Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress.

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well-known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVß3 and αVß5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and MMP-13 induced by excessive mechanical stress. In addition, the protein expression of IL1-ß and MMP-13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress-induced integrin signaling in chondrocytes by using western blotting. The levels of p-FAK, p-ERK, p-JNK, and p-p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVß3 and αVß5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL-1ß, TNF-α, MMP-3, and MMP-13 through phosphorylation of FAK and MAPKs.

Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells.

Cleft lip and palate is the most common congenital anomaly in the orofacial region. Autogenous iliac bone graft, in general, has been employed for closing the bone defect at the alveolar cleft. However, such iliac bone graft provides patients with substantial surgical and psychological invasions. Consequently, development of a less invasive method has been highly anticipated. Stem cells from human exfoliated deciduous teeth (SHED) are a major candidate for playing a significant role in tissue engineering and regenerative medicine. The aim of this study was to elucidate the nature of bone regeneration by SHED as compared to that of human dental pulp stem cells (hDPSCs) and bone marrow mesenchymal stem cells (hBMSCs). The stems cells derived from pulp tissues and bone marrow were transplanted with a polylactic-coglycolic acid barrier membrane as a scaffold, for use in bone regeneration in an artificial bone defect of 4 mm in diameter in the calvaria of immunodeficient mice. Three-dimensional analysis using micro CT and histological evaluation were performed. Degree of bone regeneration with SHED relative to the bone defect was almost equivalent to that with hDPSCs and hBMSCs 12 weeks after transplantation. The ratio of new bone formation relative to the pre-created bone defect was not significantly different among groups with SHED, hDPSCs and hBMSCs. In addition, as a result of histological evaluation, SHED produced the largest osteoid and widely distributed collagen fibers compared to hDPSCs and hBMSCs groups. Thus, SHED transplantation exerted bone regeneration ability sufficient for the repair of bone defect. The present study has demonstrated that SHED is one of the best candidate as a cell source for the reconstruction of alveolar cleft due to the bone regeneration ability with less surgical invasion.

Experimental Tooth Movement Into New Bone Area Regenerated by Use of Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: The aim of this study was to examine experimental tooth movement into regenerated bone in alveolar cleft with mesenchymal stem cells and a granulated carbonated hydroxyapatite scaffold. DESIGN: An artificial bone defect was created bilaterally in upper incisor regions of beagle dogs to simulate alveolar clefts in patients with cleft palate. The mesenchymal stem cells derived from the iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite into the bone defect area. Carbonated hydroxyapatite alone was transplanted on the control side. Six months after the transplantation, multi-bracket appliances were attached to the lateral incisors and canines on both sides of the maxilla to exert an orthodontic force of 100 × g using an elastic chain. The distance between lateral incisor and canine was measured, and standardized x-ray images were taken every month. The tissue after tooth movement was evaluated by histological observation. RESULTS: The experimental tooth movement, accompanied by resorption of regenerated bone and new bone formation, was achieved on the experimental and control sides. Although there was no difference in the amount of tooth movement obtained on the experimental and control sides during the 6-month experimental period, the rate of tooth movement varied on the control side; whereas, the rate was consistent on the experimental side. Root resorption of the tooth was observed on the control side in one dog. CONCLUSION: It is suggested that mesenchymal/carbonated hydroxyapatite transplantation therapy has great potential as a new treatment modality for bone regeneration in patients with cleft palate.

Cranial bone regeneration after cranioplasty using cryopreserved autogenous bone by a programmed freezer with a magnetic field in rats.

BACKGROUND: The purpose of this study was to develop a bone tissue bank using a programmed freezer with a magnetic field. Parietal bones were removed from rats and used for organ culture examination (non-cryopreserved, cryopreserved with a magnetic field (CAS) and cryopreserved without a magnetic field group). Next, other parietal bones were used for histological examination. The cryopreserved bones by a CAS freezer and dried bones were transplanted respectively. Control bones were replanted without cryopreservation. Animals were sacrificed at 4, 8, 12 and 24 weeks after surgery. After organ culture, the isolated osteoblasts from parietal bones which were cryopreserved by a CAS freezer can survive and proliferate as much as non-cryopreserved group. Histological examinations showed new bone formation in control and CAS group. These results suggest that bone tissue cryopreservation by CAS freezer can be successfully used for bone grafting which may be a novel option for regeneration medicine.

Factors related to stability following the surgical correction of skeletal open bite.

OBJECTIVES: If a skeletal anterior open bite malocclusion is treated by orthognathic surgery directed only at the mandible, the lower jaw is repositioned upward in a counter-clockwise rotation. However, this procedure has a high risk of relapse. In the present study, the key factors associated with post-surgical stability of corrected skeletal anterior open bite malocclusions were investigated. MATERIAL AND METHODS: Eighteen orthognathic patients were subjected to cephalometric analysis to assess the dental and skeletal changes following mandibular surgery for the correction of an anterior open bite. The patients were divided into two groups, determined by an increase or decrease in nasion-menton (N-Me) distance as a consequence of surgery. Changes in overbite, the displacements of molars and positional changes in Menton were evaluated immediately before and after surgery and after a minimum of one year post-operatively. RESULTS: The group with a decreased N-Me distance exhibited a significantly greater backward positioning of the mandible. The group with an increased N-Me distance experienced significantly greater dentoalveolar extrusion of the lower molars. CONCLUSIONS: A sufficient mandibular backward repositioning is an effective technique in the prevention of open bite relapse. In addition, it is important not to induce molar extrusion during post-surgical orthodontic treatment to preserve stability of the surgical open bite correction.

Regulation of basic helix-loop-helix transcription factors Dec1 and Dec2 by RORα and their roles in adipogenesis.

DEC1 and DEC2, members of the basic helix-loop-helix superfamily, are involved in various biological phenomena including clock systems, cell differentiation and metabolism. In clock systems, Dec1 and Dec2 expression are up-regulated by the CLOCK:BMAL1 heterodimer via E-box (CACGTG), exhibiting a circadian rhythm in the suprachiasmatic nucleus (SCN), the central circadian pacemaker and other peripheral tissues. In this study, using assays of luciferase reporters, electrophoretic mobility shift and chromatin immunoprecipitation, we identified novel nuclear receptor response elements, ROR response elements (RORE), in Dec1 and Dec2 promoters. These ROREs responded to the transcriptional activator RORα, but not to the repressor REVERBα, although the Bmal1 promoter responded to both RORα and REVERBα. Therefore, RORα, but not REVERBα, is involved in the regulation of Dec1 and Dec2 expression without significantly affecting their rhythmicity. Since RORα, DEC1 and DEC2 reportedly suppressed adipogenic differentiation, we examined expression of Rorα, Dec1, Dec2 and other clock-controlled genes in differentiating 3T3-L1 adipocytes. The results suggested that RORα suppresses adipogenic differentiation at a later stage of differentiation by RORE-mediated stimulation of Dec1 and Dec2 expression.

Synthesis of hyaluronan and superficial zone protein in synovial membrane cells modulated by fluid flow.

Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied.

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field.

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of -150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.

Open bite as a risk factor for orthodontic root resorption.

The purpose of the present study was to clarify the prevalence and degree of root resorption induced by orthodontic treatment in patients with and without open bite. One hundred and eleven patients treated with multibracket appliances were retrospectively selected from the patients and divided into non-open bite (NOB) and open bite (OB) groups. The severity of root resorption and the root shape were classified into five groups on periapical radiographs before and after treatment. Moreover, only in the OB group, all teeth were sub-divided into functional and hypofunctional ones that are occluding and non-occluding. As the results of multiple linear regression analysis of patient characteristics and clinical variables with the number of overall root resorption, the independent variables that were found to contribute significantly to root resorption were bite and abnormal root shape. The prevalences of root resorption evaluated in the number of patients were significantly higher in OB group than in NOB group, and those in the number of teeth were significantly higher in OB group than in NOB group, in particular anterior and premolar teeth. The prevalence of resorbed teeth with abnormal root shapes was also significantly higher in OB group than in NOB group. On the other hand, in OB group, the prevalences of root resorption and teeth with abnormal root shape were significantly greater in hypofunctional teeth than in normal functional teeth. There are more teeth with root resorption and abnormal root shape in open bite cases than in normal bite cases, and more teeth with abnormal root shapes and root resorption in hypofunctional teeth than in functional teeth.

Changes in the lip-line in asymmetrical cases treated with isolated mandibular surgery.

OBJECTIVE: The aim of this study was to investigate changes in the lip-line in asymmetrical cases treated with mandibular osteotomy alone. DESIGN: Retrospective study. SETTING: Hiroshima University Institute of Biomedical & Health Sciences, Hiroshima, Japan METHODS: The subjects in this study consisted of 30 patients with an altered lip-line inclination who underwent isolated mandibular osteotomy (bilateral or unilateral sagittal split ramus osteotomy or intraoral vertical ramus osteotomy) as part of their surgical correction. Frontal cephalograms and facial photographs, taken at the first examination and after treatment, were used to measure changes in the inclination of the lip-line and cant of the occlusal plane, as well as the lateral deviations of hard and soft tissue Menton. RESULTS: Inclination of the lip-line after active treatment was significantly improved compared with that before treatment. Menton on the hard and soft tissues after active treatment also experienced a significant improvement compared with its position at first examination. CONCLUSIONS: One-jaw mandibular osteotomy is able to improve the inclination of the lip-line even in the presence of an occlusal cant. The inclination of the lip-line is corrected in association with sufficient lateral movements of Menton on the mandible.

Enhanced increase in pharyngeal airway size in Japanese class II children following a 1-year treatment with an activator appliance.

OBJECTIVE: The aim of this study was to assess the upper airway soft-tissue structures in Class IIJapanese children following activator treatment by means of cephalometric analysis. STUDY DESIGN: A lateral cephalometric radiograph was taken of each patient at Ti (prior to the placement of the activator; mean age: 11 years, 5 months) and T2 (after 1 year of activator treatment; mean age: 12 years, 5 months). Tracings of the lateral cephalometric radiographs were made on acetate paper, and several soft-tissue points and contours of the tongue, soft palate, hyoid, and pharynx were digitised. RESULTS: The sizes of the oropharynx and hypopharynx were significantly smaller in Class II patients than in Class I patients. Moreover, significant differences were observed in the sizes of the tongue and soft palate between Class I and II patients at the age of 12. In terms of the ratio of change relative to the initial values between the 2 skeletal patterns, the width and dimension of the pharyngeal airway in Class II patients showed considerable increases after activator use. CONCLUSION: The pharyngeal airway soft tissue structures of Class I patients using the activator exceeded normal growth after 1 year. This finding suggests that correction ofskeletal Class II discrepancies by the activator in orthodontic treatment could reduce the risk of respiratory problems, such as severe snoring, obstructive sleep apnoea, and excessive daytime sleepiness in the future.

Construction of orthodontic setup models on a computer.

INTRODUCTION: Orthodontic setup models are usually limited to the display of teeth, with no information about the roots. The purpose of this article is to present a method for visualizing the tooth roots in setup models by integrating information from cone-beam computed tomography and a laser scanner. The reproducibility of the integration was evaluated. METHODS: The records of 5 patients were used in this study. Three-dimensional digital models were generated from the dental casts. Tooth models were generated from the cone-beam computed tomography slices. The 3-dimensional models were superimposed on the crowns of the teeth in the tooth models and integrated. The integrated 3-dimensional tooth model and 3-dimensional setup model were registered. The reproducibility of the integration was evaluated for each tooth. Unpaired Student t tests were performed on the data between the anterior and posterior teeth, and between the right and left teeth. RESULTS: The discrepancy among the integrated 3-dimensional models at the final positions after we used this technique was 0.025 ± 0.007 mm. There was a significant difference in the distance between the anterior and posterior teeth (P <0.05). However, the average distances between the anterior and posterior teeth were small: 0.023 ± 0.007 and 0.028 ± 0.007 mm, respectively. No significant difference was found between the right and left teeth (P = 0.831). CONCLUSIONS: The methods presented in this study provide a reproducible visualization of tooth roots in virtual setup models by registering accurate crown models to cone-beam computed tomography scans.

Association between root resorption incident to orthodontic treatment and treatment factors.

The purpose of this study was to clarify the prevalence and degree of root resorption induced by orthodontic treatment in association with treatment factors. The files of 243 patients (72 males and 171 females) aged 9-51 years were randomly selected from subjects treated with multi-bracket appliances. The severity of root resorption was classified into five categories on radiographs taken before and after treatment. The subjects were divided into extraction (n = 113 patients, 2805 teeth) and non-extraction (n = 130 patients, 3616 teeth) groups and surgical (n = 56 patients, 1503 teeth) and non-surgical treatment (n = 187 patients, 4918 teeth) groups. These subjects were also divided into two or three groups based on the duration of multiloop edgewise archwire (MEAW) treatment, elastic use, and total treatment time: 0 month (T1; n = 184 patients, 4831 teeth), range 1-6 months (T2; n = 37 patients, 994 teeth), more than 6 months (T3; n = 22 patients, 596 teeth); range 0-6 months (n = 114 patients, 3016 teeth) more than 6 months (n = 129 patients, 3405 teeth); range 1-30 months (n = 148 patients, 3913 teeth) and more than 30 months (n = 95 patients, 2508 teeth). The prevalence of overall and severe root resorption evaluated by the number of subjects and teeth was compared with a chi-square test. A Student's t-test for unpaired data was used to determine any statistically significant differences. The prevalence of severe root resorption based on the number of teeth was significantly higher in the group with extractions (P < 0.01). Longer use of a MEAW appliance and elastics also produced a significantly higher prevalence of root resorption (P < 0.05). On the other hand, the prevalence of severe root resorption was not significantly different between the subjects treated with or without surgery, but there was a significant increase when treatment time was prolonged (P < 0.05). A significant difference was found in the amount of root movement of the upper central incisors and the distance from their root apices to the cortical bone surface (P < 0.05). These are regarded as essential factors in the onset of root resorption. These results indicate that orthodontic treatment with extractions, long-term use of a MEAW appliance and elastics, treatment time, and distance of tooth movement are risk factors for severe root resorption.

Uprighting of severely impacted mandibular second molars: a case report.

INTRODUCTION: The incidence of mandibular first and second molar impaction is increasing but still recorded as rare. Treatment methods involving uprighting, extraction, or autologous tooth transplantation have been described. AIM: The present study describes the uprighting of 3 impacted mandibular second molars presenting with eruptive disorders. METHODS: The application of limited and appropriate orthodontic therapy completed treatment in 11 months, 5 months, and 2 years and 3 months, respectively. Although no absolute anchorage in the form of miniscrews was required, no significant anchorage demands were considered necessary. Although the third molar tooth germs were identified and preserved in each case, no adverse influence on the uprighting of the second molars was encountered. RESULTS: The favourable molar repositioning results were likely due to the youth of the 3 patients as the third molars were in early development and bone remodelling was marked. Furthermore, no problems related to anchorage or alveolar bone loss were identified after treatment. CONCLUSION: The results indicated the benefits of limited orthodontic treatment and early intervention for the uprighting of impacted mandibular second molars.

Superficial zone protein affects boundary lubrication on the surface of mandibular condylar cartilage.

We examined the localization and boundary lubricating function of superficial zone protein (SZP) on the surface of mandibular condylar cartilage. Chondrocytes were separated from the surface layer of mandibular condylar cartilage of 6- to 9-month-old female pigs. A cyclic tensile strain of 7% or 21% cell elongation was applied to the cultured chondrocytes. Gene expression levels of cartilage matrix proteins and secretory phospholipase A(2) (sPLA(2)) were quantified by real-time polymerase chain reaction analysis. The friction coefficient of the mandibular condylar surface was measured by a friction tester before and after treatment with 0.1 U/ml sPLA(2). Significantly higher mRNA levels of SZP and type I collagen were found in chondrocytes from the superficial layer than in those in the other layers. The SZP mRNA level was up-regulated by cyclic tensile strain of 7% and 21% cell elongation. Cyclic tensile strain of 21% cell elongation up-regulated the sPLA(2) mRNA level. The friction coefficient of the condylar surface was increased significantly by treatment with sPLA(2). The removal of SZP from the surface layer of mandibular condylar cartilage by sPLA(2) resulted in a significant increase in the friction coefficient on the surface of articular cartilage.

Changes in cortical bone mineralization in the mouse mandible with regenerated condyle.

The objective of this study was to evaluate the effects of condylectomy and of a functional appliance on the mineral content, density, and area of the mandibular cortical bone in a growing mouse model. In the condylectomy group, a unilateral condylectomy was performed on the right side of the mouse mandible. In the condylectomy + appliance group, a functional appliance was used to reposition the mandible after the unilateral condylectomy. All mice were killed 4 wk after surgery. Each mandible was then subjected to analyses of cortical bone mineral content (CRT_CNT), cortical bone density (CRT_DEN), and cortical bone area (CRT_A) by peripheral quantitative computed tomography (pQCT). The CRT_CNT, CRT_DEN, and CRT_A values were significantly lower in the condylectomized mandible (right side) than in the non-condylectomized mandible (left side). However, in the condylectomized animals in which a functional appliance was used, the CRT_CNT, CRT_DEN, and CRT_A values became higher than those in the mice treated with condylectomy alone. No significant differences were found in the CRT_CNT, CRT_DEN, and CRT_A between non-condylectomized (left side) mandibles, mandibles treated with condylectomy + functional appliance (right side), and control mandibles. It was thus shown that a functional appliance used to reposition the condylectomized mandible forward in a symmetric position induced improvement of the cortical bone in a mouse model in terms of cortical bone mineral content, density, and area.

In-vitro and in-vivo study of periodontal ligament cryopreserved with a magnetic field.

INTRODUCTION: The purpose of this study was to examine the effect of a new cryopreservation method with a magnetic field on periodontal regeneration in vitro and in vivo. METHODS: Human periodontal ligament cells were frozen in 10% dimethyl sulfoxide by using a programmed freezer with a magnetic field. Cells were cryopreserved for 3 days at -150°C. Immediately after thawing, collagen type I and alkaline phosphatase gene expression were determined by real-time polymerase chain reaction. Incisors were extracted from 15-week-old Wistar rats and cryopreserved or dried for 3 days. Then the incisors were replanted into the same sockets. Ninety days after transplantation, they were observed under light microscopy. RESULTS: There was no difference in the messenger RNA expression of collagen type I between the cryopreserved and the control groups. The expression of alkaline phosphatase messenger RNA in the cryopreserved group was slightly decreased compared with the control group. There was no progressive root resorption in the teeth that were replanted immediately (control group) or cryopreserved. However, there was widespread root resorption and ankylosis in the dried teeth. CONCLUSIONS: These results show that a magnetic field programmed freezer can be successfully used for cryopreservation of teeth.

Treatment of a case of skeletal class II malocclusion with temporomandibular joint disorder using miniscrew anchorage.

At the present time, there are no reports in the literature on the treatment of temporomandibular joint disorder (TMD) by intrusion of molars using mini-screws. This case report describes the treatment for a female patient, aged 19 years seven months, with a TMD and an excessive lower anterior facial height. Overjet and overbite were +5.0 mm and +0.5 mm, respectively. The patient had a history of orthodontic treatment in which her first premolars were all extracted. During the first orthodontic treatment, a clockwise mandibular rotation was observed as a result of the increase of posterior dentoalveolar height. She had temporomandibular joint (TMJ) pain during mouth opening and complained of difficulty in eating due to masticatory dysfunction. The pretreatment Schuller views of both TMJ showed a posterior condyle position. In order to correct the overjet, molar relationship and the mandibular condyle position, a miniscrew was inserted into the palatal region of the upper first molar to intrude the upper posterior teeth. As the upper molars were intruded, the overjet was decreased, and a class I molar relationship was achieved by a counterclockwise mandibular rotation. After one year of treatment, an acceptable occlusion was achieved, and the condyle moved into centric position in the glenoid fossa. The patient's teeth continued to be stable, and she had no pain in TMJ after a retention period of three years. The result of this treatment showed that molar intrusion using miniscrew anchorage is effective for treatment of a TMD patient with a posterior condyle position.

Inhibitory effects of orally administrated liposomal bovine lactoferrin on the LPS-induced osteoclastogenesis.

Bovine lactoferrin (bLF) modulates the production of proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, and may thus control alveolar bone destruction associated with periodontitis. In this study, the effects of bLF on mRNA expression in lipopolysaccharide (LPS)-stimulated osteoblasts (OBs) and on LPS-induced osteoclastogenesis were examined. The inhibitory effects of oral administration of liposomal-bLF (L-bLF), which improved the robustness of bLF to digestive enzymes, on alveolar bone resorption using LPS-induced periodontitis rat model are also reported. Three groups of 7-week-old male Wistar rats were treated with L-bLF (L-bLF group), bLF (bLF group), or the vehicle (control group) in drinking water (n=6 in each group). On day 7, LPS was topically applied into the gingival sulcus. Number of osteoclasts and immunoexpression of TNF-alpha were analyzed. The bLF inhibited the upregulation of TNF-alpha-mRNA- and upregulation of receptor activator of NF kappaB (RANKL)-mRNA expression and eliminated downregulation of osteoprotegerin (OPG)-mRNA expression in LPS-stimulated OBs and reduced LPS-induced osteoclastogenesis in co-culture with primary OBs and bone marrow cells. In the control group, the number of osteoclasts increased after LPS treatment. The number of osteoclasts that appeared along the alveolar bone margin was significantly reduced (P<0.01) in the L-bLF but not in the bLF group. Furthermore, L-bLF suppressed upregulation of TNF-alpha immunoexpression in periodontal tissue and TNF-alpha and interleukin (IL)-1 beta-mRNA level in gingival tissue. The results of this study indicate that oral administration of L-bLF significantly reduces alveolar bone resorption induced by LPS stimulation through inhibition of TNF-alpha production and modulation of RANKL/OPG balance in OBs. It is suggested that L-bLF could be a potent therapeutic and preventive agent for attenuating alveolar bone destruction in periodontitis patients.

Activation of the hypoxia-inducible factor-1 in overloaded temporomandibular joint, and induction of osteoclastogenesis.

Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats. Here we demonstrate for the first time that hypoxia-inducible factor-1alpha (Hif-1alpha) is activated in mature chondrocytes of temporomandibular joint-osteoarthritis (TMJ-OA) model rat by mechanical overload, and that activated Hif-1 in chondrocytes can induce osteoclastogenesis via repression of osteoprotegerin (Opg) expression. In rat TMJs, degeneration of the condylar cartilage became prominent in proportion to the duration of overloading. Hif-1alpha expression was observed specifically in mature and hypertrophic chondrocytes, and Hif-1alpha-positivity, level of Vegf expression, and tartrate-resistant acid phosphatase (TRAP)-positive cell numbers all increased in the same manner. When ATDC5 cells induced differentiation by insulin were cultured under hypoxia, Hif-1alpha induction was observed in mature stage, but not in immature stage. Inductions of Hif-1-target genes showed a similar expression pattern. In addition, expression of Opg decreased in hypoxia, and Hif-1alpha played a role, in part, in its regulation.