Roman J. Krawetz

Objectives: Autoantibodies targeting extracellular matrix proteins are emerging as critical contributors to arthritis pathogenesis, yet their presence across different arthropathies remains incompletely characterized. This study investigated systemic and local expression of proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP), alongside their corresponding autoantibodies, in healthy controls and patients with OA, RA or JIA. Methods: Serum and SF samples were analysed by ELISA from control subjects, and OA, RA and JIA patients. Results: Serum PRG4 levels were significantly decreased in RA compared with controls and OA, while JIA patients showed elevated levels. Serum COMP was significantly elevated in OA and JIA compared with controls and RA. In SF, OA showed decreased PRG4 compared with all other groups, while COMP levels were highest in OA and controls. Anti-PRG4 autoantibodies were significantly elevated in all disease groups compared with controls in serum, with RA and JIA also showing increased elevation in SF. Anti-COMP autoantibodies were significantly increased in RA and JIA serum and elevated in all disease groups in SF compared with controls. Notably, a positive correlation between PRG4 protein and anti-PRG4 antibodies was observed specifically in OA SF, suggesting local antigen-driven immune responses. Conclusion: These findings demonstrate that autoantibodies against PRG4 and COMP are present across arthropathies with distinct patterns reflecting different pathogenic mechanisms. The detection of these autoantibodies provides mechanistic insights into disease progression and potential biomarkers for identifying differential pathway activation/inflammatory subgroups within arthropathies.

Focal articular cartilage defects lack intrinsic regenerative capacity and can progress to osteoarthritis as no effective treatment exists to slow, stop or reverse cartilage degeneration. Cellular senescence, characterized by elevated p21 (CDKN1A) expression, impairs chondrocyte proliferation and cartilage repair. We hypothesized that targeted p21 suppression via lentiviral delivery would enhance cartilage regeneration. Lentiviral shRNA vectors targeting p21 or non‐sense controls with a tdTomato reporter were constructed. In vitro, murine synovial progenitor cells and MC3T3E1 preosteoblasts were transduced and p21 mRNA was quantified by qRT‐PCR. Cell cycle analysis was performed using flow cytometry. In vivo, immunocompromised (B6.Cg‐Prkdc ^scid /SzJ) and immunocompetent (C57BL6) mice received full‐thickness cartilage defects followed by intraarticular p21 shRNA or nonsense control injection. Cartilage repair was assessed by Safranin O staining and tissue cytometry quantified lentiviral transduction and p21 knockdown in chondrocytes. In vitro, p21 shRNA achieved ~80% reduction in p21 mRNA and increased G2M phase cells. In vivo, p21 shRNA treatment significantly improved cartilage repair in both strains. Tissue cytometry revealed 90% transduction efficiency with p21 ⁺ cells reduced from ~90% to ~30%. Spearman correlation showed significant negative correlation between p21 expression and repair outcomes. Hepatic off‐target transduction was minimal in uninjured animals but increased in injured animals, yet morphological hepatotoxicity was not observed. p21 knockdown via lentiviral shRNA effectively promotes cartilage regeneration, supporting the concept of targeting p21 and/or the p21 pathway as a therapeutic strategy for cartilage repair.

Osteoarthritis (OA) is a painful, debilitating disease with no cure or treatments that can predictably stop/reverse its progression. Treatment is particularly difficult since articular cartilage lacks intrinsic repair capacity, despite mesenchymal stem cells (MSCs) being present in the joint with robust chondrogenic potential. While heterogeneity exists among MSC subtypes within human synovium, it remains unclear which populations can regenerate cartilage or impact OA progression. We clonally isolated MSCs from normal and OA patient synovium using indexed flow cytometry, then characterized them through differentiation assays and quantitative proteomics. MSC clones were transplanted into a xenograft rat OA model and evaluated by histology and immunofluorescence. We identified heterogeneity in putative MSCs within and between patient groups and their repair capacity in the rat model. However, traditional cell surface markers could not distinguish these subtypes, highlighting the need for single-cell level understanding. Using unbiased proteomics, we identified CD47 as a novel MSC marker. CD47 ^Hi cells demonstrated robust treatment efficacy in the rat OA model and directly contributed to new articular cartilage formation. Characterizing MSC subtypes is essential for identifying candidates appropriate for clinical investigation and exploiting functional MSCs for cartilage regeneration strategies.

Proteoglycan 4 (PRG4, lubricin) is a glycoprotein serving as a boundary lubricant in joints and immune-privileged tissues. Although PRG4 coatings prevent bacterial adhesion, potential bactericidal activity remains unclear. Given that antimicrobial heparin-binding peptides act via membrane disruption, the C-terminal heparin-binding region of PRG4 may possess similar properties. We tested full-length PRG4 (PRG4-F) and its C-terminal fragment (PRG4-C) generated by tryptase in a post-infection E. coli killing assay across 5–50 µg/mL. Neither PRG4-F nor PRG4-C reduced bacterial viability, indicating absence of direct bactericidal activity. PRG4’s antimicrobial function therefore appears limited to anti-adhesion mechanisms. Despite structural similarities to antimicrobial peptides, PRG4-C likely lacks amphipathicity required for membrane disruption, a feature that may minimize host cytotoxicity in immune-privileged tissues.

Osteoarthritis (OA) is a painful and debilitating disease which has no cure and there are no treatments which can predictably stop/reverse its progression. Treating this disease is particularly difficult since the articular cartilage lacks intrinsic repair capacity even though mesenchymal stem cells (MSCs) are present in the joint environment and have robust chondrogenic potential. We have previously shown that there is heterogeneity of MSC sub-types within the human synovium, yet it remains unclear if any of these MSC types can regenerate cartilage and/or impact OA disease progression. Therefore, we have undertaken this study focusing on clonally derived MSC populations derived from the synovium of normal and OA patients to characterize if any MSC populations can positively impact OA disease trajectory in a rat model of OA. MSCs were clonally isolated by indexed flow cytometry, expanded in culture and then characterized for differentiation capacity and by quantitative proteomics. MSC clones were then transplanted into a xenograft rat OA model and treatment effect was determined by histology and immunofluorescence outcomes. We identified heterogeneity in putative MSCs derived from within and between patient groups (normal vs. OA) and the ability of these cells to effect repair in a rat OA model. However, these different sub-types of MSCs could not be distinguished by traditional cell surface markers showing the need for a better understanding of these populations at the single cell level. Using an unbiased proteomics approach, CD47 was identified a novel marker of human MSCs. Using the same rat model of OA, CD47Hi expressing cells were found to have robust treatment efficacy and directly contributed to the formation of new articular cartilage tissue. Characterizing MSCs is essential to understand which sub-types are appropriate for further clinical investigation. If OA patients still have functional MSCs in their synovium, then it is possible these cells can be exploited for cartilage regeneration / OA treatment strategies.

Objective: Articular cartilage has limited regenerative capacity due to its lack of innervation, vascularization, and lymphatic vessels. As cartilage is devoid of nerves, injuries often go unnoticed until degeneration leads to pain, reduced function, and ultimately osteoarthritis (OA). Treatment options for cartilage injury, both surgical and nonsurgical, depend on factors like defect size, shape, depth, location, and patient age. Stem cells, particularly their ability to differentiate into chondrocytes, hold promise for cartilage repair, but no therapies have yet gained clinical approval. Recently, induced pluripotent stem cells (iPSCs) have emerged as a potential solution for cartilage regeneration. However, post-transplantation tumorigenesis remains a significant concern. To mitigate this risk, robust quality and safety protocols are needed, alongside safety mechanisms to control iPSC behavior after transplantation. Design: The iCaspase9 (iCasp9) cell suicide system offers a promising solution, enabling selective elimination of genetically modified cells via apoptosis. We previously demonstrated that the efficiency of iCasp9-mediated killing increases in a p21 mutant background. Since p21 mutations also enhance cartilage repair, we investigated iCasp9-engineered p21−/− and wildtype (p21+/+) iPSCs in a mouse cartilage injury model. Results: Without iCasp9 activation, both p21−/− and p21+/+ iPSCs formed tumors post-transplantation. In contrast, mice treated with the iCasp9 activator AP20187 showed no tumors. Both p21−/− and p21+/+ iPSCs demonstrated similar cartilage regeneration. Conclusions: These findings suggest that iCasp9-mediated elimination of iPSCs can effectively mitigate tumor risks while preserving their therapeutic potential for cartilage repair.