Contractile fibroblasts , often referred to as myofibroblasts, have been originally credited with generating most of the contractile forces and tissue deformations in skin wounds (Gabbiani et al. 1971; Rudolph 1979; Rudolph et al. 1992; Gabbiani 1998; Daimon et al. 2013). Although other actin isoforms, such as smooth muscle γ-actin and skeletal muscle α-actin, have been implicated in force generation (Tomasek et al., 2013), the α-smooth muscle actin isoform is currently considered to be the most useful marker of the myofibroblastic phenotype (see Fig. 4.​5) (Hinz et al. 2012). These cells are highly elongated and display densely bundled actin microfilaments at their perimeter. Perhaps the most widely used phenotype of myofibroblasts is “stress fibers,” filaments containing α-smooth muscle actin (Gabbiani et al. 1971; Desmoulière et al. 2005). Following wound closure, myofibroblasts disappear by apoptosis (Desmoulière et al. 1995; van Beurden et al. 2005). As we have done in earlier chapters, we will use the term myofibroblast to identify cells that have been shown to stain with an antibody to the α-smooth muscle actin isoform .

Identification of contractile cells in injured peripheral nerves has historically lagged behind the evidence for contraction with skin wounds. In an early study, contractile cells were identified in the uninjured perineurium (Ross and Reith 1969). Although myofibroblasts were individually identified in early studies with injured peripheral nerves, the role of these cells in the healing process was not investigated (Badalamente et al. 1985). Myofibroblasts, in the form of a thick sheet (capsule) surrounding peripheral nerve stumps resulting from transection, appear to have been first observed as late as 2000 (see Fig. 4.​6) (Chamberlain et al. 2000a; Soller et al. 2012).

The differentiation of the fibroblast to the contractile fibroblast is regulated by transforming growth factor beta1 (TGFβ1), the ED-A splice variant of cellular fibronectin, and the presence of mechanical tension (Desmoulière et al. 2005). Although myofibroblasts are considered responsible for normal wound closure during healing of acute wounds in dermis, tendon, bone, cartilage, more recently in peripheral nerves, as well as in other injured organs, they are also considered primary actors in disfiguring scar contractures, as well as playing several other roles in pathological conditions (see Fig. 4.​7) (Follonier Castella et al. 2010; Van De Water et al. 2013) .

Organized assemblies of myofibroblasts appear in contracting wounds, both in skin (Fig. 4.​5) (Hinz and Gabbiani 2003) and (Fig. 4.​6) peripheral nerves (Chamberlain et al. 2000a; Soller et al. 2012). Considering that a macroscopic force of about 0.1 N is required for skin wound closure in the rabbit (Higton and James 1964) and that the force generated by an individual cell is about 1–10 nN (Freyman et al. 2001a; Harley et al. 2008), it appears necessary to apply forces from as many as 10⁸–10⁹ cells during wound contraction to close a skin wound in the rodent. The required macroscopic force appears to be generated by assemblies of myofibroblasts, organized to apply cooperative forces and provide for a “mechanically coherent” wound (Yannas 1998).

Assemblies of contractile cells are known to be held together by stress fibers in neighboring myofibroblasts that are joined together at the sites of cadherin-type intercellular adherens junctions (AJs) (Hinz et al. 2004). AJs are dense plaques found under the plasma membrane of fibroblasts in granulation tissue of wounds as well as in scar-like tissues (Welch et al. 1990). Maturation processes take place in AJs during healing; for example, it has been shown that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in wounds undergoing contraction (Hinz et al. 2004). Intercellular mechanical coupling of stress fibers via AJs improves contraction of collagen gels by myofibroblasts. There is evidence that local contractile events are mechanically coordinated by AJs, via synchronization of periodic intracellular Ca2+ oscillations between physically contacting myofibroblasts (Follonnier et al. 2008, 2010; Goodbout et al. 2013). It appears very likely that these coordinated associations among myofibroblasts are responsible for generation of macroscopic contraction forces across a closing wound .

Orientation of long axes of contractile cells is a distinctive feature of such assemblies. Axial orientation appears to be mechanically required for generation of a directed macroscopic contractile force. This feature was demonstrated in vitro, with fibroblasts incubated inside a collagen scaffold, applying forces to the struts of the matrix along their long axis, causing them to buckle (Freyman et al. 2001b; Harley et al. 2008). In the guinea pig skin wound, the long axes of myofibroblasts have been shown, based on transmission electron microscopic observation, to be highly oriented in the plane of the epidermis (Murphy et al. 1990). In an earlier study, myofibroblasts showed cytoplasmic microfilaments that were aligned along the direction of their long axes; in this study, contractile cells were aligned parallel to the plane of the epidermis and the applied macroscopic forces appeared to be consistent with the observed reduction in wound area in the plane of the epidermis (Baur et al. 1984). The orientation of long axes of myofibroblasts inside a contracting skin wound in the guinea pig (Fig. 8.5) is consistent with these observations. The simplest mechanical field that corresponds to such an array of cells is the plane stress field.

Contractile forces in skin wounds are applied not only in the plane of the epidermis but also out of plane. Observations of deformation of subdermal tissues in a closed skin wound in the guinea pig support the existence of mechanical forces that act immediately below the scar (Fig. 8.6; see area at bottom right labeled deformed subdermal tissue). This cross-sectional view of an initially dermis-free defect in the guinea pig skin shows the final stage of defect closure. In this micrograph, closely apposed dermal edges are separated by a small mass of scar. The micrograph obtained with polarized light shows highly deformed birefringent fibrous tissues, almost certainly stretched collagen fibers, connecting the proximal adipose layer at the base of the wound with distal scar mass (Troxel 1994). The photographic evidence of mechanical tension inside the closed-skin defect highlights the importance of subdermal tissues during wound closure (Fig. 8.6). There is a sharp contrast between these compliant subdermal tissues in the guinea pig wound and the dermis in the rabbit ear wound model which adheres tenaciously to the underlying cartilage layer (Joseph and Dyson 1966; Goss 1980, 1992; Goss and Grimes 1972, 1975; Mustoe et al. 1991). This comparison suggests the possibility that the mechanical compliance of subdermal tissues is a significant factor in the magnitude of contraction forces generated in skin wounds.

Mechanical forces that contribute to wound closure in skin are not limited to those that deform by contraction. Observations made with guinea pig skin wounds have shown that other tissue movements, including an initial expansion of the dermal edges, as well as translational perilesional movement of wound edges, also participate in the closure process. However, the overall deformation pattern, as reported by tattooing squares on the skin of the dorsal area of a guinea pig, has clearly shown the presence of a reduction in skin wound area consistent with a principal deformation axis along the dorsal–lateral (DL) axis for this defect. The deformations amounted to strains between − 11 and − 67 % (contractions) along the DL axis, as well as lesser strains of 17 to 36 % (extensions, possibly including translations) along the anterior–posterior axis (Fig. 8.7, Troxel 1994). These observations were nearly identical to the deformation pattern observed earlier in a guinea pig skin wound using tattoo marks (Straile 1959) .

Mathematical models of the contractile force and the deformation during wound closure in skin and peripheral nerve wounds are presented below. The models are not designed to provide accurate representations of data. Instead, they provide a rough overview of the cellular variables that control contraction in skin and nerve wounds and are qualitatively consistent with the evidence presented in this chapter.