Background. The molecular mechanisms underlying non-union bone fractures largely remain elusive. Recently, spatial transcriptomics approaches for musculoskeletal tissue samples have been developed requiring direct placement of histology sections on barcoded slides. However, Formalin-Fixed-Paraffin-Embedded (FFPE) bone sections have been associated with limited RNA quality and read depth compared to soft tissue. Here, we test spatial transcriptomics workflows based on transcriptomic probe transfer to characterize molecular features discriminating non-union and union bone fractures in mice. Method. Histological sections (n=8) used for spatial transcriptomics (Visium CytAssist FFPE; 10x Genomics, n=4 on glass slides, n=4 on hydrogel-coated slides) were obtained from a fracture healing study in female 20-week-old C57BL/6J mice receiving either a femur osteotomy (0.7mm) or a segmental defect (2.4mm) (license 22/2022, Grisons CH). Sequence alignment and manual segmentation of different tissues (bone, defect region/callus, bone marrow, muscle) were performed using SpaceRanger and LoupeBrowser (10x Genomics). Differential gene expression was performed using DESeq2 (Seurat) followed by Gene-Set-Enrichment-Analysis (GSEA) of Gene Ontology (ClusterProfiler). Group comparison of quality measures was done using a Welch's t-test. Results are given as mean±standard deviation. Result. The quality measures, mean counts, and genes per spot, were significantly ~10× higher for sections on hydrogel slides (counts: 4700±1796, genes: 2389±1170) compared to glass slides (counts: 463±415, genes: 250±223). In challenging tissues like cortical bone, we reached high counts+genes in comparison to published data. Direct comparison of a non-union and union section showed a total of 432 differentially regulated genes, 538 in the defect region/callus. GSEA revealed differential regulation of pathways involved in muscle organ morphogenesis, cartilage development and endochondral ossification. Conclusions. Optimized spatial transcriptomics workflows based on transcriptomic probe transfer enable for improved read depth in musculoskeletal tissue enabling the characterization of molecular features discriminating non-union and union bone fractures. Acknowledgements. AO Foundation (AOTRAUMA), SNSF (PhD salary)

Introduction. Tendon ruptures represent one of the most common acute tendon injuries in adults worldwide, affecting millions of people anually and becoming more prevalent due to longer life expectancies and sports activities. Current clinical treatments for full tears are unable to completely restore the torn tendons to their native composition, structure and mechanical properties. To address this clinical challenge, tissue-engineered substitutes will be developed to serve as functional replacements for total tendon ruptures that closely resemble the original tissue, restoring functionality. Method. Water borne polyurethanes (WBPU) containing acrylate groups, specifically polyethylene glycol methacrylate (PEGMA) or 2-hydroxyethyl methacrylate (HEMA), were combined with mouse mesenchymal stem cells (MoMSCs) and heparin sodium to formulate bioinks for the fabrication of scaffolds via extrusion-based 3D bioprinting. Result. The biocompatibility of acrylated-WBPUs was confirmed in 2D with MoMSCs using lactate dehydrogenase assay, DNA assay and live/dead assays. Cell-laden scaffolds were 3D-bioprinted by encapsulating MoMSCs at varying cell densities within the acrylated WBPUs. The resulting 3D structures support cell viability and proliferation within the scaffolds, as confirmed by live/dead assay, lactate dehydrogenase assay and DNA assays. Differentiation studies in the 3D-bioprinted scaffolds demonstrated the phenotype transition of MoMSCs toward tenocytes through gene expression and protein deposition analysis. The inclusion of sodium heparin in the bioinks revealed increased synthesis of matrix assembly proteins within the 3D-bioprinted constructs. Conclusion. The developed bioinks were biocompatible and printable, supporting cell viability within the 3D-bioprinted scaffold. The fabricated cell-laden constructs sustained cell proliferation, differentiation, and tissue formation. The addition of heparin sodium enhanced tissue formation and organization, showing promising results for the regeneration of tendon total ruptures. Principio del formularioThis work was supported by the Spanish State Research Agency (AEI) under grant No CPP2021-008754. The authors would like to thank their partners in the project, which are in charge of the synthesis of heparin sodium and acrylated-WBPUs

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

Introduction. The most frequent diagnosis in young adults undergoing total hip arthroplasty (THA) is osteonecrosis of the femoral head (ONFH), an evolving and disabling condition with an increasing prevalence worldwide. Treatment of ONFH remains a challenge mainly because of a lack of understanding of the disease's pathophysiological basis. This study investigated the biological processes that could be affected by ONFH by comparing the microstructure, histological characteristics and transcriptomic profile of trabecular bone from the femoral head (FH) and the intertrochanteric region (IT) of patients suffering from this condition. Method. A total of 18 patients with idiopathic ONFH undergoing THA in our institution were included. Trabecular bone explants were taken intraoperatively from the FH and the IT of patients. Bone microstructure was examined by micro-computed tomography (micro-CT). After bone sectioning, histological features were studied by hematoxylin and eosin staining. Differential gene expression was investigated using a microarray platform. Result. Micro-CT imaging showed higher trabecular separation and lower trabecular thickness and bone volume in trabecular bone from the FH than from the IT. Histological staining revealed that the number of osteoblasts on the bone surface and the percentage of empty lacunae were higher in trabecular bone from the FH. Transcriptome analysis identified a differential signature in trabecular bone from the FH compared to the IT. The gene ontology analyses of the genes overexpressed in trabecular bone from the FH revealed a range of enriched biological processes related to cell division and immune response. In contrast, most downregulated transcripts were involved in bone formation. Conclusion. This study identified changes in the microarchitecture, histological features and transcriptomic signature of trabecular bone from the FH of patients with idiopathic ONFH, which might underlie the pathophysiology of this condition. This work was supported by PI22/00939 grant from ISCIII-FEDER-MICINN-AES and Luis Alvarez grant from IdiPAZ

Introduction. Bioactive glasses (BGs) promote osteogenic differentiation of bone progenitor cells by releasing therapeutically active ions. The well-described 45S5-BG (in mol%: SiO. 2. 46.13; P. 2. O. 5. 2.60; CaO 26.91; Na. 2. O 24.35) was supplemented with CaF. 2. and NaF being added to the batch at nominal 5 (F5-BG) and 25 mol% (F25-BG), respectively. While the effect on physical and chemical properties has already been characterized, the biological properties require further studies. This study investigates the effects of fluoride-supplemented BGs on the osteogenic and angiogenic properties of human bone marrow mesenchymal stromal cells (BMSCs) in vitro. Method. BMSCs were co-cultured with melt-derived 45S5-BG, F5-BG, or F25-BG in ascending concentrations (1, 2 and 3 mg/ml). At 7 days, cell number was determined by 4,6-diamidine-2-phenylindole (DAPI) staining and cell viability by fluorescein diacetate (FDA) assay. The osteogenic potential of the BGs was evaluated through alkaline phosphatase (ALP) gene expression and activity, along with bone morphogenetic protein-2 (BMP2) gene expression and protein concentration. Vascular endothelial growth factor (VEGF) gene expression and protein concentration assessed angiogenic potential. As control, BMSCs were cultured without BG exposure. Result. All BGs significantly promoted cell number and viability, with F25-BG showing the highest count at 3 mg/ml. Osteogenic markers showed a significant decrease in ALP gene expression and activity, especially at higher concentrations. All BG groups demonstrated increased BMP2 protein concentration and gene expression compared to the control, with higher BG and fluoride concentrations correlating with greater increases in BMP2. VEGF gene expression increased in all analysed BGs. The fluoride-free BG group had the highest VEGF protein concentrations, while the F25 BG group showed the highest VEGF gene expression. Conclusion. The fluoride-substituted BGs exhibit excellent cytocompatibility, enhance BMSC proliferation and positively affect BMP2 gene expression and levels, suggesting their potential for osteogenic differentiation. Further research is necessary to assess their proangiogenic effect and potential advantages over 45S5-BG

Introduction. Diabetes mellitus type 2 (DMT2) patients often develop Achilles tendon (AS) degeneration. The ZDF rat model is often used to study DMT2. Hence, this study investigated whether tenocytes isolated from diabetic and non diabetic ZDF rats respond differentially to normo- (NG) and hyperglycemic (HG) conditions in the presence of tumor necrosis (TNF)α. Method. AS tenocytes isolated from adult diabetic (fa/fa) or lean (fa/+) Zucker Diabetic Fatty (ZDF) rats were treated with 10 ng/mL TNFα either under NG or HG conditions (1 g/L versus 4.5 g/L glucose). Tendons were characterized histopathologically using Movin score. Tenocyte survival, metabolic activity, gene and/or protein expression of the main tendon extracellular matrix (ECM) component collagen type 1, the myofibroblast marker alpha smooth muscle actin (αSMA, Acta2), complement regulatory factors, the antioxidant defense enzyme heme oxygenase-1 (Hmox1), suppressors of cytokine signaling (Socs)1 and Soc3 were analyzed. Result. Tendons of diabetic rats showed significantly higher Movin score values suggesting tendon degeneration. Tenocyte vitality remained high, but metabolic activity was impaired by HG conditions, irrespectively of tenocyte origin. Higher amounts of αSMA were visualized in tendons/cells of diabetic rats or those exposed to TNFα. Collagen type 1 protein and gene expression was suppressed by TNFα (NG), but only in cells of non diabetic animals. The anaphylatoxin receptor C3aR was higher expressed in tenocytes from diabetic animals. CD46 was suppressed by TNFα (NG) in cells of diabetic rats. Hmox1, Socs1 and Socs3 were induced by HG, but only in tenocytes of diabetic rats (4 h). Conclusion. The response of tenocytes to TNFα depends on glucose supply and cell origin suggesting their irreversible impairment in DMT2

Introduction. Bereft of their optimal tissue context, cells lose their phenotype, function and therapeutic potential during in vitro culture. Despite the fact that in vivo cells are exposed simultaneously to multiple signals, traditional ex vivo cultures are monofactorial. With these in mind, herein we assessed the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human tenocyte, skin fibroblast and bone marrow mesenchymal stromal cell cultures. Methods. Thermal imprinted was used to pattern (groove depth: 2,000 nm, groove width: 2,000 nm, line width: 2,000 nm) polydimethylsiloxane substrates of different rigidity (50 kPa, 130 kPa, 1,000 kPa). Grooved and planar substrates were subsequently coated with collagen type I and used to culture the aforementioned cell populations without and with macromolecular crowding (100 μg/ml carrageenan). After 3, 7 and 14 days in culture, cell morphology, viability, metabolic activity, proliferation, protein synthesis and deposition and gene expression analyses were conducted. Results. None of the variables assessed affected cell viability, metabolic activity and proliferation. Surface topography was found to be a potent regulator of cell morphology. Macromolecular crowding significantly increased extracellular matrix deposition, albeit in globular manner independently on whether grooved or planar substrates were used, possibly due to the low dimensionality of the grooves. Gene expression analysis made apparent that the 130 kPa and the 1,000 kPa grooved substrates under macromolecular crowding conditions maintained human tenocyte phenotype and directed human bone marrow mesenchymal stromal cells towards tendon-like lineage, respectively. None of the conditions assessed dramatically affected human skin fibroblast fate. Conclusions. Collectively, our data indicate that the physicochemical in vitro microenvironment modulators assessed herein are capable of maintaining human tenocyte phenotype and differentiating human bone marrow mesenchymal stromal cells towards tenogenic lineage, but not in trans-differentiating human skin fibroblasts

Introduction. Healthy tendons are mainly composed of aligned collagen hierarchically organized from collagen fibrils to fiber bundles with a scarce cellular population mainly composed of tenocytes and tendon stem/progenitor cells. However, injured tendon acquires a fibrotic state characterized by a loss of ECM alignment and increased cellularization. The lack of reliable 3D models that recreate the organization and microenvironment of healthy and diseased tendons is one of the main obstacles faced by the scientific community. Method. To recreate the architecture of healthy and diseased tendons, electrospun nanofiber scaffolds with anisotropic and isotropic nanotopography were developed. These scaffolds were coated with a shell consisting of cell-laden hydrogels encapsulating human adipose-derived stem cells (hASCs) to include the living component. To show the versatility of the system, extracellular vesicles (EVs) were encapsulated in the hydrogel as biological cues. The living fibers were characterized by microscopy and morphological analysis. The morphology and phenotype of cells was evaluated using microscopy, gene expression analysis and immunostainings for tendon markers. Results. Scaffolds mimicked the native hierarchical structure of tendons and size of tendon fascicles. hASCs showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, the bioengineered living fibers supported the tenogenic differentiation of stem cells over time, as indicated by the sustained expression of tenogenic and extracellular matrix markers. Finally, the hydrogel layer acted not only as a hydrated biomimetic environment adequate for cell encapsulation but also as a carrier and delivery system of EVs to cells, which improved their tenogenic commitment. Conclusion. We bioengineered composite living fibers made of hierarchically organized electrospun fibers, coated with hydrogel encapsulating hASCs and biofunctional EVs. These provide an in vitro system to recreate tendon, allowing for the study of the effects of biophysical cues in tendon microenvironments and the influence of biologics on cells behavior. Acknowledgments. CP21/00136, PI22/01686, CA22170, 10.54499/2020.03410.CEECIND/CP1600/CT0013, 10.54499/2022.05526.PTDC

Introduction. Articular cartilage has a low self-regeneration capacity. Cartilage defects have to be treated to minimize the risk of the onset of osteoarthritis. Bioactive glass (BG) is a promising source for cartilage tissue engineering. Until now, conventional BGs (like BG1393) have been used, mostly for bone regeneration, as they are able to form a hydroxyapatite layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to study the effect of 3D printed hydrogel scaffolds supplemented with spheres of the BG CAR12N to improve the chondrogenesis of mesenchymal stem cells (MSCs). Method. Based on our new glass composition (CAR12N), small BG spheres (25-40 µm) were produced and mixed with hydrogel and primary human (h) MSCs. Grid printed scaffolds were cultivated up to 21 days in expansion or chondrogenic differentiation medium. Macroscopical images of the scaffolds were taken to observe surface changes. Vitality, DNA and sulfated glycosaminoglycan (GAG) content was semiquantitatively measured as well as extracellular matrix gene transcription. Result. It was possible to print grid shaped hydrogel scaffolds with BG spheres and hMSCs. No significant changes in scaffold shape, surface or pore size were detected after 21 days in culture. The BG spheres were homogeneously distributed inside the grids. Vitality was significantly higher in grids with CAR12N spheres in comparison to those without. The DNA content remained constant over three weeks, but was higher in the sphere containing scaffolds than in those without BG spheres. GAG content in the grids increased not only with additional cultivation time but especially in grids with BG spheres in chondrogenic medium. Aggrecan and type II collagen gene expression was significantly higher grids cultured in the chondrogenic differentiation medium. Conclusion. This developed 3D model, is very interesting to study the effect of BG on hMSCs and to understand the influence of leaking ions on chondrogenesis

Introduction. Tendinopathies represent a significant health burden, causing inflammation, pain, and reducing quality of life. The pivotal role of macrophages (Mφ) characterized by their ability to differentiate into proinflammatory (M1) or anti-inflammatory (M2) phenotypes depending on the microenvironment, has gained significant interest in tissue inflammation research. Additionally, existing literature states that the interplay between tenocytes and immune cells during inflammation involves unidentified soluble factors (SF). This study aimed to investigate the effect of extracellular vesicles (EVs) and SF derived from polarized Mφ on tendon cells to provide deeper insights of their potential therapeutic applications in the context of inflammation. Method. Human monocytes were isolated from blood donor buffy coats and differentiated into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, EVs were isolated from the conditioned media from polarized Mφ and comprehensively characterized. In parallel, the elution media containing SF was collected. Furthermore, the EVs and SF were released independently onto tenocytes from human donors, previously induced with IL-1β to simulate an inflammatory environment. Finally, mRNA levels of tendon-related markers were evaluated by qPCR after the exposure to these EVs and SF. Result. Notably, the study found that the viability of the cells was not affected by the exposure to EVs nor SF, indicating their potential safety for therapeutic use. Moreover, the mRNA content of tendon-derived cells was evaluated following exposure to Mφ-EVs and SF revealing alterations in gene expression. Interestingly, a significant increase in the expression of tenomodulin was observed in tendon cells treated with Mφ-EVs. Conclusion. These results mark a significant advancement in understanding the interplay between Mφ and tenocytes at a molecular level. To fully understand the underlying causes of Mφ-EVs effects, and its potential clinical application in tendon inflammatory diseases, further comprehensive research is required. Acknowledgments. Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Introduction. Osteoarthritis (OA) is a predominant chronic degenerative disease exerting a deep impact on quality of life and healthcare systems. Recent evidences suggest that pyroptosis, a programmed cell death characterized by inflammatory cytokine release, may play a significant role in modulating OA pain. The aim of the study is to investigate the potential role of extracellular vesicles derived from umbilical cord Wharton's jelly (WJ-MSC EVs) in the attenuation of the pyroptotic process on human chondrocytes (hOAC) pre-treated with synovial fluid in a 3D in vitro model. Method. EVs isolated by tangential filtration of the conditioned medium of WJ-MSCs were characterized for: morphology by TEM, surface markers by WB and size by NTA. Confocal microscopy was used to identify PKH26-labelled EVs and monitor their incorporation into hOACs. The hOACs from surgical waste material of patients undergoing knee replacement, expanded, encapsulated in alginate beads were pre-treated with synovial fluid for 24 h (SF) and subsequently co-incubated with WJ-MSC EVs. We examined viability (CCK-8), metabolic activity (MTT), nitrite production (Griess) activation of the pyroptotis (IF), DNA quantification (PicoGreen) and gene expression levels of extracellular matrix (ECM) components (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. Result. WJ-MSC EVs increased hOACs viability and metabolic activity. The production of nitrites is significantly decreased compared sample group treated with SF. WJ-MSC EVs inhibited inflammasomes NLRP3 (nucleotide-binding domain, leucine-rich repeat pyrin domain containing 3) activation. The ECM catabolic genes decreased compared to the inflamed SF group for ADAMTS-5 and MMP-1. Conclusion. Our results supported the potential use of WJ-MSC EVs as a cell-free strategy for OA, overcoming the side effects of cell-therapy. Moreover, WJ-MSC EVs are able to mitigate SF-treated hOACs pyroptotic death, attenuate ECM degradation and oxidative stress counteracting the inflamed status in OA development and progression

Aims. This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously. Methods. Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes. Results. WGCNA revealed critical gene modules for OB and OP, identifying the Toll-like receptor (TLR) signalling pathway as a common factor. TLR2 was the most significant gene, with a pronounced expression in macrophages. Elevated TLR2 expression correlated with increased adipose accumulation, inflammation, and osteoclast differentiation, linking it to OP development. Conclusion. Our study underscores the pivotal role of TLR2 in connecting OP and OB. It highlights the influence of TLR2 in macrophages, driving both diseases through a pro-inflammatory mechanism. These insights propose TLR2 as a potential dual therapeutic target for treating OP and OB. Cite this article: Bone Joint Res 2024;13(10):573–587

Aims

This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels.

Aims. Rotator cuff tear (RCT) is the leading cause of shoulder pain, primarily associated with age-related tendon degeneration. This study aimed to elucidate the potential differential gene expressions in tendons across different age groups, and to investigate their roles in tendon degeneration. Methods. Linear regression and differential expression (DE) analyses were performed on two transcriptome profiling datasets of torn supraspinatus tendons to identify age-related genes. Subsequent functional analyses were conducted on these candidate genes to explore their potential roles in tendon ageing. Additionally, a secondary DE analysis was performed on candidate genes by comparing their expressions between lesioned and normal tendons to explore their correlations with RCTs. Results. We identified 49 genes in torn supraspinatus tendons associated with advancing age. Among them, five age-related genes showed DE in lesioned tendons compared to normal tendons. Functional analyses and previous studies have highlighted their specific enrichments in biological functions, such as muscle development (e.g. myosin heavy chain 3 (MYH3)), transcription regulation (e.g. CCAAT enhancer binding brotein delta (CEBPD)), and metal ion homeostasis (e.g. metallothionein 1X (MT1X)). Conclusion. This study uncovered molecular aspects of tendon ageing and their potential links to RCT development, offering insights for targeted interventions. These findings enhance our understanding of the mechanisms of tendon degeneration, allowing potential strategies to be made for reducing the incidence of RCT. Cite this article: Bone Joint Res 2024;13(9):474–484

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Aims. This study explored the shared genetic traits and molecular interactions between postmenopausal osteoporosis (POMP) and sarcopenia, both of which substantially degrade elderly health and quality of life. We hypothesized that these motor system diseases overlap in pathophysiology and regulatory mechanisms. Methods. We analyzed microarray data from the Gene Expression Omnibus (GEO) database using weighted gene co-expression network analysis (WGCNA), machine learning, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify common genetic factors between POMP and sarcopenia. Further validation was done via differential gene expression in a new cohort. Single-cell analysis identified high expression cell subsets, with mononuclear macrophages in osteoporosis and muscle stem cells in sarcopenia, among others. A competitive endogenous RNA network suggested regulatory elements for these genes. Results. Signal transducer and activator of transcription 3 (STAT3) was notably expressed in both conditions. Single-cell analysis pinpointed specific cells with high STAT3 expression, and microRNA (miRNA)-125a-5p emerged as a potential regulator. Experiments confirmed the crucial role of STAT3 in osteoclast differentiation and muscle proliferation. Conclusion. STAT3 has emerged as a key gene in both POMP and sarcopenia. This insight positions STAT3 as a potential common therapeutic target, possibly improving management strategies for these age-related diseases. Cite this article: Bone Joint Res 2024;13(8):411–426

Aims. The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice. Methods. Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results. The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion. Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis. Cite this article: Bone Joint Res 2024;13(7):321–331

Aims

Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

Aims. To assess the alterations in cell-specific DNA methylation associated with chondroitin sulphate response using peripheral blood collected from Kashin-Beck disease (KBD) patients before initiation of chondroitin sulphate treatment. Methods. Peripheral blood samples were collected from KBD patients at baseline of chondroitin sulphate treatment. Methylation profiles were generated using reduced representation bisulphite sequencing (RRBS) from peripheral blood. Differentially methylated regions (DMRs) were identified using MethylKit, while DMR-related genes were defined as those annotated to the gene body or 2.2-kilobase upstream regions of DMRs. Selected DMR-related genes were further validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to assess expression levels. Tensor composition analysis was performed to identify cell-specific differential DNA methylation from bulk tissue. Results. This study revealed 21,060 hypermethylated and 44,472 hypomethylated DMRs, and 13,194 hypermethylated and 22,448 hypomethylated CpG islands for differential global methylation for chondroitin sulphate treatment response. A total of 12,666 DMR-related genes containing DMRs were identified in their promoter regions, such as CHL1 (false discovery rate (FDR) = 2.11 × 10. -11. ), RIC8A (FDR = 7.05 × 10. -4. ), and SOX12 (FDR = 1.43 × 10. -3. ). Additionally, RIC8A and CHL1 were hypermethylated in responders, while SOX12 was hypomethylated in responders, all showing decreased gene expression. The patterns of cell-specific differential global methylation associated with chondroitin sulphate response were observed. Specifically, we found that DMRs located in TESPA1 and ATP11A exhibited differential DNA methylation between responders and non-responders in granulocytes, monocytes, and B cells. Conclusion. Our study identified cell-specific changes in DNA methylation associated with chondroitin sulphate response in KBD patients. Cite this article: Bone Joint Res 2024;13(5):237–246

Aims

The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies.