gms | German Medical Science

Objectives: Osteoarthritis (OA) still represents a major issue to modern orthopaedics and the quest to identify OA mitigating biologics is in great demand. Chondromodulin-I (Cnmd) and Tenomodulin (Tnmd) homologous genes encode type II transmembrane glycoproteins whose C-terminal domains are deposited in the extracellular matrix (ECM) of in particular cartilage and tendon tissues, respectively. Double knockout animals show no profound developmental phenotype; however, changes in articular cartilage during natural aging and OA progression have not been investigated so far. In order to circumvent possible compensatory mechanisms, in this study, knee joints of Cnmd/Tnmd knockout animals were subjected to OA-related analyses. In addition, to test the putative mechanosensitive role of Cnmd an in vitro three-dimensional (3D) model with primary human chondrocytes was established.

Methods: Knee joints from skeletally mature, 6-month-old (n=4) and aged, 20-month-old (n=5) controls and aged, 20-month-old Cnmd/Tnmd (n=5) double knockout mice were subject to cryoembeding and cryosectioning. H&E, DMMB and Safranin-O stainings were carried out using standard histological protocols. Scoring was performed by two independent observers (in a blind manner) using OARSI-modified system for mice. To establish a relevant 3D model for biomechanical studies, human primary chondrocytes (n=1 donor) were cultivated in 2D and in 3D polyurethane (PU) porous cylinders. Chondrocyte metabolic/proliferative activity and survival were assessed at 4 hours, day 1, 3, 5 and 7 by Resazurin and Live/Dead staining, correspondingly. Next, mRNA expression levels of Collagen I (alpha 1) and II (alpha 1), Aggrecan, Sox 9, Cnmd and GAPDH were investigated by semi-quantitative PCR at day 5 (positive control: mRNA from human cartilage tissue).

Results and conclusion: H&E, DMMB and Safranin-O stainings showed stronger intensity in the 6-month-old versus the 20-month-old groups. The WT young animals also demonstrated healthy and well-organized knee cartilage tissues, while aged control animals presented cartilage alterations encompassing mild surface abrasion, occasional vertical clefts and lesser chondrocyte density. Interestingly, aged double mutants exhibited more pronounced changes, namely loss of cartilage thickness, large lesions with exposure of subchondral bone and frequent vertical clefts. These observations were confirmed by the scoring system results. Next, pilot experiments in vitro revealed that the human chondrocytes attached, survived and propagated successfully in the 3D PU niche. The gene expression analysis suggested sustained chondrocyte molecular phenotype, as well as Cnmd expression in 3D. Hence, this PU/chondrocyte scaffold model can be further subjected to dynamic biomechanical stimulation protocols to study Cnmd mechanoresponsiveness in detail. Altogether, decoding Cnmd roles in aged-related OA can lead to development of novel disease-modifying drugs.