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.

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies. Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685

Introduction. Exercise increases tendon collagen synthesis and cross-link formation. Exercise also increases the expression of TGF-β. 1. TGF-β. 1. may contribute to the upregulation of tendon collagen synthesis during exercise, but this relationship has not been established in vivo. The purpose of this study was to evaluate the effects of TGF-β. 1. receptor inhibition on tendon collagen. Materials and Methods. Male Wistar rats were divided into sedentary (SED, n = 9) or exercised (RUN, n=15) groups. Exercised animals completed four days of treadmill exercise (60 minutes/days). The peritendinous space of one Achilles tendon was injected with LY-364947 (ALK5 inhibitor; INHIB) while the opposite leg was injected with a vehicle (SHAM). Injections were given daily after each exercise bout. ERK and Smad 2/3 phosphorylation was evaluated by Western blotting. Collagen I and III gene expression were evaluated via qRT-PCR. Tendon hydroxyproline and hydroxylyslpyridinoline (HP) cross-linking were assayed via HPLC. A longitudinal section of tendon was stained with H&E for evaluation of cell numbers and fibril organization. Results. Phosphorylation of ERK increased by 2.5-fold in both legs given LY-364947 (p<0.05) but was not influenced by exercise (p>0.05). Smad 3 phosphorylation was reduced (p<0.05) in tendons treated with LY-364947. Neither type I nor type III collagen gene expression was affected by TGF-β. 1. receptor inhibition or exercise (p>0.05). Collagen content was not altered by either exercise or LY-364947 (p>0.05). HP cross-linking was 3-fold lower in the RUN-INHIB when compared to the RUN-SHAM tendon (p<0.05). No effect of inhibitor on HP was noted in the sedentary animals. Cell density was greater (p<0.05) in the Achilles tendon of exercised animals (SED: 7.5 cell/100 μm. 2. , RUN: 10.3 cell/100 μm. 2. ) but was not influenced by TGF-β. 1. receptor inhibition (p>0.05). Fiber structure scores were 45% lower (p<0.07) in SED animals treated with inhibitor but normal in RUN animals given inhibitor. Discussion. The changes in ERK and Smad phosphorylation suggest that LY-364947 was effective at altering TGF-β. 1. signaling. Our data suggest that neither acute exercise nor TGF-β. 1. receptor inhibition altered collagen gene expression. In contrast, TGF-β. 1. appears to be important for regulating Achilles tendon cross-link formation during exercise training and inhibition of TGF-β. 1. impacts fiber structure

Dynamic compressive loading of cartilage can support extracellular matrix (ECM) synthesis whereas abnormal loading such as disuse, static loading or altered joint biomechanics can disrupt the ECM, suppress the biosynthetic activity of chondrocytes and lead to osteoarthritis. Interactions with the pericellular matrix are believed to play a critical role in the response of chondrocytes to mechanical signals. Loading of intact cartilage explants can stimulate proteoglycan synthesis immediately while the response of chondrocytes in tissue engineering constructs dependent on the day of culture. In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage regeneration after surgical treatment, it is essential to understand how ECM accumulation influences the loading response. This study explored how construct maturity affects regulation of ECM synthesis of chondrocytes exposed to dynamic loading and unraveled the molecular correlates of this response. Human chondrocytes were expanded to passage 2, seeded into collagen scaffolds and cultured for 3, 21, or 35 days before exposure to a single loading episode. Dynamic compression was applied at 25% strain, 1 Hz, in 9 × 10 minute-intervals over 3h. Gene expression and protein alterations were characterized by qPCR and Western blotting. Proteoglycan and collagen synthesis were determined by radiolabel-incorporation over 24 hours. Maturation of constructs during culture significantly elevated ECM deposition according to histology and GAG/DNA content and chondrocytes redifferentiated as evident from raising COL2A1 and ACAN expression. Loading of d3 constructs significantly reduced proteoglycan synthesis and ACAN expression compared to controls while the identical loading episode stimulated GAG production significantly (1.45-fold, p=0.016) in day 35 constructs. Only in mature constructs, pERK1/2 and its immediate response gene FOS were stimulated by loading. Also, SOX9 protein increased after loading only in d21 and d35 but not in d3 constructs. Interestingly, levels of phosphorylated Smad 1/5/9 protein declined during construct maturation, but no evidence was obtained for load-induced changes in pSmad 1/5/9 although BMP2 and BMP6 expression were stimulated by loading. Selected MAPK-, calcium-, Wnt- and Notch-responsive genes raised significantly independent of construct maturity albeit with a generally weaker amplitude in d3 constructs. In conclusion, construct maturity determined whether cells showed an anabolic or catabolic response to the same loading episode and this was apparently determined by a differential SOX9 and pERK signaling response on a background of high versus low total pSmad1/5/9 protein levels. Next step is to use signaling inhibitors to investigate a causal relationship between Smad levels and a beneficial loading response in order to design cartilage replacement tissue for an optimal mechanical response for in vivo applications

Objectives. Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. Methods. We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay. Results. The expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter. Conclusions. Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing. Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154–161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1

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

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6

Mesenchymal stromal cells (MSC) are multipotent, self-renewing cells that are an attractive cell source for cartilage regeneration strategies. While articular chondrocytes form stable cartilage-like tissue under chondrogenic in vitro conditions, a still unsolved problem of chondrocyte production from MSC is their endochondrol development leading to the formation of transient instead of stable articular cartilage. In order to identify relevant molecular determinants of chondrocyte redifferentiation versus MSC chondrogenesis and hypertrophy, this study assessed the differential expression of members of the transforming growth factor β (TGF-β) -superfamily, their receptors and antagonists between differentiating MSC and human articular chondrocytes (HAC). Chondrogenesis of human MSC and redifferentiation of HAC was induced in micromass pellet culture. Gene expression of MSC (n=5) and HAC (n=5) was compared using a transcriptome analysis on Illumina platform. Functional regulation of relevant candidate molecules was assessed in independent MSC and HAC populations by qRT-PCR. Smad signalling during chondrogenic differentiation was analysed by immunohistochemistry and Western Blotting. BMP signalling in both populations was modulated by co-treatment with BMP-4/7 or an inhibitor of Smad1/5/9 signalling. Proteoglycan and DNA content, collagen type II and -X deposition, gene expression of chondrogenic and hypertrophic markers as well as alkaline phosphatase (ALP) activity were quantitatively assessed at different time points. In HAC, TGF-β receptor 2 and 3 (TGFBR2/3) were up-regulated to significantly higher levels than in MSC. BMP4, expressed during HAC expansion, was suppressed while CHL2 and CHRD levels raised. In MSC, BMP4 and BMP7 were induced while TGFBR2 and TGFBR3 were down-regulated. Staining for pSmad1/5/9 in HAC demonstrated positive cells dispersed throughout the pellets at day 3 and 5 while lower pSmad1/5/9 immunostaining was observed in MSC. In HAC and MSC pellets pSmad staining decreased during chondrogenesis, in line with Western Blot results. Medium supplementation with BMP-4/7 did not improve cartilaginous matrix deposition by MSC but raised ALP-activity. When Smad1/5/9 phosphorylation was blocked in MSC culture by dorsomorphin treatment (day 14–42) COL2A1 and COL10A1 expression decreased significantly and collagen type II and type X deposition were reduced. ALP activity dropped to 12 % of control levels. Inhibition of pSmad1/5/9 signalling was unattractive to shift chondrogenesis of MSC away from endochondral development since it unpaired SOX9 expression and strongly reduced cartilaginous matrix deposition along with hypertrophy. Thus no simple correlation exists between beneficial pSmad2/3 versus unwanted pSmad1/5/9 signalling during MSC chondrogenesis

Introduction. Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1). 2. (α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1). 3. which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ. Materials and Methods. Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [. 14. C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml). Results. There was an increase in both COL1A1 and COL1A2 gene expression when stimulated with TGFβ in both cell types. In equine tenocytes the gene expression ratio of COL1A1:COL1A2 increased from 1.73 ± 0.75 to 7.87 ± 2.9 (p=0.003) when stimulated with 100 ng/ml of TGFβ3. TGFβ upregulated collagen (I) protein in both cell types. In equine tenocytes (n=3) when stimulated with 100 ng/ml of TGFβ3, the α1:α2 protein chain ratio increased from 1.93 ± 0.54 to 3.02 ± 0.32 (p=0.059) in comparison with serum-starved cells, which alongside the changes in gene expression, may be indicative of collagen (I) homotrimer production. Discussion. There were biosynthetic alterations in collagen production, and putative collagen (I) homotrimer when equine tenocytes were stimulated with 100 ng/ml TGFβ3. Future work will focus isolating different collagens by repeated differential salt precipitation. The level of TGFβ receptors and Smad signaling molecules will be also analysed using RT-qPCR and western blotting

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

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect.

Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1.

Objectives

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis.