Human dermal fibroblast‐derived extracellular matrix reduces postinflammatory hyperpigmentation after fractional carbon dioxide laser facial resurfacing in Asians

Extracellular matrix (ECM) components promote the development of skin wounds by providing biological scaffolds and regenerative microenvironments.


| INTRODUC TI ON
Extracellular matrix (ECM) components, such as collagens, fibronectin, glycosaminoglycans, or proteoglycans, play a significant role in wound healing by providing structural scaffolds. 1 Fibronectin constitutes a temporary matrix in the wound bed immediately after hemostasis and stimulates the migration and adhesion of fibroblasts, keratinocytes, and endothelial cells via integrin receptors. 1 ECM components also regulate signal transduction during wound healing by interacting with neutrophils, monocytes/macrophages, fibroblasts, endothelial cells, and keratinocytes, and by modulating cytokines and growth factors. 1 For example, decorin and collagen VI activate autophagy, which negatively regulates the inflammatory response in the wound healing process to prevent excessive inflammation from tissue damage. 2 Several ECM products derived from neonatal foreskin (Apligraf®), human amnion/chorion membrane (Epifix®), animal (Integra®), or cadavers (AlloDerm®) are commercially available for promoting skin wounds or diabetic foot ulcers by providing biological scaffolds and regenerative microenvironment. The application of ECM sheets derived from human adipose tissue resulted in an enhanced reepithelialization rate, more organized granulation tissue, and higher microvasculature density compared to the control in a fullthickness skin defect rat model. 3 In this study, we aimed to evaluate the beneficial effect of human dermal fibroblast-derived ECM after fractional carbon dioxide laser resurfacing. We measured several biophysical skin parameters to investigate the anti-melanogenic, anti-inflammatory, and rejuvenating effects of the ECM after fractional carbon dioxide laser treatment.

| Treatment
The right and left sides of the face were randomized to the treatment allocation in a 1:1 ratio. One unblinded investigator created a randomization list using a random number table and kept in a restricted place until the study data were unblinded for statistical analysis. The unblinded investigator allocated two types of serum accordingly to the participants, instructing the application of appropriate serum on the right and left sides of the face.
Prior to the procedure, the treatment areas were gently cleaned with a mild cleanser. After applying topical anesthetic cream (EMLA cream, 2.5% lidocaine, and 2. Two types of serum, one containing the ECM components (5 mg/ mL) and placebo, which had an identical appearance except for the label indicating the right or left side, were provided to the participants for posttreatment care. The participants were instructed to apply the serum to the allocated side 2-4 times daily for 2 weeks. The sera were composed of the same ingredients, except one serum contained ECM. The other ingredients used were water, 2,3-butanediol, dipropylene glycol, 1,2-hexanediol, glycerine, butylene glycol, hydrolyzed hyaluronic acid, sodium hyaluronate copolymer, sodium hyaluronate, sodium acetylated hyaluronate, hydrolyzed sodium hyaluronate, pentylene glycol, madecassoside, tromethamine, allantoin, betaine, panthenol, polyglyceryl-10 laurate, carbomer, xanthan gum, glyceryl acrylate/acrylic acid copolymer, PVM/MA copolymer, propanediol, ethylhexylglycerine, and disodium EDTA. The participants were advised to avoid sun exposure and use broad-spectrum sunscreens during the study period.

| Assessments
All assessments of skin parameters and overall improvement were completed by blinded investigators. In vivo skin parameters were measured at baseline and after four and 12 weeks of treatment. The participants were required to clean their faces with a mild cleanser and wait in the ambient environment for at least 15 min before measurement. Antera 3D® (Miravex, Dublin, Ireland) was used to determine pigmentation (average melanin [M av ]), erythema (average erythema [E av ]), texture (Ra, Rq, and texture score), and pore size (total pore volume, mean pore volume, total pore area, and mean pore area) on both cheeks. Wrinkle parameters (length, average width, average depth, maximum depth, and volume) were measured on marionette's lines and nasolabial folds using the Antera 3D®.

| Statistical analysis
All data were analyzed using the SPSS Statistics software (version 20.0; IBM Corp., Armonk, NY, IL). The differences in the measurements at baseline, week 4, and week 12 in each group were analyzed using a paired t-test. Student's t-test was used to determine statistical differences in the measurements between the ECM and control groups. A p value of less than 0.05 was set to indicate statistical significance. All results are presented as mean ± standard deviation Appendix.

| RE SULTS
Of the 15 participants enrolled in the study, 14 participants completed the study, except for one who withdrew before week 4 due to personal reasons ( Figure SS1). The mean age was 45.1 ± 9.7 years (ranging from 28 to 57 years). The male to female ratio was 7:7.

| Melanin and erythema
In the ECM group, M av at week 12 was lower than that at baseline (40.31 ± 6.00 vs. 42.12 ± 6.07, p = 0.013), while that of the control group was higher than that at baseline (44.14 ± 6.01 vs. 41.65 ± 6.33,  In comparison with the control group, the dermal density of the ECM group was not significantly different at weeks 4 and 12.

| Texture
The parameters for skin texture (texture score, Ra, and Rq) were improved in the ECM group at week 12 compared with baseline (texture score; 18.36 ± 11.94 vs. 24.79 ± 12.83, p = 0.017) but that of the control group showed no significant change at week 12 (texture score; 20.21 ± 12.89 vs. 24.07 ± 17.21, p = 0.088) ( Figure 2B). There

| Transepidermal water loss
An improvement in TEWL was reported in the ECM group at week 12 compared to baseline (8.36 ± 4.23 vs. 10.67 ± 3.40, p = 0.021) ( Figure 2C). However, no statistically significant difference in TEWL was observed between the two groups.

| Elasticity and pore
In the ECM group, the elasticity R2 parameter increased at 0.681 ± 0.104 at week 12). There was no significant difference in elasticity between the two groups at weeks 4 and 12. For pore parameters, neither group showed any significant improvement after treatment.  (Figure 2(g,i)). However, statistically significant differences in the parameters of the marionette lines or nasolabial folds were not found between the two groups at weeks 4 and 12.

| Investigator's global assessment and patient's satisfaction score
The mean IGA score was greater in the ECM group than in the control

| Adverse events
None of the participants complained of treatment-related adverse events such as bleeding, infection, pruritus, paresthesia, allergic reaction, acne, or reactivation of herpes simplex virus throughout the study period.

| DISCUSS ION
In this double-blind, randomized, vehicle-controlled, split-face study, a marked reduction in melanin after 12 weeks of fractional carbon dioxide laser resurfacing treatment was demonstrated in the treatment side of the face where human dermal fibroblast-derived ECM was applied as post-laser care, while the control side showed a significant increase in melanin, namely, postinflammatory hyperpigmentation (PIH). PIH, defined as a light-to-dark brown-colored hyperpigmented macule or patch that is left behind when erythema subsides, is one of the most common adverse events after laser procedures, especially in Asians with Fitzpatrick skin phototypes III-IV. Previous studies have reported varying incidences of PIH after laser treatment from 7.1% to 92%, depending on the type of laser devices. [4][5][6] Although the pathogenesis of PIH has not yet been clearly demonstrated, inflammation during the post-laser wound healing process might stimulate melanin production. Cutaneous inflammation causes the generation of eicosanoids, cytokines, and inflammatory mediators such as interleukin (IL)-1α and 6, tumor necrosis factorα, endothelin-1, and stem cell factor, which results in increased activity, hyperplasia, and hypertrophy of melanocytes. 7 We found a clue for the anti-melanogenic property of the ECM due to the change in erythema after treatment. A previous in vivo model of PIH demonstrated an inflammatory phase, which initiates soon after skin damage and lasts for 8-12 weeks, followed by melanogenesis, which is accelerated after 3 weeks and reaches a peak after 8 weeks. 8 We also found an elevation of erythema in the control group after 4 weeks of treatment. However, the ECM group showed no significant change in erythema after 12 weeks, and the change in erythema level from baseline was greater in the control group than in the ECM group after 4 weeks of treatment ( Figure 1C).
Thus, ECM might have anti-inflammatory properties that inhibit the production of pro-melanogenic signals and prevent the occurrence of PIH after laser treatment.
A component analysis of the ECM product revealed a high proportion of proteoglycans, collagens, glycosaminoglycans, laminins, and growth factors (tumor growth factorβ and platelet-derived growth factor) ( Figure SS2). During the proliferative phase of the wound healing process, a temporary matrix in the wound bed is replaced by collagen (mainly types I and III), elastin, proteoglycans, glycosaminoglycans, and noncollagenous proteins that are synthesized by fibroblasts to form granulation tissue. 1 These ECM components might suppress inflammatory responses to prevent excessive inflammation causing tissue damage. Decorin and collagen VI activates autophagic signaling pathways to negatively regulate inflammatory response in the wound healing process. 2,9 Biglycan, a small leucine-rich proteoglycan, triggers an anti-inflammatory response via toll-like receptor 4/CD44-mediated proautophagic activity in macrophages. 10 Autophagy induction also promotes polarization of macrophages toward the M2 phenotype to alleviate inflammatory response and promote tissue repair. 11 Chondroitin sulfate has an anti-inflammatory activity by limiting NF-κB signaling and inhibiting IL-1β-induced expression of pro-inflammatory genes, such as IL-6, nitric oxide synthase 2, and prostaglandin E2 synthase. 12 Heparin/ heparan sulfate inhibit the lectin pathway via inhibiting the activity of serine protease. 13 Multiple treatment sessions are required for the fractional laser to result in clinical improvement in elasticity, texture, or wrinkles. 14 Similarly, we did not find significant improvement in the parameters for skin barrier restoration and rejuvenation, such as TEWL, elasticity, dermal density, texture, pore, and various parameters of nasolabial folds, compared with the control. Though F I G U R E 3 Clinical photographs before laser treatment, and after 12 weeks of treatment with extracellular matrix component serum application on the treatment side for 2 weeks. several parameters of the enlisted above were demonstrated to improve only in the ECM group, the rejuvenating effects of the ECM were not clearly demonstrated in this study. The small number of participants enrolled in the study might have contributed to the lack of statistical significance in skin parameters between the two groups. In addition, treatment with fractional carbon dioxide laser creates numerous microthermal zones that are replaced with newly synthesized collagen but leaves the unaffected skin between the microthermal zones. Contrary to the full-thickness skin defect tested in the study by Lee et al., 3 only a small portion of the damaged skin is created after the fractional carbon dioxide laser, which might result in a minimal chance for the ECM to promote wound healing rate and neocollagenesis. Multiple treatment sessions or a higher density of microthermal zones could demonstrate the role of ECM in promoting neocollagenesis more clearly than in our study. 15,16 Dermal density indicates the intensity of reflected echoes of ultrasonic waves generated between adjacent structures. 17 The current study indicates that dermal density is a sensitive parameter for skin rejuvenation, as it significantly increased after a single session of fractional laser treatment. While there was no significant difference in the increase in dermal density between the ECM and control groups, subgroup analysis of 11 participants aged ≤55 years revealed a greater increase in dermal density in the ECM group than in the control group (data not shown). Aging is a major risk factor for wound healing, affecting inflammatory response, reepithelialization, collagen synthesis, and angiogenesis. 18 The limitations of the present study were the small sample size and lack of immunohistochemical analysis to demonstrate the biochemical events of wound healing. Measuring the levels of inflammatory cells or cytokines could validate the anti-inflammatory effects of the ECM. Although autophagic induction is considered to play an important role in the anti-inflammatory activity of ECM, a detailed demonstration of its mechanism was not indicated in our study.
In conclusion, our study is the first to demonstrate the role of human dermal fibroblast-derived ECM in preventing PIH after fractional carbon dioxide laser resurfacing in Asians. We suggest that post-laser care with human dermal fibroblast-derived ECM may be used as adjunctive therapy after fractional carbon dioxide resurfacing to minimize adverse events. Further studies are needed to reveal the underlying mechanism of the anti-inflammatory action of the ECM in wound healing.

AUTH O R CO NTR I B UTI O N
Kwang-Sik Kook and Jun Park made substantial contributions to conception and design and acquisition of data. Bark-Lynn Lew made significant contributions to conception and study design and were involved in revising the manuscript critically for important intellectual content. Sang-Min Choi and Soon-Hyo Kwon made substantial contributions to conception and design, acquisition of data, and analysis and interpretation of data. Sang-Min Choi and Soon-Hyo Kwon involved in drafting the manuscript and revising it critically for important intellectual content.

ACK N OWLED G M ENTS
None.

FU N D I N G I N FO R M ATI O N
This study was supported by Amorepacific Research Funding, funded by Skin Science Research Foundation (2021).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.