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.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

“…With the hanging drop method, around 15 dishes with 9 cm diameters are needed, each containing 50 spheroids to achieve the same number of spheroids. The conventional hanging drop method is widely used for spheroid production [2,4,14,16,19]. However, this culture has some further limitations.…”

“…These cell-cell communications might stabilize the differentiated phenotype of primary cells. Spheroids can be produced using many cell types and techniques [1] including tenocytes and ligamentocytes [2][3][4]. They can be used as 3D tumor models for the screening of agents and strategies effective to inhibit tumorigenesis [1,5].…”

Harnessing Thermogel Actuation for Driving Directional Stromal Cell Communication and Migration into Columnar Arrays

“…This is problematic as the complexity inherent in tissue-scale structures demands su cient time, along with various other microenvironment factors [15], for development in culture. Moreover, within the domain of spheroid cultures, studies on elongation morphogenesis [16], tissue repair [17], or cell cluster migration [18,19] have documented the occurrence of cell collectives moving synchronously in columnar patterns in vitro. However, instances of coordinated reshaping are limited to short time frames, usually spanning hours to days, after which isotropic spheroid disaggregation or disorder tends to manifest.…”