Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile

Abstract

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Keywords: ACL-derived fibroblasts; Connexin 43; Cyclic strain; Mechanostimulation; Mohawk; Myodulin; Spheroids; Tendon extracellular matrix; Uniaxial stretch.

Fig. 1

Graphical abstract. After dissection of the anterior cruciate ligament (ACL), 2 mm² tissue pieces were placed in a culture flask as an explant culture. After few days, fibroblasts emigrated out of the ACL. In the first approach, fibroblasts were seeded directly into a 4 cm² PDMS silicone elastomeric chamber as a monolayer (2D) culture and after 24 h of adherence, the stretch program (14% stretch, 0.3 Hz for 48 h) started. In the second approach, spheroids (sph) (3D culture) were assembled using the hanging drop method and after 48 h of adherence they were stretched with the Cell-Stretcher X6 (blue double-headed arrow: direction of stretching). Downstream analyses included vitality Assay, immunocytochemical staining, CyQuant Assay, dimethylmethyleneblue (DMMB) Assay and relative gene expressions of ligament-related components were performed after 2 days of stimulation

Fig. 2

Monolayer culture of unstimulated (a, d, g, j, m) and stimulated (b, e, h, k, n) ACL-derived fibroblasts after 48 h of 14% stretch. The vitality assay (a, b) showed live (green) and dead (red) cells and based on living cells the colonized area could be calculated (c). The phalloidin Alexa 488/ DAPI staining (d, e) depicted the F-actin (green) and cell nuclei (blue); the elongation of the stress fibers was measured (f). The immunocytochemical staining showed the proliferation marker ki67 (g, h, red) and the amount of ki67+ cells was calculated (i). Paxillin (j, k) is depicted in red, collagen type I (m, n) in green and alpha smooth muscle actin (αSMA [m, n]) in red. The cell nuclei were stained with DAPI in blue. Mean number of focal adhesion sites per cell (l) and the relative protein expression of collagen type I (col 1) and αSMA (o) were determined versus controls which were normalized. Three independent experiments were performed with significances (*) of p ≤ 0.05 and (****) of p ≤ 0.0001. The stretch direction is indicated with the double-headed arrows (b, e, h, k, n). Scale bars 100 µm (a, b), 50 µm (d, e, g, h, j, k, m, n)

Fig. 3

Evaluation of the cell response to mechanical stretch in the monolayer culture. Cell proliferation assay (a), a calculation of the sGAG content (b), and the measurement of nuclear shape, especially the mean values of the perimeter of the nucleus (c) and mean values of the roundness of the nuclei (d) were measured and calculation of number of cell nuclei using DAPI staining (e). Three independent experiments were performed with significances (*) of p ≤ 0.05, (**) of p ≤ 0.01 and (***) of p ≤ 0.001

Fig. 4

Expression levels of genes coding for ligament-related components in the monolayer culture. Relative gene expression of collagen type I (COL1A1), decorin (DCN), tenascin C (TNC), connexin 43 (CNX), Mohawk (MKX), and tenomodulin (TNMD) were shown with standard deviation. The red line depicts the normalized level of unstimulated controls. Five independent experiments were performed with significances (*) of p ≤ 0.05

Fig. 5

Unstimulated (a, c, e, g, i) and stimulated (b, d, f, h, j) ACL-derived fibroblasts emigrating from spheroids after 48 h and 14% stretch at 0.3 Hz. The vitality assay (a, b) showed live (green) and dead (red) cells. The phalloidin Alexa 488/ DAPI staining (c, d) depicted the F-actin (green) and cell nuclei (blue). The immunocytochemical staining visualized the proliferation marker ki67 (e, f) in red, paxillin (g, h) in red, collagen type I (i, j) in green and αSMA (i, j) in red. Small inserts showed a higher magnification (e2, f2, g2, h2, i2, j2) of the cells at the border of the spheroids (sph). The cell nuclei were stained with DAPI in blue. Scale bars 100 µm (a, b), 50 µm (c–j). The stretch direction is indicated with the double-headed arrows (b, d, f, h, j)

Fig. 6

Evaluation of the cell response to mechanical stretch in spheroid-derived fibroblasts. Mean values of the cell proliferation assay (a) and of the sGAG content (b) were shown. Additionally, the spheroid diameters were determined (c). Three independent experiments were performed with significances (**) p ≤ 0.01 and (****) of p ≤ 0.0001

Fig. 7

Expression level of genes coding for ligament-related components in spheroid-derived fibroblasts. Relative gene expression of collagen type I (COL1A1), decorin DCN, tenascin C (TNC), connexin (CXN) 43, Mohawk (MKX), and tenomodulin (TNMD) were shown with standard deviation. The red line depicts the normalized level of unstimulated controls. Five independent experiments were performed with significances (**) of p ≤ 0.001 and (****) of p ≤ 0.0001