Streaming of Proteolytic Enzyme Solutions for Wound Debridement: A Feasibility Study

Tali Yaakobi, PhD; Dalit Roth, MSc; Yoram Chen, BSc; Amihay Freeman, PhD

Disclosures

Wounds. 2004;16(6) 

In This Article

Materials and Methods

The streaming system was comprised of a feeding reservoir, connecting tubing, peristaltic pump (MP4 Minipulse 3, Gilson, France), a plastic applicator designed to direct the flow onto the treated site, and collecting vessel (Figure 1).

Shown here are the components of an enzyme streaming system.

The study was performed on groups of six 4- to 8-weeks-old (30-40g body weight) male and female white mice, on groups of six mature 2- to 3-months-old (200-250g body weight) Charles-River male rats, on one adult male New-Zealand white (NZW) rabbit (3kg body weight), and on pig skin samples freshly removed from a large, white, male pig (34kg body weight). The mice and rats were anesthetized with 0.1mL of 1.25-percent tribromoethanolin saline per 10g body weight, and the rabbit was sedated by ketamine and anaesthetized with thiopenton sodium. The areas to be treated on all animals were shaved, and the animals were placed on a jack and lifted until the applicator was tightened to the surface of the posteriolateral aspect of their backs. The fresh pig skin samples were mounted on a plastic O-ring and then fastened to the applicator.

All enzymes tested were lyophilized powders supplied by Sigma (Sigma-Aldrich Chemicals, St. Louis, Missouri, USA). The enzymes were utilized as received without further purification. 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 ethylenediaminetetraacetic 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).

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.

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.

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 posteriolateral 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 posteriolateral 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.

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

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