Minimum Reactants Required for Synthesis of Skin and Peripheral Nerves; Extension to Tissues of Other Organs

It is a report of the insoluble substances that went into the reactor (i.e., the anatomically well-defined defect) and the insoluble products that were eventually synthesized in it.

Reaction diagram A omits information about any endogenous soluble substances (e.g., cytokines and growth factors) that have regulated the function of cells involved in synthesis of the products. This omission is deliberately made to simplify the presentation of the diagram. It is justified by postulating that the initial concentrations of soluble regulators (cytokines and growth factors) inside the defect are identical in all investigations in which the same defect was studied. We will refer to this condition as the uniform cytokine field for a particular anatomically well-defined defect; in general, it describes the time-dependent changes of each of the soluble substances at each spatial location inside the defect. This information, which includes but is not limited to cell–cell signaling, is incredibly complex and is usually unavailable but we do not propose to use it in any systematic manner in this chapter.

As a first approximation, we will assume that the uniform cytokine field, i.e., the initial chemical background of the defect, is a constant for a given anatomically well-defined defect. There is justification in assuming that the initial conditions of the healing process in a defect depend predominantly on the type of defect, a constant for the regeneration processes that we have examined in preceding chapters. If so, this constant background can be omitted from reaction diagrams without affecting our judgment on the relative simplicity of protocols used by different investigators who studied the same defect. The reaction diagram includes reference to soluble reactants (e.g., cytokines) that have been added but omits reference to any soluble products that may have been synthesized during the process. As the products of main interest in these processes are the (insoluble) tissues that are synthesized, such omission does not affect a conclusion on the relative simplicity or effectiveness of a protocol. Previously, a myriad of authors describing biological processes have selectively omitted information such as, for example, the temperature and the pH levels of an in vivo study; both of these parameters are widely considered, with very few exceptions, to have remained at physiological levels during each of several studies conducted. As they comprise a common background in different investigations, these parameters are often omitted, even from a detailed discussion of the experimental conditions.

It is important to clearly distinguish between a reaction diagram and a chemical equation. As used here, reaction diagrams are shorthand descriptions of a protocol used by the investigators. They do not contain stoichiometric information and should, therefore, be considered somewhat analogous to qualitative, unbalanced chemical equations. The symbols employed on both sides of the arrow in a reaction diagram simply identify a given protocol, namely, the addition of a reactant or the synthesis of a product, not the relative masses of reactants or products. It is a qualitative tally of what went in the defect and what came out.

A reaction diagram based on data reported in the literature usually does not describe the simplest process by which a tissue or organ can be prepared. However, comparison of several reaction diagrams can lead to an irreducible reaction diagram, i.e., a description of the simplest known process by which the synthesis has been achieved. For example, let us hypothesize that tissue C, synthesized as shown in Dg. A previously, can also be synthesized by a simpler route:

$$ \text{cell type A}\to \text{tissue C}\quad \quad (\text{Dg}\text{. B} ) $$

If Dg. B is indeed the simplest route reported in the literature, the diagram representing it will be considered to be the irreducible reaction diagram for synthesis of C. In a comparison of two reaction diagrams for synthesis of the same tissue or organ, the one using fewer reactants will obviously be considered simpler. Between two protocols that make use of the same number of reactants, that which employs fewer cell types will be considered simpler; also, in vitro conditions will be considered to be simpler than in vivo conditions.

Once identified, the irreducible diagram suggests the minimal conditions known for synthesis of the product(s). Such an identification obviously cannot be used to assert that even simpler conditions cannot or will not be discovered at a later time when future investigators may come to understand much more clearly the nature of these synthetic processes. However, the irreducible process does contain the answer to the following important question: Based on the data available to us today, which reactants are required to be added (necessary) in order to synthesize a given tissue or organ? Discussion of mechanism, appearing in later chapters can then focus on the simplest known synthetic route.

7.1.2 Approximations Underlying the Use of Reaction Diagrams

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.

7.1.3 Tabulation of Reaction Diagrams

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).

Table 7.1
Reaction diagrams for synthesis of skin and of its tissues



Reaction conditions and structure of product


Response of culture medium or defect in the absence of reactants (negative control for all reaction diagrams)


Cell-free medium (in vitro) → no tissues synthesized

Negative control for all reactions in culture (in vitro); no tissues synthesized


In vivo dermis-free defect 
$\xrightarrow{\text{in vivo}}$
epithelialized dermal scar

Negative control for all reactions in the adult dermis-free defect (in vivo); epithelialized dermal scar synthesized

Billingham and Reynolds 1952; Billingham and Medawar 1955; several other authors; see Chap. 2

A. Epidermis (E)


KC + FB → E

Lethally irradiated FB could be replaced with medium from FB culture; keratinizing epithelium synthesized

Rheinwald and Green 1975a, b; Green et al. 1979, 1981; O’Connor et al. 1981; Regauer and Compton 1990



KC cultured in defined medium, pH 5.6–5.8 and optimal KC density; keratinizing epithelium synthesized

Eisinger et al. 1979; Peehl and Ham 1980; Tsao et al. 1982


KC + DRT → E

KC cultured on DRT; keratinizing epithelium synthesized

Boyce and Hansbrough 1988


KC + CBL → E

KC cultured on collagenous bilayer; keratinizing epithelium synthesized

Bosca et al. 1988; Tinois et al. 1991


KC + FB + L-DRT → E

Keratinocytes and fibroblasts cultured with modified (laminated) DRT; keratinizing epithelium synthesized

Cooper et al. 1991, 1993; Boyce et al. 1993


KC + FB + COG → E

KC cultured with FB in collagen gel; keratinizing epithelium synthesized

Nolte et al. 1993, 1994; Hansbrough et al. 1994b

B. Basement membrane (BM)


KC + COG → E · BM

KC cultured on collagen gel; typically reported evidence for synthesis of hemidesmosomes, lamina lucida, and lamina densa

Mann and Constable 1977; Hirone and Taniguchi 1980; David et al. 1981; Cook and Van Buskirk 1995


KC + CBL → E · BM

KC cultured on collagenous bilayer; hemidesmosomes, lamina lucida, lamina densa, and occasionally anchoring fibrils, synthesized

Bosca et al. 1988; Tinois et al. 1991


$\xrightarrow{\text{in vivo}}$
E · BM

KC formed epidermis in vitro and was grafted; lamina lucida, lamina densa, and anchoring fibrils synthesized

Woodley et al. 1988; Aihara 1989; Carver et al. 1993b; Cooper et al. 1993: Orgill et al. 1998


KC + FB + COG → E · BM

KC added to precultured collagen gel and fibroblasts; hemidesmosomes, lamina lucida, lamina densa, anchoring fibrils synthesized

Chamson et al. 1989; Harriger and Hull 1992; Okamoto and Kitano 1993; Marinkovich et al. 1993; Tsunenaga et al. 1994; Smola et al. 1998; Stark et al. 1999


KC + FB + L-DRT 
$\xrightarrow{\text{in vivo}}$
E · BM

KC added to precultured fibroblasts in modified (laminated) DRT, then grafted; lamina lucida, lamina densa, and anchoring fibrils synthesized

Cooper and Hansbrough 1991; Cooper et al. 1993; Boyce et al. 1993


KC + FB + NY → E · BM

KC added to precultured fibroblasts in nylon mesh; anchoring filaments, lamina densa, anchoring fibrils, nidogen synthesized

Contard et al. 1993; Fleischmajer et al. 1995



KC cultured in defined medium; hemidesmosomes, anchoring filaments, lamina lucida, lamina densa synthesized; collagen VII synthesized but not secreted

Rosdy et al. 1993


KC + FB + PGL 
$\xrightarrow{\text{in vivo}}$
E · BM(?)

KC added to precultured fibroblasts in nylon mesh, then grafted; continuous laminin; type IV collagen not reported

Hansbrough et al. 1993


KC + FB + COG 
$\xrightarrow{\text{in vivo}}$
E · BM

KC cultured on a collagen gel with FB, then grafted; hemidesmosomes, lamina lucida, lamina densa, anchoring fibrils synthesized

Hansbrough et al. 1994b; Nolte et al. 1994



KC cultured on collagen film; no BM synthesized

Cook and Van Buskirk 1995


KC + PL → No BM

KC cultured on surface of plastic dish; no BM synthesized

Cook and Van Buskirk 1995


$\xrightarrow{\text{in vivo}}$
E · BM

Uncultured KC seeded into DRT, then grafted; continuous laminin, collagen VII (anchoring fibrils), α6β4 integrin (hemidesmosomes) synthesized

Compton et al. 1998

C. Dermis (D)


$\xrightarrow{in vivo}$

Cell-free DRT grafted; synthesis of thick, vascularized dermis with quasirandomly oriented collagen fibers; no BM; no dermo-epidermal junction

Yannas 1981; Yannas et al. 1981, 1982a, b; Orgill 1983; Orgill et al. 1996; Orgill and Yannas 1998; Compton et al. 1998


KC + FB + COG → No D

KC cultured on a collagen gel with FB; no dermis synthesized

Bell et al. 1981a, 1983; Hull et al. 1983a


KC + FB + COG 
$\xrightarrow{\text{in vivo}}$
E · BM · D

KC cultured in vitro on collagen gel with FB, then grafted; thick dermis with basketweave pattern and vascularization was synthesized

Bell et al. 1983; Hull et al. 1983b; Hansbrough et al. 1994b; Nolte et al. 1994


KC + CBL → No D

KC cultured on collagenous bilayer; no dermis synthesized

Bosca et al. 1988; Tinois et al. 1991


KC + DRT → No D

KC cultured with DRT; no dermis synthesized

Boyce et al. 1988


$\xrightarrow{\text{in vivo}}$
No D

KC cultured, then grafted; KC sheet detached from surface of defect; no dermis synthesized

Aihara 1989; Ogawa et al. 1990; Carver et al. 1993b; Cooper et al. 1993; Orgill et al. 1998


KC + FB + L-DRT 
$\xrightarrow{\text{in vivo}}$
E · BM · RR · D

FB and KC cultured on laminated DRT, then grafted; dermis with basket-weave pattern synthesized

Cooper and Hansbrough 1991; Cooper et al. 1993; Boyce et al. 1993


KC + FB + L-DRT → No D

FB and KC cultured on laminated DRT; no dermis synthesized

Boyce et al. 1993


KC + FB + PGL → No D

KC cultured on polyglactin mesh with FB; no dermis synthesized

Cooper et al. 1991; Hansbrough et al. 1993


KC + FB + PGL 
$\xrightarrow{\text{in vivo}}$
E · BM(?) · D

KC cultured on polyglactin mesh with FB, then grafted; dermis with capillaries synthesized

Cooper et al. 1991; Hansbrough et al. 1993

D. Partial skin (E · BM · RR · D; skin appendages missing)


KC + FB + COG 
$\xrightarrow{\text{in vivo}}$
E · BM · D

KC cultured on collagen gel with FB to synthesize epidermis, then grafted; synthesized continuous basement and thick dermis with dermal nerve fibers; no rete ridge formation (but see Parenteau et al. 1996); no elastic fibers synthesized

Bell et al. 1981b, 1983; Hull et al. 1983b; Bosca et al. 1988; English et al. 1992; Hansbrough et al. 1994b; Nolte et al. 1993, 1994


$\xrightarrow{in vivo}$
E · BM · RR · D

Uncultured (or cultured) KC seeded into DRT, then grafted; simultaneous synthesis of epidermis, BM, rete ridges with dermal papillae and dermis, including elastic fibers and dermal nerve fibers

Yannas et al. 1981, 1982a, b, 1984, 1989; Orgill 1983; Murphy et al. 1990; Compton et al. 1998; Orgill et al. 1998; Butler et al. 1998, 1999a


KC + FB + L-DRT 
$\xrightarrow{\text{in vivo}}$
E · BM · RR · D

FB and KC cultured on modified (laminated) DRT, then grafted; simultaneous synthesis of epidermis, BM, rete ridges with dermal papillae and dermis, including elastic fibers

Cooper and Hansbrough 1991; Cooper et al. 1993


$\xrightarrow{\text{in vivo}}$
E · BM · D

KC cultured on collagenous bilayer to synthesize epidermis, then grafted; synthesis of BM and dermis; dermal elastic fibers not reported; no rete ridge formation

Bosca et al. 1988; Tinois et al. 1991


KC + FB + PGL 
$\xrightarrow{\text{in vivo}}$
E · BM(?) · D

KC cultured on polyglactin mesh with FB, then grafted; laminin stained continuously; type IV collagen synthesis not reported; dermis with capillaries synthesized; dermal elastic fibers not reported; no rete ridge formation

Cooper et al. 1991; Hansbrough et al. 1993

E. Skin (E · BM · RR · D · App; skin appendages included)

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Dec 13, 2017 | Posted by in HISTOLOGY | Comments Off on Minimum Reactants Required for Synthesis of Skin and Peripheral Nerves; Extension to Tissues of Other Organs

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