Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation

Abstract

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

“…However, culture-expanded chondrocytes tend to lose their chondrogenic phenotype, resembling fibroblasts. This is manifested by reducing the production of cartilage-specific constituents such as collagen type II, aggrecan, and proteoglycan; while raising production of non-specific cartilage constituents such as collagen type I in a process called dedifferentiation 6 , 7 . Dedifferentiation is also characterised by decreased synthesis of growth factors, decreased cell proliferation, increased apoptosis, and cell senescence 7 , 8 .…”

Mechanosensory and mechanotransductive processes mediated by ion channels in articular chondrocytes: Potential therapeutic targets for osteoarthritis

“…However, articular joint immobilization may reduce HA secretion and result in decreased cartilage thickness and inhibited proteoglycan synthesis leading to ECM degeneration, which will make the articular cartilage more vulnerable to excessive mechanical stimulation. The chondrocyte phenotype was preserved when suspended clustered chondrocytes maintained a round morphology under simulated microgravity induced by RPM [ 157 ]. Progressive dedifferentiation which includes the production proportion of collagen 2/collagen 1, proteoglycan proportion of ACNA to versican (ACNA/VCAN) and a transition to fibroblast-like morphology was observed in monolayer in vitro cultured BACs except for decreased expression of the TRPV4 ion channel, which was attenuated by the RPM system that induced the suspension status of in vitro BACs [ 157 ].…”

Microtubules and Vimentin Fiber Stability during Parabolic Flights

“…The isolation of primary bovine articular chondrocytes was carried out as previously described (Wuest et al 2018), using distal interphalangeal joints (DIPs) of cattle obtained from a local butcher (food chain). Joints from cattle slaughtered on the same day were opened under aseptic conditions and the articular cartilage was removed with a surgical blade.…”

“…Since chondrocytes are known to lose their phenotype in standard 2D cell culture (Wuest et al 2018;Marlovits et al 2004;Schulze-Tanzil 2009;Tekari et al 2014;Barlič et al 2008;Diaz-Romero et al 2005;Diaz-Romero et al 2008;Kang et al 2007;Lin et al 2008;Charlier et al 2019), the cells were not expanded after isolation. Three days before the flight, the (non-passaged) cells were seeded in commercial T25 flasks.…”