Efficacy of a Biorevitalizing-Filler Solution on All Skin Aspects: 10 Years Approach through in Vitro Studies and Clinical Trials

Introduction: Skin aging is the result of many cellular dysfunctions over time particularly the fibroblasts and the keratinocytes. These dysfunctions could be decelerated by the preventive effects of some skin treatments such as Intradermal injections. NCTF 135HA has a polycomponent formulation designed to improve the efficacy of non-cross linked hyaluronic acid (as a micro-filler) on fibroblasts function. Although NCTF 135HA has been used by aesthetic practitioners since 20 years, we have analyzed all in vitro, ex vivo and in vivo studies during past 10 years in order to summarized its anti-aging effect. Methods: In these series of studies, the known effects of HA have been evaluated with this boosted formula. Collagen I synthesis, antiglycation effect and contractile forces developed by fibroblasts were studied. Fibroblasts and keratinocytes proliferations were evaluated in monolayer cells culture. The filling effect thanks to NCTF 135HA injection was ex vivo performed by fringe projection and the protective effect of NCTF 135HA against solar irradiation was performed ex vivo to study proliferation rate, elastin and collagen expression. Two clinical trials were performed on women to evaluate the anti-aging effect of NCTF 135HA injection. Hydration, firmness, radiance, wrinkles size, pore size, thickness and density of the dermis were analyzed. How to cite this paper: Robin, S., Fanian, F., Courderot-Masuyer, C., Tordjman, M., Braccini, F., Boisnic, S., Philippon, V., Vincent, A.G., Salomon, C., Manfait, M., Humbert, P. and Piccerelle, P. (2021) Efficacy of a Biorevitalizing-Filler Solution on All Skin Aspects: 10 Years Approach through in Vitro Studies and Clinical Trials. Journal of Cosmetics, Dermatological Sciences and Applications, 11, 18-37. https://doi.org/10.4236/jcdsa.2021.111003 Received: November 27, 2020 Accepted: March 5, 2021 Published: March 8, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Our knowledge of the skin as a complex, immune, multifunctional organ is constantly evolving, including our insights into the skin aging process [1]. Skin aging can be due to intrinsic and extrinsic processes, which contribute simultaneously to a progressive loss on skin integrity. Intrinsic factors were genetically determined and extrinsic factors were due to lifestyle and environment. Structural stability as well as physiological function is affected. Physiological changes in aged skin include structural and biochemical changes. The skin thins progressively over adult life at an accelerating rate [2]. The epidermis decreases in thickness particularly in women and particularly on the face, neck, chest, hands and forearms [3]. A reduction of the water content on the skin is also observed [4]. Dermis thickness decreases with age [1]. Skin aging can largely be attributed to dermal fibroblast dysfunction and a decrease in their number and biosynthetic activity. The amount of glycosaminoglycans on the dermis declines with age, as does the amount of hyaluronic acid produced by fibroblasts [5]. Aging is inevitably associated with a decrease in collagen turnover (due to a decrease in fibroblasts and their collagen synthesis) as well as elastin [6]. Reactive oxygen species (ROS) as a by-product of cellular metabolism due to oxidative stress generated by ultra-violet, tobacco, pollution exposure… ROS cause damage to critical cellular components like membranes, enzymes and DNA.
Aesthetic Medicine specialists (dermatologists, plastic surgeons, aesthetic doctors, etc.) are increasingly called to offer solutions that respond to the constant request for "eternal youth". Often not to use more heavy invasive surgical techniques such as dermabrasion, lifting surgery and laser resurfacing, today S. Robin et al. there is big tendency to use the injectable products to reduce or erase wrinkles and skin depressions, as well as to remodel certain parts of the body [7]. Intradermal multi-injection is an injection method based on multipuncture, to inject the treatment solution exactly where they have to be efficient, it means "the small quantities at the right place". It has more recently been used in aesthetic dermatology, where there has been an upturn in interest of this method due to the development of cutaneous anti-aging treatments [8].
Intradermal multi-injection is a technique that involves micro-injections of products such as hyaluronic acid, with or without vitamins, minerals and amino acids into the superficial layers of the skin [9]. This allows active and essential ingredients to come directly into contact with the dermal fibroblasts and keratinocytes that are key to the more favorable appearance of younger skin and have a beneficial effect on metabolic processes [10].
NCTF 135HA  is a CE-labelled class III medical device. Although it has been used since 1978 by the practitioners in France, it has been freely marketed in the European Union since September 2007 after obtaining his CE marking. This product is one of the pioneer products in this field and there are many similar products produced based on its formulation. NCTF 135HA  is a hyaluronic acid (HA) based dermal filler indicated for revitalization and intense hydration of tired or dull skin, the filling of superficial wrinkles and the re-plumping of mature skin or skin that lacks firmness.
As it has been used widely by aesthetic practitioners as one of the most widely used polyrevitalizing solution since decades, we have decided to analyze all in vitro, ex vivo and in vivo studies on this product during past 10 years in order to verify its proved effects on different aspect of the skin.

Product
NCTF 135 HA  contains two components: -15 mg of non-cross-linked hyaluronic acid obtained through biotechnological processes. It is a linear polysaccharide identical to the HA present in connective tissue; -Polyrevitalizing component, a biological solution developed to support the HA and its effects on the tissue. It is mainly composed of 5 groups of ingredients: vitamins, amino acids, mineral salts, coenzymes and nucleic acids.
This product is available in the form of the sterile and ready-to-use vials of 3 ml, sterilized by double filtering method.

Objectives
The objective of this study was to evaluate if the HA boosted by a polyrevitalizing component has still a positive effect on Collagen I production, the effect which has been already proved by HA alone [11]. J. Cosmetics, Dermatological Sciences and Applications

Materials and Methods
The study performed by Bioalternatives Laboratory at Gençay, France. Normal human fibroblasts from 4 donors were seeded in 12-wells plate. After one day of culture at 37˚C with 5% of CO 2 , two dilutions of NCTF 135HA  (dilutions 1/100 and 1/1000) were added or not to the wells. After 48 h of culture, the supernatant was collected for collagen I determination. Collagen I was determined by spectrophotometry using Sirius red (540 nm). Results were expressed in µg/millions of cells.

1) Obtaining and amplification of fibroblasts
The study performed by Bioexigence Laboratory at Besançon, France. Normal  [12].
Briefly the mixture is composed of: Results were expressed in arbitrary unit. GlasBox Plus curves were drawn and fitted to obtained maximum of contraction and area under the curve.

Results
As attempt, the contractile forces were significantly decreased in wrinkle fibroblasts compared to normal fibroblasts. Contractile forces were significantly increased with NCTF 135HA  compared to WF and significantly decreased compared to NF ( Figure 2). Area under the curve and maximum of contraction were significantly increased with NCTF 135HA  compared to wrinkle fibroblasts ( Figure 3).

Discussion
As the NCTF 135HA  guarantees the cells atmosphere as a protective medium without any direct effect on their biologic activities, the fibroblasts remain healthier. This product would provide the same condition as a normal cell medium which has no physiologic effect but only keep the cells in a suitable ambiance to live ( Figure 4).

Materials and Methods
Fragments of skin from 6 donors (plastic surgery) were placed in inserts of 3 cm 2 positioned in culture wells with culture medium. 10 µl of NCTF 135HA  were injected in the skin (10 µl multiple points, about 10 impacts for 3 cm 2 ) with 32G needles in comparison with control skin (untreated). Three injections were performed at days 0, 3 and 7. Photography with Life VizMicro apparatus were made before each injection for roughness analysis.
The skin fragments were fixed in formol and enclosed in paraffin after each injection for glycosaminoglycan analysis (GAGs  5).

Discussion
The non-crosslinked HA existing in the formula keeps its positive effect on Topographic analyses were performed to measure the volume and the area of the papule. The ratio volume/area is an indicator of the height of the papule.

Results
The ratio volume/area significantly decreased with time after either NCTF 135HA  or negative control (normal saline) injection. The ratio was significantly higher with NCTF 135HA  at T0 which remains higher for all study times (1, 2, 3, 4, 5, 6, 7, 8 and 24 hour after the injection) compared to negative control (p < 0.05) ( Figure 6).

Discussion
Although the study product is in liquid form, it has a filling effect on the skin surface compared to the negative control (normal saline). This effect could be explained by the effect of HA in the product.

Materials and Methods
Fragments of skin from 5 donors (plastic surgery) were maintained alive in culture medium at 37˚C and 5% CO 2 . Fragments were irradiated with UVA (8 J/cm 2 ) and UVB (2 J/cm 2 ) and treated or not with HA + Polyrevitalizing component. The product was injected with U225 meso-injector (Biophymed  ) via 2 different methods: point to point (PTP) or nappage.
After 3 days of culture, the mitotic index was evaluated by histological study (KI67). After 14 days of culture, elastin and collagen were determined with image analysis after labelling. Figure 6. Evolution of the ratio volume/area of the papules measured by Fringe Projection. There is a significant difference between the volume of NCTF versus normal saline after 4 hours which remained significant till 24 hours of experience. J. Cosmetics, Dermatological Sciences and Applications

Results
With the accelerating aging model induced by UV radiation, the collagen and elastin content diminished significantly versus the control skin (p = 0.012 and p = 0.02 respectively). However, the elastin and collagen content were significantly increased after injection of NCTF 135HA  treatment in UV induced aged skin in different skin layers (Table 2).

Objectives
The primary objective of this study was to evaluate the efficacy of NCTF 135HA  on consequences of cutaneous aging on face, neck, bust line and hands, by assessing the improvement of wrinkles and fine lines, as well as for hydration, firmness and radiance of skin after 5 sessions of treatment.
The secondary objectives were to evaluate the degree of physicians and subjects satisfaction as well as the tolerance of the product.

Materials and Methods
This "open", multicenter, prospective study was conducted in 17 centers, treating 2 or 3 patients each. Recruitment was to be stopped once 40 patients had been included. Women over the age of 18 years, with no contraindications to use of NCTF 135HA  , were eligible to take part in the study. Patients were to be freely selected by the practitioners. The clinical scoring performed by each investigator on a 4 scale base scoring system (0 = poor, 1 = average, 2 = good, 3 = excellent) for wrinkles, skin hydration, skin firmness and skin radiance.
The total duration of the study was 3 months for each patient, divided into 3 phases: − Initiation Phase: At the inclusion visit on D1, the investigator obtained the subject's written consent, once she had been given the information sheet, and the baseline skin evaluations as well as the first treatment session took place. Photographs were taken of the study zones before and then immediately after the treatment were administered. − Repair Phase: Three treatment sessions took place on D15, D30 and D45. At each session, clinical scoring and photographs were taken of the subjects study zone before the treatment. Several NCTF 135HA  administration techniques could be used, either alone or jointly, during the same treatment session such as manual multiple intradermal injection, nappage technique or injection by an injector. At the end of each treatment session, it was recommended that compression be applied to the treated area for a few minutes to prevent injection marks and that a cooling product be applied to prevent small ecchymosis.
The non-inclusion criteria were any skin disease or damage, patients with a history of autoimmune disease or receiving immunotherapy, patients with known hypersensitivity or allergy to any of the ingredients, pregnant or breastfeeding women. The investigator advised patients not to wear make-up for 12 hours following injection to avoid prolonged exposure to sunlight, UV rays or extreme cold, and not to use saunas or steam rooms for a week following injection. 2) Wrinkle score

Outcome Measures
Results of wrinkles and fine lines scoring are summarized in Table 3. The mean wrinkle score decreased significantly in repair phase (1 vs 1.8, −46%, p < 0.0001) and stabilizing phase (0.9 vs 1.8, −50%, p < 0.0001) but not in initial phase (Figure 7). Figure 7. Evolution of mean wrinkle score, tonicity score, hydration score and radiance clinical score by time from D1 to D90 after 5 sessions of multiple intradermal injections of NCTF. The evolution is significant for all parameter as early as the third injection at D30. Data presenting the evolution of the severity of wrinkles and fine lines during the experimentation. N, Subject number; D, Day; sd, Standard deviation; Min, minimum; Max, Maximum; NS, not significant; S, Significant: ** p < 0.01, *** p < 0.001.

5) Skin Radiance clinical scoring
Mean radiance score increased significantly in all phases: initial phase by 40%
The satisfaction rate by subjects reported 97%: 55% very satisfied, 42% satisfied and only 3% of subjects were unsatisfied.

Objectives
The primary objective of this study was to evaluate the efficacy of NCTF 135HA  on cutaneous aging on the face, neck and bust-line, by assessing the improvement obtained for wrinkles, as well as for skin pore size, thickness and density of the dermis and homogeneity of complexion.
The secondary objective was to evaluate the degree of satisfaction of investigator and subjects as well as the tolerance of the product.

Materials and Methods
20 women (between 35 and 65 years old) were enrolled for this study.
The total schedule duration of the study was 4 months for each patient with: − Inclusion visit at D0, the investigator obtained the subject's written consent, once she had been given the information sheet; − Skin evaluation at D0 (before injection), D15, D30, D60, D90 and D120; − Injection treatment at D0, D15, D30, D60 and D90; The evaluation criteria used were: − Macrophotography with Proscope® for the measurement of pore size in µm, − Cutaneous High Frequency Ultrasound Imageing (HFUI) to evaluate the density and the thickness of dermis; − Chromametric analysis with chromameter Konica Minolta CR/DP-400  to evaluate the skin homogeneity after the treatment. Wrinkle depth by clinical scoring (scores 0 to 4, 4 corresponding to very deep wrinkle); − Skin radiance by clinical scoring (scores 0 to 4, 4 corresponding to dull skin).

Results
1) Demographic description 20 women aged 49.3 ± 9.3 years were enrolled in this study. A good tolerance for the product was observed. Two women came out of the study, the first for medical reason with no relation with the treatment and the second for allergy.

2) Pores size
Pores size was significantly decreased as early as the second injection (D15, p < 0.05) and was continued to the end of the experiment at D30, D60, D90 and D120 (p < 0.001, Figure 8).  3) Density and thickness of the dermis The density and the thickness of the dermis were significantly increased as early as D30 (3 rd injection session, p < 0.01) and was continued to be improved atD60, D90 and D120 compared to D0 (p < 0.001, Figure 9).

4) Skin homogeneity
The skin homogeneity was measured by the difference of the L parameter (in L, a*, b* colorimetric system) between the internal and external area of the cheek. Skin homogeneity was significantly increased (delta of 2 points decreased) as early as the second injection (D15, p < 0.01) to the end of the study at D30 (p < 0.01), D60 and D90 (p < 0.05) and D120 (p < 0.001) ( Figure 10). 5) Wrinkle depth and volume Wrinkle depth measured by profilometric device (Skinstation®) was significantly decreased at D90 and D120 compared to D0 (p < 0.05). In total, the wrinkle depth was decreased by 43.32% after 5 mesotherapy treatments. By the same device, the volume of the wrinkles was significantly decreased at D60 (p < 0.05), D90 and D120 compared to D0 (p < 0.01) after NCTF 135HA  injection ( Figure  11).

6) Skin radiance
The score of skin radiance was significantly increased as early as 15 days of treatment which was continued to increase until the end of the experiment compared to D0 (p < 0.001 for all study visits). In total, the skin radiance was increased by 49.3% after 5 mesotherapy treatments at D120) ( Figure 12). Figure 9. Density and thickness of the dermis has been increased significantly as early as day 30 (after 2 injection sessions) which continued to increase till day 120. Figure 10. Skin homogeneity was increased over time as the difference of L* parameter (L* a* b* system) between two points on the external and internal part of the cheek was diminished significantly as early as day 15 it means even after 1 injection session. Figure 11. Wrinkle depth score and wrinkle volume measured by SkinStation® was diminished significantly at D90 (3 months) and D60 (2 months) respectively.

Discussion
The aim of this study was to show the anti-aging effect of NCTF 135HA  intradermal multi-injections. Skin aging can be due to intrinsic, natural and genetically determined factors, as well as extrinsic lifestyle-driven and environmental factors [1] [10]. It is characterized by a number of objective physical indicators, including skin dryness, laxity and poor elasticity, color and surface irregularity, formation of pronounced skin markings, and wrinkles of different intensity. Fibroblasts represent the major role in producing and maintaining the extracellular connective tissue that is crucial to maintaining the youthful appearance of the skin [17].
The first part of the study was dedicated to the in vitro effect of NCTF in fibroblasts for at least 11 days in culture [18]. They hypothesize that the induction of collagen I degradation by MMP-1 could be counteracted by the concomitant expression of TIMP-1 allowing sustained collagen I production. This argument has been already supported by previous studies suggesting that hyaluronic acid can induce collagen I in fibroblasts [19]. It could thus be speculated that sensitization of fibroblasts by hyaluronic acid is elicited via interaction through various cellular receptors including CD44 [20].
Wrinkle fibroblasts generated less mechanical forces compared to non-wrinkle fibroblasts of the same donors [21]. Skin is physiologically tensed. During aging, strain forces inside the skin decreases and subsequent relaxation are responsible for wrinkles formation. The in vitro study by GlasBox Plus demonstrated that the addition of NCTF 135HA  increase contractile forces generated by wrinkle fibroblasts thanks to the HA component and has probably an anti-wrinkle effect acting on collagen. It is becoming increasingly evident that the optimal function of fibroblasts is critically dependent on their reciprocal mechanical interactions with their surrounding microenvironment [22]. Mechanical tension between fibroblasts and the surrounding extracellular matrix appears to be critical for fibroblast function and normal balanced production of collagen and collagen-degrading enzymes.
Both in vivo studies have shown main advantages of biorevitalizing solutions for skin rejuvenation. Injections were easy to perform with low pain and limited side effects (one subject only was excluded in the first clinical trial on 2011).
Clinical evaluation showed a decrease in wrinkle depth after NCTF 135HA  injections. In the same time, the density and the thickness of dermis were significantly increased after several injections. An upgrade in skin tonicity, hydration and radiance were observed.

Conclusions
Microfillers are the non-cross-linked hyaluronic acid fillers which are boosted by a complex of nutrients such as vitamins, antioxydants, aminoacids, etc. in order to protect the HA component. These microfillers, previously named as "biorevitalizing solutions" have a remarkable accumulating role in preventing the signs of skin aging.
In the past few years, there are many new products in this field but the authors summarized all studies performed during last 10 years on one the pioneer product named NCTF 135HA  as a good representative for this category of products.
In Vitro, ex vivo and in vivo studies showed that this minimally invasive technique could be used in order to improve the skin quality which could have a visual effect on preventing the aging signs. The long term cohorts need to prove this hypothesis.