Dermal-epidermal Separation, Part III: Heat and Mechanical Methods

This column concludes a three-part series on techniques for epidermal-dermal separation (see Figure 1). It focuses on heat and mechanical techniques and describes the advantages and disadvantages of each. Previous installments reviewed enzyme digestion options and the use of acid, alkalis and natural salts.

Heat Separation

Epidermal-dermal separation by heat (see Table 3) was introduced in 1942 by Baumberger et al. Since increasing temperatures can soften collagen fibers, temperatures much lower than the boiling point of water may be used to separate the epidermis from the dermis.

For example, after exposure for 2 min on a slide warming table at 50°C, the epidermis was found to peel readily and completely using forceps. Complete division occurred with ease at 49.2°C, after incompleteness at 48.2°C. With temperatures above 51°C, detachment difficulty occurred. Felsher⁴ detected an influence of salts on the subsequent separation of the epidermis by heat. Heat separation is facilitated by ions, which can promote collagen swelling or depression.

In the studies of Sonderga et al.¹⁸ and Wadskov et al.,¹⁹ separation of the epidermis from the dermis in normal human skin by heat was reported possible after 10 min at 60°C.

Kassis et al.²⁰ analyzed epidermal and dermal prostaglandins by heat separation. They kept frozen biopsy specimens in a water bath for various times and found that heating at 60°C for 1 min produced a distinct separation of epidermis from dermis. As there was no significant change in Prostaglandin E1 (PGE1) activity after 1 min of heating, this heat separation technique seemed a reliable and quick method for PG analysis of skin.

Esterases are important for topical or transdermal delivery of drugs since these enzymes may catalyze the hydrolysis of pharmaceuticals containing ester bonds. Lau et al.²¹ detected esterase activity in fresh and heat-separated porcine ear skin. Esterase activity was visibly diminished in common heating separated skin (compared to fresh skin). After heating at 60°C for 1 min, neither epidermis nor the stratum corneum exhibited positive histochemical staining for esterases activity. Therefore, heat separation should not be used to study the permeation of ester-containing permeants.

Mechanical Methods

The methods above have the disadvantage of altering, to some degree, the physical and/or chemical integrity of one or both layers and may therefore be unsatisfactory for studies that require an isolated—but otherwise intact—epidermis or dermis. There is a need for techniques by which epidermis can be separated from the dermis via purely mechanical forces, to avoid chemical or thermal damage.

Stretching

Van Scott²² stretched the skin and removed the epidermis as a continuous sheet by means of a razor blade or scalpel. When skin is stretched, the epidermis slips off easily using this procedure and can be rapidly removed. Gilbert²³ and Sputt²⁴ improved the technique so skin only needed stretching to a relaxed length. However, these methods require comparatively large surgical necropsy specimens, and when scraped off with a scalpel, may lack a basal cell layer.

Suction

As with unidirectional stretching, dermal-epidermal separation has been achieved by the application of pressure gradients causing multidirectional distension. In 1878, Unna²⁵ described the separation of the epidermis from the dermis along the dermal-epidermal junction (DEJ) during dry cupping. Blank et al.²⁶ also separated the epidermis from the dermis with vacuum via bullae formation, which demonstrated DEJ separation histologically. This approach therefore became a mechanical method to separate the epidermis. In vivo separation of the complete human epidermis by suction was accomplished in 1964.

Kiistala et al.²⁷ found that suction at a pressure of 20 cm Hg applied to intact human healthy skin produced blisters within 2 hr, the roof of which consisted solely of epidermis and included the basal cell layer. They constructed a device that produced standard suction blisters of preselected size and number on human skin without discomfort. In approx. 2 hr, suction at 150 mm Hg²⁸ permitted separation of the epidermis from cadavers and certain animals. The blister roof consisted of viable, full-thickness epidermis and the clear fluid was a non-inflammatory transudate.

No scarring resulted and the suction trauma of the dermis was minimal. Thus, the suction epidermis appeared useful for tissue culture and biochemical analyses. Today, this method is widely applied in dermatological research and practice for epidermal grafting^29–31 for creating standardized wound-healing models^{32, 33} and for studying morphological, physiological or pharmacological phenomena.^{34, 35}

There is need for techniques by which the epidermis can be separated from the dermis via purely mechanical forces, to avoid chemical or thermal damage.

Overall Pros and Cons

Many techniques relying on different mechanisms have been described in this series.

Heat is simple, yet tedious. Although the heat process has been used successfully, chemical reagents are effective but disturb the electrolyte cellular equilibrium. Digestion by enzymes gives complete separation but it destroys important components. Meanwhile, mechanical division necessitates a relatively large tissue piece but has the advantage in that chemical changes do not occur. Many investigations have compared these methods and documented their different influences on the structure and function of skin biochemistry.

Baumberger, when comparing NH₄OH and heat separation methods, found that epidermal tissues treated were not killed but only temporarily loosened, and there was a marked decrease in oxygen consumption by the epidermis by both methods. However, the heat technique may be superior because the tissue is not brought into water and there is no loss of material (see Table 4).

Quantitative determinations of stratum corneum sulfhydryl and disulfide concentration revealed relatively consistent values in certain skin diseases. Various methods of separation of epidermal sulfhydryl showed its values obtained after ammonium separation resembled those following mechanical separation, while heat and enzymes appeared to alter the results significantly and were deemed not satisfactory.³⁶

Dermo-epidermal separation is an essential technique for immunoblotting studies and for the diagnostic immunofluorescence of autoimmune bullous diseases. To study the localization of basement membrane components after different separation methods, Woodley et al.³⁷ separated adult human skin using four methods. Only the basement membrane heparan sulfate proteoglycan was trypsin-sensitive.

Willsteed et al.³⁸ examined the electron microscopic appearance of specimens separated by three methods, concluding that 1M NaCl remains the most convenient and reproducible means of inducing dermal-epidermal separation through the lamina lucida.

Ohata et al.³⁹ compared the autoantigens for bullous skin diseases by immunoblotting using different dermal-epidermal separation techniques; the results suggest that EDTA-, dispase- and heat-separated skin were similar for detecting various autoantigens but heat separation is preferable because the preparation time is shorter.

To compare the effects of epidermal-dermal separation techniques of hairless mouse skin, the most suitable method was determined by kinetic studies in homogenate medium.⁴⁰ Based on data of the effects of separation methods on soluble protein yield and dermal enzymatic activity using DBMTX as a substrate, EDTA treatment appears a favorable method for epidermal separation.

To obtain rapid, high-quality epidermal-specific RNA from human skin, Trost et al.⁴¹ investigated the effect of dermal-epidermal separation methods—including ammonium thiocyanate, dispase, heat and 1M NaCl—and concluded that the fast chemical separation method with 3.8% ammonium thiocyanate is the optimal choice for producing high-quality RNA isolation from human skin.

Epidermis separation is also useful in autologous skin transplantation. The first step in isolating epidermal cells is to separate it from dermis. Recently, several methods for epidermis isolation and epidermal cell suspension were compared to select the optimum one for clinical use. NaBr (4N) method is considered as the least toxic and the most viable cells produced.⁴²

Conclusion

In conclusion, the separation method must be appropriate for the experimental project, and no single method appears superior for all purposes. Future comparative investigation should benefit the integrity of skin care, and cosmetic research based on this methodology and predictability will lead to greater utilization, as more refined knowledge should lead to enhanced research integrity.

References

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