Skin rejuvenation through topical application of indocyanine green with diffractive optical element mode of 785 nm picosecond laser in Asian females

Indocyanine green (ICG) exhibits robust absorption near 800 nm.


| INTRODUC TI ON
The appearance of fine wrinkles, enlarged pores, and pigmented lesions can cause significant distress for patients and may affect their social interactions, relationships, and overall quality of life.Aging of the skin can be categorized into two types: intrinsic aging and photoaging, each with distinct phenotypes and underlying mechanisms. 1 Photo-aging, primarily caused by chronic exposure to sunlight, is characterized by the presence of lentigines, deep wrinkles, and enlarged pore size. 1 Picosecond lasers emit ultrashort picosecond pulses that induce powerful photomechanical effects, essentially breaking down small pigment particles and enabling the treatment of pigmented lesions. 2 Picosecond lasers in diffractive optical element (DOE) mode creates a grid of focal microinjury zones by redistributing laser beams into high-fluence regions surrounded by low-fluence backgrounds.
Laser-induced optical breakdown induces dermal microinjuries, which subsequently initiate wound healing mechanisms and stimulate dermal remodeling, leading to notable clinical improvements in wrinkles and enlarged pores. 3,4docyanine green (ICG), a tricarbocyanine dye, exhibits robust absorption near 800 nm with a low incidence of adverse effects; thus, denoting some characteristics of an ideal photosensitizer. 5inical studies have reported the photo-rejuvenating effect of photodynamic therapy (PDT) on aged skin, attributed to collagen synthesis. 6,7However, studies investigating the effects of applying ICG to photo-aged skin followed by 785 nm picosecond laser irradiation have not been previously reported.Hence, the principal objective of this study was to assess the clinical effects of applying ICG in combination with a 785 nm picosecond laser in DOE mode on Asian skin.

| Photosensitizer
Topical 9.6% lidocaine 500 mL (5 MC cream, TheU Pharmaceutical Co., Seoul, Korea) was mixed with 25 mg of ICG (Diagnogreen, Taiyo Pharma Tech, Osaka, Japan).Patients applied 30 g of 0.05% ICG cream to their faces for a duration of 15 min.To protect the ICG from light exposure, the skin samples were covered with aluminum foil throughout the application period.After the designated time, any excess ICG was removed from the skin.

| Treatment device and protocol
The patients underwent treatment using a 785 nm 600 picosecond laser (HELIOS IV 785, Laseroptek, Sungnam, Korea) in DOE mode consisting of 49 microbeams in a 5-mm square-shaped treatment area.Three-thousand shots were delivered at a pulse energy of 200 mJ and frequency of 10 Hz.The Zoom handpiece was used to treat hyperpigmented lesions with settings of: spot size 3-4 mm, pulse energy 60-120 mJ, and frequency 3-7 Hz.All procedures were conducted by a single dermatologist to avoid bias in the treatment techniques.Each patient received five treatments at 1-2-week intervals.Patients were advised to avoid exposure to sunlight throughout the study.

| Investigator-evaluated outcomes
Clinical assessments were performed at the initial visit and 4 weeks after the last treatment by two blinded dermatologists.Standardized photographs were obtained using the 3D LifeViz™ System (Quantificare, Sophia Antipolis, France) and A-one Lite imaging system (Bomtec, Seoul, Korea).Wrinkles were evaluated using the Modified Fitzpatrick Wrinkle Scale (MFWS).The evaluation of pores and hyperpigmented lesions was conducted utilizing a 10-point visual analog scale (VAS).Adverse effects, such as pain, erythema, edema, itching, oozing, hypopigmentation or hyperpigmentation, and infection, were recorded.

| Statistical analysis
To evaluate the differences between variables obtained at baseline and 4 weeks after the last treatment session, the scores of the two investigators were added and the mean was calculated.
Wilcoxon signed-rank test was performed using SPSS version 28.0 (SPSS, Chicago, IL, USA).p < 0.05 was considered statistically significant.Facial wrinkles, pores, and pigmented lesions were evaluated using the 3D LifeViz™ System (Quantificare) and A-one Lite imaging system (Bomtec).Photographic evaluation was conducted before treatment and 4 weeks after the completion of the five treatments (Figures 1 and 2).Two independent blinded investigators observed a significant improvement in depth of wrinkles (1.69 vs. 1.47; p = 0.02).Additionally, pore size showed a statistically significant reduction (3.06 vs. 2.69; p = 0.034), along with significant decrease in hyperpigmentation (3.5 vs. 2.56, p = 0.036) (Table 1).Subjectively, patients reported satisfaction with general improvement (2.63 ± 0.81), as well as improvements in pigmentation (2.56 ± 0.96), pores, and skin texture (1.69 ± 0.6).Overall, nine patients answered more than 50% improvement in general improvement, eight patients answered more than 50% improvement in pigmentation, and one patient answered more than 50% improvement in pores and texture (Figure 3).

| Histopathological findings
H&E staining displayed a notable increase in the quantity of dermal collagen bundles (Figure 4).MT staining revealed increased density and thickness of collagen fibers (Figure 5).Moreover, increased expression of collagen I and III was observed throughout the papillary dermis to upper reticular dermis, along with a diffuse increased expression of STRO-1 in the dermis (Figure 6).Stereological analysis of the area fraction of collagen III showed the greatest increase, rising from 6.75% before treatment to 10.54% 4 weeks after the fifth treatment session.The area fraction of collagen I increased from 3.04% to 5.97% and from 4.28% to 5.09% for STRO-1 (Table 2).

| Patient safety
Treatments were well-tolerated by all patients.Reported side effects included pain and erythema at the treatment site.However, these skin lesions resolved within a few days with no sequelae.Adverse events of itching, oozing, or post-inflammatory hyperpigmentation were not reported.No significant pigmentation reaction resulted from ICG application, and transient aggravation was not observed.exposure. 1Photo-aging is a synonym for extrinsic aging attributable to long-term sun exposure and is histologically characterized by elastolysis and hypocollagenesis.Clinical manifestations encompass wrinkles, skin laxity and the emergence of lentigines, with previous studies substantiating the efficacy of picosecond lasers in treating these lesions. 8,9cosecond lasers emit pulse durations of 300-900 ps (10-12 s). 10 A rapid elevation in temperature is induced by the picosecond pulse, causing a powerful acoustic shock wave within the tissue chromophore. 10As a result, the tissue fracture threshold is surpassed by the tensile stress, and the chromophore is shattered through a "photomechanical effect." 10In 2012, the first picosecond laser obtained approval by the Food and Drug Administration (FDA) for pigmentation treatment and tattoo removal (Picosure, Cynosure, Westford, Massachusetts, USA). 11actional picosecond laser energy can be generated using a diffractive lens array, allowing for energy concentration within the laser microbeams, while the surrounding tissue remains unharmed. 12This procedural approach engenders micro-injury zones that histologically exhibit as vacuoles within the intraepidermal and upper dermal layers, and previous studies revealed that cystic cavitations due to LIOB correlate with the melanin index, as the target chromophore is melanin. 3,12Additionally, the release of cytokines and chemokines from keratinocytes, instigate the synthesis of new collagen to facilitate dermal remodeling.Consequently, these processes contribute to the rejuvenation of the skin. 4,13,146][17] Previous reports have discussed the molecular mechanisms responsible for the increase in collagen synthesis following PDT. 8,7Neocollagenesis, stimulated by PDT, may be induced by (1) the direct effects of PDT on fibroblasts

TA B L E 1
The mean ± standard deviation of wrinkles assessed using the MFWS, as well as the assessment of pores and hyperpigmentation on a 10-point VAS, were recorded before treatment and 4 weeks after the fifth treatment session.
through the activation of extracellular signal-regulated kinases and (2) the indirect effects of PDT on fibroblasts through the action of keratinocyte-derived cytokines, including interleukin-6, interleukin-1α, and tumor necrosis factorα. 18Inflammatory cytokines have the potential to induce matrix metalloproteinases, which in turn facilitate the degradation of aged and damaged collagen, consequently initiating the synthesis of novel collagen. 18Previous studies have documented the increased expression of type I and III procollagen following PDT in human skin. 7 "ideal" PDT agent possesses certain characteristics.First, it must exhibit high absorbance at longer wavelengths, particularly in the range of approximately 780-810 nm, as these wavelengths can penetrate deep into tissues, reaching nearly 6 mm. 19Additionally, the "ideal" PDT agent must be safe with minimal adverse effects.
ICG, a nontoxic tricarbocyanine dye, fulfills this criterion.ICG has been extensively utilized since its approval by the United States FDA in 1956 for the measurement of cardiac output and hepatic function. 19Notably, ICG can be administered intravenously and applied topically as it is highly absorbable by the skin and accumulates in the sebaceous glands via follicular pores.The penetration time is 5 to 15 min for the dye to reach a depth of 1 mm. 20ere have been no previous reports on the effect of a topical ICG with a 785 nm picoseconds laser for the treatment of photoaged skin.The selection of the 785 nm picosecond DOE mode was based on the long wavelength, allowing for deeper tissue penetration. 7,21ICG was chosen due to its maximal absorption band of approximately 800 nm, which closely matches the wavelength of 785 nm used in this study.We hypothesized that PDT with ICG and the 785 nm picosecond laser in DOE mode would allow for deeper tissue penetration and an optimal photodynamic effect, resulting in skin rejuvenation, and reduced hyperpigmentation through the laser's strong absorption of melanin.
In our study, histological findings revealed increased thickness and density of collagen fibers, accompanied by increased expression of collagen I, collagen III, and STRO-1.In particular, increased collagen III expression was observed throughout the papillary dermis to the upper reticular dermis.Collagen III is the first collagen synthesized during the early phases of wound healing and is later supplanted by collagen I, which is the predominant collagen type in the skin. 22The presence of new collagen strands detected in the specimen collected 4 weeks after treatment serves as an indicator of wound healing, illustrating the progress of skin rejuvenation after treatment. 22 addition, the expression of STRO-1 in the dermis was increased during the course of this study (Figure 3E,F).4][25] We hypothesized that the upregulation of STRO-1 expression indicates the regeneration process in the dermis; however, extensive research is necessary to confirm this hypothesis.
Importantly, there was an increase in collagen I as well as collagen III in the papillary and upper reticular dermis, accompanied by a diffuse increase of STRO-1 in the dermis.These histological findings suggest that the application of ICG facilitated deeper penetration of the picosecond laser since the microinjury zones created by the DOE handpiece are known to be generated in the upper papillary dermis and lower epidermis. 3Additionally, this correlates with the penetration depth of roughly 1 mm after application of 5-15 min of ICG. 20us, as ICG is a photosensitizer and has a high absorption near 800 nm, our findings may suggest 785 nm picosecond laser not only was absorbed by melanin absorption but also ICG, leading to deeper penetration and rejuventation to the reticular dermis.However, further histological studies with controls are required.
To the best of our understanding, our study is novel in that there have been no previous studies evaluating the effect of PDT using a picosecond laser with ICG for the treatment of photo-aged skin.
The results showed significantly improved pores, wrinkles, and hyperpigmentation, along with an overall increase in patient subjective improvement.Importantly, no prolonged adverse effects were reported.
This study has three primary limitations.
This prospective clinical study was approved by the Institutional Review Board of Hallym University Sacred Hospital, Korea (IRB No. 2022-11-004) and was conducted in accordance with the Declaration of Helsinki.The study was registered on the Clinical Research Information Service (KCT0009169).Written informed consent was obtained from all participants.Patients with wrinkles, enlarged pores, and pigmented lesions in at least two facial areas (forehead and cheek) were enrolled in this trial between December 2022 and February 2023.Patients who had received oral isotretinoin treatment within the past 6 months, had concurrent facial infection or dermatitis, were pregnant, or had a history of photodermatosis were excluded from the study.Additional treatments for wrinkles, enlarged pores, or pigmented lesions were prohibited during the study period.Patients were advised to maintain their usual skin care regimen throughout the study.

A 2 -
mm punch biopsy of the posterior auricular area was performed at baseline and 4 weeks after the fifth session on two patients who provided written consent.Hematoxylin and eosin (H&E) staining along with Masson's trichrome (MT) staining were conducted on tissue samples.Immunohistochemical staining of collagen I, collagen III and STRO-1 was conducted to evaluate dermal collagen characteristics.The area fraction was quantified using ImageJ software (NIH, Bethesda, MD, USA) by analyzing the size of each tissue and adjusting the threshold to capture only the stained particles.The percentage of the stained particles was divided by the percentage of the tissue size to calculate the area fraction.

A
total of 16 female volunteers were enrolled in this study.The mean patient age was 58.44 ± 5.24 years (range: 51-66 years).Concerning the Fitzpatrick skin type, 4 (25%) were classified as Fitzpatrick type III and 12 (75%) as Fitzpatrick type IV.
Skin aging arises as a consequence of both intrinsic factors, including chronological aging, and extrinsic factors, notably UVB radiation F I G U R E 1 Clinical photographs before (A, C) and after (B, D) treatment.(A, C) Hyperpigmentation on the right cheek (red arrows) before treatment.(B, D) Improvement of hyperpigmented lesion (yellow arrows) after treatment.

F I G U R E 2
Clinical photographs before (A, C) and after (B, D) treatment.(A, C) Visible wrinkles and enlarged pores before treatment (yellow arrows).(B, D) Improvement of wrinkles and pore size after treatment (white arrows).

F I G U R E 3 F I G U R E 4
Patient's subjective improvement in general appearance, pigmentation, pores, and skin texture were assessed 4 weeks after the fifth treatment session.Hematoxylin and eosin staining.(A) Pre-treatment.(B) Post-treatment.Areas of improvement are indicated by black arrows (Magnification: 200×, scale bar: 0.05 mm).

F I G U R E 6
First, it was a single center study and the sample size was relatively small.Second, the study included only Fitzpatrick skin types III and IV; thus, limiting the generalizability of the findings.Third, a split-face design was not employed, and a control group was not established.However, the histological observations, which demonstrated an increase in collagen I, collagen III, and STRO-1 post-treatment, compensated for this limitation.Nevertheless, further studies comparing alternative modalities are warranted.In conclusion, combination treatment with ICG application and 785 nm picosecond laser is an effective and safe therapeutic approach for the photo-aged skin of Asians with Fitzpatrick skin type III and IV.F I G U R E 5 Masson's trichrome staining.(A) Pre-treatment.(B) Posttreatment.Areas of improvement are indicated by black arrows (Magnification: 200×, scale bar: 0.05 mm).Immunohistochemical staining for collagen I (A, B), collagen III (C, D), and STRO-1 (E, F). (A, C, E) Pretreatment.(B, D, F) Post-treatment.Areas of improvement are indicated by black arrows (Magnification: 100×, scale bar: 0.1 mm).