Regulation of articular cartilage homeostasis is a complex process in which biologic and mechanical factors are involved. Hyperactivation of Wnt signaling, associated with osteoarthritis (OA), could jeopardize the protective anabolic effect of physiological loading. Here, we investigated the role of excessive Wnt signalling in cartilage molecular responses to loading. Human cartilage explants were harvested from hips of donors without OA. The Wnt agonist CHIR99021 was used to activate Wnt signalling 24 hours before cartilage explants were subjected to a loading protocol consisting of 2 cycles of 1 hour of 10% compression at 1 Hz, followed by 1-hour free swelling. Mechano-responsiveness was evaluated using the expression of type II collagen, aggrecan and MMP-13. Expression of known target genes TCF-1 and c-JUN was evaluated as positive control for Wnt and mechanical stimulation, respectively. In the absence of loading, CHIR99021 decreased the expression of the cartilage anabolic genes type II collagen and aggrecan, and increased the levels of MMP-13, corroborating that Wnt hyperactivation disrupts cartilage homeostasis. In the absence of Wnt hyperactivation, the applied loading protocol, representative for a physiologic stimulation by mechanical loading, led to an increase in type II collagen and aggrecan levels. However, when cartilage explants were subjected to mechanical stimulation in the presence of CHIR99021, the expression of cartilage anabolic genes was decreased, indicating changes to the cells’ mechano-responsiveness. Interestingly, mechanical stimulation was able to reduce the expression levels of MMP-13 compared to the condition of CHIR stimulation without loading. Hyperactivation of Wnt signaling switches the anabolic effect of physiologic compressive loading towards a potential catabolic effect and could contribute to the development and progression of OA

Aims. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. Methods. A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively. Results. miR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing. Conclusion. BMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing. Cite this article: Bone Joint Res 2021;10(12):744–758

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

Fragility fractures are skeletal complications associated with type 2 diabetes (T2D) causing disability, hospitalization, impaired quality of life, and increased mortality. Increased circulating sclerostin and accumulation of advanced glycation end-products (AGEs) are two potential mechanisms underlying low bone turnover and increased fracture risk. We have recently shown that T2D affects the expression of genes controlling bone formation (SOST and RUNX2) and that accumulation of AGEs is associated with impaired bone formation in T2D. We hypothesized that Wnt/B- catenin target genes are down-regulated in bone of T2D subjects as a consequence of decreased SOST and AGEs accumulation. To this end, we studied gene expression in extracts of bone samples obtained from femoral heads of 14 subjects with relatively well-controlled T2D (HbA1c 6.5±1.7%) and 21 control, non-diabetic postmenopausal women (age >65 years) undergoing hip replacement. There were no differences in age (73.2± .8 vs. 75.2±8.5 years) or BMI (27.7±5.6 vs. 29.9±5.4 kg/m2) between control and T2D groups, respectively. Expression of LEF1 mRNA was significantly lower in T2D compared to non-diabetic subjects (p=0.002), while DKK1 was not different between groups (p=0.108). Correlation analysis showed that DKK1 (r2=0.038; p=0.043) and HbA1c (r2=0.503; p=0.048) increased with age in T2D. COL1A1 mRNA trended lower in T2D compared to controls (p=0.056). Bone volume (9,333 ± 1,443 vs. 15,53 ± 2,442 mm2; p=0.048), mineralized volume (9,278 ± 1,418 vs. 15,45 ± 2,444 mm. 2. ; p=0.048) and BV/TV (0,2125 ± 0,03114 vs. 0,3719 ± 0,03196 %; p=0.002) measured by bone histomorphometry were lower in T2D compared to controls. Our data show that even in patients with relatively good glycemic control, T2D decreases expression of Wnt/B-catenin target genes andCOL1A1, associated with decreased bone density. These results may help understand the mechanisms underlying bone fragility in T2D

Introduction. Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Scx), Mohawk (Mkx), Tenomodulin (Tnmd)) in rat primary tendon-derived cells (TDCs) and SCX-transduced human mesenchymal stem cells (hMSC-Scx cells), as a tendon progenitor cell line (kindly provided Dr. Docheva). The roles of TGF-beta signaling in tenogenesis have been elucidated. The purpose of the study was to evaluate the effect of TGF-beta signaling on tenogenic genes and relationship between both two signalings in rat TDCs and hMSC-Scx cells. Materials and Methods. Cell cultures. TDCs were harvested from the Achilles tendons of 6 week-old SD rats and the 3rd passage TDCs were used. To evaluate the effect of TGF-beta signaling, TGF-beta 1 protein and SD208 (TGF-beta receptor inhibitor) were utilized. To assess the effect of Wnt/beta-catenin signaling on TGF-beta signaling, BIO (Wnt/b-catenin activator) was utilized. Quantative RT-PCR. TDCs and hMSC-Scx cells were cultured with TGF-beta1 or SD208. Total RNA was isolated from the cells and cDNA was synthesized. Expression of Axin2, as a target gene for Wnt/beta-catenin signaling, as well as tenogenic genes was quantified and normalized by Gapdh. Western blotting. Each cells were cultured with BIO. Whole cell lysates were used for immunoblotting with antibodies against beta-actin and phosphorylated Smad2/Smad3 (p-Smad2/3), which indicates activation of TGF-beta signaling. Band intensity was quantified and normalized by beta-actin. Statistical analysis. Two groups were compared by unpaired Student”s t-test. Multiple groups were analyzed by one-way analysis of variance (ANOVA). (P <0.05). Results. Scx expression was increased by TGF-beta1 and decreased by SD208 in a dose-dependent manner in TDCs. However, TGF-beta1 and SD208 showed no significant effect on Mkx and Tnmd expression in TDCs and hMSC-Scx cells. BIO treatment decreased p-Smad2/3 proteins, while TGF-beta1 and SD208 had no effect on Axin2 expression in both cells. Discussion. Activation of TGF-b signaling induced Scx expression, independent of Wnt/beta-catenin signaling. Activation of Wnt/beta-catenin signaling suppressed p-Smad2/3 amounts for TGF-beta signaling and further Scx expressions. Taken together, activation of Wnt/beta-catenin signaling antagonizes Scx expression induced by TGF-beta signaling. Identification of compounds, which control Wnt/beta-catenin and/or TGF-beta signaling, may lead to developments of novel therapeutic options to facilitate regeneration of injured tendons

To overcome the severely limited regenerative capacity of cartilage, bone marrow mesenchymal stromal cells (MSCs) are an attractive cell source that is accessible less invasively and in higher quantity than articular chondrocytes (ACs). However, current in vitro chondrogenic protocols induce MSCs to form transient cartilage reminiscent of growth plate cartilage that becomes hypertrophic and is remodeled into bone. In contrast, under the same conditions, ACs form stable articular-like cartilage. Developmental studies in mice have revealed that TGF-beta/BMP, Wnt, and Hedghog/PTHrP signaling are the major regulators of both, articular cartilage and endochondral bone formation. While the differential regulation of TGF-beta/BMP and Hedgehog/PTHrP in endochondral MSC versus AC chondral differentiation is established knowledge, little is known about Wnt in these cells. Aim of this study was therefore to compare in vitro levels of Wnt network components in MSC-derived endochondral versus AC-derived articular cartilage. Whole genome expression data comparing human MSCs and ACs at days 0 and 28 of in vitro chondrogenesis were screened for differential expression of Wnt ligands, receptors, co-receptors, activators/inhibitors and signaling molecules. Expression of the most strongly differentially regulated Wnt network genes was studied in detail during in vitro chondrogenesis of MSCs vs ACs via qPCR at days 0, 7, 14, 21, 35, and 42. During early chondrogenesis, most Wnt components were expressed at low levels in both MSCs and ACs, with two exceptions. MSCs started into chondrogenesis with significantly higher levels of the non-canonical ligand WNT5A. ACs on the other hand expressed significantly higher levels of the canonical antagonist FRZB on day 0. During advancing and late chondrogenesis, MSCs downregulated WNT5A but still expressed it at significantly higher levels at day 42 than ACs. Strong regulation was also evident for WNT11 and the receptor PTK7 which were both strongly upregulated in MSCs. Unlike MSCs, ACs barely regulated these non-canonical Wnt genes. With regard to canonical signaling, only the transcription factor LEF1 showed strong upregulation in MSCs, while FZD9 and FRZB were only slightly upregulated in late MSC chondrogenesis. Again, these genes remained unregulated in ACs. Our data suggest that a dynamic Wnt network regulation may be a unique characteristic of endochondral MSC differentiation while during AC chondral differentiation Wnt expression remained rather low and stable. Overall, mRNA of the non-canonical Wnt network components were stronger regulated than canonical factors which may indicate that primarily non-canonical signaling is dynamic in endochondral differentiation. Next step is to assess levels of active and total beta-catenin, the canonical Wnt mediator, and to use Wnt antagonists to establish a causal relationship between Wnt signaling and endochondral differentiation

Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells. DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software. We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways. In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair

In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by Real Time-PCR and normalized with the expression of S29. Percentage of phosphorylated Smad2, Smad1 and JNK were determined through western blot. TGF-β1 content was quantified by sandwich ELISA; MMP-13 and GAG by western blot and DMMB assay, respectively. At least three biological replicates were used. ANOVA test was used for parametric analysis; Kruskal-Wallis and Mann-Whitney post hoc test for non-parametric. Preliminary data show that compression increased collagen II expression in control, but not in CHIR99021 and FB-fs pre-treated group (Fig. 1A-B). This was associated with downregulation of β1-integrin expression, which is the main collagen receptor and further regulates collagen II expression, suggesting inhibition of Erk1/2 pathway. A trend of increase expression of collagen type X after mechanical loading was observed in CHIR and FB-fs group. ALK-1 and ALK-5 showed a trend toward stronger upregulation in CHIR99021 group after compression, suggesting the activation of both Smad1/5/8 and Smad 2/3 pathways. To further investigate pathways leading to these different mechano-responses, the phosphorylation levels of Smad1 and Smad2, Erk1/2 and JNK proteins are currently being studied. Preliminary results show that Smad2, Smad1 and JNK protein levels increased in all groups after mechanical loading, independently of an increase in TGF-β1 expression or content. Compression further increased phosphorylation of Smad2, but not of Smad1, in all groups

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

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

Wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of RANL in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particletreated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Osteosarcoma is the most common primary bone tumour worldwide. This disease presents a formidable challenge to the orthopaedic surgeon, with a mortality rate of 30 per cent, even after surgical clearance. Aberrant Wnt signalling has been implicated in the pathogenesis osteoblastic tumours. The objective of this study is 2 fold- to investigate if osteosarcoma does indeed demonstrate aberrant Wnt signaling, and if so, does osteosarcoma respond to a novel Wnt inhibitor(ETC159). This can potentially lead to the development of a new adjuvant treatment modality for osteosarcoma. A novel Wnt signaling pathway protein antibody (YJ5) was used in immunihistochemistry staining of clinical osteosarcoma samples. A Wnt high osteosarcoma cell line(SJSA-1) was then implanted subcutaneously in a mouse model. These mice were treated with a novel PORCN inhibitor, ETC 159 for a period of 4 weeks in a two-arm randomised control study. The results of treatment were evaulated by clinical outcome parameters as well as immunohstochemistry. 100 per cent of clinical osteosarcoma samples demonstrated increased WLS expression and Wnt protein expression. SJSA-1 showed no significant decrease in tumour volume after 30 days of drug treatment (3070 SD 625 mm3 vs 3480 SD 433 mm3 p= 0.605 and 2060 SD 209 vs 1677 SD 213 mm3 p=0.219 respectively). Significantly, SJSA-1 demonstrated increased tumour necrosis in the treatment arm(30–60 percent increase across all samples p < 0 .005) Treated tumours also demonstrated markedly less angiogenesis compared to the non treatment arm. Osteosarcoma demonstrates aberrant Wnt signaling in a large percentage of cases. The use of a novel PORCN inhibitor ETC 159 for the treatment of Osteosarcoma has a marked effect on tumour necrosis. Our results suggest that ETC159 may cause tumour necrosis by inhibiting angiogenesis within the tumour. Further evaluation and understanding of the mechanism of Wnt singaling in regulating tumour pathogenesis may hold the potential for developing a curative therapeutic drug for this deadly disease

Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods. Gene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature. Results. A group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and transforming growth factor beta (TGF-β) signalling pathways. We limited the loading-responsive genes obtained via the osteocyte transcriptome signature to identify a subgroup of genes that are highly relevant to osteocytes, as the mechanics-responsive osteocyte signature in OA. Based on WGCNA, we found that this signature was highly co-expressed and identified three clusters, including early, late, and persistently responsive genes. Conclusion. In this study, we identified the mechanics-responsive osteocyte signature in OA-lesioned subchondral bone. Cite this article: Bone Joint Res 2022;11(6):362–370

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

Objectives. Osteoporosis is a systemic bone metabolic disease, which often occurs among the elderly. Angelica polysaccharide (AP) is the main component of angelica sinensis, and is widely used for treating various diseases. However, the effects of AP on osteoporosis have not been investigated. This study aimed to uncover the functions of AP in mesenchymal stem cell (MSC) proliferation and osteoblast differentiation. Methods. MSCs were treated with different concentrations of AP, and then cell viability, Cyclin D1 protein level, and the osteogenic markers of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2) were examined by Cell Counting Kit-8 (CCK-8) and western blot assays, respectively. The effect of AP on the main signalling pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin was determined by western blot. Following this, si-H19#1 and si-H19#2 were transfected into MSCs, and the effects of H19 on cell proliferation and osteoblast differentiation in MSCs were studied. Finally, in vivo experimentation explored bone mineral density, bone mineral content, and the ash weight and dry weight of femoral bone. Results. The results revealed that AP significantly promoted cell viability, upregulated cyclin D1 and increased RUNX2, OCN, ALP, and BMP-2 protein levels in MSCs. Moreover, we found that AP notably activated PI3K/AKT and Wnt/β-catenin signalling pathways in MSCs. Additionally, the relative expression level of H19 was upregulated by AP in a dose-dependent manner. The promoting effects of AP on cell proliferation and osteoblast differentiation were reversed by H19 knockdown. Moreover, in vivo experimentation further confirmed the promoting effect of AP on bone formation. Conclusion. These data indicate that AP could promote MSC proliferation and osteoblast differentiation by regulating H19. Cite this article: X. Xie, M. Liu, Q. Meng. Angelica polysaccharide promotes proliferation and osteoblast differentiation of mesenchymal stem cells by regulation of long non-coding RNA H19: An animal study. Bone Joint Res 2019;8:323–332. DOI: 10.1302/2046-3758.87.BJR-2018-0223.R2

Aims. Non-coding microRNA (miRNA) in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) may promote neuronal repair after spinal cord injury (SCI). In this paper we report on the effects of MSC-EV-microRNA-381 (miR-381) in a rodent model of SCI. Methods. In the current study, the luciferase assay confirmed a binding site of bromodomain-containing protein 4 (BRD4) and Wnt family member 5A (WNT5A). Then we detected expression of miR-381, BRD4, and WNT5A in dorsal root ganglia (DRG) cells treated with MSC-isolated EVs and measured neuron apoptosis in culture by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. A rat model of SCI was established to detect the in vivo effect of miR-381 and MSC-EVs on SCI. Results. We confirmed an interaction between miR-381 and BRD4, and showed that miR-381 overexpression inhibited the expression of BRD4 in DRG cells as well as the apoptosis of DRG cells through WNT5A via activation of Ras homologous A (RhoA)/Rho-kinase activity. Moreover, treatment of MSC-EVs rescued neuron apoptosis and promoted the recovery of SCI through inhibition of the BRD4/WNT5A axis. Conclusion. Taken altogether, miR-381 derived from MSC-EVs can promote the recovery of SCI through BRD4/WNT5A axis, providing a new perspective on SCI treatment. Cite this article: Bone Joint Res 2021;10(5):328–339

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

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