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.
Highlights
Tendon and ligaments are mechanosensitive tissues with a very similar histoarchitecture
Mechanically induced effects have not been investigated in detail in anterior cruciate ligament (ACL)-derived fibroblasts and directly compared between 2D cultures and cells emigrating from 3D spheroid cultures
The aim of the present study was to characterize the effect of cyclic uniaxial stretch on ACL-derived fibroblast 2D monolayer culture as well as on cells emigrating from 3D spheroid cultures
Summary
Tendon and ligaments are mechanosensitive tissues with a very similar histoarchitecture. It has been reported that mechanical strain promotes fibroblast adhesion, growth and increased ECM production on PDMS chambers (Cui et al 2015; Wang et al 2015). It is still unclear which mechanical stretching parameters like amplitude, frequency, and number of cycles stimulate fibroblasts in a way that they produce a maximum of ECM proteins. The aim of the present study was to characterize the effect of cyclic uniaxial stretch on ACL-derived fibroblast 2D monolayer culture as well as on cells emigrating from 3D spheroid cultures. Cells within spheroids are surrounded by their own freshly produced tissue-specific ECM, and the intimate cell-ECM interaction might prime the cells and, thereby, stabilize their tissue-specific expression profile
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