Comparative late effects of hemostats on wound healing at 14 and 30 days – an in-vivo animal study

Introduction: Hemostatic agents are commonly used during surgery or trauma care to achieve hemostasis in case of excessive bleeding. Previous studies demonstrated that hemostatic agents may exert advantageous as well as negative effects on wound healing depending on agent and length of application. Commonly employed agents are oxidized non-regenerated cellulose (ONRC), oxidized regenerated cellulose (ORC), and gelatin-based agents (GELA). This study therefore aimed to evaluate the effects of different hemostats on wound healing properties at 14 and 30 days post-wounding in a mouse model. Methods: Three different hemostatic agents ONRC, ORC and GLEA were tested. Experimental wounds were introduced in C57BL/6J mice (n=192) and compared to a sham control group without hemostat introduction. After termination, tissue samples were obtained at days 14 and 30 after surgery. Effects were assessed by comparing immunohistochemistry of Ki-67 and alpha-smooth muscle actin (α-SMA) and qPCR of vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and basic fibroblast growth factor-2 (FGF-2) between investigational groups. Results: After 14 days, no significant differences were observed between the control and hemostatic groups regarding proliferation (Ki-67 activity), contraction (α-SMA), VEGF and FGF-2 expression. TGF-ß expression was significantly inhibited by ORC (0.7, 95% CI [0.5821; 0.8363], p=0.047) compared to Sham, but not significantly altered by GELA and ONRC compared to the sham group. After 30 days, Ki-67 activity was significantly increased by GELA (286.5, 95% CI: [213.2; 504.8], p =0.02) in epidermal cells, while α-SMA activity was significantly increased by GELA (380.7, 95% CI: [302.4; 616.2], p=0.038) and ORC (405.2; 95% CI: [276.4; 672.1], p=0.032). VEGF expression was significantly inhibited by GELA (0.30, 95% CI [0.2011; 0.3774], p=0.003) and ORC (0.33, 95% CI [0.2099; 0.4285], p=0.013) compared to sham, while TGF-ß expression was significantly activated by ONRC (95%CI [-2.920; -0.2213], p =0.02) compared to the control group. No significant difference in FGF-2 activity between the control and the hemostats groups could be measured. Conclusions: All tested hemostats showed no adverse effects during the early remodeling phase (up to Day 14), indicating safe short-term biocompatibility when left in situ. However, clear material-specific differences emerged during the late remodeling phase (by Day 30): while GELA supported epidermal proliferation and contraction, and ORC promoted contraction, ONRC was associated with increased TGF-β expression, suggesting a profibrotic shift. These findings underscore the importance of hemostat selection not only for intraoperative bleeding control but also for long-term wound remodeling and scar quality, particularly in patients at risk of delayed or compromised wound healing and cases where hemostats are left in situ.