Epidermis

2.5.3.1 Epidermal structure

The epidermis is the outermost layer of the skin, and is an animal’s first barrier to external threats. The epidermis consists of viable cells that grow and reproduce to produce a cornified layer of keratin at the skin surface. During conventional processing the layers of cornified cells are destroyed by the action of alkaline sulfide presumably by the same mechanism that destroys keratin in the wool or hair fibre. The process of removing the epidermis exposes the smooth grain surface, which forms the visible surface of the crust leather, and therefore needs to be completed to form a quality

The efficiency of penetration of “extra-dermal” agents is affected by the keratin of the cornified cells and the lipid filled extracellular space (Al-Amoudi et al., 2005). The extracellular structure has been recently investigated using Cryo-electron microscopy (Al-Amoudi et al., 2005). This work showed that the extracellular space contains multiple lipid bi-layers which probably confer much of the waterproofing to the skin. If a depilatory agent were to be applied from the grain side then it would have to pass either through these bi-layers or down the hair root shaft in order to reach its target.

On the other hand, it has been observed that, strips of cornified material can be peeled from the underlying dermis during enzyme depilation (Yates, 1968b). This indicates that adhesion of the epidermal cells to the underlying extracellular matrix could be important in the enzyme depilation process. The adhesion of epithelial cells in the epidermis is a special case, and two different kinds of adhesion are observed; the adhesion of “like” cells to one another to maintain the cells in a two dimensional sheet and the adhesion of that sheet of cells to the underlying extracellular matrix (Bell, 1988). The epidermis is therefore a structure that acts as a barrier to a grain side applied enzyme depilatory.

2.5.3.2 Epidermal cell adhesion

Cells have a polysaccharide rich coating called the glycocalyx. which is most important in the process of cell adhesion and can be readily stained with ruthenium red (Bell, 1988),(Bongrand, 1988). It consists mainly of proteoglycans although some glycoproteins such as fibronectin are present. The cell surface is not flat but is covered with small protrusions about 100Å apart on average, this distance being somewhere between the length of the glycoprotein side chains and glycoprotein lateral chains (Bongrand, 1988).

Cells attach to each other and the substratum by means of footpads and thin retraction fibres that connect the cell to the foot pads. After treatment with a proteolytic agent the thin retraction fibres are cleaved leaving the footpads behind (Rollins et al., 1982). The footpad material left behind is known as substratum attached material (SAM), and is made up of fibronectin as a major component, the protein actin, which confers to the cell the ability to move over a surface, and various glycosaminoglycans (GAGs)

(Rollins et al., 1982). In particular SAM contains the GAG’s chondroitin-4-sulfate and un-sulfated chondroitin and little dermatan sulfate (Rollins et al., 1982).

The adhesion of cells is essentially regulated by receptor ligand interactions (Bell, 1988). For example, one of the biological activities of laminin is to provide a site for binding of cellular receptors (Timpl et al., 1990). The strength of these interactions, which involve multiple non-covalent bonds between specific proteins and polysaccharides, is influenced by pH. Low pH can disrupt the interactions and release the adhesion (Bell, 1988). As both receptors and ligands lie within the glycocalyx of different cells, in order for adhesion to occur the glycocalyx of each cell will have to contact the other and may become compressed in the process. The strength of binding is thus a balance between the repulsive force of the negatively charged oligosaccharides present in the glycocalyx of each cell, and the binding strength between the receptors and ligands(Bell, 1988). The protein epitopes for adhesion interactions can vary between 3 to 10 amino acids in length (Reichardt, 1999). In particular the sequence arginine-glycine-aspartate (RGD) (also found is arginine-glycine-aspartate-serine RGDS) is important, as it functions as a cell attachment site for many extracellular matrix proteins (Reichardt, 1999). If the abolition of these adhesive forces alone was sufficient to achieve wool loosening, then perhaps specifically targeting this sequence with a highly specific protease could achieve depilation with little other damage.

Fibroblasts bind to fibronectin through a complex of polysaccharides that bind to a specific tri-peptide on fibronectin (Bell, 1988). Receptors for laminin are also found on cells that interact with the basal lamina. It is likely that many macromolecules of the extracellular matrix have specific receptors on the cells with which they interact (Bell, 1988). The exact nature of all the possible interactions between cells is complicated and include interactions with a range of proteins such as: integrins, CD44, and syndecan. These proteins (and others) are known to interact specifically with extracellular material and may well be involved in the cell-extracellular matrix adhesion interaction (Reichardt, 1999).

Overall the impression is that many and varied specific interactions are involved in cell adhesion. Of special interest is the difference in interaction between cell-cell adhesion and cell-matrix adhesion of epithelial cells. Flattened cells attached to the substratum

have many attachment sites, some of which, especially those near the edge of the cell are disrupted on exposure to proteases. When this occurs the cells become more rounded and susceptible to shear force and detachment (Rollins et al., 1982). When "Dispase" (a specific commercial proteolytic enzyme with broad proteolytic activity against ECM proteins) is used, cell-matrix interactions are specifically disrupted resulting in the release of the cells as a sheet (Paul et al., 2001). Clearly, therefore, targeting the specific adhesion interactions of interest could result in the release of epidermal cells without compromising other proteins in the skin.

Heparan sulfate cannot be removed from SAM by exposure to the enzymes trypsin, and testicular hyaluronidase or the chaotroph guanidine hydrochloride. In contrast, hyaluronic acid and chondroitin sulfates can be removed by hyaluronidase and guanidine hydrochloride. Interestingly, removal of these components by enzymes does not appear to affect cell adhesion (Rollins et al., 1982). It can therefore be inferred that GAGs do not affect the strength of adhesion. Presumably the proteins within the GAG matrix are responsible.

The adhesion of cells to the substratum appears to occur by a different mechanism compared to cell-cell adhesion (Roberts & Brunt, 1985). A possible model for the cell substratum adhesion has been proposed where heparan sulfate initially binds to “cold insoluble globulin” already present in the substratum before aggregating into large complexes. Concomitantly, fibronectins bind to the developing interaction in a cooperative manner (Rollins et al., 1982). As these cell adhesion interactions age the quantity of heparan sulfate in the adhesive material decreases (Rollins et al., 1982).

To summarise, it appears that although GAGs are involved in causing cell adhesion, fibronectin seems to be the major structural component of the “adhesive”. An enzyme depilatory which hydrolyses fibronectin would therefore be a possible target for inclusion in an enzyme depilatory reagent mix. However, since many proteins are involved in the interactions between the epidermal cells and the extracellular matrix multiple proteolytic specificities are likely to be required in order to achieve complete separation of the cells and extracellular matrix.