Human outgrowth knee fibroblasts from patients undergoing total knee arthroplasty exhibit a unique gene expression profile and undergo myofibroblastogenesis upon TGFβ1 stimulation

Abstract

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasties (TKAs). Currently, treatment options are limited. Therefore, an in vitro model of knee-related myofibroblastogenesis is valuable to facilitate investigation of the arthrofibrotic process, diagnostic and therapeutic options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissues from the knees of four patients undergoing primary TKAs for osteoarthritis. From these tissues, we isolated primary cells by the outgrowth method and subsequently characterized these cells in the absence and presence of the pro-myofibroblastic cytokine, transforming growth factor beta 1 (TGFβ1). Light microscopy of knee outgrowth cells revealed spindle-shaped cells, and immunofluorescence (IF) analysis demonstrated staining for the fibroblast-specific markers TE-7 and vimentin (VIM). These knee outgrowth fibroblasts differentiated readily into myofibroblasts as reflected by enhanced α-smooth muscle actin (ACTA2) mRNA and protein expression and increased mRNA expression of collagen type 1 (COL1A1) and type 3 (COL3A1) with collagenous matrix deposition in the presence of TGFβ1.Outgrowth knee fibroblasts were more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stromal/stem cells (MSCs). While outgrowth knee fibroblasts isolated from three anatomical regions in four patients exhibited similar gene expression, these cells are distinct from other fibroblastic cell types (i.e., Dupuytren's fibroblasts) as revealed by RNA-sequencing. In conclusion, our study provides an in vitro myofibroblastic model of outgrowth knee fibroblasts derived from patients undergoing

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

“…RNA‐seq analysis of primary knee outgrowth fibroblasts derived from patients undergoing primary TKA (pTKA) has been recently performed and described in great detail elsewhere 40 . These data have been deposited in the Gene Expression Omnibus of the National Institute for Biotechnology Information (GSE185333).…”

“…All experiments were performed using plastic tissue culture flasks and plates. Passage four and five knee outgrowth fibroblasts were used for all experiments, as was done in our recent study that defines the transcriptional profile and myofibroblast differentiation potential of these cells 40 …”

“…RNA-seq analysis of primary knee outgrowth fibroblasts derived from patients undergoing primary TKA (pTKA) has been recently performed and described in great detail elsewhere. 40 These data have been deposited in the Gene Expression Omnibus of the National Institute for Biotechnology Information (GSE185333). To examine BET gene expression in knee outgrowth fibroblasts, normalized gene counts were obtained where expression values for each gene were normalized to 1 million reads and corrected for gene length (fragments per kilobase of transcript per million mapped reads, FPKM).…”

“…Detailed descriptions of specimen collection, processing, and acquisition of primary knee outgrowth fibroblasts from the posterior capsule (PC) of three patients (i.e., 030, 256, and 565) undergoing pTKA has been previously provided. 40 Cells were grown in advanced minimum essential medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (R&D Systems), 1% penicillin/streptomycin (Thermo Fisher Scientific), and 1% glutamax (Thermo Fisher Scientific) in cell culture incubators at 37°C, 95% humidity, 21% O 2 , and 5% CO 2 conditions to establish outgrowth cell cultures. All experiments were performed using plastic tissue culture flasks and plates.…”

“…Western blot analysis was performed as previously described. [40][41][42] Briefly, cells were lysed with radioimmunoprecipitation (Thermo Fischer Scientific) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fischer Scientific) and proteins quantified via the DC Protein Assay Kit II (Bio-Rad). Proteins were resolved in SDS-PAGE gels and transferred to a nitrocellulose membranes.…”