In vitro and in vivo biocompatibility evaluation of a novobiocin stimulus-responsive poly(ethylene glycol)-based hydrogel designed for soft tissue regeneration

In vitro and in vivo biocompatibility evaluation of medical devices designed for soft tissue healing and regeneration is mandatory for new biomaterials targeted toward human application. To this end, a novel stimulus-responsive eight-arm poly(ethylene glycol) (PEG)-based hydrogel for soft tissue regeneration was subjected to preclinical safety evaluation. While in vitro safety testing included determination of half-maximal inhibitory concentrations by cell index impedance measurements, an in vivo pilot rat study focused on detectable signs of oral mucosal inflammation. In a hydrogel with a total volume of 40 mu L, the following concentrations of the constituents were released upon hydrogel dissolution in 1mL body fluid: PEG, 19 mu M; gyrase B, 152 mu M; and coumermycin, 76 mu M, while the dissolving agent novobiocin was effective in a range from 50 to 100 mu M. Concerning the components' biological effects, half-maximal inhibitory concentrations of 806 and 95 mu M for human oral mucosal gingival fibroblasts and gingival keratinocytes were calculated for novobiocin, proving gingival keratinocytes more susceptible to substance effects. Furthermore, with respect to coumermycin concentrations, half-maximal inhibitory concentrations of 10 and 3 mu M for gingival fibroblasts and gingival keratinocytes, respectively, were obtained, and thereby revealed coumermycin to be toxic in vitro. In vivo, submucosal hard palatal implantation of standard hydrogels showed no evidence of inflammation at any time point under study, which was supported by immunohistochemistry analysis. In order to realize the potential of such a promising stimulus-responsive hydrogel biomaterial concept for soft tissue wound healing and/or regenerative applications, the crosslinker coumermycin and its competitor novobiocin should be replaced by more innocuous alternative protein-ligand pairs.