Osteonectin bidirectionally regulates osteoblast mineralization.

OBJECTIVE: The aim of this study was to investigate whether Osteonectin/Secreted protein acidic and rich in cysteine (ON/SPARC) had a two-way dose-dependent regulatory effect on osteoblast mineralization and its molecular mechanism. METHODS: Initially, different concentrations of ON were added in osteoblasts, and the gene of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) were detected using reverse-transcription quantitative polymerase chain reaction (RT-PCR). Secondly, based on the above results, the Optima and inhibitory concentration of ON for osteoblast mineralization were determined and regrouped, the Control group was also set up, and the gene detections of Collagen 1 (Col 1), Discoidin domain receptor 2 (DDR2) and p38 mitogen­activated protein kinase were added using RT-PCR. In the third stage of the experiment, osteoblasts were pretreated with 0.4Mm ethyl-3,4-dihydroxybenzoate (DHB) (a specific inhibitor of collagen synthesis) for 3 h before adding the optima SPARC, the gene and protein expressions of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 were detected by RT­qPCR and western blot analysis, and the mineralized nodules were observed by alizarin red staining. RESULTS: The results showed that the expression of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 genes and proteins in osteoblasts were significantly enhanced by 1 ug/ml ON, 100 ug/ml ON or 1 ug/ml ON added with 3,4 DHB significantly inhibited the expressions of DDR2, P38 and the above-mentioned mineralization indexes, and significantly reduced the formation of mineralized nodules. CONCLUSION: This study suggested that ON had a bidirectional dose-dependent regulatory effect on osteoblast mineralization, and the activation of P38 pathway by collagen binding to DDR2 was also an important molecular mechanism.

Identification of Diagnostic Markers in Synovial Tissue of Osteoarthritis by Weighted Gene Coexpression Network.

Osteoarthritis (OA) is a serious threat to human health. However, the etiology and pathogenesis of the disease are not fully understood. Most researchers believe that the degeneration and imbalance of articular cartilage, extracellular matrix, and subchondral bone are the fundamental causes of osteoarthritis. However, recent studies have shown that synovial lesions may precede cartilage, which may be an important precipitating factor in the early stage of OA and the whole course of the disease. This study aimed to conduct an analysis based on sequence data from the Gene Expression Omnibus (GEO) database to investigate the presence of effective biomarkers in the synovial tissue of osteoarthritis for the diagnosis and control of OA progression. In this study, the differentially expressed OA-related genes (DE-OARGs) in osteoarthritis synovial tissues were extracted in the GSE55235 and GSE55457 datasets using the Weighted Gene Co-expression Network Analysis (WGCNA) and limma. Least-Absolute Shrinkage and Selection Operator (LASSO) algorithm was used to select the diagnostic genes based on the DE-OARGs by glmnet package. 7 genes were selected as diagnostic genes including SAT1, RLF, MAFF, SIK1, RORA, ZNF529, and EBF2. Subsequently, the diagnostic model was constructed and the results of the Area Under the Curve (AUC) demonstrated that the diagnostic model had high diagnostic performance for OA. Additionally, among the 22 immune cells of the Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and the 24 immune cells of the single sample Gene Set Enrichment Analysis (ssGSEA), 3 immune cells and 5 immune cells were different between the OA and normal samples, respectively. The expression trends of the 7 diagnostic genes were consistent in the GEO datasets and the results of the real-time reverse transcription PCR (qRT-PCR). The results of this study demonstrate that these diagnostic markers have important significance in the diagnosis and treatment of OA, and will provide further evidence for the clinical and functional studies of OA.

The clinical effect of trochanteric slide osteotomy combined with a cementless femoral conical stem in total hip replacement for the treatment of Crowe type IV developmental dysplasia of the hip.

BACKGROUND: Total hip replacement (THR) for Crowe type IV developmental dysplasia of the hip (DDH) is still challenging due to specific joint deformities and the high incidence of post-operative complications. OBJECTIVE: This study aimed to evaluate the clinical effect of trochanteric slide osteotomy (TSO) combined with a cementless femoral conical stem in THR for the treatment of Crowe type IV DDH. METHODS: Thirty-one total hip replacements (26 patients) with Crowe type IV DDH were performed using TSO combined with a cementless femoral conical stem. Surgical outcomes were evaluated using leg length discrepancy (LLD), Harris hip score, and post-operative complications. RESULTS: The average pre-operative LLD was 51 mm (range 46-58 mm), decreasing to an average of 10 mm (range 8-12 mm) post-operatively. As a result, the post-operative incidence of the Trendelenburg sign significantly decreased compared with the pre-operative incidence (P< 0.05). Bony union was identified in 26 hips (83.9%), fibrous union in four (12.9%), and non-union in one (3.2%). No acetabular or femoral component loosening, dislocation, or deep infection around the component was found in any of the patients during the follow-up period (27 to 39 months). The average Harris hip score improved from 63.0 ± 3.0 (range 58-69) to 93.3 ± 2.0 (range 91-96). CONCLUSION: TSO combined with a cementless conical stem in THR is an appropriate option for patients with high congenital hip dislocation.

Discoidin domain receptor 2 activation of p38 mitogen-activated protein kinase as an important pathway for osteonectin-regulating osteoblast mineralization.

OBJECTIVE: The present study aimed to determine the role of the discoidin domain receptor 2 (DDR2) in the osteonectin (ON) regulation of osteoblast mineralization through the activation of p38 mitogen-activated protein kinase (MAPK). METHODS: Four groups were established: the ON group, the inhibitor group, the Ddr2-small interfering ribonucleic acid (siRNA) group, and the control group. Osteoblasts from the parietal bones of neonatal Sprague-Dawley rats were isolated and cultured. In the ON group, 1 µg/mL ON was added to the osteoblasts. The gene expressions of collagen 1 (Col 1) and Ddr2 were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the inhibitor group, the osteoblasts were added to WRG-28 (a specific DDR2 inhibitor), and in the Ddr2-siRNA group, the osteoblasts were transfected with Ddr2-siRNA. The gene and protein expressions of DDR2, bone sialoprotein, osteocalcin, osteopontin, and p38 MAPK were determined using RT-qPCR and western blot analysis. Alizarin red staining and transmission electron microscopy were used to detect mineralization. RESULTS: The results showed that ON enhanced the osteoblast Col 1 and Ddr2 gene expressions, while the use of a Ddr2-siRNA/DDR2-blocker decreased the OPN, BSP, OCN, and P38 gene and protein expressions and reduced osteoblast cellular activity and mineralized nodules. CONCLUSION: The present study demonstrated that DDR2 activation of p38 MAPK is an important approach to ON-regulating osteoblast mineralization.

Osteonectin regulates the extracellular matrix mineralization of osteoblasts through P38 signaling pathway.

Osteonectin binds strongly to type I collagen and hydroxyapatite and plays a crucial role in extracellular matrix mineralization. Previous studies have also shown that p38 signaling pathway is an important regulator for osteoblast mineralization. This study focused on the role of osteonectin in regulating extracellular matrix mineralization via the p38 signaling pathway. Osteoblasts were isolated and cultured from parietal bones of neonatal Sprague-Dawley rats. The gene and protein expressions of noncollagen proteins (BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin), p38 mitogen-activated protein kinase, and SIBLINGs (Small Integrin-Binding LIgand N-linked Glycoproteins) members (DMP1, dentine matrix protein 1, DSPP, dentin sialophosphoprotein, and MEPE, matrix extracellular phosphoglycoprotein) were detected by reverse-transcription quantitative polymerase chain reaction and western blot analysis. Alizarin red staining, intracellular calcium assay, and transmission electron microscopy were used to detect mineralization. Initially, by adding osteonectin at different concentrations in osteoblasts and detecting the above mineralization indexes, 1 µg/ml was determined to be the optima osteonectin concentration, which significantly increased gene expressions of BSP, OPN, OCN, DMP1, MEPE, DSPP, and p38 in osteoblasts, p38 and p-p38 protein expressions were also significantly increased, mineralized nodules were significantly enhanced; when added with SB203580 (a specific inhibitor for p38) these effects were inhibited. Furthermore, osteoblasts transfected with Ad-p38 also significantly upregulated the protein and gene expressions of noncollagens and SIBLINGs members, whereas transfection of p38-rhRNA showed the opposite effect. Our data suggest that osteonectin regulates the extracellular matrix mineralization of osteoblasts through the P38 signaling pathway.