Background Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. Methods Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin’s grade 0–2, 23–83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. Results Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. Conclusion CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.

Pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic calcification disorder, is predominantly caused by inactivating mutations in ABCC6. The encoded protein, ABCC6, is a hepatic efflux transporter and a key regulator of extracellular inorganic pyrophosphate (PPi). Recent studies demonstrated that deficiency of plasma PPi, a potent endogenous calcification inhibitor, is the underlying cause of PXE. This study examined whether restoring plasma PPi levels by INZ-701, a recombinant human ENPP1 protein, the principal PPi-generating enzyme, prevents ectopic calcification in an Abcc6 −/− mouse model of PXE. Abcc6 −/− mice, at 6 weeks of age, the time of earliest stages of ectopic calcification, were injected subcutaneously with INZ-701 at 2 or 10 mg/kg for 2 or 8 weeks. INZ-701 at both doses increased steady-state plasma ENPP1 activity and PPi levels. In the 8-week treatment study, histopathologic examination and quantification of the calcium content in INZ-701-treated Abcc6 −/− mice revealed significantly reduced calcification in the muzzle skin containing vibrissae, a biomarker of the calcification process in these mice. The extent of calcification corresponds to the local expression of two calcification inhibitors, osteopontin and fetuin-A. These results suggest that INZ-701 might provide a therapeutic approach for PXE, a disease with high unmet needs and no approved treatment.

Background Post-traumatic osteoarthritis (PTOA) does not currently have clinical prognostic biomarkers or disease-modifying drugs, though promising candidates such as dexamethasone (Dex) exist. Many challenges in studying and treating this disease stem from tissue interactions that complicate understanding of drug effects. We present an ex vivo human osteochondral model of PTOA to investigate disease effects on cartilage and bone homeostasis and discover biomarkers for disease progression and drug efficacy. Methods Human osteochondral explants were harvested from normal (Collins grade 0–1) ankle talocrural joints of human donors (2 female, 5 male, ages 23–70). After pre-equilibration, osteochondral explants were treated with a single-impact mechanical injury and TNF-α, IL-6, and sIL-6R ± 100 nM Dex for 21 days and media collected every 2–3 days. Chondrocyte viability, tissue DNA content, and glycosaminoglycan (sGAG) percent loss to the media were assayed and compared to untreated controls using a linear mixed effects model. Mass spectrometry analysis was performed for both cartilage tissue and pooled culture medium, and the statistical significance of protein abundance changes was determined with the R package limma and empirical Bayes statistics. Partial least squares regression analyses of sGAG loss and Dex attenuation of sGAG loss against proteomic data were performed. Results Injury and cytokine treatment caused an increase in the release of matrix components, proteases, pro-inflammatory factors, and intracellular proteins, while tissue lost intracellular metabolic proteins, which was mitigated with the addition of Dex. Dex maintained chondrocyte viability and reduced sGAG loss caused by injury and cytokine treatment by 2/3 overall, with donor-specific differences in the sGAG attenuation effect. Biomarkers of bone metabolism had mixed effects, and collagen II synthesis was suppressed with both disease and Dex treatment by 2- to 5-fold. Semitryptic peptides associated with increased sGAG loss were identified. Pro-inflammatory humoral proteins and apolipoproteins were associated with lower Dex responses. Conclusions Catabolic effects on cartilage tissue caused by injury and cytokine treatment were reduced with the addition of Dex in this osteochondral PTOA model. This study presents potential peptide biomarkers of early PTOA progression and Dex efficacy that can help identify and treat patients at risk of PTOA.

apparent integrity of the cartilage structure. Showed the most evident changes in the extremes of the condyle. However, at 20 DAI, the changes were significant, showed clearly clusters in the superficial and middle zone of the cartilage, and the evident loss of PGs contained. In addition, the expression of collagen I (Col-I), and MMP-3 and MMP-13 were increased, mainly around to the clusters. In contrast, although S samples showed a little increased in the expression of Col I, and reduction of PGs contained at 5 DAI. That changes were not significant in comparing with normal samples. Conclusions:The new OA mouse model results less invasive, due to the mice can walk normally, and its lesion permits follows a kinetic from the early to the late stages of OA. In addition, all the changes identified, are congruent with others OA models, such as some presents in rat, and even can to be scored using the OARSI histopathology recommendations.

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