Steroid-induced osteonecrosis of femoral head (SONFH) is a disease characterized by femoral head collapse and local pain caused by excessive use of glucocorticoids. Peroxisome proliferator-activated receptor-γ (PPARγ) is mainly expressed in adipose tissue. Wnt/β-catenin, AMPK and other related signaling pathways play an important role in regulating adipocyte differentiation, fatty acid uptake and storage. Bone marrow mesenchymal cells (BMSCs) have the ability to differentiate into adipocytes or osteoblasts, and the use of hormones upregulates PPARγ expression, resulting in BMSCs biased towards adipogenic differentiation. The increase of adipocytes affects the blood supply and metabolism of the femoral head, and the decrease of osteoblasts leads to the loss of trabecular bone, which eventually leads to partial or total ischemic necrosis and collapse of the femoral head. MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression by inhibiting the transcription or translation of target genes, thereby affecting cell function and disease progression. Studies have shown that miRNAs affect the progression of SONFH by regulating PPARγ lipid metabolism-related signaling pathways. Therefore, it may be an accurate and feasible SONFH treatment strategy to regulate adipogenic-osteoblast differentiation in BMSCs by targeted intervention of miRNA differential expression to improve lipid metabolism. In this paper, the miRNA-mediated PPARγ-related signaling pathways were classified and summarized to clarify their effects on lipid metabolism in SONFH, providing a theoretical reference for miRNA targeted therapy of SONFH, and then providing scientific evidence for SONFH precision medicine.
MicroRNAs/physiology* ; PPAR gamma/metabolism* ; Femur Head Necrosis/metabolism* ; Humans ; Signal Transduction/physiology* ; Lipid Metabolism/physiology* ; Animals ; Cell Differentiation ; Mesenchymal Stem Cells/cytology* ; Glucocorticoids/adverse effects*

OBJECTIVE: To investigate the biocompatibility of porous WE43 magnesium alloy scaffolds manufactured by 3D printing technology and to observe its effect in treating femoral defects in New Zealand white rabbits. METHODS: In vitro cytotoxicity test was performed using bone marrow mesenchymal stem cells from Sprague Dawley (S-D) rats. According to the different culture media, the cells were divided into 100% extract group, 50% extract group, 10% extract group and control group. After culturing for 1, 3 and 7 days, the cell activity of each group was determined by cell counting kit-8 (CCK-8). In the in vivo experiment, 3.0-3.5 kg New Zealand white rabbits were randomly divided into three groups: Experimental group, bone cement group and blank group, with 9 rabbits in each group. Each rabbit underwent surgery on the left lateral femoral condyle, and a bone defect with a diameter of 5 mm and a depth of 6 mm was created using a bone drill. The experimental group was implanted with WE43 magnesium alloy scaffolds, the bone cement group was implanted with calcium sulfate bone cement, and the blank group was not implanted. Then 4, 8 and 12 weeks after surgery, 3 rabbits in each group were euthanized by carbon dioxide anesthesia, and the femur and important internal organs were sampled. Micro-computed tomography (Micro-CT) scanning was performed on the left lateral femoral condyle. Sections of important internal organs were prepared and stained with hematoxylin-eosin (HE). Hard tissue sections were made from the left lateral femoral condyle and stained with methylene blue acid fuchsin and observed under a microscope. RESULTS: In the cytotoxicity test, the cell survival rate in the 100% extract group was higher than that in the control group (140.56% vs. 100.00%, P < 0.05) on 1 day of culture; there was no statistically significant difference (P>0.05) in cell survival rate among the groups on 3 days of culture; the cell survival rate in the 100% extract group was lower than that in the control group (68.64% vs. 100.00%, P < 0.05) on 7 days of culture. Micro-CT scanning in the in vivo experiment found that most of the scaffolds in the experimental group had been degraded in 4 weeks, with very few high-density scaffolds remaining. In 12 weeks, there was no obvious stent outline. In 4 weeks, a certain amount of gas was generated around the WE43 magnesium alloy scaffold, and the gas was significantly reduced from 8 to 12 weeks. Hard tissue sections showed that a certain amount of extracellular matrix and osteoid were generated around the scaffolds in the experimental group in 4 weeks. In the bone cement group, most of the calcium sulfate bone cement had been degraded. In 8 weeks, the osteoid around the scaffold and its degradation products in the experimental group increased significantly. In 12 weeks, new bone was in contact with the scaffold around the scaffold in the experimental group. There was less new bone in the bone cement group and the blank group. CONCLUSION The porous WE43 magnesium alloy scaffold fabricated by 3D printing process has good biocompatibility and good osteogenic properties, and has the potential to become a new material for repairing bone defects.
Animals ; Rabbits ; Printing, Three-Dimensional ; Alloys/chemistry* ; Tissue Scaffolds/chemistry* ; Magnesium/chemistry* ; Rats, Sprague-Dawley ; Biocompatible Materials ; Mesenchymal Stem Cells/cytology* ; Femur/surgery* ; Rats ; Absorbable Implants ; Male ; Bone Regeneration ; Tissue Engineering/methods* ; Cells, Cultured

OBJECTIVE: To study the effect of electro- acupuncture at Zusanli acupoint in regulating perioperative cell immune functions in rats. METHODS: Forty-two SD rats were divided into blank control group (=6), model group (=18), and electroacupuncture group (=18). The rats in the latter two groups underwent thigh incision and femoral dissection under anesthesia; the rats in electro-acupuncture group received electro-acupuncture at bilateral Zusanli acupoint for 15 min before anesthesia and 1 h after the surgery. The rats in the model group and electro-acupuncture group were sacrificed at 6 h, 24 h, and 72 h after the operation and blood samples were taken from the ventricle for analyzing CD3, CD4, and CD8 T cell subpopulations and calculation of CD4/CD8 using flow cytometry. ELISA was used to detect the levels of interleukin-1 (IL-1) and IL-6. RESULTS: The CD3 T cell subpopulation was significantly lower in the model group and electro-acupuncture group than in the blank group at 6 h and 24 h after the operation. At 72 h after the operation, CD3 subpopulation levels still remained low in the model group, but recovered the control level in electro-acupuncture group. At each time point of measurement, CD3 level was significantly lower in the model group than in the electro-acupuncture group. CD4 level in the model group was significantly lowered at 6 h and 24 h after the operation, and recovered the control level at 72 h. In the electro-acupuncture group, CD4 level was significantly lowered at 6 h after the operation, but recovered the control level at 24 h. At 24 h and 72 h, the levels of CD4 were significantly lower in the model group than in the electro-acupuncture group. CD8 level underwent no significant changes after the operation in either the model group or electro-acupuncture group. CD4/CD8 was significantly lowered at 24 h and 72 h after the operation in the model group but showed no significant variation in the electro-acupuncture group. Compared with that in the control group, IL-1 level was significantly lowered in both the model group and electroacupuncture group at 6 h, 24 h, and 72 h after the operation, and was significantly lower in the model group than in the electroacupuncture group at these time points. IL-6 level increased significantly in the model group and the electro- acupuncture group at 6 h and 24 h. at 72 h, IL-6 level was obviously lowered in the electro-acupuncture group but remained elevated in the model group. CONCLUSIONS Electro-acupuncture alleviates postoperative immune suppression and promotes recovery of the immune function in rats, suggesting a protective effect of electro-acupuncture at Zusanli acupoint on cellular immune function after surgery.
Acupuncture Points ; Animals ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Electroacupuncture ; methods ; Femur ; surgery ; Flow Cytometry ; Humans ; Immunity, Cellular ; Perioperative Period ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; cytology ; immunology

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.
Animals ; Blood Cells/*cytology ; Bone Morphogenetic Protein 2/genetics ; *Cell- and Tissue-Based Therapy ; Femur Head Necrosis/metabolism/*pathology/*therapy ; Gene Expression Regulation ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Osteonecrosis/*pathology/*therapy ; PPAR gamma/genetics ; Rabbits ; Transplantation, Homologous

To investigate the effects of Danshensu on bone formation in ovariectomized rats.Thirty female SD rats were randomly divided into three groups with 10 rats in each:blank control group, model control group and Danshensu group. The osteoporosis model was induced by bilateral ovariectomy and rats in Danshensu group were fed with Danshensu 12.5 mg·kg·dby gavage after ostroporosis model induced. All animals were sacrificed after 90 days. The bone mineral density (BMD) of the whole body, femur and lumbar vertebra was measured by dual energy X-ray absorptiometry. The biomechanical properties of femur were measured by AG-IS mechanical universal testing machine. Serum osteocalcin and bone alkaline phosphates (BALP) levels were measured by ELISA. The number of osteoblasts of proximal femoral metaphysis was counted with light microscopy after HE staining.Compared with blank control group, BMD, biomechanical properties of femur, serum osteocalcin and BALP levels and the number of osteoblasts were decreased in model control group (<0.05 or<0.01). While compared with model control group, BMDs of the whole body, femur and lumbar vertebra, the elastic modulus, maximum load, yield strength, breaking point load of femur, the serum levels of osteocalcin and BALP, and the number of osteoblasts were significantly improved in Danshensu group (<0.05 or<0.01).Danshensu can improve bone quality by increasing bone density, improving biomechanical properties, promoting the expression of osteogenesis-related factors, and increasing the number of osteoblasts.
Alkaline Phosphatase ; blood ; drug effects ; Animals ; Biomechanical Phenomena ; drug effects ; Bone Density ; drug effects ; Cell Count ; Female ; Femur ; anatomy & histology ; cytology ; drug effects ; Lactates ; pharmacology ; Lumbar Vertebrae ; anatomy & histology ; drug effects ; Osteoblasts ; drug effects ; Osteocalcin ; blood ; drug effects ; Osteogenesis ; drug effects ; Osteoporosis ; drug therapy ; Ovariectomy ; Rats ; Rats, Sprague-Dawley

BACKGROUNDAppropriate expression and regulation of the transcriptome, which mainly comprise of mRNAs and lncRNAs, are important for all biological and cellular processes including the physiological activities of bone microvascular endothelial cells (BMECs). Through an intricate intracellular signaling systems, the transcriptome regulates the pharmacological response of the cells. Although studies have elucidated the impact of glucocorticoids (GCs) cell-specific gene expression signatures, it remains necessary to comprehensively characterize the impact of lncRNAs to transcriptional changes.

METHODSBMECs were divided into two groups. One was treated with GCs and the other left untreated as a paired control. Differential expression was analyzed with GeneSpring software V12.0 (Agilent, Santa Clara, CA, USA) and hierarchical clustering was conducted using Cluster 3.0 software. The Gene Ontology (GO) analysis was performed with Molecular Annotation System provided by CapitalBio Corporation.

RESULTSOur results highlight the involvement of genes implicated in development, differentiation and apoptosis following GC stimulation. Elucidation of differential gene expression emphasizes the importance of regulatory gene networks induced by GCs. We identified 73 up-regulated and 166 down-regulated long noncoding RNAs, the expression of 107 of which significantly correlated with 172 mRNAs induced by hydrocortisone.

CONCLUSIONSTranscriptome analysis of BMECs from human samples was performed to identify specific gene networks induced by GCs. Our results identified complex RNA crosstalk underlying the pathogenesis of steroid-induced necrosis of femoral head.

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Femur Head ; cytology ; Gene Expression Profiling ; Glucocorticoids ; pharmacology ; Humans ; Oligonucleotide Array Sequence Analysis ; Osteonecrosis ; genetics ; RNA, Messenger ; genetics ; RNA, Untranslated ; genetics ; Transcriptome ; drug effects ; genetics

OBJCETIVETo investigate the method of separation of culture of bone microvascular endothelial cells (BMECs) of human femoral head in vitro.

METHODSFrom October 2013 to January 2014,15 femoral heads without pathologic change from patients resected during hip replacement were selected involving 2 males and 13 females with a mean age of 71.2 years old ranging from 38 to 92. Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion. Cells were isolated by the methods of enzymic digestion and density gradient centrifugation,purified by differiential attachment. The characteristics of cells was observed by inverted microscope. vWF and CD31 immunofluorescence analysis was applied for identification of cells.

RESULTSThe number of cells was positively correlated with patients' age after 24 hours in primary culture. The older patients had the less cells numbered. After 4 to 5 days' culture, primary cells appeared short spindle,polygon shaped and cobblestone-like morphology. After 7 to 10 days' culture, primary cells proliferated densely, became fusion, arranged in swirl, and contact inhibition appeared significantly. Immunofluorescence staining revealed the cells were 100% positive for vWF and CD31, and it showed that the cultured cells were BMECs.

CONCLUSIONIt was a simple, steady, effective method with good reproducibility, by which highly purified human BMECs can be obtained.

Adult ; Aged ; Aged, 80 and over ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Femur Head ; blood supply ; Humans ; Male ; Microvessels ; cytology ; Middle Aged

OBJECTIVETo observe the effect of Huogu II formula compatible with different channel ushering drugs on the homing of bone marrow stem cells of osteonecrosis of the femoral head (ONFH) induced by liquid nitrogen freezing in rabbits and discuss the mechanism for preventing and treating ONFH.

METHODThe ONFH model was established by liquid nitrogen freezing of 84 rabbits. They were randomly assigned to the model group and the Huogu II formula group and groups of Huogu II formula combining with Achyranthis Bidentatae Radix, Asari Radix et Rhizoma, Angelicae Pubescentis Radix, Platycodonis Radix. The remaining 14 rabbits were sham-operated. During the course of ONFH modeling, all of the rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor (rhG-CSF)(30 microg x kg(-1) x d(-1), for consecutively 7 days). Meanwhile, normal saline and decoction of the formulae were orally administrated respectively. WBC was counted in peripheral blood before and after the injection of rhG-CSF. HE stainings at the 2nd and the 4th weeks after the modeling were adopted to observe histopathological changes, vascular morphology was observed by ink perfusion, BrdU and SDF-1 were determined by immunohistochemical assay in femoral heads of the left hind leg.

RESULTCompared with the sham-operated group, the Huogu II formula group showed decrease in the ratio of empty lacuna and increase in vessel area, number of BrdU positive cells and SDF-1 level. In comparison with the model group, the Achyranthis Bidentatae Radix group displayed decreasing empty lacuna ratio and increasing vessel area at the 4th week and increasing SDF-1 at the 2nd week; the Platycodonis Radix group revealed a notably increasing empty lacuna ratio and a sharp decrease in the number of BrdU positive cells at 4th week; Asari Radix et Rhizoma and Angelicae Dubescentis Radix groups showed no remarkable change.

CONCLUSIONHuogu II formula can promote the directional homing of bone marrow stem cell to the necrosis area. Channel ushering drug achyranthes can further boost above effects of Huogu II formula.

Animals ; Bone Marrow Cells ; cytology ; Bromodeoxyuridine ; metabolism ; Chemokine CXCL12 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Femur Head Necrosis ; blood ; drug therapy ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Humans ; Leukocyte Count ; Male ; Rabbits ; Stem Cells ; cytology ; drug effects

OBJECTIVETo investigate the effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae (Ach) on bone marrow stem cells (BMSCs) homing to necrosis area after osteonecrosis of the femoral head (ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH.

METHODSThe animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg. Forty-eight Japanese White rabbits were randomly assigned to sham-operated group, model group, Huogu II group, and Huogu II plus Ach group, with 12 rabbits in each. During the course of ONFH animal model establishment, all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor [rhG-CSF, 30 μg/(kg·day) for continuous 7 days]. Meanwhile, normal saline and decoction of the two formulae were administrated by gavage, respectively. White blood cells (WBC) were counted in peripheral blood before and after injection of rhG-CSF. Materials were drawn on the 2nd and 4th weeks after model built; bone glutamine protein (BGP) and bone morphogenetic protein 2 (BMP2) levels in serum were tested. Histopathologic changes were observed by hematoxylin and eosin (HE) staining. BMP2 mRNA levels were detected with in situ hybridization (ISH) staining. 5-Bromo-2'-deoxyuridine (BrdU) and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg.

RESULTSCompared with the shamoperated group, the ratio of empty lacuna, serum BGP, and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly. However, in comparison with the model group, the empty lacuna ratio of Huogu II group and Huogu II plus Ach group decreased obviously in addition to the levels of serum BGP and BMP2, and the expressions of BMP2 mRNA, BrdU, and SDF-1 increased significantly. Above changes were particularly obvious in Huogu II plus Ach group. BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in Huogu II group significantly exceeded their counterparts. On the 2nd week, only in Huogu II plus Ach group that the BrdU counting rose significantly. On the 4th week, empty lacuna rate and serum BMP2 level in Huogu II plus Ach group exceeded those in Huogu II group distinctively.

CONCLUSIONSTo a certain extent, the medicinal guide Ach improves the preventive and therapeutic effects of Huogu II Formula on experimental ONFH model. The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.

Achyranthes ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Bone Morphogenetic Protein 2 ; blood ; genetics ; Bromodeoxyuridine ; metabolism ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Femur Head ; drug effects ; pathology ; Femur Head Necrosis ; blood ; genetics ; pathology ; therapy ; Gene Expression Regulation ; drug effects ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Humans ; Leukocyte Count ; Male ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Radioimmunoassay ; Stem Cell Transplantation ; Stem Cells ; cytology ; drug effects

OBJECTIVETo review the recent developments in the mechanisms of glucocorticoids induced osteonecrosis of femoral head (ONFH) and introduce a new theory of ONFH.

DATA SOURCESBoth Chinese- and English-language literatures were searched using MEDLINE (1997 - 2011), Pubmed (1997 - 2011) and the Index of Chinese-language Literature (1997 - 2011).

STUDY SELECTIONData from published articles about mechanisms of glucocorticoids induced ONFH in recent domestic and foreign literature were selected. Data extraction Data were mainly extracted from 61 articles which are listed in the reference section of this review.

RESULTSGlucocorticoids are steroid hormones secreted by the adrenal cortex that play a pivotal role in the regulation of a variety of developmental, metabolic and immune functions. However, high dose of exogenous glucocorticoids usage is the most common non-traumatic cause of ONFH. Glucocorticoids can affect the metabolisms of osteoblasts, osteoclasts, bone marrow stromal cells and adipocytes which decrease osteoblasts formation but increase adipocytes formation and cause ONFH finally.

CONCLUSIONSGlucocorticoids affect the differentiation of mesenchymal stem cells, through activating or inhibiting the related transcript regulators of osteogenesis and adipogenesis. At last, the size and volume of mesenchymal stem cells derived adipocytes will increase amazingly, but the osteoblasts will be decreased obviously. In the meantime, the activity of the osteoclasts will be activated. So, these mechanisms work together and lead to ONFH.

Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Femur Head ; cytology ; Glucocorticoids ; metabolism ; pharmacology ; Humans ; Osteonecrosis ; etiology ; metabolism ; Stromal Cells ; drug effects ; metabolism