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

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

Hoffa's fracture in a five-year-old child diagnosed and treated with the assistance of arthroscopy: A case report.

BACKGROUND: Hoffa's fracture is a coronal-oriented fracture of the femoral condyle. It is rarely observed in pediatric patients that isolated coronal fracture of the medial femoral condyle accompanies an intact lateral femoral condyle. Only a few cases involving Hoffa's fracture of the medial femoral condyle have been reported in patients with undeveloped skeletons. Such a fracture cannot be observed by routine imaging examinations, thus resulting in possible misdiagnosis and further treatment challenges. CASE SUMMARY: A 5-year-old boy with Hoffa's fracture of the medial femoral condyle suffered from right knee pain and severe swelling after being hit by a heavy object. The patient was misdiagnosed and initially treated in a local primary healthcare center. No improvement in his right knee's extension was observed following conservative treatment for 2 wk. The patient was transferred to our hospital, re-diagnosed using arthroscopy, and underwent open reduction and internal fixation. The therapeutic outcome was satisfactory with the screws removed 7 mo after fixation. At the final follow-up of 40 mo, the range of motion in the knee had recovered. There was no varus-valgus instability. CONCLUSION: Hoffa's fracture is rarely seen in children aged 5 years, let alone in the medial condyle, and can easily be misdiagnosed due to limited physical and imaging examinations. Suspected Hoffa's fracture in preschool children should be confirmed based on arthroscopic findings. Open reduction and internal fixation should be performed to protect the articular surface and prevent long-term complications.

Percutaneous endoscopic transforaminal discectomy for the treatment of L5-S1 lumbar disc herniation and the influence of iliac crest height on its clinical effects.

The present study aimed to explore the clinical effects of percutaneous endoscopic transforaminal discectomy using a transforaminal endoscopic spine system (TESSYS) technique for the treatment of L5-S1 lumbar disc herniation and to analyse the influence of iliac crest height on these clinical effects. The clinical data of 76 patients with L5-S1 single-segment disc herniation treated with TESSYS at The Second Affiliated Hospital and Third Affiliated Hospital of Xi'an Jiaotong University between January and December 2016 were retrospectively analysed. Patients were divided into the following three groups according to the positional relation between the highest point of the iliac crest and the L4 and L5 pedicles in the lateral lumbar, as determined by X-ray: Group I, iliac crest height below the upper edge horizontal line of the L5 pedicle (n=42); group II, iliac crest height between the lower edge horizontal line of the L4 pedicle and the upper edge horizontal line of the L5 pedicle (n=29) and group III, iliac crest height above the lower edge horizontal line of the L4 pedicle (n=5). Changes in the postoperative visual analogue scale (VAS) pain score and Oswestry disability index (ODI) of the lower back and lower limbs were observed, and the effects were compared among the three groups. The mean operating time was 86.5±13.5 min. A single patient experienced cerebrospinal fluid leakage due to a mild tear of the dura mater during the operation, which improved after symptomatic treatment. The same operation was repeated in one patient due to the recurrence of disc herniation. In all patients, the VAS pain score and ODI of the lower back and lower limbs at 1 week and 1, 3 and 12 months following the operation were significantly lower than those before the operation (all P<0.05). Furthermore, the postoperative VAS pain score and ODI of the lower back and lower limbs were poorer in group III (L5-S1 lumbar disc herniation complicated with high iliac crest) than in groups I and II (P<0.05). These results suggested that TESSYS was effective in treating lumbar disc herniation. Whether the iliac crest is higher than the lower edge horizontal line of the L4 pedicle is suggested to be one of the factors influencing the outcome of the operation.

Resveratrol prevents steroid-induced osteonecrosis of the femoral head via miR-146a modulation.

The purpose of this study was to investigate the possible use of resveratrol (Res) to reverse abnormal osteogenesis/osteoclastogenesis activity that occurs during femoral head osteonecrosis and to explore the detailed mechanisms. Application of Res to bone marrow-derived mesenchymal stem cells in vitro promoted survival, inhibited apoptosis, and downregulated expression of reactive oxygen species expression. Moreover, Res application was associated with elevated microRNA-146a (miR-146a) expression, osteogenic differentiation, and suppressed osteoclastic differentiation, which were markedly reversed by miR-146a inhibitor. Histopathological observations and micro-computed tomography scanning results indicated that the Res-treated group had lower incidence of osteonecrosis and better bone microstructure than the untreated group. Res inhibited osteoclastogenesis through altering the levels of sirtuin1 (Sirt1), nuclear transcription factor-κB (NF-κB), and receptor activator of NF-κB ligand (RANKL). Simultaneously, Res treatment improved bone formation and increased ß-catenin and runt-related transcription factor 2 (Runt2) expression levels, while reducing forkhead box class O (FOXO) family protein levels. The results of our study suggest that Res prevents steroid-induced osteonecrosis by upregulating miR-146a, and thereby stabilizes osteogenesis/osteoclastogenesis homeostasis via Wnt/FOXO and Sirt1/NF-κB pathways.

Panax notoginseng saponins protect rabbit bone marrow stromal cells from hydrogen peroxide-induced apoptosis.

OBJECTIVE: To investigate the effects of Panax notoginseng saponins (PNSs) on hydrogen peroxide-induced apoptosis in rabbit bone marrow stromal cells (BMSCs). METHODS: BMSCs were isolated from 2-month-old New Zealand rabbits and cultured with different doses of PNSs to determine the most effective dose of PNSs by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assay. The most effective dose of PNSs was used in subsequent experiments. Apoptosis of BMSCs was induced by hydrogen peroxide (100 micromol/L). BMSCs in PNSs group were also pretreated with PNSs before hydrogen peroxide exposure. Reactive oxygen species (ROSs) levels were measured by using 2',7'-dichlorodihydrofluorescein diacetate. Apoptosis rate of BMSCs was observed by flow cytometry after staining with Annexin V-fluorescein isothiocyanate/propidium iodide. The protein expression of Bax in BMSCs was analyzed by Western blotting. Activity of caspase-3 was measured by spectrofluorometry. RESULTS: The most effective dose of PNSs was 0.1g/L. PNSs at dose of 0.1g/L markedly reversed the augmentation of ROS level, decreased the apoptosis rate of, and the Bax expression and activity of caspase-3 in BMSCs treated with hydrogen peroxide (P<0.01). CONCLUSION: PNSs can protect cultured rabbit BMSCs from hydrogen peroxide-induced apoptosis by decreasing oxidative stress, Bax expression and caspase-3 activity.

[Total arthroscopic internal drainage technique for the treatment of popliteal cyst].

OBJECTIVE: To compare the clinical effects of total arthroscopic internal drainage and arthroscopic combined with posterior small incision in the treatment of popliteal cyst. METHODS: From January 2015 to January 2017, 60 patients with popliteal cyst were treated, including 29 males and 31 females, aged 30 to 65(47.8±2.5) years old, with a course of disease (8.5±4.2) months. Among them, 30 cases received total arthroscopic internal drainage for popliteal fossa cyst(total arthroscopic group), 30 cases received arthroscopic combined with posterior small incision for popliteal fossa cyst(arthroscopic combined with small incision group). The operation time, intraoperative bleeding volume, incision length, Rauschning and Lindgren grade 0 recovery rate and Lysholm score were compared between the two groups. RESULTS: Twenty-nine patients in total arthroscopy group were followed up, and 28 patients in arthroscopy combined with small incision group were followed up for 8 to 20(12.8±2.1) months. Operation time: total arthroscopic group(45.32±5.71) min, arthroscopic combined small incision group (44.56±3.85) min; Rauschning and Lindgren grade 0 recovery: 23 cases in total arthroscopic group, 22 cases in arthroscopic combined small incision group; postoperative Lysholm score: total arthroscopic group 84.5±11.2, arthroscopic combined small incision group 83.2±12.7; there was no significant difference between the two groups(P>0.05). Intraoperative bleeding volume: total arthroscopic group(5.32±1.25) ml, arthroscopic combined small incision group(20.75±8.18) ml; incision length: total arthroscopic group (1.51±0.34) cm, arthroscopic combined small incision group (7.34±0.75) cm; the difference between the two groups was significant(P<0.05). At the last follow-up, the knee joint was examined by magnetic resonance imaging, and no recurrence of cyst was found. CONCLUSIONS: Total arthroscopic internal drainage and arthroscopic combined with posterior small incision technique for popliteal fossa cyst with intra-articular lesions have the same clinical effect, but less trauma and faster recovery.

[Effects of different doses of puerarin on vascular endothelial cell proliferation in vitro].

OBJECTIVE: To explore the effects of different doses of puerarin on proliferation of cultured vascular endothelial cells in vitro. METHODS: The hearts of three new-born SD rats 5 days old were mechanically minced and enzymatically digested with collagenase and trypsin, then vascular endothelial cells were counted, washed and resuspended in Dulbecco's minimum essential medium (DMEM) added with 20% heat inactivated fetal calf serum, then inoculated in 2% gelatin-coated tissue culture flasks. Vascular endothelial cells at passage 3 were used in the experiment. Except for the normal control group, the vascular endothelial cells were cultured with puerarin in various concentrations (0.01, 0.1, 1, 10 and 100 micromol/L) for 24 hours, and the morphology and the number of the cultured endothelial cells were observed. Methyl thiazolyl tetrazolium (MTT) colorimetry was used to determine the proliferation of the cultured vascular endothelial cells. Flow cytometry (FCM) were used to detect the proliferation index (PI) of the vascular endothelial cells and the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method. RESULTS: The cell morphology was normal under the inverted microscope in each group. The number and proliferation activities of vascular endothelial cells were significantly increased by 1, 10 and 100 micromol/L puerarin compared with those of the blank control group, especially the 100 micromol/L puerarin group, and there were no remarkable changes in with 0.1 micromol/L and 0.01 micromol/L puerarin groups. The same results were seen in the positive rate of PCNA expression and PI. CONCLUSION: Puerarin has dose-dependent effects on the proliferation activity of vascular endothelial cells.

[Arnebia root oil promotes histological change and up-regulates bFGF and it's mRNA expression in the raw surface of the rabbits].

OBJECTIVE: To explore the molecular biological mechanism of arnebia root oil in promoting wound surface healing by observing histological change and basic fibroblast growth factor(bFGF) mRNA expression in the wound surface tissues of 2 groups, as well as the wound surface healing rate. METHOD: Experimental model of incised-wound was produced on the back of 18 New Zealand albino rabbits. The wound surfaces were randomly divided into two groups, namely, experimental group and control group. The wound surfaces in the experimental group were treated by arnebia root oil and those in control group were treated by petrolatum gauze. Then raw surfaces were evaluated by the techniques of histology, histochemistry and electron microscope and the healing rates of the raw surfaces were compared between the two groups. Content of bFGF and it's mRNA expression in wound surface tissue was also measured by means of Western-blot and RT-PCR. RESULT: The wound surface healing rate in experimental group was higher than that in control group( P < 0.05). The fibroblast, collagen and blood capillaries were comparatively richer in experimental group as compared with those in control group, and similarly, the expression of bFGF mRNA was also significantly enhanced in the experimental group as compared with control group during the various periods of treatment. In addition, the changes in the expressions of bFGF and it's mRNA paralleled the changes of healing rates in the two groups. CONCLUSION: the present results showed that amebia root oil significantly can promote the healing of raw surfaces, which may be mediated by up-regulation of bFGF expression.

[Arnebia root oil promotes wound healing and expression of basic fibroblast growth factor on the wound surface in rabbits].

OBJECTIVE: To observe the promoting effect of arnebia root oils on expression of basic fibroblast growth factor (bFGF) in skin wound of rabbits and the histomorphological changes in the wound surface, and to discuss its mechanism. METHODS: Bilateral round skin wounds were made on the back of 15 rabbits. The three wounds on one side of the back of each rabbit were treated with arnebia root oils, while the three wounds on the other side were treated with vaseline in order to promote the wound healing. The histomorphology and ultrastructure under electron microscopy of the wounds, and the rate of wound healing were examined at different time. Western blotting assay was used to detect the expression of bFGF in the wound surface. RESULTS: The healing rate of the arnebia root oils-treated wounds was evidently higher than that of the vaseline-treated wounds (P<0.05). The quantities of fibroblast, collagen and capillary in the arnebia root oils-treated wounds were much more than those in the vaseline-treated wounds, and the expression of endogenous bFGF in the arnebia root oils-treated wounds was enhanced obviously as compared with that in the vaseline-treated wounds in different period of wound healing. There existed a parallel correlation between the expression level of bFGF and the rate of wound healing. CONCLUSION: The promoting effect of arnebia root oils on wound healing may be related to increasing the expression level of basic fibroblast growth factor in the skin wound.

Effect of one versus two distal locking bolts on the biomechanics of tibial interlocking nail: a comparative study using a new model.

OBJECTIVE: To investigate the impact of one versus two distal locking bolts on the mechanical properties of tibial interlocking intramedullary nails. METHODS: Twenty 9-mm titanium alloy locking nails were divided into two equal groups in which the nails were fixed with only one and two distal locking bolts, respectively. Each group was further divided into two sub-groups for compression and torsion tests separately using a universal material-testing machine. RESULTS: In the compression tests, the average maximum strength of double bolts was greater than that of the single bolt (P<0.05), but the number of the bolts did not significantly affect the results of the torsion tests in terms of the either the maximum torsion moment or angle (P>0.05). CONCLUSION: One distal bolt is sufficient for fixing stable fractures and double bolts are recommended for management of serious fractures. The model we used is convenient and economic for examining the biomechanics of the tibial nails, especially for comparative purposes.

Selection of femoral prosthesis in total hip replacement for ankylosing spondylitis.

OBJECTIVE: To investigate the implication of morphological changes of the proximal femur in femoral prosthesis selection in total hip replacement for treatment of ankylosing spondylitis (AS). METHODS: Twenty-four patients (36 hips) admitted for cementless total hip replacement (THR) because of AS were enrolled in this study along with the control group of 30 patients (45 hips) without AS. Measurement of the Singh index, canal flare index, morphological index of the cortex, cortical index and stem-canal fit was performed in the two groups on the basis of roentgenograms. RESULTS: The AS patients had more serious osteoporosis in their proximal femur. There were significant differences between the two groups in Singh index, canal flare index, morphological index of the cortex and stem-canal fit (P<0.05), but not in the cortical index (P>0.05). The AS patients were found to have severe osteoporosis in the upper femur and massive spongy bone loss, so that the morphology of the canal was almost orthostatic in the upper femur because of the thinned femoral cortex. CONCLUSION: Cemented prosthesis is recommended for AS patients with severe osteoporosis, who should be treated with revised THR to ensure good fit of the prosthesis to the canal. Cementless prosthesis, however, can still achieve good therapeutic effect in AS patients without significant morphologic changes in the proximal femurs. Computer-aided design and manufacture of individualized prosthesis better suits the AS patients than the currently available prosthesis.

Downregulation of microRNA-26a is associated with metastatic potential and the poor prognosis of osteosarcoma patients.

Accumulating evidence indicates that microRNAs are involved in multiple processes in cancer development and progression. microRNA-26a (miR-26a) has been identified as a tumor suppressor and its downregulation is associated with poor prognosis in several types of human cancer. However, the specific function of miR-26a in osteosarcoma remains unclear. In the present study, we found that the expression of miR-26a in osteosarcoma tissues and cell lines was much lower than that in the normal controls, respectively. In addition, downregulation of miR-26a more frequently occurred in osteosarcoma specimens with adverse clinical stage and with the presence of distant metastasis. Moreover, multivariate survival analyses demonstrated that loss of miR-26a is an independent prognostic factor for both disease-free and overall survival in osteosarcoma. In addition, restoration of miR-26a expression inhibited the invasion and migration in osteosarcoma cells, and miR-26a directly inhibited enhancer of zeste homolog 2 (EZH2) expression by targeting its 3'-UTR. Moreover, EZH2 was upregulated and inversely correlated with miR-26a expression in the osteosarcoma tissues. Thus, for the first time, we provide convincing evidence that downregulation of miR-26a is associated with tumor aggressiveness and tumor metastasis, and miR-26a inhibits cell migration and invasion by targeting the EZH2 gene in osteosarcoma. Thus, miR-26a is an independent prognostic marker for osteosarcoma patients.

Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: a preliminary study in vitro.

OBJECTIVE: VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH). METHODS: Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MTT assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays. RESULTS: The best MOI of BMSCs transfected with rAAV was 5 x 10(4)v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF165, and BMP(7) genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF(165)-IRES-hBMP(7) enhanced angiogenesis and osteogenesis in vitro. CONCLUSION: Recombinant adeno-associated viral vectors co-expressing the hVEGF(165) and hBMP(7) genes showed efficient gene expression ability. The VEGF(165) and BMP(7) proteins expressed from the vector have efficient biological activity in vitro.

Protective effects and mechanism of Panax Notoginseng saponins on oxidative stress-induced damage and apoptosis of rabbit bone marrow stromal cells.

OBJECTIVE: To investigate the effects and possible mechanism of Panax Notoginseng saponins (PNS) on oxidative stress-induced damage and apoptosis in bone marrow stromal cells (BMSCs). METHODS: BMSCs were isolated and cultured from 2-month-old New Zealand rabbits by the density gradient centrifugation combined with adherent method. The third passage cells were used for subsequent experiments. Oxidative stress was induced in cultured BMSCs by H(2)O(2) (0.1 mmol/L). BMSCs were pretreated with 25-200 µg/mL PNS for 4 h before H(2)O(2) treatment. Proliferation of BMSCs was observed using MTT assay. Alkaline phosphatase (ALP) activity, as an index of early osteoblastic differentiation, was determined with an ALP assay kit. Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/propidium iodide. Oxidative stress level was examined by reactive oxygen species (ROS) assay. The protein expressions of Bax, Bcl-2 and Caspase-3 in BMSCs were analyzed by Western blotting. RESULTS: PNS had different concentration-dependent effects on proliferation and osteoblast differentiation of BMSCs induced by H(2)O(2). A PNS concentration of 100 µg/mL was determined as the optimal effective concentration. PNS markedly attenuated H(2)O(2)-induced apoptosis rate from 41.91% to 14.67% (P<0.01). PNS significantly decreased ROS level induced by H(2)O(2) (P<0.01). Furthermore, pretreatment with PNS significantly reversed H(2)O(2)-induced inhibition of Bcl-2 expression and augmentation of Bax and Caspase-3 expression (P<0.01). CONCLUSION: PNS had a protective effect on oxidative stress-induced damage and apoptosis in cultured rabbit BMSCs through scavenging ROS and regulating the Bcl-2/Bax pathway.

Prokaryotic expression, purification and activity assay of recombinant vascular endothelial growth factor.

OBJECTIVE: To express human vascular endothelial growth factor (hVEGF(165)) in E. coli JM109 in the form of fusion protein by genetic engineering and test the biological activity and immunological competence of the expressed protein. METHODS: hVEGF(165) gene was subcloned by PCR and inserted into pQE30 plasmid. hVEGF(165) fusion protein was expressed in E. coli JM109 and purified by Ni(2+)-NTA. The immunological competence of the expressed protein was tested by means of Western blotting and enzyme-linked immunosorbent assay (ELISA), and its biological activity was assayed by chicken chorioallantoic membrane (CAM) and Matrigel angiogenesis assay. RESULTS: The recombinant hVEGF(165) fusion protein was successfully expressed in E. coli JM109 and its expression accounted for 30% of the total cellular protein. The purified protein presented a single band of 23 kD in SDS-PAGE. Western blotting, ELISA, CAM and matrigel angiogenesis assay showed excellent immunologic competence and biological activity of the recombinant protein. CONCLUSION: Recombinant hVEGF(165) protein with excellent biological activity has been successfully expressed in E.coli JM109, which may facilitate future study in construction of prefabricated tissue-engineered bone graft.

[Effect of estrogen on osteoprotegerin, osteoclast differentiation factor and macrophage colony stimulating factor mRNA expressions in ovariectomized rat bone tissue].

OBJECTIVE: To observe the effect of the estrogen on the mRNA expression of osteoprotegerin (OPG), osteoclast differentiation factor (ODF) and macrophage colony stimulating factor (M-CSF) in bone tissue of ovariectomized rats, and investigate the possible pathway of estrogen in preventing and treating postmenopausal osteoporosis. METHODS; Thirty healthy adult SD rats were randomly divided into sham operation group, ovariectomized group and estrogen-treated group. All rats were ovariectomized except those in the sham operation group. Bone density of the L3-L6 vertebra was detected 12 weeks after the operation. The total RNA were extracted from the femur to examine mRNA expression of OPG, ODF and M-CSF by real-time PCR. RESULTS: Estrogen increased the bone density of the ovariectomized rat lumbar vertebra and up-regulated the expression of OPG, whereas down-regulated the expression of M-CSF and lowered ODF:OPG ratio. CONCLUSION: The effect of estrogen in treating postmenopausal osteoporosis is closely correlated with the regulation of OPG and M-CSF expressions and ODF:OPG ratio.