Osteochondrosis (OC) is a common joint disease that affects developing cartilage and subchondral bone in humans, and in multiple animal species including horses. It is an idiopathic localized joint disorder characterized by focal chondronecrosis and retention of growing cartilage that can lead to the formation of fissures, subchondral bone cysts or intra-articular fragments. OC is considered a complex multifactorial disease with chondrocyte biogenesis impairment mainly due to biochemical and genetic factors. Likewise, the molecular events involved in the OC are not fully understood. Moreover, the OC pathogenesis seems to be shared across species. In particular, equine OC and human juvenile OC share some symptoms, predilection sites and clinical presentation. In this study, by using the label-free mass spectrometry approach, proteome of chondrocytes isolated from equine OC fragments has been analysed in order to clarify some aspects of cell metabolism impairment occurring in OC. Equine chondrocytes isolated from 7 healthy articular cartilages (CTRL) and from 7 osteochondritic fragments (OC) (both obtained from metacarpo/metatarsophalangeal joints) were analysed. Proteins were extracted using urea and ammonium bicarbonate buffer, reduced, alkylated and digested with Trypsin/Lys-C Mix. Peptides were analysed using Q Exactive UHMR Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). All mass spectra of label-free samples analysed was set up to search against SwissProt human database (Homo sapiens) and SwissProt horse database (Equus caballus). One-way ANOVA was used for hypothesis testing. Proteins with a ≥1.5 fold change and with a FDR adjusted p value of ≤0.05 were defined as differentially expressed. Statistical analysis evidenced 41 proteins up-regulated in OC while 18 were down-regulated with respect to the CTRL. Functional analysis showed that up-regulated proteins in OC were related to extracellular matrix degradation, lysosome, apoptotic execution phase, unfolded protein response, hyaluronan and keratan sulfate degradation, oxidative stress response and negative regulation of BMP signalling pathway. The down-regulated proteins were associated with endochondral ossification, vitamin D in inflammatory disease, Wnt signalling pathway and ECM proteoglycans. Validation assays confirmed these findings. These findings may contribute to clarify the events determining the onset and progression of both equine and human OC. Imaging MS analysis of OC and healthy cartilage to analyse lipid and metabolomic changes occurring in OC cartilage is in progress

Summary Statement. OA knee with subchondral cyst formation presented differential microstructure and mechanical competence of trabecular bone. This finding sheds light on the pivot role of subchondral cyst in OA bone pathophysiology. Introduction. Subchondral bone cyst (SBC) is a major radiological finding in knee osteoarthritis (OA), together with joint space narrowing, osteophyte and sclerotic bone formation. There is mounting evidence showing that SBC originates in the same region as bone marrow lesions (BMLs). The presence of subchondral bone cyst (SBCs), in conjunction with BMLs, was associated with the severity of pain, and was able to predict tibial cartilage lolume loss and risk of joint replacement surgery in knee OA patient. It is speculated that the presence of SBCs might increase intraosseous pressure of subchondral bone, and trigger active remodeling and high turnover of surrounding trabecular bone. Yet the exact effect of SBC on the structural and mechanical properties trabecular bone, which provides the support to overlying articular cartilage, remains to be elucidated. Therefore, this study aimed to investiate the microstructure and mechanical competence of trabecular bone of knee OA in presence or absence of SBC. Patients & Methods. A total of 20 postmenopausal women (54–87 years old) with the late-stage of primary knee OA were recruited in this study. Tibial plateau specimens were collected during joint replacement surgery. The samples were grouped for comparison according to presence or absences of SBC in micro-CT images. For micro-CT examination, a cylindrical volume of region of interest (VOI) of 10mm in diameter and 1mm in height was used to cover the trabecular bone region surrounding SBC, and then a cubic VOI of 3.5×3.5×3.5mm. 3. was applied in different anatomic locations of tibial plateau, such as medial, intermediate and lateral part, for the analyses of trabecular bone microstructure. Subsequently, two cylinders of subchondral bone specimens were drilled for each sample with micro-CT guidance from lateral portion of cystic wall along the direction of physiological loading of knee joint. The specimens were processed for micro-CT and mechanical testing using MTS 858 Mini Bionix sequentially. Each specimen was compressed in a longitudinal direction at a speed of 1mm/minute; the ultimate strength and modulus of the specimens were generated. Comparisons of microstructure and mechanical properties of trabecular bone were performed between two groups using student t test. The structure-mechanics relationship was also investigated using Pearson correlation. Results. The bone volume fraction (BV/TV, %) was significantly higher in knee OA specimens in presence of SBC (32±7%) in comparison with those in absence of SBC (16±5%, p<0.001). Meanwhile there were more plate-like trabecular bone surrounding SBC (0.78±0.61) than those without SBC (1.81±0.28, p<0.001), which was indicated by structure model index (0∼3). Furthermore, the trend in conversion of rod-like (close to 3) towards plate-like trabeculae was noticed in different locations of knee OA specimens with SBC formation. Trabecular bone around SBC presented higher modulus (73±22MPa) compared with those without SBC (45±29MPa, p=0.034). The stiffer trabecular bone in presence of SBC correlated with its plate-like morphology (r=0.696, p<0.001) as well as bone volume fraction (r=0.578, p=0.004). Conclusion. Presence of SBC was associated with conversion of trabeculae towards plate-like morphology together with the increase of mechanical competence in advanced knee OA