Hyaluronan-based hydrogels as dermal fillers: The biophysical properties that translate into a "volumetric" effect

Abstract

Biophysical and biochemical data on hyaluronan (HA)-based dermal fillers strongly support their optimal use and design to meet specific requisites. Here, four commercially available (in Europe) HA "volumetric" fillers, among the most used in the clinical practice, have been characterized in vitro. Analyses revealed the highest amounts of water-soluble HA reported so far and provided hydrodynamic data for these soluble polymeric fractions. Volumetric gels exhibit a wide range of rigidity with most of them showing G' values around 200-300Pa. They greatly differ in cohesivity. 1mL of gel hydrates up to 2.4-3.2mL. The products completely solubilize due to Bovine Testicular Hyaluronidase (BTH)'s action, thus predicting in vivo complete resorption. For the first time, filler degradation due to reactive oxygen species (ROS) was studied by rheological measurements and a rank in stability was established. Studies using Human Dermal Fibroblasts (HDF) indicated a positive biological response to the HA networks. Further, gel capacity to prompt collagen I, elastin and aquaporin3 synthesis was demonstrated, thus suggesting a positive effect on skin elasticity and hydration, besides the physical volumetric action. The findings are the first wide assessment of features for the volumetric class of HA-fillers and include first data on their resistance to degradation by ROS and biological effects on HDF. The study represents a valuable contribution to the understanding of HA-fillers, useful to optimize their use and manufacture.

Fig 1. Results of the gel composition analyses.

The total HA concentration in the gels, as indicated in the package inserts, is shown in the first row. The amount of water-soluble HA in each gel (mg/mL), based on our analyses, is then reported. In Italics, values significantly different (p<0.05). The amount of water-insoluble HA, derived from the previous data, is finally indicated. In Italics, values significantly different (p<0.05).

Fig 2. Results of the SEC-TDA characterization of the water-soluble fraction of the gels.

(a) Overlap of the chromatographic profiles (RI signal): R_L (circles); J_V (triangles); T_RH4 (×); A_SV (squares). (b) Complete chromatographic report: weight average molar mass (M_w), numeric average molar mass (M_n), polydispersity index (M_w/M_n), intrinsic viscosity ([η]), hydrodynamic radius (R_h). For T_RH4, the data resulting from the analysis of the two distributions are reported. The high molecular weight fraction represents the 53±1% w/w of the total soluble HA amount. (c) Overlap of representative Mark-Houwink-Sakurada curves for (on the left) a linear HA control (stars), R_L (circles), J_V (triangles), T_RH4 II distribution (×) and A_SV (squares) and for (on the right) a linear HA control (empty and filled squares) and T_RH4 I distribution (×).

Fig 3. Filler hydration capacity in physiological medium.

(a) Hydration capacity (volumetric expansion), at equilibrium, for the diverse gels. Data indicate the volume reached by 1mL of each gel when allowed to reach equilibrium in PBS. (b) Hydration capacity for the water-insoluble HA contained in each gel. Data indicate the volume reached, at equilibrium, by 1mg of water-insoluble HA.

Fig 4. Rheological characterization.

G’ values (a) and complex viscosity values (b) for the gels, as a function of the frequency. Measurements were performed at 37°C.

Fig 5. Degradation of the gels in the presence of the H₂O₂/Cu²⁺ system.

(a) Reduction of G’ during 15 minutes of incubation with the degrading system. For each filler, the trend of the G’ value during incubation with water under the same conditions (control) is also reported. (b) Residual G’ (% compared to the control) at 5, 8, 10 and 15 minutes of incubation with the ROS generating system. Measurements were performed at 37°C.

Fig 6. Gel cohesivity.

(a) Images of the gels captured at 15 seconds after starting the test. (b) Cohesivity score assigned to the gels at diverse time intervals according to the Gavard-Sundaram Cohesivity Scale [33].

Fig 7. Western blotting analyses.

Western blotting image (a) and densitometric analysis (b) of Collagen I, Elastin and Aqp3 expression in HDF after 48h of incubation with the gels. Actin was used to normalize the results. Protein expression is reported as fold increase compared to ctr (untreated cells).