Modulating Rheological Properties via Non-Cross-Linked Phase in Biphasic Hyaluronic Acid Fillers

Hyaluronic acid (HA) based soft tissue augmentation materials are high-value-added products that have been used as filling materials in many areas such as the chin, face, and hands for many years. In our study, the synthesis methodology that utilizes a less toxic cross-linker to obtain stiffer gels with a high storage modulus (G') leads to much safer dermal fillers. For this purpose, citric acid (CA) was used as a green cross-linker. By altering the amount of CA, a series of non-cross-linked gels were prepared and reconstituted with a size-reduced cross-linked phase to prepare biphasic hyaluronic acid fillers with different mechanical and rheological characteristics. The effect of steam sterilization on the biphasic gels' physical properties was examined. No toxic waste was encountered after steam sterilization in the biphasic gels. Non-cross-linked HA fractions with a concentration of 23 mg/mL and containing 4% (w/w), 6% (w/w), 8% (w/w), 10% (w/w), and 12% (w/w) amounts of CA were prepared. 1,4-Butanediol diglycidyl ether (BDDE) was used at 6% (w/w) concentration for cross-linked hyaluronic acid (CL-HA) gel synthesis. The BP-E-10-S sample was determined as the optimal group. Rheological results of the BP-E-10-S sample such as elastic modulus (G', Pa), loss modulus (G″, Pa), complex viscosity (η*), and phase angle (○) were measured 388.76 ± 13.91, 88.89 ± 6.22, 12.69 ± 0.47, and 12.86 ± 0.45, respectively. Cell viability was evaluated by an MTT assay using L929 cells. <i>In vivo</i> biocompatibility analysis of the BP-E-10-S group was performed following the ISO 10993 standards. Histopathological evaluations were evaluated and concluded to be nonirritant and biocompatible with an analysis score of 1.92 following protocol and the evaluation criterion of ISO 10993-6:2016. In addition, a patent was obtained for the formulations.