Deciphering the pathogenic role of rare RAF1 heterozygous missense mutation in the late-presenting DDH.

Background: Developmental Dysplasia of the Hip (DDH) is a skeletal disorder where late-presenting forms often escape early diagnosis, leading to limb and pain in adults. The genetic basis of DDH is not fully understood despite known genetic predispositions. Methods: We employed Whole Genome Sequencing (WGS) to explore the genetic factors in late-presenting DDH in two unrelated families, supported by phenotypic analyses and in vitro validation. Results: In both cases, a novel de novo heterozygous missense mutation in RAF1 (c.193A>G [p.Lys65Glu]) was identified. This mutation impacted RAF1 protein structure and function, altering downstream signaling in the Ras/ERK pathway, as demonstrated by bioinformatics, molecular dynamics simulations, and in vitro validations. Conclusion: This study contributes to our understanding of the genetic factors involved in DDH by identifying a novel mutation in RAF1. The identification of the RAF1 mutation suggests a possible involvement of the Ras/ERK pathway in the pathogenesis of late-presenting DDH, indicating its potential role in skeletal development.

E7 peptide-functionalized Ti6Al4V alloy for BMSC enrichment in bone tissue engineering.

Ti6Al4V alloy is widely used for hip joint prostheses, however owing to its lack of biomimetic surface properties, it often suffers from poor osseointegration. It is well known that bone mesenchymal stem cells (BMSCs) play an important role in the osseointegration of the host bone and joint prostheses. One promising approach to improving the osseointegration of joint prostheses is to enrich the number of BMSCs at the periprosthetic site. Previous studies have reported that BMSC specific affinity peptide E7, can specifically enrich BMSCs. However, to date, few studies have reported the use of E7 in bone tissue engineering. In this study, we conjugated E7 peptide to Ti6Al4V alloy to fabricate a scaffold (BTS) to improve the biocompatibility of the alloy. E7 peptide efficiently improved the adhesion of BMSCs to Ti6Al4V alloy. In addition, the BTS scaffold was more conducive to osteogenesis than the RGD-functionalized and non-functionalized control scaffolds. The functional BTS scaffold could pave the way for designing functional joint prostheses, which promote osseointegration between the host bone and implant.

Inhibitory effect of quercetin on titanium particle-induced endoplasmic reticulum stress (ERS)-related apoptosis and in vivoosteolysis.

Wear particle-induced periprosthetic osteolysis is the main cause of aseptic loosening of orthopaedic implants. The aim of this study is to determine the protective effect of quercetin (QUE) against titanium (Ti) particle-induced ERS-related apoptosis and osteolysis. In this study, RAW264.7 cells were pretreated with different concentrations (40, 80, and 160 µmol/l) of QUE for 30 min and then treated with Ti particle (5 mg/ml) for 24 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. Protein and mRNA expressions of ERS-related genes were examined by western blot and real-time PCR, respectively. The release of inflammatory cytokines was detected by ELISA. Then a mouse calvarial osteolysis model was established. Histological sections of calvaria were stained with H&E or TRAP. The results showed that Ti particle reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of Ti particle were dramatically inhibited by QUE pretreatment. Interestingly, we found that QUE also significantly reduced Ti particle-induced up-regulation of the expression levels of PERK, IRE1, GRP78, CHOP, caspase-12 and caspase-3 and enhanced the down-regulation of Bcl-2. In addition, QUE decreased Ti particle-induced inflammatory cytokines release from RAW264.7 cells. Moreover, treatment with QUE markedly decreased osteoclast number. In a mouse calvarial osteolysis model, QUE inhibited Ti particle-induced osteolysis in vivo by inhibiting osteoclast formation and expressions of ERS-related genes. In conclusion, QUE can protect RAW264.7 cells from Ti particle-induced ERS-related apoptosis and suppress calvarial osteolysis in vivo.

Effect of Scutellarin inhibits collagen­induced arthritis through TLR4/NF­κB­mediated inflammation.

Scutellarin is the major effective constituent of the commonly used Chinese medicine Erigeron breviscapus. It has been applied in the clinic to treat various diseases, and is characterized by high content, a stable source, controllable quality, high efficiency and low toxicity. In addition, its potential pharmacological effects have been increasingly identified and elucidated. The present study was performed to examine the role of scutellarin on collagen­induced arthritis (CIA). Mice were injected subcutaneously with bovine collagen type II and administered scutellarin for 2 weeks by gavage 20 mg/kg/day. ELISA kits were used to measure the levels of interleukin (IL)­1ß, IL­6, tumor necrosis factor­α (TNF­α), oxidative stress markers [superoxide dismutase (SOD) and malondialdehyde (MDA)] and caspase­3/-9 activity. Bax, Bcl­2, toll like receptor 4 (TLR4) and nuclear factor (NF)­κB protein expression was analyzed using western immunoblot analyses. The present study demonstrated that scutellarin prevented CIA, and inhibited the expression of inflammation factors, IL­1ß, IL­6 and TNF­α. In addition, scutellarin reduced the levels of oxidative stress markers, SOD and MDA, as well as intercellular adhesion molecule­1 and monocyte chemoattractant protein 1 in CIA mice. Caspase­3/-9, Bax/Bcl­2, TLR4 and NF­κB protein expression were reduced in CIA mice following scutellarin treatment. The results of the current study suggest a novel effect of scutellarin involving the inhibition of TLR4/NF­κB­mediated inflammation.

Calcitonin protects chondrocytes from lipopolysaccharide-induced apoptosis and inflammatory response through MAPK/Wnt/NF-κB pathways.

Calcitonin (CT) is an anti-absorbent, which has long been used for treatment of osteoporosis. However, little information is available about the effects of CT on osteoarthritis (OA). This study was mainly aimed to explore the effects of CT on the treatment of OA, as well as the underlying mechanisms. Chondrocytes were isolated from immature mice and then were incubated with lipopolysaccharide (LPS), CT, small interfering (si) RNA against bone morphogenetic protein (BMP)-2, and/or the inhibitors of MAPK/Wnt/NF-κB pathway. Thereafter, cell viability, apoptosis, nitric oxide (NO) and inflammatory factors productions, and expression levels of cartilage synthesis protein key factors, cartilage-derived morphogenetic protein (CDMP) 1, SRY (sex-determining region Y)-box 9 protein (SOX9), and MAPK/Wnt/NF-κB pathways key factors were determined. CT significantly reversed LPS-induced cell viability decrease, apoptosis increase, the inflammatory factors and NO secretion, the abnormally expression of cartilage synthesis proteins and the activation of MAPK/Wnt/NF-κB pathways (P<0.05). In addition, we observed that administration of the inhibitors of MAPK/Wnt/NF-κB pathways statistically further increased the levels of CDMP1 and SOX9 (P<0.05). Suppression of BMP-2 decreased the levels of CDMP1 and SOX9 and activated MAPK/Wnt/NF-κB pathways, and could partially abolish CT-modulated the expression changes in CDMP1 and SOX9, and MAPK/Wnt/NF-κB pathways key factors (P<0.05). The results showed that CT protects chondrocytes from LPS-induced apoptosis and inflammatory response by regulating BMP-2 and thus blocking MAPK/Wnt/NF-κB pathways.

Long non-coding RNA Sox4 promotes proliferation and migration by activating Wnt/ß-catenin signaling pathway in osteosarcoma.

Osteosarcoma is a bone tumor without effective treatment in the world. Recently, long non-coding RNAs (lncRNAs) are considered as essential regulators in cancer progression. LncSox4 plays crucial roles in liver tumor-initiating cells self-renewal and tumor initiation. However, the effect of lncSox4 in osteosarcoma remains largely unclear. Quantitative real-time PCR (qRT-PCR) and Northern blot were performed to detect lncSox4 expressions in osteosarcoma. The functions of lncSox4 in osteosarcoma were determined using cell viability and migration assays. In addition, the proteins associated with lncSox4 were further evaluated by western blot. We found that lncSox4 was expressed highly in U-20S and Mg63 cells and osteosarcoma tumor tissues (all P < 0.001). LncSox4 silencing attenuated but lncSox4 overexpression promoted cell viability (all P < 0.001) and migration (P < 0.01) in the Mg63 cells. Next, we found lncSox4 regulation of osteosarcoma is involved in ß-catenin, and overexpression of lncSox4 could stable ß-catenin expression. Further, Wnt agonist CID11210285 completely abolished the decrease of Mg63 cells viability induced by lncSox4 silencing when cells cultured for 3 and 4 days (both P < 0.01), while Wnt inhibitor IWP-3 abolished the increase of Mg63 cells viability induced by overexpression of lncSox4 after treatment for 2 (P < 0.01), 3 (P < 0.001) and 4 (P < 0.01) days. Our study offers evidence for the first time that lncSox4 plays a positive role in osteosarcoma development and progression, and could act as a potential prognostic and therapy biomarker.

In Vitro Bioactivity Study of RGD-Coated Titanium Alloy Prothesis for Revision Total Hip Arthroplasty.

Total hip arthroplasty (THA) is a common procedure for the treatment of end-stage hip joint disease, and the demand for revision THA will double by 2026. Ti6Al4V (Titanium, 6% Aluminum, and 4% Vanadium) is a kind of alloy commonly used to make hip prothesis. To promote the osseointegration between the prothesis and host bone is very important for the revision THA. The peptide Arg-Gly-Asp (RGD) could increase cell attachment and has been used in the vascular tissue engineering. In this study, we combined the RGD with Ti6Al4V alloy using the covalent cross-linking method to fabricate the functional Ti6Al4V alloy (FTA). The distribution of RGD oligopeptide on the FTA was even and homogeneous. The FTA scaffolds could promote mouse osteoblasts adhesion and spreading. Furthermore, the result of RT-qPCR indicated that the FTA scaffolds were more beneficial to osteogenesis, which may be due to the improvement of osteoblast adhesion by the RGD oligopeptide coated on FTA. Overall, the FTA scaffolds developed herein pave the road for designing and building more efficient prothesis for osseointegration between the host bone and prothesis in revision THA.

The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure.

Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone-implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.