Pathogenesis Of Steroid-induced Osteonecrosis Research Articles

Double Trouble-COVID-19 and the Widespread Use of Corticosteroids: Are We Staring at an Osteonecrosis Epidemic?

BackgroundA combination of immune-mediated vascular damage and routine use of systemic corticosteroid (CS) therapy in COVID-19 may significantly increase the risk and burden of osteonecrosis (ON) after COVID-19. This narrative review explores the pathogenesis, risk factors, and possible preventive and early treatment measures for ON in COVID-19.MethodsFor this narrative review, an extensive literature search was performed using the PubMed, Medline, and Science Direct databases from January 2000 to August 2021 for relevant articles on etiopathogenesis, epidemiology, clinical manifestations, and treatment of severe acute respiratory syndrome coronavirus (SARS-CoV) infection and steroid-induced ON (SION).ResultsPathogenesis of COVID-19, utility of corticosteroids in the treatment of COVID-19, pathogenesis of SION vis-a-vis SARS-CoV infection, associated risk factors, and early diagnosis and treatment of ON following CS therapy of SARS-CoV infection were discussed.ConclusionPreliminary data of COVID-19 and similar trends from the SARS 2003 epidemic indicate that the “angiocentric” pathogenesis of SARS-CoV-2 and treatment with high-dose CS may increase the risk of ON in COVID-19 patients. Risk stratification based on CS intake during COVID-19 treatment can help identify subjects at moderate to high-risk for ON where early preventive and follow-up plans can be initiated.

Hypermethylation of Frizzled1 is associated with Wnt/\u03b2-catenin signaling inactivation in mesenchymal stem cells of patients with steroid-associated osteonecrosis

The Wnt/β-catenin signaling pathway is associated with the pathogenesis of steroid-induced osteonecrosis. Our investigation studied whether aberrant CpG island hypermethylation of the FZD1 gene was present in patients with osteonecrosis of the femoral head (ONFH), which results in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 21) and patients with new femoral neck fractures (n = 22), and then mesenchymal stem cells (MSCs) were isolated. We investigated cell viability, the transcription and translation levels of Wnt/β-catenin signaling-related genes, the extent of methylation at CpG islands of the FZD1 promoter, and the osteogenic and adipogenic differentiation abilities of MSCs from the control group and from the ONFH group treated with or without 5′-Aza-dC. According to the results, MSCs from the ONFH group showed a reduced proliferation ability, low transcription and translation levels of FZD1, inhibition of the Wnt/β-catenin signaling pathway, weakened osteogenesis and enhanced adipogenesis ability. Aberrant CpG island hypermethylation of FZD1 was observed in the ONFH group. Treatment with 5’-Aza-dC resulted in de novo FZD1 expression, reactivation of the Wnt/β-catenin signaling pathway and promotion of osteogenesis. Taken together, our study not only provides novel insights into the regulation of the Wnt/β-catenin signaling pathway in this disease but also reveals potential for the use of demethylating agents for the treatment of GC-associated ONFH.

Beneficial effect of grape seed proanthocyanidin extract in rabbits with steroid-induced osteonecrosis via protecting against oxidative stress and apoptosis.

Oxidative damage and apoptosis play dominant roles in the pathogenesis of steroid-induced osteonecrosis (ON). Grape seed proanthocyanidin extract (GSPE) demonstrates antioxidant and antiapoptotic properties. Our aim was to demonstrate the effects of GSPE in preventing steroid-induced ON in rabbits. Osteonecrosis was induced by high-dose methylprednisolone (40 mg/kg). Rabbits in the preventive medicine group were treated with 100 mg/kg/day GSPE for 14 consecutive days, and the presence or absence of ON was examined histopathologically. Oxidative damage in bone tissue was assessed by immunohistochemical staining of 8-oxo-2'-deoxyguanosine (8-OHdG), malondialdehyde (MDA) levels, and activities of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px). Apoptosis was detected via quantitative terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labelling (TUNEL) staining and activated caspase 3 immunoblotting and activity. GSPE significantly attenuated the changes of immunohistochemical staining of 8-OHdG, MDA levels, and antioxidant enzymes activities, which were caused by methylprednisolone administration. Quantitative TUNEL and caspase 3 assay showed lower apoptosis with GSPE application. Simultaneously, GSPE reduced the incidence of steroid-induced ON in an established rabbit model to 17.6 %, compared with 87.5 % in the steroid-only group. These results reveal that GSPE treatment could inhibit oxidative damage and apoptosis to exert beneficial effects on reducing the incidence of steroid-induced ON in rabbit models.

Association of toll-like receptor 4 signaling pathway with steroid-induced femoral head osteonecrosis in rats.

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.

Dexamethasone treatment alters function of adipocytes from a mesenchymal stromal cell line

Osteoporosis and osteonecrosis are associated with corticosteroid treatment, but the pathophysiologies are unclear. We hypothesized that mature adipocytes present within the bone marrow compartment play a key role in the development of both diseases. Adipocytes have recognized regulatory effects on bone viability and healing by releasing signaling molecules called adipokines. Our purpose was to evaluate whether dexamethasone alters adipokine expression in differentiated bone-marrow-derived adipocytes. Adipocytes differentiated from mouse D1 mesenchymal stromal cells were treated with dexamethasone (10−5, 10−6, 10−7, or 10−8M) or with diluent alone (controls) for up to 6days. Using real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses, six key adipokines and the transcription factor HIF-1α were evaluated. Dexamethasone treatment increased PAI-1 protein expression with increased mRNA expression at 4days, while decreasing HIF-1α mRNA expression and protein concentrations. VEGF A mRNA expression was increased at 4days for most dexamethasone concentrations, with minimal changes in protein levels. Dexamethasone increased adiponectin mRNA expression and protein levels at 4 and 6days and decreased leptin, interleukin-6, and tumor necrosis factor α mRNA expression at all time periods. Dexamethasone treatment of bone-marrow-derived adipocytes resulted in detectable changes in mRNA expression and protein levels of adipokines and HIF-1α. The detected adipokine alterations could be important early events in the pathogenesis of steroid-induced osteonecrosis and osteoporosis.

Preventive effects of the anti-vasospasm agent via the regulation of the Rho-kinase pathway on the development of steroid-induced osteonecrosis in rabbits

A number of studies have suggested that ischemia is the principal pathomechanism of osteonecrosis, however, the detailed mechanism responsible for ischemia remains unclear. We examined the effects of fasudil, an anti-vasospasm agent, on the development of steroid-induced osteonecrosis in rabbits. One group of rabbits received 15mg/kg of fasudil intravenously, which were then injected once intramuscularly with 20mg/kg of methylprednisolone (n=33), and one received methylprednisolone alone as a control (n=28). Eight rabbits from each group were sacrificed 24h after methylprednisolone injection to analyze them by the expression of endothelinA-receptor and eNOS. Two weeks after the steroid injection, the femora and humeri were examined histopathologically for the incidence of osteonecrosis. In addition, plasma from each of four osteonecrosis-positive or -negative rabbits was used for the proteomic analysis in the fasudil group. The incidence of osteonecrosis was significantly lower in the fasudil group (32%) than that in the control group (75%) (P<0.01). Immunohistochemically, endothelinA-receptor expressions levels were decreased in the smooth muscle of the bone marrow in the fasudil group in comparison to that in the control group. The eNOS expressions levels in both serum and bone marrow in the MF group were significantly higher than those in the M group (P<0.05). Based on the proteomic analysis, several proteins related to vasospasm, such as fibrinogen, thrombin, and apolipoprotein E, were identified in rabbits with osteonecrosis soon after steroid administration. This study indicates that vasospasm is one of the important factors involved in the pathogenesis of steroid-induced osteonecrosis and that the anti-vasospasm agents seem to decrease the incidence of steroid-induced osteonecrosis.

Effects of an anti-platelet drug on the prevention of steroid-induced osteonecrosis in rabbits

To investigate the effects of an anti-platelet drug (clopidogrel) on the prevention of steroid-induced osteonecrosis (ON) in rabbits. Adult male Japanese white rabbits were divided into two groups and treated as follows: one group received daily clopidogrel mixed in normal saline (AP; n = 35), the other received only normal saline (NS; n = 30). One week after the administration, all rabbits were injected once intramuscularly with 20 mg/kg of methylprednisolone acetate into the right gluteus medius muscle. Three weeks after, both the femora and humeri were examined histopathologically for the presence of ON. The platelet aggregation assay and hematological examinations were performed before and after the steroid injection. The incidence of ON in the AP group (48.5%) was significantly lower than that observed in the NS group (73.3%). The platelet aggregations in the AP group were significantly inhibited by the administration of clopidogrel. The levels of total cholesterol and triglycerides showed no significant differences between the AP and NS group. The present experimental study demonstrated that the administration of an anti-platelet drug prevented steroid-induced ON in rabbits and that platelet aggregation seems to be one of the possible factors involved in the pathogenesis of steroid-induced ON.

Gender difference in the development of steroid-induced osteonecrosis in rabbits

To investigate the incidence of steroid-induced osteonecrosis (ON) among male and female rabbits. Forty-seven adult rabbits (male, n = 24; female, n = 23) were injected once intramuscularly into the right gluteus medius muscle with 20 mg/kg of methylprednisolone acetate. Haematological examinations were performed just before and at 1 and 2 weeks after the steroid injection. Two weeks after the injection, both femora and humeri were histopathologically examined for the presence of ON, and the bone marrow fat cells were examined morphologically. Sixteen of 24 male rabbits (66.7%) and 5 of 23 female rabbits (21.7%) developed ON. There was a significant difference in the rate of incidence of ON between male and female rabbits (P = 0.0032). Haematologically, at 1 week after the steroid injection, both the mean low-density lipoprotein (LDL) and the ratio of LDL cholesterol to high-density lipoprotein cholesterol in the male rabbits were significantly higher than those in the female rabbits (P = 0.0001 for both comparisons). The bone marrow fat cells of the male rabbits [61.5 (5.6) microm] were significantly larger than those of the female rabbits [58.9 (3.7) microm; P = 0.0102]. This study indicates that gender may be an important factor in considering the pathogenesis of steroid-induced ON.

Changes of gene expression profiling and pathways related fat metabolisim in femoral head of steroid-induced osteonecrotic rats

Objective To understand the changes of gene expression profiling and pathways related to fat metabolism in femoral head of steroid-induced osteonecrotic rats in contribution to pathogenesis of steroid-induced osteonecrosis. Methods We applied lipopolysaccharide (LPS) and methylprednisolone (MPSL) to prepare a rat model of steroid-induced osteonecrosis (SIO). Rats were divided into two groups: normal (N) group and model (M) group. All rats were sacrificed after 6 weeks. Then we applied a genome wide cDNA microarray technology to analyze genes expressed in femoral head of two groups and also serum levels of cholesterol (TC) were measured in the two groups. Results (1) There were a total of 111 expression genes which were changed by a minimum of two-fold and also it was found that 18 pathways had significant changes in these differential genes by molecule annotation system (MAS) analysis in femoral head of model group compared with normal rats. (2) There were 6 upregulated genes related to fatty acid metabolism (FAM) pathway ( Acadl, Cpt2, Acaa2, Acat1, Acsl1, Hadhb ), 2 upregulated genes related to fatty acid elongation in mitochondria (FAEM) ( Acaa2, Hadhb ), 3 upregulated genes related to adipocytokine signaling pathway (ACK) ( Cpt2, Prkaa2, Acsl1 ) et al in femoral head of model group compared with normal rats. (3) Serum levels of cholesterol significantly increased 21.2% in the model group compared with the normal group (1.754 ± 0.264 mol/L vs 1.447 ± 0.142 mol/L, p Conclusions The data suggest that the upregulated genes related to fat metabolism in the pathways of femoral head of steroid-induced osteonecrotic rats might play an important role in promoting the synthesis of fatty acid and cholesterol and fat metabolism could be mediated by FAM, FAEM and ACK pathways. The changes of multiple gene expression and numerous pathways could play major roles in steroid-induced osteonecrosis pathogenesis. (This study was supported by National Natural Science Foundation of China, NSFC 30472173).

Oxidative stress on idiopathic osteonecrosis

The increasing incidence of steroid-induced osteonecrosis of the femoral head is a recent and emerging problem. In-depth pathogenesis, however, has not been well understood. We focused on oxidative stress, which was reported to be involved in many diseases, and conducted animal studies to investigate it. Our results from the rabbit model of steroid-induced osteonecrosis demonstrated that administration of steroids caused oxidative damage in bones, and the administration of glutathione reduced the incidence of osteonecrosis. We were also the first to have successfully induced osteonecrosis in rats by administering buthionine sulfoximine (BSO) . The results indicated the involvement of oxidative stress in the development of osteonecrosis and may contribute to elucidating the underlying mechanisms and prevention of the pathogenesis of steroid-induced osteonecrosis.

DNA oxidation injury in bone early after steroid administration is involved in the pathogenesis of steroid-induced osteonecrosis

Using a rabbit model, we investigated the DNA oxidation injury occurring in bone following steroid administration and focused on the relation between DNA oxidation injury and osteonecrosis. Japanese white rabbits weighing about 3.5 kg were injected with a single intramuscular dose of methylprednisolone 4 mg/kg and divided into groups consisting of 10 rabbits each, which were killed after 3, 5 and 14 days (groups A, B and C respectively). As a control, five untreated rabbits (group N) were also studied. An immunohistochemical study of the diaphysis of the proximal femur was conducted using the monoclonal antibody N45.1, which is a highly specific antibody against 8-hydroxy-2'-deoxyguanosine, an index of DNA oxidation injury. Also, using NIH Image freeware, the positive area (8-OHdG %PA) of each group was calculated and the four groups were compared. Osteonecrosis was detected only in group C (70%). N45.1 positivity was noted in bone marrow haematopoietic cells and was particularly marked in groups B and C. 8-OHdG %PA was 1.6 +/- 0.2% in group N, 2.2 +/- 0.4% in group A, 4.8 +/- 0.4% in group B and 5.1 +/- 0.5% in group C, with significantly greater oxidation injury found in groups B and C (P < 0.001). Oxidative injury was demonstrated soon after the administration of methylprednisolone in a rabbit model prior to the development of osteonecrosis. This finding may suggest new strategies to prevent steroid-induced osteonecrosis, such as the optimally timed (early) administration of antioxidant agents.

Acute and chronic stress-induced gastrointestinal injury in rats, and protection by a novel 1H636 grape seed proanthocyanidin extract (GSPE): [formula omitted], C.B. Williams, M. Milnes, J. Balmoori, X. Ye, D. Bagchi, S.J. Stohs, Creighton Univ. Health Sciences Center, Omaha, NE 68178

Oxidative damage and apoptosis play dominant roles in the pathogenesis of steroid-induced osteonecrosis (ON). Grape seed proanthocyanidin extract (GSPE) demonstrates antioxidant and antiapoptotic properties. Our aim was to demonstrate the effects of GSPE in preventing steroid-induced ON in rabbits.Osteonecrosis was induced by high-dose methylprednisolone (40 mg/kg). Rabbits in the preventive medicine group were treated with 100 mg/kg/day GSPE for 14 consecutive days, and the presence or absence of ON was examined histopathologically. Oxidative damage in bone tissue was assessed by immunohistochemical staining of 8-oxo-2’-deoxyguanosine (8-OHdG), malondialdehyde (MDA) levels, and activities of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px). Apoptosis was detected via quantitative terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labelling (TUNEL) staining and activated caspase 3 immunoblotting and activity.GSPE significantly attenuated the changes of immunohistochemical staining of 8-OHdG, MDA levels, and antioxidant enzymes activities, which were caused by methylprednisolone administration. Quantitative TUNEL and caspase 3 assay showed lower apoptosis with GSPE application. Simultaneously, GSPE reduced the incidence of steroid-induced ON in an established rabbit model to 17.6 %, compared with 87.5 % in the steroid-only group.These results reveal that GSPE treatment could inhibit oxidative damage and apoptosis to exert beneficial effects on reducing the incidence of steroid-induced ON in rabbit models.

Effect of high levels of corticosteroids on the lipids of the long bones of the mature rabbit

Corticosteroid administration is associated with the development of nontraumatic osteonecrosis in man. However, the pathogenesis of steroid-induced osteonecrosis is unknown. Osteopenia, occurring as a result of corticosteroid treatment is thought to be one factor contributing to the compromise of vascular space. We have treated rabbits daily with hydrocortisone-acetate (15 mg per 4 kg rabbit for 4 and 9 weeks, respectively). These animals progressively developed osteoporosis. Cholesterol accumulated in both the unwashed cancellous bone and the marrow-free compact bones of the treated rabbits when contrasted with age-matched controls. Elevations in marrow lipid content were also observed in the treated animals. Although none of the treated rabbits developed osteonecrosis during the short time of the study, the findings of elevated cholesterol suggest that alterations in bone cell membranes may lead to cell dysfunction and osteopenia. This osteopenia may eventually cause osteonecrosis by occlusion of subchondral vessels. The elevated marrow lipids may also contribute to the development of osteonecrosis by increasing intramedullary pressure and causing venous stasis.