A personalized nutrition plan based on genetic profile improves outcomes of facial regeneration with Platelet-Rich Fibrin liquid matrices

Aim: The importance of nutrition in the prevention of skin aging has been shown by large observational studies. However, there are no studies assessing dietary changes as adjunct procedures to aesthetic interventions. The objective of this study was to assess whether a personalized nutritional plan conveys additional benefits to platelet-rich fibrin (PRF) facial regeneration. Methods: Forty-seven healthy women (mean age 52.5 years old, SD = 7.7) were offered minimally invasive facial regeneration with the use of PRF liquid matrices, as well as a personalized nutritional plan. The nutritional plan was informed by a nutrigenetic test based on 128 polymorphisms. Horizontal forehead lines, zygomatic wrinkles or mid-cheek furrows, nasolabial folds, perioral expression wrinkles, and marionette line were assessed separately with the use of the Facial Wrinkles Assessment Scale (FWAS).


INTRODUCTION
Over the last decade, the use of autologous blood concentrates, such as platelet-rich plasma and platelet-rich fibrin (PRF), has gained importance in aesthetic medicine for dermal stimulation, augmentation, and rejuvenation [1][2][3] .Platelet-rich fibrin liquid matrices based on the low-speed centrifugation concept have shown effectiveness in facial regeneration [4,5] and are being increasingly applied in facial aesthetics either alone or in combination with other techniques.At the same time, studies of cells in the laboratory, animal models, and human trials also support roles for a variety of nutrients in preventing skin aging [6] .Large observational studies have suggested associations of vitamin C, linoleic acid, and green and yellow vegetables with younger-looking skin [7,8] , indicating the need for more holistic approaches that incorporate nutritional advice into aesthetic treatments.
Personalized nutrition plans informed by knowledge of an individual's DNA are increasingly being utilized for health as well as general well-being purposes.There are numerous studies, particularly in the fields of preventive medicine and cardiovascular diseases, showing that the specification of general dietary recommendation by the inclusion of genetic variants can have a significant impact on health parameters.At the same time, there are thousands of publications based on large cohort or other retrospective studies indicating that the exact amounts of the various nutrients can be specified, among others, by genotype results.Adherence to a nutritional plan that conforms to standard dietary guidelines as well as considers the various individual needs may improve healthy skin parameters.In this study, we sought to investigate whether a personalized nutrition plan conveys additional benefits to a minimally invasive technique for facial regeneration.

METHODS
In total, 47 otherwise healthy women offered PRF facial regeneration based on the low-speed centrifugation concept were included in the study.An ethical approval was granted (acknowledgments No. 1/26/09/2016), and the patients signed an informed consent prior to their treatment.All patients were treated by the same experienced physician (Nacopoulos C) using a standard protocol described in more detail in previous publications [4,5] .Briefly, four sessions of PRF treatments were offered with 2 to 3-week intervals.In each session, 60 mL of venous blood was collected in 10 mL PRF tubes (Orange tubes, Process for PRF, Nice, France) and centrifuged according to the following protocols: 40 mL (four tubes) for 5 min at 1300 rpm (208 g), which results in 10-11 mL of PRF, and 20 mL (two tubes) for 3 min at 700 rpm (60 g), which results in 2 mL of PRF.
A preprogramed centrifuge with a radius of 110 mm (Process for PRF, Nice France) was used to produce a total of 12-13 mL PRF of reduced density.PRF liquid matrices produced by the two abovementioned centrifugation protocols were mixed and a full-face injection technique was applied as described previously [5] .
Α personalized nutritional plan was offered by a specialized nutritionist during the treatment.A nutrigenetic test was utilized to aid personalized nutritional recommendations.In more detail, prior to the first PRF session, 5 mL of additional vein blood was collected for genomic DNA analysis using a Pure Link Genomic DNA Mini Kit Assay (Life Technologies), according to the manufacturer's instructions.

SNPs
SNPs were genotyped using the TaqMan assays, provided by Life Technologies.We performed genotyping on the Open Array™ SNP Genotyping System (Life Technologies, Carlsbad, CA, USA) using microscope slide-sized plates and the OpenArray Accufill autoloader (Life Technologies, Carlsbad, CA, USA), following the manufacturer's instructions.
Adaptation of the nutritional recommendations was assessed with a simple questionnaire provided at the time of facial reassessment.In more detail, participants were asked whether they adapted any dietary changes ("none", "some", or "all or the majority") and for how long ("1-3 weeks", "1-2 months", "3-4 months", or "5-6 months").An at least partial adaptation of recommendations for a three-month period or longer was regarded as adherence to dietary recommendations [Supplementary Table 1].
Horizontal forehead lines, zygomatic wrinkles or mid-cheek furrows, nasolabial folds, perioral expression wrinkles, and marionette line were assessed separately with the use of the Facial Wrinkles Assessment Scale (FWAS), a simple and reliable tool for the assessment of wrinkles [38] [Figure 1].Two authors (Nacopoulos C and Vesala AM) blinded to "adherence on recommended diet" scored the scales prior to initial intervention and at a follow-up visit, which took place about six months after the completion of the four PRF sessions.

Statistical analyses
Study participants were divided into two groups: those who reported an at least partial adherence to dietary recommendations for the majority of the time (Group A) and the rest (Group B).Changes in FWAS scores were compared with Mann-Whitney U Test. Unpaired t-tests were utilized for normally distributed variables and chi-squared test for nominal data.

RESULTS
In all, 43 out of 47 patients completed the study.Two patients ceased treatment, one for financial reasons and the other due to the level of perceived pain associated with treatment.Another was lost in the follow up.
The fourth patient reported a health problem (gynecological cancer) which has not been reported during the PRF treatment and was excluded from the study.
Group A (subjects who reported an at least partial adherence to dietary recommendations for the majority of the time) consisted of 28 women and Group B of the remaining 15 women [Supplementary Table 1].Demographic characteristics, total FWAS scores, and their changes for each group are presented in Table 2.
An improvement in FWAS scores was recorded in both groups.However, as shown in Table 2, improvement in total FWAS scores was statistically more significant in Group A (adherent to dietary recommendations) (Z = 2.4, P = 0.008).

DISCUSSION
Blood concentrates are increasingly being utilized as autologous products for aesthetic purposes because they contain platelets for growth factor release, fibrin scaffold for tissue remodeling, plasma proteins for collagen synthesis, white cells for inflammation and recovery, and stem cells for tissue repair and regeneration.Their outcomes regarding rejuvenation of photo-aged facial skin have been shown in small trials [4,39] .
This study was not intended to verify previous positive outcomes.In other words, whether surgeons' reported improvement in wrinkles scores is a biased or a true effect is beyond the scope of this observational study.A more appropriately designed study for this purpose has already been reported previously with positive results [4] .The current study was performed with the intention to see whether a personalized nutritional plan has an impact on PRF facial regeneration outcomes.For this reason, scorers were blinded to adherence to this plan.Thus, the additional skin aesthetics benefits conveyed by the dietary recommendations cannot be attributed to observer bias.
In addition, a relatively long-term adherence to a nutritional plan may be related to factors such as age, smoking, or smoking cessation.All patients initially visited the clinic to have a minimally invasive treatment.Personalized nutrition was offered as an add-on treatment.This may explain the relatively high percentage of the subjects (15/43) who opted not to adhere to a nutritional plan.Since a dietary change for a few days is unlikely to convey any skin changes in the long term (e.g., after six months), we only included those who sustained the recommended diet for at least half the study period.In this context, a difference between the aforementioned factors (age and smoking) is possible, can influence skin appearance and aesthetic results, and needs to be tested in pragmatic studies in which predefined strict entry and exclusion criteria are difficult to apply.Nevertheless, a statistically significant difference in relation to these factors was not noticed between our study groups.
An increasing amount of evidence suggests a protective effect of "healthy diet" on skin aging.Current dietary recommendations promote higher intakes of fruits, vegetables, fish, and PUFAs (polyunsaturated fatty acids) [40,41] .Nevertheless, several studies over the past few years have shown that what constitutes a healthy diet for an individual depends to some extent on his or her physiology and lifestyle [42][43][44][45] .Individualized nutrition advice, informed by knowledge of genetic variants, is increasingly favored over standard dietary guidelines based on population-wide averages [46] .Whether a personalized approach based on genotyping results conveys additional benefits over current nutritional recommendations in skin aesthetics, obesity, and other health issues is under study by various teams around the world.Regarding facial skin aesthetics, this study conforms to previous ones indicating a beneficial mid-or long-term effect of healthy nutrition [7,8] .In our cases, the inclusion of genetic variants helped the specification of general dietary recommendation that quite often propose a wide range of concentrations regarding macro-and micronutrients.This means that our personalized nutritional plans conformed to a "healthy diet" and the proposed optimal nutrition intake to stave off skin aging.In addition, the exact amounts of the various nutrients were further specified based on the genotype, as exemplified above.

Financial support and sponsorship
None.

Conflicts of interest
Gkouskou K is the owner of Embiodiagnostics laboratory that provides a range of comprehensive genomic services to assist individuals to reach decisions on diet and lifestyles based on their DNA profile.The

Figure 1 .
Figure1.Horizontal forehead lines, zygomatic wrinkles or mid-cheek furrows, nasolabial folds, perioral expression wrinkles, and marionette line were scored.The first case had deeper wrinkles with a medium score of 4 and despite her weight loss an improvement can been seen in all but perioral lines.In the second case an improvement from a score of 3 to a score of 2 has been marked only for forehead lines (patients have provided consent for publication of their photos).

Table 1 . Examples of single nucleotide polymorphisms and related recommendations Pathology or health issue SNP Genotype-related health effect Recommendation
SNP: Single nucleotide polymorphism.