The expression of cyclooxygenase-2 (COX-2) is a characteristic response to inflammation and can be inhibited with sodium salicylate. $TNF-{\alpha}$ plus $IFN-{\gamma}$ can induce extracellular signal-regulated kinase (ERK), IKK, $I{\kappa}B$ degradation and NF-${\kappa}B$ activation. The inhibition of the ERK pathway with selective inhibitor, PD098059, blocked cytokine-induced COX-2 expression and $PGE_2$ release. Salicylate treatment inhibited COX-2 expression induced by $TNF-{\alpha}$/$IFN-{\gamma}$ and regulated the activation of ERK, IKK and $I{\kappa}B$ degradation and subsequent NF-${\kappa}B$ activation in MC3T3E1 osteoblasts. Furthermore, antioxidants such as catalase, N-acetyl-cysteine or reduced glutathione attenuated COX-2 expression in combined cytokines-treated cells, and also inhibited the activation of ERK, IKK and NF-${\kappa}B$ in MC3T3E1 osteoblasts. In addition, $TNF-{\alpha}$/$IFN-{\gamma}$ stimulated ROS release in the osteoblasts. However, salicylate had no obvious effect on ROS release in DCFDA assay. The results showed that salicylate inhibited the activation of ERK and IKK, $I{\kappa}B$ degradation and NF-${\kappa}B$ activation independent of ROS release and suggested that salicylate exerts its anti-inflammatory action in part through inhibition of ERK, IKK, $I{\kappa}B$, $NF-{\kappa}B$ and resultant COX-2 expression pathway.
Bone morphogenetic protein-7(BMP-7), a member of the transforming growth factor superfamily, stimulates osteoblast differentiation and bone formation. There are lots of evidences supporting a direct participation of periodontal ligament(PDL) cells on periodontal tissue regeneration. The purpose of this study was to evaluate the effect of recombinant human(rh) BMP-7 on primary rat PDL cells in vitro, with special focus on the ability of bone formation. The PDL cells were cultured with rhBMP-7 at the concentration of 0, 10, 25, 50, 100 and 200ng/ml for MTT assay. We evaluated the alkaline phosphatase activity at 3 and 5 days of incubation and the ability to produce mineralized nodules of rat PDL cells at 14 days of cell culture in concentration of 0, 10, 25, 50 and 100ng/ml. The cell activity was not reduced in cells treated with BMP-7 at $10{\sim}100ng/ml$, whereas the cell activity was reduced in the concentration of 200ng/ml than the control at day 1 and 3(p<0.01). At 3 and 5 day, alkaline phosphatase activity was significantly increased in cells treated with BMP-7 at 50ng/ml and 100ng/ml(p<0.05). The area of mineralized bone nodule was greater in cells treated with BMP-7 at 50 and 100 ng/ml than the control(p<0.01). These results suggest that rhBMP-7 stimulate rat PDL cells to differentiate toward osteoblast phenotype and secretion of the extracellular matrix of rat PDL cells.
Previous studies have demonstrated an increase in bone mass and density with the use of bisphosphonate in osteoporosis. This agent acts as an inhibitor of osteoclastic activity, and results in increase of net osteoblastic activity. Currently, it has been reported that bisphosphonate has direct effect on osteoblast. This study was designed to evaluate the effect of alendronate on bone regeneration in defect of rat calvaria. The animals used for these experiments were 48 male rats, over 6-8 weeks old. They were divided into three groups according to the dose of alendronate($MK-217^{(R)}$, Merck, USA) administered. After the calvarial defects were surgically created, the rats received a peritoneal alendronate(0.25mg/kg) in group I, a peritoneal alendronate(1.25mg/kg) in group II, and a peritoneal normal saline injection in the control group. Three and six weeks later, blood was sampled and evaluated for alkaline phosphatase activity. The animals were sacrificed for histological observation and histometric analysis of the level of bone formation. The alkaline phosphatase activity was similar in three groups at 3 weeks of experiment. The activity at 6 weeks increased more than twice, compared to 3 weeks, and was slightly higher in group I than the other two groups. In histological observation, all the groups at 3 weeks, osteoblast rimming and new bone formation were observed along the defect margin. At 6 weeks, the defect was almost closed with new and more mature bone, but new bone is thinner than original bone in the central portion of defect. In histometric analysis, group I and II at 3 weeks showed significantly greater new bone formation than the control, and all the groups at 6 weeks showed similar amount of bone formation. These result suggest that alendronate administration in the dose of 0.25mg/kg and 1.25mg/kg promote osseous regeneration.
The Korean Journal of Oral and Maxillofacial Pathology
/
v.42
no.5
/
pp.111-118
/
2018
Nicotine of tobacco component has a controversial impact in the clinical outcome of dental implants. Although numerous nicotine effects on bone healing around implants have been presented, it is rarely reported in vitro study about normal human osteoblast(NHost) from oral and maxillofacial area at the surface of implants. The purpose of the present study was to evaluate the effect of nicotine on the proliferation and differentiation response of NHost to plasmatic and salivary levels of nicotine reported in smokers at the surface of screw-type plasma-sprayed titanium implants. NHosts were seeded on the surface of titanium implants and cultured for 21 days in ${\alpha}-MEM$ supplemented with 10% FBS, 50mg/ml ascorbic acid, 5mM ${\beta}$-glycerophosphate and 100nM dexamethasone. Seeded implants were exposed to various nicotine concentration(0.05-0.5mg/ml) from 1 to 21 days, and characterized for cell morphology, proliferation, differentiation, alkaline phosphatase(ALP) activity and ionized calcium concentration(Cai) of medium. Continuous exposure to higher nicotine concentration(above 0.3mg/ml) induced a dose- and time-dependent vacuolation of the cytoplasm, and a tendency to detach from the implant surface. 0.05mg/ml(lower nicotine concentration) did not cause significant effects in the cell proliferation and ALP activity. 0.1-0.2mg/ml caused evident dose-dependent effects in increased cell proliferation, ALP activity and earlier onset of matrix mineralization at levels up to 0.2mg/ml, while a dose-dependent inhibitory effect at 0.3-0.5mg/ml. Cai concentration of control group was decreased at 14 days. Increased Cai concentration at 0.1-0.2mg/ml, decreased Cai concentration at 0.3mg/ml and no change at 0.5mg/ml during the culture period were seen. It suggested that nicotine concentration could paly an role in modulating NHost activity as a contributing factor associated with proliferation and differentiation of NHost at the surface of implants.
Tissue engineering has been enhanced by advance in biomaterial nature, surface structure and design. In this paper, I report specifically vertically aligned titania ($TiO_2$) nanotube surface structuring for optimization of titanium implants utilizing nanotechnology. The formation, mechanism, characteristics of titania nanotubes are explained and emerging critical role in tissue engineering and regenerative medicine is reviewed. The main focus of this paper is on the unique 3 dimensional tubular shaped nanostructure of titania and its effects on creating epochal impacts on cell behavior. Particularly, I discuss how different cells cultured on titania nanotube are adhered, proliferated, differentiated and showed phenotypic functionality compared to those cultured on flat titanium. As a matter of fact, the presence of titania nanotube surface structuring on titanium for dental applications had an important effect improving the proliferation and mineralization of osteoblasts in vitro, and enhancing the bone bonding strength with rabbit tibia over conventional titanium implants in vivo. The nano-features of titania nanotubular structure are expected to be advantageous in regulating many positive cell and tissue responses for various tissue engineering and regenerative medicine applications.
STATEMENT OF PROBLEM. The aim of this study was to study the effects of various surface treatments to a titanium surface on the expression of Runx2 in vitro. MATERIAL AND METHODS. Human Osteosarcoma TE-85 cells were cultured on machined, sandblasted, or anodic oxidized cpTi discs. At various times of incubation, the cells were collected and then processed for the analysis of mRNA expression of Runx2 using reverse transcription-PCR. RESULTS. The expression pattern of Runx2 mRNA was differed according to the types of surface treatment. When the cells were cultured on the untreated control culture plates, the gene expression of Runx2 was not increased during the experiments. In the case of that the cells were cultured on the machined cpTI discs, the expression level was intermediate at the first day, but increased constitutively to day 5. In cells on sandblasted cpTi discs, the expression level was highest in the first day sample and the level was maintained to 5 days. In cells on anodized cpTi discs, the expression level increased rapidly to 3 days, but decreased slightly in the 5-th day sample. CONCLUSION. Different surface treatments may contribute to the regulation of osteoblast function by influencing the level of gene expression of key osteogenic factors.
Journal of Dental Rehabilitation and Applied Science
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v.20
no.1
/
pp.31-41
/
2004
The osseointegration in implant therapy is achieved following general wound healing mechanism. Platelet play a major role in wound healing process. In addition to blood clot formation, they secrete many growth factors which regulate the attachment, proliferation and differentiation of nearly all cell types. The use of these growth factors is now known to be very effective methods to improve the cellular activity. Platelet-rich plasma which is made with the newly developed technique concentrating platelets 3-folds or more is also proven to be very effective method to stimulate and accelerate the healing of bone and soft tissue. Previous study proved that platelet-rich plasma enhanced the cellular attachment by inducing fibronectin, vitronectin from osteoblast. So, this study was aimed to investigate the effect of platelet-rich plasma on the cellular proliferation and differentiation in vitro. The effect on the proliferation was evaluated by MTT assay. To evaluate autocrine and paracrine effect, conditioned medium was made and compared. By measuring alkaline phosphatase activity, the effect on the cellular differentiation was evaluated. The results were as following: The cellular proliferation of osteoblast cell line increased depending on the concentration of platelet-rich plasma and conditioned medium. The alkaline phosphatase activity increased depending on the concentration of platelet-rich plasma and conditioned medium. These findings imply that platelet-rich plasma enhance the cellular proliferation and differentiation and maximize the cellular activity by using the autocrine and paracrine effect.
Purpose: Titanium implants are widely used in the treatment of dentition defects; however, due to problems such as osseointegration failure, peri-implant bone resorption, and periimplant inflammation, their application is subject to certain restrictions. The surface modification of titanium implants can improve the implant success rate and meet the needs of clinical applications. The goal of this study was to evaluate the effect of the use of porous titanium with a chitosan/hydroxyapatite coating on osseointegration. Methods: Titanium implants with a dense core and a porous outer structure were prepared using a computer-aided design model and selective laser sintering technology, with a fabricated chitosan/hydroxyapatite composite coating on their surfaces. In vivo and in vitro experiments were used to assess osteogenesis. Results: The quasi-elastic gradient and compressive strength of porous titanium implants were observed to decrease as the porosity increased. The in vitro experiments demonstrated that, the porous titanium implants had no biological toxicity; additionally, the porous structure was shown to be superior to dense titanium with regard to facilitating the adhesion and proliferation of osteoblast-like MC3T3-E1 cells. The in vivo experimental results also showed that the porous structure was beneficial, as bone tissue could grow into the pores, thereby exhibiting good osseointegration. Conclusions: Porous titanium with a chitosan/hydroxyapatite coating promoted MC3T3-E1 cell proliferation and differentiation, and also improved osseointegration in vitro. This study has meaningful implications for research into ways of improving the surface structures of implants and promoting implant osseointegration.
Purpose: Sclerostin, an inhibitor of Wnt/${\beta}$-catenin signaling, exerts negative effects on bone formation and contributes to periodontitis-induced alveolar bone loss. Recent studies have demonstrated that serum sclerostin levels are increased in diabetic patients and that sclerostin expression in alveolar bone is enhanced in a diabetic periodontitis model. However, the molecular mechanism of how sclerostin expression is enhanced in diabetic patients remains elusive. Therefore, in this study, the effect of hyperglycemia on the expression of sclerostin in osteoblast lineage cells was examined. Methods: C2C12 and MLO-Y4 cells were used in this study. In order to examine the effect of hyperglycemia, the glucose concentration in the culture medium was adjusted to a range of levels between 40 and 100 mM. Gene expression levels were examined by quantitative reverse transcription-polymerase chain reaction and Western blot assays. Top-Flash reporter was used to examine the transcriptional activity of the ${\beta}$-catenin/lymphoid enhanced factor/T-cell factor complex. Tumor necrosis factor-alpha ($TNF{\alpha}$) protein levels were examined with the enzyme-linked immunosorbent assay. The effect of reactive oxygen species on sclerostin expression was examined by treating cells with 1 mM $H_2O_2$ or 20 mM N-acetylcysteine. Results: The high glucose treatment increased the mRNA and protein levels of sclerostin. High glucose suppressed Wnt3a-induced Top-Flash reporter activity and the expression levels of osteoblast marker genes. High glucose increased reactive oxygen species production and $TNF{\alpha}$ expression levels. Treatment of cells with $H_2O_2$ also enhanced the expression levels of $TNF{\alpha}$ and sclerostin. In addition, N-acetylcysteine treatment or knockdown of $TNF{\alpha}$ attenuated high glucose-induced sclerostin expression. Conclusions: These results suggest that hyperglycemia increases sclerostin expression via the enhanced production of reactive oxygen species and $TNF{\alpha}$.
In clinical therapy, the current goal of dental implants is to enhance quantity and quality of osseointegration. Surface roughness and oxide structure are considered to influence the behavior of adherent cells. The purpose of this study is to evaluate the effect of different surface treatment on cellular response. The attachment and proliferation of osteoblast-like cell on sandblasted, sandblasted and etched, thermal oxidated surfaces have been compared. Sandblasting was done with $Al_2O_3$ particles(grain size of $50{\mu}m$), etching was processed with $NH_4OH$ : $H_2O_2$ : $H_2O(1:1:5)$ at $90^{\circ}C$ for 1 minute. Thermal oxidation was followed sandblasting and etching at $400^{\circ}C$, $600^{\circ}C$, $800^{\circ}C$ for 2 hours. Measurement of surface roughness after the different treatment did not show any differences of Ra value between terated surfaces. Cell attachment and proliferation were increased during experiment period, but no difference was observed. SEM evaluation revealed a similar pattern of osteoblast-like cells, well attached with dendritic extension and producing numerous matrix vesicles on cell surface. The results of this study showed that oxide layer alteration by thermal oxidation did not affect the attachment and proliferation of osteoblast-like cells. This suggests the possibility that the cellular responses are further influenced by surface roughness than titaniun oxide structure.
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