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

Tali Yaakobi, PhD;[1] Dalit Roth, MSc;[1] Yoram Chen, BSc;[2] Amihay Freeman, PhD[1]


Enzymatic digestion of the intercellular matrix for the separation of mammalian cells is a common methodology in the preparation of primary cell cultures.[1] This procedure, resulting in suspended isolated cells, has been routinely employed for the preparation of cell cultures from a wide spectrum of organs, including skin.[2] Some of the proteases used in these procedures were demonstrated to be selective in disruption of the extracellular matrix and adhesion proteins without causing cell damage.[3,4]

Furthermore, separation of dermis from epidermis[2,4,5] and the removal of necrotic or damaged tissue from wounds and burns without damage to healthy tissue were also demonstrated.[6,7] These observations paved the way to systematic exploration of the potential inherent in wound debridement by enzymes. Most of these studies employed commercially available enzymes (e.g., papain, bromelain, collagenase, trypsin, thermolysin) with only few attempts to identify or develop new enzymes, e.g., from the antarctic krill.[6–8]

Enzymes tested for effective wound debridement were mostly formulated as ointments, solutions absorbed by a wet gauze, hydrocolloids, or hydrogels.[3,6,7] Comparative studies on the efficacy of wound debridement by enzyme-containing ointments—either animal models or humans—indicated efficacy dependence on the enzyme employed: While fibrinolysin ointment was found ineffective, collagenase ointment gave some improvement, and papain-urea ointment was identified as most effective from this group.[9–12] Prolonged time was required for these treatments to deliver significant improvement, ranging from four days to three weeks with daily wound treatments for fresh ointment supply (for the first 4–7 days of treatment).[9–12]

The objective of this study was to test the feasibility of a new mode of enzyme delivery for skin treatment and wound debridement—continuous controlled streaming of enzyme solutions onto the targeted treated area. The working hypothesis was that the combination of a fresh supply of enzymes in an optimal working buffer with continuous flow would substantially shorten the time required for effective enzymatic skin treatment or wound debridement. Furthermore, the slightly pressurized stream would allow homogeneous supply of the enzyme to all parts of the treated area and may remove cells, debris, and solubilized proteins.

In this report, the feasibility of enzyme streaming and its efficacy was studied regarding intact skin digestion, removal of coagulated blood, and debridement of experimental surgical and burn wounds.

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).

Animals and tissue samples. 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 posterio-lateral aspect of their backs. The fresh pig skin samples were mounted on a plastic O-ring and then fastened to the applicator.

Enzymes. 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.



1. Ferkushny RI. Culture of Animal Cells. New York, NY: AR Liss, 1983:108.
2. Hybbinette S, Bostrom M, Lindberg K. Enzymatic dissociation of keratinocytes from human skin biopsies for in-vitro cell propagation. Exp Dermatol 1999;8:30–8.
3. Berger MM. Enzymatic debriding preparations. Ostomy Wound Manage 1993;39:61–9.
4 Normand J, Karasek MA. A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin. In-Vitro Cell Dev Biol Anim 1995;31:447–55.
5. Germain L, Guignard R, Rouabhia M, Auger A. Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or dispase. Burns 1995;21:175.
6. Falanga V. Wound bed preparation and the role of enzymes: A case for multiple actions of therapeutic agents. Wounds 2002;14:47–57.
7. Klasen HJ. A review on the nonoperative removal of necrotic tissue from burn wounds. Burns 2000;26:207–22.
8. Mekkes JR, LePoole IC, Das PK, Bos JD, Westerhof W. Efficient debridement of necrotic wounds using proteolytic enzymes derived from Antarctic krill. Wound Repair Regen 1998;6:50–7.
9. Falabella AF, Carson P, Eaglstein WH, Falanga V. The safety and efficacy of a proteolytic ointment in the treatment of chronic ulcers of the lower extremity. J Am Acad Dermatol 1998; 39:737–40.
10. Hebda PA, Flynn KJ, Dohar JE. Evaluation of the efficacy of enzymatic debriding agents for removal of necrotic tissue and promotion of healing in porcine skin wounds. Wounds 1998;10:83–96.
11. Alvarez OM, Fernandez-Obregon A, Rogers RS, et al. Chemical debridement of pressure ulcers: A prospective, randomized, comparative trial of collagenase and papain/urea formulations. Wounds 2000;12:15–25.
12. Pullen R, Popp R, Volkers P, Fusgen I. Prospective randomized double-blind study of the wound-debriding effects of collagenase and fibrinolysin/deoxyribonuclease in pressure ulcers. Age and Ageing 2002; 31:126–30.

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