Gene Expression of Fibroblasts Derived from Knee Tissue of Patients Undergoing Primary Total Knee Arthroplasty for Osteoarthritis

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasty (TKA). Current surgical and pharmacologic treatment options are limited. Therefore, an in vitro model for joint myofibroblastogenesis is valuable to investigate the arthrofibrotic process and identify diagnostic biomarkers and treatment options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissue from knees of four patients undergoing primary TKA for osteoarthritis and characterized primary outgrowth cells from these tissues in the absence and presence of transforming growth factor beta 1 (TGFβ1), a pro-myofibroblastic cytokine. Light microscopy of knee outgrowth cells revealed spindle-shaped cells while immunofluorescence (IF) established staining for the fibroblast-specific antigen TE-7 and Vimentin, which are characteristics of fibroblasts. These fibroblasts differentiate readily into myofibroblasts as highlighted by enhanced alpha smooth muscle actin (ACTA2) mRNA and protein expression and increased collagen mRNA (i.e., collagen type 1 (COL1A1) and collagen type 3 (COL3A1)) expression and collagenous matrix deposition in the presence of TGFβ1. Of note, these studies also revealed that knee-derived fibroblasts are more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stem cells. Importantly, while outgrowth fibroblasts isolated from four patients and three anatomical regions exhibit similar gene expression profiles, these knee fibroblasts form a unique gene expression cluster within the fibroblast niche as revealed by RNA-sequencing analysis. In conclusion, our study provides a fibroblast/myofibroblast model of outgrowth knee cells derived from patients undergoing primary TKA that can be employed to assess myofibroblast-related processes and test novel pharmacological strategies in vitro for arthrofibrosis. Intraoperative tissues were obtained from the posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) of four patients undergoing primary TKA (patient numbers 030, 256, 565 and 797). Cells were expanded by outgrowth culture in vitro and resulting cells were evaluated for unique fibroblast markers, morphology, myofibroblast differentiation, collagen production potential and gene expression fingerprint. Cell morphology at various cell densities and the fibroblast marker TE-7 and VIM, mesenchymal human connective tissue marker, were assessed by light microscopy and immunofluorescence (IF) microscopy, respectively. Expression of ACTA2 (myofibroblast marker) assessed by IF, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), and western blotting (WB) in the absence or presence of TGFβ1 (10 ng/ml) to evaluate myofibroblast differentiation. Expression of COL1A1 and COL3A1 genes and collagen deposition were evaluated in the absence or presence of TGFβ1 (10 ng/ml) by qPCR and picrosirius red staining respectively. RNA-seq analysis was employed for gene expression profiles of knee fibroblasts.