In vitro toxicity assessment of crosslinking agents used in hyaluronic acid dermal filler.

Hyaluronic acid (HA) dermal fillers are produced by crosslinking HA with agents, such as 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether (PEGDE) to acquire desired properties. Thus, the safety evaluation of these crosslinkers is needed at the cellular level. In the present study, cell viability, cytotoxicity, membrane integrity, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory responses were evaluated in the human keratinocyte cell line, HaCaT and human dermal fibroblast cell line, HDF in response to treatment with the crosslinkers. In both the cell lines, BDDE significantly decreased cell viability at 100-1000 ppm, while PEGDE showed a decrease at 500-1000 ppm. In HaCaT cells, BDDE markedly increased cytotoxicity (lactate dehydrogenase release) at 100-1000 ppm, but PEGDE showed an increase at 500-1000 ppm. Cells treated with BDDE (100 ppm) caused alteration in the integrity of cell membrane and shape. In both the cell lines, BDDE-treated cells showed significantly higher ROS levels and MMP loss than PEGDE-treated cells. Also, BDDE-treated cells exhibited higher COX-2 expression at 100 ppm. Expression of inflammatory cytokines (TNF-α, and IL-1 ß) was higher in BDDE-treated cells. Taken together, PEGDE-treated cells showed markedly lower cytotoxicity, ROS production, and inflammatory responses than BDDE-treated cells. Our data suggest that PEGDE is safer than BDDE as a crosslinker in HA dermal fillers.

In Vitro Biocompatibility Evaluation of Nine Dermal Fillers on L929 Cell Line.

OBJECTIVE: Biomaterial research for soft tissue augmentation is an increasing topic in aesthetic medicine. Hyaluronic acid (HA) fillers are widely used for their low invasiveness and easy application to correct aesthetic defects or traumatic injuries. Some complications as acute or chronic inflammation can occur in patients following the injection. Biocompatibility assays are required for medical devices intended for human use, in order to prevent damages or injuries in the host. In this study, nine HA fillers were tested in order to evaluate their cytotoxicity and their effects on L929 cell line, according to the UNI EN ISO 10993 regulation. METHODS: Extracts were prepared from nine HA fillers, and MTS viability assay was performed after 24 h, 48 h, and 72 h of exposure of cells to extracts. Cells cultured with HA filler extracts were monitored for up to 72 h, counted, and stained with haematoxylin/eosin in order to evaluate the cell proliferation rate and morphology. RESULTS: None of the filler tested showed a cytotoxic effect. Two samples showed a higher vitality percentage and higher cell number while two samples showed a lower vitality percentage and lower cell number at 72 h. CONCLUSION: Data obtained suggest that although examined fillers are not cytotoxic, they show different effects on the in vitro cell proliferation rate. In vitro studies of medical devices could lead to important implications since these could aid to predict effects about their in vivo application. These easy and rapid assays could be useful to test new materials intended for human use avoiding animal tests.

Effectiveness of Retrobulbar Hyaluronidase Injection in an Iatrogenic Blindness Rabbit Model Using Hyaluronic Acid Filler Injection.

BACKGROUND: Blindness caused by soft-tissue filler injection is the most tragic complication, with no standard treatments until recently. Retrobulbar hyaluronidase injection has been proposed as the treatment, but its effectiveness in visual compromise remains to be determined. The authors aimed to determine the effectiveness of retrobulbar hyaluronidase using soft-tissue filler in an iatrogenic blindness animal model. METHODS: New Zealand White rabbits were used to simulate the hyaluronic acid-associated vascular occlusion model. A volume of 0.7 to 1.6 ml of hyaluronic acid filler was injected into the internal carotid artery to create a retinal artery occlusion. The rabbits were administered retrobulbar hyaluronidase (3000 IU) at different postobstruction time points (5 and 10 minutes). No intervention was given to the control group. Fundus photography was performed before and immediately after the filler injection and immediately after the administration of retrobulbar hyaluronidase. Electroretinography was performed after 60 minutes to confirm the retinal reperfusion and electrophysiologic function. RESULTS: All of the experimental eyes recorded total occlusion after hyaluronic acid injection. Three eyes with a completely occluded retinal artery following retrobulbar hyaluronidase treatment showed improved retinal reperfusion by fundus photography and corresponding electroretinography. Despite administration of the retrobulbar hyaluronidase injection, one completely occluded eye showed no improvement in perfusion. All of the control eyes recorded complete occlusion 1 hour after hyaluronic acid filler injection. CONCLUSIONS: Retrobulbar hyaluronidase may be an effective evidence-based treatment option for humans. Hyaluronidase concentration and injection time are the important factors for faster recovery, but additional studies are still required.

Preparation and characterization of a novel polysialic acid-hyaluronan graft copolymer potential as dermal filler.

Polysialic acid (PSA) and hyaluronan (HA) are non-immunogenic and biodegradable natural polysaccharides, but HA belongs to glycosaminoglycans and can be immediately degraded by human enzymes. In this study, we synthesized a novel PSA-HA draft copolymer to improve HA stability in vivo. This draft copolymer was characterized by SEM, element analysis and Zeta potential. Cytotoxicity assays, as well as pyrogen and hemolysis tests, were also conducted to test its biological functions further. Results showed that PSA-HA draft copolymer satisfies the medical requirement for biomaterials. In vivo degradation test proved that this copolymer can reduce the irritation rate and prolong the duration of cross-linked HA in skin. These results indicated that PSA-HA draft copolymer can be potentially used as an alternative for free HA in dermal filler (dual-phase cross-linked HA system).