Combination of fractional carbon dioxide laser with recombinant human collagen in periocular skin rejuvenation

The most visible sign of facial aging is often seen in the periocular area. However, periocular rejuvenation remains challenging due to the particularity of periocular anatomic locations.


| INTRODUC TI ON
The periocular region possesses the fine and thinnest skin, which is the earliest area of aging on the face. 1 As the periocular skin ages, collagen undergoes decreased biosynthesis and increased degradation to make the skin decrease in thickness and integrity, consequently leading to typical periorbital aging symptoms such as sagging, laxity, roughness, and wrinkles. 2,3Numerous antiaging treatments have been developed for rejuvenating the aged periocular skin, including topical agents, chemical peeling, microdermabrasion, micro-acupuncture needle, botulinum toxin, filler injection, laser therapy, radiofrequency treatment, and surgery. 4However, none of these treatments can be advocated as the standard treatment in periocular rejuvenation due to the particularity of these anatomic locations.
The method of collagen regeneration and supplementation that can restore the innate collagen deficit within aged skin offers a promising treatment to revive the aged skin.Recombinant human collagen (rhCOL) possesses the fundamental properties of natural collagen.With the advantages of being pathogen-free and having low immunogenicity, 5 it has been effectively enriched in both yeast and E. coli systems on a large scale.As a result, rhCOL can be used as a clinical treatment for periocular skin rejuvenation.
However, the direct topical application of rhCOL remains suboptimal because of the barrier function of the skin. 6Fractional-ablative CO 2 lasers (CO 2 -AFL) have been used to ablate micro-columns of the skin vertically through both the epidermis and dermis to facilitate the absorption of topical rhCOL.This technique may be adopted as the intralesional route to deliver rhCOL into aged skin and stimulate natural collagen production and dermal and epidermal remodeling. 7Herein, we designed the study to investigate the efficacy of high-fluence CO 2 -AFL with rhCOL penetration for periocular rejuvenation.

| Preparation of recombinant human collagen
Clinical grade rhCOL (24 mg:4 mL; rhCOLI: rhCOLIII was 2.38:1) was purchased from Xi'an Giant Biogene Technology Co., Ltd.It was approved for wound care by China Food and Drug Administration.
According to the manual from the manufacturer, recombinant human collagen I (rhCOLI) and collagen III (rhCOLIII) were obtained from E. coli and Pichia pastoris systems via genetic engineering technology.rhCOLI is derived from the Gly713-Ser727 fragment of human type I collagen α1 chains, while rhCOLIII is derived from human type III collagen α chains CO3A1.After a series of optimization, purification, and examination, clinical grade rhCOL was successfully synthesized with rhCOLI, and rhCOLIII constituted an ideal ratio of 2.38:1, which possessed excellent biocompatibility, low immunogenicity, and superior biological functionality.

| Subjects
The study was designed as a 3-month prospective, single-blinded, General information of the study participants, including age, education, income, menses status, smoking status, drinking status, hypertension, thyroid disease, liver disease, kidney disease, location of occupational activities, location of sports or leisure activities, and the number of hours spent on outdoor activity in a week, were collected by using questionnaires.The smoking status was stratified as a never-smoker, a former smoker, and a current smoker.The drinking status was categorized as a never-drinker, occasional drinker (2 units/month), light (weekend, 1-2 units/day, or 3-6 units/day, or daily 1-2 units/day, or 1-15 units/week), moderate (weekend 6 units/ day, or daily 3-6 units/day; 15-42 units/week), and heavy (daily 6 units/day or 42 units/week), based on the UK unit of alcohol (one drink) representing half a pint of beer or a glass of wine.Additionally, the monthly income of the patients was leveled as less than ¥5, 000, ¥5000-10 000, ¥10 000-20 000, and more than ¥20 000.

| Clinical and ultrasound assessment
All patients were assessed by two senior independent dermatologists blindly just before the treatment, and 1 month, then 3 months after the treatment, to determine the quantitative photoaging scales for periocular skin.The assessments based on the scale designed by Alexiades-Armenakas and Tierney were taken to evaluate lower eyelid aging (Table 1), 4,8 the crow's feet static grading scale was used to judge periorbital aging (Table 2), 9 and the amount of upper eyelid laxity (Figure 1) that derives from upper eyelid blepharoplasty and marginal reflex distance-1 (MRD1) 10 were applied to determine upper eyelid aging.The measurement was performed with an electronic vernier caliper (Sanliang Measuring Co., LTD, Dongguang, China) with the settings as a specification of 0-150 mm and a resolution of 0.01 mm.The photographs were taken in frontal and lateral projections using Canon 70D digital camera, which had been standardized in magnification and positioning.
Skin thickness (epidermis and dermis) was measured at the same two anatomic sites (upper and lower eyelids) three times on all patients before and 3 months after the treatment, with an eL18-4 linear array probe (frequency range, 4.0-18.0MHz) of the Philips Medical System (Philips iU22, Bothell, WA) by the same skilled physician with 12 years of experience in skin ultrasonograms.The average value of these measurements was recorded and used for further analysis.
Additionally, the treatment-related adverse reaction scales for edema, erythema, scab, and hyperpigmentation (Table 3) were recorded on Day 0 for Day 14, once in the first month, and afterward once per 3 months.The adverse reactions were classified as mild (score 1), moderate (score 2 or 3), and severe (score 4), depending on the scale scores.

| Laser technique
The treatment was designed to be single-session therapy to cover the upper and lower eyelid skins within the boundary of the bony orbit and the periorbital lateral with eyelid cilia shunned.Before applying CO 2 -AFL, a topical lidocaine/prilocaine cream (2.5% lidocaine and 2.5% prilocaine; Tongfang Co.) was used to daub the targeted area under an occlusive dressing for 40 min and washed off afterward.Then the topical proparacaine hydrochloride (15 mL: 75 mg; S.A.Alcon-couvreur N.V., Belgium) was further used with the insertion of a corneal protector.Meanwhile, the ablative fractional 10 600-nm wavelength CO 2 Ultrapulse® laser (DM-300, Dimei Optoelectronic Technology Co., LTD) was set at a pulse duration of 1000 μs and pulse energy between 18 and 22 mJ with double passes based on the extents of skin whitening reaction and slight shrinking.
Each lasered area was approximately 0.4 cm by 0.4 cm to 0.8 cm by 0.8 cm, according to different treatment sites.
However, the point density of the laser spot, which refers to the number of beams (skin hole points) per square centimeter, is fixed at 169 beams, with a diameter of 200 um and a distance of 600 um between two beams.After administering CO 2 -AFL, rhCOL was applied to the treated area on each side at 1 mL in 0 h, 0.5 h, 1 h, 4 h, and 12 h.
Then the treatment area was given wet compresses of tranexamic acid and sodium chloride sodium mixture (100 mL: tranexamic acid 1 g and sodium chloride 0.68 g, Chongqing Leimei Pharmaceutical Co.) along with the application of recombinant bovine basic fibroblast growth factor eye gel (5 g:21000 iu, Zhuhai Yisheng Biopharma Co.) twice a day for 1 week.All patients were told not to use water for 24 h following treatment and not to expose themselves to the sun but to apply moisturizer and SPF50 sunscreen lotion.

| Statistical analysis
Statistical data analysis was performed using the International Business Machines Corporation (IBM) Statistical Package for the TA B L E 1 Scale for skin aging (including lower eyelid and periorbital lateral skin).5) 4A)

| Improvement in various clinical indicators (Table
The average MRD1 was 2.77 mm 1 month after the treatment, which had no statistical difference compared with baseline (2.67 mm, p = 0.910), but was increased to 3.21 mm at 3 months, showing statistical significance compared with the baseline (20.2% increase, p < 0.001).

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The amount of upper eyelid laxity (Figure 4A) The amount of upper eyelid laxity decreased on average from 6.07 mm to 4.36 mm in 1 month after the treatment (28.2% decrease, p < 0.001) but reached an average of 4.29 mm after 3 months (29.3% decrease, p < 0.001).The statistical difference was not found in the measurements of 1 month and 3 months after the treatment (p = 1.000).

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The lower eyelid quantitative photoaging scale for skin (Figure 4B) The lower eyelid skin improved, apparently.For the skin texture, the mean scores significantly decreased from 2.85 (baseline) to (baseline) to 1.59 in 1 month (46.6% decrease, p < 0.001) and 1.57 in 3 months (47.3% decrease, p < 0.001) after the treatment.The total scores of the lower eyelid photoaging scale showed a significant decrease from a mean of 8.43 (baseline) to 5.18 (1 month after the treatment, 38.6% decrease, p < 0.001) and 5.13 (3 months after the treatment, 39.1% decrease, p < 0.001).However, no statistical differences were found in the first-and third-month follow-up visits for all scores for the lower eyelid evaluation (p = 1.000).

| The crow's feet static grading scale (Figure 4C)
The crow's feet static grading scale scores showed a significant decrease from a mean of 3.29 of the baseline to 2.07 in 1 month (37.1%, p < 0.001) and 2.14 in 3 months after the treatment (35.0%decrease, p < 0.001).Statistical differences were not found between the scores in the first-and third-month follow-up visits (p = 0.524).

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The side effects of the treatment (Figure 6) All the adverse reactions were diagnosed to be mild to moderate.The median duration of edema and scab were 5 and 6 days, respectively.A total of six patients (

| DISCUSS ION
Blepharoplasty has been attempted to resect loose eyelid skin to improve periorbital rhytids via a mild tensioning effect from tissue removal.However, the procedure cannot transform the skin quality. 11As it remains challenging to proceed with local non-surgical treatment around the periocular area and along the upper eyelid, a therapeutic modality in this study enables clinicians to safely and effectively perform periocular rejuvenation with shortened posttreatment downtime.
Collagens (Types I and III), as the major structural components of the extracellular matrix (ECM) in human skin, play a significant role in mechanical support, control of cell adhesion/differentiation/migration, regulation of ECM synthesis, reduction of the inflammatory response, and inhibition of melanogenesis in melanocytes, 12,13 and have been developed into various clinically usable biomaterials, which are widely used in cartilage regeneration, wound healing, and the repair of damaged tissues and dysfunctional organs in humans. 3,14,15As collagens extracted from an animal possess not only batch-to-batch variability and the toxicity of chemical crosslinking agents but also carry the chance of disease transfection and expensiveness of purification. 13,15Recombinant human collagen has been recently manufactured with biotechnology using both E. coli and yeast systems.This can offer more consistent and biochemically identical human collagen in a factory mode. 57][18][19][20] However, the large molecular size of the recombinant collagens limits their topical application because of the innate Note: Income per month: 1: less than ¥5, 000; 2: ¥5000-10 000; 3: ¥10 000-20 000; 4: more than ¥20 000.

TA B L E 4
Characteristics of the female study population.

F I G U R E 2
Photographs of a 45-year-old woman at baseline (A), 1 month (B), and 3 months after the treatment (C).
barrier property of the skin.Repeated injections are commonly used to deliver this kind of collagen to its targeted site, which causes a hodgepodge of various issues such as pain, maldistribution, infection, and skin necrosis. 21is study combined high-fluence CO 2 -AFL with rhCOLI and rh-COLIII permeation in periocular rejuvenation.First, the CO 2 -AFL, as a standard cosmetic procedure, uses the light emitted from the fractional CO 2 laser device to generate multiple microscopic treatment zones (MTZ), 22 which possess epidermal heat-shock protein synthesis and collagen regeneration to make skin rejuvenation last for more than 3 months. 23The MTZs are so small that peripheral normal skin tissues can recover them quickly.Second, CO 2 -AFL effectively reduces photoaging-induced textural irregularity and rhytids, 24 which subsequently achieves the skin tightening effect through its penetration into the deep layer of the dermis to stimulate fibroblast proliferation and promotes rearrangement of collagen and elastin. 25Skin ultrasonography has shown increases in the thickness and density of the dermis after AFL treatment, 26 which was consistent with the finding in our study.Third, the fractional CO 2 laser created micropores in the epidermis so that the high energy and long pulse duration (1000 μs) of the lasers effectively create sufficient ablation depth to facilitate the diffusion of rhCOL into the deep reticular dermis.
The study discovered a 20.2% improvement in 3 months but none in the first month after treatment compared to the pretreatment MRD1, implying that the treatment may improve upper eyelid ptosis through late de novo collagen synthesis.However, other periocular skin aging indices significantly improved at both the first and third month after the treatment than before the treatment, but no significant change was observed when only comparing these two months, thereby demonstrating that the combined treatment offered stable and lasting posttreatment improvement in the lower eyelid skin texture, the lower eyelid skin laxity, the lower eyelid skin rhytids, the static crow's feet, and the amount of upper eyelid laxity.Moreover, CO 2 -AFL combined with rhCOL has been more effective in effacing static wrinkles with more improvement in lower eyelid skin texture, rhytids, and cow's feet than in reducing skin laxity, which was assessed with MRD1, amount of upper eyelid laxity, lower eyelid skin laxity.We suggest that more repeated or additional treatments are needed for better improvement.Meanwhile, the skin thickness in our ultrasound analysis was increased (5.6% increase in the upper eyelid skin thickness and 3.3% increase in the lower eyelid skin thickness, p < 0.001), indirectly suggesting collagen neogenesis.
Moreover, the diffusing rhCOL through micropores created by CO 2 -AFL accelerated wound recovery so that the edema and scab  were resolved within 1 week.Shortening the downtime of the patients significantly enhanced their satisfaction and adherence to follow-up assessments.Our study showed that six patients developed mild postoperative erythema that persisted for two weeks.Three also progressed to hyperpigmentation, so the persisting erythema after CO 2 -AFL should be treated promptly to prevent subsequent hyperpigmentation.A previous study found that the risk of hyperpigmentation after CO 2 -AFL was 55.5% at 1 month and 37.5% at 3 months in Asian patients. 27Thus the risks in treating the skin of these patients with ablative lasers included the development of postprocedural erythema and post-inflammatory hyperpigmentation (PIH).Although the laser fluence used as 18-22 mJ with double passes in our study was high for obtaining the synergistic effect, the rates of PIH in our patients were only 15.4% at 1 month and 7.7% at 3 months, which were significantly lower than the numbers in other studies, indicating that the rhCOL might carry the potential to suppress melanogenesis.F I G U R E 5 Ultrasound images on the left upper eyelid of a 37-year-old woman (A, B).The total skin thickness (epidermis and dermis) was measured to be 0.119 cm before the treatment (A) and 0.133 cm after the treatment (B), with an increase of 10.5% in total skin thickness.Ultrasound images on the right lower eyelid of a 50-year-old woman (C, D).The total skin thickness (epidermis and dermis) was measured as 0.201 cm before the treatment (C) and 0.214 cm after the treatment (D), with an increase of 6.1% in total skin thickness.
In addition, the subsequent treatment of tranexamic acid and recombinant bovine basic fibroblast growth factor eye gel might also play a possible role in skin repairing and reducing PIH.

| Strengths and limitations
The self-controlled study showed several merits based on its results.This study carried several limitations.First, it could not prove the efficacy of the combination of high-fluence fractional-ablative CO 2 laser and laser-facilitated rhCOL infiltration to rejuvenate skin due to its small sample size in a single center.Second, it was a selfcontrolled study without comparing the efficacy of either laser or collagen treatment alone with that of the combined treatment.
However, this study provided a direction for our subsequent investigations, which will recruit more patients from different regions and try different CO 2 -AFL parameter setups to find the most optimal treatment regimens.

| CON CLUS ION
Our study has shown that high-fluence fractional-ablative CO 2 laser and laser-facilitated rhCOL infiltration can rejuvenate the aged skin in the periocular area with significant improvement in skin rhytids, texture, laxity, tightness, and especially in skin static rhytids.
Moreover, the treatment also benefits patients with a shorter recovery period, a higher safety profile, and the potential for more natural cosmetic outcomes.
self-controlled trial that enrolled Chinese female patients with the inclusive criteria as an age range of 30-60 years, Fitzpatrick skin types II-IV, and being admitted to the Department of Dermatology for treatment between June 2022 and August 2022.This study was approved by the Ethics Committee (No: LLSL-2022061402), and all patients signed written informed consent forms.The exclusion criteria were set as the patients with (1) cancer, coronary artery disease, diabetes mellitus, or ongoing infectious diseases, (2) a known allergy to the components used in this program, (3) pregnancy or lactation, (4) prior cosmetic procedure in the area, (5) a history of oral isotretinoin therapy within 1-year period before treatment, (6) presence of ocular diseases, and (7) any other unsuitable conditions for clinical research.

| 127 WU
et al.Social Sciences (SPSS) version 26.0.Repeated measures ANOVA and Friedman's rank test were used to analyze the changes in each clinical measurement indicator on the baseline and posttreatment.p-values<0.05 were considered statistically significant.

3 | RE SULTS 3 . 1 |
Characteristics of the participantsA total of 28 female patients were initially recruited into this study of which 26 completed the entire study protocol.The patients who did not complete follow-up were excluded from the statistical analysis.The basic characteristics and relevant clinical information of the participants are presented in Table4.Photos of the clinical improvement are shown in Figures2 and 3.

F I G U R E 1
1.70 (1 month after the treatment, 40.4% decrease, p < 0.001) and 1.67 (3 months after the treatment, 41.4% decrease, p < 0.001).For the skin laxity, the mean scores decreased from 2.61 (baseline) to 1.89 (1 month and 3 months after the treatment, 27.6% decrease, p < 0.001).For skin rhytids, the mean scores decreased from 2.98 Measurement steps of the amount of upper eyelid laxity: (A) Looking straight ahead, the pupil center vertical line is marked; (B) two points are marked on the central vertical line of the pupil, one is on the upper eyelid margin, and the other one is 7 mm above the upper eyelid margin; (C) thumbs are parallel to the eyebrow direction, stretch the upper eyelid skin to the maximum extent, maximize the upper eyelid skin, and measure the moving distance of the two points.

TA B L E 3
Scale for the treatment-related adverse reactions.by downregulating pro-inflammatory cytokines (TNFα, IL-1β, and IL-6) and upregulating anti-inflammatory cytokines (TGF-β1), thus allowing it to withstand or resist the damaging effects of inflammation, and leading to ECM assembly and wound

F I G U R E 3
Photographs of a 48-year-old woman at baseline (A), 1 month (B), and 3months after the treatment (C).TA B L E 5Improvement of periocular skin aging index.

F I G U R E 4
Improvement in periocular skin aging index indicators before and after the treatment: (A) Changes in both MRD1 and the amount of upper eyelid laxity; (B) changes in skin texture, laxity, rhytids, and total score.(C) Changes in the crow's feet static grading scores; (D) changes in the skin thickness of upper and lower eyelids.

First, we assessed
all the available aging indices around the eye in detail and innovatively proposed the upper eyelid laxity index as an additional indicator.Second, we had fully protected the eyeballs for eliciting more aggressive therapy around the upper eyelids; the treatment modality achieved promising results.Third, we combined human-like collagen and CO 2 -AFL as minimally invasive treatments to enhance the treatment effect and reduce adverse reactions in periocular rejuvenation.