Objective. We recently implicated in vivo oxidative stress in the development of osteonecrosis in a steroid-induced osteonecrosis model in the domestic rabbit. In the present experiment we devised a new non-traumatic model using the rat to investigate the relationship between oxidative stress and the development of osteonecrosis. Methods. Seven 24-week-old male Wistar rats were subcutaneously injected with the pro-oxidant buthionine sulphoximine (BSO) 500 mg/kg for 14 consecutive days (group B) and eight rats received injections of vehicle (physiological saline; group N). The rats in both groups were killed after 14 days, and their bilateral femurs were examined histopathologically. Blood levels of reduced glutathione (GSH), total cholesterol (T-cho) and triglycerides (TG) were also determined. Results. GSH was significantly decreased in group B compared with group N (P<0.01). No significant differences were found in T-cho or TG. Osteonecrosis was not detected in any animal in group N in contrast to five of seven animals in group B (P<0.05). Conclusion. BSO is an inducer of oxidative stress, in particular interfering with the synthesis of GSH in vivo. In the present study, GSH levels were markedly reduced by BSO, whereas neither T-cho nor TG was significantly changed. The high rate of osteonecrosis noted in group B suggests that oxidative stress alone may be sufficient to promote the development of osteonecrosis at certain sites.

Objective To investigate the relationship between transient oxidative stress and the development of osteonecrosis in a rat model. Methods A total of 160 male Wistar rats (24 weeks old) were injected only once with the pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO) (500 mg/kg given intraperitoneally) and were killed 12 hours (group A), 1 day (group B), 3 days (group C), 4 days (group D), 5 days (group E), 7 days (group F), or 14 days (group G) after administration (n = 20 per group). Twenty untreated rats were used as a control group (group N). Femurs were examined histopathologically for the presence of osteonecrosis, and reduced glutathione (GSH) in liver tissue was measured as an index of oxidative stress. Results GSH decreased rapidly after BSO administration. Significant decreases were noted in groups A and B as compared to group N (P < 0.0001 and P = 0.0007, respectively), confirming the development of transient extreme oxidative stress soon after BSO administration. The histopathologic study revealed osteonecrosis in 10% of the rats in group E, 35% of the rats in group F, and 40% of the rats in group G. Conclusion Transient extreme oxidative stress was confirmed to induce osteonecrosis in this model. Since preparation of this model is extremely simple and because rats are well suited to genetic studies, this model may be of use in elucidating the pathophysiology of femoral head osteonecrosis in future studies.

Persistent inflammation is well known to promote the progression of arthropathy. mesenchymal stem cells (MSCs) have been shown to possess anti-inflammatory properties and tissue differentiation potency. Although the experience so far with the intraarticular administration of mesenchymal stem cell (MSC) to induce cartilage regeneration has been disappointing, MSC implantation is now being attempted using various surgical techniques. Meanwhile, prevention of osteoarthritis (OA) progression and pain control remain important components of the treatment of early-stage OA. We prepared a shoulder arthritis model by injecting monoiodoacetate (MIA) into a rat shoulder, and then investigated the intraarticular administration of MSC from the aspects of the cartilage protective effect associated with their anti-inflammatory property and inhibitory effect on central sensitization of pain. When MIA was administered in this rat shoulder arthritis model, anti-Calcitonin Gene Related Peptide (CGRP) was expressed in the joint and C5 spinal dorsal horn. Moreover, expression of A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a marker of joint cartilage injury, was similarly elevated following MIA administration. When MSC were injected intraarticularly after MIA, the expression of CGRP in the spinal dorsal horn was significantly deceased, indicating suppression of the central sensitization of pain. The expression of ADAMTS 5 in joint cartilage was also significantly inhibited by MSC administration. In contrast, a significant increase in the expression of TNF-α stimulated gene/protein 6 (TSG-6), an anti-inflammatory and cartilage protective factor shown to be produced and secreted by MSC intraarticularly, was found to extend to the cartilage tissue following MSC administration. In this way, the intraarticular injection of MSC inhibited the central sensitization of pain and increased the expression of the anti-inflammatory and cartilage protective factor TSG-6. As the least invasive conservative strategies possible are desirable in the actual clinical setting, the intraarticular administration of MSC, which appears to be effective for the treatment of pain and cartilage protection in early-stage arthritis, may achieve these aims.

Peri-implant infection is a serious complication in surgical procedures involving implants. We conducted an in vitro study to determine whether the use of a fluorinated diamond-like carbon (F-DLC) coating on a titanium alloy surface can prevent peri-implant infection. After applying the F-DLC, we evaluated its antibacterial and cytotoxic properties. The coating groups, containing controlled fluorine concentrations of 5.44%, 17.43%, 24.09%, and 30%, were examined for the presence of Staphylococcus aureus and Escherichia coli according to ISO 22196 for the measurement of antibacterial activity on plastics and other nonporous surfaces. Biological toxicity was evaluated using Chinese hamster V79 cells according to ISO 10993-5 for the biological evaluation of medical devices. In the control group, populations of S. aureus and E. coli substantially increased from 2.4 × 10⁴ to (1.45 ± 1.11) × 10⁶ colony-forming units (CFUs) and from 2.54 × 10⁴ to (4.04 ± 0.44) × 10⁶ CFUs, respectively. However, no bacteria colonies were detected in any F-DLC group with a fluorine concentration of ≥ 17.43%. In the biological toxicity study, an F-DLC coating with a fluorine concentration of 30% showed a colony formation rate of 105.8 ± 24.1%, which did not differ significantly from the colony formation rate of 107.5 ± 31.1% in the nontoxic control group. An F-DLC coating on titanium alloy discs showed excellent in vitro antibacterial activity with no biological toxicity.

Mitochondrial injury has recently been implicated in the pathogenesis of glucocorticoid-induced osteonecrosis. Using cultured osteocytes and a rabbit model, we investigated the possibility that taurine (TAU), which is known to play a role in the preservation of mitochondrial function, might also prevent the development of osteonecrosis. To reduplicate the intraosseous environment seen in glucocorticoid-induced osteonecrosis, dexamethasone (Dex) was added to MLO-Y4 cultured in 1% hypoxia (H-D stress environment). An in vitro study was conducted in which changes in mitochondrial transcription factor A (TFAM), a marker of mitochondrial function, and ATP5A produced by mitochondria, induced by the presence/absence of taurine addition were measured. To confirm the effect of taurine in vivo, 15 Japanese White rabbits were administered methylprednisolone (MP) 20 mg/kg as a single injection into the gluteus muscle (MP+/TAU− group), while for 5 consecutive days from the day of MP administration, taurine 100 mg/kg was administered to 15 animals (MP+/TAU+ group). As a control 15 untreated rabbits were also studied. The rabbits in each of the groups were sacrificed on the 14th day after glucocorticoid administration, and the bilateral femora were harvested. Histopathologically, the incidence of osteonecrosis was quantified immunohistochemically by quantifying TFAM and ATP5A expression. In the rabbits exposed to an H-D stress environment and in MP+/TAU− group, TFAM and ATP5A expression markedly decreased. With addition of taurine in the in vitro and in vivo studies, the expression of TFAM and ATP5A was somewhat decreased as compared with Dex−/hypoxia− or MP−/TAU− group, while improvement was noted as compared with Dex+/hypoxia+ or MP+/TAU− group. In rabbits, the incidence of osteonecrosis was 80% in MP+/TAU− group, in contrast to 20% in the taurine administered group (MP+/TAU+), representing a significant decrease. Since taurine was documented to exert a protective effect on mitochondrial function by inhibiting the mitochondrial dysfunction associated with glucocorticoid administration, we speculated that it might also indirectly help to prevent the development of osteonecrosis in this context. Since taurine is already being used clinically, we considered that its clinical application would also likely be smooth.

The purpose of the role of antioxidant enzymes and mitochondria in the developmental mechanism of steroid-associated osteonecrosis in the femur. In the present study Japanese white rabbits (mean weight 3.5kg) were injected into the gluteus with methylprednisolone (MP) 20mg/kg, and killed after 3 days (MP3 group), 5 days (MP5 group), and 14 days (MP14 group) (n=3 each). As a Control group (C group) Japanese white rabbits not administered MP were used. In experiment 1, the expression of the antioxidant enzymes Superoxide dismutade (SOD) and catalase was compared in liver, kidney, heart, humerus, and femur in C group, and the presence/absence of mitochondria transcription factor A (TFAM) expression was determined by Western blotting (WB) and used to evaluate the number of mitochondria and their function. In experiment 2, the presence/absence of necrosis was determined by immunohistochemistry, while changes in the expression of SOD, catalase, and TFAM in the femur after steroid administration were determined by Western blotting (WB). In experiment 1, intense expression of all of SOD, catalase, and TFAM was found in the liver, kidney, and heart as compared to the humerus and femur. In experiment 2, the expression of all of SOD, catalase, and TFAM in MP3 and MP5 groups was decreased on WB as compared with C group, while in MP14 group a tendency to improvement was seen. Accordingly, steroid-associated mitochondrial injury and redox failure are concluded to be important elements implicated in the pathogenesis of osteonecrosis.