Different antigen detection from osteoarthritic cartilage samples disaggregated via classic enzymatic, mechanic, and combined protocols: preliminary findings

Joint preservation is the preferred treatment strategy for knee chondropathies in active patients to improve pain, restore activity, and delay arthroplasty. Autologous chondrocyte implantation (ACI), a procedure that involves cell harvesting, culturing, and re-implanting, can be considered a treatment for early osteoarthritis affecting younger patients. However, this cell-based approach has limited success, presumably because the chondrocytes dedifferentiate when cultured, acquiring a fibroblast- and/or mesenchymal-like phenotype [1]. Chemical and mechanical treatments are promptly employed to preserve the chondrocyte phenotype in vitro [2]. This work aims to evaluate the ability to obtain chondrocytes with different protocols (classical enzymatic, mechanical, and combined protocols) from osteoarthritic cartilage samples, testing their phenotype and viability immediately after the procedure and during culture. Human knee osteoarthritic cartilage samples were obtained from surgical discards of patients enrolled in the study, with the approval of the Ethics Committee for Human Experimentation (CESU) of the University of Urbino (session no. 84 of 25 July 2024), and written informed consent was obtained before sample collection. Explanted tissue was finely minced (~1 mm) using scalpel blades; then minced tissue was pretreated or not with 1 h Pronase E (0.4% w/v) and split in:1) enzymatically digested samples, using collagenase (1 or 3 mg/ml) for 5 h and from 16 h to 20 h; 2) samples disaggregated with disposable tissue dissociators (Medicons P and white, CTSV s.r.l.); 3) samples treated with the combination of mechanical dissociators, and 1 ml collagenase (for 45 min). The studied antibodies against human surface antigen were as follows: a) positive MSC markers: CD106-FITC (1.G11B1), CD90-PE (Thy-1/310) b) negative MSC markers: CD45-PerCP Cy5.5 (2DI), CD34-APC (8G12) and c) classic fibronectin receptor markers: CD29-PE (VJ1/14), CD49b-AF488 (P1E6-C5) and CD44-PE (J.173). Preliminary results reveal a greater antigenic density for CD90, CD49b, and CD44 molecules in cells obtained by mechanical protocols or with minimal enzymatic manipulation with respect to only the enzymatic procedure. Furthermore, the differently obtained chondrocytes will be assessed for EV release capacity since EVs can be affected by prolonged collagenase digestion during the isolation of the cells. This work has been funded by the European Union - NextGenerationEU, Mission 4, Component 1, under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041 -VITALITY - CUP H33C22000430006