Functional regulation of osteoblastic MC3T3E-1 cells by hyperbaric oxygen treatment.

OBJECTIVES: The purpose of the present study was to examine the influence of hyperbaric oxygen (HBO) on the function of osteoblastic MC3T3-E1 cells. DESIGN: Murine MC3T3-E1 cells were exposed to HBO treatment (at 2.5 absolute atmospheric pressure with 100% oxygen, 90 min per day) for 28 days. Alkaline phosphatase (ALP) staining, activity, and calcium (Ca) content were measured. Gene expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1α (HIF-1α), type 1 collagen (COL1), and osteocalcin (OCN) was assessed using real-time quantitative polymerase chain reaction after a single HBO exposure for 1.5, 6, and 12 h. Furthermore, adenosine triphosphate (ATP) levels were measured using a luminescent cell viability assay. RESULTS: ALP activity and Ca content were higher in the HBO group compared to those in the control group. Gene expression of bFGF, COL1, and OCN was upregulated in the HBO group; however, that of VEGF and HIF-1α significantly decreased in the HBO group in comparison with that in the control group. ATP levels were significantly higher in the HBO group compared to those in the control group. CONCLUSIONS: These findings suggest that HBO accelerates bone formation by increasing the ATP levels of osteoblasts, and bFGF can act as a substitute for VEGF in vascularization by HBO application.

High-frequency near-infrared diode laser irradiation suppresses IL-1ß-induced inflammatory cytokine expression and NF-κB signaling pathways in human primary chondrocytes.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are common inflammation-associated cartilage degenerative diseases. Recent studies have shown that low-level diode laser treatment can reduce inflammatory cytokine expressions in cartilage. We recently reported that high-frequency low-level diode laser irradiation attenuates matrix metalloproteinases (MMPs) expression in human primary chondrocytes. However, the molecular mechanism underlying the effect of high-frequency low-level diode laser on chondrocytes remains unclear. Therefore, we aimed to elucidate the effect of high-frequency low-level diode laser irradiation on inflammatory cytokine expression in human primary chondrocytes. Normal human articular chondrocytes were treated with recombinant interleukin-1 beta (IL-1ß) for 30 min or 24 h and irradiated with a high-frequency NIR diode laser at 8 J/cm2. The expression of IL-1ß, interleukin-6, and tumor necrosis factor-alpha was assessed using western blot analysis. To evaluate the nuclear factor-kappa B (NF-κB) signaling pathway, the phosphorylation, translocation, and DNA-binding activity of NF-κB were detected using western blot analysis, immunofluorescence analysis, electrophoretic mobility shift assay, and enzyme-linked immunosorbent assay analysis. High-frequency low-level diode laser irradiation decreased inflammatory cytokine expression in IL-1ß-treated chondrocytes. Moreover, high-frequency low-level diode laser irradiation decreased the phosphorylation, nuclear translocation, and DNA-binding activity of NF-κB in the IL-1ß-treated state. However, irradiation alone did not affect NF-κB activity. Thus, high-frequency low-level diode laser irradiation at 8 J/cm2 can reduce inflammatory cytokine expressions in normal human articular chondrocytes through NF-κB regulation. These findings indicate that high-frequency low-level diode laser irradiation may reduce the expression of inflammatory cytokines in OA and RA.

Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model.

BACKGROUND: Porphyromonas gingivalis (Pg) infection causes periodontal disease and exacerbates rheumatoid arthritis (RA). It is reported that inoculation of periodontopathogenic bacteria (i.e., Pg) can alter gut microbiota composition in the animal models. Gut microbiota dysbiosis in human has shown strong associations with systemic diseases, including RA, diabetes mellitus, and inflammatory bowel disease. Therefore, this study investigated dysbiosis-mediated arthritis by Pg oral inoculation in an experimental arthritis model mouse. METHODS: Pg inoculation in the oral cavity twice a week for 6 weeks was performed to induce periodontitis in SKG mice. Concomitantly, a single intraperitoneal (i.p.) injection of laminarin (LA) was administered to induce experimental arthritis (Pg-LA mouse). Citrullinated protein (CP) and IL-6 levels in serum as well as periodontal, intestinal, and joint tissues were measured by ELISA. Gut microbiota composition was determined by pyrosequencing the 16 s ribosomal RNA genes after DNA purification of mouse feces. Fecal microbiota transplantation (FMT) was performed by transferring Pg-LA-derived feces to normal SKG mice. The effects of Pg peptidylarginine deiminase (PgPAD) on the level of citrullinated proteins and arthritis progression were determined using a PgPAD knockout mutant. RESULTS: Periodontal alveolar bone loss and IL-6 in gingival tissue were induced by Pg oral infection, as well as severe joint destruction, increased arthritis scores (AS), and both IL-6 and CP productions in serum, joint, and intestinal tissues. Distribution of Deferribacteres and S24-7 was decreased, while CP was significantly increased in gingiva, joint, and intestinal tissues of Pg-inoculated experimental arthritis mice compared to experimental arthritis mice without Pg inoculation. Further, FMT from Pg-inoculated experimental arthritis mice reproduced donor gut microbiota and resulted in severe joint destruction with increased IL-6 and CP production in joint and intestinal tissues. The average AS of FMT from Pg-inoculated experimental arthritis was much higher than that of donor mouse. However, inoculation of the PgPAD knockout mutant inhibited the elevation of arthritis scores and ACPA level in serum and reduced CP amount in gingival, joint, and intestinal tissues compared to Pg wild-type inoculation. CONCLUSION: Pg oral infection affected gut microbiota dysbiosis and joint destruction via increased CP generation.

Effects of hyperbaric oxygen treatment on calvarial bone regeneration in young and adult mice.

OBJECTIVE: The present study aimed to investigate the effects of hyperbaric oxygen (HBO) treatment on calvarial bone regeneration in young and adult mice. METHODS: Calvarial defects of 6.0 mm diameter were created in sixteen 3-week (young) and sixteen 32-week old (adult) mice. The mice were divided into two groups of eight animals each (HBO-treated and control). The 90-min HBO treatment at 2.5 absolute atmospheric pressure and 100 % oxygen was performed for five days a week for 12 weeks. After 2-weeks from the operation, micro-computerized tomography and video microscopy were used to evaluate the regenerated bone volume and microcirculation every two weeks. The protein concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in exudates of the calvarial tissue field were measured at 1, 2, 3, and 4 weeks after surgery. After 12 weeks, histochemical examination of regenerated calvarial bone was conducted. RESULTS: Regenerated bone was formed earlier in young mice than in adult mice treated with HBO. HBO stimulates angiogenesis in the periosteum around regenerated bone area in both young and adult mice at 2 weeks. VEGF concentrations in the calvarial tissue field were lower in the HBO group than in the control 1 week after operation, although bFGF were higher till the 2nd week in the HBO group than in the control. CONCLUSIONS: HBO accelerates bone regeneration earlier in young mice than in adult mice. In the HBO-treated group, bFGF expression was promoted at an early stage, although the expression of VEGF was inhibited.

Effects of Nd:YAG low-level laser irradiation on cultured human osteoblasts migration and ATP production: in vitro study.

Low-level laser therapy has become one of the fastest growing fields of medicine in recent years. Many in vivo and in vitro studies have shown that laser irradiation activates a range of cellular processes in a variety of cell types and can promote tissue repair. However, few in vitro experiments have evaluated the effects of laser irradiation on cells in real time. The purpose of this study was to examine the effects of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on the migration of cultured human osteoblasts. A dedicated 96-well plate was used, and confluent cultures of the human osteoblast-like cell line, Saos-2, were injured with a wound maker. The wounded cells were then exposed to the Nd:YAG laser (wavelength of 1064 nm) for 60 s at 0.3 W (10 pps, 30 mJ). The total energy density was about 10.34 J/cm2. Images of the wounds were automatically acquired inside the CO2 incubator by the IncuCyte ZOOM™ software. In addition, after laser irradiation, the production of adenosine triphosphate (ATP) was measured using the CellTiter-Glo™ Luminescent Cell Viability Assay. Migration of cells from the border of the original scratch zone was accelerated by laser irradiation. In addition, compared with the control group, significant enhancement of ATP production was observed in the irradiated group. The present study showed that Nd:YAG laser irradiation (wavelength of 1064 nm, 0.3 W, 10 pps, 30 mJ, 10.34 J/cm2, irradiation time 60 s) may contribute to the regeneration of bone tissues owing to enhanced osteoblast cell migration.

A case of tooth autotransplantation after long-term cryopreservation using a programmed freezer with a magnetic field.

This case report describes the treatment of a skeletal Class III malocclusion with autotransplantation of a cryopreserved tooth. To gain an esthetic facial profile and good occlusion, extraction of bimaxillary premolars and surgical therapy were chosen. The patient had chronic apical periodontitis on the lower left first molar. Although she did not feel any pain in that region, the tooth was considered to have a poor prognosis. Therefore, we cryopreserved the extracted premolars to prepare for autotransplantation in the lower first molar area because the tooth would probably need to be removed in the future. The teeth were frozen by a programmed freezer with a magnetic field (CAS freezer) that was developed for tissue cryopreservation and were cryopreserved in -150°C deep freezer. After 1.5 years of presurgical orthodontic treatment, bilateral sagittal split ramus osteotomy was performed for mandible setback. Improvement of the facial profile and the occlusion were achieved in the retention phase. Six years after the initial visit, the patient had pain on the lower left first molar, and discharge of pus was observed, so we extracted the lower left first molar and autotransplanted the cryopreserved premolar. Three years later, healthy periodontium was observed at the autotransplanted tooth. This case report suggests that long-term cryopreservation of teeth by a CAS freezer is useful for later autotransplantation, and this can be a viable technique to replace missing teeth.

Effects of low-intensity pulsed ultrasound on the expression and activity of hyaluronan synthase and hyaluronidase in IL-1ß-stimulated synovial cells.

The purpose of this study is to examine effects of low-intensity pulsed ultrasound (LIPUS) on metabolism of hyaluronan (HA) in synovial membrane cells stimulated by IL-1ß. Rabbit knee synovial membrane cell line, HIG-82, was cultured in medium with the presence or absence of 1 ng/mL IL-1ß, and after 4 h the cell was exposed to LIPUS for 15 min. The mRNA levels of HA synthase (HAS) 2,3, hyaluronidase (HYAL) 2, and cyclooxygenase (COX)-2 were examined by real-time PCR analysis. Concentrations of HA and PGE2 were quantified by use of enzyme linked immunosorbent assay (ELISA). The COX-2 level was analyzed by western blotting. Gene levels of HAS2 and HAS3 in IL-1ß-stimulated cells were up-regulated significantly (p < 0.01) by LIPUS. HYAL2 mRNA was up-regulated by the treatment with IL-1ß, whereas down-regulated significantly (p < 0.01) by the following LIPUS exposure. Furthermore, IL-1ß stimulation enhanced COX-2 and PGE2 expression as compared to the untreated control, and IL-1ß-induced COX-2 and PGE2 expression was inhibited by LIPUS. These results suggest that LIPUS enhanced HA synthesis and inhibited HYAL2 expression, leading to the accumulation of high-molecular weight HA. Therefore, LIPUS stimulation may be a better candidate as medical remedy to treat inflammatory joint diseases accompanied with HA degradation in synovial fluid.