Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

Abstract

Introduction: Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures.

Methods: PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 10³/cm²) or multilayer cultures (density 8.5 × 10⁵/cm²) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR.

Results: Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased.

Conclusion: The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

Keywords: Aseptic loosening; Bone remodeling; Fibroblast-like cells; Implant material; Transwell culture.

Fig. 1

TRAP staining of PPFs (a) and SFs (b) cultivated in monolayer culture. No TRAP-positive cells are seen. The cells show a fibroblast-like, fusiform shape with a blue stained nucleus. Scale bar = 100 µm

Fig. 2

Immunostaining with the fibroblast marker S100A4 of multilayer cultures of PPFs (a and b) and SFs (c and d) on day 10 (a and c) and 21 (b and d). Scare bar = 20 µm

Fig. 3

Relative mRNA expression of M-CSF (a), TNF-α (b), OPG (c), RANKL (d), ALP (e), COL1A1 (f), TRAP (g), cathepsin K (h), and MMP-13 (i) in PPF monolayer culture (PPF) and PPF transwell culture (PPF TW) on day 10 (d10) and day 21 (d21). Horizontal bars represent group medians and error bars IQR. p values are indicated with * = <0.05 and ** = <0.01

Fig. 4

Relative mRNA expression of M-CSF (a), TNF-α (b), OPG (c), RANKL (d), ALP (e), COL1A1 (f), TRAP (g), cathepsin K (h), and MMP-13 (i) SF monolayer culture (SF) and SF transwell culture (SF TW) on day 10 (d10) and day 21 (d21). Horizontal bars represent group medians and error bars IQR. p values are indicated with * = <0.05 and ** = <0.01

Fig. 5

Relative mRNA expression of M-CSF (a), TNF-α (b), OPG (c), RANKL (d), ALP (e), COL1A1 (f), cathepsin K (g), and MMP-13 (h) in DF monolayer culture (DF) and DF transwell culture (DF TW) on day 10 (d10) and day 21 (d21). Horizontal bars represent group medians and error bars IQR. p values are indicated with * = <0.05 and ** = <0.01

Fig. 6

RANKL/OPG ratios of PPF, SF, and DF are presented. a PPF monolayer culture (PPF) and PPF transwell culture (PPF TW) on day 10 (d10) and day 21 (d21). b SF monolayer culture (SF) and SF transwell culture (SF TW) on day 10 (d10) and day 21 (d21). c DF in DF monolayer culture (DF) and DF transwell culture (DF TW) on day 10 (d10) and day 21 (d21). P values are indicated ** = <0.01