2.1 Clinical study

A total of 22 female subjects of approximately 52.2 years of age that satisfied the purposes of the study were recruited in the 4-week trial. The study comparatively evaluated the cosmetic effects of SCAI-HA and HMWHA by monitoring the following dermatological parameters: skin hydration, transepidermal water loss (TEWL), skin elasticity, eye wrinkles (crow's feet), facial sagging, and skin density. The subjects were selected based on (i) age, (ii) appearance of eye wrinkles (crow's feet), and (iii) lack of any acute or chronic conditions including skin disorders. The following factors were considered in subject exclusion: pregnancy, psychological disorders, administration of immunosuppressants within 3 months, administration of systemic steroid or radiation therapy within 1 month, presence of facial lesions that may hinder measurements, atopic dermatitis, extreme skin sensitivity or allergies to cosmetics, pharmaceutics, and light, chemical peel, or skin treatment within 3 months. Randomized clinical studies of SCAI-HA and HMWHA was conducted with IRB approval (P2108-2373) in accordance with the Code of Ethics based on the Helsinki Declaration, applicable human testing guidelines, and relevant regulations. The clinical cosmetic efficacy studies were conducted between August 2021 and October 2021 at PNK Skin Research Center in Seoul, South Korea. All recruited subjects have provided written informed consent. SCAI-HA (experimental) and HMWHA (control) products for clinical studies were provided as clear viscous solutions with the following compositions: SCAI-HA–0.5% SCAI-HA, 2.0% 1,2-hexanediol, 97.5% water; HMWHA–0.5% sodium hyaluronate, 2.0% 1,2-hexanediol, 97.5% water. The efficacy assessments were performed as a randomized double-blind study to compare the SCAI-HA- and HMWHA-treated facial areas. A random number table was generated to indiscriminately assign the experimental and control groups on the left and right sections of the face of each subject. The subjects were trained to clean and dry their hands before the application of equivalent amounts of each solution (single pump). The products, stored at room temperature in the dark, were administered twice a day for 4 weeks. Throughout the study, participants were instructed to avoid the use of cosmetic products containing any active ingredients (e.g., whitening, anti-wrinkle, drying) that could influence the measurement of dermatological parameters. Subjects made 3 visits for clinical evaluation: before product application, after 2 weeks of product application, and 4 weeks of product application.

2.2 Instrumental analysis

To ensure the accurate assessment of the dermatological parameters, all subjects were accommodated in a controlled environment at 20–24 °C and 40–60% humidity for 30 min before instrumental analysis. Repeated measurements were taken in the same area of the face each time. All measurements and experiments were performed in triplicate and presented as their average unless noted otherwise.

2.3 Skin hydration

Hydration levels of the skin were measured using the Corneometer CM825 (Courage Khazaka electronic GmbH, Germany) on each cheek of the subjects. The corneometer assesses the level of skin hydration as a measure of the change in electric permittivity. The skin hydration levels of each side of the subjects' face were measured at 0, 2, and 4 weeks of product application. Measurements were performed in triplicate and the average was used for analysis and interpretation.

2.4 Transepidermal water loss (TEWL)

The transepidermal water loss of each subject was measured on both cheeks using a Vapometer (Delfin Technologies Ltd, Finland). TEWL was presented by a numerical value signifying the lost mass of water (g) in a specific area (m²) per hour (h) in the form of the following unit: g/m²h. The TEWL of each side of the subjects' face was measured after 0, 2, and 4 weeks of product application.

2.5 Skin elasticity

The skin elasticity of the subjects was measured using the Cutometer MPA580 (Courage Khazaka electronic GmbH, Germany) on each cheek. Cutometer applies a constant negative pressure of 450 mbar with 2 s of on-time and 2 s of off-time to monitor the corresponding changes of the skin tissue. R2 was used as a measure of the skin's ability to resist deformation and return to its original form as a parameter representing its elasticity. The skin elasticity of each side of the subjects' face was measured after 0, 2, and 4 weeks of product application.

2.6 Wrinkles (crow's feet)

Wrinkles in the crow's feet region of the subjects were evaluated using PRIMOS CR Small Field (Canfield Imaging Systems, USA) by taking images of the region of interest. These images are then analyzed to determine the Ra value as a measure of average roughness in μm. The eye wrinkles of each side of the subjects' face were assessed after 0, 2, and 4 weeks of product application.

2.7 Facial sagging

Facial sagging of the clinical subjects was evaluated by topographically imaging the sides of their face. Topographs were taken with F-ray (BEYOUNG Co., Ltd.) on each cheek of the subjects. The resultant images were then analyzed via image pro® plus (Media Cybernetics, Rockville) to determine the angles (°) representing the degree of facial sagging in each patient. Facial sagging of each side of the subjects' face was measured after 0, 2, and 4 weeks of product application.

2.8 Skin density

Skin Scanner DUB USB (TPM taberna pro medicum, Germany) was used to evaluate the skin density of subjects using dermal ultrasonic imaging. Colored two-dimensional images of the ultrasound intensity were generated at different amplitudes. These images were then analyzed to calculate the dermal density of skin tissue. Measurements were performed in areas surrounding the eyes on each side. Skin density was presented as Density (%). The skin density of each side of the subjects' face was measured after 0, 2, and 4 weeks of product application.

2.9 In vitro study

For the in vitro experiments, the products were diluted in the appropriate media to final [HA] concentrations of 0.003, 0.006, and 0.012% (w/v). The normal human dermal fibroblasts (NHDF) cell line was maintained in FBM media containing 0.1% insulin, 0.1% rhFGF B, 0.1% GA 1000, and 2% FBS. The NHDF cells were grown and maintained at 37°C in a humidified atmosphere with 5% CO₂. It should be noted that the in vitro HA samples did not contain 1,2-hexanediol. Before evaluating their effect on cellular collagen synthesis, the cytotoxicity of the HA products was determined against NHDF cells.

2.10 Cellular collagen synthesis

NHDF cells were seeded onto a 24-well plate (20 000 cells per well) and incubated for 24 h. Thereafter, the cells were incubated in serum-free FBM containing the diluted HA products [i.e., SCAI-HA and HMWHA; 0.003, 0.006, 0.012% (w/v)] for 24 h. The supernatant was obtained from each well and its procollagen content was quantified using a procollagen type I C-peptide (PIP) EIA kit. The microplate reader was used to measure the absorbance at 450 nm. L-ascorbic acid (0.001%) was used as a positive control.

3.1 Skin hydration

Acknowledged for its ability to pertain moisture, the effects of HA in both forms, SCAI-HA and HMWHA, on skin hydration were investigated. The Corneometer measures hydration levels directly on the skin based on changes in electric permittivity.¹³ An applied scatter field penetrates the first skin layer and the level of hydration is determined by the detected changes in the dielectric constant. Larger values denote greater hydration levels. Measurements were taken at the same facial area on both sides (Table 1). The data were analyzed by referring to the patient number and the random number table generated for assigning the experimental and control groups (SCAI-HA and HMWHA, respectively). Before product application, skin hydration levels of the SCAI-HA and HMWHA groups started at 67.2 ± 9.3 and 66.7 ± 9.0, respectively. After2 weeks, these values increased to 70.7 ± 9.0 and 70.4 ± 9.3; then 74.9 ± 8.3 and 74.2 ± 8.3 after 4 weeks of applying the corresponding HA product. Dermal application of SCAI-HA led to 5.1% and 11.4% improvements in skin hydration at week 2 and 4, respectively. Similarly, skin hydration improved by 5.6% and 11.2% from HMWHA at Weeks 2 and 4, respectively. Both SCAI-HA and HMWHA elicited statistically significant increases in skin hydration after 2 and 4 weeks of application. SCAI-HA's ability to improve skin hydration, however, was comparable to that of HMWHA.

3.2 Transepidermal water loss (TEWL)

TEWL is a measure of the amount of water that passively diffuses from the dermis and epidermis through the stratum corneum to the skin surface.¹⁴ It is often considered a critical parameter of the skin barrier representing its integrity. As a dermatological parameter, TEWL is a sensitive measure that is affected by the surrounding environment and local skin properties. Kottner et al. reported that TEWL is subject to substantial intra-individual variability with the following parameters proposed as key influencing factors: (i) permeation path lengths dependent on corneocyte size and epidermal cell layer depth and (ii) quantity and composition of intercellular lipid bilayers.^14-17 Therefore, it is critical to make TEWL measurements at the same position under consistent environmental conditions after a period of skin accommodation. To evaluate the effects of SCAI-HA and HMWHA on skin barrier function, the TEWL of the subjects was assessed before and after 2 and 4 weeks of product application (Table 2). Dermal application of SCAI-HA and HMWHA resulted in TEWL reduction, denoting improvements in skin barrier function: (i) SCAI-HA treatment decreased TEWL from 13.0 ± 3.2 g/m²h to 10.7 ± 2.1 and 10.2 ± 2.2 g/m²h after two and 4 weeks, respectively, (ii) HMWHA treatment decreased TEWL from 11.5 ± 2.2 g/m²h to 10.3 ± 2.1 and 9.9 ± 1.9 g/m²h after two and 4 weeks, respectively. It should be noted that SCAI-HA induced a greater decrease in TEWL at magnitudes of statistical significance (17.6 and 22.1%) after 2 and 4 weeks of application, compared with HMWHA (10.0 and 13.2%).

3.3 Skin elasticity

Elasticity is a key physical parameter of the skin denoting its ability to retain its form and recover shape when stretched or deformed.¹⁸ Skin elasticity is a measure of its structural integrity and condition as skin aging and damage lead to noticeable decreases in skin elasticity.¹⁹ Reduction of skin elasticity is reportedly associated with elastin-fiber degradation, collagen fragmentation, and protein aggregation. Moreover, substantial loss of skin elasticity can result in wrinkle formation and facial sagging.^{20, 21} The effects of SCAI-HA and HMWHA were directly evaluated on the cheeks of the subjects using Cutometer MPA580 (Table 3). Skin elasticity was presented as a measure of R2 (Ua/Uf), the ratio of total recovery of skin on normal pressure after deformation.²² Ua is the total recovery of the skin toward its original position after a second under normal pressure; Uf is the maximal distension of the skin into the probe at the end of the vacuum period.²³ Larger R2 values denote greater skin elasticity. Starting from 0.633 ± 0.056, the R2 values increased to 0.658 ± 0.058 and 0.684 ± 0.054 after 2 and 4 weeks of SCAI-HA application; HMWHA-treated groups exhibited similar magnitudes of change from 0.633 ± 0.053 to 0.659 ± 0.058 and 0.681 ± 0.048. Such improvements in R2 values were statistically significant indicating the products' ability to improve skin elasticity through dermal application. No substantial distinction was observed between the two products' ability to increase skin elasticity.

3.4 Wrinkles

Lacking a standardized classification, wrinkles or rhytides are associated with textural changes in skin caused by intrinsic and extrinsic aging.²⁴ These features are characterized as discernible folds, ridges, or creases of the skin. The gradual loss and disorganization of collagen and elastin fibers, connective tissue that provides underlying support for skin, cause these rhytides.²⁵ Misrepair-accumulation aging theory suggests that wrinkles develop from incorrect repairs of injured elastin and collagen fibers.^{26, 27} Signs of aging are often first manifested in the periorbital region in the form of crow's feet (Figure 1B).²⁸ Therefore, the average roughness [Ra (μm); Figure 1A] of the crow's feet area was assessed as a measure of the severity of wrinkles before and after product application. Both SCAI-HA and HMWHA demonstrated the ability to attenuate wrinkles in the periorbital region to similar extents. Treatment of SCAI-HA reduced the average roughness from 18.7 ± 3.4 μm to 18.2 ± 3.5 and 17.9 ± 3.3 μm after two and 4 weeks of product application, respectively, while HMWHA reduced the average roughness from 18.7 ± 3.5 μm to 18.1 ± 3.4and 18.1 ± 3.4 μm. Although SCAI-HA exhibited slightly greater improvements in wrinkle reduction, these differences were determined to be not statistically significant.

3.5 Facial sagging

Facial sagging, despite its self-explanatory terminology, is a poorly understood dermatological and morphological parameter due to its complexity.²⁹ Relaxation of retaining facial ligaments and its consequential laxity have previously been regarded as the main cause of facial sagging.³⁰ Recent efforts, however, aim to reframe the etiology of facial sagging by emphasizing Mendelson's theory that repetitive facial movements lead to (i) development of laxity in skin, subcutaneous tissue, superficial musculoaponeurotic system, and superficial expression muscles, and (ii) facial skeleton remodeling.³¹ The amalgamation of these etiological factors is hypothesized to ultimately result in facial sagging. Hence, facial sagging can serve as a dermatological parameter representing skin health at a deeper level. To evaluate the facial sagging of subjects, topographic images were taken on both sides of the subjects' faces (Figure 2B) treated with either SCAI-HA or HMWHA by forming elongated circles to connect a set of points based on absolute 3D coordinates (x, y, z).³² From these topographs, facial sagging was evaluated by an analytical method described by Saito et al. using the Image-pro® plus software.³³ Facial sagging was represented as angles (°) depicting facial sagging in the cheek region (Figure 2A); larger angles denoting greater facial sagging and vice versa. Starting from an angle of 33.9 ± 5.3°, the administration of SCAI-HA resulted in a decrease in angle to 33.0 ± 5.0° and 32.4 ± 5.1° after two and 4 weeks, respectively. HMWHA also induced a reduction in angle to a lesser degree: from 33.0 ± 5.1°–32.6 ± 5.1° and 32.5 ± 5.1°. Our data indicate that both SCAI-HA and HMWHA could noticeably improve facial sagging, while SCAI-HA's ability to attenuate facial sagging was greater than that of HMWHA with statistical relevance.

3.6 Skin density

Loss of skin density is a characteristic of intrinsic skin aging that leads to thinner skin and reduced elasticity. With age, the junction between the epidermis and dermis, composed of dermal papillae containing vessels that supply the epidermis with nutrients, undergoes deterioration where the depth and density of the papillae are reduced.³⁴ This leads to a substantial decrease in dermal density; hence, skin density is often considered a dermatological parameter representing skin health. Herein, the dermal density of the subjects was measured via high-frequency ultrasound imaging (Figure 3).³⁵ Before product application, the average skin densities of the subjects were 7.79 ± 1.66% and 7.87 ± 1.28% for the SCAI-HA and HMWHA-designated groups, respectively. After two and 4 weeks of SCAI-HA dermal application, the measured skin density was improved to 8.63 ± 1.78% and 9.07 ± 2.00%, respectively. Two and 4 weeks of applying HMWHA also led to improvements in skin density to 8.52 ± 1.36% and 8.89 ± 1.40%. The observed improvements in skin density were determined to be statistically significant. SCAI-HA's ability to increase skin density, however, was not determined to be substantially greater than that of HMWHA.

3.7 Collagen synthesis

Collagen is a major component of the extracellular matrix responsible for dermatological structure and function, which contributes to skin firmness and elasticity. Collagen type I accounts for ca. 70% of the collagen in the skin.³⁶ Therefore, the effect of SCAI-HA and HMWHA on the cellular synthesis of Procollagen type I C-peptide (PIP) in NHDF cells was evaluated via ELISA (Figure 4). Ascorbic acid (0.001%) was used as a positive control.³⁷ At concentrations 0.003–0.012%, SCAI-HA increased the production of PIP by 17.9%–57.9%. Oppositely, HMWHA did not noticeably alter the cellular synthesis of collagen. It should be noted that neither SCAI-HA nor HMWHA exhibited notable cytotoxicity against NHDF cells in the abovementioned range of concentrations (Figure S4).

In summary, SCAI-HA demonstrated superior dermatological properties to HMWHA in the following aspects: (i) attenuation of TEWL, (ii) reduction of facial sagging, (iii) promotion of cellular collagen synthesis. SCAI-HA also exhibited cosmetic efficacy concerning the remainder of the tested parameters to extents comparable to HMWHA. A closer examination of these experimental results can lead to the exploration of potential relationships and lack thereof among these parameters. First, despite its enhanced ability to reduce TEWL, SCAI-HA's effect on augmenting skin hydration levels was comparable to that of HMWHA. Based on the conceptual connections between skin hydration and TEWL, it may be expected that a greater reduction in TEWL would lead to a greater increase in skin hydration; greater skin barrier function, indicating an increased capacity to retain moisture in the skin, could be expected to lead to improved hydration. Our findings, however, did not support this idea: while SCAI-HA's capacity for reducing TEWL was better than that of HMWHA, their capacities for increasing skin hydration were comparable. Previous research indicates the complex relationship between skin hydration and TEWL. For instance, although a strong inverse correlation is reported between age and skin hydration,^{38, 39} the proportionality between age and TEWL remains controversial with numerous reports supporting the lack of correlation.⁴⁰ It is possible that although the difference between SCAI-HA and HMWHA's effect on TEWL may be statistically significant, this variance is insufficient to noticeably affect skin hydration at the clinical level.

Another unexpected observation made from this study was that SCAI-HA exhibited an improved capacity for attenuating facial sagging, but not increasing skin elasticity. Skin elasticity and facial sagging are closely associated, as the skin's ability to retain its form by resisting external forces contributes to its overall elasticity. The drooping of facial features is a consequence of the deterioration of skin structure leading to the loosening of subcutaneous tissue, superficial musculoaponeurotic system, and superficial expression muscles. Remodeling of the facial skeleton resulting in decreased facial volume and fat mass have also been indicated as a key contributor to facial sagging. Considering the multifaceted etiology of facial sagging, HA's mechanism of action leading to its improved attenuation of facial sagging could stem from a number of these potential pathways including its ability to promote cellular collagen synthesis. As a key component of the extracellular matrix, collagen manifests substantial structural and functional contributions to the skin. Furthermore, its synergistic association with elastin leads to the stabilization of collagenous fibrils via intra- and intermolecular crosslinking imparting shape and firmness to skin.^{41, 42} In 2018, Shute et al. reported that collagen peptides significantly increased fibroblast elastin synthesis and inhibited elastin degradation.⁴³ Furthermore, reports indicate that the density of retinacula cutis (RC), a subcutaneous network of collagenous fibers responsible for attaching the deep surface of the dermis to the underlying deep fascia, exhibited a negative correlation with facial sagging.⁴⁴ In short, the increased collagen synthesis brought upon by HA leading to greater elastin-levels and RC complexity and density may present a potential dermatological pathway leading to notable improvements in facial sagging. Going a step further, SCAI-HA's enhanced ability to attenuate facial sagging could stem from SCAI-HA's increased promotional effects toward cellular collagen synthesis relative to HMWHA.

In this work, we aimed to first focus on the distinction in the cosmetic efficacy of SCAI-HA and HMWHA by assessing their short-term effects on the studied parameters; these short-term properties indicate the value of SCAI-HA. Based on the currently available data, the long-term effects of this conformation of HA can be expected to be more significant. Statistically significant differences were observed in the cosmetic efficacy of SCAI-HA and HMWHA within 4 weeks for three highlighted dermatological parameters: TEWL, facial sagging, and cellular collagen synthesis. Achieved in such a short period of time, this difference in efficacy would most likely be augmented in a longer trial. In addition, mechanistic studies regarding the improved cosmetic efficacy of SCAI-HA relative to HMWHA would be valuable in understanding the biochemical and physical properties of the novel HA conformation leading to such enhancements. A larger clinical study with a longer time frame would provide us with more reliable and valuable information regarding the long-term effects of the product. Despite these limitations, the current study sufficiently demonstrates the improved cosmetic efficacy of SCAI-HA relative to its original counterpart, HMWHA.

As for the monitoring of side effects, researchers were to take notes of any observable dermatological reactions that may have appeared following the application of our products. Through the duration of this study, however, no adverse reactions were reported. This was expected as hyaluronic acid is an API often found in cosmetic and medical products that is generally recognized as safe by the FDA.