iNOS expression and osteocyte apoptosis in idiopathic, non-traumatic osteonecrosis.

BACKGROUND AND PURPOSE: Non-traumatic osteonecrosis is a progressive disease with multiple etiologies. It affects younger individuals more and more, often leading to total hip arthroplasty. We investigated whether there is a correlation between inducible nitric oxide synthase (iNOS) expression and osteocyte apoptosis in non-traumatic osteonecrosis. PATIENTS AND METHODS: We collected and studied 20 human idiopathic, non-traumatic osteonecrosis femoral heads. Subchondral bone samples in the non-sclerotic region (n = 30), collected from osteoarthritis patients, were used as controls. Spontaneously hypertensive rats were used as a model for osteonecrosis in the study. We used scanning electron microscopy, TUNEL assay, and immunohistochemical staining to study osteocyte changes and apoptosis. RESULTS: The morphology of osteocytes in the areas close to the necrotic region changed and the number of apoptotic osteocytes increased in comparison with the same region in control groups. The expression of iNOS and cytochrome C in osteocytes increased while Bax expression was not detectable in osteonecrosis samples. Using spontaneously hypertensive rats, we found a positive correlation between iNOS expression and osteocyte apoptosis in the osteonecrotic region. iNOS inhibitor (aminoguanidine) added to the drinking water for 5 weeks reduced the production of iNOS and osteonecrosis compared to a control group without aminoguanidine. INTERPRETATION: Our findings show that increased iNOS expression can lead to osteocyte apopotosis in idiopathic, non-traumatic osteonecrosis and that an iNOS inhibitor may prevent the progression of the disease.

Soluble epoxide hydrolase inhibitor can protect the femoral head against tobacco smoke exposure-induced osteonecrosis in spontaneously hypertensive rats.

Exposure to tobacco smoke (TS) has been considered a risk factor for osteonecrosis of the femoral head (ONFH). Soluble epoxide hydrolase inhibitors (sEHIs) have been found to reduce inflammation and oxidative stress in a variety of pathologies. This study was designed to assess the effect of sEHI on the development of ONFH phenotypes induced by TS exposure in spontaneously hypertensive (SH) rats. SH and normotensive Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or TS (80 mg/m3 particulate concentration) 6 h/day, 3 days/week for 8 weeks. During this period, sEHI was delivered through drinking water at a concentration of 6 mg/L. Histology, immunohistochemistry, and micro-CT morphometry were performed for phenotypic evaluation. As results, TS exposure induced significant increases in adipocyte area, bone specific surface (BS/BV), and trabecular separation (Tb.SP), as well as significant decreases in bone mineral density (BMD), percent trabecular area (Tb.Ar), HIF-1a expression, bone volume fraction (BV/TV), trabecular numbers (Tb.N), and trabecular thickness (Tb.Th) in both SH and WKY rats. However, the protective effects of sEHI were mainly observed in TS-exposed SH rats, specifically in the density of osteocytes, BMD, Tb.Ar, HIF-1a expression, BV/TV, BS/BV, Tb.N, and Tb.SP. Our study confirms that TS exposure can induce ONFH especially in SH rats, and suggests that sEHI therapy may protect against TS exposure-induced osteonecrotic changes in the femoral head.

Curcumin prevents osteocyte apoptosis by inhibiting M1-type macrophage polarization in mice model of glucocorticoid-associated osteonecrosis of the femoral head.

Inflammation is a contributing factor in osteocyte apoptosis, which is strongly associated with the development of glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). Curcumin is a naturally derived drug that regulates immunity and inhibits inflammation. This study aimed to examine the capacity of curcumin to prevent osteocyte apoptosis and GA-ONFH, while elucidating possible mechanisms of action. C57/BL6 female mice were divided into control, GA-ONFH, and curcumin-treated GA-ONFH groups. We determined the effect of curcumin on the polarization of RAW264.7 and the apoptosis of MLO-Y4 cells. We found that curcumin reduced the infiltration of M1-type macrophages in the femoral heads and alleviated systemic inflammation in GA-ONFH models. Additionally, curcumin decreased the apoptosis of osteocytes in the femoral heads and the ratio of GA-ONFH in mice. Further, in vitro curcumin intervention inhibited M1-type polarization via the Janus kinase1/2-signal transducer and activator of transcription protein1 (JAK1/2-STAT1) pathway. Taken together, this study demonstrates that curcumin is effective in preventing osteocyte apoptosis and the development of GA-ONFH in a mouse model. Curcumin prevents inflammatory-mediated apoptosis of osteocytes in part through inhibition of M1 polarization through the JAK1/2-STAT1 pathway. These findings provide novel insights as well as a potential preventive agent for GA-ONFH. This article is protected by copyright. All rights reserved.

Paraoxonase 1 gene polymorphisms in patients with osteonecrosis of the femoral head with and without cerebral white matter lesions.

Cerebral white matter lesions (WML) are present in more than 50% of patients with osteonecrosis of the femoral head (ONFH). Paraoxonase 1 (PON1) gene product is a detoxifying and pesticide metabolizing enzyme. Genetic variants of the PON1 gene have been found to influence the occurrence and progression of WML. We examined whether two PON1 polymorphisms (M55L and R192Q) are associated with ONFH and influence the occurrence of WML. We studied 104 patients with ONFH and 113 healthy age- and sex-matched subjects. We used logistic regression models to examine associations and survival analyses (Cox proportional hazards models) to examine possible influence of alleles on age at onset of ONFH. We found no association of PON1 M55L alleles and genotypes with ONFH. The distribution of PON1 Q192R alleles (p = 0.001) and genotypes (QQ vs. QR/RR) (p = 0.004) were statistically different between controls and patients. Patients with QQ genotype had six times higher risk for WML at brain MRI (adjusted OR 5.95; 95% CI 1.30-27.03; p = 0.02). In Cox models, there was a significant association of allele Q with risk for ONFH indicating a possible dose effect (HR = 1.43; 95%CI = 1.04-1.97; p for trend = 0.03). We conclude that individuals with PON1 192QQ genotype may have increased risk for ONFH and WMLeOn.

TLR4 stimulation and corticosteroid interactively induce osteonecrosis of the femoral head in rat.

We previously reported that a toll-like receptor 4 signaling contributes to the development of osteonecrosis of the femoral head. Also, oxidative stress is suggested to be one of the possible pathogenesis of osteonecrosis of the femoral head. A recent study showed that toll-like receptor 4 signaling leads to oxidative stress. The aim of the present study was to evaluate whether toll-like receptor 4 stimulation and subsequent corticosteroid treatment lead to the development of osteonecrosis of the femoral head in rat, and oxidative stress is associated with it. Male Wistar rats were randomly divided into four treatment groups: Saline + Saline, Saline + Methylprednisolone, Lipopolysaccharide + Saline, Lipopolysaccharide + Methylprednisolone. Osteonecrosis of the femoral head at 14 days after the treatment was observed in 1 of 10 Lipopolysaccharide + Saline, and 5 of 10 Lipopolysaccharide + Methylprednisolone treated rats. However, it was not observed at all in the Saline + Saline and Saline + Methylprednisolone treated groups. Glutathione peroxidase activity in the liver at 1 day after the treatment was significantly increased when treated with lipopolysaccharide. However, methylprednisolone treatment reduced the activity. On the other hand, glutathione peroxidase activity in the femur did not change in any intergroup. In conclusion, the present study showed that toll-like receptor 4 stimulation by lipopolysaccharide administration strengthen incidence of corticosteroid-induced osteonecrosis of the femoral head, however, concomitant oxidative stress via toll-like receptor 4 signaling may not contribute to the development of osteonecrosis of the femoral head in rats.

Synthesis of active high mannose-type lipoprotein lipase in human adipose tissues.

Lipoprotein lipase (LPL) activity in the retroperitoneal adipose tissue of a patient with Cushing's syndrome and in the subcutaneous adipose tissue of a patient with aseptic necrosis of the femoral head was higher than that in the corresponding tissues of the control subjects. The amount of [35S]methionine incorporated into LPL was also higher in these patients than in control subjects. However, the ratio of activity and amount of radioactivity in the LPL of patients was identical to that of control subjects, indicating that LPL synthesized in the adipose tissues of patients had a normal specific activity. LPL with Mr = 57000 was composed of two types of subunits: one type was partially endo H-sensitive, yielding a product with Mr = 55000, and the other was totally endo H-sensitive, yielding a product with Mr = 52000. Both retroperitoneal and subcutaneous adipose tissues of control subjects contained nearly equal amounts of partially and totally endo H-sensitive subunits. In the retroperitoneal adipose tissue of a patient with Cushing's syndrome, 8% of subunits were partially endo H-sensitive and 92% were totally endo H-sensitive. In the subcutaneous adipose tissue of a patient with aseptic necrosis of the femoral head, 21% of subunits were partially endo H-sensitive and 79% were totally endo H-sensitive. The 24-h treatment of subcutaneous adipose tissue of a control subject with 1 mM 1-deoxymannojirimycin (dMM) caused the synthesis of active, but totally endo H-sensitive, LPL. Thus, in human adipose tissue, the processing of one oligosaccharide chain of an LPL subunit to a complex type chain in the trans Golgi was not necessary for the expression of activity.

Apoptosis--a significant cause of bone cell death in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head usually affects young individuals and is responsible for up to 12% of total hip arthroplasties. The underlying pathophysiology of the death of the bone cells remains uncertain. We have investigated nitric oxide mediated apoptosis as a potential mechanism and found that steroid- and alcohol-induced osteonecrosis is accompanied by widespread apoptosis of osteoblasts and osteocytes. Certain drugs or their metabolites may have a direct cytotoxic effect on cancellous bone of the femoral head leading to apoptosis rather than purely necrosis.

A case-control study of association between life-style, alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 genotype and idiopathic osteonecrosis of the femoral head.

A case-control study of idiopathic osteonecrosis of femoral head (ION) was carried out to investigate the association between workload intensity, smoking, drinking, ADH2 and ALDH2 genotype and other factors and development of ION by comparing 43 cases (34 males and 9 females) without history of systemic corticosteroid use with 86 matched controls (68 males and 18 females). Univariate analysis by conditional logistic regression for males showed statistically significant odds ratios (OR) for heavier workload (OR = 4.661), cumulative alcohol consumption (OR trend per drink-years = 1.015), ALDH2(1/1) (OR = 3.310), consumption of green tea less than 3 or 4 times/week (OR = 2.705), body weight over 60 kg at examination (OR = 0.413), or body mass index of over 25 at examination (OR = 0.208). Multivariate analysis based on a hierarchically well-formulated model strategy for males revealed that heavier workload (OR = 5.785, 95% confidence interval (CI): 1.211, 27.635) and cumulative alcohol consumption (OR = 1.016, 95% CI: 1.005, 1.026) remained as the significantly associated risk factors for ION after adjusting the remaining confounders. The reason for the insignificant multivariate OR of ALDH2(1/1) may be due to the strong confounding of alcohol consumption on the association between ALDH2 genotype and ION. For females, the small sample size made it impossible to produce any meaningful univariate analysis.

Expression of 11ß-HSD in steroid-induced avascular necrosis of the femoral head.

The aim of the present study was to investigate the association between glucocorticoid receptors and steroid­induced avascular necrosis of the femoral head (SANFH). Healthy New Zealand rabbits were randomly divided into 3 groups (n=16/group); the normal (no treatment), control (horse serum injections) and treatment (horse serum and methylprednisolone injections) groups. Methylprednisolone and horse serum injections were used to establish a SANFH model in rabbits; magnetic resonance imaging (MRI) and histopathological analysis were used to evaluate the SANFH rabbit model. Total cholesterol and triglyceride contents in the blood of SANFH rabbits were determined. The protein expression levels of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and type 2 (11ß-HSD2) were determined using western blotting. The total cholesterol and triglyceride levels in the blood of methylprednisolone­treated rabbits were significantly increased compared with the control and normal groups, which provides evidence in support of the metabolic disorder theory. Based on the results obtained from western blotting, the expression levels of 11ß-HSD1 protein were increased, whereas the expression levels of 11ß-HSD2 protein were decreased following injection. The results of the present study indicate that 11ß-HSDs are important in the development of SANFH. Furthermore, 11ß-HSDs may be important targets for preventing the development of ANFH in patients treated with steroids, which has a significant reference value for the use of steroids in clinical practice.

Heparanase role in the treatment of avascular necrosis of femur head.

BACKGROUND: Idiopathic avascular necrosis (AVN) of bone causes significant morbidity in adults although the pathophysiology is unknown. The present treatment options include systemic biphosphonate therapy and local bone drilling decompression, ameliorating the healing process and their by render the weight bearing femur head less vulnerable to collapse. In the present study we demonstrate the involvement of heparanase in AVN and in the acceptable treatments. METHODS: 56 female rats were studied. In 8 control rats AVN was induced by ligamentum teres ligation of the right femur while the left femur remained intact. In the rest of the rats, in addition to right femur AVN, treatment was added by subcutaneous biphosphonate therapy, femoral head drilling or combination of the treatments. All rats were scarified after 6weeks. Immunostaining of the femur heads were performed to heparanase, tissue factor pathway inhibitor (TFPI), tissue factor (TF) and hematoxylin-eosin. RESULTS: Staining of heparanase, TFPI and TF were most prominent in the bone-marrow tissue of the femur heads. Staining by hematoxylin-eosin revealed damaged femur heads with prominent heparanase and TFPI staining in the femur with AVN compared to the contra lateral side of the same rat. No difference was found in the TF staining between the two sides. In the drilling and / or biphosphonate therapy groups, in contrast to the control group, femur heads were preserved with no significant difference in heparanase and TFPI staining between the two sides. CONCLUSIONS: Heparanase and TFPI are locally elevated in the process of AVN and are normalized by the acceptable treatments. Inhibition of heparanase by heparins can potentially improve the nowadays therapy modalities.

Alcohol-associated femoral head necrosis.

Fifty-seven patients with femoral head necrosis (FHN), treated since 1976 at the Orthopaedic Hospital of the Invalid Foundation, Helsinki, were studied. The incidence of alcohol-associated FHN was 29% (N = 15, 13 male) and of idiopathic FHN 11.5% (N = 10, 6 male). Nine patients with alcohol-associated FHN were heavy drinkers, four were alcoholics and two were moderate drinkers at the time of the onset of the symptoms. At follow-up 80% of the alcohol-associated cases showed increased gamma-glutamyltransferase (GGT) activity in the serum. Our findings among alcohol-associated cases support previous reports according to which disturbed liver function is an important factor in the aetiology of FHN. Because of GGT's rather high sensitivity in the detection of heavy drinking in FHN, it is recommended to be used as a diagnostic aid in suspected FHN.

Type XI collagen-degrading activity in human osteoarthritic cartilage.

Homogenates of 6 samples of human osteoarthritic cartilage were shown to degrade exogenous type XI collagen. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the cleavage products generated by each homogenate were similar, and they were identical to those obtained by cleavage of the substrate with purified gelatinase. Enzyme activity, which was inhibited by EDTA, was greater in extracts of fibrillated osteoarthritic cartilage than in extracts of grossly normal cartilage from the same joint or in extracts of cartilage from joints with osteonecrosis. Activation with APMA enhanced digestion, but breakdown was apparent in extracts of fibrillated osteoarthritic cartilage even without APMA. Enzymatic degradation of type XI collagen could play a significant role in the turnover of articular cartilage in health and disease states.

Synovial fluid cartilage metabolism marker concentrations in osteonecrosis of the femoral head compared with osteoarthrosis of the hip.

OBJECTIVE: Biochemical aspects of the etiology of articular cartilage degeneration in osteonecrosis of the femoral head have not been investigated extensively. We analyzed biochemical conditions in the hip joint cavity, regarding articular cartilage turnover in osteonecrosis of the femoral head (ONFH). METHODS: Cartilage metabolism markers in synovial fluid were measured. Synovial fluid (SF) samples were collected from 19 ONFH cases and 17 control hips with osteoarthrosis (OA). Concentrations of carboxy-terminal type II procollagen peptide (pCOL-II-C), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured. In addition, concentrations of unsaturated disaccharides of hyaluronic acid (delta di-HA), chondroitin 4-sulfate (delta di-4S), and chondroitin 6-sulfate (delta di-6S) in SF were measured by high performance liquid chromatography. RESULTS: The mean SF concentration of pCOL-II-C was higher on ONFH than in OA. The mean SF MMP-3 level was higher in ONFH than in OA, while the mean SF TIMP-1 level was the same in the 2 groups. The SF concentration of delta di-HA and the delta di-6S/delta di-4S ratio were higher in ONFH than in OA. CONCLUSION: Higher concentrations of cartilage metabolism markers in ONFH compared with OA SF suggest elevated cartilage turnover in the former disease.