Tailoring hyaluronic acid hydrogels: Impact of cross-linker length and density on skin rejuvenation as injectable dermal fillers and their potential effects on the MAPK signaling pathway suppression

Hyaluronic acid (HA) hydrogels, obtained through cross-linking, provide a stable 3D environment that is important for controlled delivery and tissue engineering applications. Cross-linking density has a significant impact on the physicochemical properties of hydrogels, including their shape stability, mechanical stiffness and macromolecular diffusivity. However, often cross-linking chemistries require photoinitiator and catalyst that may be toxic and cause unwanted tissue response. Here, we prepared a series of HA hydrogel with varying cross-linker length and cross-linking density, which can be obtained by altering the feed ratio of three different cross-linkers from small molecules to macromolecules (e.g., 1,4-butanediol diglycidyl ether (BDDE), ferulic acid (FA), pluronic (PLU)), to ameliorate skin wrinkles in mice models. HA cross-linked with FA and PLU exhibited enzyme and temperature-dependent sol-to-gel phase transition, respectively, and the gels possess good injectability. In vitro test confirmed that HA hydrogels co-cultured with RAW 264.7 and HDF cells showed good biocompatibility. In particular, HA cross-linked with PLU stimulated the growth of HDF cells and HaCaT cells. HA cross-linked with PLU suppressed the expression levels of proteins involved in collagen degradation including mitogen-activated protein kinases (ERK, JNK, p38) and matrix metalloproteases (MMP-1, MMP-3, and MMP-9) resulting in increased deposition of Collagen I. The free-flowing sols of HA hydrogel precursors are subcutaneously injected into the back of BALB/c mice and form stable gels at the dermis layer and found to be non-toxic. More importantly, HA hydrogel cross-linked with PLU showed an enhanced anti-wrinkling effect in the wrinkled mice model. Thus, properties of HA hydrogels such as injectability, biocompatibility, and good anti-wrinkling effect altered through varying cross-linking density must be considered in the context of soft tissue engineering applications.