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].

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 CO₂, two dilutions of NCTF 135HA^Ò (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.

Collagen I was significantly increased by 132.2% and 114.5% in the presence of NCTF 135HA^Ò diluted at 1/100 and 1/1000 compared to control group (244.9 ± 120.1, 226.2 ± 97.6 versus 105.5 ± 28.6 µg/millions of cells respectively; p < 0.05).

The non-cross-linked hyaluronic acid has a positive effect on the contractile force of aged fibroblasts. This study was designed to evaluate if this molecule could maintain this characteristic after combination with the polyrevitalizing complex.

1) Obtaining and amplification of fibroblasts

The study performed by Bioexigence Laboratory at Besançon, France. Normal aged fibroblasts (NF) and wrinkle fibroblasts (WF) were obtained from an operative waste (lifting) of a woman (63 years old). Classical explantation technic is used to the extraction of normal fibroblasts and wrinkle fibroblasts. Explants were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal calf serum, 40 mg/l of gentamicin and 2 mg/l of fungizone (DMEMc), in an incubator at 37˚C, 5% CO₂ and 95% air. In few days, fibroblasts migrated outside the explants. Culture medium was changed twice a week and cells are subcultured by trypsinization when confluence is reached. Cells were used under 10 passages.

2) Study of contractile forces in GlasBox^Plusâ device

The GlasBox^Plusâ device is composed of a cell chamber with eight rectangular culture wells in which dermal equivalents develop. Two opposite beams hung down into each well at a distance of 27 mm apart (Figure 1). The dermal equivalent is attached to this sensor through a grid directly etched on the lower part of each beam. The beams have an optical system to detect their own deformation. This deformation is proportional to the contractile forces developed by dermal equivalent. The signal is collected by a computer, which include a specific program to give directly the forces in real time.

Fibroblasts are resuspended just before confluence with trypsin-EDTA (1X) solution. Fibroblasts are embedded three-dimensionally in hydrated collagen gels using a modified version of the technique developed by Bell et al. [12]. Briefly the mixture is composed of: 6 volumes of 1.76X (DMEMc, NaHCO₃, NaOH, antibiotics), 3 volumes rat tail type I collagen (2 mg/ml) and 1 volume of cellular suspension (8 × 10⁵ cells/ml). This device is a reference for measuring the function of the fibroblasts (contractile forces) since 15 years [13] [14] [15] [16]. The dermal equivalent mixture is poured into the rectangular culture plate of the GlasBox^Plusâ and polymerized in less than 30 min at 37˚C. Culture medium with and without NCTF 135 HA^Ò is added and the GlasBox^Plusâ is then placed into a humidified incubator at 37˚C, and forces measurements are started after 30 minutes of stabilization, for 24 hours.

Results were expressed in arbitrary unit. GlasBox^Plusâ curves were drawn and fitted to obtained maximum of contraction and area under the curve.

As attempt, the contractile forces were significantly decreased in wrinkle fibroblasts compared to normal fibroblasts. Contractile forces were significantly increased with NCTF 135HA^Ò 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^Ò compared to wrinkle fibroblasts (Figure 3).

As the NCTF 135HA^Ò 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).

The objective of this study was to evaluate if the combination of HA and polyrevitalizing component has still an effect on the glycomanioglycans content of the skin as the previous studies demonstrated that hyaluronic acid lonely increases this content [11].

Fragments of skin from 6 donors (plastic surgery) were placed in inserts of 3 cm² positioned in culture wells with culture medium. 10 µl of NCTF 135HA^Ò were injected in the skin (10 µl multiple points, about 10 impacts for 3 cm²) 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). The GAGs of the skin (with hyaluronic acid) were stained in blue by Hale staining in histologic section. A semi-quantitative scoring of the intensity of staining in epidermis, superficial, mild and deep dermis was made (scores 0: no staining, 1: light staining, 2: moderate staining, 3: important staining and 4: very important staining).

A significant decrease of roughness score was observed after 3 days of treatment compared to day 0 (0.28 ± 0.15 versus 0.40 ± 0.16, p < 0.05).

No statistical significant difference was observed for GAGs content in the epidermis and superficial dermis level compared to baseline (day 0) after injection of NCTF 135HA^Ò. However, after 2 injections at day 0 and 3, the GAGs content seems to be increased in the deep dermis compared to Day 0 (Table 1) (Figure 5).

The non-crosslinked HA existing in the formula keeps its positive effect on GAGs contents even by adding this biorevitalizing component. There is no data supporting that the presence of this component increase this positive effect versus a pure non-crosslinked HA.

Fragments of skin from 8 donors (plastic surgery) were maintained alive at 37˚C in phosphate buffer solution. 200 µl of NCTF 135HA^Ò or physiological serum were injected into the dermis. Acquisitions with fringe projection were performed before, just after the injection (T0), every hour during 8 h and after 24 h. 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.

Data presenting the anti-AGE production with NCTF 135HA^Ò after 1 and 7 days with Collagen and Albumin. AGE, Advanced Glycated End-Product; D, Day.

The ratio volume/area significantly decreased with time after either NCTF 135HA^Ò or negative control (normal saline) injection. The ratio was significantly higher with NCTF 135HA^Ò 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).

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.

The objectives of this study were to evaluate if the injection by an automated device through different injection methods (point to point or nappage) would have any result on the collagen and elastin contents in the different levels of the skin.

Fragments of skin from 5 donors (plastic surgery) were maintained alive in culture medium at 37˚C and 5% CO₂. Fragments were irradiated with UVA (8 J/cm²) and UVB (2 J/cm²) and treated or not with HA + Polyrevitalizing component. The product was injected with U225 meso-injector (Biophymed^Ò) 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.

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^Ò treatment in UV induced aged skin in different skin layers (Table 2).

The primary objective of this study was to evaluate the efficacy of NCTF 135HA^Ò 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.

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^Ò, 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.

Data presenting the mitotic index after 3 days of culture of skin fragments irradiated or not (UVA and UVB) and with and without cocktail. Elastin and Collagen were evaluated after 14 days of culture. UV, Ultraviolet; HA, hyaluronic acid.

- Stabilization Phase: The last treatment session took place on D75, with clinical scoring and photographs of the subjects study zones before the treatment.

- The end-of-study assessment visit took place on D90, where the last series of evaluations as well as photos was taken.

Several NCTF 135HA^Ò 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.

Results were assessed by clinical scoring and using photographs taken with a high-quality digital camera. Primary outcome measures, scored 0 to 3 at each visit, were as follows:

- Severity of wrinkles and fine lines (0 = none, 1 = not very marked, 2 = marked, 3 = very marked);

- Skin tonicity (0 = poor, 1 = average, 2 = good, 3 = excellent);

- Skin hydration (0 = poor, 1 = average, 2 = good, 3 = excellent);

- Skin radiance (0 = poor, 1 = average, 2 = good, 3 = excellent);

- Secondary outcome measures were as follows:

- Assessment of ease of injection by the investigator (0 = very satisfied, 1 = satisfied, 2 = unsatisfied, 3 = very unsatisfied);

- Investigator’s satisfaction with the product’s delivery form (0 = very satisfied, 1 = satisfied, 2 = unsatisfied, 3 = very unsatisfied);

- Improvement of skin appearance at the end of treatment assessed by the investigator (0 = no improvement, 1 = little improvement, 2 = improvement, 3 = marked improvement, 4 = very marked improvement);

- Overall satisfaction of the investigator at the end of treatment (0 = very satisfied, 1 = satisfied, 2 = unsatisfied, 3 = very unsatisfied);

- Overall satisfaction of the patient at the end of treatment (0 = very satisfied, 1 = satisfied, 2 = unsatisfied, 3 = very unsatisfied);

- Any adverse events observed following the injections (erythema, edema, ecchymosis) were reported at each visit and rated 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe).

1) Demographic description

40 female subjects with an average age of 51.5 years (29 to 80 years) were recruited by 17 practitioners with different specialties (dermatologists, aesthetic medicine specialists and plastic surgeons). These patients had obligatory the established wrinkles on the face and/or neck and/or bust-line according to the visual clinical scoring. Hands were treated only in one patient.

Subjects received all injections and underwent all control assessments scheduled by the study protocol over a period of 3 months. Two withdrawals were recorded (subjects 16 and 21) because of “lost to follow up”. There were no cases of signs of local skin intolerance warranting postponement or cancellation of the next injection. Subjects skin which was otherwise healthy with wrinkles was treated in the following zones: 47% on the face only, 12% on the face, neck and bust-line, 12% on the face and neck, 10% on the face and bust-line, 10% on the bust-line only, 3% on the neck and bust-line, 3% on the neck and hands and 3% on the neck only.

2) Wrinkle score

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).

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.

3) Skin Tonicity

Mean tonicity score increased significantly in repair phase (1.9 vs 1.0, +80%, p < 0.05) and stabilizing phase (2.1 vs 1.0, 110%, p < 0.05) but not in initial phase (Figure 7).

4) Skin Hydration clinical scoring

Mean hydration score increased significantly in all phases: initial phase by 30% (1.3 vs 1.0, p < 0.05), repair phase by 110% (2.1 vs 1.0, p < 0.05) and stabilizing phase by 130% (2.3 vs 1.0, p < 0.05) (Figure 7).

5) Skin Radiance clinical scoring

Mean radiance score increased significantly in all phases: initial phase by 40% (1.4 vs 1.0, p < 0.05), repair phase by 110% (2.1 vs 1.0, p < 0.05) and stabilizing phase by 130% (2.3 vs 1.0, p < 0.05) (Figure 7).

6) Subjects satisfaction score

A visible skin improvement was observed by 97% of subjects: 18% Very marked improvement, 37% Marked improvement, 42% Improvement, 3% Slight improvement and 0% No improvement.

The satisfaction rate by subjects reported 97%: 55% very satisfied, 42% satisfied and only 3% of subjects were unsatisfied.

The primary objective of this study was to evaluate the efficacy of NCTF 135HA^Ò 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.

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^Ò 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).

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^Ò 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).