Similar results were obtained by streaming of same solutions on rat, rabbit, and pig skins (data not shown).

Streaming of active enzyme solutions was essential to obtain these effects. Streaming of buffer solution without enzymes was ineffective. Furthermore, streaming of enzyme solution for a few minutes to fill the system followed by flow arrest also resulted in no visual change.

The specific activity of all streamed enzyme solutions remained stable (>85%) throughout the three-hour application period. The minor loss of input activity was most probably caused by autodigestion.

Effect of enzyme streaming on experimental wounds. Effective removal of fresh blood clots was readily achieved by streaming of trypsin and collagenase mixture (3mg/mL each) for three hours onto freshly made cuts with smooth surface cleaning regardless of their shape (compare Figures 4A and C with Figures 4B and D).

Controlled enzymatic streaming for burn wound debridement was also readily achieved by two hours streaming of several protease combinations, including collagenase/thermolysin mixture (3mg/mL and 1.5mg/mL, respectively) (Figure 5B), trypsin/papain mixture (4mg/mL and 2mg/mL) (Figure 5C), and trypsin/collagenase mixture (3mg/mL each) (Figure 5D).

Debridement with streamed enzymes, e.g., papain or pepsin (2mg/mL and 3mg/mL, respectively), for two hours resulted in smooth healing (compare Figure 6A with Figure 6B; photographs taken 20 days post-burn induction).

Discussion

The overall objective of this study was feasibility and efficacy demonstration of enzyme streaming for skin treatment and wound debridement as an alternative and highly effective mode of enzyme application. The working hypothesis was that continuous streaming will minimize handling and provide higher efficacy and more rapid debridement. The continuous fresh supply of the enzyme dissolved in solutions buffered for optimal activity would result in effective distribution and access to all parts of the treated area as well as in better control of the process. Moreover, the continuous stream could help in washing away digested material resulting in faster erosion and a shortened procedure. This flow system may also allow step-by-step treatment with different solutions, such as pretreatment with antibiotics, enzymatic digestion, and wound cleaning.

The feasibility of this approach was demonstrated on lab animals by studies on skin treatment and wound debridement. Our results have clearly demonstrated technical feasibility and efficacy of streaming of enzyme solution. The time required for effective treatment was on a scale of few hours, substantially shorter than the several days/weeks required for treatment with enzyme-containing ointments.

The simplicity of this method carries potential for wound bed management by the sequential streaming of solutions for softening, cleaning, debriding, and washing wounds in order to prepare them for optimal healing or subsequent
procedures.