Journal of Periodontology; Copyright 2014
DOI: 10.1902/jop.2014.140386
Effect of Porphyromonas Gingivalis Lipopolysaccharide on Bone Marrow Mesenchymal Stem Cell Osteogenesis on a Titanium Nanosurface *†
‡
Helin Xing D.D.S. Ph.D. , Yoichiro Taguchi D.D.S., Ph.D. , Satoshi Komasa D.D.S., †
‡
§
‡
Ph.D. , Isao Yamawaki D.D.S. , Tohru Sekino Ph.D. ,Makoto Umeda D.D.S., Ph.D. , Joji Okazaki D.D.S., Ph.D. *
†
Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, China.
†
Department of Removable Prosthodontics and Occlusion, Osaka Dental University, Japan. ‡
§
Department of Periodontology, Osaka Dental University, Osaka, Japan.
Advanced Hard Materials, the Institute of Scientific and Industrial Research, Osaka University, Japan. Background: Titanium (Ti) dental implants have been widely used for prosthetic reconstruction
of dentition. Unfortunately, peri-implantitis can result in failure of dental implant osseointegration. Lipopolysaccharides (LPS) acts as a chronic inflammatory stimulus and maintains peri-implant under inflammation, worsening the prognosis for implant osseointegration. The purpose of this study was to determine the effects of 10 M NaOH-modified titanium surface with nanonetwork structure on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in the context of Porphyromonas gingivalis (P. gingivalis) LPS exposure. Methods: Titanium disks treated with 10 M NaOH solution and control were incubated with BMMSCs and then exposed to P. gingivalis LPS (0, 0.1 or 1 μg/mL). The effects of the modified nanonetwork structure on osteogenic differentiation of rat BMMSCs were evaluated in the context of different concentrations of P. gingivalis LPS exposure.
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Results: Rat BMMSCs on the 10 M NaOH-modified titanium surface with nanonetwork structure had higher levels of osteogenesis-related gene expression and significantly greater cell proliferation, ALP activity, extracellular matrix deposition and mineralization than cells on the untreated titanium surfaces, in all the groups with different doses of P. gingivalis LPS exposure. Conclusion: The 10 M NaOH-modified titanium surface with nanonetwork structure has better endotoxin tolerance under P. gingivalis LPS exposure than non-modified surface.
KEY WORDS Bone marrow mesenchymal stem cells, Porphyromonas gingivalis, Lipopolysaccharide, osseointegration, titanium
Since Branemark 1introduced the concept of osseointegration fifty years ago, titanium (Ti) implants have been regarded as important appliances for prosthetic dental reconstruction. Previous studies have demonstrated the medium- and long-term success of dental implants.2, 3 However, limited data exist on the failure of medium- and longterm dental implants. Medium- and long-term failure of implant osseointegration has generally been attributed to occlusal overload4 and peri-implantitis.5, 6 Clinicians can easily control overload, because it ultimately depends on the number and location of occlusal contacts, whereas peri-implantitis requires a specific therapeutic approach.7 Peri-implantitis is an inflammatory lesion around a dental implant, resulting in loss of supporting bone.8 Gram-negative anaerobes, such as Porphyromonas gingivalis (P. gingivalis)9, have a variety of virulence factors, including proteases, fimbriae, lipopolysaccharide (LPS) and capsule, that make them potent pathogens in the development of peri-implantitis. Among these, LPS acts as a chronic stimulus, maintaining peri-implant inflammation and thereby worsening the prognosis for implant osseointegration10 and influencing the behavior of cells such as fibroblasts, osteoblasts and lymphocytes.11 Bone marrow mesenchymal stem cells (BMMSCs) are multipotent and represent a particularly attractive source for tissue engineering. BMMSCs are the first cells to colonize biomaterial surfaces after implantation and they have osteogenic differentiation capacity. Some studies have used BMMSCs as a source of osteogenic cells for bone repair.12,13 The role of BMMSCs in osseointegration through their differentiation into osteoblasts depends on whether their growth and differentiation is affected by LPS exposure. However, little is known about the interactions between LPS and BMMSCs. Therefore, investigation into the effects of LPS on the proliferation and osteogenic differentiation of BMMSCs is necessary. 2
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DOI: 10.1902/jop.2014.140386
We recently reported that the nano-network structures with TNS nanofeatures induced by alkali etching markedly enhanced cell adhesion, proliferation, and osteogenesis. These effects were most pronounced when the concentration of NaOH was 10 M, suggesting its great potential in improving the clinical performance of bone implants.14 To better understand whether modified surfaces could tolerate LPS exposure, this study investigated the effects of 10 M NaOH-modified titanium surface with nanonetwork structure on the proliferation and osteogenic differentiation of BMMSCs, in the context of P. gingivalis LPS exposure.
MATERIALS AND METHODS Disk Preparation Titanium disks (15 mm in diameter and 1 mm thick) of grade 2 commercially pure titanium were prepared by machining.‡ After ultrasonic cleaning, these disks were immersed in 10 M NaOH solution in the flask and placed in an oil bath maintained at 30°C for 24 h. Unprocessed titanium disks were used as controls. The solution in each flask was replaced with ion exchange water (200 mL), and this procedure was repeated until the solution reached a conductivity of 5 μS/cm. The disks were then dried at room temperature. The disks were classified as titania nanosheet (TNS) or titanium (Ti) group. Surface Characterization The TNS and Ti disks were plated on 24-well tissue culture plates and 1 mL phosphatebuffered saline (PBS) containing commercially available P. gingivalis LPS (0, 0.1 or 1 μg/mL) § was added to each well. The specimens were labeled (TNS, CON), (TNS, 0.1), (TNS, 1), (Ti, CON), (Ti, 0.1) or (Ti, 0.1), according to the concentration of LPS. The disk surfaces were examined by scanning electron microscopy (SEM; S-4800) and atomic force microscopy (AFM; SPM-9600). ¶ Protein Adsorption Assay Bovine serum albumin, fraction V# was used as a model protein. Three hundred µL of protein solution (1 mg/mL protein in saline) containing P. gingivalis LPS (0, 1, or 10 mg/mL) was pipetted onto the TNS and Ti disks. After incubation for 1, 3, 6 and 24 h at 37°C, the amount of removed albumin and the total amount of albumin inoculated was quantified using a microplate reader at 562 nm, using a 96-well microplate reader (SpectraMax M5). **
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Cell Culture Animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Osaka Dental University (Approval No. 13-02039). Rat BMMSCs were obtained from the femurs of 8-week-old Sprague–Dawley rats. BMMSCs were maintained in growth medium containing minimal essential medium, †† 10% fetal bovine serum, ‡‡ and Antibiotic–Antimycotic Mixed Stock Solution, §§ and cultured in a humidified atmosphere with 5% CO2 at 37°C. After 3 days, the medium containing the nonadherent cells was replaced and thereafter the medium was changed every 3 days. When rat BMMSCs were seeded on the disks, after cultured 7 days, the medium was removed and replaced with differentiation medium containing 10% fetal bovine serum, Antibiotic–Antimycotic Mixed Stock Solution and osteogenic supplements: 10 mM βglycerophosphate, corbic acid¶¶ and 10 nM dexamethasone. ## This medium was changed every 3 days. Cell Proliferation Assay Cell proliferation was measured using the CellTiter-Blue Cell Viability Assay **** according to the manufacturer’s protocol. Rat BMMSCs were seeded on the TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL) and incubated for 1, 3 or 7 days. Fluorescence was recorded at 560/590 nm using a 96-well microplate reader (SpectraMax M5). †††† The difference between the two optical densities was defined as the proliferation value. Alkaline Phosphatase Staining and Activity Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). After culturing for 1 or 2 weeks, the cells were washed and fixed, and ALP staining was performed with BCIP/NBT alkaline phosphatase (ALP) color development substrate‡‡‡ for 15 min. ALP activity of the BMMSCs incubated with LPS was examined at 1 and 2 weeks using an ALP luminometric ELISA kit. §§§ ALP activity was evaluated as the amount of p-nitrophenol released in the enzymatic reaction and measured at 405 nm using the microplate reader (SpectraMax M5).
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Extracellular Matrix Mineralization Extracellular matrix (ECM) mineralization by rat BMMSCs was evaluated with Alizarin Red staining. Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). After culturing for 3 or 4 weeks, the cells were stained with Alizarin Red for 10 min at room temperature. Cell monolayers were washed with distilled water until no more color appeared, and images were acquired. Calcium deposited in the ECM was measured after dissolution with 10% formic acid. The amount of calcium was quantified using a Calcium E-test Kit. ¶¶¶ After 3 or 4 weeks of culture with LPS, the absorbance of the reaction products was measured at 610 nm using the microplate reader (SpectraMax M5). ### The concentration of calcium ions was calculated from the absorbance relative to a standard curve. Osteocalcin ELISA The sandwich enzyme immunoassay used in this study was specific for rat osteocalcin and measured its levels directly in cell culture supernatant after 3 and 4 weeks of culture with P. gingivalis LPS (Rat Osteocalcin ELISA Kit DS), ****** according to the manufacturer’s instructions. Gene Expression Gene expression was evaluated using the real-time TaqMan RT-PCR assay. †††††† Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL) and cultured for 3 or 7 days. Total RNA was isolated using the RNeasy Mini Kit.‡‡‡‡ 10 µL of RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit.§§§§ Expression of genes including bone sialoprotein (BSP), osteonectin (ON), runtrelated transcription factor 2 (RUNX2), collagen type 1 (COL-1), Receptor action of NF kappa B Ligand (RANKL), osteoprotegerin (OPG), interleukin-1β (IL-1β), interleukin6 (IL-6) and interleukin-10 (IL-10) was quantified using the StepOne Plus Real-Time PCR System. The reactive gene expression rate was calculated using the ΔΔCt 14 method in each group, assuming the gene expression rate of the negative control group.
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Statistical Analysis Data were analyzed using SPSS 19.0 software (SPSS).¶¶¶¶ Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine significance. Values of p < 0.01 were considered significant.
RESULTS Surface Properties of Specimens Figure 1 shows surface views of (TNS, CON), (TNS, 0.1), (TNS, 1), (Ti, CON), (Ti, 0.1) and (Ti, 0.1). Porous network structures were formed in the TNS group. LPS particles adhered similarly to the surfaces in both groups, and there were more LPS particles in specimens with higher LPS concentration. Our results demonstrated that the quantity of LPS particles increased with higher concentration of P. gingivalis LPS from 0.1 μg/mL to 1 μg/mL, and that the LPS particles were irregular in shape and less than 300 nm in size. Three-dimensional AFM images also showed LPS particles deposited on the surface of LPS-treated specimens, changed the nanoscale character of experiments ‘surface. (Figure 1).AFM showed a similar LPS particle appearance with uniform roughness (Ra: average roughness and Rz: maximum height) (Table 1). Effects of NaOH Modified Surface on Protein Adsorption and Cell Proliferation Under LPS Exposure To test the protein adsorption capacity, the amount of bovine serum albumin protein adsorbed on the disks after 1, 3, 6 and 24 h of incubation was assayed (Figure 2A-C). More protein was adsorbed onto the TNS group disks than that onto the Ti group disks at each time point both without LPS exposure (Fig2A) and with LPS exposure( P. gingivalis LPS 0.1 and 1 μg/mL) at each time point (Fig 2B and C).Cell proliferation on the disks with indicated treatments during the first 7 days of incubation was assessed (Figure 2D-E). As expected, the highest proliferation at each time point was in the samples without LPS exposure. In addition, there were significant differences in cell proliferation between the TNS and Ti groups at each LPS concentration at 1, 3 and 7 days. Osteogenic Effects of NaOH Modified Surface Under LPS Exposure As shown in Figure 3A and B, rat BMMSCs produced ALP on both substrates as early as 1 week after incubation and production increased with time. As expected, LPS treatment reduced the osteogenesis in both TNS and Ti groups. There were significant 6
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differences in ALP production between the TNS and Ti groups at each LPS concentration at each time point. (Figure 3C). In addition, ECM mineralization was assessed by Alizarin Red staining (Figure 4A and B). The TNS group produced abundant mineralization nodules that were larger than those of the Ti group exposed to the same LPS concentration. In addition, the mineralization dots differed in appearance according to the concentration of LPS (Figure 4A and B), indicating that ECM mineralization in the TNS group was significantly greater than in the Ti group (Figure 4C). Accordingly, osteocalcin production was lower with increased LPS concentration, however it is significantly higher in TNS groups in each pair of LPS treatment, as shown in Figure 4D. Furthermore, osteogenesis-related genes, including BSP (A), ON (B), RUNX2(C) and COL-1(D), was assessed by quantitative RT-PCR. The similar substrates explored in this study induced different mRNA expression levels at 3 and 7 days. Generally, the TNS group induced higher mRNA levels than the Ti group. Within the TNS and Ti groups, the experiments without LPS treatment induced the highest mRNA levels for all osteogenesis-related genes. For RANKL, which is osteoclastic, the TNS group displayed lower mRNA induction by LPS than the Ti group. In contrast, the TNS group had higher mRNA levels for OPG than the Ti group, although LPS dose dependently reduced its expression. Anti-inflammation Effects of NaOH Modified Surface Under LPS Exposure Figure 6 showed the expression of inflammatory cytokine genes, including IL-1ß, IL-6 and IL-10, was assessed by quantitative RT-PCR. The gene expression of IL-1ß (A) and IL-6(B) showed that the TNS group induced lower mRNA levels than the Ti group. Within both groups, mRNA levels were higher with increased concentration of LPS. In Figure 6 C, the expression of anti-inflammatory inflammatory cytokine gene IL-10 was also assessed by quantitative RT-PCR. The TNS group and Ti group explored in this study induced different mRNA expression levels at 3 and 7 days. Generally, the TNS group induced higher mRNA levels than the Ti group. Within the TNS and Ti groups, the experiments without LPS treatment induced the highest mRNA levels for IL-10.
DISCUSSION To our knowledge, the present study is the first report testing the effects of 10 M NaOH-modified titanium surfaces with nanonetwork structure on osteogenesis and antiinflammation in the context of LPS exposure. The modified titanium surface of the TNS group showed better endotoxin tolerance than the untreated surface of the Ti group in the growth and osteogenic differentiation of rat BMMSCs. 7
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It has been reported that the implant surface exerts nanoscale topographic control over cell behavior and that certain combinations of nanofeatures can improve osseointegration18-20 and can influence differentiation of BMMSCs into osteoblasts.12,13 Contaminants, such as bacterial debris, may remain on implant surfaces after sterilization procedures and may inhibit osseointegration.21 Gram-negative anaerobes, such as Porphyromonas gingivalis (P. gingivalis)9,22, have a variety of virulence factors, including proteases, fimbriae, lipopolysaccharide (LPS) and capsule, that make them potent pathogens in the development of peri-implantitis. Among these, LPS acts as a chronic stimulus, maintaining peri-implant inflammation and thereby worsening the prognosis for implant osseointegration.10 Osseointegration is crucial for the early fixation and long-term success of dental implants. Three stages of osteogenic differentiation can be distinguished: proliferation, matrix development/maturation, and mineralization.26 Four osteogenic markers, BSP, osteonectin, RUNX2 and COL-1, were selected to analyze differentiation activities. BSP expression is one indicator of the onset of terminal osteoblastic differentiation.27 Osteonectin is a glycoprotein abundantly expressed in bone undergoing active remodeling.28 RUNX2 is an essential transcription factor that is expressed in the early stages of osteogenic differentiation.29 COL-1 accounts for 90% of the total protein in the organic matrix of bone. It not only provides the structural framework with viscoelastic properties but also defines compartments for ordered mineral deposition.30 In contrast, RANKL is produced by osteoblasts and binds to RANK on osteoclast precursors, inducing osteoclast maturation. However, this process is offset by osteoblastic production of OPG, which serves as a decoy receptor for RANKL, thereby inhibiting osteoclast differentiation. Bone remodeling is tightly controlled by RANKL and OPG, and the ratio of RANKL/OPG is considered a good marker for bone resorption.32 It has been demonstrated that excessive micromotion at the bone-implant interface-a key determinant in implant failure-can promote RANKL and inhibit OPG, thereby inducing accelerated peri-implant bone resorption.33 Our study here revealed that NaOH modified surface can promote osteogenesis in all of these steps. Further studies are needed to explore how the modified surface favors an osteogenic gene expression profile, which would further provide clues for refinement. LPS induces the production of inflammatory cytokines, such as IL-1ß, IL-6 and IL8. These cytokines and LPS induce alveolar bone resorption and loosening of implants36 IL-10 acts in the opposite fashion by downregulating the activity of cells and suppressing further cytokine production, leading to an anti-inflammatory/anti-wound healing effect37. We found that LPS increased inflammatory cytokines. However, TNS 34,35
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group induced higher IL10 mRNA levels than the Ti group, which is in contrast with the inflammatory IL-1ß and IL-6. These findings are consistent with our hypothesis. The present study is only a preliminary study in vitro, and further studies are needed to explore its feasibility in vivo, furthermore, additional data from experimental studies with a clinically applicable model, as well as clinical trials, are needed to validate this method. In summary, NaOH modified surface promote osteogenesis at least in two manners. On one hand, it promotes cell esteogenic differentiation; on the other hand, it inhibits the inflammatory response. The underlying mechanism remains largely unknown, especially how the mechanical change senses the cells to alter the gene expression profile. Further studies are undergoing.
CONCLUSION Our study here highlights that the 10M NaOH treated titanium surfaces with nanonetwork structure had better endotoxin tolerance than the untreated titanium surfaces, in the context of P. gingivalis LPS exposure, suggesting an anti-inflammatory potential of 10 M NaOH-modified titanium surface nanonetwork in future clinical application. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (B) for young researcher (24792345 to Y T, 26861664 to S K) from the Japan Society for the Promotion of Science. The authors declare that they have no conflicts of interest. This work was supported by a Grant-in-Aid for Scientific Research (24792345, 26861664) from the Japan Society for the Promotion of Science. The authors declare that they have no conflicts of interest.
REFERENCES 1. Brånemark PI, Hansson BO, Adell R,Breine U, et al. Osseointegrated titanium implants in the treatment of the edentulous jaw. Experience from a 10-year period. Scand J Plast Reconstr Surg Suppl. 1977; 16:1-132. 2. Dam HG, Najm SA, Nurdin N, et al. A 5- to 6-year radiological evaluation of titanium plasma sprayed/sandblasted and acid-etched implants: results from private practice. Clin Oral Implants Res 2014;25:159–165.
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3. Francetti L, Azzola F, Corbella S, et al. Evaluation of clinical outcomes and bone loss around titanium implants with oxidized surface: six-year follow-up results from a prospective case series study. Clin Implant Dent Relat Res 2014;16:81–88. 4. Naert I, Duyck J, Vandamme K. Occlusal overload and bone/implant loss. Clin Oral Implants Res 2012;23 Suppl 6:95–107. 5. Bobia F, Pop RV. Periimplantitis. Aetiology, diagnosis, treatment. A review from the literature. Curr Health Sci J 2010;36:171–175. 6. Mombelli A, Lang NP. The diagnosis and treatment of peri-implantitis. Periodontol 2000 1998;17:63– 76. 7. Giannelli M, Bani D, Tani A, et al. In vitro evaluation of the effects of low-intensity Nd:YAG laser irradiation on the inflammatory reaction elicited by bacterial lipopolysaccharide adherent to titanium dental implants. J Periodontol 2009;80:977–984. 8. Pontoriero R, Tonelli MP, Carnevale G, et al. Experimentally induced peri-implant mucositis. A clinical study in humans. Clin Oral Implants Res 1994;5:254–259. 9. Nelson SK, Knoernschild KL, Robinson FG, et al. Lipopolysaccharide affinity for titanium implant biomaterials. J Prosthet Dent 1997;77: 76–82. 10. Irshad M, Scheres N, Crielaard W, et al. Influence of titanium on in vitro fibroblast-Porphyromonas gingivalis interaction in peri-implantitis. J Clin Periodontol 2013;40:841–849. 11. Barao VA, Mathew MT, Yuan JC, et al. Influence of corrosion on lipopolysaccharide affinity for two different titanium materials. J Prosthet Dent 2013;110:462–470. 12. Zhao L, Wang H, Huo K, et al. The osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates. Biomaterials 2013;34:19–29. 13. Zhang W, Li Z, Liu Y, et al. Biofunctionalization of a titanium surface with a nano-sawtooth structure regulates the behavior of rat bone marrow mesenchymal stem cells. Int J Nanomedicine 2012;7:4459–4472. 14. Xing H, Komasa S, Taguchi Y, et al. Osteogenic activity of titanium surfaces with nanonetwork structures. Int J Nanomedicine 2014;9:1741–1755. 15. Finke B, Luethen F, Schroeder K, et al. The effect of positively charged plasma polymerization on initial osteoblastic focal adhesion on titanium surfaces. Biomaterials 2007;28:4521–4534. 16. Fan H, Cook JA. Molecular mechanisms of endotoxin tolerance. J Endotoxin Res 2004;10:71–84.
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17. Nomura F, Akashi S, Sakao Y, et al. Cutting Edge: Endotoxin Tolerance in Mouse Peritoneal Macrophages Correlates with Down-Regulation of Surface Toll-Like Receptor 4 Expression. J Immunol 2000;164:3476–3479. 18. Mendonca G, Mendonca DB, Aragao FJ, et al. The combination of micron and nanotopography by H(2)SO(4)/H(2)O(2) treatment and its effects on osteoblast-specific gene expression of hMSCs. J Biomed Mater Res A 2010;94:169–179. 19. Zhao L, Mei S, Chu PK, et al. The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions. Biomaterials 2010;31:5072–5082. 20. Kubo K, Tsukimura N, Iwasa F, et al. Cellular behavior on TiO2 nanonodular structures in a microto-nanoscale hierarchy model. Biomaterials 2009;30:5319-5329. 21. Bonsignore LA, Colbrunn RW, Tatro JM, et al. Surface contaminants inhibit osseointegration in a novel murine model. Bone 2010;49:923–930. 22. Shibli JA, Melo L, Ferrari DS, et al. Composition of supra- and subgingival biofilm of subjects with healthy and diseased implants. Clin Oral Implants Res 2008;19:975–982. 23 Vandrovcova M, Hanus J, Drabik M, et al. Effect of different surface nanoroughness of titanium dioxide films on the growth of human osteoblast-like MG63 cells. J Biomed Mater Res A 2012;100:1016–1032. 24 Zhang H, Bremmell K, Kumar S, et al. Vitronectin adsorption on surfaces visualized by tapping mode atomic force microscopy. J Biomed Mater Res A 2004;68:479–488. 25. Xu Z1, Zhang YL, Song C et al. Interactions of hydroxyapatite with proteins and its toxicological effect to zebrafish embryos development. PLoS One. 2012; 7(4):e32818 26 Yamamoto Y, Ohsaki Y, Goto T, et al. Effects of Static Magnetic Fields on Bone Formation in Rat Osteoblast Cultures. J Dent Res 2003;82:962–966. 27 Guo J, Padilla RJ, Ambrose W, et al. The effect of hydrofluoric acid treatment of TiO2 grit blasted titanium implants on adherent osteoblast gene expression in vitro and in vivo. Biomaterials 2007;28:5418–5425. 28 Sila-Asna M, Bunyaratvej A, Maeda S, et al. Osteoblast Differentiation and Bone Formation Gene Expression in Strontium-inducing Bone Marrow Mesenchymal Stem Cell. Kobe J Med Sci 2007;53:25–35. 29 Masaki C, Schneider GB, Zaharias R, et al. Effects of implant surface microtopography on osteoblast gene expression. Clin Oral Implants Res 2005;16:650–656.
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30 Kruger TE, Miller AH, Wang J. Collagen scaffolds in bone sialoprotein-mediated bone regeneration. Scientific World Journal 2013;3:812718. 31 Wlodarski KH, Reddi AH. Alkaline Phosphatase as a Marker of Osteoinductive Cells. Calcif Tissue Int 1986;39:382–385. 32 Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 2008;473:139–146. 33 Stadelmann VA, Terrier A, Pioletti DP. Microstimulation at the bone-implant interface upregulates osteoclast activation pathways. Bone 2008;42:358–364. 34 Nakashima T, Kobayashi Y, Yamasaki S, et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun 2000;275:768–775. 35 Jiang Y. Bacteria Induce Osteoclastogenesis via an Osteoblast-Independent Pathway. Infect Immun 2002;70:3143–3148. 36 Greenfield EM, Bi Y, Ragab AA, al et. Does endotoxin contribute to aseptic loosening of orthopedic implants? J Biomed Mater Res B Appl Biomater 2005;72:179–185. 37. Brodbeck WG1, Voskerician G, Ziats NP et. al. In vivo leukocyte cytokine mRNA responses to biomaterials are dependent on surface chemistry. J Biomed Mater Res A. 2003 Feb 1; 64(2):320-9.
Correspondence author: Helin Xing, Department of Removable Prosthodontics and Occlusion, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 5731121, Japan, Tel: +81-72-864-3084, Fax: +81-72-864-3184, E-mail: [email protected] Submitted June 26, 2014; accepted for publication October 26, 2014. Figure 1. Scanning electron micrographs of TNS and Ti disks treated with 1 mL/well of phosphate-buffered saline containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). The upper pictures at a lower magnification of ×10 000 show the overall microscale topography. The lower pictures at a higher magnification of ×50 000 reveal the nanoscale texture. Table1. Ra: average roughness; Rz: maximum height.
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Figure 2(A-C) Assay of protein adsorption to disks treated with different concentrations of P. gingivalis LPS after 1, 3, 6 and 24 h incubation in bovine serum albumin. P. gingivalis LPS μg/ml (A), P. gingivalis LPS 0.1μg/ml (B), P. gingivalis LPS 1 μg/ml(C). Figure 2(D-F). Bone marrow mesenchymal stem cell proliferation on TNS and Ti samples after 1, 3 and 7 days of incubation with different concentrations of P. gingivalis LPS, as measured by the CellTiter-Blue Cell Viability Assay. P. gingivalis LPS μg/ml (D), P. gingivalis LPS 0.1μg/ml (E), P. gingivalis LPS 1 μg/ml(F). *p
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Effect of Porphyromonas Gingivalis Lipopolysaccharide on Bone Marrow Mesenchymal Stem Cell Osteogenesis on a Titanium Nanosurface *†
‡
Helin Xing D.D.S. Ph.D. , Yoichiro Taguchi D.D.S., Ph.D. , Satoshi Komasa D.D.S., †
‡
§
‡
Ph.D. , Isao Yamawaki D.D.S. , Tohru Sekino Ph.D. ,Makoto Umeda D.D.S., Ph.D. , Joji Okazaki D.D.S., Ph.D. *
†
Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, China.
†
Department of Removable Prosthodontics and Occlusion, Osaka Dental University, Japan. ‡
§
Department of Periodontology, Osaka Dental University, Osaka, Japan.
Advanced Hard Materials, the Institute of Scientific and Industrial Research, Osaka University, Japan. Background: Titanium (Ti) dental implants have been widely used for prosthetic reconstruction
of dentition. Unfortunately, peri-implantitis can result in failure of dental implant osseointegration. Lipopolysaccharides (LPS) acts as a chronic inflammatory stimulus and maintains peri-implant under inflammation, worsening the prognosis for implant osseointegration. The purpose of this study was to determine the effects of 10 M NaOH-modified titanium surface with nanonetwork structure on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in the context of Porphyromonas gingivalis (P. gingivalis) LPS exposure. Methods: Titanium disks treated with 10 M NaOH solution and control were incubated with BMMSCs and then exposed to P. gingivalis LPS (0, 0.1 or 1 μg/mL). The effects of the modified nanonetwork structure on osteogenic differentiation of rat BMMSCs were evaluated in the context of different concentrations of P. gingivalis LPS exposure.
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Results: Rat BMMSCs on the 10 M NaOH-modified titanium surface with nanonetwork structure had higher levels of osteogenesis-related gene expression and significantly greater cell proliferation, ALP activity, extracellular matrix deposition and mineralization than cells on the untreated titanium surfaces, in all the groups with different doses of P. gingivalis LPS exposure. Conclusion: The 10 M NaOH-modified titanium surface with nanonetwork structure has better endotoxin tolerance under P. gingivalis LPS exposure than non-modified surface.
KEY WORDS Bone marrow mesenchymal stem cells, Porphyromonas gingivalis, Lipopolysaccharide, osseointegration, titanium
Since Branemark 1introduced the concept of osseointegration fifty years ago, titanium (Ti) implants have been regarded as important appliances for prosthetic dental reconstruction. Previous studies have demonstrated the medium- and long-term success of dental implants.2, 3 However, limited data exist on the failure of medium- and longterm dental implants. Medium- and long-term failure of implant osseointegration has generally been attributed to occlusal overload4 and peri-implantitis.5, 6 Clinicians can easily control overload, because it ultimately depends on the number and location of occlusal contacts, whereas peri-implantitis requires a specific therapeutic approach.7 Peri-implantitis is an inflammatory lesion around a dental implant, resulting in loss of supporting bone.8 Gram-negative anaerobes, such as Porphyromonas gingivalis (P. gingivalis)9, have a variety of virulence factors, including proteases, fimbriae, lipopolysaccharide (LPS) and capsule, that make them potent pathogens in the development of peri-implantitis. Among these, LPS acts as a chronic stimulus, maintaining peri-implant inflammation and thereby worsening the prognosis for implant osseointegration10 and influencing the behavior of cells such as fibroblasts, osteoblasts and lymphocytes.11 Bone marrow mesenchymal stem cells (BMMSCs) are multipotent and represent a particularly attractive source for tissue engineering. BMMSCs are the first cells to colonize biomaterial surfaces after implantation and they have osteogenic differentiation capacity. Some studies have used BMMSCs as a source of osteogenic cells for bone repair.12,13 The role of BMMSCs in osseointegration through their differentiation into osteoblasts depends on whether their growth and differentiation is affected by LPS exposure. However, little is known about the interactions between LPS and BMMSCs. Therefore, investigation into the effects of LPS on the proliferation and osteogenic differentiation of BMMSCs is necessary. 2
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We recently reported that the nano-network structures with TNS nanofeatures induced by alkali etching markedly enhanced cell adhesion, proliferation, and osteogenesis. These effects were most pronounced when the concentration of NaOH was 10 M, suggesting its great potential in improving the clinical performance of bone implants.14 To better understand whether modified surfaces could tolerate LPS exposure, this study investigated the effects of 10 M NaOH-modified titanium surface with nanonetwork structure on the proliferation and osteogenic differentiation of BMMSCs, in the context of P. gingivalis LPS exposure.
MATERIALS AND METHODS Disk Preparation Titanium disks (15 mm in diameter and 1 mm thick) of grade 2 commercially pure titanium were prepared by machining.‡ After ultrasonic cleaning, these disks were immersed in 10 M NaOH solution in the flask and placed in an oil bath maintained at 30°C for 24 h. Unprocessed titanium disks were used as controls. The solution in each flask was replaced with ion exchange water (200 mL), and this procedure was repeated until the solution reached a conductivity of 5 μS/cm. The disks were then dried at room temperature. The disks were classified as titania nanosheet (TNS) or titanium (Ti) group. Surface Characterization The TNS and Ti disks were plated on 24-well tissue culture plates and 1 mL phosphatebuffered saline (PBS) containing commercially available P. gingivalis LPS (0, 0.1 or 1 μg/mL) § was added to each well. The specimens were labeled (TNS, CON), (TNS, 0.1), (TNS, 1), (Ti, CON), (Ti, 0.1) or (Ti, 0.1), according to the concentration of LPS. The disk surfaces were examined by scanning electron microscopy (SEM; S-4800) and atomic force microscopy (AFM; SPM-9600). ¶ Protein Adsorption Assay Bovine serum albumin, fraction V# was used as a model protein. Three hundred µL of protein solution (1 mg/mL protein in saline) containing P. gingivalis LPS (0, 1, or 10 mg/mL) was pipetted onto the TNS and Ti disks. After incubation for 1, 3, 6 and 24 h at 37°C, the amount of removed albumin and the total amount of albumin inoculated was quantified using a microplate reader at 562 nm, using a 96-well microplate reader (SpectraMax M5). **
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Cell Culture Animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Osaka Dental University (Approval No. 13-02039). Rat BMMSCs were obtained from the femurs of 8-week-old Sprague–Dawley rats. BMMSCs were maintained in growth medium containing minimal essential medium, †† 10% fetal bovine serum, ‡‡ and Antibiotic–Antimycotic Mixed Stock Solution, §§ and cultured in a humidified atmosphere with 5% CO2 at 37°C. After 3 days, the medium containing the nonadherent cells was replaced and thereafter the medium was changed every 3 days. When rat BMMSCs were seeded on the disks, after cultured 7 days, the medium was removed and replaced with differentiation medium containing 10% fetal bovine serum, Antibiotic–Antimycotic Mixed Stock Solution and osteogenic supplements: 10 mM βglycerophosphate, corbic acid¶¶ and 10 nM dexamethasone. ## This medium was changed every 3 days. Cell Proliferation Assay Cell proliferation was measured using the CellTiter-Blue Cell Viability Assay **** according to the manufacturer’s protocol. Rat BMMSCs were seeded on the TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL) and incubated for 1, 3 or 7 days. Fluorescence was recorded at 560/590 nm using a 96-well microplate reader (SpectraMax M5). †††† The difference between the two optical densities was defined as the proliferation value. Alkaline Phosphatase Staining and Activity Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). After culturing for 1 or 2 weeks, the cells were washed and fixed, and ALP staining was performed with BCIP/NBT alkaline phosphatase (ALP) color development substrate‡‡‡ for 15 min. ALP activity of the BMMSCs incubated with LPS was examined at 1 and 2 weeks using an ALP luminometric ELISA kit. §§§ ALP activity was evaluated as the amount of p-nitrophenol released in the enzymatic reaction and measured at 405 nm using the microplate reader (SpectraMax M5).
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Extracellular Matrix Mineralization Extracellular matrix (ECM) mineralization by rat BMMSCs was evaluated with Alizarin Red staining. Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). After culturing for 3 or 4 weeks, the cells were stained with Alizarin Red for 10 min at room temperature. Cell monolayers were washed with distilled water until no more color appeared, and images were acquired. Calcium deposited in the ECM was measured after dissolution with 10% formic acid. The amount of calcium was quantified using a Calcium E-test Kit. ¶¶¶ After 3 or 4 weeks of culture with LPS, the absorbance of the reaction products was measured at 610 nm using the microplate reader (SpectraMax M5). ### The concentration of calcium ions was calculated from the absorbance relative to a standard curve. Osteocalcin ELISA The sandwich enzyme immunoassay used in this study was specific for rat osteocalcin and measured its levels directly in cell culture supernatant after 3 and 4 weeks of culture with P. gingivalis LPS (Rat Osteocalcin ELISA Kit DS), ****** according to the manufacturer’s instructions. Gene Expression Gene expression was evaluated using the real-time TaqMan RT-PCR assay. †††††† Rat BMMSCs were seeded on TNS and Ti disks at a density of 4 × 104 cells/cm2 in normal culture medium (1 mL/well). After a 24-h culture for cell adherence, the medium was replaced with a medium containing P. gingivalis LPS (0, 0.1 or 1 μg/mL) and cultured for 3 or 7 days. Total RNA was isolated using the RNeasy Mini Kit.‡‡‡‡ 10 µL of RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit.§§§§ Expression of genes including bone sialoprotein (BSP), osteonectin (ON), runtrelated transcription factor 2 (RUNX2), collagen type 1 (COL-1), Receptor action of NF kappa B Ligand (RANKL), osteoprotegerin (OPG), interleukin-1β (IL-1β), interleukin6 (IL-6) and interleukin-10 (IL-10) was quantified using the StepOne Plus Real-Time PCR System. The reactive gene expression rate was calculated using the ΔΔCt 14 method in each group, assuming the gene expression rate of the negative control group.
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Statistical Analysis Data were analyzed using SPSS 19.0 software (SPSS).¶¶¶¶ Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine significance. Values of p < 0.01 were considered significant.
RESULTS Surface Properties of Specimens Figure 1 shows surface views of (TNS, CON), (TNS, 0.1), (TNS, 1), (Ti, CON), (Ti, 0.1) and (Ti, 0.1). Porous network structures were formed in the TNS group. LPS particles adhered similarly to the surfaces in both groups, and there were more LPS particles in specimens with higher LPS concentration. Our results demonstrated that the quantity of LPS particles increased with higher concentration of P. gingivalis LPS from 0.1 μg/mL to 1 μg/mL, and that the LPS particles were irregular in shape and less than 300 nm in size. Three-dimensional AFM images also showed LPS particles deposited on the surface of LPS-treated specimens, changed the nanoscale character of experiments ‘surface. (Figure 1).AFM showed a similar LPS particle appearance with uniform roughness (Ra: average roughness and Rz: maximum height) (Table 1). Effects of NaOH Modified Surface on Protein Adsorption and Cell Proliferation Under LPS Exposure To test the protein adsorption capacity, the amount of bovine serum albumin protein adsorbed on the disks after 1, 3, 6 and 24 h of incubation was assayed (Figure 2A-C). More protein was adsorbed onto the TNS group disks than that onto the Ti group disks at each time point both without LPS exposure (Fig2A) and with LPS exposure( P. gingivalis LPS 0.1 and 1 μg/mL) at each time point (Fig 2B and C).Cell proliferation on the disks with indicated treatments during the first 7 days of incubation was assessed (Figure 2D-E). As expected, the highest proliferation at each time point was in the samples without LPS exposure. In addition, there were significant differences in cell proliferation between the TNS and Ti groups at each LPS concentration at 1, 3 and 7 days. Osteogenic Effects of NaOH Modified Surface Under LPS Exposure As shown in Figure 3A and B, rat BMMSCs produced ALP on both substrates as early as 1 week after incubation and production increased with time. As expected, LPS treatment reduced the osteogenesis in both TNS and Ti groups. There were significant 6
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differences in ALP production between the TNS and Ti groups at each LPS concentration at each time point. (Figure 3C). In addition, ECM mineralization was assessed by Alizarin Red staining (Figure 4A and B). The TNS group produced abundant mineralization nodules that were larger than those of the Ti group exposed to the same LPS concentration. In addition, the mineralization dots differed in appearance according to the concentration of LPS (Figure 4A and B), indicating that ECM mineralization in the TNS group was significantly greater than in the Ti group (Figure 4C). Accordingly, osteocalcin production was lower with increased LPS concentration, however it is significantly higher in TNS groups in each pair of LPS treatment, as shown in Figure 4D. Furthermore, osteogenesis-related genes, including BSP (A), ON (B), RUNX2(C) and COL-1(D), was assessed by quantitative RT-PCR. The similar substrates explored in this study induced different mRNA expression levels at 3 and 7 days. Generally, the TNS group induced higher mRNA levels than the Ti group. Within the TNS and Ti groups, the experiments without LPS treatment induced the highest mRNA levels for all osteogenesis-related genes. For RANKL, which is osteoclastic, the TNS group displayed lower mRNA induction by LPS than the Ti group. In contrast, the TNS group had higher mRNA levels for OPG than the Ti group, although LPS dose dependently reduced its expression. Anti-inflammation Effects of NaOH Modified Surface Under LPS Exposure Figure 6 showed the expression of inflammatory cytokine genes, including IL-1ß, IL-6 and IL-10, was assessed by quantitative RT-PCR. The gene expression of IL-1ß (A) and IL-6(B) showed that the TNS group induced lower mRNA levels than the Ti group. Within both groups, mRNA levels were higher with increased concentration of LPS. In Figure 6 C, the expression of anti-inflammatory inflammatory cytokine gene IL-10 was also assessed by quantitative RT-PCR. The TNS group and Ti group explored in this study induced different mRNA expression levels at 3 and 7 days. Generally, the TNS group induced higher mRNA levels than the Ti group. Within the TNS and Ti groups, the experiments without LPS treatment induced the highest mRNA levels for IL-10.
DISCUSSION To our knowledge, the present study is the first report testing the effects of 10 M NaOH-modified titanium surfaces with nanonetwork structure on osteogenesis and antiinflammation in the context of LPS exposure. The modified titanium surface of the TNS group showed better endotoxin tolerance than the untreated surface of the Ti group in the growth and osteogenic differentiation of rat BMMSCs. 7
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It has been reported that the implant surface exerts nanoscale topographic control over cell behavior and that certain combinations of nanofeatures can improve osseointegration18-20 and can influence differentiation of BMMSCs into osteoblasts.12,13 Contaminants, such as bacterial debris, may remain on implant surfaces after sterilization procedures and may inhibit osseointegration.21 Gram-negative anaerobes, such as Porphyromonas gingivalis (P. gingivalis)9,22, have a variety of virulence factors, including proteases, fimbriae, lipopolysaccharide (LPS) and capsule, that make them potent pathogens in the development of peri-implantitis. Among these, LPS acts as a chronic stimulus, maintaining peri-implant inflammation and thereby worsening the prognosis for implant osseointegration.10 Osseointegration is crucial for the early fixation and long-term success of dental implants. Three stages of osteogenic differentiation can be distinguished: proliferation, matrix development/maturation, and mineralization.26 Four osteogenic markers, BSP, osteonectin, RUNX2 and COL-1, were selected to analyze differentiation activities. BSP expression is one indicator of the onset of terminal osteoblastic differentiation.27 Osteonectin is a glycoprotein abundantly expressed in bone undergoing active remodeling.28 RUNX2 is an essential transcription factor that is expressed in the early stages of osteogenic differentiation.29 COL-1 accounts for 90% of the total protein in the organic matrix of bone. It not only provides the structural framework with viscoelastic properties but also defines compartments for ordered mineral deposition.30 In contrast, RANKL is produced by osteoblasts and binds to RANK on osteoclast precursors, inducing osteoclast maturation. However, this process is offset by osteoblastic production of OPG, which serves as a decoy receptor for RANKL, thereby inhibiting osteoclast differentiation. Bone remodeling is tightly controlled by RANKL and OPG, and the ratio of RANKL/OPG is considered a good marker for bone resorption.32 It has been demonstrated that excessive micromotion at the bone-implant interface-a key determinant in implant failure-can promote RANKL and inhibit OPG, thereby inducing accelerated peri-implant bone resorption.33 Our study here revealed that NaOH modified surface can promote osteogenesis in all of these steps. Further studies are needed to explore how the modified surface favors an osteogenic gene expression profile, which would further provide clues for refinement. LPS induces the production of inflammatory cytokines, such as IL-1ß, IL-6 and IL8. These cytokines and LPS induce alveolar bone resorption and loosening of implants36 IL-10 acts in the opposite fashion by downregulating the activity of cells and suppressing further cytokine production, leading to an anti-inflammatory/anti-wound healing effect37. We found that LPS increased inflammatory cytokines. However, TNS 34,35
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group induced higher IL10 mRNA levels than the Ti group, which is in contrast with the inflammatory IL-1ß and IL-6. These findings are consistent with our hypothesis. The present study is only a preliminary study in vitro, and further studies are needed to explore its feasibility in vivo, furthermore, additional data from experimental studies with a clinically applicable model, as well as clinical trials, are needed to validate this method. In summary, NaOH modified surface promote osteogenesis at least in two manners. On one hand, it promotes cell esteogenic differentiation; on the other hand, it inhibits the inflammatory response. The underlying mechanism remains largely unknown, especially how the mechanical change senses the cells to alter the gene expression profile. Further studies are undergoing.
CONCLUSION Our study here highlights that the 10M NaOH treated titanium surfaces with nanonetwork structure had better endotoxin tolerance than the untreated titanium surfaces, in the context of P. gingivalis LPS exposure, suggesting an anti-inflammatory potential of 10 M NaOH-modified titanium surface nanonetwork in future clinical application. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (B) for young researcher (24792345 to Y T, 26861664 to S K) from the Japan Society for the Promotion of Science. The authors declare that they have no conflicts of interest. This work was supported by a Grant-in-Aid for Scientific Research (24792345, 26861664) from the Japan Society for the Promotion of Science. The authors declare that they have no conflicts of interest.
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30 Kruger TE, Miller AH, Wang J. Collagen scaffolds in bone sialoprotein-mediated bone regeneration. Scientific World Journal 2013;3:812718. 31 Wlodarski KH, Reddi AH. Alkaline Phosphatase as a Marker of Osteoinductive Cells. Calcif Tissue Int 1986;39:382–385. 32 Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 2008;473:139–146. 33 Stadelmann VA, Terrier A, Pioletti DP. Microstimulation at the bone-implant interface upregulates osteoclast activation pathways. Bone 2008;42:358–364. 34 Nakashima T, Kobayashi Y, Yamasaki S, et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun 2000;275:768–775. 35 Jiang Y. Bacteria Induce Osteoclastogenesis via an Osteoblast-Independent Pathway. Infect Immun 2002;70:3143–3148. 36 Greenfield EM, Bi Y, Ragab AA, al et. Does endotoxin contribute to aseptic loosening of orthopedic implants? J Biomed Mater Res B Appl Biomater 2005;72:179–185. 37. Brodbeck WG1, Voskerician G, Ziats NP et. al. In vivo leukocyte cytokine mRNA responses to biomaterials are dependent on surface chemistry. J Biomed Mater Res A. 2003 Feb 1; 64(2):320-9.
Correspondence author: Helin Xing, Department of Removable Prosthodontics and Occlusion, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 5731121, Japan, Tel: +81-72-864-3084, Fax: +81-72-864-3184, E-mail: [email protected] Submitted June 26, 2014; accepted for publication October 26, 2014. Figure 1. Scanning electron micrographs of TNS and Ti disks treated with 1 mL/well of phosphate-buffered saline containing P. gingivalis LPS (0, 0.1 or 1 μg/mL). The upper pictures at a lower magnification of ×10 000 show the overall microscale topography. The lower pictures at a higher magnification of ×50 000 reveal the nanoscale texture. Table1. Ra: average roughness; Rz: maximum height.
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Figure 2(A-C) Assay of protein adsorption to disks treated with different concentrations of P. gingivalis LPS after 1, 3, 6 and 24 h incubation in bovine serum albumin. P. gingivalis LPS μg/ml (A), P. gingivalis LPS 0.1μg/ml (B), P. gingivalis LPS 1 μg/ml(C). Figure 2(D-F). Bone marrow mesenchymal stem cell proliferation on TNS and Ti samples after 1, 3 and 7 days of incubation with different concentrations of P. gingivalis LPS, as measured by the CellTiter-Blue Cell Viability Assay. P. gingivalis LPS μg/ml (D), P. gingivalis LPS 0.1μg/ml (E), P. gingivalis LPS 1 μg/ml(F). *p
