Development of osteoarthritis (OA) correlates with epigenetic alteration in chondrocytes. H3K27me3 demethylase UTX is known to regulate tissue homeostasis, but its role in the homeostasis of articulating joint tissue is poorly understood. Forced UTX expression upregulated H3K27me3 enrichment at the Sox9 promoter region to inhibit key extracellular matrix (ECM) molecules, like e.g. type II collagen, aggrecan, and glycosaminoglycans in articular chondrocytes. Utx loss in vitro altered the H3K27me3-binding epigenomic landscape, which contributes to mitochondrial activity, cellular senescence, and cartilage development. Functional target genes of Utx comprise insulin-like growth factor 2 (Igf2) and polycomb repressive complex 2 (PRC2) core components Eed and Suz12. Specifically, Utx deletion promoted Tfam transcription, mitochondrial respiration, ATP production and Igf2 transcription, but inhibited Eed and Suz12 expression. Igf2 inhibition or forced Eed or Suz12 expression increased H3K27 trimethylation and H3K27me3 enrichment at the Sox9 promoter, compromising Utx loss-induced ECM overproduction. Overexpression of Utx in murine knee joints aggravated OA development, including articular cartilage damage, synovitis, osteophyte formation, and subchondral bone loss. Transgenic mice with a chondrocytespecific Utx knockout develop thicker articular cartilage as compared to wild-type controls and show fewer gonarthrotic symptoms during destabilized medial meniscus- and collagenase-induced joint injury. In summary, UTX represses chondrocytic activity and accelerates cartilage degradation during OA, while Utx loss promotes cartilage integrity through epigenetic stimulation of mitochondrial biogenesis and Igf2 transcription. This highlights a novel noncanonical role of Utx that regulates articular chondrocyte anabolism and OA development

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

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Cranial cruciate ligament (CrCL) disease/rupture causes pain and osteoarthritis (OA) in dogs. α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 and kainate (KA)-1 glutamate receptors (GluR) and the excitatory amino acid transporter-1 (EAAT-1) and EAAT-3 are expressed in joint tissues from OA patients and rodent arthritis models and represent potential therapeutic targets. Objectives. To evaluate glutamate signalling in canine diseased and normal CrCL and meniscus by immunohistochemistry (IHC). Methods. Surgical waste (CrCL, n=5 and medial meniscus, n=3) were obtained from canines with CrCL disease (RCVS ethics approval:2017/14/Alves) and normal analogous tissues (n=2). IHC optimization was performed for rabbit polyclonal (AMPA-2:ab52176, KA-1:ab67402, EAAT-1:ab416) and monoclonal (EAAT-3:ab124802) antibodies from Abcam. IHC was optimised over antibody dilutions from 1:100 to 1:5000 alongside equivalent IgG isotype controls (ab37415 and ab172730) and negative controls (TBS/Tween buffer without primary antibodies). IHC staining was compared in diseased and normal tissues and disclosed with 3,3’-Diaminobenzidine (DAB). Results. Specific immunostaining was observed for all primary antibodies, at concentrations between 2.0×10. −4. mg/mL to 1.0×10. −2. mg/mL, depending on the tissue and primary antibody. All GluR and transporters were expressed in the cellular membrane, in the normal and diseased CrCL and meniscus. Healthy CrCL showed a well-organized microstructure, with normal positively labelled ligamentocytes, whereas diseased CrCL microstructure was disrupted, with many positively stained fibroblastic cells in the epiligamentous region and evident neovascularization, indicative of ongoing repair. The normal and diseased meniscal tissues showed similar chondrocytes-like cells labelling and microstructure. Negative controls demonstrated no labelling. Conclusions. GluR and transporters expression is altered in canine diseased CrCLs, implicating glutamate signalling in this pathology. Since AMPA/KA GluR antagonists alleviate joint degeneration in post-traumatic OA in rodent models, they may be useful for the treatment of CrCL disease in dogs, as well as translated to other veterinary and human orthopaedic diseases

Introduction. Articular cartilage injuries have a limited potential to heal and, over time, may lead to osteoarthritis, an inflammatory and degenerative joint disease associated with activity-related pain, swelling, and impaired mobility. Regeneration and restoration of the joint tissue functionality remain unmet challenges. Stem cell-based tissue engineering is a promising paradigm to treat cartilage degeneration. In this context, hydrogels have emerged as promising biomaterials, due to their biocompatibility, ability to mimic the tissue extracellular matrix and excellent permeability. Different stimulation strategies have been investigated to guarantee proper conditions for mesenchymal stem cell differentiation into chondrocytes, including growth factors, cell-cell interactions, and biomaterials. An interesting tool to facilitate chondrogenesis is external ultrasound stimulation. In particular, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to have a role in regulating the differentiation of adipose mesenchymal stromal cells (ASCs). However, chondrogenic differentiation of ASCs has been never associated to a precisely measured ultrasound dose. In this study, we aimed to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D hydrogel. Materials and Methods. Human adipose mesenchymal stromal cells at 2∗10. 6. cells/mL were embedded in a hydrogel ratio 1:2 (VitroGel RGD®) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm. 2. , duty cycle: 20%, pulse repetition frequency: 1 kHz, stimulation time: 5 min) in order to assess its influence on cell differentiation. Hydrogel-loaded ASCs were cultured and differentiated for 2, 7, 10 and 28 days. At each time point cell viability (Live&Dead), metabolic activity (Alamar Blue), cytotoxicity (LDH), gene expression (COL2, aggrecan, SOX9, and COL1), histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated respect to a non-stimulated control. Results. Histological analysis evidenced a uniform distribution of ASCs both at the periphery and at the center of the hydrogel. Live & Dead test evidenced that the encapsulated ASCs were viable, with no signs of cytotoxicity. We found that LIPUS induced chondrogenesis of ASCs embedded in the hydrogel, as demonstrated by increased expression of COL2, aggrecan and SOX9 genes and proteins, and decreased expression of COL1 respect to the non-stimulated control. Conclusions. These results suggest that the LIPUS treatment could be a valuable tool in cartilage tissue engineering, to push the differentiation of ASCs encapsulated in a 3D hydrogel

Introduction and Objective. Traditionally, osteoarthritis (OA) has been associated mostly with degradation of cartilage only. More recently, it has been established that other joint tissues, in particular bone, are also centrally involved. However, the link between these two tissues remains unclear. This relationship is particularly evident in post-traumatic OA (PTOA), where bone marrow lesions (BMLs), as well as fluctuating levels of inflammation, are present long before cartilage degradation begins. The process of bone-cartilage crosstalk has been challenging to study due to its multi-tissue complexity. Thus, the use of explant model systems have been crucial in advancing our knowledge. Thus, we developed a novel patellar explant model, to study bone cartilage crosstalk, in particular related to subchondral bone damage, as an alternative to traditional femoral head explants or cylindrical core specimens. The commonly used osteochondral explant models are limited, for our application, since they involve bone damage during harvest. The specifics aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response and mechanical stimulation to determine the subsequent developments of PTOA. Materials and Methods. Lewis rats (n=48) were used to obtain patellar and femoral head explants which were harvested under an institutional ethical approval license. Explants were maintained in high glucose media (containing supplements), under sterile culture conditions. Initially, we characterised undamaged patellar explants and compared them with the commonly used femoral head. First, tissue viability was assessed using an assay of metabolic activity and cell damage. Second, we created chemical and mechanical damage in the form of IL-1B treatment, and mechanical stimulation, to replicate damage. Standard biochemical assays, histological assays and microstructural assays were used to evaluate responses. For chemical damage, explants were exposed to 10ng/ml of IL-1B for 24 hours at 0, 1, 3 and 7 days after harvesting. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles, for 7 days. Contralateral patellae served as controls. In both groups, sGAG, ADAMTS4, and MMP-13 were measured as an assessment of representative cartilage responses while ALP, TRAP and CTSK were assessed as a representative of bone responses. In addition to this, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out. Results. Our results confirm that the patellar explant system is an excellent ex vivo model system to study bone-cartilage crosstalk, and one which does not induce any bone damage at the time of tissue harvest. We successfully established culture conditions to maintain viability in these explants for up to 28 days. Rat IL-1B treatment resulted in increased both proteoglycan content and bone metabolism markers after 7 days when compared with the controls. To confirm this finding, qualitative immunohistochemical staining showed chondrocytes increased expression of MMP13 after treatment with IL-1B. Furthermore, we observed that the levels of ADAMTS4 decreased in 48 hours after IL-1B exposure. Contrastingly IL-1B treatment had the opposite effect on CTSK markers when compared with the control. Mechanically compressed patellae showed a decrease in compressive moduli from day 3 to day 7, suggesting that tissue remodelling may have taken place as a compensatory mechanism in response to damage. In addition, MMP13 release decreased over 48 hours after mechanical compression, while TRAP levels were increased compared with the control. Conclusions. Thus, we successfully demonstrated that IL-1B and mechanical stimulation affects both bone and cartilage tissues independently in this system, which may have relevance in the understanding of bone-cartilage crosstalk after injury and how this is involved in PTOA development

Treatment of comminuted intraarticular calcaneal fractures remains controversial and challenging. Anatomic reduction with stable fixation has demonstrated better outcomes than nonoperative treatment of displaced intraarticular fractures involving the posterior facet and anterior calcaneocuboid joint (CCJ) articulating surface of the calcaneus. The aim of this study was to investigate the biomechanical performance of three different methods for fixation of comminuted intraarticular calcaneal fractures. Comminuted calcaneal fractures, including Sanders III-AB fracture of the posterior facet and Kinner II-B fracture of the CCJ articulating calcaneal surface, were simulated in 18 fresh-frozen human cadaveric lower legs by means of osteotomies. The ankle joint, medial soft tissues and midtarsal bones along with the ligaments were preserved. The specimens were randomized according to their bone mineral density to 3 groups for fixation with either (1) 2.7 mm variable-angle locking anterolateral calcaneal plate in combination with one 4.5 mm and one 6.5 mm cannulated screw (Group 1), (2) 2.7 mm variable-angle locking lateral calcaneal plate (Group 2), or (3) interlocking calcaneal nail with 3.5 mm screws in combination with 3 separate 4.0 mm cannulated screws (Group 3). All specimens were biomechanically tested until failure under axial loading with the foot in simulated midstance position. Each test commenced with an initial quasi-static compression ramp from 50 N to 200 N, followed by progressively increasing cyclic loading at 2Hz. Starting from 200 N, the peak load of each cycle increased at a rate of 0.2 N/cycle. Interfragmentary movements were captured by means of optical motion tracking. In addition, mediolateral X-rays were taken every 250 cycles with a triggered C-arm. Varus deformation between the tuber calcanei and lateral calcaneal fragments, plantar gapping between the anterior process and tuber fragments, displacement at the plantar aspect of the CCJ articular calcaneal surface, and Böhler angle were evaluated. Varus deformation of 10° was reached at significantly lower number of cycles in Group 2 compared to Group 1 and Group 3 (P ≤ 0.017). Both cycles to 10° plantar gapping and 2 mm displacement at the CCJ articular calcaneal surface revealed no significant differences between the groups (P ≥ 0.773). Böhler angle after 5000 cycles (1200 N peak load) had significantly bigger decrease in Group 2 compared to both other groups (P ≤ 0.020). From biomechanical perspective, treatment of comminuted intraarticular calcaneal fractures using variable-angle locked plate with additional longitudinal screws or interlocked nail in combination with separate transversal screws seems to provide superior stability as opposed to variable-angle locked plating only

Displaced intraarticular calcaneal fractures are debilitating injuries with significant socioeconomic and psychological effects primarily affecting patients in active age between 30 and 50 years. Recently, minimally and less invasive screw fixation techniques have become popular as alternative to locked plating. The aim of this study was to analyze biomechanically in direct comparison the primary stability of 3 different cannulated screw configurations for fixation of Sanders type II-B intraarticular calcaneal fractures. Fifteen fresh-frozen human cadaveric lower limbs were amputated mid-calf and through the Chopart joint. Following, soft tissues at the lateral foot side were removed, whereas the medial side and Achilles tendon were preserved. Reproducible Sanders type II-B intraarticular fracture patterns were created by means of osteotomies. The proximal tibia end and the anterior-inferior aspect of the calcaneus were then embedded in polymethylmethacrylate. Based on bone mineral density measurements, the specimens were randomized to 3 groups for fixation with 3 different screw configurations using two 6.5 mm and two 4.5 mm cannulated screws. In Group 1, two parallel longitudinal screws entered the tuber calcanei above the Achilles tendon insertion and proceeded to the anterior process, and two transverse screws fixed the posterior facet perpendicular to the fracture line. In Group 2, two parallel screws entered the tuber calcanei below the Achilles tendon insertion, aiming at the anterior process, and two transverse screws fixed the posterior facet. In Group 3, two screws were inserted along the bone axis, entering the tuber calcanei above the Achilles tendon insertion and proceeding to the central-inferior part of the anterior process. In addition, one transverse screw was inserted from lateral to medial for fixation of the posterior facet and one oblique screw – inserted from the posterior-plantar part of the tuber calcanei – supported the posterolateral part of the posterior facet. All specimens were tested in simulated midstance position under progressively increasing cyclic loading at 2 Hz. Starting from 200N, the peak load of each cycle increased at a rate of 0.1 N/cycle. Interfragmentary movements were captured by means of optical motion tracking and triggered mediolateral x-rays. Plantar movement, defined as displacement between the anterior process and the tuber calcanei at the most inferior side was biggest in Group 2 and increased significantly over test cycles in all groups (P = 0.001). Cycles to 2 mm plantar movement were significantly higher in both Group 1 (15847 ± 5250) and Group 3 (13323 ± 4363) compared to Group 2 (4875 ± 3480), P = 0.048. Medial gapping after 2500 cycles was significantly bigger in Group 2 versus Group 3, P = 0.024. No intraarticular displacement was observed in any group during testing. From biomechanical perspective, screw configuration implementing one oblique screw seems to provide sufficient hindfoot stability in Sanders Type II-B intraarticular calcaneal fractures under dynamic loading. Posterior facet support by means of buttress or superiorly inserted longitudinal screws results in less plantar movement between the tuber calcanei and anterior fragments. On the other hand, inferiorly inserted longitudinal screws seem to be associated with bigger interfragmentary movements

Background. As the number of ceramic THR bearings used worldwide is increasing, the number of implants that experience off-normal working conditions, e.g. edge loading, third bodies in the joint, soft tissues laxity, dislocation/subluxation of the joint, increases too. Under all such conditions the bearing surfaces can be damaged, leading eventually to a limitation of the expected performances of the implant. Methods. We characterised the damage resistance of different bearing surfaces (alumina matrix composite BIOLOXdelta, alpha-alumina BIOLOXforte, zirconia 3Y-TZP, oxidized zirconium alloy Zr-2.5Nb, CoCr-alloy) by scratch tests performed following the European standard EN 1071–3:2005. Also the scratch hardness of same materials has been assessed. Results. The Lc1 value (i.e., the load for the onset of a scratch) measured for BIOLOXdelta is about fivefold the one measured for the oxidized zirconium alloy (OXZr) surface and about tenfold the Lc1 measured for the CoCr alloy. The height of ridges along the scratch edges due to plastic flow in the composite ceramic BIOLOXdelta are only 21% in height than in CoCr, and only a small fraction (0.04%) of the height of ridges measured on OXZr surfaces. The scratch hardness of the metal samples tested (CoCr, OXZr) results one order of magnitude lower than the ones of ceramics. This behavior is not influenced by of the presence of the coating on OXZr surface. Conclusions. The transformation toughened ceramics tested (BIOLOXdelta, 3Y-TZP) are the materials that exhibit the higher resistance to scratching. Ridges at scratch edges are lower in ceramics than in coated or uncoated metals. The result show the superior scratch resistance behavior of toughened ceramics for THR wear couples with respect to coated or bare alloys. Level of Evidence. Level 1

There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4). Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for in-vivo experiments to create a novel model of atrophic non-union with stable fixation

Several studies explored the biological effects of low frequency low energy pulsed electromagnetic fields (PEMFs, Igea Biophysics Laboratory, Carpi, Italy) on human body reporting different functional changes. In the orthopedic field, PEMFs have been shown to be effective in enhancing endogenous bone and osteochondral repair, incrementing bone mineral density, accelerating the process of osteogenic differentiation and limiting cartilage damage. Much research activity has focused on the mechanisms of interaction between PEMFs and membrane receptors such as adenosine receptors (ARs). In particular, PEMF exposure mediates a significant upregulation of A. 2A. and A. 3. ARs expressed in various cells or tissues involving a reduction of most of the pro-inflammatory cytokines. In tissue engineering for cartilage repair a double role for PEMFs could be hypothesized: in vitro by stimulating cell proliferation, colonization of the scaffold and production of tissue matrix; in vivo after surgical implantation of the construct by favoring the anabolic activities of the implanted cells and surrounding tissues and protecting the construct from the catabolic effects of inflammation. Of particular interest is the observation that PEMFs, through the increase of ARs, enhance the working efficiency of the endogenous modulator adenosine, producing a more physiological effect than the use of exogenous drugs. This observation suggests the hypothesis that PEMFs could be considered a non-invasive treatment with a low impact on daily life. In conclusion, PEMFs represent an important approach in the pharmacological field providing excellent therapeutic results in various inflammatory diseases and in particular in the functional recovery of the damaged joint tissues

Mesenchymal stromal cells (MSCs) are promising candidate for cell therapy in osteoarthritis (OA) patients since that they exert anti-inflammatory, immunomodulatory, anti-fibrotic and anti-hypertrophic effects in the joint tissues. However, little is known about the OA milieu factors that could enhance the migration and tissue specific engraftment of exogenously injected MSC for successful regenerative cell therapy. GMP-clinical grade adipose stromal cells (ASC) were evaluated both in normoxic and hypoxic (2%O. 2. ) conditions, with or without OA synovium milieu. We found that both OA synovial fluids and OA synoviocytes derived conditioned medium (CM) contain approximately the same amounts of different cytokines/chemokines (i.e. IL6, CXCL8, CXCL10, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11). ASC migration was significantly increased by both OA synovium milieu and not affected by normoxic or hypoxic condition. We identify that ASC migration was mainly influenced by different macrophage chemokines (i.e. CCL2, CCL3, CCL4). In hypoxic condition basal GMP-ASC showed an increase of CXCR3 and CCR3, a decrease of CCR1 and CCR5 receptors, while CXCR1, CXCR4, CXCR7, CCR2 and IL6R were not modulated. The addition of OA synovium milieu induced CCR3, CXCR3 and IL6R and decreased CCR1 and not affected CCR2, CCR5, CXCR1, CXCR4, CXCR7 in hypoxic condition. Our data demonstrated that GMP-ASC chemotaxis was mainly induced by macrophage chemokines. Moreover, we evidenced that hypoxia, as better condition to mimic the OA milieu, affected some GMP-ASC cytokine/chemokine receptors, suggesting the involvement of specific chemokine-receptor axis

There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4). Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for in-vivo experiments to create a novel model of atrophic non-union with stable fixation

Pulsed Electromagnetic Fields (PEMFs) promote joint tissue anabolic activities, particularly in cartilage and bone. Here we investigated the effect of selected PEMFs (75Hz, 1.5mT, 1.3msec) in a differentiating model of murine myoblasts (C2C12) in vitro. C2C12 were seeded at 5×10. 3. cells/cm. 2. in 4 well plates and left to adhere for 24h. Subsequently, cells were either maintained in growth medium (GM) or induced towards myogenic differentiation in low-serum conditions, with and without PEMF exposure, for 4 days. Morphological analysis, myotube formation and fusion index (FI) were assessed with fluorescence microscopy techniques. Metabolic activity was determined by MTT; moreover, a multiplex cytokine array (RayBiotech) allowed cell supernatant molecule quantification. Cells exposed to PEMFs in GM acquired a distinctive elongated morphology, with increased bi-nuclear figures (3.2-fold FI increase over PEMF-unexposed cells) and displayed a significantly higher metabolic activity (+31%, p<0.05 over PEMF-unexposed cells). PEMF exposure increased metabolic activity also under myogenic differentiation (+15% over PEMF-unexposed differentiating cells, p<0.05), with the formation of long, thick polynuclear myotubes, suggesting a role of PEMFs in enhancing myogenesis (7.7-fold FI increase over PEMF-unexposed cells). 4-day culture supernatants revealed the presence of several myokines (KC/CXCL1, LIX, MCP-1, TIMP-1). Preliminary analysis showed a 1.16-fold increase (n=2) of LIX and, notably, a 1.91-fold increase (n=2) of TNF-RI, in cell supernatants of PEMF-exposed over PEMF-unexposed cells. Collectively, these results suggest that PEMF may successfully be applied in models of muscle cell trauma to optimise muscle fibre repair, by fine-tuning the release of myokines, promoting myoblast proliferation and myotube formation

Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features in the progression of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral bone integrity diminishment remained elusive. This study was undertaken to characterize behavior and intracellular signaling of subchondral mesenchymal stem cells (SMSCs) and bone-marrow MSCs (BMMSCs) in OA knees isolated from patients with end-stage knee OA underwent total knee arthroplasty. The SMSCs isolated from subchondral bone explants expressed remarkable surface antigens CD73, CD105, CD90, CD166, CD44, CD29, instead of MHC II, CD45, and CD31. The cell cultures exhibited high proliferation capacity concomitant with low population doubling time compared to those of BMMSCs. Incubation in differentiation media, the SMSCs showed high osteogenic and chondrogenic lineage commitment and low adipogenic differentiation potential. They also exhibited high expression of embryonic stem cell marker OCT3/4, osteogenic factors Wnt3a, β-catenin and microRNA-29a (miR-29a) in conjunction with low expression of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNFα, and MMP3. Loss of miR-29a function lowered HDAC4 level, mineralized matrix accumulation and osteogenic marker expression of SMSCs. miR-29a reduced HDAC4 translation through targeting the 3”-untranslated region of HDAC4, which concomitantly sustained Wnt3a and β-catenin signaling. Collectively, high osteogenic lineage commitment existed in the SMSCs in OA knee microenvironment. miR-29a modulation of HDAC4 and Wnt3a signaling contributed to the increases in osteogenesis. This study shines a light no the biological role of MSCs in subchondral compartment in the end-stage OA development and highlights a new source of MSCs for joint tissue repair

Summary. Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair. Introduction. As compared to articular chondrocytes (AC), nasal septum chondrocytes (NC) proliferate faster and have a higher and more reproducible capacity to generate hyaline-like cartilaginous tissues. Moreover, the use of NC would allow reducing the morbidity associated with the harvesting of cartilage biopsy from the patient. The objective of the present study was to demonstrate safety and feasibility in the use of tissue engineered cartilage graft based on autologous nasal chondrocytes for the repair of articular defect in goats. Methods. Isolated autologous NC and AC from 6 goats were expanded and GFP-labelled before seeding 4×10. 4. cells/cm. 2. on a type I/III collagen membrane (Chondro-Gide®, Geistlich). After 2 weeks of chondrogenic differentiation 2 NC- and 2 AC-based grafts were implanted into chondral defects (6mm diameter) of the same posterior stifle joint. Repair tissue was harvested after 3 or 6 months and the decalcified samples evaluated according to O'Driscoll. Furthermore, samples from the surrounding fat pad, ligament, synovium, tendon and patellar cartilage were harvested and isolated cells tested for GFP-positivity after expansion using FACS. Results. No surgical complication or signs of inflammation occurred in any of the animals. GFP-positive cells were detectable in the repair tissue, indicating the contribution of the implanted cells to the newly formed cartilage. The O'Driscoll score of the repair tissue increased from 8.6 and 7.6 after 3 months to 14.1 and 12.4 after 6 months for nasal and articular grafts, respectively. Surrounding tissues showed no or very low (fat pad 0–0.36%) migration of the grafted cells. Conclusion. Our results demonstrate the use of NC as safe and feasible for tissue engineering approaches in articular cartilage repair. The repair tissue-quality generated by NC-grafts was demonstrated to be at least comparable to that of AC-grafts, thus opening the way for clinical test of a novel therapeutic strategy

Background. Epigenetic regulation of gene transcription affects metabolism of chondrocytes and synovial fibroblasts and is associated with the prevalence of osteoarthritis (OA) of knees. Histone lysine demethylase (KDMs) reportedly modulates tissue homeostasis and deterioration. This study investigated whether KMD6a inhibitor treatment affected the joint injuries in the progression of OA. Methods. Collagenase-induced OA knees in mice were intra-articular administered with KDM6a inhibitor GSK-J4. Walking patterns and footprints of affected animals were detected by Catwalk. Articular cartilage injury was quantified by OARSI scoring; and subchondral bone microstructure was analysed by μCT imaging. Histopathology and mRNA expression of cartilage, fibrosis and bone matrices in joint micro-compartments were detected by histomorphometry and quantitative RT-PCR. Methylation states of chondrogenic transcription factor SOX9 promoter was detected by methylation-specific PCR and chromatin immuno-precipitation. Results. Declined KDM6a expression and SOX9 gene transcription was associated with the pathogenesis of collagenase-induced joint injures. GSK-J4 administration dose-dependently improved gait profiles and footprint characteristics of affected feet and alleviated histopathology of severe cartilage degradation, synovial inflammation, fibrotic matrix accumulation and subchondral bone microarchitecture deterioration in injured joints. Treatment with GSK-J4 decreased expression of fibrogenic factor (TGF-β1, PLOD2 and TIMP) and restored expression of cartilage and bone matrices (collagen II, I, aggrecan, and osteocalcin). KDM6a inhibitor curtailed the hypomethylation of SOX9 promoter and lysine 27 of histone H3 (H3K27) and restored SOX9 mRNA and protein levels in joint tissues. Conclusions. KDM6a enhanced SOX9 promoter and H3K27 hypomethylation that accelerated the progression of OA. KDM6a inhibitor had mitigated effects on SOX9 promoter demethylation thereby restored SOX9 signaling and stabilised homeostasis of cartilage, synovium and subchondral bone compartments in affected joints. This study sheds a new light on the KDM6a-mediated epigenetic dysfunction in OA joints and has a perspective that pharmaceutical KDM6a inhibitor has therapeutic potential for OA knee pathogenesis. Level of evidence. II

Osteoarthritis (OA) is no longer considered a cartilage-centric disease with remodelling of other joint tissues now recognized. While understudied, entheseal pathology is considered a secondary OA feature. A pivotal role for proteinase-activated receptor 2 (PAR2) in OA has been demonstrated previously in cartilage and subchondral bone at early time points, however the entheseal role of PAR2 has not been reported. OA was induced by destabilization of the medial meniscus (DMM) in wild type (WT) and PAR2 deficient (KO) animals. At 4 weeks and one year post surgery, knee joints were harvested for histological analysis. Medial collateral ligament (MCL) width was measured by 2D planimetry analysis. Immunohistochemistry was used to characterize the MCL and anterior cruciate ligament (ACL). Data were expressed as mean±SEM (n=4–6/group) and analysed using Student's t-test, with p<0.05 as the criterion of significance. MCL width increased between 4 weeks and 1 year in WT DMM (0.24 ±0.07 vs 0.40 ±0.008mm respectively, p<0.001). Interestingly, a significant reduction in MCL was observed in KO compared with WT at 1 year (0.23 ±0.005 vs 0.40 ±0.008mm respectively, p <0.001) post-DMM. Further characterization of DMM WT MCL and ACL at 4 weeks showed the presence of F4/80. +. cells in addition to IL-33 and histamine. At one year post-surgery, a cellular infiltrate was observed in MCL DMM WT but absent in KO mice. Histological evaluation revealed an absence of F4/80. +. cells but the presence of a PAR2. +. population, subsequently identified as hypertrophic-like chondrocytes (RUNX2) and chondrocytes-like cells (SOX9). Deletion of PAR2 affords long-term protection against ligament remodelling and demonstrates a critical role for this receptor in both OA joint pathology and ligament injuries. While PAR2 appears to be a credible therapeutic target in OA entheseal pathology, further understanding of the molecular mechanism regulated by this receptor will be required

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion. Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue. It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro

Objectives

Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes.