p-Coumaric acid suppresses reactive oxygen species-induced senescence in nucleus pulposus cells.

p-Coumaric acid (PCA) is a phenolic acid that is widely present in numerous plants and human diets. Studies have demonstrated the antioxidant and anti-senescence effects of PCA in different cell types. However, the anti-senescence effects of PCA in nucleus pulposus (NP) cells have remained to be determined. In the present study, reverse transcription-quantitative PCR was used to measure the gene expression of Cyclooxygenase-2 (Cox-2), inducible nitric oxide synthase (iNOS), p53, p16, aggrecan and collagen-2 in NP cells. Immunofluorescence staining was used to evaluate the protein expression of p53, p16 and collagen-2 in NP cells. In addition, cell cycle of NP cells was measured by flow cytometry. ß-galactosidase staining were used to investigate the senescence of NP cells. Preliminary results indicated that PCA suppressed ROS-induced senescence in NP cells via both the p16 and p53 pathways. NP cells were pretreated with PCA at a concentration of 10 or 50 µg/ml prior to stimulation with 200 µM hydrogen peroxide (H2O2). Pretreatment with PCA significantly inhibited H2O2-induced cell cycle arrest in a dose-dependent manner. PCA also reduced the gene expression of Cox-2, iNOS, p53 and p16 induced by H2O2. By contrast, aggrecan and collagen-2 expression in NP cells was upregulated after PCA treatment. Furthermore, PCA suppressed H2O2-induced changes in the protein expression of p16, p53 and collagen-2. H2O2 stimulation of NP cells increased senescence-associated ß-galactosidase (SA-ß-gal) activities, while PCA treatment markedly reversed these SA-ß-gal activities. Collectively, the present results indicated that PCA attenuated H2O2-induced oxidative stress and cellular senescence, suggesting a potential therapeutic utility of PCA in intervertebral disc degeneration.

Transformation of acellular dermis matrix with dicalcium phosphate into 3D porous scaffold for bone regeneration.

Animal derived biomaterials have attracted much attentions in treating large size bone defect due to their excellent biocompatibility and potent bioactivities offered by the biomacromolecules and growth factors contained in these materials. Dermis-derived matrix (ADM) has been used as skin grafts and wound dressings for decades, however its application in bone tissue engineering has been largely limited as ADM possesses a dense structure which does not support bone tissue ingrowth. Recently, we have successfully fabricated porous scaffold structure using an ADM with the aid of micronization technique. When integrated with inorganic components such as calcium phosphate, ADM could be transformed to bone graft substitutes with desirable osteogenic properties. While purified and chemically cross-linked collagen has lost its natural structure, our ADM successfully preserved natural tropocollagen structure, as well as other bioactive components. A composite scaffold was fabricated by incorporating dicalcium phosphate (DCP) microparticles into ADM microfibers and freeze-dried to form a highly porous structure. Unlike conventional ADM materials, this scaffold possesses high porosity with interconnected pores. More importantly, our evaluation data demonstrated that it performed much more effective in treating critical bone defects in comparison with best commercial product on the market. In a head-to-head comparison with a commercial bone graft material Bongold®, the ADM/DCP scaffold showed superior osteogenic capacity by filling the defect with well-organized new bone tissue in a rabbit radius segmental defect model. Put together, our results exhibited a novel bone graft substitute was developed by circumventing processing barriers associated with natural ADM, which offers another novel bone graft substitute for bone regeneration.

The Effect of MMP-2 Inhibitor 1 on Osteogenesis and Angiogenesis During Bone Regeneration.

Bone regeneration is a popular research focus around the world. Recent studies have suggested that the formation of a vascular network as well as intrinsic osteogenic ability is important for bone regeneration. Here, we show for the first time that matrix metalloproteinase (MMP) 2 inhibitor 1 (MMP2-I1) has a positive role in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) and angiogenesis of human vascular endothelial cells (HUVECs). MMP2-I1 activated the p38/mitogen-activated protein kinase signaling pathway to promote the osteogenesis of hBMSCs, and promoted the angiogenesis of HUVECs via the hypoxia-inducible factor-1α signaling pathway. We also found that MMP2-I1 enhanced bone formation using a rat tibial defect model and prevented bone loss using an ovariectomy-induced mouse model of osteoporosis. Data from the mouse model demonstrated that MMP2-I1 generated more type H vessels (CD31hiEmcnhi) when preventing bone loss. These results provide important insights into the regulatory effects of MMP2-I1 on bone regeneration.

The pro-inflammatory effect of NR4A3 in osteoarthritis.

NR4A3 is a member of nuclear receptor subfamily 4, which is an important regulator of cellular function and inflammation. In this study, high expression of NR4A3 in human osteoarthritis (OA) cartilage was firstly observed. To explore the relationship between NR4A3 and OA, we used a lentivirus overexpression system to simulate its high expression and study its role in OA. Additionally, siRNA-mediated knockdown of NR4A3 was used to confirm the findings of overexpression experiments. The results showed the stimulatory effect of IL-1ß on cartilage matrix-degrading enzyme expression such as MMP-3, 9, INOS and COX-2 was enhanced in NR4A3-overexpressed chondrocytes and decreased in NR4A3-knockdown chondrocytes at both mRNA and protein levels, while IL-1ß-induced chondrocyte-specific gene (collagen 2 and SOX-9) degradation was only regulated by NR4A3 at protein level. Furthermore, overexpression of NR4A3 would also enhance EBSS-induced chondrocytes apoptosis, while knockdown of NR4A3 decreased apoptotic level after EBSS treatment. A pathway study indicated that IL-1ß-induced NF-κB activation was enhanced by NR4A3 overexpression and reduced by NR4A3 knockdown. We suggest that NR4A3 plays a pro-inflammatory role in the development of OA, and we also speculate that NR4A3 mainly regulates cartilage matrix-degrading gene expression under inflammatory conditions via the NF-κB pathway.

LncRNA TUG1 promotes the development of osteosarcoma through RUNX2.

The lncRNA taurine-upregulated gene 1 (TUG1) is known to serve a role as an oncogene in the development of a number of human malignancies. However, the functionality of TUG1 in osteosarcoma remains poorly characterized. Therefore, the aim of the present study was to explore the role of TUG1 in osteosarcoma. TUG1 expression in tumor tissues, adjacent healthy tissues and plasma from 40 osteosarcoma patients and 40 healthy controls was detected using reverse transcription-quantitative PCR. Receiver operating characteristic curves were used to analyze the diagnostic value of TUG1 for osteosarcoma while the prognostic value of TUG1 for osteosarcoma was analyzed using the Kaplan-Meier method. TUG1 expression vectors and siRNAs were transfected into MG-63 and U2OS osteosarcoma cell lines, and the effects on osteosarcoma cell viability, migration and invasion were tested using Cell Counting kit-8 and Transwell assays. The effects of TUG1 overexpression on runt-related transcription factor 2 (RUNX2) expression were also detected using western blotting. TUG1 expression was found to be significantly higher in osteosarcoma tissues compared with adjacent healthy tissues, and in the plasma of osteosarcoma patients compared with healthy controls. TUG1 expression also exhibited significant diagnostic and prognostic value for osteosarcoma. TUG1 overexpression and knockdown respectively increased and reducedosteosarcoma cell viability, migration and invasion. In addition, TUG1 overexpression upregulated RUNX2 expression. These results suggest that lncRNA TUG1 may promote the development of osteosarcoma by modulating RUNX2 and TUG1 expression, which can serve as prognostic and diagnostic markers for this malignancy.