Professor University of California Irvine, United States
Introduction:: Biomaterial wound dressings can enhance healing and reduce scarring by interacting with and modulating the function of host cells infiltrating the biomaterial. Macrophages (Mϕ) and fibroblasts play crucial roles in wound healing and scar formation, participating in inflammation, healing, and tissue regeneration. Here, we examined host cell responses to gelatin-methacryloyl (GelMA) hydrogels with varying crosslinking levels applied to murine skin wounds using single cell RNA sequencing (scRNA-seq). Higher crosslinked, stiffer hydrogels (hi-GelMA) induced more inflammation and fibrosis than lower crosslinked, softer ones (lo-GelMA), skewing Mϕs towards pro-inflammatory activation and creating a highly oxidative environment. Increased inflammation resulted in enhanced fibrotic dermis and pro-fibrotic fibroblast activity. Our findings show that the mechanical and degradation characteristics of biomaterials significantly influence wound healing and suggest the potential for novel wound dressings to reduce scarring and promote tissue regeneration.
Materials and Methods:: Low (3 kPa) and high (150 kPa) crosslinked GelMA gels were prepared by tuneable crosslinking with UV light and applied to full-thickness 5mm wounds of p50 mice. Wounds were covered with Tegaderm, assessed at 3-, 5-, 10-, and 30-days post-wounding, with scar area measured at day 30. Wound healing was histologically evaluated at days 3, 5, and 10, and scRNA-seq profiling was performed at day 5. RNA-seq findings were validated via RNA probe staining on histological sections at the gel-Mϕ interphase. In-vitro gel-Mϕ interaction assays using bone marrow derived Mϕ (BMDM) measured cytokine activation using ELISA, metabolic reprogramming with phasor autofluorescence NADH lifetime imaging, phagocytosis assay via confocal microscopy, and ECM receptor expression by qPCR.
Results, Conclusions, and Discussions:: We found that GelMA treatment reduced scar size, with lo-GelMA resulting in the smallest scars. The crosslinking/stiffness of the hydrogel affected the immune and fibroblast response to the wound, with lo-GelMA promoting pro-healing polarization and smaller scars compared to hi-GelMA, which induced more inflammation and fibrosis. We identified distinct Mϕ populations, with hi-GelMA inducing more hyper-inflammatory/oxidative Mϕs and lo-GelMA inducing more phagocytic and pro-healing Mϕs. Histological analysis revealed that the oxidative Mϕs directly accumulate at the high-crosslinked GelMA border, and cell-cell communication analysis of scRNA-seq revealed that these Mϕs transmit enhanced inflammatory and pro-fibrotic signals to fibroblasts. In turn, the pro-fibrotic fibroblasts reciprocally signal to these Mϕs, including inflammatory signals of CCLs and CXCLs and the key Mϕ fusion signal – RANKL. Promoting the foreign body response phenotype by Mϕ fusion and expression of Dcstamp marker. Conclusions: Our study emphasizes the significance of biomaterial properties and the mechanical-cellular interplay in biomaterial-enhanced wound healing and tissue engineering. It reveals how biomaterials modulate the immune response through direct contact and propagate their influence on stromal cells through cell-cell communication, impacting all wound healing stages and scar formation. These insights enhance our comprehension of cell-matrix and cell-cell nteractions and offer guidance for the development of novel biomaterials.
