During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast its origins, functions and molecular regulation will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.

To evaluate whether ␣-smooth muscle actin (␣-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of ␣-SMA, with that of lung fibroblasts (LFs), expressing high levels of ␣-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of ␣-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for ␣-SMA. In a second approach, we measured the isotonic contraction of SCF-and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGF␤1 increased ␣-SMA expression and lattice contraction by SCFs to the levels of LFs; TGF␤-antagonizing agents reduced ␣-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with ␣-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with ␣-cardiac and ␤-or ␥-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased ␣-SMA expression is sufficient to enhance fibroblast contractile activity. INTRODUCTIONEarly during healing of an open wound, resident dermal fibroblasts proliferate from the wound margin and migrate into the provisional matrix composed of a fibrin clot. About 1 week after wounding, the provisional matrix is replaced by neo-formed connective tissue, known as granulation tissue, essentially composed of small vessels, extracellular matrix, and fibroblastic cells that become activated and modulate into myofibroblasts. The main feature of myofibroblasts is represented by an important contractile apparatus similar to that of smooth muscle (Gabbiani et al., 1971), and in particular by the neo-expression of ␣-smooth muscle actin (␣-SMA), the actin isoform typical of vascular smooth muscle cells (Skalli et al., 1986). Myofibroblasts are recognized to play a central role in closing the wound tissue, through their capacity to produce a strong contractile force (for review see Grinnell, 1994;Rønnov-Jessen et al., 1996;Powell et al., 1999;Serini and Gabbiani, 1999), possibly generated within stress fibers, similar to those present in cultured fibroblasts (Skalli et al., 1986;Serini and Gabbiani, 1999). Because wound contraction takes place when de novo expressed ␣-SMA is incorporated in stress fibers (Darby et al., 1990), it has been suggested that this actin isoform plays an important role in granulation tissue contraction (for review see Serini and Gabbiani, 1999). Although stress fibers have been proposed to function as contractile organelles (Burridge, 1981;Katoh et al., 1998) and their presence has been correlated with the production of isometric tension (Harris et al., 1981), at p...

After the first description of the myofibroblast in granulation tissue of an open wound by means of electron microscopy, as an intermediate cell between the fibroblast and the smooth muscle cell, the myofibroblast has been identified both in normal tissues, particularly in locations where there is a necessity of mechanical force development, and in pathological tissues, in relation with hypertrophic scarring, fibromatoses and fibrocontractive diseases as well as in the stroma reaction to epithelial tumors. It is now accepted that fibroblast/myofibroblast transition begins with the appearance of the protomyofibroblast, whose stress fibers contain only beta- and gamma-cytoplasmic actins and evolves, but not necessarily always, into the appearance of the differentiated myofibroblast, the most common variant of this cell, with stress fibers containing alpha-smooth muscle actin. Myofibroblast differentiation is a complex process, regulated by at least a cytokine (the transforming growth factor-beta1), an extracellular matrix component (the ED-A splice variant of cellular fibronectin), as well as the presence of mechanical tension. The myofibroblast is a key cell for the connective tissue remodeling that takes place during wound healing and fibrosis development. On this basis, the myofibroblast may represent a new important target for improving the evolution of such diseases as hypertrophic scars, and liver, kidney or pulmonary fibrosis.

We have examined the role of mechanical tension in myofibroblast differentiation using two in vivo rat models. In the first model, granulation tissue was subjected to an increase in mechanical tension by splinting a full-thickness wound with a plastic frame. Myofibroblast features, such as stress fiber formation, expression of ED-A fibronectin and alpha-smooth muscle actin (alpha-SMA) appeared earlier in splinted than in unsplinted wounds. Myofibroblast marker expression decreased in control wounds starting at 10 days after wounding as expected, but persisted in splinted wounds. In the second model, granuloma pouches were induced by subcutaneous croton oil injection; pouches were either left intact or released from tension by evacuation of the exudate at 14 days. The expression of myofibroblast markers was reduced after tension release in the following sequence: F-actin (2 days), alpha-SMA (3 days), and ED-A fibronectin (5 days); cell density was not affected. In both models, isometric contraction of tissue strips was measured after stimulation with smooth muscle agonists. Contractility correlated always with the level of alpha-SMA expression, being high when granulation tissue had been subjected to tension and low when it had been relaxed. Our results support the assumption that mechanical tension is crucial for myofibroblast modulation and for the maintenance of their contractile activity.