Five female patients aged 37 to 67 years old (mean age 50.8 years) agreed and provided informed consent to participate in this treatment and analysis.

All patients were non-smokers with Fitzpatrick phototypes I or II, had no history of prior aesthetic procedures in the treated areas, and had not been exposed to excessive sun exposure in the four weeks preceding the study.

The analysis was conducted over a period of 8 weeks. To compare the efficacy of the neuromodulator (BoNTA) alone or in combination with a biorevitalizing solution (NCTF®135HA, FILLMED, France) on the same patient, both sides of the face and neck were treated with BoNTA (Dysport®, Ipsen, France) while only one side was additionally injected with NCTF®135HA. Baseline evaluation was performed at D0, followed by the first injection of NCTF®135HA on one side of the face and neck. BoNTA injections were performed on both sides at D15, two weeks after the initiation of NCTF®135HA. This staggered schedule was based on the clinical rationale that early dermal revitalization may improve skin hydration, fibroblast activity, and overall dermal density before the onset of neuromodulator-induced muscle relaxation. As BoNTA typically reaches its peak effect approximately 14 days post-injection, this sequencing was intended to ensure that muscle relaxation occurred in a potentially improved dermal environment. The authors acknowledge that this sequential design limits the ability to fully isolate the individual contribution of each treatment to the observed outcomes. Additional NCTF®135HA sessions were administered on the same side at D30 and D45 to maintain a continuous regenerative stimulus throughout the observation period. Final evaluation was conducted at D60 (Figure 1). The average procedure duration was 10 - 15 minutes, and no cosmetic care was performed during the study period.

Schematic representation of the split-face and split-neck study design conducted over 8 weeks in five subjects. One side of the face and neck received three sessions of NCTF®135HA injections at D0, D30, and D45, while botulinum toxin (BoNTA) was injected on both sides at D15. Clinical and instrumental evaluations were performed at baseline and at D60.

Figure 1. Study design and treatment protocol.

BoNTA (Dysport®, Ipsen, France) was reconstituted with 2.5 mL of normal saline (0.9% NaCl) per vial. For crow’s feet, injections were administered superficially at three points per side: Point 1, 1 cm lateral to the orbital rim directly below the tail of the brow; Point 2, 1 cm lateral to the orbital rim in line with the lateral canthus; and Point 3, 1 cm lateral to the orbital rim and 1 cm below Point 2. Each point received 10 IU, for a total of 30 IU per side. For platysma injections, bands were marked during dynamic contraction. Two rows of three injection points were placed 1 cm apart along each band, with 5 IU administered per point, for a total of 30 IU per side.

NCTF®135HA (FILLMED, France) was administered from a 3 mL vial using a 3 mL syringe and a two-step technique. After antiseptic cleaning of the face and neck, the first step used a 30G × 12 mm needle at an angle of less than 10 degrees to deliver very superficial epidermal micro-injections approximately 0.5 cm apart across the treatment area, with only the bevel of the needle inserted and gentle pressure applied to the plunger throughout. The second step used a 32G × 4 mm needle at a 45-degree angle, with rapid intradermal injections to a depth of no more than 2 mm, ensuring intradermal placement of the product. Both steps were applied across the full surface of one side of the face and neck.

Inclusion Criteria

Eligible participants were adults aged 30 to 65 years with visible moderate-to-severe horizontal neck lines and lateral canthal lines at rest or during animation, corresponding to a Grade 2 or 3 on the respective scoring scales. Participants were required to provide written informed consent and to adhere to the treatment and follow-up schedule. Women of childbearing potential were required to have a negative pregnancy test prior to enrollment and to use effective contraception throughout the study.

Exclusion Criteria

Exclusion criteria included prior treatment with botulinum toxin, dermal fillers, or energy-based devices in the target areas within the preceding six months; active skin infection or inflammation at injection sites; history of hypertrophic scarring or keloid formation; known hypersensitivity to any component of the study products; neuromuscular disorders such as myasthenia gravis or Lambert-Eaton syndrome; use of medications interfering with neuromuscular transmission (e.g., aminoglycosides); pregnancy or breastfeeding; unrealistic aesthetic expectations; and any planned surgical intervention during the study period.

Crow’s feet severity was assessed using the Bazin Crow’s Feet Wrinkle Scale (BCFWS), a seven-point scale ranging from 0 (no wrinkle) to 6 (very deep wrinkles), with lower scores indicating greater improvement. Neck wrinkle severity was assessed using the Bazin Photographic Visual Score, a seven-point scale ranging from 0 (no wrinkle) to 6 (very deep wrinkles), with lower scores indicating greater improvement. Skin radiance was evaluated using a five-point clinical scoring scale ranging from 0 (very dull skin) to 4 (very radiant skin), with higher scores indicating greater improvement. Skin hydration, firmness, and homogeneity were each assessed using a nine-point clinical scoring scale (0 = none, 1 - 3 = light, 4 - 6 = moderate, 7 - 9 = important), with higher scores indicating greater severity; improvements are therefore reflected by lower scores at follow-up relative to baseline. Treatment efficacy was also assessed using the Global Aesthetic Improvement Scale (GAIS), a seven-point scale ranging from −3 (very much worse) to +3 (very much improved), with 0 indicating no change and higher scores reflecting greater perceived improvement.

Before and after photos were documented and assessed by VISIA® Skin Analysis.

For all quantitative data, the mean and median were determined. The Student t-test was used for comparative analysis.

Both crow’s feet and neck wrinkle progression were assessed. The wrinkles-scoring results are presented in Figure 2 and Figure 3.

For crow’s feet, mean wrinkle scores decreased from 2.7 at baseline (D0) to 1.0 at D60 on the side treated with the combination of BoNTA + NCTF®135HA, whereas the side treated with BoNTA alone showed a smaller reduction from 2.7 to 2.0 over the same period. The difference in score reduction between the two treatments was statistically significant (Δp = 0.02), indicating greater improvement with the combined approach (Figure 2).

Mean clinical scores for crow’s feet wrinkles at baseline (D0) and D60. The side treated with BoNTA + NCTF®135HA showed a greater reduction in wrinkle severity compared with the BoNTA-only side. The difference between treatments was statistically significant (Δp = 0.02).

Figure 2. Clinical scoring of crow’s feet wrinkles.

A similar pattern was observed for neck wrinkles. The mean clinical score decreased from 3.4 at D0 to 1.5 at D60 on the BoNTA + NCTF®135HA side, corresponding to a 55.8% reduction. In contrast, the BoNTA-only side showed a more modest improvement from 3.4 to 2.6, corresponding to a 23.5% reduction. These findings indicate a greater reduction in neck wrinkle severity with the combined treatment compared with BoNTA alone (Figure 3).

Mean clinical scores for neck wrinkles at baseline (D0) and D60. A greater reduction in wrinkle severity was observed on the side treated with BoNTA + NCTF®135HA compared with BoNTA alone, corresponding to reductions of 55.8% and 23.5%, respectively.

Figure 3. Clinical scoring of neck wrinkles.

After clinical evaluation, improvements in several skin quality parameters were observed, particularly on the side treated with the combined approach.

Skin radiance, evaluated using a clinical score ranging from 0 (very dull skin) to 4 (very radiant skin), increased from 1.6 at D0 to 2.8 at D60 on the side treated with BoNTA + NCTF®135HA (p = 0.005). A smaller improvement was observed on the BoNTA-only side, where the score increased from 1.6 to 1.8 over the same period (Figure 4(a)).

Similarly, the mean hydration score increased markedly on the side treated with BoNTA + NCTF®135HA, rising from 3.4 at D0 to 5.8 at D60 (mean delta +2.4). In comparison, the BoNTA-only side showed a more modest increase from 3.4 to 4.0 (mean delta +0.6). The difference between the two treatments was statistically significant (p = 0.02) (Figure 4(b)).

Skin firmness (Figure 4(c)) showed a slight increase on both treated sides, with a more pronounced improvement on the side treated with BoNTA + NCTF®135HA. Skin homogeneity (Figure 4(d)) remained stable on the BoNTA-only side, while a modest improvement was observed on the combined-treatment side.

Photo analysis using the VISIA® Skin Analysis system (Canfield Scientific, USA) revealed greater improvements in several skin quality parameters on the side treated with BoNTA + NCTF®135HA compared with the BoNTA-only side (Figure 5).

Changes in clinical skin quality scores between baseline (D0) and D60 for the side treated with BoNTA + NCTF®135HA and the BoNTA-only side. (a) Skin radiance score; (b) Skin hydration score; (c) Skin firmness score; (d) Skin homogeneity score. Greater improvements were observed on the side treated with the combined approach.

Figure 4. Clinical scoring of skin quality parameters.

Changes in VISIA® skin analysis parameters between baseline and follow-up for the side treated with BoNTA + NCTF®135HA compared with BoNTA alone. Parameters evaluated included spots, wrinkles, texture, pores, UV spots, brown spots, red areas, and porphyrins. Negative values represent improvement (reduction in the parameter score). Greater improvements were observed on the side treated with the combined approach.

Figure 5. VISIA® skin quality parameters.

The Spot parameter, reflecting the quantification of skin discolorations and pigment irregularities, showed a marked reduction on the combined-treatment side (Δ = −19.4) compared with a smaller reduction on the BoNTA-only side (Δ = −2.8). This difference between treatments was statistically significant (p = 0.02), indicating improved overall skin uniformity with the addition of NCTF®135HA.

The Wrinkle parameter also showed a greater decrease on the combined-treatment side (Δ = −22.0) compared with the BoNTA-only side (Δ = −3.2) (p = 0.07).

Similarly, skin texture improved on the side treated with BoNTA + NCTF®135HA (Δ = −12.6), whereas no improvement was observed on the BoNTA-only side, where the parameter slightly increased (Δ = +3.2). This difference between treatments was statistically significant (p = 0.003).

Additional VISIA® parameters showed a similar trend. The Pore parameter slightly increased on the BoNTA-only side while remaining stable or improving on the combined-treatment side. UV spot counts decreased on the BoNTA + NCTF®135HA side, whereas minimal changes were observed with BoNTA alone. Brown spots showed an increase on the BoNTA-only side but remained more stable on the combined-treatment side. Red areas and porphyrins decreased more markedly on the combined-treatment side compared with the BoNTA-only side.

Standardized VISIA® photographic overlays corroborated these quantitative findings. A visible reduction in pigmented spot density, wrinkle mapping intensity, and surface irregularities was observed on the BoNTA + NCTF®135HA-treated side, whereas minimal changes were detected on the BoNTA-only side.

Representative standardized clinical photographs and VISIA® imaging overlays from two participants are presented in Supplementary Figure S1. These images illustrate the changes observed between baseline and follow-up, including a visible reduction in pigmented spots, wrinkle mapping intensity, and surface irregularities on the side treated with BoNTA + NCTF®135HA. These visual observations are consistent with the quantitative improvements measured by VISIA® analysis.

Treatment efficacy was assessed using the Global Aesthetic Improvement Scale (GAIS) by both the physician and the participants at D60.

As shown in Figure 6(a), the physician reported greater aesthetic improvement on the side treated with BoNTA + NCTF®135HA, with a mean GAIS score of 2 (much improved) compared with 1 (improved) on the BoNTA-only side. However, this difference did not reach statistical significance.

Patient-reported outcomes showed a significantly greater perceived aesthetic improvement with the combined treatment. At D60, the mean GAIS score reported by participants was 2.2 for the BoNTA + NCTF®135HA side versus 1 for the BoNTA-only side (p = 0.0003) (Figure 6(b)).

Assessment of aesthetic improvement at D60 using the Global Aesthetic Improvement Scale (GAIS). (a) Physician-reported GAIS scores comparing BoNTA + NCTF®135HA and BoNTA alone; (b) Patient-reported GAIS scores showing greater perceived improvement on the side treated with the combined approach.

Figure 6. Global Aesthetic Improvement Scale (GAIS) evaluation.

The treatment protocol was well tolerated by all participants, with no reports of systemic adverse events or functional impairments. Specifically, there were no documented cases of ptosis, facial asymmetry, muscle weakness, or dysphagia on either the treated or control sides. Regarding local injection-site reactions, the safety profile was favorable: only one patient experienced mild ecchymosis at two injection points in the periorbital region. This bruising was localized, did not require medical intervention, and resolved spontaneously within five days. No significant differences in pain levels were reported between the BoNTA-only side and the combined treatment side.