Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502

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.

RNA-Seq or whole transcriptome shotgun sequencing has been adopted in the last years as a reference technique to determine the presence and the quantity of different species of RNA in determined biological samples, thanks to it allows the identification every single RNA species transcribed from a reference genome. Meta-profiling takes advantage of the public availability of an increasing set of RNA-Seq data produced by different laboratories to summarize the expression levels of the different RNA species of many samples according to their biological context, giving the opportunity to perform comparisons on the gene expression profiles of different tissues by integrating data derived from a high number of studies. By using Genevestigator™; a platform which integrates RNA-Seq data into meta-profiles, we have performed a comparison between the gene expression profiles of bone, cartilage, muscle tendon and skin by means of interrogating its database with different gene sets and families with relevance to the function of the tissues of the musculoskeletal system. The collagen gene family and genes coding for proteoglycans, matrix metalloproteinases and tissue inhibitors of metalloproteinases, mechanotransduction-related proteins and signalling pathways involved in tissue development and differentiation have been analysed. Hierarchical clustering for every gene set was performed for the understanding the differences and similarities between the different tissues included in the analyses. The results of this study will help to improve our understanding of the musculoskeletal system, and will help to identify new biomarkers and signalling pathways of specific relevance for the bone, cartilage, muscle and tendon

Introduction. PIEZO mechanoreceptors are increasingly recognized to play critical roles in fundamental physiological processes like proprioception, touch, or tendon biomechanics. However, their gating mechanisms and downstream signaling are still not completely understood, mainly due to the lack of effective tools to probe these processes. Here, we developed new tailor-made nanoswitches enabling wireless targeted actuation on PIEZO1 by combining molecular imprinting concepts with magnetic systems. Method. Two epitopes from functionally relevant domains of PIEZO1 were rationally selected in silico and used as templates for synthesizing molecularly imprinted nanoparticles (MINPs). Highly-responsive superparamagnetic zinc-doped iron oxide nanoparticles were incorporated into MINPs to grant them magnetic responsiveness. Endothelial cells (ECs) and adipose tissue-derived stem cells (ASCs) incubated with each type of MINP were cultured under or without the application of cyclical magnetomechanical stimulation. Downstream effects of PIEZO1 actuation on cell mechanotransduction signaling and stem cell fate were screened by analyzing gene expression profiles. Result. Nanoswitches showed sub-nanomolar affinity for their respective epitope, binding PIEZO1-expressing ECs similarly to antibodies. Expression of genes downstream of PIEZO1 activity significantly changed after magnetomechanical stimulation, demonstrating that nanoswitches can transduce this stimulus directly to PIEZO1 mechanoreceptors. Moreover, this wireless actuation system proved effective for modulating the expression of genes related to musculoskeletal differentiation pathways in ASCs, with RNA-sequencing showing pronounced shifts in extracellular matrix organization, signal transduction, or collagen biosynthesis and modification. Importantly, targeting each epitope led to different signaling effects, implying distinct roles for each domain in the sophisticated function of these channels. Conclusion. This innovative wireless actuation technology provides a promising approach for dissecting PIEZO-mediated mechanobiology and suggests potential therapeutic applications targeting PIEZO1 in regenerative medicine for mechanosensitive tissues like tendon. Acknowledgements. EU's Horizon 2020 ERC under grant No. 772817 and Horizon Europe under grant No. 101069302; FCT/MCTES for PD/BD/143039/2018, COVID/BD/153025/2022, 10.54499/2020.03410.CEECIND/CP1600/CT0013, 10.54499/2022.05526.PTDC, 10.54499/UIDB/50026/2020, 10.54499/UIDP/50026/2020, and 10.54499/LA/P/0050/2020

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator. Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10. 6. /mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment. Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect. This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation

Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns. Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6. We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to MAPK and transforming growth factor beta (TGF-β). Respective genes, such as TGF-β (Tgfb1) and its receptor (Tgfbr) were found to be upregulated, and negative MAPK regulators were found to be downregulated, both by microarray analysis and qRT-PCR. Accordingly, we saw a C5aR1- and TLR2-dependent phosphorylation of p38 MAPK. Interestingly, this effect was enhanced and prolonged by costimulation of both receptors. An additive effect of C5aR1 and TLR2 was also seen regarding Cxcl10 levels, which were enhanced compared to C5aR1 or TLR2 stimulation alone. This study shows that C5aR1 and TLR2 interact in osteoblasts, not only physically but also functionally, regarding downstream signaling and target genes. Those data strongly imply a synergistic interplay between the receptors, through which osteoblasts possibly contribute to inflammatory reactions affecting bone

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. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation. Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594–601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1

Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test, gene expression profile, histology and immunohistochemistry. Results showed the maintenance and preservation, at time 0 and after 40 days of culture, of the tissue viability, biological activity, as well as molecular markers, i.e. several key genes involved in the complex interactions between the tumour cells and bone able to drive cancer progression, cancer aggressiveness and metastasis to bone. A good tis sue morphological and microarchitectural preservation with the presence of lacunar osteolysis, fragmented trabeculae locally surrounded by osteoclast cells and malignant cells and an intense infiltration by tumour cells in bone marrow compartment in all examined samples. Histomorphometrical data on the levels of bone resorption and bone apposition parameters remained constant between T0 and T40 for all analysed patients. Additionally, immunohistochemistry showed homogeneous expression and location of CDH1, CDH2, KRT8, KRT18, Ki67, CASP3, ESR1, CD8 and CD68 between T0 and T40, thus further confirming the invasive behaviour of breast cancer cells and indicating the maintaining of the metastatic microenvironment. The novel tissue culture, set-up in this study, has significant advantages in comparison to the pre-existent 3D models: the tumour environment is the same of the clinical scenario, including all cell types as well as the native extracellular matrix; it can be quickly set-up employing only small samples of breast cancer bone metastasis tissue in a simple, ethically correct and cost-effective manner; it bypasses and/or decreases the necessity to use more complex preclinical model, thus reducing the ethical burden following the guiding principles aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes; it can allow the study of the interactions within the breast cancer bone metastasis tissue over a relatively long period of up to 40 days, preserving the tumour morphology and architecture and allowing also the evaluation of different biological factors, parameters and activities. Therefore, the study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumour subtypes so predicting patient specific responses

Summary Statement. Paraspinal muscle contain higher proportion of slow-twich fibers. The fixation of the rat tail induced transition of muscle fiber types in the paravertebral muscles characterised by the decrease in the proportion of the slow type myosin heavy chain. Introduction. Lumbar degenerative kyphosis often accompanies back pain, easy fatigability, fatty degeneration and atrophy of back muscles. There are two types of skeletal muscle fibers according to oxidative activities: slow-twich (Type 1) and fast-twitch (Type 2) fibers. Type 2 fibers were subdivided into three types: Type 2A, 2B and 2D/X. Each fiber type primarily expresses a specific isoform of myosin heavy chain (MHC). It has been known that back muscles contain higher proportion of MHC type 1. However, the impact of kyphosis on the proportion of fiber types in the paravertebral muscles has not been fully understood. The aim of this study is to analyze the transition of muscle fiber types after kyophotic or straight fixation using a rat tail model. Methods. A rat tail was fixed in straight or kyphotic position (straight or kyphosis group) by a custom-made external fixator and wires. A group of animals which underwent only pierced wounds in their tails served as control. The gene expression profiles of isoforms of MHCs in dorsal coccygeal muscles were analyzed by quantitative RT-PCR. The fiber types of muscles were assessed using SDS-PAGE. Band densities of silver-stained gel were quantified. Results. At first, the gene expression profiles of MHCs and protein expression in the dorsal coccygeal muscles were compared with tibilis anterior and gastrocunemius muscles. Higher proportion of MHC type 1 gene and protein expression were confirmed in the dorsal coccygeal muscles than tibialis anterior and gastrocuneimus muscles. MHC type 2B protein expression was not detected in dorsal coccygeal muscles. Next, coccygeal muscles after straight or kyphotic fixation were analyzed and compared with control. Gene expression of MHC type 1 was decreased at 7 and 28 days after fixation in straight and kyphosis group. The significant difference was seen at 28 days in kyphosis group. The band densities of MHC protein type 1 and 2A plus 2D/X were decreased in both straight and kyophosis groups at 28 days after fixation while sample volume was adjusted by wet wight of dissected coccygeal muscles. The mean proportion of MHC protein type 1 separated by SDS-PAGE were decreased in straight and kyphosis group. The difference was significant in straight group. Discussion. Our results demonstrated that the fixation of the rat tail induced transition of muscle fiber types in the paravertebral muscles characterized by the decrease in the proportion of the MHC type 1. Back muscles are required to contract continuously to keep posture. Slow-twitch fibers in back muscle contribute for continuous contraction. Slow-twitch fibers utilise energy efficiently by oxidative process while fast-twitch fibers mainly consume glucose through glycolysis producing lactate acid. Not only decreased amount of MHC but also decreased proportion of MHC type 1 might be the reason of easy fatigability in lumbar degenerative kyphosis. The limitations of this study is the difference between human paravertebral and rat coccygeal muscles and short duration of observation

Abstract. Objective. Mesenchymal stem cells (MSCs) and chondrocytes have both been crucial in trials for cartilage repair, and there has been growing interest into their respective secretomes owing to their role in chondrogenic crosstalk. This has been studied by in vitro co-culture studies, yet the optimal ratio of seeding MSCs in co-culture has been understudied. Methods. Our study utilised 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=5). To investigate whether a large proportion of MSCs could be stimulated by a small number of chondrocytes, we seeded these MSCs at increasing logarithmic ratios to the number of chondrocytes at 1:1, 10:1, and 100:1. The AMSCs were phenotyped by a panel of MSC surface markers in flow cytometry, and allowed to undergo trilineage differentiation. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenesis. Experiments were performed in triplicate. Results. The AMSCs had CD105, CD73, CD90, and heterogenous CD34 expression but not CD45, CD14, CD19, and HLA-DR expression in flow cytometric phenotyping, and demonstrated differentiation into chondrogenic, osteogenic, and adipogenic lineages. The chondrogenic gene expression profiles from co-cultures of larger MSC-to-chondrocyte ratio such as 10:1 and 100:1 were significantly lower than the expression profile of the 1:1 co-culture. No significant difference was observed between the 10:1 and 100:1 co-cultures. Conclusion. These findings suggest that the optimal ratio of co-culturing MSCs and chondrocytes approaches 1:1, and that seeding at larger ratios would diminish the overall chondrogenic expression and crosstalk involved. This therefore has implications in the limited efficacy of MSCs in in vitro co-culture studies or in existing trials of intra-articular and subchondral MSC injections, owing to a suboptimal in situ ratio of MSCs and chondrocytes. 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

Summary Statement. A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction. Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo. Methods. hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global gene expression profiles were evaluated prior and after bioreactor culture. Bone development was investigated using biochemical and histological methods, and by micro-computed tomography (μCT) imaging over the duration of bioreactor culture and after 12-week subcutaneous implantation in immunodeficient mice. Results. Progenitors with high proliferation potential, expressing typical mesenchymal surface antigens were successfully derived from three hiPSC lines. Differences in mesenchymal surface antigens expression and global gene expression profiles of progenitors from different lines corresponded to their differentiation abilities toward the osteogenic, chondrogenic and adipogenic lineages. Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into the culture medium as compared to static culture. Dense bone matrix formation was evidenced by the positive staining of collagen, osteopontin, bone sialoprotein and osteocalcin. In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce. μCT revealed a significant increase in bone structural parameters, evidencing mineralization of the deposited bone tissue during the 5-week culture in bioreactors. Osteogenesis and bone tissue formation were comparable between hESCs, hiPSCs and hMSCs (3). Bioreactor cultivation resulted in repression of genes involved in proliferation and tumorigenesis, and upregulation of genes associated with osteogenesis and bone development. Engineered bone tissue displayed stable phenotype after 12-week implantation in vivo, with cells of human origin, ingrowing vasculature and osteoclasts, suggesting an initiation of tissue remodeling. Discussion/Conclusion. Our biomimetic strategy opens the possibility to construct an unlimited quantity of patient-specific bone grafts for personalised applications, and to generate qualified experimental models to study bone biology under normal and pathological conditions, as well as test new drugs using selected pools of hiPSC lines

Osteoarthritis (OA) is the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of hypertrophic markers known from the growth plate. Although physiological mechanical loading can support cartilage formation and maintenance, mechanical overload represents one major risk factor for OA development. To date, little is known on how an OA-like hypertrophic chondrocyte phenotype alters the response of cartilage tissue to mechanical loading. The aim of this study was to investigate whether a hypertrophic phenotype change of chondrocytes affects the response to physiological mechanical loading and to reveal differences compared to normal control cartilage. Cartilage replacement tissue was generated using human articular chondrocytes (normal control cartilage, n=3–5) or human mesenchymal stromal cells which develop a hypertrophic phenotype similar to the one observed in OA (OA cartilage model, n=3–6). Cells were seeded in a collagen type I/III carrier and attached to a beta-TCP bone replacement phase, building an osteochondral unit for simulation of natural conditions. After 21 and 35 days of chondrogenic (re)differentiation, a single physiological mechanical compression episode (1 Hz, 25 %, 3 h) was applied, imitating three hours of normal walking in ten-minute intervals. Proteoglycan and collagen synthesis, gene expression and activation of signaling pathways were assessed. Cartilage replacement tissue of both groups had similar proteoglycan and collagen type II content as well as hardness properties. During (re)differentiation, both cell types showed a comparable upregulation of the chondrogenic marker genes COL2A1 and ACAN. As expected, hypertrophic marker genes (COL10A1, ALPL, MEF2C, IBSP) were only upregulated in the OA cartilage model. Mechanotransduction in both tissues was confirmed by load-induced activation of pERK1/2 signaling. While the 3 h loading episode significantly increased proteoglycan synthesis in normal control cartilage at day 35, the same protocol resulted in a suppression of proteoglycan and collagen synthesis in the OA cartilage model, which was accompanied by a downregulation of COL2A1 gene expression. In addition, hypertrophic marker genes COL10A1, ALPL and IBSP were significantly reduced after loading. Along lower load-induced SOX9 mRNA and protein stimulation in the OA cartilage tissue, a weaker induction of mechanosensitive BMP2, BMP6, FOS and FOSB gene expression was observed. While stable cartilage showed anabolic effects after physiological loading, the hypertrophic chondrocytes reacted with a reduced extracellular matrix synthesis. This could be explained by a lower mechanoinduction of the BMP signaling cascade and insufficient SOX9 stimulation. Progressive OA development could thus be influenced by a reduced mechanocompetence of osteoarthritic chondrocytes

Bone regeneration using a scaffold-based tissue engineering approach involves a spectrum of overlapping processes, which are driven by cell-to-cell, cell-to-extracellular matrix (ECM) and cell-to-biomaterials interactions. Traditionally, the study of osteogenesis potential of tissue-engineered constructs (TECs) in vitro only considers the osteoblasts- or mesenchymal cells (MSCs)-to-biomaterials interactions. However, this poorly recapitulates the process of bone regeneration under physiological conditions. In this study, a growth factors free co-culture model, comprising osteoblasts and monocytes was established to allow for the study of the osteogenesis potential of a TEC taking into consideration osteoblasts-to-monocytes and cells-to-biomaterials interactions. Scaffolds made of medical-grade polycaprolactone (mPCL) were fabricated by means of melt electrospinning writing technique. Subsequently, scaffolds were coated with a thin layer of calcium phosphate (CaP) by means of chemical deposition. Scaffolds with CaP coating were seeded with human-derived primary osteoblasts and monocytes and cultured for up to nine weeks. At several time-points, cells were evaluated for alkaline phosphatase and tartrate-resistant acid phosphatase activity. Additionally, cell morphology was observed through fluorescence microscopy and osteoblastic- and osteoclastic-related gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction. The simultaneous presence of osteoblasts and monocytes and CaP accelerated cell matrix formation on scaffolds. Quantitative gene expression profile showed similar findings. Whereby, osteoblastic- and osteoclastic-related gene expression was highest in the PCL/CaP co-culture groups compared to other groups. This indicated synergistic effects of soluble factors secreted by cells and solubilized inorganic components from the scaffolds in promoting matrix deposition

There is a growing socio-economic need (i.e. “ageing society”) for effective and reproducible strategies to repair musculoskeletal tissue. In particular, acute tendon injury and chronic tendinopathies remain clinically challenging and novel treatment modalities are urgently needed. Tendons resemble a connective tissue rich in highly organized collagen fibers, displaying a remarkably high tensile strength. However, partly due to the low number of cells and their more or less avascular nature tendons heal relatively slowly. Ultimately, tendon regeneration encompasses the full restoration of the biological, biochemical and biomechanical properties, which are often impaired by endogenous healing cascades. Usually, a connective scar tissue forms at the injury site and the replaced tissue does not function adequately at high strain levels, increasing the chance of re-rupture. Despite significant advancements in tissue regeneration and engineering strategies, the clinical impact for the regeneration of tendon remains limited. For the development of novel methods to repair tendons we need to pin down the molecular and cellular mechanisms amenable to modulate endogenous (or exogenous) cell behaviour towards functional tissue regeneration. By comparing the gene expression profile of Achilles tendon tissue harvested from young-mature and old mice we demonstrate profound changes in the expression of ECM-related proteins and a previously unknown role of Secreted protein acidic and rich in cysteine (Sparc; also known as BM-40 or osteonectin) in tendons. Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties potentially drives adipogenic differentiation of tendon stem and progenitor cells (TDSPCs) and consequently lipid accretion in tendons. Generally, the fate of stem/ progenitor cells is largely determined by stimuli from the stem cell niche. In tendons, we describe a novel cellular barrier, most likely preventing the leakage of blood-borne products into the tendon proper. We propose that this “blood-tendon barrier” is part of the stem cell niche in tendons controlling TDSCP fate, preventing erroneous differentiation. By investigating the developmental programs driving tendon tissue formation and on the other hand the mechanisms contributing to the senescence of tendons, ultimately resulting in decreased quality of tendons in the elderly, novel targets for clinical intervention potentially can be discovered

With around 20–40% of our bodyweight, skeletal muscles are the biggest organ complex of the human body. Being a metabolic active tissue, muscle mass, function and fibertype composition is highly regulated in a tight spatial-temporal manner. In geriatric patients, it is essentially important to understand the underlying mechanisms of the age related losses of fiber size and total number of fibers, as well as fibertype shifting. To date, there have been few studies dealing with gene expression profiling of skeletal muscles, mostly focusing on age related differences in whole-muscle specimen. Being carried out on mouse or rat limb muscles, most other studies do not represent the conditions of human muscle, due to the differences in fibertype composition. Our study provides a fibertype-specific approach for whole-genome expression analysis in human skeletal muscle. 22 fresh frozen biceps brachii and quadriceps femoris muscle samples were acquired from the muscle bank of the Friedrich-Baur-Institut, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany. Consecutive cross-sections were used for immunohistochemical myosine-heavy-chain-staining and individual fibers were acquired by laser-capture-microdissection. Around 100 cells of each fibertype of each biopsy were dissected, reversely transcribed, pre-amplified and labeled for microarray analysis. Fiber type-specific gene expression was analyzed with ANOVA. Correction for multiple testing was performed using the Benjamini-Hochberg procedure with a conservative threshold and the pathway analysis was carried out using the Ingenuity Pathway Analysis program (QIAGEN). By comparing type I vs. type IIa, type I vs. type IIx and type IIa vs. type IIx, we could identify 2855, 2865 and 510 differentially expressed genes. As expected, many differentially regulated genes belong to functional groups like cytoskeleton, muscle contraction and energy metabolism, proving the feasibility of our study. However, many genes that are involved in the response to oxidative stress were also differently regulated, showing distinct mechanisms of the different fiber types, of coping with oxidative stress. In consensus with available literature, the relative proportion of type I fibers seemed to increase with age. Despite higher levels of oxidative stress, type I fibers seem to have more efficient antioxidative mechanisms in comparison to type IIa and IIx fibers, which might explain the higher vulnerability of members of the type II family to oxidative stress. Furthermore, genes that are involved in fibertype specification were also regulated differently. However, we could not verify an age-specific activation of pathways involved in fibertype shifting. Whether fibertype shifting is solely due to disproportionate loss of type II fibers, or also in vivo - transdifferentiation of fibers, has to be investigated further

Introduction. The two-dimensional (2D) monolayer culture paradigm has limited translational potential to physiological systems; chondrocytes and tenocytes in monolayer lose expression of hallmarks of differentiated status (dedifferentiation). Qualitative assessment of three-dimensional (3D) cultures in musculoskeletal biology relative to native tissues has been limited. An understanding of prevailing gene regulatory networks is required to define whether 3D culture systems faithfully restitute the native tissue phenotype (redifferentiation). Using a systems biology approach to explore the gene networks associated with de- and re-differentiation may define targetable regulators associated with phenotypic plasticity of adult musculoskeletal cells. Materials and Methods. Global transcriptomic and proteomic profiling of matrix-depleted chondrocytes and tenocytes from the rat was performed for each of three conditions (native tissue, monolayer at passage three, or tissue-appropriate 3D cultures). Differential analysis of mRNA and protein abundance, gene ontology annotation, pathway topology impact analysis, and derivation of common mechanistic networks was undertaken to define consensus expression profiles, signalling pathways, and upstream regulators for de- and re-differentiation in each cell type. Results. Principal component analysis demonstrated a convergence of gene expression profiles in monolayer, including the expression of musculoskeletal progenitor markers scleraxis (Scx) and Mohawk (Mkx). Three-dimensional culture systems failed to demonstrate parity with native tissue and incited the expression of Il-6 and Ptgs2 (COX2). The CCN-family member Ctgf (CCN2), and the marker of skeletal differentiation Grem1 (gremlin 1), were consistently differentially abundant in de- and re-differentiation at both the mRNA and protein level. Pathway topology impact analysis defined PI-3K/Akt as the common signalling pathway in de- and re-differentiation. Discussion. Historically, the terms de- and re-differentiation have been used with no mechanistic definition. Additionally, there is no standardised phenotype for 3D cultures to benchmark novel progress in bioengineering. Consensus upstream regulators yielded a unified mechanistic network for chondrocyte and tenocyte phenotypes in three conditions. The PI-3K/Akt signalling pathway has been implicated in a range of physiological activities including dedifferentiation, proliferation, matrix synthesis, and cell survival. Pathway analysis suggests that the PI-3K/Akt signalling pathway may contribute to the de- and re-differentiation phenotypes for both chondrocytes and tenocytes and represents a rational target for further network-level analysis

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

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

Aim

Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage.

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.