Evaluation of Papain–Urea for Enzymatic Debridement of Coagulation Zones Following Ablative Fractional Laser Treatment

ABSTRACT

Background

Ablative fractional CO₂ laser (10,600 nm) treatment creates an array of microscopic treatment zones composed of an ablation zone (AZ) surrounded by a denatured coagulation zone (CZ). The CZ is believed to play a functional role in skin tightening, posttreatment inflammation, and laser‐assisted drug delivery. This study investigates the viability of enzymatic post‐processing to remove the CZ without affecting the surrounding tissue.

Methods

Ex vivo skin samples were treated with either control, papain, urea, or papain–urea solutions before being covered with occlusive dressing and incubated at 37°C for 1 h. Tissue viability as well as AZ and CZ geometries were assessed histologically.

Results

Treatment with all three experimental solutions resulted in a decrease in CZ. The largest average reduction in CZ area was observed in the papain–urea group (44%, p < 0.001), followed by the papain (14%, p < 0.001) and urea (11%, p < 0.001) only groups. Only the papain–urea group showed a significant increase in AZ (14%) and changes in lesion geometry.

Conclusion

This exploratory study of enzymatic post‐processing with papain–urea highlighted the potential of selectively removing the CZ after treatment with ablative fractional laser therapy. If results can be translated to in vivo studies, these findings could expand the use of high‐fluence CO₂ laser therapy with functional implications for lowering posttreatment recovery time, providing clinicians more control over skin tightening, and enabling a broader range of laser‐assisted drug delivery.

1. Introduction

Fractional laser resurfacing, also known as fractional photothermolysis (FP), is a widely used technique for skin remodeling. First introduced by Manstein and colleagues [1] in 2004, the modality involves creating a fractionated pattern of microscopic treatment zones (MTZs) with the use of laser beams at wavelengths highly absorbed by water in the epidermis and dermis. The therapeutic benefit of FP comes from the ensuing inflammatory processes triggered by the presence of thermally damaged tissue interspersed throughout healthy tissue [1, 2]. The damaged skin recruits a wide variety of metalloproteinases, immune cells, stem cells, and fibroblasts to remodel the extracellular matrix of the skin, resulting in the controlled replacement of pretreatment collagen and elastin [3, 4, 5]. FP is currently used for a variety of esthetic applications, including the treatment of photoaged skin, wrinkles, discoloration, and the revision of acne, burn, and surgical scars [6, 7, 8, 9, 10].

There exists a growing field of research dedicated to broadening the applications of FP. Each MTZ represents a channel into the dermis, providing a direct route for transcutaneous drug delivery [11, 12, 13, 14]. Topical application of many drugs is limited by the hydrophobic stratum corneum, which limits passive diffusion into the skin to molecules smaller than 500 Da [15, 16]. By disrupting the stratum corneum, laser‐assisted drug delivery (LADD) overcomes the size barrier, allowing for the penetration of a greater range of charged and large molecules [12]. Additionally, in accordance with Fick's first law of diffusion, microporation of the skin decreases the skin's diffusion coefficient (Km), further increasing the influx and bioavailability of small and uncharged molecules [17, 18].

The major benefit of transcutaneous drug delivery is its ability to deliver possibly harmful or cytotoxic agents directly to the target tissue while avoiding the side effects of systemic administration and first‐pass metabolism [12, 14, 19, 20, 21]. The efficacy of cutaneous drug delivery has been clinically demonstrated for treatment of conditions like non‐melanoma skin cancers and precancerous lesions, vitiligo, alopecia, scarring, and hemangiomas, along with experimental investigations into the effects of transdermal vaccination [11, 18]. Furthermore, transcutaneous drug delivery often requires a lower dose than systemic treatment, potentially decreasing healthcare spending [22].

The effects of molecular size, lipophilicity, and drug dosage, along with the functional contributions of lesion architecture in LADD, are in the process of being understood [23, 24, 25]. The open channel, or ablation zone (AZ) of each MTZ is surrounded by a layer or denatured collagen referred to as the coagulation zone (CZ) (Figure 1). While “cold” laser modalities that ablate while producing little to no CZ exist (e.g., Er:YAG) [25, 26, 27], fractional CO₂ laser resurfacing remains the gold standard for esthetic treatment [28]. Although the CZ in fractional CO₂ laser treatment is believed to be the source of a longer recovery time and inflammation compared with cold laser modalities, the increase in skin tightening is attributed to the denatured collagen burden [29, 30, 31, 32].

Figure 1.

Human ex‐vivo skin treated with an ablative fractional laser, the coagulation zone (CZ) is defined as the cuff of thermally degraded collagen surrounding the empty ablation zone (AZ) left by treatment. When stained with NBTC, the AZ appears as a blank space surrounded by a white, disorganized CZ. (A) Displays a horizontal view of a microscopic treatment zone (MTZ) at a depth of 100 µm with color overlay indicating the CZ and AZ. (B) A representative image of an MTZ at a depth of 100 µm from an enzymatic debridement treatment along with color overlay indicating the CZ and AZ.

It is the purpose of this paper to demonstrate a novel method of noninvasively removing the CZ after aFP treatment with a CO₂ laser, herein referred to as post‐processing. The method outlined here was inspired by the burgeoning field of enzymatic debridement of burn wounds, resulting in the selective digestion and removal of unwanted, denatured tissue from wound beds [33, 34]. We chose to test the efficacy of papain, a cysteine protease whose proteolytic properties on denatured collagen and fibrin were identified as early as 1879 [35]. As papain is relatively ineffective alone, urea was added to activate the enzyme. Multiple studies have demonstrated the efficacy of papain–urea products for tissue debridement in both lab and clinical settings and the combination has historically been used as a topical agent [36, 37].

Post‐processing promises to increase the versatility of CO₂ lasers by making the CZ a tunable side effect, obviating the need for a secondary laser system to achieve the same effect.

Given the CZ's role in posttreatment inflammation, this technique may hold functional utility in decreasing downtime after aFP treatment and provide clinicians more control over the skin‐tightening process. Finally, post‐processing could become a valuable research tool for exploring the role of the CZ in transdermal drug delivery, allowing for a direct comparison between laser treatment with and without the presence of a CZ.

2. Materials and Methods

2.1. Experimental Design

Human abdominal skin was obtained under Massachusetts General Hospital IRB#2017P000027 from a single female patient undergoing abdominoplasty and was stored at −80°C before the experiment. Bulk tissue was thawed to ambient room temperature before beginning interventions. For each treatment condition, one tissue sample was sectioned into a 3.75 × 3.75 cm full‐thickness square, and subcutaneous fat was trimmed to the level of the dermis horizontally. Ex vivo skin underwent laser treatment and solutions of PBS, papain, urea, and papain–urea were liberally applied with a cotton swab according to experimental group. The entire treatment zone was then covered by a nonadhesive dressing saturated in the same solution and an occlusive bandage (Tegaderm) was put in place to keep the area moist and prevent the solution from reaching the edge of the tissue. Samples were then placed in a 37.5°C incubator for 1 h before removal of both dressings and thoroughly rinsed with PBS to quench the reaction. Samples were immediately processed for histological analysis.

2.2. Laser Parameters

Laser ablation was performed with a 10,600 nm Ultrapulse CO₂ laser (Lumenis Inc. Yokneam, Israel) and DeepFX microscanner (Lumenis Inc. Yokneam, Israel) over a 1 cm × 1 cm treatment area. The laser was set at 50 mJ with 5% density at 300 Hz resulting in 196 (14 × 14) individual laser lesions per experimental group. For the histologic analysis, 10 out of 196 laser lesions per experimental group were evaluated. Care was taken to irradiate on a flat surface to ensure uniform penetration depths and even spatial distribution of holes.

2.3. Enzymatic Solutions

For the control group, 1× phosphate buffered solution (PBS) was used. For the groups containing papain, a commercially available papain‐latex in a buffered aqueous suspension, 2× crystalized, ≥ 16 U/mg protein (Sigma‐Aldrich, P3125) was used. For the papain–urea treatment group, powdered urea (Sigma‐Aldrich, U5378) was added to the papain–latex suspension at a 2:1 molar ratio (3.5 mg/mL) with minimal change in volume. The urea solution contained the same concentration of urea in PBS. All solutions were used at room temperature and custom solutions were prepared day of use.

2.4. Histology

An 8 mm punch biopsy was obtained from the center of each treatment area, embedded horizontally in optimal cutting temperature compound (O.C.T., Tissue‐Tek), and stored at −20°C until processing. Tissue samples were then mounted in a cryostat and serially cut in 20 µm increments with the first cut containing > 40% of the tissue sample defined as 0 µm to a depth of 500 µm. Frozen sections were placed on microscope slides and stained with nitro‐blue tetrazolium chloride (NBTC) in a dark incubator for 1 h before being dehydrated, fixed, and cover‐slipped.

2.5. Imaging

All brightfield imaging was conducted using a digital slide scanner (NanoZoomer S60; Hamamatsu). Each of the six slides in the four groups contained four serial 20 µm slices per slide, resulting in a total of 24 whole slide color images.

2.6. Quantification

Image analysis was conducted manually by two blinded experts with the use of NDP.view 2 software. Area measurements were chosen over comparison of lesion diameter because of the asymmetry and variability introduced by the enzymatic debridement process. Ten laser treatment zones per sample were selected at random for measuring the external borders of the AZ and the CZ. The area of the AZ was identified by outlining the ablated tissue surrounded by the CZ. The area of the CZ was defined by tracing the interface between thermally denatured and healthy tissue and subtracting the area of the AZ. The region of interest measurement was calculated manually with NDP.view 2 software. If no holes or CZ were present at a given depth, the CZ and AZ were recorded as zero. Each experimental group comprised a single tissue sample containing 196 MTZs. We selected 10 representative treatment zones per sample and conducted continuous analysis at 20 µm intervals from a depth of 0 to 500 µm, yielding a total of 250 microscopic observations per experimental group.

2.7. Statistical Analysis

Statistical analysis was performed in GraphPad Prism Version 9, and for all sample groups, a Shapiro–Wilks test was performed to determine normality, followed by a one‐way ANOVA test and a Tukey's post‐hoc test to determine if the measurements taken from each of the four categorical groups were significantly different from each other.

3. Results

3.1. Comparison of the AZ Between Groups as a Function of Depth

For all four groups, the AZ thickness was normally distributed. Therefore, a one‐way ANOVA test was used to determine whether the AZ area was significantly different between treatment groups. Only for the papain–urea group, the mean AZ was significantly different (Figure 2I). The average AZ area in the control group was 0.0091 ± 0.004 mm² as compared to 0.0089 ± 0.006 mm² for papain‐only, 0.0103 ± 0.022 mm² for papain–urea, and 0.0084 ± 0.004 mm² for urea only. Compared to the control, the mean AZ area increased by 14% in the papain–urea group and decreased by 2% and 7% in the papain‐only and urea‐only group. The papain–urea group has a significant increase in AZ compared to all other groups. The distribution of the AZ area as a function of depth (Figure 2E–H) shows a significant increase of the AZ for the papain–urea group for depths up to 140 µm.

Figure 2.

Analysis of the ablation zone (AZ). Representative histology images at 100 µm depths for (A) control, (B) papain‐only, (C) papain–urea, and (D) urea only treatment groups. (E–H) Scatter plots represent AZ area plotted as a function of depth at 20 µm intervals. (I) A bar graph of mean AZ area by experimental group across all depths. Statistical significance was expressed as *p < 0.05 and **p < 0.001. Comparison of coagulation zone (CZ) area between groups as a function of depth.

For all four groups, the CZ thickness was normally distributed. Therefore, a one‐way ANOVA test was used to determine whether the CZ area was significantly different between treatment groups. The test revealed a significant decrease in CZ area between the control and all three treatment groups (Figure 3E). The mean CZ area in the control group was 0.051 ± 0.024 mm² as compared to 0.044 ± 0.014 mm² for papain‐only, 0.028 ± 0.014 mm² for papain–urea, and 0.045 ± 0.013 mm² for urea only. Compared to control, the mean CZ area decreased by 44% (p < 0.001) in the papain–urea group, 14% (p < 0.001) in the papain‐only group, and 11% (p < 0.001) in the ureal‐only group. For distribution of area as a function of depth, see Figure 3. The distribution of the CZ area as a function of depth (Figure 3A–D) shows a significant decrease of the CZ for the papain–urea group for depths up to 140 µm.

Figure 3.

(A–D) The scatter plots represent coagulation zone (CZ) area plotted as a function of depth at 20 µm intervals. (E) A bar graph of the mean CZ area by the experimental group across all depths is shown. Statistical significance was expressed as *p < 0.05 and **(p < 0.001.

3.2. Comparison of Lesion Geometry Between Groups

The experiment's results reveal distinct variations in lesion geometry across different treatment groups, as depicted in Figure 4. The control group exhibits a lesion geometry consistent with existing literature, characterized by a cone‐shaped AZ that narrows with increasing depth, and a CZ that maintains a relatively uniform thickness, slightly thickening beyond 200 µm depth. The papain‐only and urea‐only treatment groups demonstrate similar ablation and CZs compared to the control group. The most pronounced alteration is observed in the papain–urea treatment group, where the AZ is significantly enlarged, and the CZ is notably diminished at the surface level up to 140 µm in depth. Beyond 140 µm in depth, the AZ narrows significantly while the CZ slightly tapers of in thickness.

Figure 4.

The average ablation and coagulation zone width are presented in a way that represents the lesion geometry for the groups (A) control, (B) papain‐only, (C) papain–urea, and (D) urea only. Next to the lesion geometry are representative images at 0, 100, 200, 300, 400, and 500 µm depth.

4. Discussion

Experimental results of this exploratory study demonstrate the effectiveness of enzymatic post‐processing in ex vivo skin samples. Therefore, post‐processing could be a viable option as a minimally invasive technique for the selective removal or reduction of the CZ and the resulting increase in AZ area. Samples treated with the papain–urea solution exhibited the most pronounced alteration in the CZ area relative to the control. A notable observation was the reduction in the CZ above 140 µm, likely attributed to the partial digestion of the dense denatured tissue ring at the surface. This hypothesis is corroborated by the significantly larger AZ area at the surface in the papain–urea group compared to the control. Below 140 µm, the AZ area was significantly reduced relative to the control, which may be due to the gravitational pull causing partially digested tissue to migrate deeper into the tissue, leading to its accumulation. This phenomenon might have been mitigated if the denatured tissue had been removed from the individual ablative lesions via vacuum rather than by simply rinsing with PBS. Distinguishing partially digested tissue from undigested CZ tissue is challenging, but it appears that the deeper regions of the lesion remained unaffected by the enzymatic treatment. This could be explained by the limited penetration of the enzymatic solution into the ablative lesions due to surface tension. Although skin samples treated with either papain or urea alone showed statistically significant differences in CZ compared to the control, there was no significant change in lesion geometry or AZ, emphasizing that the combination of these two components is necessary to achieve meaningful digestive effects.

This pattern is consistent with our hypothesis that the combined papain–urea group would display the largest decrease in CZ and increase in AZ. Papain, a cysteine protease derived from papaya latex, was chosen as a relatively selective enzyme with a proclivity for denatured tissue and collagen [38]. The enzyme's specificity is due to the absence of the α1‐antitripsine plasmatic antiprotease on injured or denatured tissue, allowing for targeted proteolysis [39]. Papain's wide pH stability and temperature range are well suited to physiologic conditions in clinical practice, making it a suitable candidate for experimentation [34]. The synergistic effects of papain and urea, used together in the debridement of burn and chronic wound beds, is likely due to urea's ability to stabilize the cysteine protease in solution, decreases in enzyme aggregation, and potentially reveal additional cleavage sites [34, 40]. The slight CZ reduction in the papain‐only group supports this hypothesis; however, the statistically significant effect of the urea‐alone group is surprising. This difference from control may stem from its hydrolytic character and ability to alter the tertiary structure of denatured collagen, allowing for more effective mechanical debridement in the posttreatment washing process.

The potential clinical benefits of post‐processing have yet to be demonstrated in vivo; however, several clinical applications seem plausible. Extended periods of posttreatment erythema pose a major burden for patients pursuing elective esthetic procedures, along with multiple inflammation‐associated adverse events including acneiform eruptions, hyperpigmentation, various infectious processes, and hypertrophic scarring [41]. By proactively removing the cellular debris left by aFP, one could expect a decreased inflammatory burden and accelerated transition out of the inflammatory phase. However, it is essential to ensure that partially digested tissue migrating into deeper treatment zones does not exacerbate inflammation. Therefore, removing this tissue, potentially using a vacuum system, may be necessary. Post‐processing may also result in shorter downtime after cosmetic treatment with a possible decreased side‐effect profile [31, 42]. If a reduction in healing time is demonstrated clinically, post‐processing may become a routine adjuvant to esthetic fractional laser procedures.

Another benefit may lie in the modulation of skin‐tightening effects. The extent of skin tightening following CO₂ laser treatment ranges from 20% to 60% from baseline and is believed to correlate directly with the size of the CZ [29, 30, 31, 32, 43]. While skin‐tightening may be invaluable in esthetic procedures, treatment of abnormal fibrotic lesions like hypertrophic, burn, and keloid scars for laser debulking rely on higher energy levels to create MTZs, resulting in larger CZs and unwanted contraction [44, 45]. In locations where excessive skin tightening may distort surrounding structures or limit range of motion, avoiding unwanted contracture may expand the utility of fractional CO₂ laser therapy [43].

Another avenue for exploration is the use of post‐processing on LADD. Several studies suggest that the CZ significantly increases the absorption of small hydrophilic molecules and acts as a reservoir for continuous low‐dose drug delivery, making the CZ an asset in LADD [23, 46, 47]. However, it has been demonstrated that the uptake of lipophilic molecules was significantly limited in MTZs with a substantial CZ [24]. Therefore, post‐processing may enhance laser‐assisted delivery of lipophilic molecules, including glucocorticoids and drugs in liposomal formulations.

Moreover, the denatured collagen barrier that makes up the CZ likely acts as a size‐limiting barrier for the delivery of biologics into the dermis [46, 48, 49, 50]. There is an emerging field of research that has successfully demonstrated the topical delivery of large molecules such as targeted monoclonal antibodies, siRNA, viral vectors, and entire cells through LADD [13, 51, 52]. The introduction of topical immunomodulatory biologics via LADD in combination with enzymatic debridement could transform the treatment of cutaneous inflammatory conditions and allow for local, selective targeting of inflammatory mediators without effecting systemic immune status. Given the investment necessary to purchase several lasers to produce different ablative lesion geometries, post‐processing may be a cost‐effective alternative, enabling clinics to use existing technology for novel applications.

The major limitation of this paper is that it only establishes proof of concept on a tissue sample of a single subject with one application of 10 individual laser lesions per treatment group. As a result, variability between laser applications, which could potentially influence the outcomes, has not been assessed. This experiment did not include any formal attempt at optimization of the papain urea solution concentration, incubation time, consistency of topical solution, or application procedures. The lack of optimization is likely reflected in the variability of effect size seen within samples which may be decreased with better technique. The ex vivo nature of this study also presents inherent limitations, as it lacks critical physiological factors present in vivo that could significantly impact clinical outcomes. In particular, the absence of blood flow which is essential for tissue repair, immune defense, and potentially influencing inflammatory responses post‐CZ removal. Additionally, the clinical benefits of removing the CZ through enzymatic debridement and its implications for transcutaneous drug delivery are all theoretical and have yet to be tested in vivo or in animal models. Multiple dynamic factors including the generation of fibrin plugs, the effects of cellular influx posttreatment, the possibility of pain, and the likelihood of bleeding with disruption of the CZ's barrier function have to be studied [53]. Also, the study's reliance on tissue from a single subject limits our understanding of interindividual variability, as factors like skin type, age, and genetic differences could influence treatment response in a clinical setting. These limitations underscore the need for in vivo studies to better evaluate the clinical feasibility. A potential approach to address these questions could involve a clinical trial on the abdomens of subjects undergoing abdominoplasty, helping to answer questions that cannot be resolved through ex vivo experiments while also mitigating risk. Additionally, the development of papain–urea as a commercial product will likely face challenges. In 2008, the Federal Drug Administration (FDA) ordered companies to stop marketing products containing papain because the enzyme was historically sold without FDA investigation and reports emerged about the possibility of an associated hypersensitivity reaction. However, it is unclear whether papain bears any greater risk of allergic reaction than other approved topical enzyme treatments and it is commonly used in countries outside the United States [54].

5. Conclusion

This exploratory study demonstrated that enzymatic debridement is an effective method for selectively removing the CZ generated by aFP. Post‐processing is a noninvasive, relatively simple method for removing the nonviable tissue left behind after laser skin treatment in ex vivo skin. Removal of the CZ may have functional implications on posttreatment inflammation, long‐term skin remodeling, and could expand the uses of CO₂ fractional laser treatment. While this study establishes the efficacy of papain–urea treatment, further studies are necessary to evaluate the effects of post‐processing on transcutaneous drug delivery, inflammation, and esthetic outcomes.

Conflicts of Interest

The authors declare no conflicts of interest.