Xiaobo Feng

MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis

MicroRNAs (miRNAs) have recently been recognized to have a role in human orthopedic disorders. The objective of our study was to explore the expression profile and biological function of miRNA-17-5p (miR-17-5p), which is well known to be related to cancer cell proliferation and invasion, in osteoblastic differentiation and in cell proliferation. The expression levels of miR-17-5p in the femoral head mesenchymal stem cells of 20 patients with non-traumatic osteonecrosis (ON) and 10 patients with osteoarthritis (OA) were examined by quantitative reverse transcription-PCR (qRT–PCR). Furthermore, the interaction between miR-17-5p and SMAD7 was observed. We found that in non-traumatic ON samples the level of mature miR-17-5p was significantly lower than that of OA samples (P=0.0002). By targeting SMAD7, miR-17-5p promoted nuclear translocation of β-catenin, enhanced expression of COL1A1 and finally facilitated the proliferation and differentiation of HMSC-bm cells. We also demonstrated that restoring expression of SMAD7 in HMSC-bm cells partially reversed the function of miR-17-5p. Together, our data suggested a theory that dysfunction of a network containing miR-17-5p, SMAD7 and β-catenin could contribute to ON pathogenesis. The present study prompts the potential clinical value of miR-17-5p in non-traumatic ON.

TNF-a mediated inflammatory macrophage polarization contributes to the pathogenesis of steroid-induced osteonecrosis in mice

The phenotypic polarization of macrophages are involved in steroid-induced osteonecrosis (ON). This study tried to investigate the detrimental and beneficial roles of M1/M2 macrophages associated with TNF-a in ON. Mice ON model was induced by the injection of methylprednisolone. After that, flow cytometry technique, immunohistochemistry, immunofluorescence, ELISA, and RT-PCR methods were used to investigate the expression pattern of macrophages and the expression of inflammatory cytokines. During the progression of ON, massive chronic inflammatory cells infiltrated into the necrotic zone, represented by the infiltration of macrophages. In the early stage of ON, there was high TNF-a activity; and a large population of M1 macrophages infiltrated into the necrotic zone. On the contrary, the expression of TNF-a gradually decreased; simultaneously, a larger M2 cell population presented in the necrotic zone in the late stage of ON. The increased M2 macrophages could be beneficial for resolving inflammation and promoting tissue repair, confirmed by the histologic findings of appositional new bone formation around the necrotic bone. Thus, it showed that TNF-a-mediated alteration of M1/M2 macrophage polarization contributed to the pathogenesis of steroid-induced osteonecrosis. M1-polarized macrophages appeared to be disruptive in the early stage of ON, while M2-polarized macrophages played an important role in the late stage during the pathogenesis of ON.

Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro

BackgroundTo study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro.MethodsA Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting.ResultsZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells.ConclusionsZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

ε-Polylysine and next-generation dendrigraft poly-L-lysine: chemistry, activity, and applications in biopharmaceuticals

Polylysine is an important class of polyamino acids with a broad spectrum of applications in biomedical research and development. It can be divided into two classes, α-polylysine and ε-polylysine, the former is synthesized by artificial chemical synthesis and has limited applications due to its high toxicity, and the latter is produced by microbial synthesis as a class of natural polymers and is widely used in various food, medicinal, and electronics products. Another major class of synthetic polymers is dendrimers (after linear, cross-linked, and branched polymers). Dendrigraft poly-L-lysine (DGL) has the favorable properties of polylysine and dendrimers, with a broad spectrum of applications in drug discovery and development, including drug delivery, gene carriers, diagnostic imaging, diagnostics, biosensors, and special cancer therapies (such as boron neutron capture therapy and photodynamic therapy). As there are still some problems with the development of DGL, further research is warranted for its broad applications.

Establishment and characterization of a novel osteosarcoma cell line: CHOS

Osteosarcoma has a well‐recognized bimodal distribution, with the first peak in adolescence and another in the elderly age‐group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58‐year‐old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well‐characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle‐shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real‐time polymerase chain reaction (RT‐PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co‐express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients.

Mid-term outcomes of primary constrained condylar knee arthroplasty for severe knee deformity

SummaryThis study aimed to examine the clinical and radiographic outcomes of primary total knee arthroplasy (TKA) with use of NexGen® Legacy® Constrained Condylar Knee (CCK) prosthesis for severe knee deformity. Clinical data of 46 patients (48 knees in total, aged 61 years on average) with severe knee deformity who underwent TKA with NexGen® Legacy® CCK prosthesis between December 2007 and February 2012 were retrospectively analyzed. There were 34 knees with severe valgus with incompetent medial collateral ligament, 11 knees with severe flexion contracture with inability to achieve knee balancing in flexion and extension by posterior soft tissue release, 2 knees with Charcot arthritis with severe varus and bone loss, and 1 with traumatic osteoarthritis with severe varus and ligamentous instability. The mean duration of follow-up was 71 months (range 40–90 months). The New Knee Society scoring (NKSS) system and the Hospital for Special Surgery (HSS) score were used to evaluate the functional and clinical outcomes. Visual Analogue Scale (VAS) was used for pain measurement and Knee Society criteria for evaluation of radiological images. The results showed that, in the total 48 knees, 1 case of loosening due to short-stem tibial component at 3 months post-operatively underwent revision. The 6-year prosthesis survival rate in this cohort was 97.9%. There was no component infection occurring within 6 years. Significant post-operative improvements were found in NKSS and HSS scores. Patient satisfaction was significantly increased. Pain score was decreased significantly. Total functional score was improved from 31.46±11.43 to 86.42±8.87, range of motion (ROM) from 42.42°±23.57° to 95.31°±23.45° and the flexion contracture from 5.31°±7.87° to 0.92°±1.80°. Preoperative radiographic study showed excessive valgus (≥7°) in 37 knees, and varus deformity in 3 knees. Post-operative femorotibial alignment was valgus 3.88°±1.76° in 48 knees. Antero/posterior (A/P) view of X-ray films showed 4 radiolucent lines (RLL) in 48 tibial components. It was concluded that TKA with CCK is effective for the treatment of the severe unstable knee that cannot be balanced by soft tissue.

A novel study on the mechanisms of drug release in PLGA-mPEG microspheres with fluorescent drug

Abstract The purpose of this research was to proof the microspheres release mechanism by a novel method-detecting and comparing the drugs fluorescent changes on the microspheres surface. Fluorescein sodium (FS, 0.4 kDa) and fluorescein isothiocyanate-bovine serum albumin (FITC-BSA, 66.8 kDa) were employed as model drugs. FS and FITC-BSA were encapsulated into PLGA-mPEG microspheres through double emulsion evaporation method, and the drug-loaded microspheres in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, confocal laser scanning microscopy (CLSM), BCA assay kit, and UV–vis spectrophotometry. FS-loaded microspheres revealed a severe initial burst release, followed by a sustained release, and we could observe a bright fluorescent on the microspheres surface during the early release period under the CLSM. The bright fluorescent gradually faded out in the later period as only 1~2% FS was remained after 14 days release. FITC-BSA-loaded microspheres revealed a typical tri-phase release profile, and we observed a weak fluorescent on the microspheres surface after the initial burst release, and the fluorescent came bright again after an obvious erosion appeared on the microspheres surface. In the later release stage, the fluorescent gradually faded out as the fast release of FITC-BSA.

Relation between the development of osteoporosis and osteonecrosis following glucocorticoid in a rabbit model.

Background: There has been a recent increase in the number of patients suffering from bone and joint diseases, as a consequence of corticosteroids administration. There are more patients treated with low dose of GCs under long-term conditions in clinical, such as effect of GCs on Rheumatoid arthritis, Crohns disease and Asthma patients. Hence, it was difficult for doctor to determine which problem occur first – OP or ON; however, there was no clinical report previously in the literature, and there was no effective animal model of OP and ON about low dose GCs. This study was conducted to develop rabbit models of glucocorticoid (GC)-induced femoral head ON and OP and to investigate the temporal relationship between the occurrence of the two events following administration of glucocorticoids. Materials and Methods: Fifty six, 6 months old female rabbits were randomly divided into the GC group and control group (C). Rabbits received gluteal injections of methylprednisolone sodium succinate once a day for 4 weeks, while normal saline solution in the control group. Rabbits were sacrificed at 0, 2, 4, and 8 weeks. Hip magnetic resonance imaging was performed before the rabbits were sacrificed. Serum calcium (Ca), phosphorus (P), total cholesterol, and triglyceride levels were also measured. The bone mineral density (BMD) of femoral head and the femoral shaft were measured by dual-energy X-ray absorptiometry. The trabecular parameters of the femur and the 4th lumbar vertebrae (L4) were measured with a micro-computed tomography (μ-CT). Also, the femoral head was stained with hematoxylin-eosin staining. Results: At 4 weeks in the GC group, the BMD of the femur reduced 33% and 22% in the femoral head and shaft; there was irregular intermediate to high T2-weighted images signals; μ-CT showed microfractures and cystic changes in the femoral head and L4 at 4 weeks. At 8 weeks in the GC group, the classical “line-like sign” indicating ON of the femoral head was observed in 64.3% of the rabbits. Conclusion: A rabbit model of GC-induced OP and ON was developed by repetitive injection with small doses of GCs in the gluteal region. OP was observed at 4 weeks while ON developed at 8 weeks and followed a clear temporal pattern.

The Influence of Tetracycline Inducible Targeting Rat PPARγ Gene Silencing on the Osteogenic and Adipogenic Differentiation of Bone Marrow Stromal Cells

Peroxisome proliferator-activated receptor γ (PPARγ) has been considered as the master regulator for adipogenesis of bone marrow stromal cells (BMSCs). However, there are few reports regarding the effect of PPARγ gene silencing on osteogenic and adipogenic differentiation in rat BMSCs, and no reports about tissue targeting and conditional knockdown of PPARγ gene. In this study, we construct rat PPARγ gene shRNA Tet-on lentiviral vector, the lentiviral vector facilitated tetracycline (which has the characteristics of bone targeting)-inducible knockdown specific to PPARγ gene, and transfect it into BMSCs, the silencing effects induced by tetracycline is significant. The expression of the adipogenic factors adipocyte determination and differentiation-dependent factor 1 (ADD1) and recombinant CCAAT/enhancer binding protein alpha (C/EBPα) were decreased as measured by RT-PCR and Western blot assay following PPARγ silencing. In contrast, expression of the osteogenic genes encoding collagen I and Cbfa1/Runx2 were increased. In adipogenic medium, PPARγ-shRNA transfection reduced the lipid droplet count as measured by Oil red O staining when compared to the control groups. In osteogenic medium, PPARγ-shRNA increased the activity of alkaline phosphatase and the amount of calcium deposition as measured by Alizarin red S staining. These results suggest that the rat PPARγ gene shRNA Teton lentiviral vector decreases adipogenic differentiation and promotes osteogenic differentiation in BMSCs induced by tetracycline.

Long non-coding RNA BDNF-AS modulates osteogenic differentiation of bone marrow-derived mesenchymal stem cells

For patients with osteoporosis, the inability of osteogenic differentiation is the key reason for bone loss. In this study, we investigated the expression and function of long non-coding RNA BDNF-AS in mesenchymal stem cell-derived osteogenic differentiation. Mouse bone marrow-derived mesenchymal stem cells (BMMSCs) were cultured in vitro and induced toward osteogenic differentiation. Quantitative real-time PCR (qRT-PCR) was used to evaluate gene expressions of BDNF-AS and BDNF during osteogenic differentiation. BMMSCs were also extracted from ovariectomized (OVX) mice. The dynamic change of BDNF-AS in OVX-derived BMMSCs during osteogenic differentiation was also evaluated. Lentivirus was used to upregulate BDNF-AS in BMMSCs. The effects of BDNF-AS upregulation on BMMSCs’ proliferation and osteogenic differentiation were then evaluated. In addition, qRT-PCR and western blot were applied to further examine the effect of BDNF-AS upregulation on osteogenesis-associated signaling pathways, including BDNF, OPN, and Runx2, in osteogenic differentiation. BDNF-AS was downregulated, whereas BDNF was upregulated in osteogenic differentiation of BMMSCs. Among OVX-derived BMMSCs, BDNF-AS expression was upregulated during osteogenic differentiation. Lentivirus-induced BDNF-AS upregulation promoted BMMSCs self-proliferation but inhibited osteogenic differentiation, as demonstrated by proliferation, alizarin red staining, and alkaline phosphatase activity assays, respectively. QRT-PCR and western blot demonstrated that BDNF, OPN, and Runx2 were downregulated by BDNF-AS upregulation in the differentiated BMMSCs. BDNF-AS is dynamically regulated in osteogenic differentiation. Upregulating BDNF-AS inhibits osteogenesis, possibly through inverse regulation on BDNF and osteogenic signaling pathways.