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Bioactivity of nanostructure on titanium surface modified by chemical processing at room temperature
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Journal of Prosthodontic Research 56 (2012) 170–177 www.elsevier.com/locate/jpor
Original article
Bioactivity of nanostructure on titanium surface modified by chemical processing at room temperature Satoshi Komasa DDSa,*, Yoichiro Taguchi DDS, PhDb,1, Hisataka Nishida DDS, PhDc,1, Masahiro Tanaka DDS, PhDa,1, Takayoshi Kawazoe DDS, PhDa,1 a
Department of Fixed Prosthodontics and Occlusion, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan b Department of Periodontology, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan c Department of Operative Dentistry, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Received 25 August 2011; received in revised form 24 November 2011; accepted 7 December 2011 Available online 20 May 2012
Abstract Purpose: Recently, there has been considerable interest in finding novel applications and functions for existing dental materials. We found that, at room temperature and atmospheric pressure, titanium oxide spontaneously generates nanostructures very similar to the ‘‘nanotubes’’ created by TiO2 sputtering. The aim of this study was to evaluate the ability of this surface to affect the cellular osteogenic differentiation response. Methods: Titanium disks without and with a ‘nanosheet’ deposited on their surface were used as the control and test groups, respectively. Cell culture experiments were performed with SD rat bone marrow cells, which were seeded into microplate wells and cultured in media designed to induce osteogenic differentiation. We measured alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, calcium deposition and Runx2 gene expression to assess the levels of differentiation. Results: After 14 and 21 days, cellular ALP activity was significantly higher in the test group than in the control group. After 28 days, cells in the test group also showed significantly more calcium deposition and OCN production than those in the control group. There was significantly different expression of Runx2 mRNA in the test group compared to the control group after 3 days of culture. Conclusion: In conclusion, these data suggest that titanium implants modified by the application of nanostructures promote osteogenic differentiation, and may improve the biointegration of these implants into the alveolar bone. # 2011 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. Keywords: Nanostructure; Osseointegration; Mesenchymal stem cell
1. Introduction There has been a concerted effort amongst materials scientists and clinicians worldwide to improve the performance of dental implants, with the aim of accelerating and maintaining their integration into hard and soft tissues and/or extending their range of application. The surface characteristics of the implant material affect the rate and extent of osseointegration [1]. Vandrovcova´ and Bacˇa´kova´ [2] have recently reviewed the growing evidence that surface-modified materials are highly
* Corresponding author. Tel.: +81 72 864 3111; fax: +81 72 864 3000. E-mail addresses: [email protected] (S. Komasa), [email protected] (Y. Taguchi), [email protected] (H. Nishida), [email protected] (M. Tanaka), [email protected] (T. Kawazoe). 1 Tel: +81 72 864 3111; fax: +81 72 864 3000.
attractive for adhesion, growth and osteogenic differentiation of cells, and thus promote the integration of the implant into the bone tissue and maintain its secondary stability. A recent advance in dental implant research is the modification of the surface of the implant material at the nano-meter level [3,4]. Techniques that provide an increased surface area and finer surface roughness may yield better tissuetitanium mechanical interlocking [5]. However, more importantly, such nanoscopic features are also believed to directly affect osteogenic cell behavior around implant fixtures possessing non-conventional surfaces, creating a biomimetic relationship between alloplastic surfaces and host tissues through the replication of the natural cellular environment at the nanometer level [3,4,6]. Low dimensional TiO2 nanostructures have attracted much attention in recent years because these nanostructures take the form of nanotubes [7], nanofibers [8], and nanowires [9].
1883-1958/$ – see front matter # 2011 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. doi:10.1016/j.jpor.2011.12.002
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Compared to bulk materials or nanoparticles, TiO2 nanotubes have a high specific surface area available for the adsorption of a dye sensitizer, and provide channels for enhanced electron transfer, thereby helping to increase the efficiency of solar cells [7]. The structures used in this study are nano-structures similar to the TiO2 nanotubes created by titania deposition using the process of TiO2 sputtering [7], and are named titania nanosheets (TNS). Rat bone marrow (RBM) cells are multipotent cells that are able to self-renew and differentiate into precursors of several tissues, including osteoprogenitor cells. They are involved in the normal remodeling and reparative mechanisms of bone, and play a central role in the osseointegration process [10]. RBM cells are therefore an ideal tool to investigate the interaction between alveolar bone cells and the implant surface. If TNS promotes osteogenic differentiation, it would improve the biointegration of dental implants into the alveolar bone. However, the bioactivity of the TNS structure was not revealed. The aim of the present study was to investigate the cell behavior of the TNS structure, and evaluate the ability of these modified surfaces to affect osteogenic differentiation of RBM cells and potentially further increase the success rate of titanium implants. 2. Materials and methods 2.1. Sample preparation In the test group, titanium disks that have been treated to produce titanium nanosheets (TNS) on their surfaces were used as the experimental material. Unprocessed titanium disks were used as the control group. Titanium disks (15 mm diameter) were punched from sheets of 1 mm thickness grade 2 unalloyed titanium (Daido Steel, Osaka, Japan). These disks were immersed in 10 M NaOH (aq), which, in turn, was placed for 24 h in an oil bath maintained at 30 8C. The solution in each flask was replaced and treated with distilled water (200 mL), and this procedure was repeated until the solution reached a conductivity of 5 mS/cm. Samples were then dried at room temperature. The specimen surface topography was qualitatively evaluated using a scanning electron microscope (SEM, S4000, SHIMADZU, Kyoto, Japan) and a scanning probe microscope (SPM, SPM-9600, SHIMADZU). Scanning electron microscopy with EDX-analysis (EDX, S-2700, Hitachi Instrument, Inc., Tokyo, Japan) was used for elemental analysis of the titanium surface of the test and control groups. 2.2. Cell culture RBM cells were isolated from the femurs of 7-week-old Sprague-Dawley rats. This study was performed under the Guidelines for Animal Experimentation at Osaka Dental University (Approval No. 11-03038). Briefly, rats were euthanized using 4% isoflurane, and the bones were aseptically excised from the hind limbs. The proximal end of the femur and the distal end of the tibia were clipped. A 21-gauge needle (TERUMO, Tokyo, Japan) was inserted into the hole in the
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knee joint of each bone, and the marrow was flushed from the shaft with culture medium (Eagle’s minimal essential medium, EMEM; Wako Pure Chemical Industries, Ltd, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Life Technologies Corp., Carlsbad, CA, USA), penicillin (500 U/mL) (Cambrex Bio Science Walkersville Inc., Walkersville, MD, USA), streptomycin (500 mg/mL) (Cambrex Bio Science Walkersville Inc.), and fungizone (1.25 mg/mL) (Cambrex Bio Science Walkersville Inc.). The resulting marrow pellet was dispersed by trituration, and the cell suspensions from all bones were combined in a centrifuge tube. RBM cells were cultured in 75 cm2 culture flasks (Falcon, Becton Dickinson Labware, NJ, USA) with culture medium. At confluence, cells were removed from flasks by trypsinization, washed twice with PBS, resuspended in culture medium and seeded at a cell density of 4 104 cells/cm2 into 24-well tissue culture plates (Falcon) containing test or control titanium disks. The cells were cultured at 37 8C in a humidified 5% CO2/95% air atmosphere. 2.3. Cell differentiation The cells on test and control titanium disks were incubated until they reached confluence. The medium was then removed and replaced with differentiation medium containing 10% FBS and osteogenic supplements (10 mM b-glycerophosphate; Wako), 80 mg/mL of ascorbic acid (Nacalai Tesque Inc., Kyoto, Japan), and 10 nM dexamethasone (Nacalai Tesque Inc.). This differentiation medium was replaced every second day. 2.4. Alkaline phosphatase activity After 14 and 21 days of culture, cells were washed with PBS, lysed with 200 mL of 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) and the lysate was transferred to a microcentrifuge tube containing a 5 mm hardened steel ball. Tubes were agitated on a shaker (Mixer Mill Type MM 301, Retsh Gmbh & Co., Haan, Germany) at 29 Hz for 20 s to homogenize the sample. ALP activity was measured using the Alkaline Phosphatase Luminometric ELISA Kit (Sigma) according to the manufacturer’s protocol. The reaction was terminated with 3 N NaOH to a final concentration of 0.5 N NaOH and pnitrophenol production was measured by absorbance at 405 nm using a 96 well microplate reader (SpectraMax1 M5, Molecular Device Inc., Sunnyvale, CA, USA). DNA content was measured using the PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer’s protocol. To normalize ALP activity, the amount of ALP was normalized to the amount of DNA in the cell lysate. 2.5. Osteocalcin ELISA analysis The sandwich enzyme immunoassay used in this study is specific for rat osteocalcin (OCN) and can measure its levels directly in cell culture supernatant after 28 days of culture. The OCN levels in cell culture supernatant were measured using a
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Fig. 1. Scanning electron microscope (SEM) images of the surface topography of the titanium disks in the control and test groups. The control group displayed relatively smooth surface features, whereas, in the test group, nanostructures formed by NaOH treatment of the titanium surface caused a substantial increase in surface area and roughness.
commercially available ELISA Kit (Rat Osteocalcin ELISA Kit DS, DS Pharma Biomedical Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. 2.6. Mineralization Calcium deposited in the extracellular matrix was measured after dissolution with 10% formic acid. The amount of calcium was quantified using a Calcium E-test Kit (Wako Pure Chemical Industrials Ltd). After 28 days of culture, 1 mL Calcium E-Test reagent and 2 mL kit buffer were added to 50 mL of collected medium, and the absorbance of the reaction products was measured at 610 nm using a 96 well microplate reader (SpectraMax1 M5). The concentration of calcium ions was calculated from the absorbance value relative to a standard curve. 2.7. Real-time PCR analysis After 3 days of culture, total RNA was extracted from the cells and cDNA was synthesized from 1 mg of RNA using a High Capacity cDNA Archive Kit (Applied Biosystems Inc., Foster City, CA, USA). Runx-2 mRNA expression was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using a StepOne PlusTM Real-Time RT-PCR System (Applied Biosystems). In a Fast 96-well Reaction Plate (0.1 mL well volume; Applied Biosystems), 10 mL of Taqman Fast Universal PCR Master Mix, 1 mL of the Runx2 primer (Taqman Gene Expression Assays), 2 mL of sample cDNA, and 7 mL of DEPC water (Nippongene) were added to each well. The plate was subjected to 40 reaction cycles of 95 8C for 1 s, and 60 8C for 20 s. The reactive gene expression rate was calculated employing the DDCt method [11] in each group assuming the gene expression rate of the negative control group. 2.8. Statistical analysis All experiments were performed in triplicate. All data are described as the mean SD. In all analyses, statistical significance was determined by Student’s t-test.
Fig. 2. SPM images of the test and control group samples. Surface roughnesses (Ra values) were almost the same between the test and control groups.
3. Results 3.1. Sample analysis Scanning electron microscopy (SEM) of the titanium surfaces after modifying in NaOH at 30 8C showed a network structure at a nanometer level. Fig. 1 shows SEM images of the relatively smooth surface features of the untreated titanium surface, whereas the chemically treated titanium surface of the test group displays nanometer level fine network structure, as also shown in Pattanayak et al.’s research [12]. The surface morphology and the roughness values of the TNS were obtained by SPM, as shown in Fig. 2. The SPM image showed that there were a large number of nanonodules, about 100 nm
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Fig. 3. Elemental analysis of the titanium surfaces in the control and test groups. A scanning electron microscope system with EDX-analysis functionality was used to analyze the chemical composition of the test and control disks. Analysis showed that the surface composition of the disks was largely unchanged by the formation of the titanium nanostructures.
Fig. 4. Alkaline phosphatase (ALP) activity, measured at 14 and 21 days of culture in the test and control groups using an ALP ELISA assay. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
across, on the TNS titanium surface. The surface roughnesses (Ra values) were 44.4 and 47.8 nm for the TNS and the control group, respectively. The EDX results of the TNS and the control group are shown in Fig. 3. The TNS yielded a slight Na signal, in addition to the signals of Ti, Al and Si.
3.2. Alkaline phosphatase activity Cell differentiation was assessed by measuring the activity of the differentiation marker, ALP, in the test and control groups at 14 and 21 days. At both time points, ALP activity was significantly higher in the cells of the test group compared with the control group (Fig. 4). Fig. 5 shows the amount of DNA of
the test and control group. There was no significant difference in the amount of DNA in the test and control groups. 3.3. Osteocalcin production Fig. 6 shows the production of OCN in the test and control groups at 28 days. The presence of OCN in the supernatant of the test group was significantly higher than that in the control group. 3.4. Mineralization Fig. 7 shows calcium deposition in the extracellular matrix of RBM cells in the test and control groups at 28 days. Ca
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Fig. 5. The amount of DNA expressed after 14 and 21 days of culture in the test and control groups. At days 14 and 21, there were no significant differences in the amount of DNA between the test and control groups.
deposition was cumulative in the culture wells, so that measured levels normally increased with exposure time. As shown in the figure, significantly more calcium was deposited by cells in the test group at 28 days than by cells in the control group. 3.5. Runx2 mRNA expression Runx2 is a transcription factor involved in the early stages of osteogenic differentiation. As shown in Fig. 8, Runx-2 activity was significantly higher in the test group than in the control group at 3 days of culture. 4. Discussion Osseointegration of dental implants is dependent on their surface characteristics, including both their surface topography properties and chemical composition [4,13]. Modifying the surface characteristics of the implant produces altered RBM cell responses to it, and can change its interaction with the surrounding hard tissue. This study investigated whether RBM cells respond differently to titanium implant material that had undergone chemical surface modification. We found that the expression of the Runx-2 transcription factor (at an early time point) and RBM cell differentiation markers such as ALP and OCN (at later time points) was elevated in samples containing a TNS modified titanium disk, in comparison to an unmodified, polished titanium disk. We also found that calcium deposition in the extracellular matrix of the RBM cells was increased in the
TNS modified disk. Our results suggest that the TNS structure promotes RBM cell differentiation and activation, which augments calcium deposition. We believe that this nanostructure could effectively augment the biointegration of titanium implant materials by accelerating the bone tissue response to them. Various methods are used to prepare TiO2 nanotubes [14]. Tubes with diameters of 70–100 nm were produced using a replication method [7]. However, obtaining nanotubes with smaller diameters is complicated because the dimension of the tubes is controlled by the pore size of the template prepared using porous materials such as alumina. Kasuga et al. demonstrated that TiO2 nanotubes with a diameter of 8 nm and a length of 100 nm are formed by treatment of Ti with 10 M NaOH aqueous solution for 20 h at 110 8C and without the need for templates or replication [7]. Recently, we showed that nanotube and TNS structures could be obtained on a titanium metal surface by treatment with 10 M NaOH aqueous solution at 30 8C [15], and we employed that method here to create the TNS structures on the test disks. Recent research has shown that treatment with NaOH aqueous solution produces a rough nanoscopic surface [16], and SEM images of our test disks demonstrate that the TNS modified surface has good surface roughness without cracks. Svanborg et al. showed that although a surface could appear smooth at the micrometer scale, it could have considerable roughness at the nanometer scale [17]. Other studies have also shown that nanostructure surface modification causes significant differences in surface appearance in SEM images [5,18,19], notably including titanium disks modified with alkali solution [20]. These
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Fig. 6. Osteocalcin production after 28 days of culture in the test and control groups, as measured using a sandwich enzyme immunoassay. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
differences in surface nanostructure are known to modulate osteogenic differentiation and mineralization of titanium implant materials [3,4]. In SPM analysis, the surface roughnesses of TNS were similar to those of the control groups. However, nanonodules of about 100 nm diameter were created on TNS. Matthew reported that mesenchymal stem cells were induced to an osteoblastic lineage on titanium surface with 100–120 nm [21]. In EDX analysis, the presence of Na in the TNS was confirmed. Kasuga et al. reported that NaOH treatment leads to the formation of a Ti–O–Na layer on the titanium surface [7]. Kasuga et al formed nanostructures using treatment for 20 h at 60 8C with 10 M NaOH aqueous solution, but found that these structures did not form at 30 8C [7]. Treatment at 110 8C leads to the formation of needle-shaped products when using 5– 10 M NaOH aqueous solutions, but no products form at less than 5 M NaOH, demonstrating that dilute NaOH aqueous solution is a poor medium for the formation of nanotubes, and justifying the use of 10 M NaOH solution in our protocol. In the present study, room temperature modification is used to make TNS on the titanium surface. Previous surface modification studies [7] prepared TNT at high temperatures, which involves hazardous hot caustic solutions, and is hence much more difficult for clinicians. The effects of surface modification on the ability of multipotent mesenchymal stem cells (MSCs) to differentiate into osteogenic cells and produce paracrine factors are not known. The present work evaluated the possible effects of surface modification on MSC differentiation toward an osteogenic lineage. MSCs will exhibit surface dependent
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Fig. 7. Calcium deposition after 28 days in the test and control groups was estimated using a Calcium-E test kit (Wako). Calcium concentrations were extrapolated from a standard curve. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
Fig. 8. Expression of Runx-2 mRNA after 3 days of culture. Runx-2 mRNA was quantified by real-time RT-PCR. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
changes as shown in the various osteoblast models examined previously here we are interested in, whether they will differentiate into osteoblasts in a substrate-dependent manner [22]. RBM cells proliferate and differentiate into a phenotype that expresses bone cell markers and forms mineralized nodules in vitro. PCR analyses for the most specific and common markers of osteogenic induction were performed on bone marrow cells
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grown on titanium implant materials. We showed here that different surface nano-topographies altered the expression of key osteogenic regulatory genes such as Runx-2. Runx-2 mediates several early osteogenic gene responses for cellular adhesion [23]. Several lines of evidence show that surface modification leads to high expression of Runx2 mRNA [23– 25]. The data from this study suggests that the elevated Runx-2 mRNA expression in cells grown on the nanostructured titanium surface is a causative factor in the differentiation of RBM cell differentiation into osteogenic cells. ALP activity, OCN production and calcium deposition were all elevated by TNS. Importantly, the functional phenotypes expressed in the middle and late stages of culture, such as ALP activity and mineralization, were considerably increased. A great deal of research has shown the ALP activation effect of surface modified materials [22,26–36]. There is also significant evidence that nanostructured surface modification increases Ca deposition [22,26,28,30,36], and our results support these conclusions. Our measurements of OCN production are also in agreement with previous studies. A few researchers have shown that nanostructured surfaces enhance OCN production [28,37]. Osteogenic differentiation may be stimulated directly by surface topography on the nanometer scale influencing cell morphology. Improvement of surface topography may be indirect: the adsorption of proteins or ions may act as a bridge between the nanosurface structure and the cells [22]. Indeed, all implant surfaces are immediately covered with a layer of protein from the in vitro culture medium or in vivo biological fluids, and this interface modulates the cascade of cellular response and behavior. The composition, stability and thickness of this layer are highly dependent on the implant surface properties [22]. Titanium implants have become an essential treatment modality in reconstructive surgeries in the orthopedic and dental fields. However, there is always a clinical demand that patient morbidity and treatment complications be reduced, and that outcome predictability and treatment indications be maximized. Therefore, considerable efforts have been made to develop new technologies to modify the surface of titanium to assist its biointegration into the bone [7]. The modification method used here is useful and easily accomplished because the incubation in NaOH required is at room temperature and requires no template [38]. 5. Conclusion In conclusion, our investigation of different implant surface nanostructures demonstrated that modifying the implant surface at the nanometer scale leads to the regulation of osteogenic differentiation of bone marrow cells and enhances mineralization. We conclude that further development of advanced implant materials using nanotechnology will improve osseointegration. Acknowledgments The authors would like to thank Dr. Toru Sekino from Tohoku University for making the ‘‘Nano sheet’’ and for helpful suggestions.
The authors also would like to thank Prof. Shoji Takeda and Dr. Yoshiya Hashimoto for their encouragement and helpful suggestions. We are also grateful to the members of the Department of Fixed Prosthodontics and Occlusion, Department of Periodontology and the Department of Biomaterials for their kind advice and assistance. This study was partly supported by a Research Promotion Grant (11-04) and Oral Implant Research Grant (11-05) from Osaka Dental University and a Grant-in-Aid for Scientific Research (23792303) from the Japan Society for the Promotion of Science.
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Journal of Prosthodontic Research 56 (2012) 170–177 www.elsevier.com/locate/jpor
Original article
Bioactivity of nanostructure on titanium surface modified by chemical processing at room temperature Satoshi Komasa DDSa,*, Yoichiro Taguchi DDS, PhDb,1, Hisataka Nishida DDS, PhDc,1, Masahiro Tanaka DDS, PhDa,1, Takayoshi Kawazoe DDS, PhDa,1 a
Department of Fixed Prosthodontics and Occlusion, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan b Department of Periodontology, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan c Department of Operative Dentistry, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Received 25 August 2011; received in revised form 24 November 2011; accepted 7 December 2011 Available online 20 May 2012
Abstract Purpose: Recently, there has been considerable interest in finding novel applications and functions for existing dental materials. We found that, at room temperature and atmospheric pressure, titanium oxide spontaneously generates nanostructures very similar to the ‘‘nanotubes’’ created by TiO2 sputtering. The aim of this study was to evaluate the ability of this surface to affect the cellular osteogenic differentiation response. Methods: Titanium disks without and with a ‘nanosheet’ deposited on their surface were used as the control and test groups, respectively. Cell culture experiments were performed with SD rat bone marrow cells, which were seeded into microplate wells and cultured in media designed to induce osteogenic differentiation. We measured alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, calcium deposition and Runx2 gene expression to assess the levels of differentiation. Results: After 14 and 21 days, cellular ALP activity was significantly higher in the test group than in the control group. After 28 days, cells in the test group also showed significantly more calcium deposition and OCN production than those in the control group. There was significantly different expression of Runx2 mRNA in the test group compared to the control group after 3 days of culture. Conclusion: In conclusion, these data suggest that titanium implants modified by the application of nanostructures promote osteogenic differentiation, and may improve the biointegration of these implants into the alveolar bone. # 2011 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. Keywords: Nanostructure; Osseointegration; Mesenchymal stem cell
1. Introduction There has been a concerted effort amongst materials scientists and clinicians worldwide to improve the performance of dental implants, with the aim of accelerating and maintaining their integration into hard and soft tissues and/or extending their range of application. The surface characteristics of the implant material affect the rate and extent of osseointegration [1]. Vandrovcova´ and Bacˇa´kova´ [2] have recently reviewed the growing evidence that surface-modified materials are highly
* Corresponding author. Tel.: +81 72 864 3111; fax: +81 72 864 3000. E-mail addresses: [email protected] (S. Komasa), [email protected] (Y. Taguchi), [email protected] (H. Nishida), [email protected] (M. Tanaka), [email protected] (T. Kawazoe). 1 Tel: +81 72 864 3111; fax: +81 72 864 3000.
attractive for adhesion, growth and osteogenic differentiation of cells, and thus promote the integration of the implant into the bone tissue and maintain its secondary stability. A recent advance in dental implant research is the modification of the surface of the implant material at the nano-meter level [3,4]. Techniques that provide an increased surface area and finer surface roughness may yield better tissuetitanium mechanical interlocking [5]. However, more importantly, such nanoscopic features are also believed to directly affect osteogenic cell behavior around implant fixtures possessing non-conventional surfaces, creating a biomimetic relationship between alloplastic surfaces and host tissues through the replication of the natural cellular environment at the nanometer level [3,4,6]. Low dimensional TiO2 nanostructures have attracted much attention in recent years because these nanostructures take the form of nanotubes [7], nanofibers [8], and nanowires [9].
1883-1958/$ – see front matter # 2011 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. doi:10.1016/j.jpor.2011.12.002
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Compared to bulk materials or nanoparticles, TiO2 nanotubes have a high specific surface area available for the adsorption of a dye sensitizer, and provide channels for enhanced electron transfer, thereby helping to increase the efficiency of solar cells [7]. The structures used in this study are nano-structures similar to the TiO2 nanotubes created by titania deposition using the process of TiO2 sputtering [7], and are named titania nanosheets (TNS). Rat bone marrow (RBM) cells are multipotent cells that are able to self-renew and differentiate into precursors of several tissues, including osteoprogenitor cells. They are involved in the normal remodeling and reparative mechanisms of bone, and play a central role in the osseointegration process [10]. RBM cells are therefore an ideal tool to investigate the interaction between alveolar bone cells and the implant surface. If TNS promotes osteogenic differentiation, it would improve the biointegration of dental implants into the alveolar bone. However, the bioactivity of the TNS structure was not revealed. The aim of the present study was to investigate the cell behavior of the TNS structure, and evaluate the ability of these modified surfaces to affect osteogenic differentiation of RBM cells and potentially further increase the success rate of titanium implants. 2. Materials and methods 2.1. Sample preparation In the test group, titanium disks that have been treated to produce titanium nanosheets (TNS) on their surfaces were used as the experimental material. Unprocessed titanium disks were used as the control group. Titanium disks (15 mm diameter) were punched from sheets of 1 mm thickness grade 2 unalloyed titanium (Daido Steel, Osaka, Japan). These disks were immersed in 10 M NaOH (aq), which, in turn, was placed for 24 h in an oil bath maintained at 30 8C. The solution in each flask was replaced and treated with distilled water (200 mL), and this procedure was repeated until the solution reached a conductivity of 5 mS/cm. Samples were then dried at room temperature. The specimen surface topography was qualitatively evaluated using a scanning electron microscope (SEM, S4000, SHIMADZU, Kyoto, Japan) and a scanning probe microscope (SPM, SPM-9600, SHIMADZU). Scanning electron microscopy with EDX-analysis (EDX, S-2700, Hitachi Instrument, Inc., Tokyo, Japan) was used for elemental analysis of the titanium surface of the test and control groups. 2.2. Cell culture RBM cells were isolated from the femurs of 7-week-old Sprague-Dawley rats. This study was performed under the Guidelines for Animal Experimentation at Osaka Dental University (Approval No. 11-03038). Briefly, rats were euthanized using 4% isoflurane, and the bones were aseptically excised from the hind limbs. The proximal end of the femur and the distal end of the tibia were clipped. A 21-gauge needle (TERUMO, Tokyo, Japan) was inserted into the hole in the
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knee joint of each bone, and the marrow was flushed from the shaft with culture medium (Eagle’s minimal essential medium, EMEM; Wako Pure Chemical Industries, Ltd, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Life Technologies Corp., Carlsbad, CA, USA), penicillin (500 U/mL) (Cambrex Bio Science Walkersville Inc., Walkersville, MD, USA), streptomycin (500 mg/mL) (Cambrex Bio Science Walkersville Inc.), and fungizone (1.25 mg/mL) (Cambrex Bio Science Walkersville Inc.). The resulting marrow pellet was dispersed by trituration, and the cell suspensions from all bones were combined in a centrifuge tube. RBM cells were cultured in 75 cm2 culture flasks (Falcon, Becton Dickinson Labware, NJ, USA) with culture medium. At confluence, cells were removed from flasks by trypsinization, washed twice with PBS, resuspended in culture medium and seeded at a cell density of 4 104 cells/cm2 into 24-well tissue culture plates (Falcon) containing test or control titanium disks. The cells were cultured at 37 8C in a humidified 5% CO2/95% air atmosphere. 2.3. Cell differentiation The cells on test and control titanium disks were incubated until they reached confluence. The medium was then removed and replaced with differentiation medium containing 10% FBS and osteogenic supplements (10 mM b-glycerophosphate; Wako), 80 mg/mL of ascorbic acid (Nacalai Tesque Inc., Kyoto, Japan), and 10 nM dexamethasone (Nacalai Tesque Inc.). This differentiation medium was replaced every second day. 2.4. Alkaline phosphatase activity After 14 and 21 days of culture, cells were washed with PBS, lysed with 200 mL of 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) and the lysate was transferred to a microcentrifuge tube containing a 5 mm hardened steel ball. Tubes were agitated on a shaker (Mixer Mill Type MM 301, Retsh Gmbh & Co., Haan, Germany) at 29 Hz for 20 s to homogenize the sample. ALP activity was measured using the Alkaline Phosphatase Luminometric ELISA Kit (Sigma) according to the manufacturer’s protocol. The reaction was terminated with 3 N NaOH to a final concentration of 0.5 N NaOH and pnitrophenol production was measured by absorbance at 405 nm using a 96 well microplate reader (SpectraMax1 M5, Molecular Device Inc., Sunnyvale, CA, USA). DNA content was measured using the PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer’s protocol. To normalize ALP activity, the amount of ALP was normalized to the amount of DNA in the cell lysate. 2.5. Osteocalcin ELISA analysis The sandwich enzyme immunoassay used in this study is specific for rat osteocalcin (OCN) and can measure its levels directly in cell culture supernatant after 28 days of culture. The OCN levels in cell culture supernatant were measured using a
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Fig. 1. Scanning electron microscope (SEM) images of the surface topography of the titanium disks in the control and test groups. The control group displayed relatively smooth surface features, whereas, in the test group, nanostructures formed by NaOH treatment of the titanium surface caused a substantial increase in surface area and roughness.
commercially available ELISA Kit (Rat Osteocalcin ELISA Kit DS, DS Pharma Biomedical Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. 2.6. Mineralization Calcium deposited in the extracellular matrix was measured after dissolution with 10% formic acid. The amount of calcium was quantified using a Calcium E-test Kit (Wako Pure Chemical Industrials Ltd). After 28 days of culture, 1 mL Calcium E-Test reagent and 2 mL kit buffer were added to 50 mL of collected medium, and the absorbance of the reaction products was measured at 610 nm using a 96 well microplate reader (SpectraMax1 M5). The concentration of calcium ions was calculated from the absorbance value relative to a standard curve. 2.7. Real-time PCR analysis After 3 days of culture, total RNA was extracted from the cells and cDNA was synthesized from 1 mg of RNA using a High Capacity cDNA Archive Kit (Applied Biosystems Inc., Foster City, CA, USA). Runx-2 mRNA expression was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using a StepOne PlusTM Real-Time RT-PCR System (Applied Biosystems). In a Fast 96-well Reaction Plate (0.1 mL well volume; Applied Biosystems), 10 mL of Taqman Fast Universal PCR Master Mix, 1 mL of the Runx2 primer (Taqman Gene Expression Assays), 2 mL of sample cDNA, and 7 mL of DEPC water (Nippongene) were added to each well. The plate was subjected to 40 reaction cycles of 95 8C for 1 s, and 60 8C for 20 s. The reactive gene expression rate was calculated employing the DDCt method [11] in each group assuming the gene expression rate of the negative control group. 2.8. Statistical analysis All experiments were performed in triplicate. All data are described as the mean SD. In all analyses, statistical significance was determined by Student’s t-test.
Fig. 2. SPM images of the test and control group samples. Surface roughnesses (Ra values) were almost the same between the test and control groups.
3. Results 3.1. Sample analysis Scanning electron microscopy (SEM) of the titanium surfaces after modifying in NaOH at 30 8C showed a network structure at a nanometer level. Fig. 1 shows SEM images of the relatively smooth surface features of the untreated titanium surface, whereas the chemically treated titanium surface of the test group displays nanometer level fine network structure, as also shown in Pattanayak et al.’s research [12]. The surface morphology and the roughness values of the TNS were obtained by SPM, as shown in Fig. 2. The SPM image showed that there were a large number of nanonodules, about 100 nm
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Fig. 3. Elemental analysis of the titanium surfaces in the control and test groups. A scanning electron microscope system with EDX-analysis functionality was used to analyze the chemical composition of the test and control disks. Analysis showed that the surface composition of the disks was largely unchanged by the formation of the titanium nanostructures.
Fig. 4. Alkaline phosphatase (ALP) activity, measured at 14 and 21 days of culture in the test and control groups using an ALP ELISA assay. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
across, on the TNS titanium surface. The surface roughnesses (Ra values) were 44.4 and 47.8 nm for the TNS and the control group, respectively. The EDX results of the TNS and the control group are shown in Fig. 3. The TNS yielded a slight Na signal, in addition to the signals of Ti, Al and Si.
3.2. Alkaline phosphatase activity Cell differentiation was assessed by measuring the activity of the differentiation marker, ALP, in the test and control groups at 14 and 21 days. At both time points, ALP activity was significantly higher in the cells of the test group compared with the control group (Fig. 4). Fig. 5 shows the amount of DNA of
the test and control group. There was no significant difference in the amount of DNA in the test and control groups. 3.3. Osteocalcin production Fig. 6 shows the production of OCN in the test and control groups at 28 days. The presence of OCN in the supernatant of the test group was significantly higher than that in the control group. 3.4. Mineralization Fig. 7 shows calcium deposition in the extracellular matrix of RBM cells in the test and control groups at 28 days. Ca
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Fig. 5. The amount of DNA expressed after 14 and 21 days of culture in the test and control groups. At days 14 and 21, there were no significant differences in the amount of DNA between the test and control groups.
deposition was cumulative in the culture wells, so that measured levels normally increased with exposure time. As shown in the figure, significantly more calcium was deposited by cells in the test group at 28 days than by cells in the control group. 3.5. Runx2 mRNA expression Runx2 is a transcription factor involved in the early stages of osteogenic differentiation. As shown in Fig. 8, Runx-2 activity was significantly higher in the test group than in the control group at 3 days of culture. 4. Discussion Osseointegration of dental implants is dependent on their surface characteristics, including both their surface topography properties and chemical composition [4,13]. Modifying the surface characteristics of the implant produces altered RBM cell responses to it, and can change its interaction with the surrounding hard tissue. This study investigated whether RBM cells respond differently to titanium implant material that had undergone chemical surface modification. We found that the expression of the Runx-2 transcription factor (at an early time point) and RBM cell differentiation markers such as ALP and OCN (at later time points) was elevated in samples containing a TNS modified titanium disk, in comparison to an unmodified, polished titanium disk. We also found that calcium deposition in the extracellular matrix of the RBM cells was increased in the
TNS modified disk. Our results suggest that the TNS structure promotes RBM cell differentiation and activation, which augments calcium deposition. We believe that this nanostructure could effectively augment the biointegration of titanium implant materials by accelerating the bone tissue response to them. Various methods are used to prepare TiO2 nanotubes [14]. Tubes with diameters of 70–100 nm were produced using a replication method [7]. However, obtaining nanotubes with smaller diameters is complicated because the dimension of the tubes is controlled by the pore size of the template prepared using porous materials such as alumina. Kasuga et al. demonstrated that TiO2 nanotubes with a diameter of 8 nm and a length of 100 nm are formed by treatment of Ti with 10 M NaOH aqueous solution for 20 h at 110 8C and without the need for templates or replication [7]. Recently, we showed that nanotube and TNS structures could be obtained on a titanium metal surface by treatment with 10 M NaOH aqueous solution at 30 8C [15], and we employed that method here to create the TNS structures on the test disks. Recent research has shown that treatment with NaOH aqueous solution produces a rough nanoscopic surface [16], and SEM images of our test disks demonstrate that the TNS modified surface has good surface roughness without cracks. Svanborg et al. showed that although a surface could appear smooth at the micrometer scale, it could have considerable roughness at the nanometer scale [17]. Other studies have also shown that nanostructure surface modification causes significant differences in surface appearance in SEM images [5,18,19], notably including titanium disks modified with alkali solution [20]. These
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Fig. 6. Osteocalcin production after 28 days of culture in the test and control groups, as measured using a sandwich enzyme immunoassay. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
differences in surface nanostructure are known to modulate osteogenic differentiation and mineralization of titanium implant materials [3,4]. In SPM analysis, the surface roughnesses of TNS were similar to those of the control groups. However, nanonodules of about 100 nm diameter were created on TNS. Matthew reported that mesenchymal stem cells were induced to an osteoblastic lineage on titanium surface with 100–120 nm [21]. In EDX analysis, the presence of Na in the TNS was confirmed. Kasuga et al. reported that NaOH treatment leads to the formation of a Ti–O–Na layer on the titanium surface [7]. Kasuga et al formed nanostructures using treatment for 20 h at 60 8C with 10 M NaOH aqueous solution, but found that these structures did not form at 30 8C [7]. Treatment at 110 8C leads to the formation of needle-shaped products when using 5– 10 M NaOH aqueous solutions, but no products form at less than 5 M NaOH, demonstrating that dilute NaOH aqueous solution is a poor medium for the formation of nanotubes, and justifying the use of 10 M NaOH solution in our protocol. In the present study, room temperature modification is used to make TNS on the titanium surface. Previous surface modification studies [7] prepared TNT at high temperatures, which involves hazardous hot caustic solutions, and is hence much more difficult for clinicians. The effects of surface modification on the ability of multipotent mesenchymal stem cells (MSCs) to differentiate into osteogenic cells and produce paracrine factors are not known. The present work evaluated the possible effects of surface modification on MSC differentiation toward an osteogenic lineage. MSCs will exhibit surface dependent
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Fig. 7. Calcium deposition after 28 days in the test and control groups was estimated using a Calcium-E test kit (Wako). Calcium concentrations were extrapolated from a standard curve. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
Fig. 8. Expression of Runx-2 mRNA after 3 days of culture. Runx-2 mRNA was quantified by real-time RT-PCR. Data are mean SD, n = 3. * denotes a statistically significant difference ( p < 0.05, calculated by Student’s t-test).
changes as shown in the various osteoblast models examined previously here we are interested in, whether they will differentiate into osteoblasts in a substrate-dependent manner [22]. RBM cells proliferate and differentiate into a phenotype that expresses bone cell markers and forms mineralized nodules in vitro. PCR analyses for the most specific and common markers of osteogenic induction were performed on bone marrow cells
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grown on titanium implant materials. We showed here that different surface nano-topographies altered the expression of key osteogenic regulatory genes such as Runx-2. Runx-2 mediates several early osteogenic gene responses for cellular adhesion [23]. Several lines of evidence show that surface modification leads to high expression of Runx2 mRNA [23– 25]. The data from this study suggests that the elevated Runx-2 mRNA expression in cells grown on the nanostructured titanium surface is a causative factor in the differentiation of RBM cell differentiation into osteogenic cells. ALP activity, OCN production and calcium deposition were all elevated by TNS. Importantly, the functional phenotypes expressed in the middle and late stages of culture, such as ALP activity and mineralization, were considerably increased. A great deal of research has shown the ALP activation effect of surface modified materials [22,26–36]. There is also significant evidence that nanostructured surface modification increases Ca deposition [22,26,28,30,36], and our results support these conclusions. Our measurements of OCN production are also in agreement with previous studies. A few researchers have shown that nanostructured surfaces enhance OCN production [28,37]. Osteogenic differentiation may be stimulated directly by surface topography on the nanometer scale influencing cell morphology. Improvement of surface topography may be indirect: the adsorption of proteins or ions may act as a bridge between the nanosurface structure and the cells [22]. Indeed, all implant surfaces are immediately covered with a layer of protein from the in vitro culture medium or in vivo biological fluids, and this interface modulates the cascade of cellular response and behavior. The composition, stability and thickness of this layer are highly dependent on the implant surface properties [22]. Titanium implants have become an essential treatment modality in reconstructive surgeries in the orthopedic and dental fields. However, there is always a clinical demand that patient morbidity and treatment complications be reduced, and that outcome predictability and treatment indications be maximized. Therefore, considerable efforts have been made to develop new technologies to modify the surface of titanium to assist its biointegration into the bone [7]. The modification method used here is useful and easily accomplished because the incubation in NaOH required is at room temperature and requires no template [38]. 5. Conclusion In conclusion, our investigation of different implant surface nanostructures demonstrated that modifying the implant surface at the nanometer scale leads to the regulation of osteogenic differentiation of bone marrow cells and enhances mineralization. We conclude that further development of advanced implant materials using nanotechnology will improve osseointegration. Acknowledgments The authors would like to thank Dr. Toru Sekino from Tohoku University for making the ‘‘Nano sheet’’ and for helpful suggestions.
The authors also would like to thank Prof. Shoji Takeda and Dr. Yoshiya Hashimoto for their encouragement and helpful suggestions. We are also grateful to the members of the Department of Fixed Prosthodontics and Occlusion, Department of Periodontology and the Department of Biomaterials for their kind advice and assistance. This study was partly supported by a Research Promotion Grant (11-04) and Oral Implant Research Grant (11-05) from Osaka Dental University and a Grant-in-Aid for Scientific Research (23792303) from the Japan Society for the Promotion of Science.
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