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.

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.

Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo. Results. HDAC4 markedly improved the survival rate and biofunction of chondrocytes. RNA-seq analysis of the EP and HDAC4 groups showed that HDAC4 induced 2,668 significant gene expression changes in chondrocytes (1,483 genes upregulated and 1,185 genes downregulated, p < 0.05), and ribosomes exhibited especially large increases. The results were confirmed by RNA-seq of the EP versus mutated HDAC4 groups and the validations in vitro and in vivo. Conclusion. The enhanced ribosome pathway plays a key role in the mechanism by which HDAC4 improves the survival rate and biofunction of chondrocytes. Cite this article: Bone Joint Res 2023;12(7):433–446

Aims. To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65

Tendinopathy can commonly occur around the foot and ankle resulting in isolated rupture, debilitating pain and degenerative foot deformity. The pathophysiology and key cells involved are not fully understood. This is partly because the dense collagen matrix that surrounds relatively few resident cells limits the ability of previous techniques to identify and target those cells of interest. In this study, we apply novel single cell RNA sequencing (CITE-Seq) techniques to healthy and tendinopathic foot/ankle tendons. For the first time we have identified multiple sub-populations of cells in human tendons. These findings challenge the view that there is a single principal tendon cell type and open new avenues for further study. Healthy tendon samples were obtained from patients undergoing tendon transfer procedures; including tibialis posterior and FHL. Diseased tendon samples were obtained during debridement of intractable Achilles and peroneal tendinopathy, and during fusion of degenerative joints. Single cell RNA sequencing with surface proteomic analysis identified 10 sub-populations of human tendon derived cells. These included groups expressing genes associated with fibro-adipogenic progenitors (FAPs) as well as ITGA7+VCAM1- recently described in mouse muscle but, as yet, not human tendon. In addition we have identified previously unrecognised sub-classes of collagen type 1 associated tendon cells. Each sub-class expresses a different set of extra-cellular matrix genes suggesting they each play a unique role in maintaining the structural integrity of normal tendon. Diseased tendon harboured a greater proportion of macrophages and cytotoxic lymphocytes than healthy tendon. This inflammatory response is potentially driven by resident tendon fibroblasts which show increased expression of pro-inflammatory cytokines. Finally, identification of a previously unknown sub-population of cells found predominantly in tendinopathic tissue offers new insight into the underlying pathophysiology. Further work aims to identify novel proteins targets for possible therapeutic pathways

Aims. Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis. Methods. Minus RNA sequencing, fluorescence in situ hybridization, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Chondrocytes were then stimulated to secrete exosomal miR-9-5p by cyclic tensile strain. Intra-articular injection of exosomal miR-9-5p into the model induced by destabilized medial meniscus (DMM) surgery was conducted to alleviate OA progression. Results. Tensile strain could decrease the expression of circStrn3 in chondrocytes. CircStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively ‘sponging’ miRNA-9-5p targeting Kruppel-like factor 5 (KLF5), indicating that the decrease in circStrn3 might be a protective factor in mechanical instability-induced OA. The tensile strain stimulated chondrocytes to secrete exosomal miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblast differentiation by targeting KLF5. Intra-articular injection of exosomal miR-9-5p alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Conclusion. Taken together, these results demonstrate that reduction of circStrn3 causes an increase in miR-9-5p, which acts as a protective factor in mechanical instability-induced OA, and provides a novel mechanism of communication among joint components and a potential application for the treatment of OA. Cite this article: Bone Joint Res 2023;12(1):33–45

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro. Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction. BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction. This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood. The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair. Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee. The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process

During aging, tendons demonstrate substantial disruptions in homeostasis, leading to impairments in structure-function. Impaired tendon function contributes to substantial declines quality of life during aging. Aged tendons are more likely to undergo spontaneous rupture, and the healing response following injury is impaired in aged tendons. Thus, there is a need to develop strategies to maintain tendon homeostasis and healing capacity through the lifespan. Tendon cell density sharply declines by ∼12 months of age in mice, and this low cell density is retained in geriatric tendons. Our data suggests that this decline in cellularity initiates a degenerative cascade due to insufficient production of the extracellular matrix (ECM) components needed to maintain tendon homeostasis. Thus, preventing this decline in tendon cellularity has great potential for maintaining tendon health. Single cell RNA sequencing analysis identifies two changes in the aged tendon cell environment. First, aged tendons primarily lose tenocytes that are associated with ECM biosynthesis functions. Second, the tenocytes that remain in aged tendons have disruptions in proteostasis and an increased pro-inflammatory phenotype, with these changes collectively termed ‘programmatic skewing'. To determine which of these changes drives homeostatic disruption, we developed a model of tenocyte depletion in young animals. This model decreases tendon cellularity to that of an aged tendon, including decreased biosynthetic tenocyte function, while age-related programmatic skewing is absent. Loss of biosynthetic tenocyte function in young tendons was sufficient to induce homeostatic disruption comparable to natural aging, including deficits in ECM organization, composition, and material quality, suggesting loss biosynthetic tenocytes as an initiator of tendon degeneration. In contrast, our data suggest that programmatic skewing underpins impaired healing in aged tendons. Indeed, despite similar declines in the tenocyte environment, middle-aged and young-depleted tendons mount a physiological healing response characterized by robust ECM synthesis and remodeling, while aged tendons heal with insufficient ECM

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug. Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip. The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions. In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging. Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1. Cre. -mGmT and Dmp1. Cre. -COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1. Cre. -Mfn2. f/f. mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs. Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1. Cre. -mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1. Cre. -COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain. Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging

We previously reported that osteoblasts at the curve apex in adolescent idiopathic scoliosis (AIS) exhibit a differential phenotype, compared to non-curve osteoblasts(1). However, the Hueter-Volkmann principle on vertebral body growth in spinal deformities (2) suggests this could be secondary to altered biomechanics. This study examined whether non-curve osteoblasts subjected to mechanical strain resemble the transcriptomic phenotype of curve apex osteoblasts. Facet spinal tissue was collected perioperatively from three sites, (i) the concave and (ii) convex side at the curve apex and (iii) from outside the curve (non-curve) from six AIS female patients (age 13–18 years; NRES 19/WM/0083). Non-curve osteoblasts were subjected to strain using a 4-point bending device. Osteoblast phenotype was determined by RNA sequencing and bioinformatic pathway analysis. RNAseq revealed that curve apex osteoblasts exhibited a differential transcriptome, with 1014 and 1301 differentially expressed genes (DEGs; p<0.05, fold-change >1.5) between convex/non-curve and concave/non-curve sites respectively. Non-curve osteoblasts subjected to strain showed increased protein expression of the mechanoresponsive biomarkers COX2 and C-Fos. Comparing unstimulated vs strain-induced non-curve osteoblasts, 423 DEGs were identified (p<0.05, fold-change >1.5). Of these DEGs, only 5% and 6% were common to the DEGs found at either side of the curve apex, compared to non-curve cells. Bioinformatic analysis of these strain-induced DEGs revealed a different array of canonical signalling pathways and cellular processes, to those significantly affected in cells at the curve apex. Mechanical strain of AIS osteoblasts in vitro did not induce the differential transcriptomic phenotype of AIS osteoblasts at the curve apex

Aims. Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Methods. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot. Results. DC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001). Conclusion. Exosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA. Cite this article: Bone Joint Res 2022;11(9):652–668

In a recent phase 2 superiority clinical trial we demonstrated that a single dose of 60mg of the human monoclonal antibody denosumab inhibits osteolytic lesion activity in patients undergoing revision total hip arthroplasty (THA), demonstrating proof of biological efficacy for this clinical application. Here, we examined the effect that denosumab has on disease biology at the osteolysis tissue level. Osteolytic tissue taken from the prosthesis-bone lesion interface at revision surgery in patients with osteolysis (n=10 participants that had received a single 60 mg dose of denosumab 8 weeks prior to revision surgery and n=10 that had received placebo) was examined for total genetic message activity and protein levels using whole genome sequencing and mass spectrometry, respectively. The top five upregulated enriched pathways with denosumab treatment included inflammatory response, myeloid cell activation, myeloid leukocyte migration, neutrophil and granulocyte activation (p<6.26 × 10. −28. ). Cell morphogenesis was amongst the most downregulated pathways (p<3.42 ×10. −23. ). Finally, comparison of the trial mRNA and protein data versus mouse single cell RNA sequencing data of the same pathway blockade in mouse tibia showed the same direction of effect, suggesting that giving the drug causes then cells responsible for osteolysis to disperse into a more immature form (128 of 189 genes (z=4.87, P<0.0001) disease and functional pathways at the mRNA level and 10 of 11 (z=2.72, P=0.0065) at the protein level). In this first-in-man study we identify multiple genes and pathways within periprosthetic osteolysis tissue that are affected by denosumab treatment. The dominant pathways involved upregulation of innate inflammatory signaling and downregulation of cell morphogenesis. We also found enrichment of similar disease and functional pathways at both the mRNA and protein levels versus mRNA pathway enrichment found in mouse osteomorphs. These data provide the first human data of the mechanistic effect of denosumab treatment on inflammatory osteolytic lesion activity after joint replacement that is necessary to support its clinical application. ∗Winner of The Bone & Joint Journal prize∗

Aims. Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods. Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results. Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion. These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients. Cite this article: Bone Joint Res 2023;12(10):657–666

Abstract. Objectives. Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype. Results. Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNAseq revealed that there were 2,085 highly significant upregulated genes in 3D matrices compared to TCP. Notably, >90% of highly expressed genes (including FOSB, FOS and TNFAIP6) were shared in all hydrogels. Gene ontology confirmed the TNF signalling pathway among the most significantly represented. Protein-protein interaction predictions identified TNF-alpha/NF-kappa B and AP1 pathways as differentially influenced by the hydrogel environment. Using inhibitors to these pathways, NFkB, but not AP1, impacted on the upregulation of TNFAIP6 and CXCL10 in 3D culture. Conditioned media from these studies was added to cultures of human monocytes with distinct changes in the resulting macrophage phenotype. MSCs in a 3D environment promoted a greater acquisition of the M2 repair macrophage phenotype and impacted on the numbers of pro-inflammatory M1 macrophages. Conclusion. These data provide further evidence that the immunomodulatory action of human MSCs can be influenced by the surrounding structural environment. These observations have significance for understanding the events that following skeletal injury and the potential to be exploited in preconditioning MSCs for cell therapy

Aim. While metagenomic (microbial DNA) sequencing technologies can detect the presence of microbes in a clinical sample, it is unknown whether this signal represents dead or live organisms. Metatranscriptomics (sequencing of RNA) offers the potential to detect transcriptionally “active” organisms within a microbial community, and map expressed genes to functional pathways of interest (e.g. antibiotic resistance). We used this approach to evaluate the utility of metatrancriptomics to diagnose PJI and predict antibiotic resistance. Method. In this prospective study, samples were collected from 20 patients undergoing revision TJA (10 aseptic and 10 infected) and 10 primary TJA. Synovial fluid and peripheral blood samples were obtained at the time of surgery, as well as negative field controls (skin swabs, air swabs, sterile water). All samples were shipped to the laboratory for metatranscriptomic analysis. Following microbial RNA extraction and host analyte subtraction, metatranscriptomic sequencing was performed. Bioinformatic analyses were implemented prior to mapping against curated microbial sequence databases– to generate taxonomic expression profiles. Principle Coordinates Analysis (PCoA) and Partial Least Squares-Discriminant Analysis were utilized to ordinate metatranscriptomic profiles, using the 2018 definition of PJI as the gold-standard. Results. After RNA metatranscriptomic analysis, blinded PCoA modeling revealed accurate and distinct clustering of samples into 3 separate cohorts (infected, aseptic, and primary joints) – based on their active transcriptomic profile, both in synovial fluid and blood (synovial anosim p=0.001; blood anosim p=0.034). Differential metatranscriptomic signatures for infected versus noninfected cohorts enabled us to train machine learning algorithms to 84.9% predictive accuracy for infection. Multiple antibiotic resistance genes were expressed, with high concordance to conventional antibiotic sensitivity data. Conclusions. Our findings highlight the potential of metatranscriptomics for infection diagnosis. To our knowledge, this is the first report of RNA sequencing in the orthopaedic literature. Further work in larger patient cohorts will better inform deep learning approaches to improve accuracy, predictive power, and clinical utility of this technology