Objectives. Osteoporosis is a chronic disease. The aim of this study was to identify key genes in osteoporosis. Methods. Microarray data sets GSE56815 and GSE56814, comprising 67 osteoporosis blood samples and 62 control blood samples, were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in osteoporosis using Limma package (3.2.1) and Meta-MA packages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify biological functions. Furthermore, the transcriptional regulatory network was established between the top 20 DEGs and transcriptional factors using the UCSC ENCODE Genome Browser. Receiver operating characteristic (ROC) analysis was applied to investigate the diagnostic value of several DEGs. Results. A total of 1320 DEGs were obtained, of which 855 were up-regulated and 465 were down-regulated. These differentially expressed genes were enriched in Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways, mainly associated with gene expression and osteoclast differentiation. In the transcriptional regulatory network, there were 6038 interactions pairs involving 88 transcriptional factors. In addition, the quantitative reverse transcriptase-polymerase chain reaction result validated the expression of several genes (VPS35, FCGR2A, TBCA, HIRA, TYROBP, and JUND). Finally, ROC analyses showed that VPS35, HIRA, PHF20 and NFKB2 had a significant diagnostic value for osteoporosis. Conclusion. Genes such as VPS35, FCGR2A, TBCA, HIRA, TYROBP, JUND, PHF20, NFKB2, RPL35A and BICD2 may be considered to be potential pathogenic genes of osteoporosis and may be useful for further study of the mechanisms underlying osteoporosis. Cite this article: B. Xia, Y. Li, J. Zhou, B. Tian, L. Feng. Identification of potential pathogenic genes associated with osteoporosis. Bone Joint Res 2017;6:640–648. DOI: 10.1302/2046-3758.612.BJR-2017-0102.R1

Dupuytren's disease (DD) is a fibroproliferative soft tissue disease affecting the palmar fascia of the hand causing permanent and irreversible flexion contracture. Aberrant fibrosis is likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in soft tissue fibrosis in diseases such as DD is not well established. Therefore, we conducted a comprehensive multi-omic study investigating the epigenetic profiles that influence gene expression in DD pathology. Using control (patients undergoing carpal tunnel release) and diseased fibroblasts (patients undergoing Dupuytren's fasciectomy), we conducted ATAC-seq to assess differential chromatin accessibility between control and diseased fibroblasts. Additionally, ChIP-seq mapped common histone modifications (histone H4; H3K4me3, H3K9me3, H3K27me3, H4K16Ac, H4K20Me3) associated with fibrosis. Furthermore, we extracted RNA from control and DD tissue and performed bulk RNA-seq. ATAC-seq analysis identified 2470 accessible genomic loci significantly more accessible in diseased fibroblasts compared to control. Comparison between diseased and control cells identified numerous significantly different peaks in histone modifications (H4K20me3, H3K27me3, H3K9me3) associated with gene repression in control cells but not in diseased cells. Pathway analysis demonstrated a substantial overlap in genes being de-repressed across these histone modifications (Figure 1). Both, ATAC-seq and ChIP-seq analysis indicated pathways such as cell adhesion, differentiation, and extracellular matrix organisation were dysregulated as a result of epigenetic changes. Moreover, de novo motif enrichment analysis identified transcription factors that possibly contributed to the differential gene expression between control and diseased tissue, including HIC1, NFATC1 and TEAD2. RNA-seq analysis found that these transcription factors were upregulated in DD tissue compared to control tissue. The current epigenetic study provides insights into the aberrant fibrotic processes associated with soft tissue diseases such as DD and indicates that epigenetic-targeted therapies may be an interesting viable treatment option in future. For any figures or tables, please contact the authors directly

Objectives. Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes. Methods. Through a combined approach of gene expression analysis and web-based searching of NFAT1 binding sequences, 25 candidate target genes that displayed aberrant expression in Nfat1. -/-. AC at the initiation stage of OA, and possessed at least four NFAT1 binding sites in the promoter of each gene, were selected and tested for NFAT1 transcriptional activities by chromatin immunoprecipitation (ChIP) and promoter luciferase reporter assays using chondrocytes isolated from the AC of three- to four-month-old wild-type mice or Nfat1. -/-. mice with early OA phenotype. Results. Chromatin immunoprecipitation assays revealed that NFAT1 bound directly to the promoter of 21 of the 25 tested genes encoding cartilage-matrix proteins, growth factors, inflammatory cytokines, matrix-degrading proteinases, and specific transcription factors. Promoter luciferase reporter assays of representative anabolic and catabolic genes demonstrated that NFAT1-DNA binding functionally regulated the luciferase activity of specific target genes in wild-type chondrocytes, but not in Nfat1. -/-. chondrocytes or in wild-type chondrocytes transfected with plasmids containing mutated NFAT1 binding sequences. Conclusion. NFAT1 protects AC against degradation by directly regulating the transcription of target genes in articular chondrocytes. NFAT1 deficiency causes defective transcription of specific anabolic and catabolic genes in articular chondrocytes, leading to increased matrix catabolism and osteoarthritic cartilage degradation. Cite this article: M. Zhang, Q. Lu, T. Budden, J. Wang. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone Joint Res 2019;8:90–100. DOI: 10.1302/2046-3758.82.BJR-2018-0114.R1

Abstract. Objective. SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between mesenchymal stromal cells (MSCs) and chondrocytes by comparing SOX gene expression in their co-culture and respective monocultures. Methods. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in flow cytometry and were allowed to undergo trilineage differentiation for subsequent histological investigation. SOX5, SOX6, and SOX9 expression of co-cultures and monoculture controls were quantified by TaqMan quantitative real-time PCR. Experiments were performed in triplicate. Results. AMSC phenotype was evidenced by the expression of CD105, CD73, CD90 & heterogeneous CD34 but not CD45, CD14, CD19 & HLA-DR in flow cytometry, and also differentiation into chondrogenic, osteogenic and adipogenic lineages with positive Alcian blue, Alizarin Red and Oil Red O staining. The expression of SOX5, SOX6, and SOX9 was greater in observed co-cultures than would be expected from an expression profile modelled from monocultures. Conclusions. These findings provide evidence for the upregulation of SOX family transcription factors expression during the co-culture of MSCs and chondrocytes, suggesting an active induction of chondrogenic differentiation and change of cell fate amidst a microenvironment that facilitates cell-contact and paracrine secretion. This provides insight into the chondrogenic potential and therapeutic effects of MSCs preconditioned by the chondrocyte secretome (or potentially chondrocytes reinvigorated by the MSC secretome), and ultimately, cartilage repair. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Glucocorticoid excess is shown to deteriorate bone tissue integrity, increasing the risk of osteoporosis. Marrow adipogenesis at cost of osteogenesis is a prominent feature of this osteoporosis condition. Epigenetic pathway histone deacetylase (HDAC)-mediated histone acetylation regulates osteogenic activity and bone mass. This study is aimed to figure out what role of acetylated histone reader bromodomain-containing protein 4 (BRD4) did play in glucocorticoid-induced osteoporosis. Bone-marrow mesenchymal stem cells were incubated in osteogenic medium with or without 1 μM dexamethasone. Mineralized matrix and adipocyte formation were probed using von Kossa and Nile Red O staining, respectively. Osteogenic and adipogenic marker expression were quantified using RT-PCR. The binding of acetylated histone to promoter of transcription factors were detected using chromatin immunoprecipitation-PCR. Bone mineral density and microstructure in osteoporotic bone were quantified with microCT system. Glucocorticoid repressed osteogenic transcription factor Runx2 expression and mineralized matrix formation along with a low level of acetylated lysine 9 at histone 3 (H3K9ac), whereas BRD4 signaling and adipocytic formation were increased in cell cultures. BRD4 knockdown reversed the H3K9ac enrichment in Runx2 promoter and osteogenesis, but downregulated adipogenic differentiation. Silencing BRD4 attenuated H3K9ac occupancy in forkhead box P1 (Foxp1) relevant to lipid metabolism upon glucocorticoid stress. Foxp1 interference downregulated adipogenic activities of glucocorticoid-treated cells. In vivo, treatment with BRD4 inhibitor JQ-1 compromised the glucocorticoid-induced bone mineral density loss, spare trabecular structure, and fatty marrow, as well as improved biomechanical properties of bone tissue. Taken together, BRD4-mediated Foxp1 pathways drive mesenchymal stem cells shifting toward adipocytic cells rather than osteogenic cells to aggravates excessive marrow adipogenesis in the process of glucocorticoid-induced osteoporosis. Pharmacological inhibition of BRD4 signaling protects bone tissue from bone loss and fatty marrow in glucocorticoid-treated mice. This study conveys a new molecular insight into epigenetic regulation of osteogenesis and adipogenesis in osteoporotic skeleton and highlight the remedial effect of BRD4 inhibitor on glucocorticoid-induced bone loss

SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between and preconditioning of mesenchymal stem cells (MSCs) with chondrocytes through comparing SOX gene expression in their co-culture and respective monocultures. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Samples were handled according to the 2004 UK Human Tissue Act. Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in flow cytometry and were allowed to undergo trilineage differentiation for subsequent histological investigation. SOX5, SOX6, and SOX9 expression of co-cultures and monoculture controls were quantified by TaqMan quantitative real-time PCR. Experiments were performed in triplicate. AMSC phenotype was evidenced by the expression of CD105, CD73, CD90 & heterogenous CD34 but not CD45, CD14, CD19 & HLA-DR in flow cytometry, and also differentiation into chondrogenic, osteogenic and adipogenic lineages with positive Alcian blue, Alizarin Red and Oil Red O staining. The expression of SOX5, SOX6, and SOX9 were greater in observed co-cultures than would be expected from an expression profile modelled from monocultures. The findings provides evidence for the upregulation of SOX family transcription factors expression during the co-culture of MSCs and chondrocytes, suggesting an active induction of chondrogenic differentiation and change of cell fate amidst a microenvironment that facilitates cell-contact and paracrine secretion. This provides insight into the chondrogenic potential and therapeutic effects of MSCs preconditioned by the chondrocyte secretome (or potentially chondrocytes reinvigorated by the MSC secretome), and ultimately, cartilage repair

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes. A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading. In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13

As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis. Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and flow cytometry were performed to ensure the specific role of KLF4. Finally, TiPs-induced mice cranial osteolysis model was established, and micro-CT scanning and immunohistochemistry assay were performed to reveal the role of p110δ and KLF4 in vivo. Here, we found that p110δ was upregulated in TiPs-stimulated macrophages. The inhibition of p110δ or knockdown of p110δ could significantly dampen the TiPs-induced secretion of TNFα and IL-6. Further mechanistic studies confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. Additionally, we explored the upstream regulators and confirmed that Krüppel-like factor 4 (KLF4) was the transcription repressor of p110δ. Apart from that, KLF4, targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. By the establishment of TiPs-induced mice cranial osteolysis model, we found that KLF4 knockdown exacerbated TiPs-induced osteolysis which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis

Knee osteoarthritis (KOA) diagnosis is based on symptoms, assessed through questionnaires such as the WOMAC. However, the inconsistency of pain recording and the discrepancy between joint phenotype and symptoms highlight the need for objective biomarkers in KOA diagnosis. To this end, we study relationships among clinical and molecular data in a cohort of women (n=51) with Kellgren-Lawrence grade 2–3 KOA through Support Vector Machine (SVM) and a regulation network model (RNM). Clinical descriptors (i.e., pain catastrophism (CA); depression (DE); functionality (FU); joint pain (JP); rigidity (RI); sensitization (SE); synovitis (SY)) are used to classify patients. A Youden's test is performed for each classifier to determine optimal binarization thresholds for the descriptors. Thresholds are tested against patient stratification according to baseline WOMAC data from the Osteoarthritis Initiative, and the mean accuracy is 0.97. For our cohort, the data used as SVM inputs are KOA descriptors, synovial fluid (SL) proteomic measurements (n=25), and transcription factors (TF) activation obtained from RNM [2] stimulated with the SL measurements. The relative weights after classification reflect input importance. The performance of each classifier is evaluated through AUC-ROC analysis. The best classifier with clinical data is CA (AUC = 0.9), highly influenced by FU and SE, suggesting that kinesophobia is involved in pain perception. With SL input, leptin strongly influences every classifier, suggesting the importance of low-grade inflammation. When TF are used, the mean AUC is limited to 0.608, which can be related to the pleomorphic behaviour of osteoarthritic chondrocytes. Nevertheless, FU has an AUC of 0.7 with strong importance of FOXO downregulation. Though larger and longitudinal cohorts are needed, this unique combination of SVM and RNM shall help to map objectively KOA descriptors. Acknowledgements: Catalan & Spanish governments 2020FI_b00680; STRATO-PID2021126469ob-C21-2, European Commission (MSCA-TN-ETN-2020-Disc4All-955735, ERC-2021-CoG-O-Health-101044828). ICREA Academia

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Introduction. Despite the high regenerative capacity of bone, large bone defects often require treatment involving bone grafts. Conventional autografting and allografting treatments have disadvantages, such as donor site morbidity, immunogenicity and lack of donor material. Bone tissue engineering offers the potential to achieve major advances in the development of alternative bone grafts by exploiting the bone-forming capacity of osteoblastic cells. However, viable cell culture models are essential to investigate osteoblast behavior. Three-dimensional (3D) cell culture systems have become increasingly popular because biological relevance of 3D cultures may exceed that of cell monolayers (2D) grown in standard tissue culture. However, only few direct comparisons between 2D and 3D models have been published. Therefore, we performed a pilot study comparing 2D and 3D culture models of primary human osteoblasts with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation. Patients and Methods. Primary human osteoblasts were extracted from femoral neck spongy bone obtained during surgery procedures. Primary human osteoblasts of three donor patients were cultured in monolayers and in three different 3D culture models: 1) scaffold-free cultures, also referred to as histoids, which form autonomously after multilayer release of an osteoblast culture; 2) short-term (10-day) collagen scaffolds seeded with primary human osteoblasts (HOB); 3) long-term (29-day) collagen scaffolds seeded with HOB. Expression levels of transcription factors RUNX2 and osterix, both involved in osteoblast differentiation, were investigated using quantitative PCR and immunohistochemical staining. Furthermore, markers of osteogenic differentiation were evaluated, such as alkaline phosphatase activity, osteocalcin expression, and mineral deposition, as well as the expression of collagen type I and fibronectin extracellular matrix proteins. Results. Cells of the same origin, which were cultivated in different culture models, showed varying expression levels with regard to transcription factors RUNX2 and osterix as well as osteogenic markers. Increased levels of transcription factor RUNX2 and the extracellular matrix protein fibronectin were observed in all 3D cell culture models compared to monolayers. Furthermore, long-term cultivated histoids showed increased levels of osteogenic late-stage marker osteocalcin and transcription factor osterix. Additionally, long-term collagen scaffolds seeded with HOB showed elevated levels of osteocalcin compared to monolayers and short-term scaffolds. Moreover, alkaline phosphatase activity and mineralization capacity were increased in histoids. Conclusion. Considering the complex biochemical interactions of cells with surrounding cells and the extracellular matrix in vivo, important biological properties are disregarded when cells are only studied in 2D study models. Hence, we compared different 3D HOB cell culture models to 2D HOB monolayers with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation in vitro. Our pilot study indicated that three-dimensional study models may promote osteogenic differentiation in vitro. Additionally, a beneficial effect of longer culture duration on osteogenic differentiation was observed. Hence, our findings emphasise the importance of dimension and culture duration when studying osteoblast function. Subsequent studies with higher sample sized may lead to the development of viable primary human osteoblast cell culture models for bone tissue engineering. Summary. Three-dimensional cell culture models of primary human osteoblasts (HOB), including collagen scaffolds and scaffold-free cultures, were compared to HOB monolayers with regard to osteogenic differentiation. Our study indicated that three-dimensional study models may promote osteogenic differentiation of HOB in vitro

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a crucial transcription factor to maintain cellular redox homeostasis, but is also affecting bone metabolism. As the association between Nrf2 and osteoporosis in elderly females is not fully elucidated, our aim was to shed light on the potential contribution of Nrf2 to the development of age-dependent osteoporosis using a mouse model. Female wild-type (WT, n=18) and Nrf2-knockout (KO, n=12) mice were sacrificed at different ages (12 weeks=young mature adult, and 90 weeks=old), morphological cortical and trabecular properties of femoral bone analyzed by micro-computed tomography (µCT), and compared to histochemistry. Mechanical properties were derived from quasi-static compression tests and digital image correlation (DIC) used to analyze full-field strain distribution. Bone resorbing cells and aromatase expression by osteocytes were evaluated immunohistochemically and empty osteocyte lacunae counted in cortical bone. Wilcoxon rank sum test was used for data comparison and differences considered statistically significant at p<0.05. When compared to old WT mice, old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), and cortical bone fraction (Ct.Ar/Tt.Ar). Surprisingly, these parameters were not different in skeletally mature young adult mice. Metaphyseal trabeculae were thin but present in all old WT mice, while no trabecular bone was detectable in 60% of old KO mice. Occurrence of empty osteocyte lacunae did not differ between both groups, but a significantly higher number of osteoclast-like cells and fewer aromatase-positive osteocytes were found in old KO mice. Furthermore, female Nrf2-KO mice showed an age-dependently reduced fracture resilience when compared to age-matched WT mice. Our results confirmed lower bone quantity and quality as well as an increased number of bone resorbing cells in old female Nrf2-KO mice. Additionally, aromatase expression in osteocytes of old Nrf2-KO mice was compromised, which may indicate a chronic lack of estrogen in bones of old Nrf2-deficient mice. Thus, chronic Nrf2 loss seems to contribute to age-dependent progression of female osteoporosis

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Objectives. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Methods. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab. Results. Low mRNA expression of PIEZO1 in unloaded cells was upregulated 5-fold following 1-hr of mechanical load (p=0.003). In addition, the transcription factor NFATc1, a known regulator of Piezo1 mechanotransduction, was also upregulated by load (2.4-fold; p=0.03). Y201 cells differentiated in gels expressed the osteocyte marker, SOST. Yoda1 upregulated PIEZO1 (1.7-fold; p=0.057), the early mechanical response gene, cFOS (4-fold; p=0.006), COL1A1 (3.9-fold; p=0.052), and IL-6 expression (7.7-fold; p=0.001). Discussion. This study reveals PIEZO1 as an important mechanosenser in osteocytes. Piezo 1 mediated increases in the bone matrix protein, type I collagen, and IL-6, a cytokine that drives inflammation and bone resorption. This provides a direct link between mechanical activation of Piezo 1, bone remodelling and inflammation, which may contribute to mechanically-induced joint degeneration in osteoarthritis. Mechanistically, we hypothesise this may occur through promoting Ca2+ influx and activation of the NFAT1 signalling pathway

Histone modifications critically contribute to the epigenetic orchestration of bone development - in part by modifying accessibility of genes to transcription factors. Based on the previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue development, we here analyzed the molecular and cellular mechanisms of Mysm1-p53 interplay in bone development. The bone phenotype of 4–5 week-old Mysm1-/- (MKO), Mysm1-/-p53-/- (DKO) and corresponding wildtype (WT) mice was determined using µCT and histology. Primary osteoblasts, mesenchymal stem cells (MSCs) and osteoclasts were isolated from long bones to assess cell proliferation, differentiation, apoptosis and activity. Statistics: one-way ANOVA, p<0.05. MKO mice displayed an osteopenic bone phenotype compared to WT (BV/TV: 5.7±2.9 vs. 12.5±4.2, TbN: 1.3±0.6 vs. 2.7±0.7 1/mm, respectively), and these effects were abolished in DKO mice (BV/TV: 17.8±2.6, TbN: 3.7±0.4 1/mm). MKO mice compared to WT also showed both in vitro and in vivo disturbed osteoclast formation (in vitro: 1.5±1.2 vs. 9.9±1.8 OcN/mm2, in vivo OcN/BPm: 1.4±1.0 vs. 3.0±0.7 cells/mm, respectively) accompanied by increased apoptosis and DNA damage; additional p53 knockout attenuated these effects (7.8±1.8 OcN/mm2 and OcN/BPm: 2.2±1.0 cells/mm). Primary osteoblasts from both MKO and DKO mice showed decreased expression of the transcription factor Runx2 and of the osteogenic markers. ChIP-Seq analysis revealed direct binding of Mysm1 to Runx2 promoter regions in osteoblasts, implying that Mysm1 here regulates osteogenic differentiation. In contrast, MKO-MSCs differentiation did not differ from WT, but DKO-MSCs displayed a significantly increased expression of Alpl, Bglap and Runx2. The different effects of Mysm1-/- in MSCs and osteoblasts presumably resulted from the lower expression level of Mysm1 in MSCs in comparison to mature osteoblasts. Thus, our data demonstrate that H2A deubiquitinase Mysm1 is essential for the epigenetic control of bone development via distinct mechanisms: 1) In osteoclasts, Mysm1 is involved in maturation of osteoclast progenitors and osteoclast survival. 2) In osteoblasts, Mysm1 directly controls Runx2 expression, thereby explaining osteopenic phenotype of MKO mice. 3) In MSCs, Mysm1 may play an inferior role due to low expression level. However, loss of p53 increases Runx2 expression during MSC differentiation, leading to normal bone formation in DKO mice

Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Methods. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on mRNA level via qRT-PCR and protein level via Western-Blotting. Chondrogenesis was evaluated by histology at d42 and expression of chondrogenic markers like COL2. Hypertrophic development was characterized by ALP activity and expression of hypertrophic markers like COL10. Results. Hypertrophic development, characterized by upregulation of COL10, high COL10/COL2 ratios and ALP activity, was confirmed in MSC and absent in AC. MSC started into differentiation with less SOX9 before induction, while higher RUNX2/3 was observed compared to AC. During MSC chondrogenesis SOX9 and MEF2C steadily increased on mRNA and protein level. Surprisingly, although RUNX2 mRNA level increased in MSC over 42 days, RUNX2 protein remained undetectable. During AC redifferentiation, SOX9 levels remained high on mRNA and protein level while RUNX2/3 and MEF2C remained low. Conclusion. After expansion and before applying chondrogenic stimuli, a chondrogenic priming with more SOX9 and lower RUNX2/3 was found in AC. In contrast osteochondral priming with higher RUNX2/3 and lower SOX9 levels was observed in MSC which could set the stage for endochondral development, leading to hypertrophy. Dynamic regulation of RUNX2/3 and MEF2C at lower SOX9 background levels separated MSC from AC differentiation over 42 days. Adjusting transcription factor levels in MSC could be essential for creating a protocol leading to diminished hypertrophy of MSC during chondrogenesis

Summary Statement. Ischaemic preconditioning protected skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection was associated with increased Nrf2 signalling. Introduction. Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to a period of prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. While much is known about the ability of ischaemic preconditioning to protect myocardial tissue against ischaemia-reperfusion injury, its potential to confer benefit in an orthopaedic setting by protecting skeletal muscle remains relatively unexplored to date. One mechanism by which ischaemic preconditioning may induce protection is through a reduction in oxidative stress. Reactive oxygen species (ROS) are generated both during prolonged ischaemia and also upon reperfusion by infiltrating neutrophils, thereby leading to an increase in oxidative stress. The transcription factor, NF-E2-related factor 2 (Nrf2), is a key regulator of the cells response to oxidative stress as it regulates the expression of a network of anti-oxidant/detoxifying enzymes. Nrf2 signalling has recently been shown to protect against ischaemia-reperfusion injury in both a kidney cell line and in liver biopsies, indicating that this transcription factor may play a key role in the protection provided by ischaemic preconditioning. To date, the involvement of Nrf2 in the response of skeletal muscle to ischaemia-reperfusion has not been investigated. Thus, the aims of this study were to investigate the ability of ischaemic preconditioning to protect skeletal myotubes against ischaemia-reperfusion and to determine the role of Nrf2 signalling in this protection. Materials & Methods. C2C12 mouse myoblasts were maintained at 37. o. C in a humidified atmosphere of 95% air and 5% CO. 2. in DMEM containing 20% FBS. When cultures were approximately 90% confluent, myoblasts were differentiated to myotubes by changing to DMEM supplemented with 2% horse serum and culturing for 7–10 days. Differentiated myotubes were then exposed to simulated ischaemia for 4h (1% O. 2. ) followed by 2h reoxygenation (21% O. 2. ). To precondition myotubes, cells were subjected to 30 min of simulated ischaemia followed by 1 hour reoxygenation prior to the prolonged ischaemic event. Cell survival was assessed by lactate dehydrogenase release. Changes in Nrf2 expression were assessed using real-time PCR, Western blotting and immunofluorescence. Changes in sequestosome-1 (SQSTM1), catalase (CAT), glutathione S-transferase theta-1 (GSTT1), heme oxygenase-1 (HO-1) expression were assessed using a combination of real-time PCR and Western blotting. Results. Preconditioned myotubes showed greater viability both after 4h of ischaemia, and after 4h ischaemia followed by 2h of reoxygenation. This increase in cell viability was associated with increased Nrf2 expression. In addition, increased expression of SQSTM1, and the antioxidant enzymes, CAT, GSTT1 and HO-1 was observed in preconditioned myotubes. Discussion. Our findings indicate that ischaemic preconditioning can protect skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection is associated with increased Nrf2 signalling indicating that this transcription factor may play a role in mediating the protection induced by ischaemic preconditioning. By modulating the response of skeletal muscle to ischaemia, ischaemic preconditioning has the potential to limit reperfusion injury, which in turn, may lead to improvements in outcome following orthopaedic surgery

Mesenchymal stromal cells (MSCs) have been intensively researched in the orthopaedic field since they hold great promise for aiding the regeneration of musculoskeletal tissues. While there are a range of postulated surface markers to identify MSCs, currently there are no known cell markers that predict in vivo osteochondral potency. Runt-related transcription factor 2 (Runx2) is considered as an essential transcription factor in osteoblast differentiation [1] and has been shown to physically interact with retinoblastoma protein (pRb), which leads the loss of osteoblast proliferation and the activation of genes concerning terminal differentiation of osteoblasts [2]. The aim of this study was to use adenoviral-mediated gene overexpression/knockdown to investigate the interplay between Runx2 and pRb during in vitro osteogenic differentiation of human bone marrow (hBM)-MSCs. A first generation human adenovirus (hAd) serotype 5 dE/E3 carrying the gene of interest (Runx2 or shRNA-Runx2) were propagated and amplified in AD-293 cells, and purified over successive CsCl gradients. A second generation hAd serotype 5 carrying the gene of interest (Rb1) was generated. High efficiency single or double transduction of undifferentiated hBM-MSCs was achieved using lanthofection [3]. The transduced hBM-MSCs were then differentiated in osteogenic medium (OM) and osteogenic potency was assessed by quantification of alkaline phosphatase (ALP) activity (day 14) and Alizarin red staining (day 28). In addition, cell cultures were assessed for absorbance at OD 450nm, correlating to the refractive index of calcified areas, at days 0, 7, 14, 21 and 28 [4]. Quantitative RT-PCR was used to confirm expression of target genes following viral transduction. Basal medium was used as a control. Untransduced hBM-MSCs cultures grown in OM demonstrated peak calcium deposition at day 28, while the overexpression of either Runx2 or Rb1 accelerated peak calcium deposition to day 21. Consistent with this, Runx2 overexpression increased ALP activity of hBM-MSCs cultured in OM, while Rb1 overexpression enhanced ALP activity of hBM-MSCs cultured in both basal and osteogenic conditions. Co-expression of Runx2 and Rb1 did not further increase ALP activity compared to cells transduced with Runx2 or Rb1 alone. Alizarin red staining revealed that overexpression of either Runx2 or Rb1 increased mineral deposition in hBM-MSCs under basal conditions, although mineralisation was not enhanced above that of untransduced cells when cultured in OM. However, mineralisation was markedly enhanced above levels in untransduced cells when Runx2 and Rb1 were co-expressed in hBM-MSCs grown under both basal and osteogenic conditions. This study demonstrates an important stimulatory role of pRb in enhancing ALP activity of hBM-MSCs in the absence of osteogenic clues. However, pRb overexpression alone is insufficient to enhance mineralisation, requiring the co-expression of Runx2 in hBM-MSCs. The crucial nature of Runx2 for osteogenic differentiation of hBM-MSCs was demonstrated since knockdown of Runx2 prevented both mineral deposition and the increased ALP activity observed in untransduced cells grown in OM. Interestingly, overexpression of Rb1 could not compensate for the knockdown of Runx2 since Rb1 overexpression did not recover either mineral deposition or ALP activity in hBM-MSCs where Runx2 expression was inhibited

Objectives. Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Methods. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. Results. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. Conclusions. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred. Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J. Russell, C. L. Mendias. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear. Bone Joint Res 2017;6:57–65. DOI: 10.1302/2046-3758.61.BJR-2016-0232.R1