Increased circulating microRNA-21 level as a potential indicator for predicting a higher risk of incident fragility fractures.

CONTEXT: As a common disease in the elderly, osteoporosis clearly increases the risk of fractures, leading to higher mortality, but the current markers to estimate the risk of fractures are limited. MicroRNA-21 (miR-21) may play an important role in osteoporosis, but the link of this biomarker with fractures was undetermined. OBJECTIVES: We aimed to investigate the association between miR-21 levels and the presence of fragility fractures. METHODS: A total of 200 patients were recruited and miR-21 was collected from baseline serum. The correlation between miR-21 and the fracture risk assessment tool (FRAX) score was analyzed. The incidence of fragility fractures was presented by Kaplan-Meier analysis, and Cox regression analysis was utilized to evaluate risk factors. The diagnostic value of miR-21 was conducted by the area under curve (AUC). RESULTS: The FRAX score was significantly associated with miR-21 level (p<0.001). According to the 50th percentile of miR-21 content in the overall distribution, the cumulative incidence of fragility fractures was significantly higher in patients with higher miR-21 levels than those with lower levels (75.4, 95 % CI: 69.0-81.8 vs. 59.2, 95 % CI: 42.1-76.3, p<0.001). The results of the Cox regression analysis showed that the miR-21 level was an independent risk factor linked to the incidence of fracture (p=0.005). The optimal cut-off value of the miR-21 was 6.08, and the AUC for predicting fracture was 0.718 (95 % CI, 0.645-0.790). CONCLUSIONS: This study showed that miR-21 has optimal diagnostic performance in the discrimination of fragility fracture, and the circulating miR-21 level in predicting the risk of fragility fracture may have a certain value.

Mechanism of Methylprednisolone-Induced Primary Cilia Formation Disorder and Autophagy in Osteoblasts.

OBJECTIVE: To study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid-induced avascular necrosis of the femoral head (SANFH). METHODS: Osteoblasts were isolated from rabbit bones and treated with 1 µM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg-5, Atg-12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP-RFP-LC3 lentivirus. RESULTS: Methylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg-5, Atg-12 and OFD1 mRNAs and proteins in a time-dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time-dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg-5 and Atg-12, were found to be negatively correlated with IFT20, with an average correlation coefficient of -0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of -0.53. CONCLUSION: Methylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.

Hypoxia-induced resistance to cisplatin-mediated apoptosis in osteosarcoma cells is reversed by gambogic acid independently of HIF-1α.

In vitro evidence of hypoxia-induced resistance to cisplatin (CDDP)-mediated apoptosis exists in human osteosarcoma (OS). Gambogic acid (GA) is a promising chemotherapeutic compound that could increase the chemotherapeutic effectiveness of CDDP in human OS cells by inducing cell cycle arrest and promoting apoptosis. This study examined whether GA could overcome OS cell resistance to CDDP. Hypoxia significantly reduced levels of CDDP-induced apoptosis in the OS cell lines MG63 and HOS. However, combined treatment with GA and CDDP revealed a strong synergistic action between these drugs, and higher protein levels of the apoptosis-related factor Fas, cleaved caspase-8 and cleaved caspase-3 and lower expression of hypoxia-inducible factor (HIF)-1α are detected in both cell lines. Meanwhile, drug resistance was not reversed by exposure to the HIF-1α inhibitor 2-methoxyestradiol. These findings strongly suggest that hypoxia-induced resistance to CDDP is reversed by GA in OS cells independently of HIF-1α. Furthermore, in vivo studies using xenograft mouse models revealed that combination therapy with CDDP and GA exerted increased antitumor effects by inducing apoptosis. Taken together, our results demonstrate that GA may be a new potent therapeutic agent useful for targeting human OS cells.

BMP7 enhances the effect of BMSCs on extracellular matrix remodeling in a rabbit model of intervertebral disc degeneration.

Intervertebral discs (IVDs) provide stability and flexibility to the spinal column; however, IVDs, and in particular the nucleus pulposus (NP), undergo a degenerative process characterized by changes in the disc extracellular matrix (ECM), decreased cell viability, and reduced synthesis of proteoglycan and type II collagen. Here, we investigated the efficacy and feasibility of stem cell therapy using bone marrow mesenchymal stem cells (BMSCs) over-expressing bone morphogenetic protein 7 (BMP7) to promote ECM remodeling of degenerated IVDs. Lentivirus-mediated BMP7 over-expression induced differentiation of BMSCs into an NP phenotype, as indicated by expression of the NP markers collagen type II, aggrecan, SOX9 and keratins 8 and 19, increased the content of glycosaminoglycan, and up-regulated ß-1,3-glucuronosyl transferase 1, a regulator of chondroitin sulfate synthesis in NP cells. These effects were suppressed by Smad1 silencing, indicating that the effect of BMP7 on ECM remodeling was mediated by the Smad pathway. In vivo analysis in a rabbit model of disc degeneration showed that implantation of BMSCs over-expressing BMP7 promoted cell differentiation and proliferation in the NP, as well as their own survival, and these effects were mediated by the Smad pathway. The results of the present study indicate the beneficial effects of BMP7 on restoring ECM homeostasis in NP cells, and suggest potential strategies for improving cell therapy for the treatment of disc diseases.

Suppressed invasive and migratory behaviors of SW1353 chondrosarcoma cells through the regulation of Src, Rac1 GTPase, and MMP13.

Chondrosarcoma is the second frequent type of primary bone cancer. In response to stress to the endoplasmic reticulum, activation of eIF2α-mediated signaling is reported to induce apoptosis. However, its effects on invasive and migratory behaviors of chondrosarcoma have not been understood. Focusing on potential roles of Src kinase, Rac1 GTPase, and MMP13, we investigated eIF2α-driven regulation of SW1353 chondrosarcoma cells. In particular, we employed two chemical agents (salubrinal, Sal; and guanabenz, Gu) that elevate the level of eIF2α phosphorylation. The result revealed that both Sal and Gu reduced invasion and motility of SW1353 chondrosarcoma cells in a dose dependent manner. Live imaging using a fluorescent resonance energy transfer (FRET) technique showed that Sal and Gu downregulated activities of Src kinase as well as Rac1 GTPase in an eIF2α dependent manner. RNA interference experiments supported an eIF2α-mediated regulatory network in the inhibitory role of Sal and Gu. Partial silencing of MMP13 also suppressed malignant phenotypes of SW1353 chondrosarcoma cells. However, MMP13 was not regulated via eIF2α since administration of Sal but not Gu reduced expression of MMP13. In summary, we demonstrate that eIF2α dependent and independent pathways regulate invasion and motility of SW1353 chondrosarcoma cells, and inactivation of Src, Rac1, and MMP13 by Sal could provide a potential adjuvant therapy for combating metastatic chondrosarcoma cells.

Angelica Sinensis attenuates inflammatory reaction in experimental rat models having spinal cord injury.

This study was aimed to evaluate the effect of Angelica Sinensis on experimental rat models in which spinal cord injury was induced by studying different factors. Different factors causing inflammation play a key role in pathophysiology of SCI. Here three groups of rats (n=15, each was used). These included a sham control group where only laminectomy was performed, SCI group where SCI was induced and AS/SCI group where although SCI was induced but Angelica Sinensis was also administered to study its effect and draw a comparison with control. The expression of I-kBα and NF-kB p65 was also studied using western blotting and after recording optical density (OD) values of western blots. MPO activity was used to measure the effect of 20 mg/kg Angelica Sinensis. The levels of proinflammatory cytokines TNF-α, IL-1ß and IL-6 were also studied. As compared with SCI group and sham control it was observed that Angelica Sinensis significantly reduced the expression of I-kBα and NF-kB p65, (P<0.05), while MPO activity was also significantly reduced. Proinflammatory cytokine level was also reduced in treated group as compared to both other groups. On the basis of this study we concluded that the use of 20 mg/kg Angelica Sinensis in rat models can attenuate the secondary damage caused by SCI and thus help in controlling the pathology of SCI in rats.

Matrine inhibits the growth and induces apoptosis of osteosarcoma cells in vitro by inactivating the Akt pathway.

Matrine, a natural product, has been demonstrated to be a promising chemotherapeutic drug for some cancers. Using flow cytometric analysis of the cell cycle and apoptosis, we found that matrine inhibited the proliferation and induced apoptosis in the human osteosarcoma (OS) cell lines MG63, HOS, U2OS, and SAOS2 in vitro in a dose-dependent manner. We therefore assessed the role of the serine/threonine kinase Akt in the regulation of matrine-mediated cell growth inhibition and apoptosis induction in human OS cell lines. After treatment for 48 h, matrine induced G0/G1-stage cell cycle arrest in MG63, U2OS, and SAOS2 cells associated with an increase in the expression of p27(Kip1) and a decrease in the expression of Akt, glycogen synthase kinase 3 (GSK3)-ß (Ser9), and cyclin D1. Furthermore, the pro-apoptotic factor Bax was upregulated. Overall, our findings suggest that matrine may be an effective anti-osteosarcoma drug due to its ability to inhibit proliferation and induce apoptosis in OS cells, possibly through the involvement of Akt signaling.

Distinctive subcellular inhibition of cytokine-induced SRC by salubrinal and fluid flow.

A non-receptor protein kinase Src plays a crucial role in fundamental cell functions such as proliferation, migration, and differentiation. While inhibition of Src is reported to contribute to chondrocyte homeostasis, its regulation at a subcellular level by chemical inhibitors and mechanical stimulation has not been fully understood. In response to inflammatory cytokines and stress to the endoplasmic reticulum (ER) that increase proteolytic activities in chondrocytes, we addressed two questions: Do cytokines such as interleukin 1 beta (IL1ß) and tumor necrosis factor alpha (TNFα) induce location-dependent Src activation? Can cytokine-induced Src activation be suppressed by chemically alleviating ER stress or by applying fluid flow? Using live cell imaging with two Src biosensors (i.e., cytosolic, and plasma membrane-bound biosensors) for a fluorescence resonance energy transfer (FRET) technique, we determined cytosolic Src activity as well as membrane-bound Src activity in C28/I2 human chondrocytes. In response to TNFα and IL1ß, both cytosolic and plasma membrane-bound Src proteins were activated, but activation in the cytosol occurred earlier than that in the plasma membrane. Treatment with salubrinal or guanabenz, two chemical agents that attenuate ER stress, significantly decreased cytokine-induced Src activities in the cytosol, but not in the plasma membrane. In contrast, fluid flow reduced Src activities in the plasma membrane, but not in the cytosol. Collectively, the results demonstrate that Src activity is differentially regulated by salubrinal/guanabenz and fluid flow in the cytosol and plasma membrane.

Extensor tendon injury due to repetitive wrist dorsiflexion: morphological study of extensor retinaculum and extensor tendon.

Most etiological studies of extensor tendon injury were based on the normal anatomy of extensor tendon and extensor retinaculum of the wrist. Further understanding of the morphological changes of the extensor tendon and extensor retinaculum during wrist dorsiflexion might contribute to improved and more accurate understanding of the etiology. The morphology of the extensor tendon of the mid-finger and the fourth compartment of the wrist extensor retinaculum was studied by sonography, and the anatomy was studied in 15 extremities from 11 young male cadavers. Compared with anatomical images, ultrasonography provides similar morphological observations of the extensor retinaculum of the wrist and extensor tendon. Ultrasonography findings revealed that as the dorsiflexion angle changed, the extensor retinaculum of the wrist formed different shaped trochleas. The trochlea guides the rotation of the extensor tendon at the wrist, but it does not form a sharp corner with the extensor tendon; thus, the extensor tendon is not compressed. As the dorsiflexion angle increased from 0° to 60°, the length of the trochlea gradually decreases. The shortening of the trochlea length will lead to a smaller frictional contact area between the extensor tendon and the extensor retinaculum. Consequently, the friction is centralized. During wrist dorsiflexion, the extensor retinaculum provides a trochlea for the extensor tendon. Extensor tendon injury of repetitive wrist dorsiflexion might be caused by centralized friction at the small contact area.

A comparison of osteocyte bioactivity in fine particulate bone powder grafts vs larger bone grafts in a rat bone repair model.

The osteogenic potential for bone grafts is based on numbers and activities of cells that survive transplantation. In this study, we compared the bioactivity of osteocytes in 300-500 µm fine particulate bone powder grafts to 2 mm larger bone grafts in a rat radial defect model. Expression levels of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-ß1), alkaline phosphatase (ALP), and collagen I were semi-quantified by both immunohistochemistry and RT-PCR at days 1 and 4, as well as weeks 1, 2, 4, 6 and 10 post-transplantation. Within two weeks post-transplantation, more cells stained positively for BMP-2, TGF-ß1, ALP, and collagen I within the bone grafts and in the surrounding tissues in the group transplanted with the fine particulate bone powder grafts than in those with larger bone grafts (P<0.05). The mRNA levels of all four markers in the group transplanted with fine particulate bone powder graft peaked earlier and were expressed more highly than in the larger bone graft group, suggesting that fine particulate bone powder grafts provide more viable and active osteocytes to accelerate bone defect healing than larger bone grafts.

Curcumin promotes the spinal cord repair via inhibition of glial scar formation and inflammation.

Spinal cord injury (SCI) is a serious clinical situation without any effective therapy to date. Traumatic SCI triggers a complex pathological process including inflammatory response and glial scar formation. In this study, we demonstrated that curcumin, a natural product which functions as an anti-inflammatory agent, inhibited the activation of signal transducer and activator of transcription-3 and NF-kappa B in the injured spinal cord. Curcumin treatment greatly reduced the astrogliosis in SCI mice and significantly decreased the expression of IL-1ß and NO, as well as the number of Iba1(+) inflammatory cells at the lesion site. Notably, more residual axons and neurons were protected and significantly improved functional recovery was observed in the curcumin-treated mice, compared to the mice without curcumin treatment. These findings indicate that curcumin promotes spinal cord repair through inhibiting glial scar formation and inflammation and suggests the therapeutic potential of curcumin for SCI.

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway.

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G2/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.

Surgical treatment of cervicothoracic junction spinal tuberculosis via combined anterior and posterior approaches in children.

BACKGROUND: Cervicothoracic junction spinal tuberculosis (CJST) in children is uncommon, especially when accompanied by a huge abscess. However, its consequences can be severe. Because of the special anatomic location of the cervicothoracic junction, surgical treatment is difficult and rarely reported. The aim of this clinical study was to assess the effectiveness of combined anterior and posterior approaches for focal debridement, decompression, allografting and anterior instrumentation in the treatment of CJST in children. METHODS: Ten pediatric CJST patients underwent focal debridement and cord decompression through combined anterior and posterior approaches. Then an appropriate allograft and titanium plate were applied to reconstruct the spine. The patients were asked to wear head-neck-chest braces for six months and received regular anti-tubercular drugs therapy for 12 months. RESULTS: The patients were followed-up for an average of 26 months (range, 15-32 months). There was no recurrent tuberculous infection. The bone grafts incorporated well and the instrumentation was stable. Cervical and thoracic kyphosis was successfully corrected from 40° (range, 30-52°) before the operation to 18° (range, 12-26°) post-operation. Neurological function was improved in all patients. CONCLUSIONS: Combined anterior and posterior approaches for focal debridement, decompression, bone allografting and anterior instrumentation provided an effective means of treatment in children of CJST with a huge abscess in the posterior part of the vertebral body.

The fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats.

The aim of the study was to investigate the fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats. The iliac bone of male inbred DA rats was harvested and used as the larger bone grafts and also prepared as fine particulate (granulated) bone powders (300-500µm size particles) for transplantation into radial defects in female rats. The presence and relative amounts of genes specific to the sex-determining region of the Y-chromosome (Sry) originating from the bone grafts were evaluated by polymerase chain reaction and by in situ hybridization, respectively. Additional samples were evaluated histologically. In the larger bone grafts, the expression of Sry decreased relatively early, disappeared by 1 week, reappeared at 4 weeks and continued to increase with time. In the fine particulate bone powders, Sry was detected all the time and its expression was statistically greater than in the larger bone grafts at each time point. Both bone grafts provided donor cells to repair the defects. The donor cells seemed to function differently between the two groups. The fine particulate bone powders contained more living osteocytes in comparison with the larger bone grafts and may accelerate the healing of bone defects compared with conventional autografts.

A cell-penetrating peptide suppresses inflammation by inhibiting NF-κB signaling.

Nuclear factor-κB (NF-κB) is a central regulator of immune response and a potential target for developing anti-inflammatory agents. Mechanistic studies suggest that compounds that directly inhibit NF-κB DNA binding may block inflammation and the associated tissue damage. Thus, we attempted to discover peptides that could interfere with NF-κB signaling based on a highly conserved DNA-binding domain found in all NF-κB members. One such small peptide, designated as anti-inflammatory peptide-6 (AIP6), was characterized in the current study. AIP6 directly interacted with p65 and displayed an intrinsic cell-penetrating property. This peptide demonstrated significant anti-inflammatory effects in vitro and in vivo. In vitro, AIP6 inhibited the DNA-binding and transcriptional activities of the p65 NF-κB subunit as well as the production of inflammatory mediators in macrophages upon stimulation. Local administration of AIP6 significantly inhibited inflammation induced by zymosan in mice. Collectively, our results suggest that AIP6 is a promising lead peptide for the development of specific NF-κB inhibitors as potential anti-inflammatory agents.

Gambogic acid inhibits the growth of osteosarcoma cells in vitro by inducing apoptosis and cell cycle arrest.

The natural product gambogic acid (GA) has been demonstrated to be a promising chemotherapeutic drug for some cancers because of its ability to induce apoptosis and cell cycle arrest. Until now, no studies have looked at the role of GA in osteosarcoma. In this study, we observed the effects of GA on the growth and apoptosis of osteosarcoma cells in vitro. We found that GA treatment inhibits the proliferation of osteosarcoma cells by inducing cell cycle arrest. Moreover, we found that GA induces apoptosis in MG63, HOS and U2OS cells. Furthermore, we showed that GA treatment elevates the Bax/Bcl-2 ratio. GA mediated the G0/G1 phase arrest in U2OS cells; this arrest was associated with a decrease in phospho-GSK3-ß (Ser9) and the expression of cyclin D1. Similarly, in MG63 cells, GA mediated G2/M cell cycle arrest, which was associated with a decrease in phospho-cdc2 (Thr 161) and cdc25B. Overall, our findings suggest that GA may be an effective anti-osteosarcoma drug because of its capability to inhibit proliferation and induce apoptosis of osteosarcoma cells.

Anti-tumor effects of dihydroartemisinin on human osteosarcoma.

Dihydroartemisinin (DHA) exhibits antitumor activity against a wide spectrum of cancer cells. However, whether DHA has anti-tumor effect on human osteosarcoma cells remains unknown. This study aims to investigate the anti-tumor activity of DHA and the underlying mechanisms in human osteosarcoma cell lines with different p53 mutation statuses. Four human osteosarcoma cell lines were treated with different concentrations of DHA. Then, cell proliferation was determined by the CCK-8 viability assay; apoptosis and cell cycle progression were evaluated by flow cytometry; protein expression was analyzed by western blot assay; and NF-kB activity was examined by luciferase assay. The results demonstrated that DHA treatment could inhibit the proliferation of four osteosarcoma cell lines in a dose-dependent manner. P53 wild-type osteosarcoma cells were more sensitive to DHA. Moreover, the percentage of apoptotic cell and cell arrest in G2/M phase was increased upon DHA treatment in a dose-dependent manner. Mechanistically, DHA activated caspase-3, caspase-8, and caspase-9; upregulated the expression of Bax, FAS, and cyclin D1; downregulated the expression of Bcl-2, Cdc25B, and cyclin B1; and inhibited the activity of NF-кB. In conclusion, DHA has significant anticancer effects against human osteosarcoma cells, which include induction of apoptosis and cell cycle arrest. The p53 gene may play a certain role in the DHA-induced human osteosarcoma apoptosis and cell cycle arrest. DHA is a novel anti-osteosarcoma drug candidate that merits further study.

Effect of morselized bones on binding of the canine hydroxyapatite-coated femoral stem.

BACKGROUND: Non-cement femoral stems are recognized in clinical use, but there are still some problems. The aim of this research was to make non-cement femoral stems to be press-fit with the medullary cavity. METHODS: Twenty-four healthy adult mongrel dogs were randomly divided into experimental and control groups. In the right hip joint, an artificial femoral bone replacement surgery was conducted. For the experimental group, the replacement surgery of hydroxyapatite (HA)-coated femoral stems was done, while autogeneous morselized bone was implanted into the medullary cavity. For the control group, morselized bone was not implanted. At postoperative 1, 3, 6 months, a test for interfacial shear characteristics was conducted in the MTS810 Tester. The comparison between the two groups' bone-prostheses in shear strength for their interface from shearing destruction was made. A histological observation to check prosthesis-bone interface contact ratios and bone growth was carried out. RESULTS: For the experimental group, shear strength was 0.317 MPa in 1 month, 1.447 MPa in 3 months, and 1.621 MPa in 6 months. For the control group, shear strength was 0.195 MPa in 1 month, 1.023 MPa in 3 months, and 1.483 MPa in 6 months. The difference was statistically significant. Stereomicroscope-based observation showed that the number of trabecular bones in the experimental group was larger than that of the control group, and bone growth of the former group was better than that of the latter group. Inverted microscopic observation showed that the binding degree between the prosthesis and trabecular bone of the experimental group was higher than that of the control group. Comparatively, the experimental group's trabecular bone had more stromal cells. CONCLUSIONS: The morselized bones can effectively improve the biological bonding strength and bone-contact ratios in the short term for the HA-coated femoral stem and accelerate the bonding process. The use of morselized autogenous bones played a good role in bone in-growth of the femoral bone stem surface.