Extracorporeal shockwave relieves endothelial injury and dysfunction in steroid-induced osteonecrosis of the femoral head via miR-135b targeting FOXO1: in vitro and in vivo studies.

Injury and dysfunction of endothelial cells (ECs) are closely related to the pathogenesis of steroid-induced osteonecrosis of the femoral head (ONFH), while MicroRNAs (miRNAs) play an essential role in the processes. Extracorporeal shockwave treatment (ESWT) has been used in the non-invasive treatment of various diseases including musculoskeletal and vascular disorders. In particular, ESWT with low energy levels showed a beneficial effect in ischemic tissues. However, there has been no comprehensive assessment of the effect of ESWT and miRNAs on steroid-induced ONFH. In the present study, we investigated the role and mechanism of ESWT and miRNAs both in vitro and in vivo. Using a steroid-induced ONFH rat model, we found that ESWT significantly enhances proliferation and angiogenesis as well as alleviates apoptosis. In two types of ECs, ESWT can promote cell proliferation and migration, enhance angiogenesis, and inhibit apoptosis. Notably, our study demonstrates that miR-135b is downregulated and modulated forkhead box protein O1 (FOXO1) in ECs treated with dexamethasone. Remarkably, both miR-135b knockdown and FOXO1 overexpression reversed the beneficial effect of ESWT on ECs. Additionally, our data suggest that ESWT activates the FOXO1-related pathway to impact proliferation, apoptosis, and angiogenesis. Taken together, this study indicates that ESWT relieves endothelial injury and dysfunction in steroid-induced ONFH via miR-135b targeting FOXO1.

Liver injury after methylprednisolone pulse therapy in multiple sclerosis is usually due to idiosyncratic drug-induced toxicity rather than autoimmune hepatitis.

BACKGROUND: In patients with multiple sclerosis (MS), development of hepatic injury has been sporadically reported after methylprednisolone (MP) pulse therapy. Some studies suggest autoimmune hepatitis, while other studies reported direct hepatotoxicity as a cause for hepatic injury. Here, we studied the pathological mechanism of such liver injury in patients with MS. METHODS: From 2005 to 2016, eight patients with MS developed liver injury after MP pulse therapy. Their average age was 38 years (range: 28-49 years, all female). Autoimmune antibodies were measured and a liver biopsy was performed in seven patients. RESULTS: Liver injury developed within two weeks in two patients and later (30-90 days after MP) in six patients. No hepatitis-related autoantibody or hepatitis virus were found. All cases were classified as hepatocellular injury and none as cholestatic or mixed. A liver biopsy in five cases revealed centrilobular necrosis with lobular infiltrates of inflammatory cells, suggesting drug-induced acute hepatitis. The biopsy findings in another case suggested a residual stage of acute hepatitis. Only one patient showed portal expansion with periportal fibrosis, suggesting autoimmune hepatitis. All patients recovered spontaneously or with only hepatoprotective drugs, although one patient with possible autoimmune hepatitis recovered slowly. CONCLUSION: Liver injury develops usually later than two weeks after MP treatment. The prognosis is good in most cases and rarely autoimmune hepatitis may be involved.

Glucocorticoid Enhanced the Expression of Ski in Osteonecrosis of Femoral Head: The Effect on Adipogenesis of Rabbit BMSCs.

Glucocorticoid (GC)-induced osteonecrosis has been considered as the most serious side effect in long-term or over-dose steroid therapy. The decreased bone mass and increased marrow fat tissue demonstrated that GC can destroy the normal differentiation of bone marrow mesenchymal stem cells (BMSCs), which accelerates adipogenesis but not osteogenesis. However, the underlying mechanisms are still unclear. Ski, an evolutionary conserved protein, is a multifunctional transcriptional regulator that involved in regulating signaling pathways associated with adipogenesis differentiation, but the concrete function remains unclear. In this work, we first established a methylprednisolone (MPS)-induced osteonecrosis of femoral head (ONFH) rabbit model, in which the expression of Ski, PPAR-γ, and FABP4 was up-regulated compared with control group, and then we induced the isolated BMSCs from rabbit with dexamethasone (Dex) in vitro and the results showed that the Ski expression was up-regulated by Dex in a dose- and time-dependent manner. Therefore, we demonstrated that the expression of Ski was up-regulated in glucocorticoid-related osteonecrosis disease in vivo and in vitro. Moreover, the adipogenesis differentiation capacity of BMSCs was enhanced after induced by Dex, which was identified by Oil Red O staining, and the up-regulated PPAR-γ and FABP4 expression. To further study the function of Ski in BMSC after induced by Dex, Ski specific small interfering RNA (Ski-siRNA) was used. Results showed that knockdown of Ski obviously decreased adipogenesis differentiation evident by Oil Red O staining, and the expression of PPAR-γ and FABP4 was down-regulated simultaneously. Collectively, our findings suggest that Ski increased significantly during glucocorticoid-induced adipogenic differentiation of BMSCs, and the expression level was consistent with adipogenic-related proteins including PPAR-γ and FABP4. Based on the above data, we believe that Ski might become a new molecule in the treatment of GC-induced ONFH and our study could provide a basis for further study on the detailed function of Ski in ONFH.

MicroRNA-34a alleviates steroid-induced avascular necrosis of femoral head by targeting Tgif2 through OPG/RANK/RANKL signaling pathway.

The study aims to investigate the effect of microRNA-34a (miR-34a) targeting Tgif2 on steroid-induced avascular necrosis of femoral head (SANFH) by regulating OPG/RANK/RANKL signaling pathway. SD rats were divided into normal control and model (RNAKL rat models) groups. The model group was further assigned into model control, negative control, miR-34a mimics and miR-34a inhibitors groups. QRT-PCR was applied to detect miR-34a, Tgif2, OPG, RANK and RNAKL mRNA expressions. Femoral head tissues were collected for Micro-CT scanning and HE staining. QRT-PCR and Western blotting were used to detect expressions of miR-34a, Tgif2, OPG, RANK, RANKL and Runx2, OPN and OC in bone tissues. Dual-luciferase reporter gene assay was used to testify the target relationship between miR-34a and Tgif2. Compared with the normal control group, the model group showed increased Tgif2, RANK and RANKL mRNA expressions, but decreased miR-34a and OPG mRNA expressions. Tgif2 mRNA expression was negatively correlated with miR-34a and OPG mRNA expressions. Micro-CT showed cystic degeneration of femoral head, with decreased bone volume/total volume (BV/TV), bone surface area/bone volume and trabecular number in the model control group compared with the normal control group. Compared with the model control group, the miR-34a mimics group showed increased BV/TV and trabecular thickness and Runx2, OPN and OC expressions, while the parameters decreased in the miR-34a inhibitors group. Compared with the normal control group, the other groups showed increased Tgif2, RANK and RANKL expressions but decreased miR-34a and OPG expressions. Compared with the model control group, Tgif2, RANK and RANKL expressions decreased and miR-34a and OPG expressions increased in the miR-34a mimics group, while the miR-34a inhibitors group had a reverse trend in contrast to the miR-34a mimics group. Tgif2 is a target gene of miR-34a. In conclusion, miR-34a can alleviate SANFH through targeting Tgif2 and further regulating OPG/RANK/RANKL signaling pathway. Impact statement miR-34a can alleviate SANFH through targeting Tgif2 and further regulating OPG/RANK/RANKL signaling pathway, which can be used as a new theoretical basis for SANFH treatment.

Protective effects of molecular hydrogen on steroid-induced osteonecrosis in rabbits via reducing oxidative stress and apoptosis.

BACKGROUND: The objective of this study was to investigate the protective effects of molecular hydrogen, a novel and selective antioxidant, on steroid-induced osteonecrosis (ON) in a rabbit model. METHODS: Sixty rabbits were randomly divided into two groups (model group and hydrogen group). Osteonecrosis was induced according to an established protocol of steroid-induced ON. Rabbits in the hydrogen group were treated with intraperitoneal injections of molecular hydrogen at 10 ml/kg body weight for seven consecutive days. Plasma levels of total cholesterol, triglycerides, soluble thrombomodulin(sTM), glutathione(GSH) and malondialdehyde(MDA) were measured before and after steroid administration. The presence or absence of ON was examined histopathologically. Oxidative injury and vascular injury were assessed in vivo by immunohistochemical staining of 8-hydoxy-2-deoxyguanosine(8-OHdG) and MDA, and ink artery infusion angiography. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to measure apoptosis. RESULTS: The incidence of steroid-induced ON was significantly lower in hydrogen group (28.6%) than that in model group (68.0%). No statistically differences were observed on the levels of total cholesterol and triglycerides. Oxidative injury, vascular injury and apoptosis were attenuated in the hydrogen group compared with those in the model group in vivo. CONCLUSIONS: These results suggested that molecular hydrogen prevents steroid-induced osteonecrosis in rabbits by suppressing oxidative injury, vascular injury and apoptosis.

Effect of Resveratrol on Preventing Steroid-induced Osteonecrosis in a Rabbit Model.

BACKGROUND: Prevention of osteonecrosis (ON) has seldom been addressed. The purpose of this study was to evaluate the effect of resveratrol on preventing steroid-induced ON in rabbits. METHODS: Seventy-two rabbits were divided into four groups: (1) NEC (ON) group: thirty rabbits were treated with lipopolysaccharide (LPS) once, then with methylprednisolone (MPS) daily for 3 days; (2) PRE (prevention) group: thirty rabbits were given one dose of LPS, then MPS daily for 3 days, and resveratrol on day 0 and daily for 2 weeks; (3) RES (resveratrol) group: six rabbits were given resveratrol for 2 weeks but without LPS/MPS; (4) CON (control) group: six rabbits were given alcohol for 2 weeks but without LPS/MPS. Levels of plasma tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), thrombomodulin (TM), vascular endothelial growth factor (VEGF), maximum enhancement (ME) by magnetic resonance imaging, and ON incidence were evaluated. RESULTS: The PRE group had a lower ON incidence than the NEC group, but with no significant differences at 2 weeks and 12 weeks. The RES and CON groups did not develop ON. TM and VEGF were significantly higher in the NEC group compared with the PRE group at weeks 1, 2, and 4 (TM: 1 week, P = 0.029; 2 weeks, P = 0.005; and 4 weeks, P = 0.047; VEGF: 1 week, P = 0.039; 2 weeks, P = 0.021; 4 weeks, P = 0.014), but the difference disappeared at 12 weeks. The levels of t-PA and PAI-1 were not significantly different between the NEC and PRE groups. The TM, t-PA, PAI-1, and VEGF concentrations in the RES and CON groups did not change over time. Compared to the baseline, ME in the NEC group decreased significantly (P = 0.025) at week 1, increased significantly (P = 0.021) at week 2, and was decreased at week 12. The variance was insignificant in the PRE group. CONCLUSIONS: Resveratrol may improve blood supply to bone in a rabbit model of ON of the femoral head via anti-inflammatory effects to protect the vascular endothelium and reduce thrombosis.

Sclerostin antibody and interval treadmill training effects in a rodent model of glucocorticoid-induced osteopenia.

Glucocorticoids have a beneficial anti-inflammatory and immunosuppressive effect, but their use is associated with decreased bone formation, bone mass and bone quality, resulting in an elevated fracture risk. Exercise and sclerostin antibody (Scl-Ab) administration have both been shown to increase bone formation and bone mass, therefore the ability of these treatments to inhibit glucocorticoid-induced osteopenia alone or in combination were assessed in a rodent model. Adult (4 months-old) male Wistar rats were allocated to a control group (C) or one of 4 groups injected subcutaneously with methylprednisolone (5mg/kg/day, 5 days/week). Methylprednisolone treated rats were injected subcutaneously 2 days/week with vehicle (M) or Scl-Ab-VI (M+S: 25mg/kg/day) and were submitted or not to treadmill interval training exercise (1h/day, 5 days/week) for 9 weeks (M+E, M+E+S). Methylprednisolone treatment increased % fat mass and % apoptotic osteocytes, reduced whole body and femoral bone mineral content (BMC), reduced femoral bone mineral density (BMD) and osteocyte lacunae occupancy. This effect was associated with lower trabecular bone volume (BV/TV) at the distal femur. Exercise increased BV/TV, osteocyte lacunae occupancy, while reducing fat mass, the bone resorption marker NTx, and osteocyte apoptosis. Exercise did not affect BMC or cortical microarchitectural parameters. Scl-Ab increased the bone formation marker osteocalcin and prevented the deleterious effects of M on bone mass, further increasing BMC, BMD and BV/TV to levels above the C group. Scl-Ab increased femoral cortical bone parameters at distal part and midshaft. Scl-Ab prevented the decrease in osteocyte lacunae occupancy and the increase in osteocyte apoptosis induced by M. The addition of exercise to Scl-Ab treatment did not result in additional improvements in bone mass or bone strength parameters. These data suggest that although our exercise regimen did prevent some of the bone deleterious effects of glucocorticoid treatment, particularly in trabecular bone volume and osteocyte apoptosis, Scl-Ab treatment resulted in marked improvements in bone mass across the skeleton and in osteocyte viability, resulting in decreased bone fragility.

MicroRNA-29a mitigates glucocorticoid induction of bone loss and fatty marrow by rescuing Runx2 acetylation.

Glucocorticoid treatment reportedly increases the morbidity of osteoporotic or osteonecrotic disorders. Exacerbated bone acquisition and escalated marrow adipogenesis are prominent pathological features of glucocorticoid-mediated skeletal disorders. MicroRNAs reportedly modulate tissue metabolism and remodeling. This study was undertaken to investigate the biological roles of microRNA-29a (miR-29a) in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Transgenic mice overexpressing miR-29a precursor or wild-type mice were given methylprednisolone. Bone mass, microarchitecture and histology were assessed by dual energy X-ray absorptiometry, µCT and histomorphometry. Differential gene expression and signaling components were delineated by quantitative RT-PCR and immunoblotting. Glucocorticoid treatment accelerated bone loss and marrow fat accumulation in association with decreased miR-29a expression. The miR-29a transgenic mice had high bone mineral density, trabecular microarchitecture and cortical thickness. miR-29a overexpression mitigated the glucocorticoid-induced impediment of bone mass, skeletal microstructure integrity and mineralization reaction and attenuated fatty marrow histopathology. Ex vivo, miR-29a increased osteogenic differentiation capacity and alleviated the glucocorticoid-induced promotion of adipocyte formation in primary bone-marrow mesenchymal progenitor cell cultures. Through inhibition of histone deacetylase 4 (HDAC4) expression, miR-29a restored acetylated Runx2 and ß-catenin abundances and reduced RANKL, leptin and glucocorticoid receptor expression in glucocorticoid-mediated osteoporosis bone tissues. Taken together, glucocorticoid suppression of miR-29a signaling disturbed the balances between osteogenic and adipogenic activities, and thereby interrupted bone formation and skeletal homeostasis. miR-29a inhibition of HDAC4 stabilized the acetylation state of Runx2 and ß-catenin that ameliorated the detrimental effects of glucocorticoid on mineralization and lipogenesis reactions in bone tissue microenvironments. This study highlighted emerging skeletal-anabolic actions of miR-29a signaling in the progression of glucocorticoid-induced bone tissue destruction. Sustaining miR-29a actions is beneficial in protecting against glucocorticoid-mediated osteoporosis.

[MODEL ESTABLISHMENT, MRI AND PATHOLOGICAL FEATURES OF EARLY STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN RABBIT].

OBJECTIVE: To establish an rabbit model of early steroid-induced avascular necrosis of the femoral head (SANFH) and evaluate its validity with MRI and pathological examination. METHODS: Twenty 6-month-old rabbits (weighing, 2-3 kg) were randomly divided into 2 groups (control group and model group), 10 rabbits in each group. Dexamethasone sodium phosphate solution (10 mg/kg) was injected into bilateral gluteus in model group, and the same amount of saline was injected in control group, every 3 days for 14 times. General observation was done after modelling. Osteonecrosis was verified by pathological observation and MRI findings at 6 weeks. RESULTS: After 6 weeks, rabbits did not show obvious changes in control group; increased hair removal, decreased food intake, and slight limp were observed in model group. The MRI results showed normal shape of the bilateral femoral head and no abnormal signals in control group; irregular shape of the bilateral femoral head and a slice of irregular abnormal signals were observed, and necrosis and cystolization of the subchondral bone and sparse changes of trabecular bone were shown in model group. General observation from coronal section of femoral head showed smooth red cartilage surface in control group; on the contrary, the cartilage surface of the femoral head became dull, thin even visible hemorrhage under articular cartilage and necrosis of the femoral head were observed. The histopathological examination indicated that trabecular bone of the femoral head in control group was massive, thick, and close and osteocytes in the bone lacunae had normal shapes. The osseous trabecular became thinner and broken; karyopyknosis of osteocytes and bone empty lacunae could be obviously seen in model. group. The rates of empty lacunae were 8.0% ± 0.5% in control group and 49.0% ± 0.3% in model group, showing significant difference (t = 21.940, P = 0.000). CONCLUSION: Establishing a model of early SANFH through injecting short-term, shock, and high dose of dexamethasone, and it can been evaluated effectively with MRI and pathological examination.

Effect of preoperative administration of methylpredonisolone and ulinastatin on tumor cell metastasis after surgical stress.

Using a rat laparotomy stress model, we conducted a comparative analysis of postoperative organ metastasis after administration of ulinastatin (UTI) or methylprednisolone (MP), which have an inhibitory effect on cytokine production. The subjects were classified into 4 groups: 1) minimal laparotomy group (C group), 2) major laparotomy group (L group), 3) preoperative MP intravenous administration + major laparotomy group (MP group), and 4) preoperative UTI intravenous administration + major laparotomy group (UTI group). Either MP or UTI was administered intravenously before surgery, and RI-labeled cells were injected into the portal vein immediately after laparotomy to collect tissue specimens in order to measure radiation dosage. Then, the concentrations of serum IL-2 and IL-6, liver interleukin 1 beta (IL-1ß) and interleukin 10 (IL-10), and liver E-selectin were measured. In addition natural killer cell, (NK cell) activation and neoplastic nodules on the liver surface at 3 weeks after surgery were also measured. The adhesion rate of malignant cells to the liver was higher in the L group than in the C group, higher in the MP group than the L group, and lower overall in the UTI group. The concentration of IL-1ß and IL-6 were decreased in the MP and UTI groups compared to the L group. IL-2 was decreased significantly in the MP group compared with the C and L groups. E-selectin expression level decreased in the UTI group compared with the L group. NK cell activation decreased in the MP group compared with the C group and L group, but no differences were observed between the UTI and L groups. The number of tumor nodules on the surface of the liver increased in the MP group compared with the L group, and decreased in the UTI group compared with the L group. Postoperative alleviation of invasive reaction was suggested in both the MP and UTI groups. However, preoperative administration of MP increased metastasis while that of UTI inhibited metastasis. MP was considered to have decreased anti-tumor immunocompetence and promoted metastasis, while UTI was considered to have inhibited the expression of adhesive molecules and decreased metastasis.

The effect of core decompression on local expression of BMP-2, PPAR-γ and bone regeneration in the steroid-induced femoral head osteonecrosis.

BACKGROUND: To investigate the efficacy of the sole core decompression surgery for the treatment of steroid-induced femoral head osteonecrosis. METHODS: The model was established by administration of steroids in combination with horse serum. The rabbits with bilateral femoral head osteonecrosis were randomly selected to do the one side of core decompression. The other side was used as the sham. Quantitative RT-PCR and western blot techniques were used to measure the local expression of BMP-2 and PPAR-γ. Bone tissues from control and operation groups were histologically analyzed by H&E staining. The comparisons of the local expression of BMP-2 and PPAR-γ and the bone regeneration were further analyzed between different groups at each time point. RESULTS: The expression of BMP-2 in the osteonecrosis femoral head with or without decompression was significantly lower than that in normal animals. BMP-2 expression both showed the decreasing trend with the increased post-operation time. No significant difference of BMP-2 expression occurred between femoral head osteonecrosis with and without decompression. The PPAR-γ expression in the femoral head osteonecrosis with and without core decompression both was significantly higher than that in control. Its expression pattern showed a significantly increased trend with increased the post-operation time. However, there was no significant difference of PPAR-γ expression between the femoral head osteonecrosis with and without decompression at each time point. Histopathological analysis revealed that new trabecular bone and a large number of osteoblasts were observed in the steroid-induced femoral head osteonecrosis with lateral decompression at 8 weeks after surgery, but there still existed trabecular bone fractures and bone necrosis. CONCLUSIONS: Although decompression takes partial effect in promoting bone regeneration in the early treatment of femoral head osteonecrosis, such an effect does not significantly improve or reverse the pathological changes of femoral head necrosis. Thus, the long-term effect of core decompression in the treatment of steroid-induced femoral head osteonecrosis is not satisfactory.

Promotion of bone repair by implantation of cryopreserved bone marrow-derived mononuclear cells in a rabbit model of steroid-associated osteonecrosis.

OBJECTIVE: Cytotherapy is an insufficient method for promoting bone repair in steroid-associated osteonecrosis (SAON), and this has been attributed to impairment of the bioactivity of bone marrow-derived stem cells (BMSCs) after pulsed administration of steroids. Cryopreserved autologous bone marrow-derived mononuclear cells (BMMNCs), which contain BMSCs, might maintain their bioactivity in vitro. This study sought to investigate the effects of cryopreserved BMMNCs, before steroid administration, on the enhancement of bone repair in an established rabbit model of SAON. METHODS: For in vitro study, bone marrow was harvested 4 weeks before SAON induction from the iliac crests of rabbits (n = 10) to isolate fresh BMMNCs, and the BMMNCs were then cryopreserved for 8 weeks. Both the fresh and the cryopreserved BMMNCs were evaluated for their bioactivity and osteogenic differentiation capacity. In addition, BMMNCs were isolated 2 weeks after SAON induction and subjected to the same evaluations. For in vivo study, cryopreserved BMMNCs were implanted into the bone tunnel during core decompression of the femur (n = 12 rabbits) after the induction of SAON, and tissue regeneration was evaluated by micro-computed tomography and histologic analyses at 12 weeks postoperation. RESULTS: In vitro, there were no significant differences in the bioactivity or ability to undergo osteogenic differentiation between fresh BMMNCs and cryopreserved BMMNCs, but after SAON induction, both features were decreased significantly. In vivo, the bone mineral density, ratio of bone volume to total volume of bone, and volume and diameter of neovascularization within the bone tunnel were significantly higher in the BMMNC-treated group compared to the nontreated control group at 12 weeks postoperation. CONCLUSION: Cryopreserved BMMNCs maintained their bioactivity and promoted bone regeneration and neovascularization within the bone tunnel after core decompression in this rabbit model of SAON.

Effect of pentoxifylline on histopathological changes in steroid-induced osteonecrosis of femoral head: experimental study in chicken.

PURPOSE: Pentoxifylline (PTX) is a derivative of methylxanthine and is used in peripheral vascular and cerebrovascular diseases for its effect on the regulation of blood circulation. We investigated whether PTX could be beneficial for femoral head osteonecrosis associated with steroid through these effects. METHODS: Sixty mature Leghorn type chickens were chosen and divided into three groups. The 25 chickens in group A were given a weekly dose of 3 mg/kg/week methylprednisolone acetate intramuscularly. Four chickens in group B died after the first drug injection and were excluded from the study. Therefore, the remaining 21 chickens in group B were additionally given 25 mg/kg/day pentoxifylline intramuscularly, along with the steroid medication as given in group A. The ten chickens in group C were not given any injections, as they were accepted as the control group. After the sacrifice of the animals at week 14, both femoral heads were taken from each animal. The animals which died along the course of the study also underwent pathological examination but were not a part of the statistical analysis. RESULTS: In this study, steroid induced femoral head osteonecrosis has been experimentally observed in chickens after high doses of corticosteroid therapy. The chickens were given pentoxifylline in order to prevent the effects of steroid on bones and bone marrow. The results showed that chickens are suitable osteonecrosis models, and that steroid causes adipogenesis and necrosis in the bone marrow and the death of the subchondral bone. CONCLUSIONS: The results of this study hint at the assumption that PTX may have a positive benefit on ONFH. PTX seems to minimise the effects of the steroid and reduce the incidence of ONFH.

The chondrotoxicity of single-dose corticosteroids.

PURPOSE: Corticosteroids are commonly injected into the joint space. However, studies have not examined the chondrotoxicity of one-time injection doses. The purpose of this study is to evaluate the effect of dexamethasone sodium phosphate (Decadron), methylprednisolone acetate (Depo-Medrol), betamethasone sodium phosphate and betamethasone acetate (Celestone Soluspan), and triamcinolone acetonide (Kenalog) on human chondrocyte viability in vitro. METHODS: Single-injection doses of each of the corticosteroids were separately delivered to human chondrocytes for their respective average duration of action and compared to controls using a bioreactor containing a continuous infusion pump constructed to mimic joint fluid metabolism. A 14-day time-controlled trial was also performed. A live/dead reduced biohazard viability/cytotoxicity assay was used to quantify chondrocyte viability. RESULTS: Over their average duration of action, betamethasone sodium phosphate/acetate solution and triamcinolone acetonide caused significant decreases in chondrocyte viability compared to control media (19.8 ± 2.9% vs. 5.2 ± 2.1%, P = 0.0025 and 10.2 ± 1.3% vs. 4.8 ± 0.9%, P = 0.0049, respectively). In the 14-day trial, only betamethasone sodium phosphate/acetate solution caused a significant decrease in chondrocyte viability compared to control media (21.5% vs. 4.6%, P < 0.001). CONCLUSIONS: A single-injection dose of betamethasone sodium phosphate and betamethasone acetate solution illustrated consistent and significant chondrotoxicity using a physiologically relevant in vitro model and should be used with caution. Given the observed chondrotoxicity of triamcinolone acetonide in a single trial, there may be some evidence that this medication is chondrotoxic. However, at 14 days, betamethasone sodium phosphate and betamethasone acetate was the only condition that caused significant cell death.

Methylprednisolone exacerbates acute critical illness-related corticosteroid insufficiency associated with traumatic brain injury in rats.

Emerging evidence demonstrates that severe illness could induce critical illness-related corticosteroid insufficiency (CIRCI) and cause poor prognosis. The purpose of this study was to test the hypothesis that methylprednisolone (MP), a synthetic glucocorticoid, promotes post-traumatic apoptosis in both the hypothalamus and pituitary, resulting in acute CIRCI and increased mortality in the acute phase of traumatic brain injury (TBI). We tested this hypothesis by measuring acute CIRCI in rats subjected to fluid percussion injury (FPI) and treated with MP (5-30mg/kg). The corticosteroid response to TBI was evaluated using the corticosterone increase index (CII), where values less than 2.5 were considered indicative of acute CIRCI. The CII of MP treated rats was comparable to that of saline treated control rats before injury but was significantly decreased in injured rats receiving high-dose MP on post-injury day 7. Similarly, the incidence of acute CIRCI was significantly higher in the high-dose MP group on post-injury day 7. Furthermore, the CII of rats that did not survive post-injury was significantly lower compared to that of survival and was indicative of acute CIRCI. We also examined apoptosis in the paraventricular nucleus (PVN) of the hypothalamus and the adenohypophysis of the pituitary, using a TUNEL assay and transmission electron microscopy (TEM). The number of TUNEL-positive cells was significantly higher in injured rats treated with high-dose MP. No TUNEL-positive cells were detected in the adenohypophysis across experimental groups at either 7 or 14days after TBI. However, autopsies performed on rats that did not survive post-injury revealed obvious apoptotic cells in the adenohypophysis. Moreover, TEM revealed morphological changes characteristic of apoptosis in both the PVN and adenohypophysis of high-dose MP treated rats. These data suggest that MP therapy for TBI could increase neuronal apoptosis in both the hypothalamus and pituitary and consequently exacerbate acute CIRCI and mortality induced by TBI.

Pentosan reduces osteonecrosis of femoral head in SHRSP.

Increased oxidative stress is considered one of the main causes of steroid-induced osteonecrosis of the femoral head (ONFH). The aim of this study was to evaluate the effects of a steroid hormone and pentosan polysulfate sodium (pentosan), a heparin analog, in stroke-prone spontaneously hypertensive rats (SHRSP) as a model of ONFH. One hundred twenty-three 13-week-old male SHRSP/Izm rats were divided into four groups: a control group (group C), pentosan-administered group (group P), steroid-administered group (group S), and group administered pentosan plus steroid (group PS). Methylprednisolone acetate, as the steroid hormone, at a dose of 4 mg (15 mg/kg) was administered at 15 weeks of age. Pentosan at a dose of 3 mg/day/kg was continuously administered intraperitoneally from 13 weeks of age for 4 weeks. Rats were sacrificed at 17 weeks of age, and heart blood and both femora were collected. Triglyceride levels were significantly lower in group PS than in group S, indicating that pentosan improves lipid metabolism. The incidence of histologic ONFH was significantly lower in group P, at 14.8% (10/71 femoral heads), than in group C, at 30.4% (17/56 femoral heads), and significantly lower in group PS, at 40.8% (29/71 femoral heads), than in group S, at 91.3% (42/46 femoral heads), indicating that pentosan markedly inhibits ONFH. Immunohistochemical staining for oxidative stress showed that the stainability was significantly lower in group PS than in group S. Pentosan seems to reduce the incidence of ONFH in SHRSP by improving lipid metabolism and decreasing oxidative stress.

Effect of rifampicin on the risk of steroid-induced osteonecrosis of the femoral head.

OBJECTIVE: To investigate the effects and possible mechanism of rifampicin on steroid-induced osteonecrosis of the femoral head (ONFH). METHODS: Bone marrow stromal cells (BMSC) separated from male rats were cultured in vitro without any treatment (Group mA), exposed to dexamethasone (Group mB), treated with rifampicin (Group mC), and exposed to dexamethasone and rifampicin simultaneously (Group mD) respectively (n = 5 in each group). After 7 days, P-glycoprotein (P-gp) activity and adipogenesis of the BMSC were evaluated. In an in vivo experiment, 80 rats were randomly divided into 4 groups (n= 20 in each group). Group A received intragastric saline for 5 weeks. Group B received intragastric saline for one week, followed by subcutaneous methylprednisolone and saline for 4 weeks. Group C received intragastric rifampicin for 5 weeks. Group D received intragastric rifampicin for one week, followed by subcutaneous methylprednisolone and rifampicin for 4 weeks. At the end of the experiment, all rats underwent analysis of P-gp activity of BMSC, P-gp expression in the femoral heads, MRI and histomorphometry of the femoral heads. RESULTS: In vitro, the P-gp activity of BMSC increased and lipid accumulation decreased significantly in Group mD, compared to Group mB. In vivo, P-gp activity and P-gp expression in Group D increased compared to Group B. The mean area of MRI abnormal signal, adipocytic variables and apoptotic cells in Group D decreased, mean percentage of the whole epiphysis made up by the epiphyseal ossification center and trabecular structure variables improved compared to those in Group B. The incidence of ONFH was lower in Group D (50%) than in Group B (80%). CONCLUSION: Rifampicin may decrease the risk of steroid-induced ONFH by enhancing P-gp activity, thus preventing steroid-induced BMSC adipogenesis.

The effect of bone marrow mononuclear cells on vascularization and bone regeneration in steroid-induced osteonecrosis of the femoral head.

OBJECTIVE: We examined whether implantation of autologous bone marrow mononuclear cells (BM-MNC) can augment neovascularization and bone regeneration in steroid-induced osteonecrosis of the femoral head. METHODS: Sixty-five 28-week-old male New Zealand white rabbits were divided into group I (left untreated, N=20), group II (core decompression, N=20) and group III (core decompression+autologous bone marrow cells implantation, N=25) after receiving an established inductive protocol for inducing steroid-associated ON. Four weeks later, these rabbits were euthanized, bilateral femora were dissected for micro-CT-based microangiography to assess vascularization, and then the osteonecrotic changes and repair processes were examined histopathologically. RESULTS: Quantitative analysis showed that new vessel formation in group III was significantly greater compared with other groups at 4 weeks after treatment. Penetrating capillary vessels number vessels number in group III (44.5+/-5.11) was significantly larger than that of group II (11.4+/-2.46) and group I (3.10+/-0.33) (p<0.01). The histologic and histomorphometric analysis revealed that the new bone volume was significantly higher in the group III than in the group I and II, 4 weeks after treatment. CONCLUSION: In this animal model, a combination of bone marrow mononuclear cells and core decompression enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration. These findings confirm the preliminary clinical results obtained in humans that the implantation of bone marrow mononuclear cells is an effective and feasible method for treating early osteonecrosis.

Comparative toxicity of 4 commonly used intravitreal corticosteroids on rat retina.

OBJECTIVE: To investigate the effects of 4 commonly used steroids (dexamethasone, triamcinolone, betamethasone, and methylprednisolone) on 50 retinas of 25 adult pigmented rats. STUDY DESIGN: Experimental animal study. PARTICIPANTS: Twenty-five pigmented Long-Evans male rats. METHODS: Each steroid drug with 2 different doses (0.025 mL and 0.050 mL) was injected into the vitreous of each eye of 5 rats. The low drug dose was injected into the right eye and the high dose was injected into the left eye. Ten eyes of 5 randomly selected rats were used as a control group and intravitreal saline was injected into these eyes. Oxidative damage and intrinsic antioxidative capacity were determined by measuring retinal malondialdehyde (MDA) and glutathione (GSH) levels, respectively. RESULTS: No statistically meaningful difference was observed in retinal GSH and MDA measurements in the low- and high-dose triamcinolone (1 and 2 mg), low-dose betamethasone (0.075 mg), and low-dose dexamethasone (0.1 mg) groups, compared with the control group. Both doses of methylprednisolone (1.6 mg and 3.2 mg), high-dose betamethasone (0.15 mg), and high-dose dexamethasone (0.2 mg) markedly altered retinal GSH and MDA levels. CONCLUSIONS: The results of our study show that the toxicity of triamcinolone is not evident even in high doses. It may be used safely. We also suggest that intravitreal use of low doses of betamethasone and dexamethasone is safer than higher doses of these drugs and both doses of methylprednisolone.