Cell-mediated contraction

Tissue contraction is a fundamental part of many important biological processes, including wound healing, in which abnormal contraction leads to fibrosis and

scarring that associate with a wide range of debilitating pathological conditions. The resident fibroblasts are believed to play a key role in controlling this process, by generating substantial contractile forces on the extracellular matrix that are in part

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regulated by the mechanical loading in the environment in which they reside. To maintain an active tensional homeostasis, fibroblasts consistently react to modify the endogenous matrix tension in the opposite direction to externally applied loads by changing in cell shape and attachment in a predictable manner (Eastwood et al., 1998, Brown et al., 1998). However, the mechanisms by which they remodel their environment are still unclear. Bell's introduction of the fibroblast-populated collagen lattice (FPCL) has become the most commonly used in vitro model to study the reciprocal and adaptive interactions that occur between fibroblasts and surrounding matrix in the tissue-like environment (Bell et al., 1979, Grinnell, 2003). To create such a pseudo-physiological 3D environment, a suspension of trypsinised

fibroblasts are added to pH neutralised type-I collagen solution with concentrated medium. After the collagen polymerises, the fibroblasts are dispersed throughout the resulting gel-like matrix, which is then allowed to free-float in the medium containing tissue culture dish. Stimulated by the serum or growth factors contained in the culture medium, the cells contract the matrix by applying force to the

neighbouring collagen fibres. Through cycles of extension and retraction, they structurally reorganise the collagen architecture down to a fraction of its original size. The speed of contraction depends on the cell type, density and collagen concentration (Tomasek et al., 2002) (Figure 1.5).

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Figure 1.5 The free-floating fibroblast-populated collagen gel contraction assay.

Collagen gel contraction assay with human conjunctival fibroblasts HTF7071 at Day0, 3 and 5 in culture medium with 10% FBS (the contracting collagen gel at the centre of the well is labelled with white circle).

There are three main cellular mechanisms proposed to be responsible for generating the FPCL contraction (Dallon and Ehrlich, 2008). The first one is cell tractional forces that are how fibroblasts generate sufficient force in order to bend individual collagen fibres bound to their surface to allow cell spreading and

migration, which relate to cell migration or locomotion. The assumption is that these tractional forces are distributed throughout the matrix via the cross-linked collagen fibres, which lead to global remodelling and contraction of the whole environment (Meshel et al., 2005, Roy et al., 1997). Nevertheless, challenging data suggested that tractional forces may not be sufficient to induce matrix contraction in vitro as well as wound closure in vivo (Ehrlich and Rajaratnam, 1990, Roy et al., 1999).

Another possible mechanism is that through differentiation into α-smooth muscle actin rich stress fibres expressing myofibroblast phenotype, the ‘modified’ cells enhance their contractility and become the ‘icon of fibrosis’ (Tomasek et al., 2002).

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The myofibroblast was first defined by Gabbiani's group in 1971 in an experimental animal model of wound healing (Gabbiani et al., 1971, Majno et al., 1971).

Subsequently, their presence has been identified in a variety of pathological connective tissue conditions including cancer and has been intensively studied (Gabbiani, 1992, Gabbiani, 1999, Desmouliere et al., 2004). However,

myofibroblasts only appear at the later stage of wound healing in vivo, and

differentiation into myofibroblasts in vitro requires specific conditions such as TGFβ, tension, and most importantly, time (Arora and McCulloch, 1994, Hinz, 2015,

Grinnell et al., 1999, Desmouliere et al., 1993). Hence the transformation of myofibroblasts is unlikely to be the reason of early matrix contraction of FPCL in vitro and early wound closure in vivo (Grinnell, 1994, Dahlmann-Noor et al., 2007).

The third mechanism of cell-mediated contraction proposed is the traction generated by cell protrusive activity without association with net cell locomotion.

Previous studies have demonstrated that through the dynamic extension and retraction of pseudopodial extensions, non-motile cells can produce local tension in the matrix that leads to contraction (Roy et al., 1997, Sawhney and Howard, 2002, Sawhney and Howard, 2004). By performing protrusions and retractions by

lamellipodia in the typical “hand-over-hand” cycle, fibroblasts can also reposition the individual collagen fibres placed on their upper surface in such case (Meshel et al., 2005). The molecular machinery contributes to the process including assembly of actin filaments, myosin activity, as well as microtubules depolymerizing (Sawhney and Howard, 2004). Furthermore, the macroscopic matrix contraction has been linked to the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology, but not cell migration (Freyman et al., 2001).

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The host laboratory have investigated the cellular mechanisms underlying force generation and matrix contraction using primary human ocular fibroblasts in the standard collagen matrix. The former studies have identified factors that affect early matrix contraction including cell size, intrinsic level of actin dynamics and genuine contractile force, dynamic cell protrusive activity, and net pericellular matrix displacement. It was reported that protrusive activity is the main cell behaviour observed within the first 24 hrs of matrix deformation (Dahlmann-Noor et al., 2007).

Furthermore, it has been proposed that fibroblasts remodel the collagen matrix by two major mechanisms, one via local active collagen fibre alignment through cellular protrusive activity, and the other through matrix degradation. We found that cells with a rounded morphology and proliferative profile display low intrinsic cellular force, whereas those with an elongated morphology express higher levels of protrusive activity that leads to efficient matrix remodelling and contraction (Martin-Martin et al., 2011).