Comparing Classical Enzymatic vs Pauci-Enzymatic Automated Mechanical Disaggregation: Chondrocyte Phenotypes and proliferative signatures

INTRODUCTION: Osteoarthritis (OA) affects over 500 million people worldwide [1], making joint preservation a priority for active patients to delay arthroplasty. Autologous Chondrocyte Implantation, which involves the harvesting, expansion, and re-implantation of cells, is a key treatment for early OA, yet its success is limited by the tendency of primary chondrocytes to de-differentiate during ex vivo expansion into chondroprogenitors (CPC) [2]. To overcome this, chemical and mechanical strategies are employed to preserve the chondrocyte phenotype. This study evaluates the efficiency of isolating chondrocytes/CPCs from human OA knee cartilage for autotransplantation, comparing two protocols: classical enzymatic digestion versus a combined approach of rapid enzymatic incubation and automated-mechanical dissociation. MATERIAL AND METHODS: Human cartilage was obtained from surgical discards under the approval of the Ethics Committee for Human Experimentation of the University of Urbino (n. 84, 25 July 2024). Explanted tissues were finely minced using scalpel blades and pretreated with Pronase E. The samples were then divided into 2 groups: a classical enzymatic group (Enzymatic) subjected to prolonged collagenase digestion and a combined group (Medi) treated with rapid collagenase incubation, followed by automated-mechanical disaggregation using Medicons (CTSV). Cellular phenotypic characterization was performed using flow cytometry. Cultured chondrocytes/CPC were characterized by classical MSC markers and fibronectin receptor markers. RESULTS: Preliminary results show that cells obtained via the Medi protocol exhibit higher antigenic density for COL2A and Aggrecan one day post-procedure, as well as higher levels of CD90, CD49b, and CD44 after three culture passages. Furthermore, qPCR analysis revealed that CCND1 and CCND3 mRNA levels were significantly downregulated in the Enzymatic group compared to those obtained by Medi. This reduction suggests decreased cell proliferation in the Enzymatic protocol, likely due to impaired progression through the G1 phase of the cell cycle. CONCLUSION: Finally, all these findings will be further confirmed and studied to establish a more useful procedure for autotransplantation purposes.