Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes

Abstract

Static compressive stress can influence the matrix, which subsequently affects cell behaviour and the cell's ability to further transform the matrix. This study aimed to assess response to static compressive stress at different stages of osteoblast differentiation and assess the cell cytoskeleton's role as a conduit of matrix-derived stimuli. Mouse bone marrow mesenchymal stem cells (MSCs) (D1 ORL UVA), osteoblastic cells (MC3T3-E1) and postosteoblast/pre-osteocyte-like cells (MLO-A5) were seeded in hydrated and compressed collagen gels. Contraction was quantified macroscopically, and cell morphology, survival, differentiation and mineralisation assessed using confocal microscopy, alamarBlue ® assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and histological stains, respectively. Confocal microscopy demonstrated cell shape changes and favourable microfilament organisation with static compressive stress of the collagen matrix; furthermore, cell survival was greater compared to the hydrated gels. The stage of osteoblast differentiation determined the degree of matrix contraction, with MSCs demonstrating the greatest amount. Introduction of microfilament disrupting inhibitors confirmed that prestress and tensegrity forces were under the influence of gel density, and there was increased survival and differentiation of the cells within the compressed collagen compared to the hydrated collagen. There was also relative stiffening and differentiation with time of the compressed cell-seeded collagen, allowing for greater manipulation. In conclusion, the combined collagen chemistry and increased density of the microenvironment can promote upregulation of osteogenic genes and mineralisation; MSCs can facilitate matrix contraction to form an engineered membrane with the potential to serve as a 'pseudo-periosteum' in the regeneration of bone defects.

An easy-to-use and versatile method for building cell-laden microfibres

“…Here, we focused on the use of a mouse bone marrow mesenchymal stem cell source (D1 cell line) and show that in the absence of osteogenic media cells show to differentiate into the osteoblastic lineage. Previous reports support this observation, where mesenchymal stem and osteoblastic cell culture within collagen gels showed to generate internal loads, inside the gels, leading to gel contraction and differentiation towards the osteoblastic lineage 21 22 23 .…”

A glance on the role of actin in osteogenic and adipogenic differentiation of mesenchymal stem cells

“…However, cells in compressed gel develop an ovoid shape and present a close network of numerous actin filaments. The actin filaments in dense gels show a greater tendency toward osteogenic differentiation as compared with hydrostatic gels [70]. Another study showed a similar result for F-actin stress fibers, which become prominent when estrogen was added under mechanical pressure, wherein, alkaline phosphatase activity, an early indicator of osteogenesis, was observed to be higher showing osteogenesis [71].…”

Cell morphology and focal adhesion location alters internal cell stress

“…It was found that this reduction in cell tension induced adipogenic rather than osteogenic differentiation, as measured by expression of liposome lipase (adipogenic) as well as ALP and CBFa1 (osteogenic). However, the interdependence between cell morphology and intracellular stress cannot be ignored as it has been shown that the disruption of the cytoskeleton through ROCK inhibition will prevent the formation of cell processes in MC3T3-E1 cells cultured on collagen substrates [75]. Cell morphology has also been shown to affect cell-generated stress, with bovine pulmonary smooth muscle cells and 3T3 mouse fibroblasts both shown to induce higher forces on micropost substrates when allowed to expand to a larger cell area [76].…”