Dual Functionalization of Hyaluronan Dermal Fillers with Vitamin B3: Efficient Combination of Bio-Stimulation Properties with Hydrogel System Resilience Enhancement

Abstract

Hyaluronic acid (HA) hydrogels are commonly used for facial dermal filling and for alternative medical aesthetic purposes. High diversity exists in commercial formulations, notably for the optimization of finished product stability, functionality, and performance. Polyvalent ingredients such as calcium hydroxylapatite (CaHA) or vitamin B3 (niacinamide) are notably used as bio-stimulants to improve skin quality attributes at the administration site. The aim of the present study was to perform multi-parametric characterization of two novel cross-linked dermal filler formulas (HAR-1 "Instant Refine" and HAR-3 "Maxi Lift") for elucidation of the various functional impacts of vitamin B3 incorporation. Therefore, the HAR products were firstly comparatively characterized in terms of in vitro rheology, cohesivity, injectability, and resistance to chemical or enzymatic degradation (exposition to H₂O₂, AAPH, hyaluronidases, or xanthine oxidase). Then, the HAR products were assessed for cytocompatibility and in vitro bio-stimulation attributes in a primary dermal fibroblast model. The results showed enhanced resilience of the cohesive HAR hydrogels as compared to JUVÉDERM^® VOLBELLA^® and VOLUMA^® reference products in a controlled degradation assay panel. Furthermore, significant induction of total collagen synthesis in primary dermal fibroblast cultures was recorded for HAR-1 and HAR-3, denoting intrinsic bio-stimulatory effects comparable or superior to those of the Radiesse^® and Sculptra^™ reference products. Original results of high translational relevance were generated herein using robust and orthogonal experimental methodologies (hydrogel degradation, functional benchmarking) and study designs. Overall, the reported results confirmed the dual functionalization role of vitamin B3 in cross-linked HA dermal fillers, with a significant enhancement of hydrogel system stability attributes and the deployment of potent bio-stimulatory capacities.

Keywords: bio-stimulation; cohesivity attributes; cross-linked dermal fillers; dermal fibroblasts; functional characterization; hyaluronic acid; hydrogel system; niacinamide; skin collagen; viscoelasticity.

Figure 1

Results of basic rheological characterization studies for the hydrogels of interest. (A) Storage modulus G′ values for the undiluted samples at 25 °C. (B) Loss modulus G″ values for the undiluted samples at 25 °C. (C) Tan delta (tan δ) values for the undiluted samples at 25 °C. Measurements were performed in triplicate and standard deviations were reported as error bars around mean values. Detailed results of the statistical analyses are presented in Table S1. Pa, Pascals.

Figure 2

Results of accelerated hydrogel degradation studies expressed as endpoint residual fractions of the rheological attributes of the samples as compared to their initial (i.e., unchallenged) values. (A) Experimental endpoint storage modulus G′ values for the challenged samples. (B) Experimental endpoint loss modulus G″ values for the challenged samples. All the samples were parallelly exposed to strong oxidant sources and to hyaluronidases. After 10 min of exposure, the samples were analyzed in oscillatory rheology at 22 °C with a frequency of 1 Hz. Measurements were performed in triplicate and standard deviations were reported as error bars around mean values. The corresponding complex viscosity η* values are presented in Figure S3. Detailed results of the statistical analyses are presented in Tables S2–S4.

Figure 3

Comparative hydrogel system biophysical characterization results. (A) System cohesivity determined by the drop-weight method (30 G needle, 12 mm·min⁻¹ extrusion). An illustration of the investigated hydrogels is presented in Figure S4. An illustration of the experimental setup is presented in Video S1 (i.e., VOLUMA^® product). (B) System cohesivity determined by the drop-weight method (18 G needle, 7.5 mm·min⁻¹ extrusion). Detailed results of the statistical analyses are presented in Table S5. (C) Injection force profiles of HAR-1 in an automated measurement setup. (D) Injection force profiles of HAR-3 in an automated measurement setup. Injectability results were presented for three distinct product syringes, corresponding to profiles N°1, N°2, and N°3. Injectability assays were performed at ambient temperature using a constant plunger rod actuation speed of 1 mm·s⁻¹. All measurements were performed in triplicate. G, gauge.

Figure 4

Comparative product bio-stimulatory effect assessments in an in vitro cell-based model. (A) Cellular viability of primary dermal fibroblasts incubated in contact with the samples for 96 h. (B) Absolute values of the total collagen produced by primary dermal fibroblasts incubated with the samples for 96 h. (C) Absolute values of the total proteins produced by primary dermal fibroblasts incubated with the samples for 96 h. Measurements were performed four times and standard deviations were reported as error bars around mean values. Significant differences with the PBS control groups were evidenced by asterisks (i.e., two asterisks “**” corresponded to a p-value between 0.001 and 0.01, four asterisks “****” corresponded to a p-value inferior to 0.0001). Photographic and mechanistic illustrations of the experimental setup are presented in Figures S6–S8. Detailed results of the statistical analyses are presented in Tables S6–S8. PBS, phosphate-buffered saline.