Although reaction diagrams greatly simplify the description of complex processes involving tissues and organs, they are rough approximations of reality and must be used with caution, as discussed below.

One of these approximations is the use of a single symbol to represent a reactant or a product, suggesting the existence of a unique state for each. This convention is normally used in the representation of chemical compounds. Yet, the normal morphology of a tissue may change significantly from one anatomical site to the next in the same organ, even though its name does not. Furthermore, as maturation (or remodeling) proceeds, a tissue in an organ can be present at various levels of differentiation. Assignment of a single symbol to a tissue does not take into account many of these variations in morphology or functional state with anatomical location or maturation time.

A few examples illustrate the degree of approximation involved in describing a tissue by a single name. The epidermis (E) synthesized by culturing keratinocytes in vitro with fibroblasts and a collagen gel (COG) is a product that can be prepared at various identifiable levels of differentiation, depending on timing and other reaction conditions (Parenteau et al. 1992, 1996). The same caution applies when cells are used as reactants in a process. The symbol KC is used below to represent keratinocytes in culture medium. Implicit in the use of a single symbol is the assumption that keratinocytes exist in a single state of differentiation. In a number of studies, however, keratinocytes have been isolated from a skin biopsy and have either been used to induce synthetic processes without further culture or else have been extensively cultured before being used. The uncultured keratinocytes typically comprise cells from all epidermal layers, representing various levels of differentiation, while cultured cells have typically been converted to a higher level of differentiation (Wille et al. 1984). The details of a synthetic process can be affected, often very significantly, by the precise state of differentiation of a cell that is used as a reactant. In the example of KC seeded into dermis regeneration template (DRT) , a nondiffusible regulator, the skin synthesized in the process had a significantly higher number of rete ridges when the keratinocytes had been cultured, than when the cells were freshly dissociated but not cultured, prior to seeding (Butler et al. 1999a). In a further example, a study of synthesis of BM in vitro in the presence of keratinocytes, fibroblasts, and a COG led to the conclusion that BM was synthesized only when keratinocytes were added to the COG that had already been cultured for a period of time with fibroblasts; a BM was not formed when keratinocytes were added to fibroblasts and the COG without first culturing the latter the two cell types together (Chamson et al. 1989).

How well differentiated a product needs to be in order to merit being referred to by unique symbolism? Most tissues discussed in this chapter have been synthesized in more than one distinguishable level of differentiation. In contrast to the symbolism of organic chemistry, where the term benzene refers to a unique compound, investigators have typically not employed standard definitions of the tissues under study. Although the vast majority of investigators agree that a tissue that displays a minimum number of well-defined morphological characteristics can be uniquely identified, a formal process of standardization of tissues based on a necessary and sufficient set of morphological and functional characteristics has not yet been developed. As an example, we often find that an investigator defines the product of a reaction as the “basement membrane” of skin if it comprises at least four distinct structural characteristics of this tissue, identified immunohistochemically in terms of the major protein constituent that is uniquely associated with each layer. In this example, these constituents are the a6b4 integrin, characteristic of hemidesmosomes; laminin, present in lamina lucida; type IV collagen, a major constituent of lamina densa; and type VII collagen, the main component of anchoring fibrils.

In addition to this process of tissue identification based on protein components, an investigator may provide ultrastructural evidence of normal organization of these macromolecular elements into a whole, functioning tissue. At the other extreme, another investigator may report synthesis of a “basement membrane” based on immunohistochemical identification of just two protein constituents, such as laminin and type IV collagen, without reporting on the presence of the other proteins commonly found in BMs or providing any ultrastructural data to document the organization of the tissue. In this example, both investigators have identified the product of the reaction as BM but have employed different criteria in assaying for it. In view of this diversity in use of identifying criteria, I have arbitrarily chosen to report synthesis of a given tissue if the investigators provided clear evidence that at least one assay of widely recognized value, such as those described in histology textbooks (Young et al. 2006; Kierszenbaum and Tres 2012), was employed in its identification. This nominal approach clearly errs on the side of inclusion of products and probably leads to irreducible reaction diagrams that are weighted excessively toward simplicity in description of reaction conditions.

To discuss synthesis of individual tissues of an organ in the actual presence of part of the organ, we will introduce the symbolism used to represent an organ in terms of its tissue components. The physical connection between two tissues in the context of an organ is represented as a dot between the symbols of adjacent tissues; use of a dot, rather than a connecting line, prevents confusion with a chemical bond. As an example, physiological skin (S) is considered below, in abbreviated fashion, to comprise only of an epidermis, a BM with hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils (BM), rete ridges with dermal papillae (RR), and a thick, vascularized dermis (D) with sensory nerves and appendages (AP); the latter derive from the epidermis during development but are located in the dermis. A completely physiological dermis with nerve fibers and appendages will, accordingly, be referred to as RR · D · AP while the physiological skin organ is ­symbolically represented as E · BM · RR · D · AP. When only appendages are missing, the representation of skin changes to E · BM · RR · D, also referred to below as partial skin (PS). Occasionally, rete ridges in a partial organ product are missing, and the symbolic representation becomes E · BM · D.

The simplified view of an organ as a “linear assembly” of tissues is occasionally partly supported by experimental evidence. An illustration is the synthesis of an epidermis attached to a physiological BM (E · BM) in a dermis-free defect (Carver et al. 1993b); even though the dermis is missing in this tissue product, the epidermis–BM bilayer, E · BM, survives for days, a sufficiently long experimental period to allow the investigators to make several useful observations about its structure. Another example is the preparation of a BM on a dermis in the absence of an epidermis (BM · D) (Guo and Grinnell 1989); here, the epidermis is missing but the BM–dermis bilayer persists over a period of time.

Nevertheless, there are often serious problems with this approximation. A tissue that has been synthesized outside its anatomical context eventually shows evidence of its instability. Such a tissue may often be tentatively connected physically to the rest of the organ; and it may be unvascularized or unsupported metabolically by the organ. For example, there is strong evidence, presented in Chap. 5, that an epidermis, synthesized in vitro without a BM or a dermis attached to it, fails to attach itself on the muscle surface of a dermis-free defect surface (Billingham and Reynolds 1952; Billingham and Russell 1956; Eldad et al. 1987; Latarjet et al. 1987; Carver et al. 1993b; Cooper et al. 1993; Kangesu et al. 1993b; Orgill et al. 1998). On the other hand, an epidermis, originally synthesized in vitro in an immature state, undergoes rapid maturation after it has been placed on a dermal substrate in vivo (Prunieras 1975; Faure et al. 1987). The evidence clearly shows that the individual tissue of an organ can be considered as a discrete, stable entity only as a rough first approximation, e.g., in the context of an experimental protocol where the question posed is whether the tissue in question can be synthesized at all, even in a state that is only temporarily stable. We recall that chemists frequently find it very valuable to include in equations symbolic representation for free radicals, most of which are very unstable species.

With few exceptions for data obtained with skin defects (see Breuing et al. 1992; Levine et al. 1993) or nerve defects (Fu and Gordon 1997), data on concentration levels of diffusible regulators in defects have rarely been reported; extensive data are a rarity. The uniform cytokine field hypothesis is expected to apply only under the initial conditions for the process, i.e., immediately after the injury and just before the addition of any reactant(s). The postulated uniformity is expected to fail soon after addition of a reactant to the defect. The exudate in the defect typically responds to addition of a reactant by a modification of its contents, the direction or extent of which strongly depends on the nature of the reactant.

Finally, the degree of relevance of an irreducible reaction diagram to the processes of remodeling in an adult or to developmental processes in a growing organism is unclear. The conditions in a healing defect, in which an inflammatory exudate is prominently present, should be anticipated to be significantly different from those at the equivalent anatomic site of a remodeling organ or a developing organism.

The collection of reaction diagrams in Tables 7.1 (skin) and 7.2 (peripheral nerves) include those in which the reactants added were well-defined and easy to reproduce in independent laboratories . Reactants that are explicitly included in the diagrams comprise dissociated (disaggregated) cells of known type, synthetic polymers of known composition, and defined components of the extracellular matrix (ECM) or nondiffusible macromolecular networks synthesized from ECM components following standard synthetic methods (ECM analogs). Processes in which tissue grafts (e.g., epidermal or dermal grafts), were employed as reactants were not included in Tables 7.1 and 7.2 since, as discussed in detail in Chaps. 5 and 6, their presence in the defect compromises the identification of the products. Diffusible regulators have not been included explicitly as reactants; as discussed above, since all in vivo processes in Tables 7.1 and 7.2 took place in a dermis-free defect or in a transected and tubulated nerve, they are considered to have taken place in the same (uniform) cytokine field. See the tables for the abbreviations used to construct reaction diagrams for skin synthesis (Table 7.1) and for peripheral nerve synthesis (Table 7.2).