Fracture healing is an issue that has not yet been fully elucidated. It is generally accepted in the literature that head trauma accelerates fracture healing and causes higher volume callus tissue. Recent studies have examined the relationship between head trauma and fracture healing more molecularly. Based on this research; the aim of this study is to show the effect of head trauma on fracture healing radiologically and histologically and to investigate the relationship between serum β-Catenin level and fracture healing with the experiment we performed on rats. A total of 36 Wistar Albino female rats with a mean age of 24 weeks were included in the study with the permission of Mersin University Animal Experiments Local Ethics Committee. Six rats in the first group were not traumatized and their blood samples were collected on the day of the experiment started, end of the third week and end of the sixth week. In the second group, only head trauma was performed and blood samples were collected at the end of the third and sixth weeks. In the third group, only open femoral fracture model was applied, blood samples were collected at the third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. In the fourth group, both head trauma and open femur fracture model were applied, blood samples were collected at the end of third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. The expression level of β-Catenin was measured by PCR from all blood samples. Direct radiographs of the third and fourth groups at 3 and 6 weeks were evaluated by two orthopedists according to Rust and Lane & Sandhu scoring system. The histomorphometric examination was performed by evaluating the Huo scoring and the ratio of fracture callus components (cartilage callus, bone callus, fibrous callus) to areas. According to PCR analysis, the change of expression of β-Catenin by weeks was not statistically significant in the first and second groups. However, a statistically significant decrease was observed in the 0–6 week interval in the third and fourth groups (p = 0.002, p <0.0001, respectively). In the radiological examination, the union scores of the rats with head trauma + femoral fracture were higher than the isolated femoral fractures at 3 weeks and 6 weeks. In histomorphometric examination, no statistically significant difference was found between head trauma + femur fracture group and isolated femur fracture group. In addition, there was no correlation between the groups in the correlation studies between radiological findings, histomorphmetric findings and PCR findings. Considering that each molecule involved in fracture healing processes has a time interval and concentration; We concluded that the expression levels of β-catenin can be repeated in smaller time periods including the early stages of fracture healing

Bone regeneration includes a well-orchestrated series of biological events of bone induction and conduction. Among them, the Wnt/β-catenin signaling pathway is critical for bone regeneration. Being involved in several developmental processes, Wnt/β-catenin signaling must be safely targeted. There are currently only few specific therapeutic agents which are FDA-approved and already entered clinical trials. A published work has shown that Tideglusib, a selective and irreversible small molecule non-ATP-competitive glycogen synthase kinase 3-β(GSK-3β) inhibitor currently in trial for Alzheimer's patients, can promote tooth growth and repair cavities. [1]Despite some differences, they are some similarities between bone and tooth formation and we hypothesise that this new drug could represent a new avenue to stimulate bone healing. In this work, we locally delivered Tideglusib (GSK3β inhibitor) in the repair of femoral cortical window defects and investigated bone regeneration. A biodegradable FDA-approved collagen sponge was soaked in GSK-3βinhibitor solution or vehicle only (DMSO) and was implanted in 1 × 2 mm unicortical defects created in femora of 35 adult wild-type male mice. Bone defect repair on control and experimental (GSK-3βinhibitor) groups was assessed after 1 week (n=22), 2 weeks (n=24) and 4 weeks (n=24) with microCT and histological analysis foralkaline phosphatase (ALP, osteoblast activity), tartrate resistant acid phosphatase (TRAP, osteoclasts), and immunohistochemistry to confirm the activation of the Wnt/β-catenin pathway. Our results showed that Tideglusib significantly enhanced cortical bone bridging (20.6 ±2.3) when compared with the control (12.7 ±1.9, p=0.001). Activity of GSK-3β was effectively downregulated at day 7 and 14 resulting in a higher accumulation of active β-catenin at day 14 in experimental group (2.5±0.3) compared to the control (1.1±0.2, p=0.03). Furthermore, the onset of ALP activity appears earlier in the experimental group (day 14, 1.79±0.28), a level of activity never reached at any end-point by the control defects. At 4 weeks treatment, we observed a significant drop in ALP in the experimental group (0.47±0.05) compared to the control (1.01±0.19, p=0.02) and a decrease in osteoclast (experimental=1.32±0.36, control=2.23±0.67, p=0.04). Local downregulation of GSK-3β by tideglusib during bone defect repair resulted in significant increase in amount of new bone formation. The early upregulation of osteoblast activity is one explanation of bone healing augmentation. This is likely the effect of upregulation of β-catenin following pharmaceutical inhibition of GSK-3β since β-catenin activation is known to positively regulate osteoblasts, once committed to the osteoblast lineage. As a GSK-3β inhibitor, Tideglusib demonstrates a different mechanism of action compared with other GSK-3β antagonists as treatment was started immediately upon injury and did not interfere with precursor cells recruitment and commitment. This indicates that tideglusib could be used at the fracture site during the initial intraoperative internal fixation without the need for further surgery. This safe and FDA-approved drug could be used in prevention of non-union in patients presenting with high risk for fracture-healing complications

Introduction: Ethanol is one of risk factors associated with osteonecrosis, it has been demonstrated that ethanol induces adipogenesis, decreases osteogenesis in bone marrow stroma cells and produces intracellular lipid deposits, resulting in the death of osteocytes. Materials and Methods: In this approach, we isolated human bone marrow stroma cells and triggered for different differentiations. Results: These cells could be induced for osteogenesis, adipogenesis, and chondrogenesis. We also evaluated cell surface markers of isolated human bone marrow stromal cells that were found to express CD29, CD49d, CD62 CD90, CD105/SH2, SH3, CD133, and CD166, but not CD31, CD34, CD45, or CD56. Discussion: We demonstrated that ethanol decreases the expression of osteogenic genes, but increases adipogenic genes expressions. Moreover, we found that ethanol decreases the beta-catenin-dependent canonical Wnt signaling pathway related gene expressions, including Wnt 3a and LRP5 genes. Interestingly, ethanol also diminishes the intra-nuclear translocation of β-catenin in human bone marrow stromal cells. Therefore, these results indicate that ethanol might decrease osteogenic gene expressions through Wnt signaling pathway

Fracture non-union can be as high as 20% in certain clinical scenarios and has a high associated socioeconomic burden. Boron has been shown to regulate the Wnt/β-catenin pathway in other bodily processes. However, this pathway is also critical for bone healing. Here we aim to demonstrate that the local delivery of boric acid can accelerate bone healing, as well as to elucidate how boric acid, via the regulationtheWnt/β-catenin pathway, impacts theosteogenic response of bone-derived osteoclasts and osteoblasts during each phase of bone repair. Bilateral femoral cortical defects were created in 32 skeletally mature C57 mice. On the experimental side, boric acid (8mg/kg concentration) was injected locally at the defect site whereas on the control side, saline was used. Mice were euthanized at 7, 14, and 28 days. MicroCT was used to quantify bone regeneration at the defect. Histological staining for ALP and TRAP was used to quantify osteoblast and osteoclast activity respectively. Immunohistochemical antibodies, β-catenin and CD34 were used to quantify active β-catenin levels and angiogenesis respectively. Sclerostin and GSK3β were also quantified and are both inhibitors of the wnt signaling pathway via degradation and inactivation of β-catenin. The boron group exhibited higher bone volume and trabecular thickness at the defect site by 28 days on microCT. ALP activity was significantly higher in boron group at 7 days whereas boron had no effect on TRAP activity. Additionally, CD34 staining revealed increased angiogenesis at 14 days in boron treated groups. β-catenin activity on immunohistochemistry was significantly higher in the boron group at 7 days, GSK3β was significantly higher in the boron group at 14 days and Sclerostin was significantly higher in the boron group at 28 days. Boron appears to increase osteoblast activity at the earlier phases of healing. The corresponding early increase in β-catenin along with ALP likely supports that boron increases osteoblast activity via the wnt/β-catenin pathway. Increased angiogenesis at 14 days could be a separate mechanism increasing bone formation independent of wnt/β-catenin activation. Neither GSK3β or Sclerostin levels correlated with β-catenin activity therefore boron likely increases β-catenin through a mechanism independent of both GSK3β and Sclerostin. The addition of this inexpensive and widely available ion could potentially become a non-invasive, cost-effective treatment modality to augment fracture healing and decrease non-union rates in high risk patients

Summary Statement. Wnt/β-catenin gene expression is altered in early osteochondrosis, particularly in chondrocytes surrounding cartilage canals, and may be associated with disease initiation and/or pathogenesis. Introduction. Osteochondrosis (OC) is a disease of articular cartilage development involving abnormal endochondral ossification along the osteochondral junction. Associated etiological factors of OC have included rapid growth rate, biomechanical trauma, abnormal collagen turnover, aberrant paracrine signaling, and altered blood supply involving cartilage canals. Wnt signaling regulates chondrocyte differentiation/maturation during pre-/post-natal cartilage development. Gene expression profiling of leukocytes has revealed aberrant expression of Wnt/β-catenin pathway in early OC. The objective of this study was to elucidate the expression of molecules associated with Wnt/β-catenin signaling in early OC using an equine model. Our hypothesis was that there would be increased expression of Wnt signaling molecules in chondrocytes adjacent to cartilage canals and the osteochondral junction in early OC lesions compared to normal controls. Patients & Methods. Osteochondral samples were obtained (IACUC-approved) from femoropatellar joints of 15 euthanised immature horses (1–6 months old). Disease status was determined based on histology of osteochondral junctions (7 early OC, 8 normal controls). Osteochondral sections were frozen in OCT for laser capture microdissection (LCM) or fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry. Chondrocytes surrounding cartilage canals and osteochondral junctions were captured using LCM. RNA isolation and reverse transcription were performed. Equine-specific β-catenin, Wnt-4, Wnt-5b, Wnt-11, Dickkopf-1(Dkk-1), Lrp-4 and -6, Axin1, Wnt inhibitory factor(WIF)-1, secreted Frizzled-related protein-1, -3, and -5(Sfrp), retinoic acid receptor-gamma(RARG), RAR-inducible serine carboxypeptidase(SC-PEP) and 18S mRNA expression was evaluated by two-step real-time qPCR. Spatial protein expression was determined by immunohistochemistry using rabbit polyclonal (β-catenin, Wnt-11) or mouse monoclonal (Wnt-4, Dkk1) primary antibodies (confirmed by Western blot). Statistical analysis of early OC vs. normal controls was performed using Wilcoxon rank sum test (p <0.05). Results. Chondrocytes adjacent to cartilage canals had significantly increased gene expression of β-catenin (p=0.026), Wnt-5b (p=0.04), Lrp6 (p=0.026), WIF-1 (p=0.026), Dkk-1 (p=0.015), Axin1 (0.041), and SC-PEP (p=0.026), and decreased expression of Wnt-11 (p=0.04), in OC vs. normal controls. OC chondrocytes along osteochondral junctions had significantly increased gene expression of β-catenin (p=0.004) and SC-PEP (p=0.026), with a trend for increased Wnt-4 (p= 0.06) and Wnt-5b (p=0.06) compared to normal controls. Immunostaining for β-catenin was moderate in deep cartilage layers, including osteochondral junction chondrocytes. Wnt-4 immunostaining was moderate along the osteochondral junction and minimal along cartilage canals. Strong Wnt-11 protein expression was apparent in superficial cartilage layers and vascular cells lining cartilage canals and osteochondral junction. Mild to moderate Dkk1 immunostaining was found along osteochondral junction. Discussion/Conclusion. Wnt/β-catenin signaling regulates cartilage differentiation during development and is important in endochondral ossification. Increased gene expression of β-catenin in OC chondrocytes may affect chondrocyte hypertrophy or induce cartilage degeneration, depending on the stage of cartilage development, as β-catenin has been shown to play a dual role in cartilage growth and degeneration. In cells surrounding cartilage canals, increased gene expression of Lrp6, a co-receptor for Wnt proteins, provides further evidence of upregulation of canonical signaling in OC. However, increased Wnt inhibitor gene expression in OC chondrocytes, including Dkk1, WIF-1, and Axin1, may be an attempt to control activation of the canonical pathway

Summary Statement. Differential expression of canonical and noncanonical Wnt signalling along cartilage canals and osteochondral junctions is dependent on age. Increased gene expression of PTHrP along cartilage canals and Ihh along osteochondral junctions suggests paracrine feedback in articular-epiphyseal cartilage. Introduction. Wnt signaling has been shown to regulate chondrocyte differentiation during pre-/post-natal cartilage development. In addition, parathyroid-related peptide(PTHrP) and Indian hedgehog(Ihh) create a negative feedback loop in growth cartilage, but less is known in articular cartilage. The objective of this study was to elucidate expression of regulatory molecules in chondrocytes surrounding cartilage canals and osteochondral junctions during neonatal and pre-adolescent development. We hypothesised there would be increased expression of canonical Wnt signalling molecules and Ihh in osteochondral junction chondrocytes compared to cartilage canal chondrocytes. In addition, we hypothesised that Wnt signaling and PTHrP expression would be greater in neonates than pre-adolescents. Patients & Methods. Osteochondral samples were obtained(IACUC-approved) from normal femoropatellar joints of 14 euthanised immature horses(6 neonates, 8 pre-adolescents). Samples were frozen in OCT for laser capture microdissection(LCM) or fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry. Chondrocytes surrounding cartilage canals and osteochondral junctions were captured using LCM. Following RNA isolation, equine-specific β-catenin, Wnt-4, Wnt-5b, Wnt-11, Dickkopf-1(Dkk-1), low-density lipoprotein receptor-related protein-4,-6(Lrp4, Lrp6), Axin1, Wnt inhibitory factor-1(WIF)-1, secreted Frizzled-related protein-1,-3,-5(sFRP), retinoic acid receptor gamma(RARG), RAR-inducible serine carboxypeptidase(SC-PEP), Ihh, PTHrP, VEGF, PDGF, MMP-13, and 18S mRNA expression levels were evaluated by two-step real-time qPCR. Following immunohistochemistry using rabbit polyclonal or mouse monoclonal primary antibodies (confirmed by Western blot), spatial tissue protein expression was scored (0–3). Statistical analysis included Wilcoxon signed rank test(paired samples) or rank sum test(unpaired samples)(P<0.05). Results. Gene expression in chondrocytes along cartilage canals was significantly higher for PTHrP, β-catenin, Lrp6, Axin1, sFRP5, RARgamma, and SC-PEP than osteochondral junctions. Conversely, gene expression of Ihh, Wnt4, Wnt11, sFRP3, and VEGF were higher in osteochondral junction chondrocytes than cartilage canals. There was higher protein expression of β-catenin, PDGF, VEGF, and MMP-13 along osteochondral junctions than cartilage canals of pre-adolescents. Neonates had higher gene expression of PTHrP, Wnt-5b, sFRP3, Lrp6, and RARG in cartilage canal chondrocytes than pre-adolescents, while Ihh, Wnt-11, Lrp4, and Dkk1 were significantly higher in pre-adolescents. Immunostaining was higher for β-catenin and Wnt-11 in pre-adolescent osteochondral junction cartilage than neonates. No differences were found between age groups for Wnt-4 immunostaining. Dkk1 protein expression was significantly higher in the middle cartilage layer of pre-adolescents than neonates. Immunostaining was greater for Ihh and PTHrP in the deep cartilage layer of pre-adolescents than neonates. PDGF, VEGF, and MMP13 protein expression was higher in the superficial cartilage layer of pre-adolescents than neonates. Discussion. Wnt/β-catenin and Ihh/PTHrP signaling regulate cartilage differentiation during development and are important in endochondral ossification. This study revealed cell-specific, age-related differences in gene/protein expression of both regulatory pathways. Cells surrounding cartilage canals typically appeared small/rounded compared to larger chondrocytes along osteochondral junctions, likely due to different developmental stages. Higher PTHrP gene expression along cartilage canals and Ihh expression along osteochondral junctions may reflect these stages, suggesting paracrine feedback in articular-epiphyseal cartilage. β-catenin signaling may induce chondrocyte hypertrophy, potentially by enhancing Ihh and MMP-13 expression. Differential expression of canonical(β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signalling(Wnt-5b, Wnt-11) and Wnt inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding cartilage canals and osteochondral junctions provides evidence of age-related interactions during postnatal development

SSX was initially identified as a melanoma associated tumour antigen MEL2 and in the SS18/SSX fusion gene of synovial sarcoma. It consists of a family of nine, highly homologous X chromosome genes, being SSX1, SSX2 and SSX4 the most commonly expressed in tumours. In normal tissue, SSX expression is restricted to germ cells, trophoblasts, and mesenchymal stem cells. In malignant cells, SSX expression is over-represented in sarcomas. SSX expression is epigenetically regulated by methylation and histone deacetylation. Aim. To investigate the oncogenic properties of SSX. Validate it as tumour target and identify lead compounds. Methods. RNAi system for conditional silencing of SSX expression, Protein analysis and Mass Spec, PCR arrays, tumour xenographs and drug library screening. Results. SSX expression peaks at the G1/S phase of the cell cycle where it interacts with histone H3, H4 and β-catenin. This interaction promotes the phosphorylation of β-catenin at tyrosine residues and the transactivation of β-catenin genes MMP2 and cyclin D1. The association of SSX and MMP2 expression resulted in an increased invasion capacity of tumour cells. In vivo, silencing of SSX in tumour xenographs significantly affected the tumour growth. These tumours were characterized by large necrotic areas, impaired vascularization and cytoplasmic β-catenin. In contrast, tumour xenographs expressing SSX were highly proliferative, vascularized and exibited nuclear β-catenin. We further screened a chemical library and have identified lead compounds that target SSX. Conclusion. Our results demonstrate that SSX is a tumour target for drug development. We hypothesise that drugs targeting of SSX could affect functions related with the mesenchymal cell phenotype having potential cytostatic effect in sarcomas and/or epithelial tumour cells that undergo a epithelial-to-mesenchymal transition prior to metastasis

Sclerostin (SOST) is an endogenous inhibitor of Wnt/β-catenin signalling pathway to impair osteogenic differentiation and bone anabolism. SOST immunotherapy like monoclonal antibody has been observed to control bone remodeling and regeneration. This study is aimed to develop a SOST vaccine and test its protective effects on estrogen deficiency-induced bone loss in mice. Gene sequences coded SOST peptide putative targeting Wnt co-receptor LRP5 were cloned and constructed into vectors expressing Fc fragment to produced SOST-Fc fusion protein. Mice were subcutaneously injected SOST-Fc to boost anti-SOST antibody. Bone mineral density, microstructure, and mechanical property were quantified using μCT scanning and material testing system. Serum bone formation and resorption markers and anti-SOST levels were measured using ELISA. SOST-Fc injections significantly increased serum anti-SOST antibody levels but reduced serum SOST concentrations. SOST-Fc vaccination significantly reduced estrogen deficiency-induced serum bone resorption markers CTX-1 increased serum bone formation marker osteocalcin. Of note, it significantly alleviated the severity of estrogen-induced loss of bone mineral density, trabecular morphometric properties, and biomechanical forces of bone tissue. Mechanistically, SOSF-Fc vaccination attenuated trabecular loss histopathology and restored immunostaining of Wnt pathway like Wnt3a, β-catenin, and TCF4 in bone tissue along with increased serum osteoclast inhibitor OPG levels but decreased serum osteoclast enhancer RANKL concentrations. Taken together, SOST-Fc vaccination boosts anti-SOST antibody to neutralize SOST and mitigates the estrogen deficiency-induced bone mass and microstructure deterioration through preserving Wnt signalling. This study highlights an innovative remedial potential of SOST vaccine for preventing osteoporosis

Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features in the progression of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral bone integrity diminishment remained elusive. This study was undertaken to characterize behavior and intracellular signaling of subchondral mesenchymal stem cells (SMSCs) and bone-marrow MSCs (BMMSCs) in OA knees isolated from patients with end-stage knee OA underwent total knee arthroplasty. The SMSCs isolated from subchondral bone explants expressed remarkable surface antigens CD73, CD105, CD90, CD166, CD44, CD29, instead of MHC II, CD45, and CD31. The cell cultures exhibited high proliferation capacity concomitant with low population doubling time compared to those of BMMSCs. Incubation in differentiation media, the SMSCs showed high osteogenic and chondrogenic lineage commitment and low adipogenic differentiation potential. They also exhibited high expression of embryonic stem cell marker OCT3/4, osteogenic factors Wnt3a, β-catenin and microRNA-29a (miR-29a) in conjunction with low expression of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNFα, and MMP3. Loss of miR-29a function lowered HDAC4 level, mineralized matrix accumulation and osteogenic marker expression of SMSCs. miR-29a reduced HDAC4 translation through targeting the 3”-untranslated region of HDAC4, which concomitantly sustained Wnt3a and β-catenin signaling. Collectively, high osteogenic lineage commitment existed in the SMSCs in OA knee microenvironment. miR-29a modulation of HDAC4 and Wnt3a signaling contributed to the increases in osteogenesis. This study shines a light no the biological role of MSCs in subchondral compartment in the end-stage OA development and highlights a new source of MSCs for joint tissue repair

Introduction. Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive micro-architectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. With a progressively aging population, osteoporosis is becoming an increasingly important public health issue. The Wnt/β-catenin pathway is a major signalling cascade in bone biology, playing a key role in regulating bone development and remodelling, with aberrations in signalling resulting in disturbances in bone mass. Objectives. To assess the effects of silencing the expression of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to 10-8M dexamethasone. Methods. Primary human osteoblasts (HOBs) were cultured in vitro and exposed to 10-8M dexamethasone over a time course of 4hr, 12hr and 24hr. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated phObs (silenced & non-silenced) were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition and osteocalcin expression as determined by pNPP assay, quantitative alizarine red staining and ELISA respectively. Results. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with a rescue effect in dexamethasone-induced bone loss in vitro. Conclusions. Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodelling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signalling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass

Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive microarchitectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signalling resulting in disturbances in bone mass. Our objectives were to assess the gene expression profile of primary human osteoblasts (HOBs) exposed to dexamethasone with a view to identifying key genes driving bone mass regulation and to assess the effects of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to dexamethasone. HOBs were cultured in vitro and exposed to 10–8M dexamethasone over a time course of 4hr, 12hr and 24hr. RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed. Microarray data was validated by quantitative real time RT-PCR. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated HOBs were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition, osteocalcin expression, along with cell proliferation and cellular apoptosis. Global changes in HOB gene expression were elicited by dexamethasone. Development associated gene pathways were co-ordinately dysregulated with the expression profile of key genes of the Wnt Pathway significantly altered. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with an increase in alkaline phosphatase activity and calcium deposition, along with increased cell proliferation and reduced cellular apoptosis. Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodeling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass

Background. Long-term glucocorticoid treatment increases incidence of osteoporotic or osteonecrotic disorders. Excessive bone loss and marrow fat accumulation are prominent features of glucocorticoid-induced osteoporosis. MicroRNA-29 (miR-29) family members reportedly modulate lineage commitment of stem cells. This study was undertaken to define the biological roles of miR-29a in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Methods. Osteoblast-specific miR-29a transgenic mice (Tg) driven by osteocalcin promoter (C57BL/6JNarl-TgOCN-mir29a) or wild-type (WT) mice were given methylprednisolone. Bone mass, trabecular and cortical bone microarchitecture were assessed by μCT. Comparative mRNA and protein expression was quantified by RT-PCR and immunoblotting. Primary bone-marrow mesenchymal cells were isolated for elucidating ex vivo osteogenic and adipogenic differentiation capacity. Results. Decremented miR-29a expression was associated with severe skeletal deterioration and excessive marrow adipogenesis in glucocorticoid-induced osteoporosis bone tissue. Tg mice had high bone mass, spacious trabecular bone and thick cortical bone microstructure. Tg mice also had modest responses to the deleterious actions of glucocorticoid on trabecular microstructure and histomorphological characteristics, mineral acquisition and attenuated marrow fat deposition and osteoclast resorption. Ex vivo, miR-29a overexpression promoted bone-marrow mesenchymal progenitor cells differentiation towards osteogenic cells and away from adipogenic lineage cells. Mechanistically, miR-29a via inhibiting histone deacetylase 4 (HDAC4) actions restored acetylation states of osteogenic regulators Runx2 and β-catenin and decreased osteoclastogenic factor RANKL and adipokine leptin expression in bone microenvironments. Conclusions. Glucocorticoid suppression of miR-29a disintegrates the homeostasis between osteogenic and adipogenic activities, thereby impairs bone formation and skeletal integrity. By suppressing HDAC4, miR-29a stabilizes Runx2 and β-catenin signalling that counteracts the adverse effects of glucocorticoid on bone mass and marrow adiposity. This study unveils the anabolic roles of miR-29a in the progression of glucocorticoid-induced bone loss. Sustained miR-29a action is beneficial for protecting against osteoporosis and excessive marrow adipogenesis. Level of evidence. I

Mice are increasingly used for fracture healing research because of the possibility to use transgenic animals to conduct research on the molecular level. Mice from both sexes can be used, however, there is no consensus in the literature if fracture healing differs between female and male mice. Therefore, the aim of the present study was to analyze the similarities and differences in endochondral fracture healing between female and male C57BL/6J mice, since this mouse strain is mainly used in bone research. For that purpose, 12-weeks-old female and male mice received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone regeneration was analyzed by biomechanical testing, µCT analysis, histology, immunohistochemistry and gene expression analysis. At day 21, male mice displayed a significantly larger fracture callus than female mice accompanied by higher number of osteoclasts, higher tissue mineral density and absolute values of bone volume, whereas relative bone volume to tissue volume ratio did not differ between the groups. Biomechanical testing revealed significantly increased bending stiffness in both fractured and intact femurs from male vs. female mice, whereas relative bending stiffness of fractured femurs related to the intact femurs did not differ. 10 days after fracture, male mice display significantly more cartilage and less fibrous tissue area in the fracture callus than female mice, whereas bone area did not differ. On the molecular level, male mice displayed increased active β-catenin expression in the fracture callus, whereas estrogen receptor α (ERα) expression was reduced. In conclusion, male mice showed more prominent cartilaginous callus formation, increased mineralization and whole callus tissue formation, whereas functional outcome after fracture did not differ from female mice. This might be due either to the heavier weight of male mice or because of differences in molecular signaling pathways

Surgical failure, mainly caused by loosening implants, causes great mental and physical trauma to patients. Improving the physicochemical properties of implants to achieve favourable osseointegration will continue to be the focus of future research. Strontium (Sr), a trace element, is often incorporated into hydroxyapatite (HA) to improve its osteogenic activity. Our previous studies have shown that miR-21 can promote the osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway. The aim of this study is to fabricate a SrHA and miR-21 composite coating and it is expected to have a favorable bone healing capability. Ti discs (20 mm diameter and one mm thickness for the in vitro section) and rods (four mm diameter and seven mm length for the in vivo section) were prepared by machining pure Ti. The Ti cylinders were placed in a Teflon-lined stainless-steel autoclave for treating at 150°C for 24 h to form SrHA layer. The miR-21 was encapsulated in nanocapsules. The miR-21 nanocapsules were mixed with CMCS powder to form a gel-like sample and uniformly coated on the SrHA modifed Ti. Osteoblast-like MG63 cells were cultured on SrHA and miR-21 modified Ti, Cell proliferation activity and osteogenesis-related gene expression were evaluated. A bone defect model was established with mature New Zealand to evaluate the osseointegration. Cylindrical holes (four mm in diameter) were created at the distal femur and tibial plateau. Each rabbit was implanted with four of the aforementioned rods (distal femur and tibial plateau of the hind legs). After implantation for one, two and three months, the rabbits were observed by X-ray and scanned using u-CT. Histological and Immunohistochemical analysis were performed to examine the osteogenic markers. A biomechanical push-in test was used to assess the bone-implant bonding strength. Both SrHA nanoparticles with good superhydrophilicity and miR-21 nanocapsules with uniform sizes were distributed evenly on the surface of the Ti. In vitro experiments revealed that the composite coating was beneficial to osteoblast proliferation, differentiation and mineralization. In vivo evaluations demonstrated that this coating could not only promote the expression of angiogenic factor CD31 but also enhance the expression of osteoblastic genes to facilitate angio-osteogenesis. In addition, the composite coating also showed a decreased RANKL expression compared with the miR-21 coating. As a result, the SrHA/miR-21 composite coating promoted new bone formation and mineralization and thus enhanced osseointegration and bone-implant bonding strength. A homogeneous SrHA and miR-21 composite coating was fabricated by generating pure Ti through a hydrothermal process, followed by adhering miR-21 nanocapsules. This coating combined the favorable physicochemical properties of SrHA and miR-21 that synergistically promoted angiogenesis, osteogenesis, osseointegration, bone mineralization and thus bone-implant bonding strength. This study provided a new strategy for surface modification of biomedical implants

Summary Statement. Dickkopf-3 is upregulated in OA cartilage and synovial tissue. In vitro studies show Dkk3 can prevent cartilage degradation and antagonise Wnt signaling. We hypothesis that Dkk3 can protect against OA-related cartilage destruction. Introduction. Our group has previously shown that Dkk3, a member of the Dkk family of Wnt antagonists, is upregulated in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The role of Dkk3 in cartilage or the factors regulating its expression are not currently understood. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFβ signalling in other cell systems. Methods. Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1,-6 (IL1, IL6), TNFα, FGF2 and oncostatin-M (OSM). Dkk3 expression was assessed following ex vivo injury of murine cartilage explants. The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)- and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/−Wnt3a, TGFβ and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/−TGFβ or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFβ signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/− Dkk3 and proteoglycan output assessed using alcian blue staining. β-catenin was silenced in primary HAC prior to TGFβ and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results. Dkk3 expression was decreased in primary HAC following IL1/OSM treatment but increased by TNFα. Dkk3 expression was decreased immediately following injury to murine explants. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonised chondrocyte Wnt signalling and Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFβ signalling, increasing TGFβ-induced TIMP3 and ADAM12 expression and TGFβ-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonised Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. β-catenin knockdown did not significantly alter TGFβ- or Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Conclusions. Dkk3 expression is increased in OA and can be regulated injury and inflammatory cytokines. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA may alter chondrocyte phenotype through alterations in miRNA activity. The ability of Dkk3 to antagonise Wnt, enhance TGFβ and antagonise Activin signalling would have multiple effects on chondrocyte activity. These results imply that Dkk3 could influence multiple OA-relevant processes, protect cartilage from degradation and be important in cartilage development and homeostasis

Fatty marrow and bone loss are prominent pathologic features of osteoporosis. DNA hypermethylation shifts mesenchymal stem cells towards adipocytes impairing bone formation. Brown adipocytes produce growth factors advantageous to osteogenesis, whereas white adipocytes secrete pro-inflammatory cytokines deleterious to bone homeostasis. We assess DNA methylation inhibitor action to brown and white adipocyte formation in marrow fat of osteoporotic skeletons. Osteoporotic skeletons in mice were induced by glucocorticoid, ovariectomy or ageing. Marrow adipose volume and bone structure were quantified using OsO4 contrast-μCT imaging. Brown and white adipocytes were probed using immunostaining, RT-PCR and primary bone-marrow mesenchymal stem cell cultures. Abundant marrow fat and spare trabecular bone existed in osteoporotic skeletons. Osteoporosis increased expressions of general adipogenic markers PPARγ2 and FABP4 and white adipocyte markers TCF21 and HOXc9, whereas expressions of brown adipocyte markers PGC-1α and UCP-1 and osteogenic markers Runx2 and osteocalcin were significantly decreased. Number of UCP-1 immunostaining-positive brown adipocytes also reduced in osteoporotic bone. In vitro, DNA methylation inhibitor 5'-aza-deoxycystidine significantly increased brown adipocyte formation and osteogenic differentiation and mitigated dexamethasone-induced white adipocyte formation in mesenchymal stem cells. 5'-aza-deoxycystidine control of brown adipogenesis and white fat formation appeared to be regulated by increasing Wnt3a/β-catenin and reducing Dkk1. Disintegrated brown adipocyte and white fat cell differentiation contribute to osteoporosis pathogenesis. Maintaining DNA hypomethylation promotes Wnt signalling and brown adipocyte differentiation facilitating osteogenic differentiation. This study shed a new light to the contribution of brown adipocytic cells to bone metabolism during osteoporosis

Summary Statement. Increased Dkk-1 signaling is associated with OA occurrence and joint microenvironment damage. Interruption of Dkk1 action is beneficial to improve OA knees. Introduction. Osteoarthritis (OA) is a leading cause of disability and healthcare financial burden for total knee arthroplasty, rehabilitation, and disability. Inappropriate mechanical stress, immunological, or biochemical regulation reportedly disturbs homeostasis among cartilage, synovium and subchondral bone microstructure that contributes to OA pathogenesis. Control of joint-deleterious factor action is an emerging strategy to ameliorate OA-induced joint deterioration. Dickkopf-1 (Dkk-1) is a potent inhibitor for Wnt/β-catenin signaling regulation of tissue development and remodeling in physiological or pathological contexts. Dkk-1 also acts as a master deleterious factor that represses osteoblast differentiation capacity and bone repair. Associations among Dkk-1 expression, chondrocyte fate, synovial fibroblast behavior or OA incidence are merit of characterization. Patients & Methods. Cartilage, synovial tissue and fluid were harvested from informed consent OA patients underwent arthroplasty and patient with knee injuries without OA changes as controls. Primary chondrocyte cultures and synovial fibroblasts were treated with inflammatory cytokines or Dkk-1 antisense oligonucleotide or monoclonal antibodies. Knees in experimental animals were subjected to anterior cruciate ligament transection- or intra-articular collagenase injection to induce OA. Joint inflammation, integrity and subchondral bone microstructure in knees as well gait profiles were quantified using 2-deoxyglucose-probed near-infrared in vivo image, µCT, catwalk and histomorphometric analyses. Results. In clinical vignettes, patients with end-stage OA knee had higher abundances of Dkk-1 in cartilage, synovial tissue, and synovial fluid compared to control patients. Disruption of DKk-1 signaling ameliorated the promoting effects of inflammatory cytokines on the survival and cartilage matrix synthesis in primary cartilage chondrocyte cultures. Of interest, Dkk-1 neutralization attenuated the excessive angiogenic activities and matrix metalloproteinase secretion in primary synovial fibroblasts of OA knees. Dkk-1 modulation of survival or metabolic activities in chondrocytes and synovial fibroblasts were through β-catenin-dependent and -independent signaling pathways. Moreover, increased Dkk-1 expression in lesion sites and sera was associated with the incidence of femoral head osteonecrosis. Loss of Dkk-1 action alleviated bone cell apoptosis in osteonecrotic bone microenvironments. In experimental OA knee models, knockdown of Dkk-1 alleviated articular cartilage damage as evidenced by improved Mankin score in OA knees. Dkk-1 disruption also alleviated the adverse effects of OA on subchondral bone exposure and loss of trabecular bone volume and mineral acquisition in injured joints. Loss of Dkk-1 function reduced joint inflammation, vessel number, leukocyte infiltration in synovium compartment of OA joint and improved gait profiles of affected limbs. Conclusion. Dkk-1 signaling is associated with the OA knee occurrence and accelerates apoptosis, matrix degradation and angiogenic activities in chondrocytes and synovial fibroblasts of OA joint. Dkk-1 interference alleviates the promoting effects of OA on cartilage, synovial and subchondral bone remodeling. Blocking the deleterious actions of Dkk-1 in joint microenvironment will be a prospective molecular regime beneficial for retarding excessive joint deterioration in OA knees