Toward facial rejuvenation; A clinical trial to assess the efficacy of nano fat grafting on wrinkles

With increasing age, patients' facial volume decrease. For this reason, nano fat grafting has recently gained popularity as adjunctive treatment to facial rejuvenation procedures. However, few quantitative studies have been conducted to investigate the impact of nano fat on facial wrinkles.


| INTRODUC TI ON
With increasing the age, patients' facial volume decreases due to a combination of skin thinning, muscle atrophy, fat volume loss, and bone thickness reduction. 1As the life-changing power of beauty is becoming more apparent and valued these days, interest in aesthetic procedures has skyrocketed among many individuals regardless of age and gender. 2,3In response to this situation, various surgical and nonsurgical treatments are being introduced every day as adjunctive treatments to facial rejuvenation procedures. 4 pursue of a more natural and long-lasting solution for skin rejuvenation, fat grafts were introduced as a facial filler for aesthetic purposes around the beginning of the 20th century. 5,6th advances in fat harvesting instruments and techniques, shortcomings of the initial procedure were diminished; thus, autologous fat grafting gained more popularity over time. 7,8However, a great breakthrough was made when Zuk et al discovered adipose-derived stem cells as a cellular component of lipoaspirates. 9Further research on these multipotent cells exhibited their great regenerative and therapeutic potential, resulting in diverse rejuvenating effects on aging skin. 10Consequently, concerning a better understanding of the cellular components of fat grafts and their potential therapeutic effects, more advanced techniques of fat grafting were introduced. 11,12Micro fat, a lipoaspirate that was introduced as an alternative to synthetic dermal fillers, consisted of viable adipocytes alongside adipose-derived stem cells; therefore, used for both lipofilling and regenerative purposes. 12 work less invasive with even finer needles, Tonnard et al. introduced a novel adipose tissue derivative, called nano fat, which consisted of adipose-derived stem cells and very small fat particles that could pass through sharp needles by using a 1.2-2.4mm diameter cannula.13 Nano fat improves skin quality and promotes tissue regeneration in scars, chronic wounds, and face rejuvenation applications.14 In recent years, there has been an increasing amount of literature on the beneficial effects of nano-fat grafting on the skin rejuvenation; however, few quantitative studies have been conducted to investigate the impact of nano-fat on facial wrinkles.15 Thus, authors conducted this study to evaluate the effect of intradermal nano fat injection on fine facial wrinkles and assess their volume, area, depth, and percent area changes over time.

| Study participants
In this clinical study, 15 volunteer subjects consist of 11 females and four males were randomly selected between patients who underwent facial depressed scar filling with nano fat referred to the Dermatology Clinic of Faghihi Hospital, Shiraz, Iran between April 2019 to March 2021.The study was conducted according to the declaration of Helsinky and registered in the Iranian Registry of Clinical Trials.The Ethics Committee of the University has approved the study protocol and written informed consent was obtained from all participants before starting the trial.
Inclusion criteria consist of adult's participants aged over 30 years, Fitzpatrick skin type III and IV, with fine facial wrinkles located in the forehead area.Among participants who met the inclusion criteria, the ones who had a history of Botox injection from 6 months before to 7 months after the procedure, under immunosuppressive treatments, previous operation or filler injection for the facial wrinkles, trauma to the face, and other comorbidities that might affect wound healing including serious skin diseases, acute infections, diabetes, and liver disease were excluded.

| Nano fat isolation
Initially, an area of excess fat mostly from the lower abdomen (the most reliable collection in males) or lateral side of the thigh (preferably in females) was selected as a donor site.Donor sites were chosen according to fat distributions and preferences of the patients.After prep and draping, a shallow-depth injection of lidocaine 2% with a fine needle of 27-gauge was performed at the entrance points of the cannula.The infiltration cannula is a blunt-tipped millimeter diameter device with multiple side openings connected to a 10 or 20-cc syringe.Subsequently, fat (nearly 10 cc) was harvested after infiltrating with about 250 cc tumescent solution including 1 mg epinephrine 1:1000, 1000 cc normal saline 0.9%, and 25-ml lidocaine 2%.The harvested fat was irrigated by physiological saline solution and then mechanically emulsified by shifting it between two 10-cc syringes that were connected using a female-to-female luer-lok connector.
After nearly 30 continuous passes, the harvested fat was changed into an emulsified liquid which is a translucent liquid that is rich in high-quality mesenchymal stem cells without any viable adipocytes.
Then, this emulsion was filtered through a sterile nylon strainer with 0.5 mm pore size to obtain nano fat that could easily pass through the sharp 1 cc needle (27G).

| Nano fat transplantation
The patient's face was anesthetized with topical lidocaine (Tehran Chemie Xyla-p Cream 30 gr, Iran) 45 min before nano fat injection.
After disinfecting the skin with betadine-impregnated gauze, the processed fat was injected intradermally with cannula Gauge 27, 1 cc in the fine wrinkles of the forehead utilizing multipoint and layering methods.Typically, 1 cc of nano fat can cover a 1 × 1 cm area.The appearance of yellowish discoloration of the skin during the injection process indicated the endpoint of the injection.To avoid missed injections into the blood vessels, the syringe plunger was aspirated prior to each injection.After this procedure, gentle local massages were applied to ensure optimal nano-fat distribution.Eventually, topical tetracycline oint 3% (Iran Nazho Pharmacy) was applied to the area and then covered by a dressing.Also, possible side effects like erythema, edema, ecchymosis and infection were explained to the patients.

| Follow-up
Follow-up visits were scheduled 3 , 7 days, and 7 months after the operation for all 15 patients.Seven months follow-up was chosen based on our patients' ease of referring to our clinic, reviewing other studies in this field, and also considering the observed peaking effect time of nano-fat between 4 and 6 months after injection. 16,17e first two visits were allocated for examining operation sites and addressing patient's complaints; and the third one was for taking photos and documentation.Images were taken by Visioface 1000D (CK electronic, manufactured in Germany) in three different views (front, left, and right), immediately before and 7 months after operation under the same condition for comparison.This device is capable of measuring volume, area, depth, and percent area as four parameters of the marked wrinkles which were quantitatively compared before and after the treatment.The authors evaluated each two wrinkles from the same view for all patients.To compare the mean of the quantitative parameters before and after intervention, the paired t-test or its non-parametrical equivalence was used.The p-value of less than 0.05 was considered as statistically significant.

| RE SULTS
A total of 15 individuals, consisting 11 females and four males, were recruited to the study and attended all three follow up visits.The mean age of the participants was 44.93 ± 12.05 (SD) years, ranging from 31 to 64.The mean volume of the wrinkles was 49.24 ± 29.9 px 3 (SD) ranging from 13.22 to a maximum of 152.31.whereas, the mean volume decreased to 37.32 ± 24.56 px 3 (SD) after 7 months.
The mean area and depth of wrinkles were also decreased by 0.74 and 0.8 respectively.Measured values of volume, depth, area, and percent area of the wrinkles before and 7 months after the intervention are summarized in Table 1.Analysis of Visio face results demonstrated that nano fat injection can significantly decrease volume, depth, area, and percent area of skin wrinkles (p <0.05).
The amount of decrease in measured values was compared between both groups (each group was considered as less and more than the mean of each variable).The results show that patients with wrinkles higher in volume, depth, and percent area (group B) experienced significantly greater improvement compared with the other group (Table 2, p-value <0.05).interestingly, gender difference was only contributed to percent area changes that were more significant in male patients (mean: 0.11 ± 0.09) than female patients (mean: 0.04 ± 0.06) with the p-value of 0.02. Figure 1 shows the evaluation of the volume, area, depth, and percent area on the same wrinkles with Visio face 1000D, before and 7 months after nano fat injection.
Additionally, Figure 2 shows the post injection area in two patients.

| DISCUSS ION
With increase in demand for facial rejuvenation among general population, more studies are aimed at improving and developing optimal aesthetic therapies to reverse age-related alterations. 18Nano fat which is rich in ASC has the potential to trigger soft tissue remodeling and result in satisfactory rejuvenative effects on aging skin. 19,20e present study investigated the effect of nano fat injection on fine facial wrinkles by analyzing the quantitative data obtained from measurement of the volume, depth, area, and percent area changes of wrinkles 7 months after intervention.According to the results of our study, fine facial wrinkles improved significantly in all measured parameters (volume, depth, area, and percent area) after nano fat grafting and demonstrated pleasing clinical results.Authors found that wrinkles with higher percent area, depth and volume loss show greater improvement after the treatment.Our results indicate that males and females benefit the same from this treatment except for the percent area parameter that was improved more significantly in male patients.2-4 weeks and maintained till 6 months.The authors concluded that facial rejuvenation with subcutaneous nano fat injections is effective and safe and also has lifting effects. 14 a part of a study by Uyulmaz et al they applied nano fat grafting method on six patients with wrinkles to evaluate its efficacy; consistent with our findings the results of their study led to the conclusion that this method has beneficial effects as wrinkles appeared less prominent. 21In a novel study by Liang et al, impact of nano fat in combination with platelet rich fibrin on facial rejuvenation was investigated and compared with hyaluronic acid injection as control.The results showed higher satisfaction rate of patients treated with nano fat-PRF, thus authors concluded it was a highly effective method for skin rejuvenation. 22In another study by Pattayadeekul for surface evaluation of wrinkles after 6 months of treatment revealed statically significant decrease in wrinkles. 23Also, in a study by Mohammed S. et al 20 patients with scars, wrinkles and dark hallows under eyes were treated with nano fat injection, among them, most satisfaction was from patients with scars and wrinkles. 24Moreover, results from a study on effects of nano fat on photoaging mice skin  revealed improvements by reducing wrinkles and increasing in dermal collagen thickness. 25Similarly, another study on chronologically aged mice skin confirmed wrinkle-reducing effects of nano fat. 26e observed improvements in skin quality after application of nano fat grafting technique could be attributed to abundant adipose tissue derived stem cells present in nonafat and its capacities.AScs have self-renewal ability and paracrine properties with different functions which leads to angiogenesis, production of dermal collagen, and tissue remodeling and regeneration. 27,28Literature suggests that they also have antiapoptotic and anti-oxidative effects. 28,29Skin histological analysis performed after using this technique in clinical studies confirms the mentioned changes in skin tissue. 14,30Taken together, through mentioned mechanisms, nano fat has the capability to smooth out wrinkles and offer a more youthful skin.
On the other hand, XIAO et al assessed the clinical impact of nano fat on superficial rhytids of 86 cases, 14 of them had wrinkles on their forehead, after 1 and 6 months post treatment and reported high satisfactory rate(above 80 percent) among both doctors and patients; however, some patients were not satisfied by the results. 31 a similar study by Fakih-Gomez et al, they treated 20 female patients by intradermal injection of nano fat in all their facial regions to assess the impact of nano fat on skin's smoothness, pores, wrinkles, erythema and hyperpigmentation. XIAO et al study, patients and doctors evaluated the changes and rated their satisfaction using standard photographs that were taken before and after the procedure. 31Fakih-Gomez et al assessed grade of skin Improvement by asking patients to fill a questionnaire in this regard sixth months after the intervention. 32consistent with our results which showed a significant reduction in wrinkles' volume after the treatment, the study of Mesguich Batel et al on four patients with peri-oral wrinkles showed not significant decrease in volume of wrinkles which was measured by standardized photography and also 3D microphotography.They concluded that clinical efficacy of this novel treatment should be investigated by additional studies. 33These conflicting results may in part be due to different study samples, location of wrinkles and their initial depth.
Ultimately, to the best of our knowledge there are few numbers of studies which investigated the effect of nonafat alone on fine wrinkles and quantified the skin improvement results.Thus, the findings of this investigation complement those of earlier studies.
Although present study illustrated nano fat grafting technique's capability to attenuate fine facial wrinkles, further studies with greater sample size with equal male to female ratio is suggested to provide more accurate literature.The present study assessed the treatment using Visio face 1000D and there were no other efficacy assessments.Moreover, an issue that was not addressed in this research was comparing patients treated with nano fat with a control group.This is an important issue for future research to assess the costeffectiveness of this novel method with other available alternative methods.

| CON CLUS ION
The evidence from this study suggests that intradermal injection of autologous nano fat into fine facial wrinkles considerably refine them in every measurable aspect.According to promising and satisfactory clinical results of this study, it strengthens the idea that nano Statistical analysis was carried out using the statistical package for social sciences (SPSS) (IBM Corp.Released 2016.IBM SPSS Statistics for Windows, Version 23.0.Armonk, NY: IBM Corp.).Quantitative variables were described using mean ± standard deviation.Measured values of each parameter before intervention were classified into two groups based on the mean of each parameter.Values less and more than the mean of each parameter were classified as group A and B. Two groups were compared by independent sample t-test.
In this regard, accumulating lines of studies in support of nano fat efficacy on facial rejuvenation has been published recently.Menkes and colleagues performed nano fat grafting in facial skin on 50 patients (2 men and 48 women) with age range of 35-65 years.They found visible (objective) and subjective improvement appeared after TA B L E 1 Comparison of the biometric characteristics of wrinkles before and seven months after the intervention according to the paired T-test.
et al, authors explored the efficacy of autologous stromal vascular fraction, which is abundant in nano fat emulsion, on infraorbital skin rejuvenation of 19 patients.They assessed the skin surface via Vi-sioScan camera (Courage+Khazaka Electronic) 3 and 6 months after treatment and compared it with the baseline parameters.The results

TA B L E 2 F I G U R E 1
Study population classification based on their wrinkles' parameters.Two groups were compared by independent sample T-test.The evaluation of the measured parameters on the same wrinkles with Visio face 1000D, before (photos 1, 2, and 3) and 7 months after nano fat injection (photos 4, 5, and 6) in two cases.

F I G U R E 2
Pre (left side) and postinjection (right side) areas in two cases.