Composite indices of femoral neck strength predicts the collapse of steroid-associated osteonecrosis of the femoral head: a retrospective study.

PURPOSES: The purpose of this study was to investigate the predictive effect exerted by composite indices of femoral neck strength (compressive strength index (CSI), bending strength index (BSI) and impact strength index (ISI) on the femoral head collapse in steroid-associated ONFH patients. METHODS: Nonoperative steroid-associated osteonecrosis of the femoral head (ONFH) patients from 2017 to 2019 were selected. The patients fell into the collapsed group and the non-collapsed group according to whether the femoral head collapsed. CSI, BSI and ISI were calculated. Moreover, bone turnover markers were measured. The statistical analysis was conducted on the predictive effects of composite indices of femoral neck strength and bone turnover index on ONFH collapse. RESULTS: A total of 62 patients were included. The mean CSI, BSI and ISI were significantly lower in the collapsed group than those in the non-collapsed group (P < 0.05). CSI, ISI,t-P1NP and ß-CTx were suggested as the protective risk factors for the femoral head collapse in ONFH patients. The ISI area under the curve values was 0. 878.The mean survival time of the hips of patients with ISI greater than 0.435 was greater (P < 0.05) than that of patients with ISI less than 0.435. CONCLUSION: The composite indices of femoral neck strength can predict steroid-associated ONFH femoral head collapse more effectively than the bone turnover markers. The ISI value of 0.435 is a potential cut-off value, lower than this value can predict the early collapse of steroid-associated ONFH.

miR-155-5p regulates mesenchymal stem cell osteogenesis and proliferation by targeting GSK3B in steroid-associated osteonecrosis.

microRNAs (miRNAs) have recently been recognized as playing an important role in bone-associated diseases. This study investigated whether the reduced miR-155-5p in steroid-associated osteonecrosis of the femoral head (ONFH) attenuated osteogenic differentiation and cell proliferation by targeting GSK3B. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 10) and patients with new femoral neck fracture (n = 10) and mesenchymal stem cells (MSCs) were isolated. The expression profile, the biological function of miR-155-5p, and the interaction between miR-155-5p and GSK3B were investigated by cell viability measurement, western blot, real-time polymerase chain reaction, luciferase reporter assay, and Alizarin Red S (ARS) staining of MSCs. The MSCs that were obtained from the femoral neck fracture group and from the steroid-associated ONFH group were transfected with or without miR-155-5p. We found that, in ONFH samples, the level of mature miR-155-5p was significantly lower than that of control samples. By inhibiting GSK3B, miR-155-5p promoted the nuclear translocation of ß-catenin, increased the expression of osteogenesis-related genes, and facilitated the proliferation and differentiation of MSCs. Restoring the expression of GSK3B in MSCs partially reversed the effect of miR-155-5p. These findings suggest that reduced miR-155-5p in steroid-associated ONFH attenuates osteogenic differentiation and cell proliferation by increased levels of GSK3B and inhibition of Wnt signaling.

Repetitive 1.6 ATA hyperbaric oxygen therapy for bilateral ARCO Stage II steroid-associated osteonecrosis of the femoral head.

Hyperbaric oxygen (HBO2) therapy has been demonstrated to have beneficial effects on the early stages of steroid-associated osteonecrosis (SAON) of the femoral head. Since high HBO2 pressure (e.g., 2.4-2.5 atmospheres absolute/ATA) has been commonly considered to have a greater ability to restore tissue oxygenation in the femoral head than low pressure (e.g., 1.6 ATA), the latter HBO2 protocol is rarely used for SAON treatment. In this paper, we present the case of a 36-year-old female diagnosed with bilateral early stage (Association for Research on Osseous Circulation, ARCO stage II) SAON caused by steroid therapy for neuromyelitis optica spectrum disorder (NMOSD). Because the patient could not endure high HBO2 pressures, the treatment pressure was adjusted to 1.6 ATA, which was the highest pressure the patient could withstand. After 20 treatment sessions, her symptoms were relieved significantly. Her visual analog score (VAS: using a 0-10 score) decreased from 7 to 2, and after 50 treatment sessions her symptoms disappeared almost completely. A significant improvement was also observed radiologically by computed tomography (CT) and magnetic resonance imaging (MRI) examinations. This case study provides a potential HBO2 treatment protocol with reduced pressure for early-stage femoral head necrosis. Further research is needed to validate this finding and explore the potential mechanism of HBO2 on SAON.

Mitochondrial stress and redox failure in steroid-associated osteonecrosis.

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.

Advances in the Pathogenesis of Steroid-Associated Osteonecrosis of the Femoral Head.

Osteonecrosis of the femoral head (ONFH) is a refractory orthopedic condition characterized by bone cell ischemia, necrosis, bone trabecular fracture, and clinical symptoms such as pain, femoral head collapse, and joint dysfunction that can lead to disability. The disability rate of ONFH is very high, which imposes a significant economic burden on both families and society. Steroid-associated osteonecrosis of the femoral head (SANFH) is the most common type of ONFH. However, the pathogenesis of SANFH remains unclear, and it is an urgent challenge for orthopedic surgeons to explore it. In this paper, the pathogenesis of SANFH and its related signaling pathways were briefly reviewed to enhance comprehension of the pathogenesis and prevention of SANFH.

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway.

Endothelial impairment and dysfunction are closely related to the pathogenesis of steroid-associated osteonecrosis of the femoral head (SONFH). Recent studies have showed that hypoxia inducible factor-1α (HIF-1α) plays a crucial role in endothelial homeostasis maintenance. Dimethyloxalylglycine (DMOG) could suppress HIF-1 degradation and result in nucleus stabilization by repressing prolyl hydroxylase domain (PHD) enzymatic activity. Our results showed that methylprednisolone (MPS) remarkably undermined biological function of endothelial progenitor cells (EPC) by inhibiting colony formation, migration, angiogenesis, and stimulating senescence of EPCs, while DMOG treatment alleviated these effects by promoting HIF-1α signaling pathway, as evidenced by senescence-associated ß-galactosidase (SA-ß-Gal) staining, colony-forming unit, matrigel tube formation, and transwell assays. The levels of proteins related to angiogenesis were determined by ELISA and Western blotting. In addition, active HIF-1α bolstered the targeting and homing of endogenous EPCs to the injured endothelium in the femoral head. Histopathologically, our in vivo study showed that DMOG not only alleviated glucocorticoid-induced osteonecrosis but also promoted angiogenesis and osteogenesis in the femoral head as detected by microcomputed tomography (Micro-CT) analysis and histological staining of OCN, TRAP, and Factor Ⅷ. However, all of these effects were impaired by an HIF-1α inhibitor. These findings demonstrate that targeting HIF-1α in EPCs may constitute a novel therapeutic approach for the treatment of SONFH.

MiR-29a-3p Inhibits Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via Targeting FOXO3 and Repressing Wnt/ß-Catenin Signaling in Steroid-Associated Osteonecrosis.

Background and Objectives: This study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis. Methods and Results: The online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid- associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and ß-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of ß-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3'UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs' proliferation, osteogenic differentiation markers' expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and ß-catenin, and inhibition of miR-29a-3p promoted translocation of ß-catenin to the nucleus. Conclusions: MiR-29a-3p can modulate FOXO3 expression and Wnt/ß-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

Delivery of MiR335-5p-Pendant Tetrahedron DNA Nanostructures Using an Injectable Heparin Lithium Hydrogel for Challenging Bone Defects in Steroid-Associated Osteonecrosis.

Corticosteroids-induced Dickkopf-1 (DKK1) upregulation and Wnt signaling inhibition result in bone metabolism disorder and steroid-associated osteonecrosis (SAON). Implanting biomaterials to regulate the Wnt pathway is a promising method to repair challenging bone defects associated with SAON. Here, tetrahedral DNA nanostructures (TDNs) are fabricated as gene carriers to deliver MiR335-5p, which targets DKK1 translation. Heparin lithium hydrogel (Li-hep-gel) is synthesized to act as a lithium and MiR@TDNs delivery agent. Finally, the repair effects on challenging bone defect in SAON using a MiR@TDNs/Li-hep-gel composite are assessed in vivo. The results reveal that MiR@TDNs are absorbed by bone mesenchymal stem cells (BMSCs) and increase cell viability and reduce apoptosis. Moreover, MiR@TDNs promote alkaline phosphatase expression and calcium nodular deposition, decrease lipid droplet expression of BMSCs, and improve vascular endothelial growth factor secretion and vascular-like structure formation in vitro. After MiR@TDNs/Li-hep-gel is implanted into the SAON model, the internal bone defect of osteonecrosis is repaired with a large area of new bone accompanied with neovascularization and reduced empty lacunae. In conclusion, MiR@TDNs/Li-hep-gel can provide dual delivery of lithium and MiR@TDNs, which synergistically upregulate the Wnt signaling pathway, enhancing bone regeneration in challenging bone defects, and can be potentially used in SAON repair.

Long-Term Retrospective Follow-Up of Fresh Osteochondral Allograft Transplantation for Steroid-Associated Osteonecrosis of the Femoral Condyles.

OBJECTIVE: No studies currently exist with long-term follow-up of use of osteochondral allografting (OCA) for treatment of steroid-associated osteonecrosis of femoral condyles in young, active patients who wish to avoid total knee arthroplasty (TKA). We evaluate the extent to which fresh osteochondral allografts can (1) prevent or postpone need for prosthetic arthroplasty and (2) maintain long-term clinically meaningful decrease in pain and improvement in function at mean 11-year follow-up. DESIGN: Twenty-five patients (33 knees) who underwent OCA transplantation for osteonecrosis of the knee between 1984 and 2013 were evaluated, including 22 females and 11 males with average age of 25 years (range, 16-48 years). Mean total allograft surface area was 10.6 cm2 (range, 4.0-19.0 cm2). Evaluation included International Knee Documentation Committee (IKDC) scores, Knee Society function (KS-F) score, and modified (for the knee) Merle d'Aubigné-Postel (18-point) score. RESULTS: OCA survivorship was 90% at 5 years and 82% at 10 years. Twenty-eight of 33 knees (85%) avoided arthroplasty and 25 of 33 knees (73%) avoided other surgical intervention. Mean IKDC pain score improved (P = 0.001) from 7.2 preoperatively to 2.8 at latest follow-up, mean IKDC function score increased (P = 0.005) from 3.3 to 6.5, and mean IKDC total score improved (P = 0.001) from 31.9 to 61.1. Mean KS-F score improved (P = 0.003) from 61.7 to 87.5. Mean modified Merle d'Aubigné-Postel (18-point) score improved (P < 0.001) from 11.4 to 15.1. CONCLUSIONS: Our findings suggest that OCA transplantation is a reasonable surgical treatment option for steroid-associated osteonecrosis of the femoral condyles, with durable long-term outcomes.

A tree shrew model for steroid-associated osteonecrosis.

Osteonecrosis is a common human disease in orthopedics. It is difficult to treat, and half of patients may need artificial joint replacement, resulting in a considerable economic burden and a reduction in quality of life. Hormones are one of the major causes of osteonecrosis and high doses of corticosteroids are considered the most dangerous factor. Because of the complexity of treatment, we still need a better animal model that can be widely used in drug development and testing. Tree shrews are more closely related to primates than rodents. As such, we constructed a successful tree shrew model to establish and evaluate steroid-associated osteonecrosis (SAON). We found that low-dose lipopolysaccharide (LPS) combined with high-dose methylprednisolone (MPS) over 12 weeks could be used to establish a tree shrew model with femoral head necrosis. Serum biochemical and histological analyses showed that an ideal model was obtained. Thus, this work provides a useful animal model for the study of SAON and for the optimization of treatment methods.

Assessment of proliferation, migration and differentiation potentials of bone marrow mesenchymal stem cells labeling with silica-coated and amine-modified superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles have been widely used for cell labeling in preclinical and clinical studies, to improve labeling efficiency, particle conjugation and surface modifications are developed, but some modified SPIONs exert side-effect on physiological activity of cells, which cannot be served as ideal cell tracker. In this study, amine-modified silica-coated SPIO (SPIO@SiO2-NH2, SPIO@S-N) nanoparticles were used to label bone marrow derived mesenchymal stem cells (BM-MSCs), then the stem cell potentials were evaluated. It was found BM-MSCs could be efficiently labeled by SPIO@S-N nanoparticles. After labeling, the BM-MSCs viability kept well and the migration ability increased, but the osteogenesis and adipogenesis potentials were not impaired. In steroid associated osteonecrosis (SAON) bone defect model, stem cell implantation was performed by injection of SPIO@S-N labeled BM-MSCs into marrow cavity locally, it was found the SPIO positive cells homed to the periphery of defect region in control group, but were recruited to the defect region in poly lactic-coglycolic acid/tricalcium phosphate (PLGA/TCP) scaffold implantation group. In conclusion, SPIO@S-N nanoparticles promoted migration while retained proliferation and differentiation ability of BM-MSCs, implying this kind of nanoparticles could be served not only an ideal tracking marker but also an accelerator for stem cell homing during tissue repair.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis.

BACKGROUND: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. OBJECTIVE: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. METHOD: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. RESULTS: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased ß-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. CONCLUSION: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

Osteogenic magnesium incorporated into PLGA/TCP porous scaffold by 3D printing for repairing challenging bone defect.

Bone defect repair is a challenging clinical problem in musculoskeletal system, especially in orthopaedic disorders such as steroid associated osteonecrosis (SAON). Magnesium (Mg) as a biodegradable metal with properly mechanical properties has been investigating for a long history. In this study, Mg powder, poly (lactide-co-glycolide) (PLGA), ß-tricalcium phosphate (ß-TCP) were the elements to formulate a novel porous PLGA/TCP/Mg (PTM) scaffolds using low temperature rapid prototyping (LT-RP) technology. The physical characterization of PTM scaffold and Mg ions release were analyzed in vitro. The osteogenic and angiogenic properties of PTM scaffolds, as well as the biosafety after implantation were assessed in an established SAON rabbit model. Our results showed that the PTM scaffold possessed well-designed bio-mimic structure and improved mechanical properties. Findings of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and micro-computed tomography (micro CT)-based angiography indicated that PTM scaffold could increase blood perfusion and promote new vessel ingrowth at 4 weeks after surgery, meanwhile, a plenty of newly formed vessels with well-architective structure were observed at 8 weeks. Correspondingly, at 12 weeks after surgery, micro-CT, histological and mechanical properties analysis showed that PTM could significant enhance new bone formation and strengthen newly formed bone mechanical properties. The mean bone volume in PTM group was 56.3% greater than that in PT group. Biosafety assessments from 0 to 12 weeks after implantation did not induce increase in serum Mg ions concentration, and immune response, liver and kidney function parameters were all at normal level. These findings suggested that the PTM scaffold had both osteogenic and angiogenic abilities which were synergistic effect in enhancing new bone formation and strengthen newly formed bone quality in SAON. In summary, PTM scaffolds are promising composite biomaterials for repairing challenging bone defect that would have great potential for its clinical translation.

Porous composite scaffold incorporating osteogenic phytomolecule icariin for promoting skeletal regeneration in challenging osteonecrotic bone in rabbits.

Steroid-associated osteonecrosis (SAON) often requires surgical core decompression (CD) in the early stage for removal of necrotic bone to facilitate repair where bone grafts are needed for filling bone defect and avoiding subsequent joint collapse. In this study, we developed a bioactive composite scaffold incorporated with icariin, a unique phytomolecule that can provide structural and mechanical support and facilitate bone regeneration to fill into bone defects after surgical CD in established SAON rabbit model. An innovative low-temperature 3D printing technology was used to fabricate the poly (lactic-co-glycolic acid)/ß-calcium phosphate/icariin (PLGA/TCP/Icariin, PTI) scaffold. The cytocompatibility of the PTI scaffold was tested in vitro, and the osteogenesis properties of PTI scaffolds were assessed in vivo in the SAON rabbit models. Our results showed that the fabricated PTI scaffold had a well-designed biomimic structure that was precisely printed to provide increased mechanical support and stable icariin release from the scaffold for bone regeneration. Furthermore, our in vivo study indicated that the PTI scaffold could enhanced the mechanical properties of new bone tissues and improved angiogenesis within the implanted region in SAON rabbit model than those of PLGA/TCP (PT) scaffold. The underlying osteoblastic mechanism was investigated using MC3T3-E1 cells in vitro and revealed that icariin could facilitate MC3T3-E1 cells ingrowth into the PTI scaffold and regulate osteoblastic differentiation. The PTI scaffold exhibited superior biodegradability, biocompatibility, and osteogenic capability compared with those of PT scaffold. In summary, the PTI composite scaffold which incorporated bioactive phyto-compounds is a promising potential strategy for bone tissue engineering and regeneration in patients with challenging SAON.

Phytomolecule icaritin incorporated PLGA/TCP scaffold for steroid-associated osteonecrosis: Proof-of-concept for prevention of hip joint collapse in bipedal emus and mechanistic study in quadrupedal rabbits.

Steroid-associated osteonecrosis (SAON) may lead to joint collapse and subsequent joint replacement. Poly lactic-co-glycolic acid/tricalcium phosphate (P/T) scaffold providing sustained release of icaritin (a metabolite of Epimedium-derived flavonoids) was investigated as a bone defect filler after surgical core-decompression (CD) to prevent femoral head collapse in a bipedal SAON animal model using emu (a large flightless bird). The underlying mechanism on SAON was evaluated using a well-established quadrupedal rabbit model. Fifteen emus were established with SAON, and CD was performed along the femoral neck for the efficacy study. In this CD bone defect, a P/T scaffold with icaritin (P/T/I group) or without icaritin (P/T group) was implanted while no scaffold implantation was used as a control. For the mechanistic study in rabbits, the effects of icaritin and composite scaffolds on bone mesenchymal stem cells (BMSCs) recruitment, osteogenesis, and anti-adipogenesis were evaluated. Our efficacy study showed that P/T/I group had the significantly lowest incidence of femoral head collapse, better preserved cartilage and mechanical properties supported by more new bone formation within the bone tunnel. For the mechanistic study, our in vitro tests suggested that icaritin enhanced the expression of osteogenesis related genes COL1α, osteocalcin, RUNX2, and BMP-2 while inhibited adipogenesis related genes C/EBP-ß, PPAR-γ, and aP2 of rabbit BMSCs. Both P/T and P/T/I scaffolds were demonstrated to recruit BMSCs both in vitro and in vivo but a higher expression of migration related gene VCAM1 was only found in P/T/I group in vitro. In conclusion, both efficacy and mechanistic studies show the potential of a bioactive composite porous P/T scaffold incorporating icaritin to enhance bone defect repair after surgical CD and prevent femoral head collapse in a bipedal SAON emu model.

Pathomorphological changes of bone marrow adipocytes in process of steroid-associated osteonecrosis.

OBJECTIVE: The role of extravascular fat deposition in pathogenesis of steroid-associated osteonecrosis (ON) still remains unclear. This study aimed to explore the pathomorphological changes of bone marrow adipocytes over time in a rabbit ON model. METHODS: Thirty-two adult rabbits were divided into control group (n=16) and steroid group (n=16). Rabbits in the steroid group were injected with venous lipopolysaccharide once and intramuscular methylprednisolone trice to induce ON. Rabbits in the control group were treated with normal saline of equal volume. 2 weeks (early stage; n=8) and 4 weeks (late stage; n=8) after the last steroid injection, animals were sacrificed, and bilateral femora were harvested. The density, diameter and area of bone marrow adipocytes were determined by histomorphometry, and ON was evaluated histopathologically. RESULTS: The adipocyte density in steroid group increased by 67.1% and 54.4% at week 2 and week 4, respectively, when compared with control group, but there was no significant difference between week 2 and week 4. The adipocyte diameter in the steroid group at week 4 was significantly larger than that in the control group, but the adipocyte diameter in the steroid group at week 2 was slightly smaller than that in the control group. The adipocyte area in the steroid group increased by 44% and 83.4% at week 2 and week 4, respectively, when compared with the control group, and the adipocyte area in the steroid group at week 4 was markedly larger than that at week 2. In the control group, there were a largest number of adipocytes with 40-50 µm in diameter. When compared with the control group, most of increased adipocytes in the steroid group at week 2 were 30-40 µm in diameter, and those at week 4 were 50-60 µm in diameter. In the steroid group, histopathological examination showed ON was found in 25% (2/8) of rabbits at week 2 and 87.5% (7/8) of rabbits at week 4. CONCLUSION: In the process of ON, extravascular fat deposition is characterized by increased small adipocytes at the early stage and hypertrophy of adipocytes at the late stage.

Aberrant CpG islands' hypermethylation of ABCB1 in mesenchymal stem cells of patients with steroid-associated osteonecrosis.

OBJECTIVE: Patients carrying an ABCB1 polymorphism have a higher risk of developing osteonecrosis of the femoral head (ONFH). We investigated whether aberrant dinucleotide CpG islands' hypermethylation of ABCB1 gene existed in mesenchymal stem cells (MSC) of patients with ONFH, which results in cell dysfunction. METHODS: Bone marrow was collected from the proximal femur of patients with glucocorticoid (GC)-associated ONFH (n = 22) and patients with new femoral neck fractures (n = 25). MSC were isolated by density gradient centrifugation. We investigated cell viability, intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), the amount of P-glycoprotein (P-gp) and ABCB1 transcripts, and methylation at CpG islands of ABCB1 promoter from both the femoral neck fractures group and the GC-associated ONFH group treated with or without the DNA methyltransferase inhibitor, 5'-Aza-2-deoxycytidine (5'-Aza-dC). RESULTS: We observed that MSC from GC-associated ONFH groups showed reduced proliferation ability, elevated ROS levels, and depressed MMP when compared with the other 2 groups. Low levels of P-gp and ABCB1 transcript, as well as ABCB1 gene hypermethylation, in patients with GC-associated ONFH were also noted. Treatment with 5'-Aza-dC rapidly restored ABCB1 expression. Analysis of general expression revealed that aberrant CpG islands' hypermethylation of ABCB1 caused sensitivity to GC and induced changes in the proliferation and oxidative stress of MSC under GC administration. CONCLUSION: These data suggest that aberrant CpG islands' hypermethylation of ABCB1 gene may be responsible for individual differences in the development of GC-associated ONFH.

Therapeutic effect of gastrin on steroid-associated osteonecrosis in rats / 上海交通大学学报（医学版）

Objective: To investigate therapeutic effect of gastrin on steroid-associated osteonecrosis (SAON) in rat model. Methods: Twenty-four SD rats were randomly divided into three groups i.e. normal control group (normal group), SAON control group (SAON group) and SAON treatment group (treatment group). SAON group and treatment group were intravenously injected with lipopolysaccharide 1 time per day (600 μg/kg) for 2 d and meanwhile intramuscularly injected with methylprednisolone 1 time per day (50 mg/kg) for 3 d. Normal group was injected with normal saline of the same volumns. After steroid injections, treatment group was injected with gastrin 1 time per day (800 μg/kg) for 14 d, while SAON group was injected with normal saline of the same volumns. After the treatment, bone trabeculas below femoral head growth plate were dissected in the rats for bone histology. Hematoxylin-eosin (H-E) staining, immunohistochemistry, fluorescence staining and Goldner's trichrome staining were applied in this study. Results: SAON model in rats was successfully established. The result of H-E staining showed that compared with SAON group, thrombus area, number and area of fat cells in the bone marrows of treatment group obviously decreased (all P<0.05). Immunohistochemistry showed that osteogenic transcription factor (Sp7) positive cells in treatment group were more than those in SAON group (P<0.01). Compared with SAON group, osteoid length and area (Goldner's trichrome staining), and bone formation rate and bone mineralization deposition rate (fluorescence staining) all significantly increased in treatment group (all P<0.01). Conclusion: Gastrin can effectively treat SAON in rats by suppressing thrombus and lipid formation and enhancing boneformation.

Therapeutic effect of gastrin on steroid-associated osteonecrosis in rats / 上海交通大学学报（医学版）

Objective·To investigate therapeutic effect of gastrin on steroid-associated osteonecrosis (SAON) in rat model. Methods·Twenty-four SD rats were randomly divided into three groups i.e. normal control group (normal group), SAON control group (SAON group) and SAON treatment group (treatment group). SAON group and treatment group were intravenously injected with lipopolysaccharide 1 time per day (600 μg/kg) for 2 d and meanwhile intramuscularly injected with methylprednisolone 1 time per day (50 mg/kg) for 3 d. Normal group was injected with normal saline of the same volumns. After steroid injections, treatment group was injected with gastrin 1 time per day (800 μg/kg) for 14 d, while SAON group was injected with normal saline of the same volumns. After the treatment, bone trabeculas below femoral head growth plate were dissected in the rats for bone histology. Hematoxylin-eosin (H-E) staining, immunohistochemistry, fluorescence staining and Goldner's trichrome staining were applied in this study. Results·SAON model in rats was successfully established. The result of H-E staining showed that compared with SAON group, thrombus area, number and area of fat cells in the bone marrows of treatment group obviously decreased (all P<0.05). Immunohistochemistry showed that osteogenic transcription factor (Sp7) positive cells in treatment group were more than those in SAON group (P<0.01). Compared with SAON group, osteoid length and area (Goldner′s trichrome staining), and bone formation rate and bone mineralization deposition rate (fluorescence staining) all significantly increased in treatment group (all P<0.01). Conclusion·Gastrin can effectively treat SAON in rats by suppressing thrombus and lipid formation and enhancing bone-formation.