In vitro inflammatory and immune response to uncrosslinked hyaluronic acid (HA) and HA fillers

Introduction

Delayed-onset nodules are a complication associated with soft tissue filler products, including hyaluronic acid (HA) fillers; however, the etiology of these events remains unclear. The role of uncrosslinked HA and crosslinked HA fillers in the response of immune cells (dendritic cells, B cells, and T cells) was evaluated with and without concurrent bacterial stimulation.

Methods

Uncrosslinked HA of varying molecular weights and crosslinked HA fillers were tested in a series of in vitro assays to evaluate 1) human monocyte activation; 2) activation, maturation, and migration of human monocyte-derived dendritic cells (MoDC); 3) T-cell activation and associated skin damage; and 4) T-cell–mediated B-cell response. For the latter assay, the HA test articles were also evaluated following stimulation by bacteria.

Results

When treated with lipopolysaccharide (LPS) as a positive control, inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-10, IL-12p70, and tumor necrosis factor [TNF]-α) released by MoDC were significantly upregulated. Treatment of the MoDC with HA (0.776, 150, and 485 kDa) did not result in stimulation of IL-1β, IL-6, IL-10, and IL-12p70, although increased TNF-α was observed with all HA test samples. The TNF-α increase was significantly lower for HA samples (1.5–2.64 pg/mL) compared with LPS (966.96–4834.18 pg/mL). Similarly, treatment of MoDC with HA samples did not result in expression of surface markers indicative of dendritic cell maturation. MoDC treated with 0.776 and 150 kDa HA, but not with 485 kDa HA, had significantly increased migration toward fetal bovine serum, but not C–C motif chemokine ligand 21 (CCL21). In the T-cell activation and skin damage assay, VYC-15L, degraded VYC-15L, HYC-24L+, and oligosacharride HA samples were negative in the T-cell proliferation and interferon-γ assays at all tested concentrations. The degraded HYC-24L + sample was borderline at the highest concentration and negative at the middle and low concentrations. Skin damage was determined to be negative for all HA samples at all concentrations. In the human monocyte activation assay, all tested uncrosslinked HA samples (5, 150, and 731 kDa) were determined to be non-sensitizing (i.e., did not activate monocyte cells). In the T-cell–mediated B-cell activation assay, treatment with HA oligosaccharides, uncrosslinked HA, and crosslinked HA fillers did not result in any consistent stimulation of any cytokines measured. A molecular weight–dependent decrease in the secreted immunoglobulin G (IgG) was observed, and this effect was more pronounced with the crosslinked HA fillers. Addition of heat-inactivated Cutibacterium acnes bacteria (HIB) to uncrosslinked HA and crosslinked HA fillers resulted in increased cytokine production similar to when HIB was tested alone. The decrease in secreted IgG observed for crosslinked HA filler samples without HIB was maintained with addition of HIB to VYC-20, VYC-25, SGD-30XP, and HYC-24+ samples. The reduction of secreted IgG observed for RSQ and VYC-15 alone was no longer apparent when HIB was added to these cultures (i.e., returned to control levels but were not upregulated).

Conclusion

The results from this study suggest that uncrosslinked HA and HA fillers themselves do not stimulate an inflammatory/immune response, regardless of molecular weight or crosslinking. However, the immune response following HIB stimulation may be impacted by the presence of HA fillers, suggesting a potential initiating role of contaminating factors such as bacteria and a modulating role of HA.