The effects of the following enzymes were investigated: bromelain (B4882, dissolved in 0.01M Tris [T1503, Sigma] pH7.5); collagenase (C01300, dissolved in 0.1M Tris pH7.6); papain (P4762, dissolved in 0.01M phosphate buffer pH6.5 containing 5mM L-Cystein [16,814-9,Sigma] and 2mM ethylenediaminetetra-acetic acid [EDTA] [E9884 Sigma]; pepsin (P7012, dissoved in 10mM HCl pH 2.9); protease type X (Thermolysin, P1512 dissolved in 10mM sodium acetate [TA948368, Merck] and 5mM calcium acetate [C1000, Sigma]); and trypsin (T1005, dissolved in 0.01M Tris pH8.6).

Intact skin treatment. Freshly prepared solutions were continuously streamed onto confined shaved skin surface areas of the anesthetized mice, rats, rabbit, or pig skin samples at a flow rate of 5 to 6mL/hour for three hours at room temperature, after which the animals were sacrificed and samples for histological examination were removed from treated areas.

Histology. Following the three hours treatment, the mice and rats were sacrificed with an overdose of chloral hydrate (Fluka Chemicals, Switzerland). and the rabbit was sacrificed with an overdose of thiopenton sodium. Full-thickness skin samples (4x15mm) were removed for histological analysis from the margins of the confined area to allow comparison of treated and nontreated areas in same slide. Tissue samples were immediately fixed in four-percent phosphate buffered formaldehyde for 48 hours, processed by routine histological procedures, and embedded in paraffin. Serial sections perpendicular to the skin surface were cut at 8 micron thickness. The sections thus obtained were stained with hematoxylin and eosin for observations.

Experimental wound models. Thermal burns, 1 to 1.5mm in depth, were induced by a direct contact of a tip of a standard soldering instrument for 30 seconds on the posterio-lateral aspect of dorsal skin of anesthetized mice and rats as previously described.10 Freshly prepared single or combination protease solutions were applied by continuous streaming onto the wound within one hour from injury for 2 to 3 hours at the same flow rate as mentioned above.

Full-thickness linear fresh cuts were made by scalpel on the posterio-lateral aspect of animal backs and immediately treated with continuous streaming of enzymes for three hours.

Photographs of treated areas were taken immediately after treatment and after seven and 20 days for assessment of the healing process.

Monitoring of streamed enzymatic activity. As proteolytic enzyme solution may lose proteolytic activity due to autodigestion, residual activity of enymes employed was routinely monitored by in-vitro biochemical assays recommended by the supplier.

Results

Effect of enzyme streaming on intact skin. Controlled streaming of enzymes could be readily and conveniently applied as a series of consecutive treatments using a multichannel pump, as demonstrated in Figure 2A, for treatments of six anesthetized rats or treatment of six different sites on a larger animal (Figure 2B). Effective digestion of different skin layers was readily achieved by streaming diluted buffered enzyme solutions for three hours. The controlled streaming of 2mg/mL papain onto mice effected digestion and removal of the outer keratinized layer (compare Figure 3A to Figure 3B). Detachment of the epidermis from the dermis was effected by a trypsin (4mg/mL) and bromelain (5mg/mL) mixture (Figure 3C). Controlled streaming of 8mg/mL trypsin solution effected complete digestion of the epidermis layer (Figure 3D). Streaming of 3mg/mL pepsin resulted in deeper penetration and collagen fiber digestion (Figure 3E). Streaming of a mixture of 3mg/mL collagenase and 1.5mg/mL thermolysin resulted in digestion similar to that shown in Figure 3D.