Cyclic stretch induces cellular and cytoskeletal alignment, enhanced proliferation and adaption of the synthetic profile of ACL-derived fibroblasts under 2D and 3D biomaterial-free and -associated conditions

Objectives Ligament fibroblasts are mechanosensitive and possess sufficient adaptability to mechanical changes to ensure the permanent load capacity of the tissue. The activation of membrane mechanoreceptors leads to the activation of specific intracellular signals (mechanotransduction) and results in cytoskeletal reorganization and adapted synthetic activity. The cellular response of fibroblasts of the anterior cruciate ligament(ACL) to cyclic mechanical stretch is still unclear. Hence, this study aims to provide a deeper insight into the reaction profile of stretched ACL fibroblasts in two-(2D) and three-dimensional(3D)

biomaterial-free and biomaterial cultures.

Methods A monolayer of 6000 lapine ACL fibroblasts per cm2 as well as 10 spheroids consisting of 25000 cells were performed and seeded into a polydimethyl siloxane chamber for 2D and 3D biomaterial-free approaches. After 24h (2D) and 48h (3D) of adherence, the chambers were stretched for 48h with 14 % uniaxial stretch and 0.3 Hz. For the 3D biomaterial-based approach 1x106 cells were seeded on scaffolds (30x4x1 mm), embroidered from polylactic acid and poly(L-lactide-co-caprolactone) threads, functionalized with gas-phase fluorination and re-fibrillated collagen foams. After 24h of dynamical precolonization, the scaffolds were stretched in a mechanostimulator for 72h with 4 % uniaxial stretch and 0.3 Hz. Vitality, distribution of F-actin fibers, alpha smooth muscle actin, paxillin and collagen type I immunoreactivity, proliferation, sulfated glycosaminoglycan (sGAG) content and relative gene expression of collagen type I, decorin, tenascin C, tenomodulin, connexin 43 and mohawk were determined.

Results and Conclusion The results from the biomaterial-free approach showed that the vitality was above 90 % not only in the unstretched 2D and 3D but also in the stretched samples. Cells and stress fibers were orientated against stretch direction in the 2D and 3D cultures. Probably due to the fact, that stretched fibroblasts were proliferating more in comparison to unstimulated, the colonized area was significant larger. Stretch induced a significant higher number of focal adhesion sites in the 2D biomaterial-free approach. The perimeter as well as the roundness of the cell nuclei of the unstimulated cells were significantly higher compared to those stimulated. In the 3D biomaterial-free approach, the relative gene expression of collagen type I, decorin, tenascin C, and connexin 43 were upregulated after cyclic loading. Stretched scaffolds showed a more homogenous cell distribution throughout the whole scaffold, whereas cell clusters were found in the unstretched controls. In addition, cell proliferation and content of sGAGs were influenced by mechanical stretch in the scaffold. The applied mechanical stimulation protocols led to rapid (48h) anabolic cellular changes in the 2D and 3D biomaterial-free as well as in the biomaterial approach and therefore, could be suitable to facilitate in vitro ligamentogenesis by ACL tissue engineering.

Stichwörter anterior cruciate ligament, fibroblasts, mechanostimulation, 2D and 3D stretch

Publication History

Article published online:
15 October 2020