Intradermal Local Injection of Autologous Cell-Free Fat Extract for the Treatment of Refractory Postinflammatory Hyperpigmentation

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Introduction
Postinfammatory hyperpigmentation (PIH) is a common dermatological condition characterized by long-term hyperpigmentation, skin barrier damage, and atrophic scarring [1].It is often a consequence of infammatory processes, resulting in a complex pathogenesis that poses challenges for conventional treatment approaches.PIH lesions, frequently observed on the face with a considerable size, cause emotional distress and have a profound impact on the quality of life of afected individuals [2,3].Unfortunately, existing therapies for PIH, including hypo-pigmenting agents, chemical peels, and laser treatments, often fail to achieve satisfactory outcomes, necessitating a prolonged treatment duration [4][5][6].Consequently, an urgent need exists to develop a comprehensive therapy that efectively addresses PIH while ensuring tolerability.
Cell-free fat extract (CEFFE) is an intriguing treatment option derived from adipose tissue through emulsifcation and centrifugation [7].It contains high concentrations of diverse soluble active proteins, including transforming growth factor (TGF)-β1, TGF-β2, interleukin (IL)-4, and vascular endothelial growth factor (VEGF), which play pivotal roles in healing and regenerative processes [2,8].Extensive research has already demonstrated the therapeutic efcacy of CEFFE in various dermatological diseases, including photo-aging, fap grafting, and the treatment of refractory wounds [9][10][11].Moreover, our unpublished fndings have confrmed its potential in ameliorating hyperpigmentation, as evidenced by positive outcomes in a zebrafsh model.
Given the regenerative properties of CEFFE and its promising track record in dermatological conditions, we hypothesize that CEFFE therapy holds considerable promise as an efective and well-tolerated treatment modality for postinfammatory hyperpigmentation.Consequently, this study aims to evaluate the efcacy and tolerability of CEFFE therapy in patients with PIH.We aspire to contribute to the development of an innovative and comprehensive therapeutic approach that addresses the unmet medical needs of individuals with PIH by investigating the efects of CEFFE on hyperpigmentation.

Subjects and Inclusion and Exclusion
Criteria.Tis study was approved by the Institutional Review and Ethics Board of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (SH9H-2020-T97-3) and registered at https://www.chictr.org.cn(ChiCTR2000039381).Fifteen patients were enrolled in this study between October 2020 and October 2021 and have provided written informed consent.Te patients were women aged 18-33 years, with postradiotherapy pigmentation, in which conventional approaches, such as topical medications or laser therapies, were inefective.A single group, repeated measures design was used for this study.
Te exclusion criteria were the following: current or planned pregnancy; the presence of a wound or broken epidermis in the focus area; serious underlying diseases, including mental disorders; known hypersensitivity to lidocaine; body mass index (BMI) <18 or >30 kg/m 2 ; therapy, such as laser, mesotherapy, and chemical peeling within the last 6 months before this study; or enrollment in other clinical trials.

CEFFE Preparation and Treatment.
Liposuction was performed using a standard cannula (3 mm) with large side holes (2 × 7 mm) after local infltration injections with modifed Klein solution.Approximately 100 mL of fat was collected from each patient.Fat tissues were washed with saline solution to remove remnant blood cells and centrifuged at 1, 200 × g for 3 min.Te middle fat layer was collected for further emulsifcation through two syringes connected by a 2 mm diameter Luer lock connector (B.Braun Medical Inc., Melsungen, Germany) and shufed 60 times.Te emulsifed fat was then centrifuged at 1, 200 × g for 5 min and separated into four layers (oil, fat, CEFFE, and cell fragment pellet).CEFFE was collected and fltered through a 0.22 µm flter (Corning Glass Works, Corning, NY, USA) and stored at −80 °C in a 1.8 mL sterilized cryopreservation tube (Termo Fisher Scientifc, Waltham, MA, USA).Approximately 10 mL of CEFFE was obtained from each patient (Supplementary Video File (available here)).
Te time points of patients receiving treatment and follow-up after enrollment are shown in Figure 1(a).Te relevant treatment frequencies refer to the platelet-rich plasma treatment for hyper-pigmentation containing melasma, acne scar, and periocular hyperpigmentation [12][13][14][15].Each patient received fve intracutaneous CEFFE injections at the lesion site with a two-week interval.Te frst injection was performed two weeks after CEFFE preparation.After the fnal injection, the patients underwent three follow-ups at 3, 6, and 12 months posttreatment.
One hour before every injection, the pigmented area was cleaned and topical anesthetic ointment comprising 25% lidocaine and 25% procaine (Ziguang, Beijing, China) was applied.After sterilization, CEFFE was intradermal injected by the nappage technique using a 32-G needle (0.1 mL per point in a linear pattern and the typical total treatment course involves injecting a volume of 8-10 mL; injection points were 1 cm apart; Figure 1(b)).

Objective Efcacy Assessment. Standard twodimensional (2D) photographs and quantitative analysis
of brown spots (BS) were conducted using VISIA-CR ® (Canfeld Scientifc, Fairfeld, NJ, USA) before treatment and at every follow-up visit (3, 6, and 12 months after the fnal treatment).Measurements were performed at fxed locations for each patient and follow-up.
Lesion color was measured by tristimulus colorimetric using a skin colorimeter CL400 probe (Courage-Khazaka, Germany) [16].Te three darkest areas of the lesion were tested for each patient.L * value indicates lightness, is correlated with the level of pigmentation of the skin, and is represented on a vertical axis with values from 0 (black) to 100 (white).a * and b * are chromaticity coordinates.Te a * value indicates the red-green component of a color and is correlated with erythema.Te b * value indicates the blueyellow component and is correlated with pigmentation and tanning.Te change in lightness was calculated as follows: where L * baseline represents the baseline lightness (before CEFFE treatment) and L * t represents the skin lightness at 3, 6, or 12 months after the fnal treatment (t � 3, 6, or 12).Te change in skin color is calculated as follows:

Dermatologic Terapy 3
Transepidermal water loss (TEWL) was measured using a Tewameter ® TM300 and used to estimate the skin barrier function [17].Measurements were taken at fxed locations for each follow-up visit.Te change in skin barrier function was calculated as follows: 2.4.Subjective Efcacy Assessment.Global aesthetic improvement in appearance during each follow-up compared to pretreatment was assessed using the Global Aesthetic Improvement Scale (GAIS) [18].GAIS scores range from −2 (much worse) to 2 (very much improved).Te satisfaction of patients at 12 months after the fnal treatment was assessed using the Likert Satisfaction Scale (LSS) [19].LSS measures specifc items utilizing a fve-point scale: very dissatisfed, dissatisfed, slightly satisfed, satisfed, or very satisfed.

Tolerance Assessments.
Te tolerance of each treatment was evaluated based on the number of injection-related events (IREs) reported by the subjects [20].During the frst 14 days after injection, local tolerability was assessed by the frequency and severity of predefned IREs.

Statistical Analysis.
Te nonparametric Friedman test was used to compare the outcome measures at the four assessment times (baseline, 3, 6, and 12 months after the fnal treatment).Statistical analysis and graphical work were performed using R (R Foundation for Statistical Computing, Vienna, Austria)."tidyverse," "ggplot," and "ggsci" packages were used to visualize the results.Continuous variables are presented as the median (frst quartile; third quartile) or mean ± standard deviation.Statistical signifcance was set at p < 0.05.

Results
Te mean ± SD age of the participants at the baseline was 22.13 ± 4.29 years (range from 18 to 33).Hyperpigmentation developed on the face and resulted following strontium 90 radiotherapy administered for the treatment of IH.Te improvement in the skin barrier function was most evident at the end of the 12-month follow-up period compared to that at the baseline (p < 0.05).

Subjective Efcacy Assessment.
According to the Global Aesthetic Improvement Scale (Figure 3), 93.33% (n � 14) of the patients showed "improved" or "very much improved" conditions 12 months after treatment.Based on the Likert satisfaction scale Table 2), 100% (n � 15) of the patients were either satisfed or very satisfed with the CEFFE treatment.
3.3.Tolerance Assessments.CEFFE treatment was well tolerated; treatment-related adverse events including contacting bruise, and stinging were transient, of mild-tomoderate severity, and localized at the injection sites.All patients experienced transient and tolerable stinging during injection, and 53.33% of the patients (n � 8) showed mild bruises after injection, which recovered within 4.13 ± 3.08 days (Table 3).Tere were no dropouts or withdrawals due to IREs in this study.To date, no serious IREs have been reported.

Discussion
PIH signifcantly impacts the quality of life of patients and satisfaction with their appearance [2].Unfortunately, current therapeutic options for PIH often fall short of meeting expectations of patients [1].In this study, we investigated the efcacy and tolerability of CEFFE as a potential treatment for PIH.
Our objective evaluation revealed that CEFFE treatment led to signifcant improvements in the general appearance and lightness of the skin, indicating its efectiveness in reducing pigmentation.Tese positive outcomes can be attributed to various factors.VEGF has been reported to inhibit tyrosinase activity, while IL-4 suppresses melanocyte formation through the JAK2-STAT6 pathway [1,21].Furthermore, CEFFE possesses antioxidant, antiapoptotic, and proangiogenic properties, as observed in our previous studies [7,11,22].Tese properties contribute to the protection of dermal fbroblasts and skin from UVB-induced photoaging [23].CEFFE treatment also improved skin texture and enhanced barrier function, which can be attributed to the stimulation of neovascularization by VEGF,  Dermatologic Terapy epidermal cell proliferation by a basic fbroblast growth factor, and fbroblast proliferation by an epidermal growth factor.Notably, the sustained reduction in skin lesions observed 12 months after CEFFE treatment suggests potential changes in the skin lesion microenvironment.Subjective evaluation by patients reporting a substantial decrease in the overall intensity of dark spots further supported the efcacy of CEFFE treatment.Tolerance to CEFFE was favorable, with only transient bruises observed postinjection, and no other patients reported adverse skin reactions.Consistent with our previous studies, CEFFE administration did not elicit immunogenicity, cytotoxicity, intradermal reactions, or acute systemic toxicity [24].
Additionally, CEFFE preparation is convenient and can be utilized for multiple treatments, suggesting its potential applicability in other refractory hyperpigmentation disorders, such as melasma, acne scars, and periocular hyperpigmentation.However, it is important to acknowledge the limitations of our study, including the small sample size and the complexity of determining the specifc factors within CEFFE that contribute to its efcacy.
In summary, our study demonstrates that CEFFE is a well-tolerated agent that efectively reduces PIH.Te objective and subjective improvements observed support its potential as a promising therapeutic option for patients with postradiotherapy PIH.Further research with larger sample sizes is necessary to validate these fndings and gain a deeper understanding of the underlying mechanisms.

Figure 2 :
Figure 2: Te change of objective assessments from the baseline to 12 months' follow-up.(a) Brown spot index.(b) Lesion lightness.(c) Lesion color.(d) Transepidermal water loss.

Table 1 :
Clinical characteristics of all 15 patients.