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.

Effects of Neodymium-Doped Yttrium Aluminium Garnet (Nd:YAG) Laser Irradiation on Bone Metabolism During Tooth Movement.

INTRODUCTION: The aim of this study is to evaluate the effects of low-level neodymium-doped yttrium aluminium garnet (Nd:YAG) laser irradiation on orthodontic tooth movement and histological examination. METHODS: Eleven male Wistar rats (aged 10 weeks) were included. To produce experimental tooth movement in rats, 10 g force was applied to maxillary first molars with nickel titanium closed coil springs. Right molars were irradiated with Nd:YAG laser on days 0, 1, 2, 3, 7, 10, 14, 17, 21 and 24, while un-irradiated left molars were used as control. Distance between mesial side of second molar and distal side of first molar was measured on µCT image during tooth movement and the rats were sacrificed 4 weeks after the initiation of tooth movement. RESULTS: The amount of tooth movement was significantly greater in the irradiation group (0.20 ± 0.06) than in the control group (0.14 ± 0.03) during the first week (P < 0.05). However, no statistically significant difference was found afterwards. There was a tendency of higher tartrate-resistant acid phosphatase (TRAP)-positive nuclei count in the pressure zones of the laser irradiation group, but it was not statistically significant. In immuno-histological examination, expressions of alkaline phosphatase (ALP) and receptor activator of nuclear factor kappa-B ligand (RANKL) were higher at the pressure site of the laser irradiation group than the control group, whereas there was no difference in osteoprotegerin (OPG) expression. CONCLUSION: The results suggest that low-level Nd:YAG laser may stimulate osteoclast and osteoblast activation and accelerate bone metabolism during tooth movement.

Effects of low-intensity pulsed ultrasound on the expression of cyclooxygenase-2 in mandibular condylar chondrocytes.

AIMS: To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1ß). METHODS: Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1ß. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1ß, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1ß-treated chondrocytes were examined in terms of the expression of p-integrin ß1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS: COX-2 mRNA level was upregulated by the treatment with IL-1ß and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin ß, and it inhibited the expression of p-ERK 1/2. CONCLUSION: LIPUS exposure inhibited IL-1ß-induced COX-2 expression through the integrin ß1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.

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.

Effects of bFGF on the Modulation of Apoptosis in Gingival Fibroblasts with Different Host Ages.

The purpose of this study was to investigate the effects of basic fibroblast growth factor (bFGF) treatment on the proliferation and apoptosis of cultured gingival fibroblasts (GFs). Human GFs were isolated from the palatal gingival tissues of 16 healthy volunteers ranging in the age from 9 to 35 years old. Cultured GFs were subjected to the analyses for cell proliferation by ELISA assay, gene expression by RT-PCR analysis, and apoptosis potency by caspase-3 assay. The cell proliferation activity and gene expression of type-I collagen and caspase-3 activity were enhanced significantly by the treatment with bFGF in cultured GFs. Furthermore, the activity of caspase-3 in cultured GFs from young subjects was significantly higher than that in GFs from adults. It is shown that bFGF significantly enhances the gene expression of type-I collagen in cultured fibroblasts from human gingival tissues. It also demonstrated that bFGF modulates the apoptosis of periodontal fibroblasts, and the effect is higher in young subjects, indicating a significant role of bFGF in the prevention of scar formation during wound healing.