Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment.

Cite this article: Bone Joint Res 2024;13(12):725–740.

Introduction. Homogenous and consistent preparations of mesenchymal stem cells (MSCs) can be acquired by selecting them for integrin α10β1 (integrin a10-MSCs). Safety and efficacy of intra-articular injection of allogeneic integrin a10-MSCs were shown in two post-traumatic osteoarthritis horse studies. The current study investigated immunomodulatory capacities of human integrin a10-MSCs in vitro and their cell fait after intra-articular injection in rabbits. Method. The concentration of produced immunomodulatory factors was measured after licensing integrin a10-MSCs with pro-inflammatory cytokines. Suppression of T-cell proliferation was determined in co-cultures with carboxyfluorescein N-succinimidyl ester (CFSE) labelled human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 and measuring the CFSE intensity of CD4+ cells. Macrophage polarization was assessed in co-cultures with differentiated THP-1 cells stimulated with lipopolysaccharide and analysing the M2 macrophage cell surface markers CD163 and CD206. In vivo homing and regeneration were investigated by injecting superparamagnetic iron oxide nanoparticles conjugated with Rhodamine B-labeled human integrin a10-MSCs in rabbits with experimental osteochondral defects. MSC distribution in the joint was followed by MRI and fluorescence microscopy. Result. The production of the immunomodulatory factors indoleamine 2,3-dioxygenase and prostaglandin E2 was increased after inflammatory licensing integrin a10-MSCs. Co-cultures with integrin a10-MSCs suppressed T-cell proliferation and increased the frequency of M2 macrophages. In vivo injected integrin a10-MSCs homed to osteochondral defects and were detected in the repair tissue of the defects up to 10 days after injection, colocalized with aggrecan and type II collagen. Conclusion. This study showed that human integrin a10-MSCs have immunomodulatory capacities and in vivo can home to the site of osteochondral damage and directly participate in cartilage regeneration. This suggests that human integrin α10β1-selected MSCs may be a promising therapy for osteoarthritis with dual mechanisms of action consisting of immunomodulation and homing to damage followed by early engraftment and differentiation into chondrocyte-like cells that deposit hyaline cartilage matrix molecules

Aims

This study aimed to define the histopathology of degenerated humeral head cartilage and synovial inflammation of the glenohumeral joint in patients with omarthrosis (OmA) and cuff tear arthropathy (CTA). Additionally, the potential of immunohistochemical tissue biomarkers in reflecting the degeneration status of humeral head cartilage was evaluated.

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.

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

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

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.

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known. In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory. AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287)

Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair. We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation. Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing. By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders. Acknowledgments. This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.

Sarcopenia is an age-related geriatric syndrome which is associated with subsequent disability and morbidity. Currently there is no promising therapy approved for the treatment of sarcopenia. The receptor activator of nuclear factor NF-κB ligand (RANKL) and its receptor (RANK) are expressed in bone and skeletal muscle. Activation of the NF-κB pathway mainly inhibits myogenic differentiation, which leads to skeletal muscle dysfunction and loss. LYVE1 and CD206 positive macrophage has been reported to be associated with progressive impairment of skeletal muscle function with aging. The study aims to investigate the effects of an anti-RANKL treatment on sarcopenic skeletal muscle and explore the related mechanisms on muscle inflammation and the polarization status of macrophages. Sarcopenic senescence-accelerated mouse P8 (SAMP8) mice at month 8 were treated intraperitoneally with 5mg/kg anti-RANKL (IK22/5) or isotype control (2A3; Bio X Cell) antibody every 4 weeks and harvested at month 10. Senescence accelerated mouse resistant-1 (SAMR1) were collected at month 10 as the age-matched non-sarcopenic group. Ex-vivo functional assessment, grip strength and immunostaining of C/EBPa, CD206, F4/80, LYVE1 and PAX7 were performed. Data analysis was done with one-way ANOVA, and the significant level was set at p≤0.05. At month 10, tetanic force/specific tetanic force, twitch force/specific twitch force in anti-RANKL group were significantly higher than control group (all p<0.01). The mice in the anti-RANKL treatment group also showed significantly higher grip strength than Con group (p<0.001). The SAMP8 mice at month 10 expressed significantly more C/EBPa, CD206 and LYVE1 positive area than in SAMR1, while anti-RANKL treatment significantly decreased C/EBPa, CD206 and LYVE1 positive area. The anti-RANKL treatment protected against skeletal muscle dysfunctions through suppressing muscle inflammation and modulating M2 macrophages, which may represent a novel therapeutic approach for sarcopenia. Acknowledgment: Collaborative Research Fund (CRF, Ref: C4032-21GF)

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

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Aims. Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. Methods. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. Results. In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. Conclusion. Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA. Cite this article: Bone Joint Res 2023;12(2):121–132

In the context of regenerative medicine for the treatment of musculoskeletal pathologies mesenchymal stromal cells (MSCs) have shown good results thanks to secretion of therapeutic factors, both free and conveyed within the extracellular vesicles (EV), which in their totality constitute the “secretome”. The portfolio and biological activity of these molecules can be modulated by both in vitro and in vivo conditions, thus making the analysis of these activities very complex. A deep knowledge of the targets regulated by the secretome has become a matter of fundamental importance and a homogeneous and complete molecular characterization is still lacking in the field of applications for the musculoskeletal system. Therefore, the aim of this work was to characterize the secretome obtained from adipose-derived MSCs (ASCs), and its modulation after pre-conditioning of the ASCs. Pre-conditioning was done by culturing cells in the presence of i) high levels of IFNγ, as proposed for the production of clinical grade secretome with enhanced regenerative potential, ii) low levels of inflammatory stimuli, mimicking conditions found in the osteoarthritis (OA) synovial fluid. Furthermore, EVs ability to migrate within cartilage, chondrocyte and synoviocytes obtained from OA patients was evaluated. The data showed that more than 50 cytokines / chemokines and more than 200 EV-microRNAs are detectable at various intensity levels in ASCs secretomes. The majority of the most abundantly present molecules are involved in the remodelling of the extracellular matrix and in the homeostasis and chemotaxis of inflammatory cells including macrophages, which in OA are often characterized by an M1 inflammatory polarization, promoting their transition to an M2 anti-inflammatory phenotype. Inflammatory priming with IFNγ and synovial fluid-like conditions were able to further increase the ability of the secretome to interact with inflammatory cells and modulate their migration. Finally, the penetration of the EVs in the cartilage explants resulted a rapid process, which begins a few minutes after administration of the EVs that are able to reach a depth of 30-40 μm in 5 hours. The same capacity for interaction was also verified in chondrocytes and synoviocytes isolated from the cartilage and synovial membrane of OA patients. Thanks to the soluble factors and EV-microRNAs, the ASCs secretome has shown a strong propensity to modulate the inflammatory and degenerative processes that characterize OA. The inflammatory pre-conditioning through high concentrations of inflammatory molecules or in conditions similar to the synovial fluid of OA patients was able to increase this capacity by increasing their chemotactic power. The microscopy data also support the hypothesis of the ability of MSC-EVs to influence the chondrocytes residing in the ECM of the cartilage and the synovial cells of the synovial membrane through active interaction and the release of their therapeutic content

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Aims

This study aimed, through bioinformatics analysis, to identify the potential diagnostic markers of osteoarthritis, and analyze the role of immune infiltration in synovial tissue.

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