Targeting Chondromodulin-I (Cnmd) for Novel Osteoarthritis Interventions: In Vivo and 3d Model Insights

Osteoarthritis (OA) is a significant challenge in orthopedics, driving the demand for pharmacological drug development to mitigate its progression. Gene knockout mouse models help us understand disease pathology and outline novel treatment strategies. The homologous genes Chondromodulin-I (Cnmd) and Tenomodulin (Tnmd) encode type II transmembrane glycoproteins, with C-terminal domains found in the extracellular matrix (ECM) of cartilage and tendon tissues, respectively. Given their similarities, these genes are thought to exhibit compensatory roles in maintaining tissue integrity. Although double-knockout animals show no major developmental abnormalities, Cnmd's and Tnmd's role in cartilage tissue during age-associated OA remains unclear. Furthermore, as Cnmd is postulated to be mechanosensitive, an appropriate 3D cartilaginous model is needed. The main goals of this study were to examine the knee joints of Cnmd/Tnmd mutant animals and to develop a 3D in vitro model to assess Cnmd's mechanosensitive functions. Knee joints from skeletally mature mice were analyzed in three groups: 6-month-old (young) controls (n=4), 20-month-old (aged) controls (n=5), and 20-month-old (aged) Cnmd/Tnmd double mutants (n=5). Cryoembedded joints were sectioned and stained with H&E, DMMB, and Safranin-O/Fast Green. Sections were scored for cartilage changes using a modified OARSI system by three blinded observers. For in vitro studies, primary human chondrocytes (n=3) were cultured in static 3D culture using a hybrid polyurethane (PU)/Dextran (De)-Hyaluronic Acid (HA) porous (PUDeHA) scaffold. Chondrocyte viability and metabolic activity were assessed on days 1, 3, and 5 via Resazurin and Live/Dead staining. Gene expression of Collagen I, Collagen II, Aggrecan, SOX9, and Cnmd was analyzed by RT-PCR, using 2D chondrocytes and human cartilage as controls. Histological staining revealed more intense DMMB and Safranin-O staining in 6-month-old mice compared to 20-month-old groups. Aged controls showed mild surface abrasions, vertical clefts, and reduced chondrocyte density. Aged double mutants displayed severe cartilage thinning, larger lesions with subchondral bone exposure, and frequent vertical clefts. OARSI scores corroborated these findings, linking Cnmd absence to more severe OA progression. In vitro experiments showed that PUDeHA scaffolds supported chondrocyte survival and proliferation, with gene analysis revealing increased CNMD and COL2A1 expression and decreased COL1A1 compared to monolayer culture. Thus, this scaffold offers a promising system for studying CNMD mechanoresponsiveness in chondrocytes under dynamic conditions. Collectively, the in vivo and 3D models can help us decipher Cnmd's role in OA and contribute to the development of novel disease-modifying therapies inspired by Cnmd's mode-of-action.