliferation, migration, and differentiation [3,12,45]. Integrins have been proved to be mechanoreceptors in some types of cells including hBMSCs [18]. Some studies indicated that micro- or nanostructure-activated integrins played an important role in osteogenic differentiation [11,14,15], and the nanostructure could facilitate clustering of integrins and provide the binding site of integrin-ligand [11]. However, it is unknown whether the micro- and nano-structures activate integrins in the same way, and we assume that micro- and nano-structures may activate integrins

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in a different way, so that the combination of the micro- and nano-structures have synergistic activation effect. Our results confirmed our assumption and revealed for the first time, that micro-patterns (S2) significantly stimulated expression of integrin subunits α5 and β1 as compared with that on the nano-structure (S1), and both the micro-structure and the nanostructure could up-regulate integrin subunit αv. However, the reason for the difference of integrin activation on different micro/nano-surface structure still needs to be investigated. Some studies have shown that when the size of the surface nano-structure of biomaterials was less than 300 nm, the cell shape constraint could be generated, which further may determine the adhesion structure for integrin localization [46,47]. Thus we speculate that one reason might be related to the difference of cell constraint condition on different micro/nano structure, and integrins could be restricted to the attachment site and spatially separated with ligand at nanoscale structure, while integrin-ligand binding was not limited on the micropattern. Some studies suggest that integrin subunit β1 can provide the most binding sites of ECM and further mediate cell adhesion on biomaterials, and integrin α5β1 is the receptor of fibronectin which plays an important role in the initial contact stage when cells are seeded on biomaterials, and integrin subunit αv could also bind to ECM and strengthen cell adhesion [48,49]. Thus, our results revealed that the microstructure had a higher promotion than the nano-structure in the early cells adhesion [48,50], and then enhance subsequent expression of early osteogenic differentiation markers [51,52]. In addition, our data indicated that the micro/nano hybrid structure could provide synergistic topographical stimulation on integrin expression, which might be attributed to the different roles of micro- and nano-structures in regulating osteogenic differentiation. As an osteoinductive growth factor, BMP2 not only induces oeteogenic differentiation, but also promotes cell adhesion and migration [53,54], which suggests that BMP2 may co-function

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with integrins in regulating both cell adhesion and osteogenic differentiation. In this study, we first investigated the effect of micro- and nano-structures on BMP2 signaling pathway with the hypothesis that the micro-structure may affect BMP2 signaling differently as compared to the nano-structure. We found that both the micro- and nano-structures could enhance BMP receptors, downstream SMADs including SMAD1 and SMAD5 in BMP2 signaling pathway. However, we indeed identified a difference between the micro- and nano-structures in activating SMADs expression. The micro-patterns (S2) significantly stimulated more expression of the commonmediator SMAD4 than the nano-structure. Moreover, an obvious synergistic effect of the micro/nano hybrid structure was observed, and the results demonstrated that the hybrid structure significantly enhanced the expression of BMP2 signaling pathway-related genes as compared to the single type of structure such as micro- or nano-structure. Furthermore, the activated SMADs may form a complex and moves into the nucleus to activate transcription factor Runx2 for promoting osteogenic differentiation [55]. More interesting is that we confirmed our hypothesis and observed a difference between the micro- and nano-structures in the activation of the expression of BMP2 receptors. The nanorod structure activated more receptor, type I (BMPR1A, BMPR1B), while the micropattern structure activated more receptor, type II (BMPR2). It is interesting to see the correlation of this result with that of the activation of integrins, in which the micro-structure significantly stimulated expression of integrin subunits α5 and β1 than the nanostructure, indicating a possible correlated activation of BMPR2 and integrin subunits α5 and β1. The difference of the activation of genes in BMP2 signaling pathways by micro- and nanostructures may explain the synergistic stimulation of the micro/nano-hybrid structure on osteogenesis as compared to micro- or nano-structure only. Some previous studies have shown that integrins played a key role in BMP2 function, such

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as stimulating the expression of osteogenesis genes and inducing matrix mineralization, and integrin subunits αv and β1 could overlap with BMP receptors BMPR1, BMPR2, suggesting that integrins could facilitate BMP2 to bind to its receptors when integrins interacted with ECM. Moreover, studies have also demonstrated that additive BMP2 enhanced the αvβ1 integrins expression [17]. However, the effect of the micro- and nano-structures on the interaction between integrins and BMP2 in BMP2 signaling pathway are unknown. Therefore, it is important to know the roles of integrins in activation of BMP2-related signaling pathway and the effect of BMP2 protein on integrins. Our results firstly showed that the integrin α5β1 expression on microstructure was higher than that on nano-structure. In addition, we found in this study that the integrin subunit β1 was inhibited by blocking BMP2 activity. The decrease of β1 expression might reduce the adhesion of cells on ECM, because β1 has been demonstrated to provide the most binding sites of ECM and play a role in the cell-materials adhesion [56]. Our result also indicates that BMP2 protein could activate some of the integrin subunits when the BMP2 signaling pathway was activated in osteogenic differentiation induced by micro- or nanostructure. It is known that cell-cell communication through gap junction plays a key role in tissue regeneration via stimulating cell differentiation [26,57]. Previous studies indicated that activated integrins could directly interact with Cx43 and induce opening of the Cx43 hemichannel, which is a major portal in cell-cell contact for the exchange of factors responsible for bone remodeling and regulation of cellular function through activating some signaling pathways [34]. In addition, some studies found that cell-cell contact could modulate BMP2 function in chondrogenic differentiation, and in turn, BMP2 could also up-regulate Cx43 expression resulting increased chondrogenic differentiation, indicating that BMP2 signaling pathway could enhance cell-cell

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communication during chondrogenic differentiation [28]. Since chondrogenic differentiation is closely related to osteogenesis, we assume that the surface topography induced activation of BMP2 signaling may also be correlated with gap junction-mediated intercellular communication in osteogenic differentiation of stem cells. Indeed we confirmed that HA bioceramics with either micropattern or nanorod structure activated both BMP2 and Cx43 expression. Furthermore, cells cultured on nano-surface (S1) showed higher Cx43 expression than that on the micro-patterned surface (S2), which was similar as the effect of nano-surface on the expression of BMPR1, indicating a possible correlated activation of cell-cell contact and BMPR1. Here we also observed that the micro/nano hybrid structure exhibited higher stimulation effect on Cx43 expression as compared to the micropattern or nanorod alone, suggesting a synergistic role of micro- and nano-structures. A possible explanation of this synergistic effect is that micro- and nano-structures play different roles in activating osteogenic signaling pathways and the micropattern could precisely control the spatial position of cells and further enhance gap junction-mediated cell-cell contact [24], and the nano-structure could promote gap junctionmediated cell-cell communication mainly via regulating cell behaviors including adhesion, spreading, migration and aggregation [58,59]. In addition, our result revealed that the Cx43 expression was partially inhibited by blocking BMP2 activity, indicating a close correlation between the activated BMP2 signaling pathway and the cell-cell communication in osteogenic differentiation. Hence, the osteogenic differentiation induced by surface structure may be firstly controlled through the activation of integrins, which then further activated cell-cell communication. The activated cell-cell contact regulates BMP2 signaling pathway so that BMPR1 could be activated on the surface of the nano-structure. Meanwhile, BMP2 may in turn modulate Cx43-related cell-cell communication.

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Based on all the results, the possible mechanism of the osteogenic differentiation of BMSCs induced by micro/nano hybrid surface structure can be described in Figure 10. When stem cells attach on the bioceramic surface, the micro- and nano-structures activate integrins including α5β1 and αvβ1, and more α5β1 is expressed on micro-structure as compared with nanostructure, while there is no significant difference of αvβ1 expression on the micro- and nanostructures. The activated integrin α5β1 further activate BMP receptor, type II (BMPR2). At the same time, integrin αvβ1 might activate BMP receptor, type I (BMPR1). Then, the downstream SMADs including SMAD1, SMAD5 and common-mediator SMAD4 are activated. The activation of SMADs further up-regulates Runx2 which promote expression of other osteogenic genes such as ALP, COL1 and OCN. On the other hand, the activated integrins promote Cx43mediated cell-cell interaction, which also enhances Runx2 expression to promote osteogenic differentiation. In addition, the activated Cx43 modulates BMP2 signaling pathway, which might up-regulate in part BMPR1 on nano-structure. Moreover, the BMP2 protein in the activated BMP2 signaling pathway does not only enhance the activation of integrin subunits, but also promote the expression of Cx43 to stimulate osteogenic differentiation. Therefore, this mechanism explains the synergistic effect of micro- and nano-structures on the activation of osteogenic differentiation of BMSCs because of the different roles of the micro- and nanostructures in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cellcell communication. Although we have described the direct effect and possible mechanism of the surface micro/nano structure on osteogenic differentiation, it is important to indicate that, generally speaking, the difference of surface micro and nano structures may also affect other surface characteristics such as the degradation rate, surface area, and the ability to adsorb proteins, which

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may also affect cell behaviors and should not be ignored. Therefore, some other material characteristics related to the surface micro/nano- structure, such as the degradation of the HA ceramics, specific surface areas, and protein adsorption were measured in our study. According to the results, the release of Ca and P ions were limited as expected for well crystalized HA, and so the chemical effect of the HA bioceramics with micro/nano surface structure could be ignored. In contrast, the surface area measurement showed that all the micro and nano surface have higher specific surface area than that of the flat surface, and the micro/nano hybrid structure revealed highest surface area among all the micro/nano structure groups. Furthermore, different micro and nano surface structures showed clearly different protein adsorption ability. The micro and nano surface structures showed higher adsorption of fibronectin than flat surface, and the micro/nano hybrid surface is clearly higher than nano and micro surfaces, which is similar as the changes of surface area with different surface structures indicating a correlation between the surface area and protein adsorption in particular the adsorption of fibronectin. Previous studies have shown that fibronectin as one of the most important cell adhesion proteins could bind to integrins for regulating cell attachment and other cellular behaviors [60]. Our results suggest that the microand nano- structures may activate integrins through two different mechanisms. One is the direct activation through direct contact with cells, and the other is the indirect activation through the adsorption of fibronectin. It is worth noting that the expression of osteogenic genes during the osteogenic differentiation will vary with the time, and high expression of different genes occurs at different stage of cell differentiation [61,62]. BMP2, as an important osteogenic factor, is usually expressed at early stage [63]. Considering the complexity of the material surface structure and its influence on cells behaviors and one of the main focus of the present study on BMP2 signaling

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pathways, we only investigated osteogenic gene expression of cells including BMP2 at a fixed time for 7 days as proposed in some literatures [64,65] with the emphasis on comparison of the bioactive effects of the different micro/ nano-structure. However, the time dependent influence of the micro- and nano- structures on the expression of osteogenic genes should be concerned in further studies.

5. Conclusions The micro/nano hybrid structure with nanorods and micropatterns were successfully fabricated on bioceramic surface. The effect of micro-, nano-, and micro/nano hybrid structures on osteogenic differentiation of BMCs was investigated. The results revealed that the micro/nano hybrid structure significantly enhanced the cell behavior including the adhesion, proliferation and osteogenic gene expression indicating a synergistic effect of micro- and nano-surface structures on stem cell behavior. Furthermore, the results revealed that the micro/nano hybrid surface structure firstly activated integrins when cells attached on bioceramics, which further activated BMP2 signaling pathway and Cx43-related cell-cell interaction, and BMP2 could also interact with Cx43 to enhance osteogenic differentiation. In addition, the activated BMP2 protein could in turn regulate some integrin subunits and Cx43 expression. The synergistic effect can be explained by the finding that the micro- and nano-surface structures activated different integrin subunits and BMP2 receptors. In addition to the direct effects of the surface structures on osteogenic differentiation by firstly activating integrins, the surface structure could also indirectly regulate integrins for subsequent cell adhesion and osteogenic differentiation by enhancing fibronectin adsorption. Our results suggest that micro- and nano-structures have different roles in regulating stem cell behavior and need to be considered in designing optimal

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biomaterials for bone tissue engineering, and the combination of the structure in different scales may lead to further enhancement of the biological activity of the bioceramics as compared to the single type of surface structure.

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Scheme 1. Preparation scheme of HA bioceramics S0-S3 with the different surface structures, including plat structure (S0), nanorod structure (S1), micropatterned structure (S2) and micropattern/nanorod hybrid structure (S3).

Figures captions Figure 1. FESEM images of the control sample S0 with a smooth ceramic surface, and the HA bioceramics with nanorod surface structure (S1), micropatterned surface structure (S2) and micropattern/nanorod hybrid surface structure (S3). The inserts in the top-right corner of S0, S1, S2 and S3 were the corresponding high magnification images, respectively. Figure 2. A) Confocal images showing cytoskeleton of hBMSCs cultured on the samples S0-S3 for 24 h; B) Quantification of cell spreading area. * p < 0.05, ** p < 0.01. Figure 3. The proliferation of hBMSCs cultured on the samples S0-S3 for 1, 3 and 7 days. A mark (*) indicates significant differences between S0 and the other samples, a mark (＆) indicates significant differences between S1 and the other samples, a mark (#) indicates significant differences between S2 and the other samples, p < 0.05. Figure 4. ALP activity of the cells cultured on the samples S0-S3 with different structure surface after 14 days: A) ALP staining. B) The quantitative results of ALP activity. * p < 0.05, ** p < 0.01. Figure 5. Osteogenic genes expression of hBMSCs cultured on S0-S3 for 7 days. A mark (*) indicates significant differences between S0 and the other samples, a mark (＆) indicates significant differences between S1 and the other samples, a mark (#) indicates significant differences between S2 and the other samples, p < 0.05.

Figure 6. Integrin expression of hBMSCs cultured on S0-S3 for 7 days. A mark (*) 1

indicates significant differences between S0 and the other samples, a mark (＆) indicates significant differences between S1 and the other samples, a mark (#) indicates significant differences between S2 and the other samples, p < 0.05.

Figure 7. Relative mRNA expressions of BMP2 signaling pathway genes BMPR1A, BMPR1B, BMPR2, SMAD1, SMAD4, and SMAD5. hBMSCs were cultured on S0-S3 for 7 days. A mark (*) indicates significant differences between S0 and the other samples, a mark (＆) indicates significant differences between S1 and the other samples, a mark (#) indicates significant differences between S2 and the other samples, p < 0.05.

Figure 8. Immunofluorescence labeling showing Cx43 expression and localization in hBMSCs cultured on S0-S3 after 10 days. (A) Immunostaining of Cx43, (B) immunostaining of cultured hBMSCs including Cx43 and cells cytoskeleton (green, Cx43; red, actin; blue, nuclei), (C) Quantification of Cx43, (D) Cx43 gene expression in hBMSCs cultured on S0-S3 after 10 days. * p < 0.05, ** p < 0.01.

Figure 9. BMP2 gene, integrins and Cx43 expression in hBMSCs cultured on S0-S3 for 4 days after blocking BMP2 activity. The control group was labeled as “- noggin”, and the experimental group was marked as “+ noggin”. * p < 0.05, ** p < 0.01. Figure 10. Summary of possible synergy mechanism of the micro/nano hybrid structure on osteogenic differentiation. Table 1. The intensity of diffraction peaks for (002) and (211) and orientation degree. Table 2. The surface area of the samples S0-S3.

2

Scheme 1.

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Figure 1.

37

Figure 2.

38

Figure 3.

39

Figure 4.

40

Figure 5.

41

Figure 6.

42

Figure 7.

43

Figure 8.

44

Figure 9.

45

Figure 10.

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Table 1. The intensity of diffraction peaks for (002) and (211) and orientation degree diffraction peak intensity sample orientation degree (002) crystal plane (211) crystal plane S0 1670 ± 119.55 5908 ± 239.10 0.28 S1 1584 ± 11.51 4682 ± 254.03 0.34 S2 1597 ± 97.37 5562 ± 198.32 0.29 S3 1604 ± 92.21 5164 ± 289.84 0.31

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Table 2. The surface area of the samples S0-S3 sample

S0

S1

S2

S3

Surface area (m2/g)

0.10 ± 0.016

0.40 ± 0.038

0.32 ± 0.029

0.47 ± 0.030

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Graphical abstract

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Statement of Significance The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro- and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cellcell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect.

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