Initial immune response to a FRET-based MMP sensor-immobilized silk fibroin hydrogel in vivo

To investigate the initial immune response to biodegradable silk fibroin (SF) hydrogels in vivo, a Förster/fluorescence resonance energy transfer (FRET)-based sensor was developed to detect matrix metalloproteinase (MMP) activity (FRET-MMPS) and immobilized to SF hydrogel. FRET-MMPS immobilized to SF hydrogel in vitro displayed intra-molecular FRET more than inter-molecular FRET, and MMP activity was detected through a decrease in FRET signal intensity. Then, the SF hydrogel modified with FRET-MMPS was implanted into mice subcutaneously, and it was observed that the FRET signal intensity decreased significantly soon (< 3 h) after implantation. Although the intensity exhibited a sharp decrease toward 24 h post-implantation, histological evaluation proved that bulk-level hydrogel degradation, such as breakdown, was mainly caused by macrophages and foreign body giant cells on a timescale of weeks. These results indicated that, immediately upon implantation, active MMPs reached the SF hydrogel and began cleaving SF networks, which might result in the loosening of the networks and then enabled immune cells, such as macrophages, to start the bulk-level hydrogel degradation. The sensor clarified the initial immune response to SF hydrogels and will provide clues for designing the biodegradation behaviors of scaffolds for regenerative medicine. STATEMENT OF SIGNIFICANCE: Silk fibroin (SF) materials are degraded gradually by the immune response. Immune cells, such as macrophages, break down implanted SF materials on a timescale of weeks or months, but the initial (< 24 h) immune response to SF materials remains unclear. In this study, SF hydrogels modified with Förster/fluorescence resonance energy transfer (FRET)-based matrix metalloproteinase (MMP) sensors were implanted in mice and within 3 h post-implantation, the SF hydrogels were degraded by MMPs. Although this molecular-level biodegradation was not correlated with the hydrogel breakdown, the MMPs were likely to loosen the SF networks to enable immune cells to infiltrate and degrade the hydrogel. This is the first study to unveil the initial stage of immune response to biomaterials.