Materials Science and Engineering C 37 (2014) 120–126

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Surface functionalization of nanoporous alumina with bone morphogenetic protein 2 for inducing osteogenic differentiation of mesenchymal stem cells Yuanhui Song a, Yang Ju a,⁎, Yasuyuki Morita a, Baiyao Xu a, Guanbin Song b a b

Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603, Japan Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People's Republic of China

a r t i c l e

i n f o

Article history: Received 18 October 2013 Received in revised form 16 December 2013 Accepted 5 January 2014 Available online 9 January 2014 Keywords: Nanoporous alumina Bone morphogenetic protein 2 Mesenchymal stem cells Proliferation Differentiation Tissue engineering

a b s t r a c t Many studies have demonstrated the possibility to regulate cellular behavior by manipulating the specific characteristics of biomaterials including the physical features and chemical properties. To investigate the synergistic effect of chemical factors and surface topography on the growth behavior of mesenchymal stem cells (MSCs), bone morphorgenic protein 2 (BMP2) was immobilized onto porous alumina substrates with different pore sizes. The BMP2-immobilized alumina substrates were characterized with scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Growth behavior and osteogenic differentiation of MSCs cultured on the different substrates were investigated. Cell adhesion and morphological changes were observed with SEM, and the results showed that the BMP2-immobilized alumina substrate was able to promote adhesion and spreading of MSCs. MTT assay and immunofluorescence staining of integrin β1 revealed that the BMP2-immobilized alumina substrates were favorable for cell growth. To evaluate the differentiation of MSCs, osteoblastic differentiation markers, such as alkaline phosphatase (ALP) activity and mineralization, were investigated. Compared with those of untreated alumina substrates, significantly higher ALP activities and mineralization were detected in cells cultured on BMP2-immobilized alumina substrates. The results suggested that surface functionalization of nanoporous alumina substrates with BMP2 was beneficial for cell growth and osteogenic differentiation. With the approach of immobilizing growth factors onto material substrates, it provided a new insight to exploit novel biofunctional materials for tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Tissue engineering is a radical new approach to repair and replace damaged or diseased tissues. It involves the use of a combination of cells, engineered scaffolds and suitable biochemical and physiochemical factors to improve or replace biological functions [1]. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues by assembling engineered 3-dimensional (3D) biocompatible scaffolds, cells, and biologically active molecules into functional structures resembling native tissues [2–11]. The major challenge now facing tissue engineering is the need of scaffold for more complex functionality, as well as both functional and biomechanical stability destined for transplantation. The ideal scaffolds provide a framework and initial support for cell attachment, proliferation, differentiation and formation of extracellular matrix (ECM) [12]. Material surface properties have significant effects on cellular behavior. Several features of the implant surface such as chemical composition, topography, roughness, and stiffness play important roles in

⁎ Corresponding author. Tel.: +81 52 789 4672; fax: +81 52 789 3109. 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.004

implant osteointegration [13,14]. Due to the biocompatibility and excellent mechanical properties, porous alumina substrates have received a great deal of attention in bone tissue engineering, since bone cells can penetrate throughout the interconnected pores and grow on their biocompatible surfaces, which would promote bone ingrowth by providing a 3D environment [15,16]. We have demonstrated that it was possible to influence cellular attachment, differentiation and mineralization of osteoblasts by changing the nanopore size of alumina [17]. Swan et al. demonstrated that nanoporous alumina with the pore size of 72 nm was favorable for osteoblast adhesion [18]. Our previous study proved that nanoporous alumina was able to promote the adhesion and proliferation of MSCs [19]. These reports indicated that nanoporous alumina could be used as an ideal cell culture scaffold in tissue engineering. Besides surface topography, local chemistry property of the substrate is considered as another important factor that affects cellular behaviors, which regulates cell–material interaction. Surface coating with growth factors is a feasible approach to change the surface chemical property of substrates. However, quick diffusion of the coated growth factors from the substrates reduced the effective time, and the frequent high-dose growth factors may result in minus effect on cell behavior. To avoid deleterious effects, surface modification by immobilizing adhesive peptides, growth factors, or hormones onto

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

biomaterial surfaces via either chemicals or a physical strategy has been introduced and been proven as a valuable method to promote desirable cell–substrate interactions and to enhance cell functions at the celltissue interface[20–26]. Bone morphogenetic proteins (BMPs) play important roles in bone and cartilage formation. Among the members of BMP family, bone morphorgenic protein 2 (BMP2) has been demonstrated to be able to stimulate osteogenic differentiation and promote bone formation [27,28]. Kim et al. proved that BMP2-immobilized polycaprolactone scaffolds were able to stimulate the osteogenic differentiation of mesenchymal stem cells (MSCs) [29]. Lack of quickly self-renewable cell source is another bottleneck in the current development of tissue engineering. MSCs are a promising cell source in therapeutic and regenerative medicine. They can be easily harvested and cultured from a wide range of tissues. Furthermore, as undifferentiated cell types, MSCs are able to differentiate into a variety of cell types, such as osteoblasts, chondroblasts, myoblasts, adipocytes and tenocytes [30–34], under certain stimulation. Due to these advantages, MSCs have been becoming an attractive cell source for the repair of damaged or defective tissues/organs in tissue engineering. In this study, the synergistic effect of substrate topography and chemical cue (BMP2), which was immobilized onto alumina substrates with different pore sizes to fabricate surface functionalized materials, on the proliferation and osteogenic differentiation of MSCs was investigated.

121

bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin. Upon reaching confluence, cells were detached with 0.05% trypsin/ 0.02% EDTA, collected by centrifugation and resuspended in DMEM. The cells were counted using a hemacytometer and approximately 1 × 105 cells were seeded onto the alumina substrate. After cells have adhered to the material, the alumina membrane was transferred to a new cell culture plate. The medium was changed every 3 days. 2.5. Cell viability MTT assay was employed to estimate viabilities of the cells on the substrates. Briefly, after MSCs were cultured on the different substrates for 7 days, the medium was changed and 200 μl of MTT (5 mg/ml) (Wako, Japan) was added and incubated at 37 °C for another 4 h. The medium containing MTT was removed and 1.5 ml dimethyl sulfoxide (DMSO) was added to each well of the plate to dissolve the formazan crystals. The optical density of the solution was measured at the wavelength of 490 nm with a microplate reader (Bio-Rad 680, USA). 2.6. Immunofluorescence staining

Nanoporous alumina substrates with the pore size of 20 and 100 nm were purchased from Whatman International Ltd, England. The substrates were ultrasonically cleaned twice in ethanol for 30 min each, and then dried at room temperature. They were stored in a vacuum oven until use. Smooth alumina purchased from Alfa Aesar was used as control.

After 2 days of culture, MSCs were fixed with 4% paraformaldehyde at room temperature for 20 min. Samples were then washed three times with PBS and permeabilized with 0.25% Triton X-100 at room temperature for 30 min. The samples were then incubated with 1% bovine serum albumin (BSA)/PBS at 37 °C for 1 h. Subsequently, goat monoclonal antibody against integrin β1 (1:200) (Santa Cruz Biotechnology) was added and kept at 4 °C for overnight. Mouse-antigoat Rhodamine-conjugated secondary antibody (1:100) (Santa Cruz Biotechnology) was added at room temperature for 10 min. Samples were rinsed three times with PBS and stained with 0.5 μM FITCconjugated phalloidin at room temperature for 2 h. The nuclei of MSCs were stained with 10 μg/ml DAPI at room temperature for 5 min, after which mounting medium (10 μl) was dispensed on the samples. The stained samples were finally observed by a confocal laser scanning microscope (Nikon, Japan).

2.2. BMP2 immobilization

2.7. Cell morphological observation and analysis

The alumina substrates were submersed in a 2 mg/ml solution of dopamine (10 mM Tris buffer, pH 8.5) overnight in the dark [35,36]. The substrates were then rinsed with distilled water to remove unattached dopamine and dried under nitrogen flow. The polydopamine-grafted substrates were then submersed in BMP2 solution (100 ng/mL) (BioVision Incorporated, USA) with 10 mM Tris buffer (pH 8.5) and incubated overnight in a humid atmosphere at room temperature [36,37]. The substrates were then washed three times with sterile PBS to remove unattached BMP2 and air-dried in a sterile environment for the following cell experiments. The polydopamine-grafted alumina substrates were denoted as PDOP-alumina; the BMP2-immobilized PDOPalumina substrates were denoted as BMP2-PDOP-alumina.

To observe adhesion and morphologies of MSCs on the different substrates, the cells were rinsed with PBS after incubation for 4 days, then fixed with 2.5% glutaraldehyde (Wako, Japan) in PBS for 1 h at room temperature. After thorough washing with PBS, the cells were dehydrated in a graded series of ethanol (70%, 80%, 90% and 99.5%) (Wako, Japan) for 15 min each and air-dried at room temperature. The fixed samples were sputter-coated with gold (Canon E-200S, Japan) and imaged by SEM. To quantify the differences in cell morphology imaged by SEM, the length and width of MSCs were measured using ImageJ software. The ratio of obtained cell length to cell width was denoted as cell elongation ratio. At least 30 cells were measured in each group.

2.3. Surface characterization

2.8. Alkaline phosphatase activity

The surface topography of the alumina substrates was imaged by scanning electron microscope (SEM; JEOL 7000FK, Japan). The chemical composition of the substrates was determined by X-ray photoelectron spectroscopy (XPS) using a Model PHI 5600 system (Perkin elmer, USA) with an Al Kα source (1486.6 eV).

The quantitative detection of alkaline phosphatase (ALP) activity was determined by an assay based on the hydrolysis of pnitrophenylphosphate to p-nitrophenol to evaluate the osteogenic differentiation of MSCs. In brief, after MSCs were cultured on the different substrates for 1, 2 and 3 weeks, 20 μl of the cell lysate was added to 100 μl working reagent. The samples were then incubated at 37 °C for 15 min and the reactions were then stopped with 80 μl sodium hydroxide. The absorbance at the wavelength of 405 nm was measured with a spectrophotometric microplate reader (BioRad 680). ALP activity was normalized to the total intracellular protein production and expressed as micromoles per milligram protein per minute.

2. Materials and methods 2.1. Nanoporous alumina

2.4. MSC cell culture MSCs were purchased from the Health Science Research Resources Bank, Japan. The cells were cultured in 25 cm2 flasks at 37 °C in a humidified incubator containing 5% CO2. The culture medium was Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal

122

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

Total protein content was measured with bicinchoninic acid (BCA) protein assay. After incubation for 1, 2 and 3 weeks, MSCs were washed with PBS and then 300 μl of a detergent-based lysis buffer (M-PER Mammalian Protein Extraction Reagent, Pierce, USA) was added to collect total cellular protein. Total protein content in the cell lysates was measured using a BCA assay kit (Pierce Chemical Co., USA). In brief, 25 μl of triton lysate was added to 200 μl of BCA working solution and the mixture was then incubated for 30 min at 37 °C. The protein concentration was determined from the absorbance at 570 nm wavelength by the microplate reader. 2.9. Real-time RT-PCR Total RNA of the cells on the different substrates was extracted using an RNeasy Mini Kit (Qiagen, USA) according to manufacturer's instructions. Reverse transcript was performed using a High Capacity RNA-to-cDNA Kit (ABI) with approximately 1.5 μg total RNA in a final 20 μl reaction volume. Real-time PCR was performed with Taqman Gene Expression Master Mix (ABI, USA) on a ABI 7300 real-time PCR system. Predesigned MGB probes of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) and integrin β1 (ITGB1, Hs00559595_m1) were used to detect relative gene expressions. Gene expression levels of integrin β1 were normalized to those of GADPH and calculated using standard curve method. 2.10. Mineralization assay Alizarin red staining was used to detect mineralization. The staining was performed after incubation for 4 weeks, as described in a previous study [38]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) and treated with 40 mM alizarin red S (pH 4.1, Sigma) for 20 min at room temperature with gentle shaking. After aspiration of unincorporated dye, the samples were washed four times with distilled water with shaking of 5 min. For quantification of staining, 10% v/v acetic acid was added to each sample and incubated for 30 min with shaking. The surface staining on the substrate was collected with acetic acid (10%, v/v) and transferred to microcentrifuge tubes. The microcentrifuge tubes

were heated to 85 °C for 10 min and then centrifuged at 20,000 g/min for 15 min. The supernatant was transferred to a new microcentrifuge tube and neutralized with 10% v/v ammonium hydroxide. The absorbance of the supernatant was measured at the wavelength of 405 nm with the microplate reader. 2.11. Statistical analysis Data are represented as the mean ± standard deviation. Statistical analysis to compare the results between two groups was carried out by unpaired Student's t-test, and a value of p b 0.05 was considered to be statistically significant. 3. Results 3.1. Surface characterization The surface topography of alumina and BMP2-immobilized alumina substrates was characterized by SEM. Fig. 1a shows the representative SEM images of smooth alumina and nanoporous alumina substrates with the pore size of 20 and 100 nm. The surfaces were flat and circular pores were homogeneously distributed on the surface. According to the SEM images, the pore sizes of the nanoporous alumina substrates were measured through ImageJ. The pore sizes of the surfaces on which cells were cultured were 20 and 100 nm in average, respectively. Fig. 1b displays the SEM images of smooth alumina and nanoporous alumina substrates grafted with dopamine and further immobilized with BMP2. The surfaces of the alumina were partially covered by a thin layer which might be composed of polydopamine film and BMP2. To confirm the successful formation of polydopamine film and immobilization of BMP2 onto the nanoporous alumina substrates, the chemical composition of the substrate (20 nm) at various stages of surface functionalization was determined by XPS (Fig. 2). Fig. 2a shows the XPS spectra of PDOP-alumina, four elements including Al, C, N and O were measured. After the immobilization of BMP2 onto the PDOPalumina substrates, higher N content was detected in BMP2-PDOPalumina compared with the dopamine treated nanoporous alumina

Fig. 1. SEM images of (a) smooth alumina and nanoporous alumina substrates with different pore diameters without surface treatment and (b) BMP2-immobilized alumina substrates.

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

123

extensions were observed (Fig. 3a, black arrows in enlarged part). Similarly, MSCs cultured on BMP2-immobilized alumina groups also showed the same trend of cell spreading, morphological elongation and filopodia protrusions (Fig. 3b, black arrows in enlarged part). However, higher elongation ratio was detected in cells cultured on the untreated alumina substrates comparing with those cultured on BMP2immobilized alumina substrates. Cell morphological elongation was also quantitatively calculated (Fig. 4). The elongation ratio of MSCs increased with the increasing pore size of both the BMP2 treated and untreated porous alumina substrates. MSCs cultured on the 20 nm sized alumina (as well as the smooth alumina) exhibited a basically isotropic configuration with an overall average elongation ratio of around 4–5 in both untreated and BMP2-immobilized groups. Whereas, cells adhered to the 100 nm sized alumina substrate showed an average elongation ratios as large as 10 and 9 in both untreated and BMP2 immobilized conditions, which were significant larger than those cultured on the smooth and the 20 nm sized alumina substrates.

3.3. Integrin β1 expression

Fig. 2. XPS spectra of (a) BMP2-PDOP-alumina substrate and (b) PDOP-alumina.

(20 nm). The reason can be explained by which BMP2 molecules contain more N element than dopamine and the existence of BMP2 on the surfaces of the nanoporous alumina increases the amount of N element (Fig. 2a). Moreover, a decrease of Al, C and O contents was also observed due to the immobilization of BMP2 onto the substrate surface. The result further confirmed the successful conjugation of BMP2 onto the nanoporous alumina surface.

3.2. Analyses of cell morphological changes In order to evaluate the effects of substrate topography and BMP2 on cell morphological changes, MSCs morphologies on different substrates were observed using SEM after 4 days of culture (Fig. 3). The results showed that among the group cultured on untreated porous alumina, MSCs adhered to 20 nm sized alumina spread better than those cultured on 100 nm sized substrate (Fig. 3a). However, with the increasing pore size, elongated cell morphology and prominent filopodia

The cytoskeletal morphologies and integrin β1 expressions of MSCs were immunostained (Fig. 5). After incubation for 2 days, MSCs cultured on BMP2-immobilized alumina substrates showed a clearly enhanced integrin β1 expression compared with those cultured on untreated alumina substrates. The expression of integrin β1 was further analyzed by quantitative real-time PCR, which proved that surface functionalized alumina (20 nm) with BMP2 could promote the expression of integrin β1 (Fig. 5c).

3.4. Cell viability To evaluate the viability of MSCs on different substrates, MTT assay was employed. Fig. 6 shows the representative absorbance of formazan crystals (dissolved in DMSO) in MSCs cultured on BMP2-immobilized and untreated alumina substrates. MSCs grown on BMP2-immobilized alumina substrates showed significantly higher cell viabilities than those grown on untreated substrates (p b 0.05), which indicated that surface functionalization of alumina substrate with BMP2 was favorable for cell growth. With the increasing pore size of the alumina membranes, cell viability was decreased gradually in both BMP2immobilized and untreated alumina substrates.

Fig. 3. SEM images of MSC morphologies on (a) smooth alumina and nanoporous alumina substrates with 20 nm and 100 nm pore diameters without surface treatment and (b) BMP2immobilized alumina substrates. Black arrows indicate membrane protrusions.

124

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

Fig. 4. Quantitative analysis of cell morphological elongation. *(p b 0.05) denotes the significant differences between 100 nm pore sized alumina group and 20 nm pore sized alumina group, smooth alumina group (before and after BMP2 immobilization). Error bars represent means ± SD, at least 30 cells were measured in each group.

Fig. 6. Effect of nanoporous alumina substrates on cell viability. MSCs were cultured on different substrates for 7 days and cell viability was measured with a MTT assay. *(p b 0.05) denotes a significant difference. Error bars represent means ± SD, n = 3.

Fig. 5. Immunofluorescence images of MSCs adhered to different substrates for 2 days: (a) smooth alumina and nanoporous alumina substrates with 20 nm and 100 nm pore diameters and (b) BMP2 treated smooth alumina and porous alumina substrates. Cells were triple stained with actin filaments (green), cell nuclei (blue) and integrin β1 (red). (c) Quantitative PCR analysis of integrin β1 gene expression. MSCs were cultured on 20 nm sized alumina substrate for 2 days. Gene expression level was normalized to GADPH and calculated using standard curve method. Error bars represent means ± SD, n = 4.

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

125

3.5. ALP activity To investigate the osteogenic differentiation of MSCs on different substrates, ALP activity was measured. ALP is a membrane enzyme commonly recognized as a marker of osteoblastic differentiation [39]. Fig. 7 shows the ALP activities of MSCs cultured on different substrates after incubation for 1, 2 and 3 weeks. Significantly higher ALP activities were detected in cells cultured on BMP2-immobilized alumina substrates than those cultured on the untreated substrates (p b 0.05). MSCs cultured on 100 nm sized alumina substrate showed a relative higher ALP activity as compared with 20 nm sized group and smooth alumina in both BMP2-immobilized and untreated alumina substrates. 3.6. Mineralization of MSCs In order to evaluate the osteogenic differentiation of MSCs, mineralization was measured by Alizarin red staining. Fig. 8 displays the quantified mineralization of MSCs on different substrates after incubation for 4 weeks. MSCs cultured on BMP2-immobilized porous alumina substrates showed significantly higher mineralization as compared with the cells cultured on untreated substrates (p b 0.05). Moreover, the highest mineralization was detected in cells cultured on BMP2immobilized 100 nm pore sized alumina substrate. It is noted that the results for the untreated nanoporous alumina substrates are the same as those shown in reference [19] since the experiments were carried out at the same time. 4. Discussion In this study, a strategy of immobilizing BMP2 onto nanoporous alumina substrates was introduced and the synergistic effect of BMP2 and alumina substrate topography on the osteogenic differentiation of MSCs was investigated. Adhesion and cell spreading have been shown to be correlated with the ability of the cell to survive and to initiate proliferation on a substrate, with increased cell spreading associated with increased cell survival and cell cycling [40]. Our results showed that MSCs exhibited faster adhesion when cultured on BMP2-immobilized alumina substrates as compared with unmodified substrates. It was demonstrated that BMP2 promoted cell adhesion and regulated cell–matrix interactions by inducing the expression of fibronectin, integrin β1 and focal adhesion kinase [41,42]. Fong et al. also reported that BMP2 stimulated the migration of human chondrosarcoma cells via increasing the expression of integrin β1 [43]. Higher level of integrin β1 expression was detected

Fig. 7. ALP activities of MSCs cultured on different substrates for 1, 2 and 3 weeks. ALP synthesized by MSCs was normalized to total intracellular protein content. Results of the ALP activities were expressed in μmol/min/mg protein. *(p b 0.05) denotes a significant difference between the untreated group and BMP2-immobilized group. Error bars represent means ± SD, n = 4.

Fig. 8. Extracellular matrix mineralization of MSCs on different substrates after 4 weeks of incubation. *(p b 0.05) indicates significant difference between the untreated group and BMP2-PDOD treated group. Error bars represent means ± SD, n = 4.

in cells cultured on BMP2-immobilized alumina substrates, which indicated that BMP2 was able to promote the expression of integrin β1. The result proved that BMP2 promoted cell adhesion was related to the expression of integrin β1. Morphological study showed that the cells seeded on BMP2immobilized substrates exhibited more spreading morphology compared to those on untreated substrates. Oh et al. demonstrated that the adhesion and elongation of cells are determined by the initial adsorption of fibronectin and albumin from the culture medium [44]. For cells cultured on untreated alumina substrates, due to fewer proteindeposited sites on larger pore sized alumina, MSCs had to elongate the morphology and extend filopodia to find protein-deposited surface, and thus eventually forming an exceedingly elongated morphology on 100 nm pore sized alumina substrate. Whereas, for cells cultured on BMP2-immobilized alumina substrates, due to the pre-deposition of BMP2 molecules on the alumina surface, more protein-deposited sites were available for cell adhesion. Akino et al. demonstrated that BMP2 was able to promote the proliferation of MSCs [45]. Park et al. reported that the proliferation of osteoblast was significantly promoted when cultured on BMP2-immobilized nanofibrous chitosan membrane [46]. Compared with cells grown on untreated alumina substrates, MSCs cultured on BMP2-immobilized alumina substrates displayed higher cell viability, which indicated that BMP2 was advantageous for cell growth. Cell interactions with extracellular matrix and other cells are mediated by integrins, which control cellular activities, such as adhesion, cell morphological changes, proliferation and differentiation, in a synergistic manner with hormones and growth factors [47,48]. Integrins are transmembrane receptors that mediate cell-matrix or cell-cell adhesion. It is reported that integrin β1 is required for cell spreading, adhesion and proliferation [49–51]. This study showed that the cells cultured on BMP2-immobilized alumina substrates have a trend of higher integrin β1 expression than those cultured on untreated nanoporous alumina substrates (p N 0.05), which implied that BMP2 may promote the expression of integrin β1. The enhanced β1 integrin expression indicated that BMP2-immobilized alumina substrates were able to cluster integrin into focal adhesion (FA) complexes, resulting in activating of focal adhesion kinase (FAK) signaling pathway to promote proliferation [52–54]. As a marker of immature osteoblast and an essential element of ossification, ALP activity was quantitatively measured to prove the early osteogenic tendency. Higher ALP activities were detected in cells cultured on BMP2-immobilized substrates as compared with those cultured on untreated alumina substrates after incubation for 1, 2 and 3 weeks, respectively. Our results clearly demonstrated that surface

126

Y. Song et al. / Materials Science and Engineering C 37 (2014) 120–126

functionalized alumina with BMP2 promoted the osteogenic differentiation of MSCs. It was attributed to the synergetic effect of surface topography and grafted BMP2 on the surface. After incubation for 3 weeks, the ALP activity of MSCs was relatively lower than those after incubation for 2 weeks in each group. This noticeable phenomenon was due to the fact that the differentiation into mature osteoblasts of MSCs was in the later stage, whereas ALP activity increased in the early stage of osteogenic differentiation and decreased in the later stage [55]. Osteoblastic differentiation from MSCs to a matured osteoblast requires a series of steps involving a number of proteins expressed at each stage. ALP is regarded as a marker for early stage of osteoblast differentiation, whereas matrix mineralization is associated with the final differentiation phase. MSCs cultured on the BMP2-immobilized substrates showed significantly higher extracellular matrix mineralization than those cultured on untreated substrates. Moreover, MSCs cultured on 100 nm sized BMP2-immobilized alumina substrate showed a relative higher ALP activity. Also the higher mineralization was detected in cells cultured on 100 nm pore sized BMP2-immobilized alumina substrate as compared with 20 nm sized group. The reason may be that cell morphology is regarded as a determinant of cell growth, cytoskeleton arrangement, and differentiation for a variety of cell types. Cells cultured on 100 nm pore sized BMP2immobilized alumina substrate showed the largest cell morphological elongation, which indicates that cells were under the largest tensile stress, and the changes in cell morphology and tensile stress affected the osteogenic differentiation of MSCs. These results are consistent with the results of nanoporous alumina substrate in our previous study [19]. Taken together, these results indicated that BMP2immobilized alumina substrates can induce the osteogenic differentiation of MSCs. 5. Conclusions In this study, the synergistic effect of surface topography and BMP2 on the growth behavior and osteogenic differentiation of MSC was investigated. BMP2 was immobilized to nanoporous alumina substrate via introducing the intermediate of dopamine. SEM characterization and XPS spectra demonstrated that BMP2 can be successfully conjugated onto the alumina substrate by employing dopamine as mediation. We proved that surface functionalization of nanoporous alumina with BMP2 was able to promote cell adhesion and cell viability. Furthermore, BMP2-immobilized alumina substrate could significantly induce the osteogenic differentiation of MSCs. This study implied a potential application of surface modification in tissue engineering. Disclosure

[5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46]

The authors report no conflicts of interest in this work. [47]

Acknowledgment This work was supported by the Japan Society for the Promotion of Science under a Grant-in-Aid for Scientific Research (A23246024). References [1] [2] [3] [4]

R. Langer, J.P. Vacanti, Science 260 (1993) 920–926. V.K. Gupta, S. Agarwal, T.A. Saleh, Water Res. 45 (2011) 2207–2212. V.K. Gupta, A. Rastogi, A. Nayak, J. Colloid Interface Sci. 342 (2010) 533–539. V.K. Gupta, R. Jain, A. Mittal, M. Mathur, S. Sikarwar, J. Colloid Interface Sci. 309 (2007) 464–469.

[48] [49] [50] [51] [52] [53] [54] [55]

A. Mittal, V.K. Gupta, A. Malviya, J. Mittal, J. Hazard. Mater. 151 (2008) 821–832. A.K. Jain, V.K. Gupta, Suhas A. Bhatnagar, Separ. Sci. Technol. 38 (2003) 463–481. V.K. Gupta, A. Mittal, A. Malviya, J. Mittal, J. Colloid Interface Sci. 335 (2009) 24–33. V.K. Gupta, R. Jain, S. Varshney, J. Hazard. Mater. 142 (2007) 443–448. V.K. Gupta, A. Mittal, L. Krishnan, J. Mittal, J. Colloid Interface Sci. 293 (2006) 16–26. V.K. Gupta, A. Mittal, L. Kurup, J. Mittal, J. Colloid Interface Sci. 304 (2006) 52–57. A. Mittal, L. Kurup Krishnan, V.K. Gupta, J. Hazard. Mater. 117 (2005) 171–178. C.M. Agrawal, R.B. Ray, J. Biomed. Mater. Res. 55 (2001) 141–150. A. Wennerberg, T. Albrektsson, B. Andersson, J. Mater. Sci. Mater. Med. 6 (1995) 302–309. L.H. Li, Y.M. Kong, H.W. Kim, Y.W. Kim, H.E. Kim, S.J. Heo, J.Y. Koak, Biomaterials 25 (2004) 2867–2875. N. Tamai, A. Myoui, T. Tomita, T. Nakase, J. Tanaka, T. Ochi, H. Yoshikawa, J. Biomed. Mater. Res. 59 (2002) 110–117. K.A. Hing, Int. J. Appl. Ceram. Technol. 2 (2005) 184–199. M. Karlsson, E. Palsgard, P.R. Wilshaw, L. Di Silvio, Biomaterials 24 (2003) 3039–3046. E.E.L. Swan, K.C. Popat, T.A. Desai, Biomaterials 26 (2005) 1969–1976. Y.H. Song, Y. Ju, G.B. Song, Y. Morita, Int. J. Nanomedicine 8 (2013) 2745–2756. K.S. Masters, Macromol. Biosci. 11 (2011) 1149–1163. T. Pompe, K. Salchert, K. Alberti, P. Zandstra, C. Werner, Nat. Protoc. 5 (2010) 1042–1050. Y. Ito, S.Q. Liu, Y. Imanishi, Biomaterials 12 (1991) 449–453. C.K. Poh, Z.L. Shi, X.W. Tan, Z.C. Liang, X.M. Foo, H.C. Tan, K.G. Neoh, W. Wang, J. Orthop. Res. 29 (2011) 1424–1430. A.G. Karakecli, C. Satriano, M. Gumusderelioglu, G. Marletta, Acta Biomater. 4 (2008) 989–996. C.A. Custodio, C.M. Alves, R.L. Reis, J.F. Mano, J. Tissue Eng. Regen. Med. 4 (2010) 316–323. H.N. Zhang, C.Y. Lin, S.J. Hollister, Biomaterials 30 (2009) 4063–4069. V. Karageorgiou, L. Meinel, S. Hofmann, A. Malhotra, V. Volloch, D. Kaplan, J. Biomed. Mater. Res. A 71A (2004) 528–537. D.S. Benoit, S.D. Collins, K.S. Anseth, Adv. Funct. Mater. 17 (2007) 2085–2093. S.E. Kim, H.K. Rha, S. Surendran, C.W. Han, S.C. Lee, H.W. Choi, Y.W. Choi, K.H. Lee, J.W. Rhie, S.T. Ahn, Macromol. Res. 14 (2006) 565–572. L. Marinucci, S. Balloni, E. Becchetti, G. Bistoni, E.M. Calvi, E. Lumare, F. Ederli, P. Locci, Ann. Biomed. Eng. 38 (2010) 640–648. A.C.W. Zannettino, S. Paton, A. Arthur, F. Khor, S. Itescu, J.M. Gimble, S. Gronthos, J. Cell. Physiol. 214 (2008) 413–421. A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Cell 126 (2006) 677–689. D.L. Morganstein, P. Wu, M.R. Mane, N.M. Fisk, R. White, M.G. Parker, Cell Res. 20 (2010) 434–444. B.Y. Xu, G.B. Song, Y. Ju, X. Li, Y.H. Song, S. Watanabe, J. Cell. Physiol. 227 (2012) 2722–2729. J.H. Waite, Nat. Mater. 7 (2008) 8–9. H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426–430. H. Lee, J. Rho, P.B. Messersmith, Adv. Mater. 21 (2009) 431. J. Park, S. Bauer, K.A. Schlegel, F.W. Neukam, K. von der Mark, P. Schmuki, Small 5 (2009) 666–671. Y. Gotoh, K. Hiraiwa, M. Nagayama, Bone Miner. 8 (1990) 239–250. S.M. Frisch, K. Vuori, E. Ruoslahti, P.Y. ChanHui, J. Cell Biol. 134 (1996) 793–799. L. Nissinen, L. Pirila, J. Heino, Exp. Cell Res. 230 (1997) 377–385. A.K. Shah, J. Lazatin, R.K. Sinha, T. Lennox, N.J. Hickok, R.S. Tuan, Biol. Cell. 91 (1999) 131–142. Y.C. Fong, T.M. Li, C.M. Wu, S.F. Hsu, S.T. Kao, R.J. Chen, C.C. Lin, S.C. Liu, C.L. Wu, C.H. Tang, J. Cell. Physiol. 217 (2008) 846–855. S. Oh, K.S. Brammer, Y.S.J. Li, D. Teng, A.J. Engler, S. Chien, S. Jin, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 2130–2135. K. Akino, T. Mineta, M. Fukui, T. Fujii, S. Akita, Wound Repair Regen. 11 (2003) 354–360. Y.J. Park, K.H. Kim, J.Y. Lee, Y. Ku, S.J. Lee, B.M. Min, C.P. Chung, Biotechnol. Appl. Biochem. 43 (2006) 17–24. K. Burridge, M. ChrzanowskaWodnicka, Annu. Rev. Cell Dev. Biol. 12 (1996) 463–518. F.G. Giancotti, Dev. Cell 4 (2003) 149–151. T.J. Rowland, L.M. Miller, A.J. Blaschke, E.L. Doss, A.J. Bonham, S.T. Hikita, L.V. Johnson, D.O. Clegg, Stem Cells Dev. 19 (2010) 1231–1240. S. Abraham, N. Kogata, R. Fassler, R.H. Adams, Circ. Res. 102 (2008) 562–570. E.A. Cavalcanti-Adam, T. Volberg, A. Micoulet, H. Kessler, B. Geiger, J.P. Spatz, Biophys. J. 92 (2007) 2964–2974. K.A. DeMali, K. Wennerberg, K. Burridge, Curr. Opin. Cell Biol. 15 (2003) 572–582. T. Shibue, R.A. Weinberg, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 10290–10295. M. Arnold, E.A. Cavalcanti-Adam, R. Glass, J. Blummel, W. Eck, M. Kantlehner, H. Kessler, J.P. Spatz, ChemPhysChem 5 (2004) 383–388. T.A. Owen, M. Aronow, V. Shalhoub, L.M. Barone, L. Wilming, M.S. Tassinari, M.B. Kennedy, S. Pockwinse, J.B. Lian, G.S. Stein, J. Cell. Physiol. 143 (1990) 420–430.

Surface functionalization of nanoporous alumina with bone morphogenetic protein 2 for inducing osteogenic differentiation of mesenchymal stem cells.

Many studies have demonstrated the possibility to regulate cellular behavior by manipulating the specific characteristics of biomaterials including th...
1MB Sizes 1 Downloads 3 Views