Impact of Isolation Procedures on the Development of a Preclinical Synovial Fibroblasts/Macrophages in an In Vitro Model of Osteoarthritis.

Abstract

Simple SummaryIn vitro models able to represent osteoarthritis (OA) synovial tissue (ST) inflammation are lacking. Therefore, we aimed to characterize OA ST and to compare mechanical and enzymatic digestion procedures to find the one that better preserve the heterogeneity of the main OA synovial cell populations: fibroblasts and macrophages. We demonstrated that typical macrophage phenotypical markers, like CD68, CD80 and CD163, were higher expressed on cells isolated with mechanical than enzymatic procedure. Moreover, we found that typical cytokines of inflammatory macrophages (i.e., TNFα) and anti-inflammatory macrophages (i.e., IL10) were also higher on mechanically isolated cells. Synovial fibroblasts were well conserved in both procedures. The definition of an OA ST model in vitro that better preserve the heterogeneity of specific cell populations represents a prerequisite for defining the real effects of new cell therapies or drugs for OA treatment, and could contribute to the reduction or avoidance of animal models.There is a lack ofin vitromodels able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts (CD55, CD73, CD90, CD106), macrophages (CD14, CD68), M1-like (CD80, CD86), and M2-like (CD163, CD206) synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFβ1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFβ1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations (fibroblasts, macrophages), fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models.