Role of adsorbed proteins on hydroxyapatite-coated titanium in osteoblast adhesion and osteogenic differentiation

Sci. Bull. DOI 10.1007/s11434-015-0753-8

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Article

Materials Science

Role of adsorbed proteins on hydroxyapatite-coated titanium in osteoblast adhesion and osteogenic differentiation Sai Wu • Xuanyong Liu • Changyou Gao

Received: 9 November 2014 / Accepted: 30 December 2014 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2015

Abstract The method of plasma-spray coating of hydroxyapatite (HA) onto pure titanium has been demonstrated to be effective to enhance the osteogenic differentiation and accelerate bone regeneration. Yet it is still a big challenge to figure out the interplay among implant surface properties, adsorbed proteins and cell-surface interactions. In this study, the plasma-sprayed HA-coated titanium (HA–Ti) surface was compared with the titanium substrate in terms of protein adsorption, cell adhesion and differentiation. The phase composition, wettability and topography were characterized. Compared to the Ti substrate, the HA–Ti had a smaller water contact angle, but larger micro-scale roughness, and showed a poorer ability to adsorb fibronectin (Fn), bovine serum albumin (BSA) and serum proteins. However, it could adsorb larger amount of recombinant human bone morphogenetic protein 2 (BMP2). The osteoblasts and bone marrow mesenchymal stem cells (BMSCs) tended to adhere on the Ti substrate. By contrast, the BMSCs cultured on the HA–Ti showed a stronger tendency toward osteogenesis differentiation. Keywords Hydroxyapatite  Implants  Protein adsorption  Fibronectin  Bone morphogenetic protein  Mesenchymal stem cells

S. Wu  C. Gao (&) Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China e-mail: [email protected] X. Liu Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China

1 Introduction Titanium and its alloys are widely used in orthopedic and dental implants [1–4], and surgical instruments [5] for decades due to their excellent mechanical strength, outstanding corrosion resistance and low density. For example, titanium has been made into implants of load-bearing areas like titanium hips and titanium knees. Hydroxyapatite (HA), as the major inorganic component of natural bone, has been increasingly designed in implant materials to promote bone integration [6–9]. However, the low mechanical strength of normal HA generally restricts its applications. The combination of inert titanium and bioactive HA is one of the most ideal implant materials for bone replacement, which is supposed to possess excellent mechanical properties and outstanding biocompatibility. Several methods of modifying HA onto titanium surface have been reported, such as chemical vapor deposition, magnetron sputtering, electro-deposition, thermal spraying, pulsed laser deposition, biomimetic coating and hot isostatic pressing [10–13]. Because of the faster production and strong bonding between the HA layer and implant, the plasma spraying method has been widely used to prepare the HA coating on titanium substrate [14, 15]. Many researches have demonstrated that the properties (crystallization and roughness, etc.) of the plasma-sprayed HA have a great influence on the behaviors of cells such as osteoblast-like cells (MG-63) [16, 17]. Although the HA interface can effectively stimulate osteogenic differentiation and enhance bone formation in vitro and in vivo, the underlying mechanisms are still needed to be substantiated. It is known that the properties of adsorbed proteins on biomaterials are of crucial importance for cell adhesion, migration, proliferation and differentiation [18]. When an implant contacts with serum or

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body fluids, its surface is first contacted with water and inorganic salts, and then the surface water layer influences the adsorption of successively arrived proteins and other molecules. At last, cells will interact with the proteins precovered surface [19–21]. Therefore, the adsorbed proteins would be highly important for modulating the subsequent cell behaviors on biomaterials surface [22–24]. The celladhesive proteins, such as fibrinogen (Fn), vitronectin and fibronectin, are the main regulation factors in the initial cell adhesion, whereas the osteogenic differentiation proteins such as the family of bone morphogenetic proteins (BMPs) are known to possess the osteoinductive activity to promote osteogenic differentiation and enhance bone formation [25]. The proteins on biomaterials are able to stimulate cell adhesion, proliferation, migration and differentiation in a protein type- and amount-dependent manner [26–30]. This implies that understanding of the protein adsorption behaviors becomes primarily essential for unveiling the cell interaction with biomaterials. Recently, Anseth and coworkers [31] reported that the adsorbed serum proteins on poly(ethylene glycol) hydrogels facilitate cell differentiation. Varghese and co-workers [29] found that conformational changes of the pendant side chains driven by hydrophobicity leaded to different bindings of proteins, which played a considerably larger role in regulating adhesion, migration and differentiation of stem cells. So far numerous theoretical and experimental studies have been conducted to explore the essence of certain proteins and their effects on cell behaviors. Wu and coworkers [32] studied the adsorption simulation of fibronectin and found that the electrostatic interaction played a dominant role between HA and proteins. Anselme and co-workers [33] showed that the HA influenced positively on cell spreading compared to b-tricalcium phosphate (bTCP) due to the higher albumin and fibronectin adsorption. The plasma-sprayed HA with doped strontium on titanium alloy can promote osteoblast differentiation and new bone formation in vitro and in vivo [34]. Although the cell adhesion, differentiation and protein adsorption on the plasma-sprayed HA have been studied individually, the insight understanding of the complicated interplay among biomaterial interface, adsorbed proteins and cell behaviors is still ongoing. Hence, in this study, attempt is made to study the protein adsorption, cell adhesion and differentiation on the HA-coated Ti substrate, and to explore their relationships. Cell-adhesive proteins such as Fn, laminin and vitronectin have a central role in promoting cell adhesion. Bone morphogenetic protein 2 (BMP-2) is a well-known growth factor that can maintain the normal function of bone, and promotes osteodifferentiation of mesenchymal stem cells and thereby bone formation [35, 36]. Therefore, Fn (for adhesion) and BMP-2 (for differentiation) were chosen as the typical functional

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proteins for the adsorption study and were further compared with the adsorption of albumin and serum proteins. Preosteoblast cells MC3T3-E1 and bone marrow mesenchymal stem cells (BMSCs) were cultured to reveal the cell adhesion, morphology and differentiation. The relationship among the HA–Ti substrate, adsorption of Fn and BMP-2, and cell responses were analyzed.

2 Materials and methods 2.1 Materials and characteristics Bovine serum albumin (BSA, fraction V, lyophilized powder), fluorescein diacetate (FDA), propidum iodide (PI) and dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich (USA). Fetal bovine serum was obtained from Gibco (USA). Bovine Fn (Catalog Number: 1030-FN) was obtained from R&D Systems (USA). Fn antibody (C6F10, sc-73611) was obtained from Santa Cruz (USA). Recombinant human BMP-2 was purchased from Sino Biological Inc. (China). Micro-bicinchoninic acid (lBCA) assay regent kits were purchased from Pierce Biotechnology, Inc. (USA). Alkaline phosphatase kit and BCA assay kit were purchased from Nanjing KeyGen Biotech. Co. Ltd. (China). The water used in the experiment was purified by a Milli-Q water system (Millipore, USA). The HA coating was fabricated by plasma spraying of HA powder onto pure titanium substrate with a dimension of 10 mm 9 10 mm 9 1 mm according to the procedures reported previously [37]. The surface morphology of triplicate samples was observed by field-emission scanning electron microscopy (FESEM, Hitachi S-4800, Japan). The phase compositions of Ti and HA–Ti were examined by X-ray diffraction (XRD, Empyrean 200895, PANalytical B.V., Netherlands). The static water contact angle was measured with a DSA 100 contact angle measuring system (Kru¨ss, Germany) using a sessile drop method (drop volume = 2 lL). Surface roughness values of the samples were measured by surface profiler (Veeco, Dektak 150, USA). Each reported value was an average of at least three independent measurements. 2.2 Amount of protein adsorption on samples The adsorption amount of proteins was measured by lBCA assay kit, using standard procedures according to the manual instructions. Briefly, the Ti and HA–Ti substrates were placed into 48-well plates, and the protein solutions (5 mg/mL BSA, 30 lg/mL Fn, 40 lg/mL BMP-2, and 10 % phosphate buffered saline (FBS)) were added into plates, respectively. After being incubated at 37 °C for 4 h

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under static condition, the samples were taken out and washed with PBS three times to remove those loosely adsorbed proteins. The samples were then exposed to 5 % sodium dodecylsulfate (SDS) at 37 °C under shaking to release the tightly adsorbed proteins. The solutions were collected, and the amounts of proteins were quantified using the lBCA assay. 2.3 Activity of Fn detected by enzyme-linked immunosorbent assay (ELISA) The arginine-glycine-aspartic acid (RGD) activity in Fn adsorbed on the substrates was assayed by ELISA using the Fn antibody clones as the first antibody and the IgG-HRP as the second antibody. Briefly, after the Ti and HA–Ti substrates were immersed in 10 % FBS solution at 37 °C for 4 h, they were washed with PBS three times. The substrates were then incubated in 5 % BSA/PBS solution for 4 h and followed by incubation in Fn antibody/5 % BSA/PBS (1:200, v:v) clones at 37 °C for 1 h. After washed with 5 % BSA/PBS three times, they were incubated in IgG-HRP/5 % BSA/PBS (1:1,000, v:v) at 37 °C for 40 min, and rinsed five times with PBS. Before 0.5 mol/L H2SO4 was added to stop the reaction, the substrates were incubated in 3,30 ,5,50 -tetramethylbenzidine (TMB) solution at 37 °C for 30 min. The solutions were distributed in triplicate to a 96-well plate (200 lL) to measure the absorbance of the mixture solution at the wavelength of 450 nm. 2.4 Cell culture MC3T3-E1 preosteoblast cells were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (China). The cells were cultured in alpha minimum essential medium (aMEM, Gibco) containing ribonucleosides and deoxyribonucleosides, supplemented with 10 % FBS, 100 U/mL penicillin and 100 lg/mL streptomycin. The cultures were maintained at 37 °C in a fully humidified atmosphere of 5 % CO2 in air. The BMSCs were isolated from the bone marrow of young adult male Sprague–Dawley rats as previously described [9]. The procedures were performed in accordance with the ‘‘Guidelines for Animal Experimentation’’ issued by the Institutional Animal Care and Use Committee, Zhejiang University. Briefly, the BMSCs were obtained from the femoral shafts of rats by flushing out with PBS. The released cells were collected into a cell culture flask (Corning, USA) containing 10 mL of aMEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 lg/mL streptomycin, and incubated in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C. After the cells reached

about 80 % confluence, they were detached and sub-cultured. The BMSCs at passage two were used in this study. Before cell seeding, the HA–Ti and Ti substrates were sterilized in a 24-well plate by immersing in 75 % ethanol solution for 2 h, and then rinsed five times with sterilized PBS (pH 7.4). The samples were transferred into a new culture plate, onto which the cells were seeded at a density of 5 9 103 per well in serum-free medium. 2.5 Protein pre-adsorption and cell adhesion Protein pre-adsorption experiments on the Ti and HA–Ti substrates were carried out in the following three conditions, and the resulted substrates were used for culture with MC3T3-E1 and BMSCs (seeding density 5 9 103 per well) in serum-free aMEM for 4 and 24 h, respectively. (1) Cell adhesion in serum-free medium: the cells were seeded directly using aMEM; (2) cell adhesion on Fn-pre-incubated samples: the Ti and HA–Ti substrates were first incubated with Fn (30 lg/mL) overnight, rinsed three times with sterilized PBS, and then immediately seeded with cells; (3) cell adhesion on serum-pre-incubated samples: the Ti and HA–Ti substrates were firstly incubated in cell culture medium (aMEM containing 10 % FBS) overnight, rinsed three times with sterilized PBS, and then immediately seeded with the cells. 2.6 Cell staining and cell morphology The seeded MC3T3-E1 cells were allowed to attach on the substrates for 4 and 24 h. At the prescribed time points, they were incubated with 5 lg/mL fluorescein diacetate (FDA, Sigma-Aldrich) for 5 min to stain live cells, and 5 lg/mL propidum iodide (PI, Sigma-Aldrich) for 30 min to stain dead cells. The images of cells were recorded under a fluorescence microscope (IX81, Olympus, Japan). The numbers of live and dead cells were obtained by analyzing the images of cells using ImageJ analysis software. To observe the morphology of BMSCs, the substrates with cells were taken out after being cultured for 24 h, and rinsed with PBS to remove the nonadherent cells. After that the substrates were fixed with 4 % paraformaldehyde solution, and followed by rinsing with PBS twice. Then, the samples were treated with 0.5 % Triton X-100/PBS to permeabilize the cells for 10 min. After rinsing three times with PBS, the samples were blocked with 1 % BSA/PBS solution for 30 min at 37 °C. Next, the samples were stained with rhodamine phalloidin (Invitrogen, USA), 40 ,6diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 1 h, and followed by rinsing three times in PBS. The cells were observed under a confocal laser scanning microscope (CLSM, Leica TCS SP5, Germany).

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For SEM observation, the cells were cultured for 24 h on the samples, rinsed with PBS three times, and then dehydrated by a graded series of ethanol (10 %, 30 %, 50 %, 70 %, 80 %, 90 % and 100 %) for 20 min at each step. Finally, the samples were dried using a critical point dryer, and observed by FESEM after gold spraying. 2.7 Alkaline phosphatase (ALP) activity The BMSCs were cultured on Ti and HA–Ti substrates for 1, 3, 5 and 7 d, respectively. The samples were washed with PBS three times and treated with 0.5 % Triton X-100/ PBS at 4 °C overnight. The ALP activity was assayed with alkaline phosphatase kit. The amount of total proteins on substrates was determined using a BCA assay kit. The ALP activity per lg of protein was reported. 2.8 Statistical analysis The experimental data are expressed as mean ± standard deviation, and the significant difference between groups was analyzed using one-way ANOVA in the Origin software. The significant difference level is set as P \ 0.05.

3 Results 3.1 Characterization of the substrates The topographic surface images of Ti and HA–Ti substrates are presented in Fig. 1a–d, showing that both the Ti and HA–Ti substrates were rough surfaces. Since the Ti

surfaces were not polished before HA coating, there were some sheet-like structures on the pristine Ti. On the HAcoated surface, there were partially melted HA particles. Under higher magnification, the structure of the HA coating appeared to be built up from micro-scale HA particles. The surface roughness of HA–Ti surface (Ra = 51.6 lm) was significantly larger than that of Ti substrate (Ra = 6.2 lm). The thickness of HA coating measured by SEM, and digital optical microscopy was approximately 200 lm (image not shown). Figure 1e presents the XRD patterns of Ti and HA–Ti substrates. The diffraction peaks of HA coating could be identified as hydroxyapatite (JCPDS No. 74-0566). The shape of sharp diffraction peaks indicated that the HA coating was well crystallized. The sharp and intensive (002) peak indicated that the HA crystals preferred to be aligned along c-axis. Furthermore, the water contact angle of Ti substrate was approximately 120° (Fig. 1f), while the HA–Ti substrate was hard to examine because the water droplet spread out immediately (Fig. 1g), showing the good wettability. 3.2 Protein adsorption on the substrates The amount of adsorbed proteins onto the HA–Ti and Ti substrates was quantified by lBCA assay. BSA had an ultra high concentration in serum plasma, and Fn had a significant role in cell adhesion and spreading, whereas BMP2 induced the osteogenic differentiation of mesenchymal stem cells. Therefore, the adsorption of BSA, Fn, BMP-2 and serum plasma, and the activity of adsorbed Fn were testified. Figure 2a shows that the amount of adsorbed

Fig. 1 (Color online) SEM images of a, b Ti and c, d HA–Ti surface of different magnifications; e XRD patterns; water contact angle picture of f Ti and g HA–Ti

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BSA, Fn and serum proteins on the Ti substrate was about two times higher than that on the HA–Ti surface. However, the HA–Ti adsorbed significantly larger amount of BMP-2 than the Ti substrate although the absolute difference in BMP-2 densities was not huge. Moreover, the ELISA result (Fig. 2b) reveals that the RGD activity of Fn adsorbed from 10 % serum was two times higher on the Ti substrate than on the HA–Ti substrate. This was basically consistent with the larger amount of Fn adsorbed on the Ti substrate, suggesting that the relative activity of Fn should not be influenced significantly on both type of surfaces. It was worth mentioning that the error bars for the pure proteins (BSA, Fn, BMP-2) were generally smaller compared with that of the FBS. The concentrations of the proteins might have a big influence on the variance between parallel samples, and the higher ones, such as the FBS (10 % serum), caused bigger variance due to the complicate protein adsorption behaviors.

between the serum-free and Fn-pre-inbubated became less significant. This result might be ascribed to the fact that cells also remodel their microenvironments by manipulating the extracellular matrix [38]. Comparatively, the serum-pre-incubated substrates had a stronger effect on promoting cell adhesion and spreading. Besides, the serumpre-incubated substrates significantly improved the number of live cells. Similarly, the adhesion of BMSCs was conducted in the same three conditions for 4 and 24 h. The FDA/PI double staining results for live/dead cells and cell numbers are shown in Fig. 4. The results were in good agreement with those of the MC3T3-E1 cell adhesion, suggesting that the cell adhesion behavior was cell typeindependent. Basically, the Fn adsorption generally facilitated the cell adhesion and spreading, whereas other types of plasma proteins should also take a positive role.

3.3 Cell adhesion

SEM images show that after being cultured for 24 h the BMSCs could attach and spread well on both the Ti and HA–Ti substrates without notable difference (Fig. 5a–d). The actin cytoskeletons were labeled by rhodamine phalloidin, and the cell nuclei were stained by DAPI for confocal microscopy observation (Fig. 5e, f). The BMSCs showed good spreading and adhesion on both substrates. However, they became more spreading out with more stressed and well-aligned actin fibers on the Ti substrate (Fig. 5e) compared with on the HA–Ti substrate (Fig. 5f). This difference in cell morphology could be attributed to both the different surface wettability (chemistry) and topography. The planar surface with appropriate wettability, like the Ti substrate, favored the cell spreading to a larger area [39]. The highly rough and hydrophilic surface, like the HA–Ti substrate, was not able to provide very strong cell-substrate interaction, leading to enhanced cell–cell interaction and thereby less spreading of cells on the substrate [40].

The cellular behaviors such as cell adhesion, migration and differentiation are highly dependent on the adsorbed proteins. Here, the cell adhesion was evaluated in serum-free medium on the substrates with different pre-treatment conditions: (1) in serum-free medium directly, (2) on Fnpre-incubated samples, and (3) on serum-pre-incubated samples. The adhesion numbers of MC3T3-E1 were measured by FDA/PI fluorescent staining for live/dead cells, and the total live/dead cell numbers were counted by ImageJ software. The results are displayed in Fig. 3. The cell number on the Ti substrate was more than 50 % larger than that on the HA–Ti substrate after being cultured for 4 and 24 h in serum-free condition (Fig. 3b, c). When the cells were cultured on the Fn and FBS-pre-incubated substrates, the overall cell numbers, in particular the live cell numbers were improved by about 30 % at 4 h (Fig. 3b). However, at 24 h (Fig. 3c), the difference of cell numbers

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Fig. 5 a–d SEM images and e, f confocal microscopy images of BMSCs adhered on Ti (a, b, e) and HA–Ti (c, d, f) after being seeded for 24 h, respectively. Actin filament (cytoskeleton) and the cell nuclei were stained red and blue colors, respectively

3.5 BMSCs differentiation The early osteogenic differentiation of BMSCs and their differentiation degree can be revealed by several characteristics such as morphology, production of marker products. ALP is one of the important marker proteins of BMSCs differentiation in the early stage. Therefore, the ALP activity was measured after the cells were incubated on both substrates for 1, 3, 5 and 7 d, respectively. As shown in Fig. 6, the ALP activity per cell was not obviously different among the tissue culture polystyrene (TCPS), Ti and HA–Ti substrates after 1 and 3 d incubation. However, at 5 and 7 d, the BMSCs cultured on the HA–Ti showed obviously higher ALP activity than those

on the TCPS and Ti substrates. This is a well-known feature for the HA biomaterials that are able to promote the osteogenesis and osteolinage differentiation of stem cells [41, 42].

4 Discussion Ti and its alloys are the most widely used biomaterials for replacing hard tissues. So far considerable efforts have been focused on the Ti implant surface technology, for example, bone-bonding ceramic coatings to promote integration [43]. HA has the similar chemical constituent as human hard tissue and is one of the most important

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bioceramics for medical and dental applications [6, 7]. No matter in which type HA forms, including granules, pure ceramic scaffolds, composite scaffolds, or coating layers, HA has shown to be effectively osteoconductive and osteoinductive, stimulating cell osteogenic differentiation and enhancing new bones formation in vivo and in vitro. Yoshikawa et al. [44] prepared interconnected porous HA ceramics showing superior osteoconduction. Chang and co-workers [45] fabricated distinct nanostructured HA ceramics, which selectively adsorbs specific proteins such as Fn and Vn in plasma, and stimulates osteoblast adhesion, growth, and osteogenic differentiation. Chung and coworkers [46] prepared a three-dimensional porous poly(D,L-lactic-co-glycolic acid)/HA composite scaffold, which can enhance new bone tissue regeneration successfully. Tang and co-workers [47] coated HA onto a Mg–Zn substrate using an electrochemical method. The HA coating improves the bioactivity of Mg–Zn substrate, achieving good cellular proliferation and differentiation. It is well known that many cell behaviors are related to the properties of adsorbed proteins on substrates. For example, Fn plays an important role in the initial cell attachment and spreading on biomaterials because it can specially bind to integrins on cell membranes [20, 48, 49]. In addition, the BMPs family (including BMP-2, BMP-4, BMP-6, and BMP-7) are able to accelerate bone repair and bone formation [25]. Particularly, BMP-2 can effectively promote cell differentiation toward osteoblast phenotype [50, 51]. Understanding the role of adsorbed proteins on the implant surfaces is still a great challenge. Our results showed that the HA coating had a more rough, but better hydrophilic surface compared to the pristine Ti substrate, resulting in the difference in protein adsorption and cell behaviors. Many previous reports have shown that the surface chemistry and surface topography play primary roles in cell adhesion and differentiation,

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which are closely correlated with the adsorbed proteins. For example, Chen and co-workers [52] found that cell adhesion and proliferation were hampered by the lotus leaflike topography, in which the adsorption of adhesive proteins was weakened. Besides the physical structure and chemical composition of biomaterials [53], the protein adsorption on materials is closely related to the physicochemical property of proteins, such as the size, charge and flexibility [54, 55]. Fn contains two almost identical subunits of about 220 kDa each, which is cross-linked by disulfide bonds in the C-terminal with a maximum length of 140 nm and average diameter of the strand of 2 nm for each subunit [56]. BMP-2 has a much smaller size of about 26 kDa with two subunits cross-linked by disulfide bond [57]. The HA–Ti surface adsorbed relatively more BMP-2 over Fn, showing selective property to the types of proteins to some extent. Consequently, it promoted BMSCs osteogenic differentiation, but hampered MC3T3-E1 and BMSCs adhesion due to the less adsorption of Fn and other serum proteins. The good consistency between the adsorbed proteins and cell behaviors substantiates the significant role of functional proteins adsorbed on the substrate. Due to the complication of protein adsorption, a tiny change of adsorbed amount, molecular weight, size, configuration or the sequence of amino acids, would produce a great variation in cell behaviors. In fact, the concentration of BMP-2 was critically low in the real blood, and thereby the selectivity of HA–Ti might take a more important role in terms of BMP-2 adsorption and subsequent differentiation of BMSCs. The cell attachment assay showed that the adherent MC3T3-E1 cell numbers on the Ti substrate were larger than those on the HA–Ti at 4 or 24 h. This result was in accordance with the larger adsorption of Fn and FBS on the Ti surface, and was substantiated by the fact that larger numbers of viable cells were found on the Fn and FBS-preincubated substrates. Besides Fn, other types of serum proteins should also play an important role in the cell adhesion. These adhesive proteins include but not limited to laminin and vitronectin, which have a smaller concentration compared with Fn but may take an effect in bridging the cell adhesion depending on the surface physiochemical properties [58]. In the serum-free culture condition, there were a larger number of dead cells compared with that in the serum-containing condition, which was generally the case as reported previously [59]. The ability of HA–Ti in promoting osteogenic differentiation of BMSCs was consistent with the previous studies [60]. Compared with the TCPS and Ti substrate, there was a sharp increase (about three times) in the ALP activity level on the HA–Ti after the BMSCs were cultured for 5 and 7 d. This result is consistent with the enhanced

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adsorption of BMP-2 and decreased adsorption of Fn on the HA–Ti, leading to a smaller number of adherent cells and more clustered cell morphology, but stronger differentiation of BMSCs toward the osteogenesis linage at the same culture conditions.

5 Conclusion The HA-coated Ti substrate was prepared by plasma spraying coating. It was built up from HA particles and had a rough but hydrophilic surface. The amounts of Fn and serum proteins adsorbed on the HA–Ti were significantly smaller than those on the Ti substrate, whereas the adsorption of BMP-2 on the HA–Ti was enhanced, revealing the selectivity of HA–Ti to the type of proteins to some extent. The phenomenon could be explained by a combination of physicochemical properties of substrates and the geometric structures of proteins. The osteoblast cells and stem cells tended to adhere with well spreading morphology on the Ti substrate, which adsorbed more Fn and other serum proteins. However, the BMSCs cultured on the HA–Ti had a higher osteogenic activity as a result of selective adsorption of BMP-2. Acknowledgments This work was supported by the National Basic Research Program of China (2011CB606203) and the National Natural Science Foundation of China (21434006, 21374097). Conflict of interest of interest.

The authors declare that they have no conflict

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