The observed downregulation in TGFβ1 concentration in the presence of DRT could result from at least two unrelated sources. There is evidence that TGFβ1 is bound nonspecifically but with great affinity on the DRT surface, to an extent of about 7 µg TGFβ1/mg DRT (Thies 2010). This result raises the question whether the bound TGFβ1 is an equally active contributor to the differentiation of myofibroblasts as is the free growth factor. There is also evidence by electron microscopy that the acetic acid-treated collagen used to fabricate DRT lacks a fraction of its native banding. Loss of collagen banding, without loss of the triple helical structure of collagen, occurs during processing of the scaffold, following swelling in acetic acid solution which is known to selectively melt most of the banding periodicity of collagen fibers at pH levels below 4.25 ± 0.30 (Sylvester et al. 1989). Lack of banding in collagen fibers has, in turn, been associated (Sylvester et al. 1989) with downregulation of the normal collagen-platelet reaction that eventually leads to degranulation (Baumgartner et al. 1976; Jang et al. 2014) and release of TGFβ1 during wound healing. In the absence of platelet degranulation, it is accordingly expected that the TGFβ1 concentration in the wound would be reduced. Although there are unsettled questions about the observed reduction in TGFβ1 concentration in the presence of DRT, its reduction in DRT-treated wounds has been confirmed repeatedly. The consequences of its reduction will be examined below.

Dispersion of assemblies of contractile cells is also observed in the presence of DRT, both in skin wounds and peripheral nerve wounds (Fig. 9.1). A partial explanation can be obtained by comparing the effects on the morphology of contractile cells inside two collagen scaffolds from the same library (Fig. 9.2). The two scaffolds were identical in structure except for the average pore size: DRT had a pore size of 40 µm while the second scaffold (previously referred to as “analog B”) had a pore size of 400 µm (Yannas 2001f). The pore size of the DRT scaffold was within the active range of 20–125 µm, where delay of contraction is significant (Yannas et al. 1989), while analog B did not delay contraction significantly and was considered inactive (Fig. 9.3). The histologic evidence shows that the cell density inside the pores of inactive analog B was much higher than in the pores of active DRT scaffold (cells, mostly fibroblasts, were not stained for αSMA in this study). Furthermore, the large pores in analog B contained thick clusters of cells, approximately 30–50 cells per pore cross section, while the much smaller pores of DRT contained only 2–5 cells per pore cross section (Yannas 2001f). The overall lower cell content of the DRT scaffold can be explained in terms of the lower cell density that characterizes wounds grafted with DRT. Furthermore, cell-scaffold binding would be expected to be much more extensive than cell–cell binding inside DRT due to a purely physical reason; the active scaffold had a lower pore size, corresponding to a higher specific surface, relative to analog B (estimated 12-fold higher specific surface in DRT than in analog B; see Yannas 2001f).

The isolated presence of cells inside DRT shows that the assemblies of myofibroblasts, based on intercellular adherens junctions (AJs), as described above for normally contracting skin wounds (Hinz et al. 2004), were dispersed in the presence of DRT. Assemblies of contractile cells appear to scale up the contractile force of individual cells to the macroscopic contractile forces that deform wound tissues and eventually close the wound. Such deformation was prominent in the presence of the regeneratively inactive analog B, where contraction takes place almost unchanged from the ungrafted control, but not in the presence of DRT where contraction was blocked (Yannas et al. 1989; Troxel and Yannas 1991; Yannas 1998). Independent studies of the forces that maintain contractile cells assembled during normal contraction suggest that cell–cell contact and consequent coordination among myofibroblasts are required for generation of mechanical forces between cells in normally contracting wounds (Follonnier et al. 2008; Follonier et al. 2010; Goodbout et al. 2013).

Disorientation of long axes of contractile cells present inside the pores of the DRT scaffold is clearly evident in photographs of myofibroblasts in skin wounds and nerve wounds (Fig. 9.1). Such disorientation stands in contrast to the high orientation observed in the absence of DRT, where long cell axes are oriented in the plane of the epidermis in skin wounds or circumferentially around the stumps of transected nerves (Fig. 8.​5; Troxel and Yannas 1991; Chamberlain et al. 2000a). The resulting randomization of force vectors contributed by individual cells is expected to lead to extensive mutual cancellation of vectors, leading to reduction of the resultant macroscopic force.

Binding of contractile cells on the DRT surface is the simplest explanation for dispersion of cell assemblies and disorientation of long axes of cells in the presence of DRT. These morphological changes in contractile cells are not observed in the presence of very similar collagen scaffolds used as controls or in the absence of DRT, suggesting that DRT possesses molecular features that distinguish it from closely related controls. Direct evidence of close approach and contact of contractile cells to the scaffold surface has been obtained by transmission electron microscopy (Fig. 9.4; Murphy et al. 1990); however, such evidence at the ultrastructural scale contains no molecular evidence that confirms cell–matrix binding.

What is the molecular evidence that binding between cells and DRT actually takes place? This question will be answered below.