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.

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.

A relative high dose of vitamin E does not attenuate unweighting-induced oxidative stress and ubiquitination in rat skeletal muscle.

We previously reported that intragastric administration of cysteine could be beneficial to prevent unweighting-induced ubiquitination and degradation of muscle protein in association with redox regulation [Ikemoto et al., Biol. Chem., 383 (2002), 715-721]. In this study, we investigated whether vitamin E, another potent antioxidative nutrient, also had beneficial effects on the muscle protein catabolism. However, daily intragastric supplementation of 1.5 or 15 mg/rat of alpha-tocopherol did not prevent weight loss of hindlimb skeletal muscle in tail-suspended rats. To elucidate the reason for the non-effectiveness of vitamin E, we further examined concentrations of oxidative stress markers, ubiquitination of muscle proteins and fragmentation of myosin heavy chain in gastrocnemius muscle of rats daily treated with 15 mg of alpha-tocopherol. Unexpectedly, vitamin E increased concentrations of glutathione disulfide and thiobarbituric acid-reactive substance and decreased glutathione level in the muscle, compared with those of vehicle treatment, indicating that vitamin E enhanced unweighting-induced oxidative stress in skeletal muscle. The vitamin E supplementation did not suppress the ubiquitination of muscle proteins and fragmentation of myosin heavy chain caused by tail-suspension. Our results suggest that supplementation of a relative high dose of vitamin E could not inhibit ubiquitin-dependent degradation of muscle protein in tail-suspended rats possibly due to its prooxidant action.

Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle.

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tail-suspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tail-suspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.