Sw982

The Santiago Hospital Ethics Committee (CAEIG-2016/258) approved the informed consent, the signature for which is mandatory for all participant donors. Articular issue samples from healthy and OA patients were, respectively, obtained from dead and total knee replacement surgery donors [77 (link)]. Primary joint cells (chondrocytes, osteoblasts, and synoviocytes), isolated from their respective articular tissues, murine chondrogenic ATDC5 (RIKEN Cell Bank, Tsukuba, Japan), human osteoblastogenic SaOs2, and human synovial-sarcoma SW982 (ATCC, Manassas, VA, USA), were initially cell-cultured in P100 plates with DMEN F12 cell medium supplemented with fetal bovine serum (FBS) at 5% (mouse cells) or 10% (human cells), 2% Glutamine, and 2% penicillin-streptomycin, plus 0.2% sodium selenite and 0.1% transferrin (mouse cells) [22 (link),23 (link),77 (link),78 (link)].
Only early cell passes (P) comprised of P₀–P₃ (primary cells) and P₅–P₃₅ (non-primary cells) were used to keep the cellular phenotype unaltered. Cells were seeded in cell plates (P12 or P100) and after 4 h, the medium was renewed with FBS deprived medium [22 (link),23 (link),77 (link),78 (link)]. All cell culture reagents were purchased from Sigma-Aldrich (Sant Louis, MO, USA) except otherwise mentioned.

Primary synovial fibroblasts (synoviocytes) were isolated from synovial tissues obtained from patients undergoing total knee replacement surgery in accordance with the guidelines of the Ethical Committee of the INIGEM Institute, as previously described (Scian et al., 2013 (link)). Primary osteoblasts were isolated from newborn-mouse calvaria using the method described by Wong and Cohn (Wong and Cohn, 1975 (link)). The Swan71 cell line (kindly provided by Dr. Gil Mor, Yale University, New Haven, USA) was obtained by telomerase-mediated transformation of a 7-week cytotrophoblast isolate. Primary cells and the cell lines Caco-2 (ATCC HTB-37™), HT-29 (ATCC HTB-38), Swan71, SW982 (ATCC HTB-93™), and hFOB (ATCC CRL-11372™) were seeded in 24-well plates (5 × 10⁴ cells per well) and inoculated with the different strains (multiplicity of infection [MOI], 100:1). Plates were centrifuged for 10 min at 170 RCF and then incubated in a 5% CO₂ atmosphere at 37°C for 1 h. To determine the total number of bacteria associated to the cells, wells were washed three times with PBS and treated for 10 min at 37°C with 0.1% Triton X-100 (in PBS). Serial dilutions of the obtained lysates were done in PBS and plated on TSA with the appropriate antibiotics, and CFU were determined. All the results were expressed in reference to the wild-type strain, defined as 100%.