Impact of defensins‐containing body cream on skin composition

Abstract

Background and Aims

Defensins are peptides capable of reactivating latent LGR6 stem cells in the basal layer. When applied topically, these peptides can reduce signs of skin aging and increase dermal thickness. This study investigates the effects of a topical defensin formulation on extremity skin composition.

Methods

An open label, single arm clinical trial was conducted on participants with dry, photoaged, or dull skin. A defensin‐containing hand and body cream was applied twice daily for 6 weeks to the hands, forearms, elbows, and knees. Photographs and objective measurements of skin hydration, viscoelasticity (VE), retraction time (RT), thickness, density/transepidermal water loss (TEWL), as well as self‐evaluation of skin quality and characteristics were obtained pre‐ and post‐ intervention.

Results

After the study period, RT decreased by 56% across all body sites (p < 0.001) and VE improved at the elbow (125%, p = 0.009) and knee (110%, p < 0.001). Skin density also increased in all 4 body sites (40%, p < 0.001), while skin thickness increased at the elbow (29%, p = 0.03) and knee (17%, p = 0.04). Skin hydration increased at the elbow, knee, and forearm by 99%, 28%, and 16%, respectively (p < 0.05), while TEWL improved at the elbow only (−39%, p = 0.02). Patients' self‐evaluations showed improvements in overall skin quality and in the domains of dryness, ashiness, wrinkling, pigmentation, redness, roughness, and discomfort (p < 0.05).

Conclusions

Following 6‐week use of a defensin‐containing cream, subjects reported significant improvement across many subjective skin domains. Similarly, objective measurements demonstrated significant improvement in skin architecture at select sites.

1. INTRODUCTION

Skin is a vital element of appearance and is subject to the process of aging characterized by exogenous (i.e., ultraviolet radiation [UV], ionizing radiation, pollution, or toxins) and endogenous insults. Of these exogeneous sources, UV radiation plays a prominent role in aging by triggering formation of reactive oxygen species, ultimately leading to the dysregulation of collagen synthesis. ¹ , ² Endogenous aging is dictated by individual genetics and metabolism, with telomeres thought to play an important role in cellular aging. ³ The combined effects of extrinsic and intrinsic processes present histopathologically as reduced epidermal thickness, a flattened dermal‐epidermal junction, weakened ability of basal cell proliferation, increased type III collagen to type I collagen ratio, and hyperplasia of the sebaceous glands. Phenotypically, these changes manifest as dryness, wrinkling, decreased elasticity, and telangiectasia and purpura formation. ⁴ , ⁵ , ⁶

Topical cosmeceuticals are designed to combat aging skin and are integral to the rejuvenation industry. More recently, topicals targeting LGR6+ stem cells in the basal epidermal layer have been of interest. These stem cells are located at the hair follicle bulge and have the potential to enhance gene pathways involved in wound healing, follicle development, epithelization, and angiogenesis. ⁷ , ⁸ In the absence of wounds, LGR6+ stem cells lay largely dormant; however, it is now known that their regenerative capacity is enhanced when exposed to alpha‐ and beta‐defensins. Defensins are cationic peptides characterized by six cysteine residues forming three intramolecular disulfide bridges. ⁹ In a murine model, application of alpha‐defensin‐5 to a third degree burn increased migration of LGR stem cells into the wound bed and augmented wound healing transcripts. ¹⁰

Skin cosmeceutical products have undergone an evolution since their inception over a decade ago. Initially consisting of plant‐based nonspecific growth factors, these products demonstrated concerns regarding side effects and downstream risks related to malignancy. Similarly, targeted growth factor cosmeceuticals and retinols demonstrate good clinical response, but stimulate terminally differentiated epidermal cells, leading to questions regarding mutation, senescence, and propensity for malignancy. With their ability to target stem cells which do not divide throughout life, defensins have translated to the cosmeceutical industry and demonstrated promising results. In a multi‐center randomized control trial, a three‐product skin regimen containing alpha‐ and beta‐defensins reduced visible pores, superficial wrinkles, oiliness, and pigmentation of the face and neck. ¹¹ Moreover, a defensin‐containing eye cream was found to improve hydration, elasticity, and skin thickness by 6%, 120%, and 28%, respectively. ¹²

While facial rejuvenation has historically garnered more attention, there is an impetus to seek a more globally youthful appearance by addressing the skin of the extremities and chest. The effect of defensins on non‐head and neck skin has not been previously investigated and its broader utility in skin rejuvenation would contribute to our understanding of the pathophysiology of skin aging. The current study aims to explore the ability of topical alpha‐ and beta‐defensins to improve the appearance and composition of aging skin of the limbs.

2. MATERIALS AND METHODS

This prospective, open label, longitudinal study was approved by Cottage Health Institutional Review Board (Santa Barbara, CA, IRB #21‐85MRX). Patients who presented with complaints of dry, photoaged, or dull skin on their arms/legs/hands were evaluated for candidacy for enrollment. Patients who were greater than 18 years of age, without any underlying interfering dermatologic or systemic disorders, and with no prior surgery on forearms/knees/hands were included. Patients meeting the following criteria were excluded: current use of topical or systemic steroids, history of systemic lupus erythematosus, rheumatoid arthritis, HIV, atopic dermatitis/eczema, recently treated skin cancer within the last 12 months, insulin‐controlled diabetes, breastfeeding, or pregnant/planning to become pregnant. Patients were able to withdraw from this study at any time.

Eligible and interested patients were enrolled and informed consent was obtained. Patients were instructed to discontinue the use of any other topical products for the duration of the study. Skincare products, DefenAge 10 Luxe Hand and Body Cream and a trial size 2‐Minute Reveal Masque (DefenAge, Progenitor Biologics LLC®, a division of MediCell Technologies) were provided to all patients free of charge. A full list of ingredients in these products can be found in Table S1. Patients completing the study received a full‐size body cream and skin care regimen for their participation.

Patients were instructed to begin the study by using the 2‐Minute Reveal Masque on their bilateral hands, forearms, elbows, and knees. Immediately after, they were instructed to begin using the body cream by applying one pump each per arm/elbow, hands, and knees in the morning, and one pump per location in the evening. Compliance was self reported by the participants and tracked with the assistance of a log. The body cream was used over a 6‐week period.

2.1. Outcome measures

2.1.1. Patient reported outcomes

Outcome measures were obtained at the pretreatment and posttreatment visit by asking participants to complete a 10‐item questionnaire on their overall satisfaction with their skin characteristics. The participants rated their overall skin quality and specific characteristics: roughness, dryness, discomfort, ashiness, wrinkling, redness, age spots, and pigmentation at both the pre‐ and post‐treatment visits. Scores were reported on a 7‐point scale for each skin condition with 0 = very poor, 4 = neutral, and 7 = outstanding. An additional questionnaire was administered at the 6‐week visit to determine additional information surrounding participant's experience and overall impressions of the body cream.

2.1.2. Photography

Photographs of the dorsal surface of the hands, extensor surface of the forearm and elbow, and ventral surface of the knee were taken at the pre and post‐intervention visits. A ruler was used to ensure photos were taken at the same location.

2.1.3. Skin structure

The DermaLab Combo® (Cortex Technology) was used to obtain objective skin structural and physiologic measurements at each body location using a variety of probes. ¹³ Data collected included transepidermal water loss (TEWL), elasticity, hydration, skin thickness, and density. Complete measurements of skin structure can be found in Table S1. Hydration was assessed using the hydration probe, which provides an assessment of the stratum corneum's water binding capacity through a nonpenetrating pin‐probe design and is expressed in microSiemens (μS) per centimeter. Skin elasticity was measured using the elasticity probe, which uses a suction chamber to measure skin elevation and subsequent retraction. The retraction time is the time in milliseconds (ms) for skin to retract 1.5 mm from full elevation. The viscoelasticity (mBar) is a calculation that combines both elevation and retraction phases. Skin thickness and density were obtained using the high‐frequency ultrasound probe. Skin thickness represents the width of the dermis in micrometers (μm). Skin density was calculated by the intensity of collagen signals in the dermis and was presented as an arbitrary intensity score (0–100). ¹³ , ¹⁴ Measurements were taken at the same location at each visit. TEWL, a widely used standardized measure of the skin barrier function, was obtained using the TEWL probe which uses relative humidity and temperature sensors to determine the vapor pressure gradient at the skin surface, with results reported in grams per square meter per hour (g/m²/h).

2.2. Statistics

Objective skin measurements and participant's self‐reported skin characteristic scores before and after the 6‐week study period were analyzed using paired one tailed t‐tests for comparison, where the participant's pre‐treatment values served as the comparison. The decision to use a one‐tailed t‐test was made a priori when defining our hypothesis for each skin parameter: that there would be a statistically significant improvement in objective and subjective skin characteristics after 6‐weeks of cream use. A p‐value of <0.05 was considered statistically significant. To address errors in DermaLab measurements, a conservative approach was used and outlier values below the 1st percentile and above the 99th percentile were excluded. All statistical analyses were performed using the STATA 15.1 software.

3. RESULTS

A total of 22 participants were enrolled in the study. Two participants experienced mild skin sensitivity and discontinued use of the cream. An additional six participants completed the 6‐week use of the cream but were unable to attend the post‐treatment visit. None of the participants lost to follow‐up reported any adverse reactions to the cream. Therefore, follow‐up data were available for 14 participants. Participant characteristics are shown in Table 1. All recruited participants were female with an average age of 60 years (range 44–75). The majority of participants were Caucasian, with 21.4% of Hispanic ethnicity. The most common skin phototype in this study was Fitzpatrick 2 (57%) followed by Fitzpatrick 3 (21%). Participant‐reported compliance was 100% for daily application of the cream, and 92.3% for twice daily application of the cream at least 50% of the time.

TABLE 1.

Participant's demographics, Fitzpatrick phototype, and compliance

ID	Ethnicity/Race	Age (Years)	Fitzpatrick score	Compliance (1× Day)	Compliance (2× Day)
1	Caucasian	68	2	100%	100%
2	Caucasian	59	2	100%	100%
3	Caucasian	68	2	100%	50%
4	Caucasian	75	3	100%	100%
5	Hispanic	47	3	100%	100%
6	Caucasian	45	2	100%	25%
7	Caucasian	44	3	100%	100%
8	Caucasian	73	3	100%	100%
9	Caucasian	65	2	100%	100%
10	Caucasian	68	2	100%	50%
11	Caucasian	45	2	100%	100%
11	Caucasian	62	2	100%	100%
13	Hispanic	56	3	100%	75%
14	Hispanic	59	4	100%	50%

3.1. Objective skin measurements

Photographs of all 4 body sites tested before and after use of the body cream are shown in Figure 1. Mean changes from baseline were calculated for all DermaLab Combo® noninvasive measurements: skin viscoelasticity, retraction time, hydration, TEWL, skin thickness, and density. For all body sites combined, skin viscoelasticity and retraction time improved significantly, by 50% (0.70 mBar, p < 0.001) and 56% (−275 ms, p < 0.001), respectively. The elbow and above knee areas demonstrated the largest improvement in viscoelasticity and retraction time. The elbow's viscoelasticity improved by 125% (0.81 mBar, p = 0.009) and the retraction time by 69% (−684 m, p = 0.003), whereas the above knee region's viscoelasticity improved by 110% (1.61 mBar, p < 0.001) and retraction time by 64% (−254 ms, p = 0.003). This information can be found in Figure 2A,B.

FIGURE 1.

Before and after 6‐week study period photographs of all four body sites. (A, B) Pre‐ and post‐hand of participant #11. (C, D) Pre‐ and post‐forearm of participant #4. (E, F) Pre‐ and post‐elbow of participant #5. (G, H) Pre‐ and post‐knee of participant #7

FIGURE 2.

Objective skin measurements before and after 6‐week study period. (A) Mean change in skin retraction time. (B) Mean change in skin viscoelasticity. (C) Mean change in skin hydration. (D) Mean change in TEWL. (E) Mean change in skin thickness. (F) Mean change in skin density. ***p < 0.05

The body cream also significantly improved participants' skin hydration in the elbow, forearm, and above knee areas (Figure 2C). There was a 31% increase in skin hydration among all sites, with the elbow (99%, 53.5 μS, p < 0.001) and above knee regions (28%, 22.4 μS, p = 0.04) showing the greatest improvement. TEWL showed a significant 39% improvement in the elbow (−4.1 g/m²/h, p = 0.02), and a trend toward improvement in the hand (19%, −3.2 g/m²/h, p = 0.2). (Figure 2D).

Participants' dermal thickness and collagen density also demonstrated significant improvements after 6 weeks of body cream use (Figure 2E,F). For all body sites, dermal thickness increased by 16% (198 μm, p = 0.002) and collagen density by 40% (9.5 points, p < 0.001). Again, the elbow and above knee sites experienced the greatest improvement in skin thickness, by 29% (334 μm, p = 0.03) and 17% (227 μm, p = 0.04), respectively. Skin density showed a significant improvement across all body sites tested, with the greatest change measured in the hand (47%, 11.7 points, p < 0.001) followed by the above knee area (45%, 10.1 points, p < 0.001).

3.2. Patient reported outcomes

Concerning participant's self‐evaluation of skin characteristics using a 1–7 scale, there was a significant average improvement of 2.1 points (p < 0.001) in the overall skin quality when comparing the pre‐ and post‐consumer questionnaire (Figure 3). The patient's self‐reported scores also show a significant mean improvement in skin roughness (+1.6 points), dryness (+2.6 points), discomfort (+1.6 points), ashiness (+2.2 points), wrinkling (+2.0 points), redness (+1.4 points), age spots (+1.6 points), and pigmentation (+1.8 points). There was no significant improvement in skin sensitivity (mean change 0.1, p = 0.4).

FIGURE 3.

Mean change in participant self‐evaluation of skin characteristics before and after 6‐week study period. Participants assigned a score with a scale of 1–7 for each skin domain, where 0 = very poor, 4 = neutral, and 7 = outstanding. ***p < 0.05

Overall, participants were highly satisfied with the body cream, as demonstrated by the post‐study questionnaire in Figure 4, with 86% stating they would recommend the cream to a friend. More than 80% of participants agreed that the body cream improved their skin firmness, clarity, dryness, texture, softness, and dullness. More than 60% of participants stated the cream helped smooth lines, diminish wrinkles, even skin tone, and correct surface imperfections.

FIGURE 4.

Results of participant questionnaire after 6‐week study period

4. DISCUSSION

Since the time of Alexander the Great, kings, physicians, and others have searched for the physical or metaphorical fountain of youth. Though tangible strides have been made in both aesthetic and holistic medicine, a solution to aging still evades modern practitioners. With regard to anti‐aging approaches to the skin, contemporary methods include the application of topical growth factors, retinols, and chemical/energy insult to induce new skin growth. Each of these approaches carries the risk of cellular senescence, uncontrolled replication, or inflammatory sequelae.

Cell shedding, proliferation, and differentiation are tightly controlled within the dermis and epidermis of the skin. Found near the follicular bulge of hair follicles, LGR6+ multipotent stem cells have been called the “master stem cell of the skin” due to their ability to differentiate into all epidermal cell types. A focus of research in wound healing has been to identify mechanisms by which fetuses heal wounds without scarring, and it has been found that in‐utero epidermal growth is controlled by LGR6+ stem cell replication and differentiation. ¹⁵ Similarly, mouse models have demonstrated accelerated wound healing in the presence of activated LGR6+ stem cells. ⁸ Stimulation of LGR6+ stem cells occurs via alpha‐ and beta‐defensin signaling peptides, which also exert pro‐immunity, antibacterial, and antitumor effects. ⁷ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹

Previous authors have investigated commercially available cosmeceuticals containing alpha‐ and beta‐defensin molecules. A previous multi‐center, double‐blind, vehicle‐controlled trial by our group, which was restricted to the face, objectively demonstrated decreased appearance of pores, increased epidermal thickness, reduced melanin, and reduction in wrinkles after 6 weeks of use. ¹¹ Importantly, these effects were observed in the absence of an inflammatory response. Similarly, a defensin‐containing eye cream subsequently showed subjective improvement in various common skin concerns but was underpowered to demonstrate objective changes in skin architecture. ¹²

The current study represents the first to evaluate objective effects of a defensin‐containing cosmeceutical product at multiple sites across the body. Following 6 weeks of twice‐daily application, patients demonstrated improved skin elasticity, with decreased retraction time across all sites and increased viscoelasticity in the knee and elbow. They were also found to have increased dermal thickness at the knee and elbow, increased skin density across all sites, increased hydration at the knee, elbow, and forearm, and decreased trans‐epidermal water loss at the elbow. These findings represent a significant improvement in objective skin architecture and physiology following 6 weeks of topical therapy. Of interest to our group was the mildly discrepant findings across body sites. In particular, the back of the hand did not respond as robustly to our intervention as did other sites. It is possible that this was due to overuse of the hands and repeat exposure throughout the day to soap and water that may remove a portion of the applied cream and influence results.

With regard to patient‐reported outcomes, global measurements demonstrated a high patient satisfaction, with 86% of patients responding that they would recommend the product to a friend. Across all measured domains, the majority of patients saw improvement following 6 weeks of use. The domains which showed the most patient‐reported improvement included skin quality, dryness, ashiness, and wrinkling. The domain with the smallest improvement was that of decreasing skin sensitivity, which is consistent with previous studies of defensin‐containing products. ¹¹ , ¹²

The current study has multiple limitations which warrant discussion. As subjects were used as their own controls, no formal control arm was built into the study protocol. As a result, subjects and practitioners were not blinded to the intervention. In addition, due to concerns of COVID‐19 exposure, a relatively small cohort was utilized, with a preponderance of Caucasian and Latin American females.

5. CONCLUSION

Following a 6‐week topical application of a defensin‐containing cosmeceutical body cream, subjects reported significant improvement across all subjective skin domains. Similarly, objective measurements demonstrated significant improvement in skin architecture at select sites, including improvement in elasticity, hydration, and skin thickness. Additional randomized and controlled studies are warranted to further elucidate the effect of defensins at multiple skin sites across the body.

CONFLICT OF INTEREST

None.

ETHICAL APPROVAL

We verify that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. Our study was approved by the Institutional Review Board of Santa Barbara.

Supporting information

Appendix S1

Eggerstedt M, Torres‐Maldonado S, Danielian A, Hwang SHJ, Echanique KA. Impact of defensins‐containing body cream on skin composition. J Cosmet Dermatol. 2023;22:620‐627. doi: 10.1111/jocd.15118

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.