Acetylated chitosan scaffolds modulate the micro-environment and promote peripheral nerve regeneration through macrophage TLR2/4 pathway activation.

Current nerve repair materials have limited capacity to actively regulate the regenerative micro-environment, lack effective intervention strategies and clarity mechanisms, resulting in insufficient tissue repair and regeneration capabilities. Acetylated chitosan (ACS) with high degree of acetylation (DA) exhibits rapid degradation properties and induces severe local inflammatory responses when subcutaneously implanted as a tissue engineering scaffold material. To balance the bio-compatibility and biodegradability of ACS, we systematically investigated the role of the DA in modulating inflammatory responses and tissue regeneration. Results showed that for ACS scaffolds with DA ≥ 50 %, the intensity of the inflammatory response increased proportionally with the DA. In a rat sciatic nerve crush repair model, ACS scaffolds with DA values of 50 % and 70 % induced moderate inflammation that was favorable for nerve regeneration. However, scaffolds with DA values of 90 % triggered excessive inflammation, impairing effective nerve repair. Mechanistically, we discovered that acetyl groups, acting as lipid a mimetics, can activate Toll-like receptor 4 (TLR4). TLR4 interacts with Adenylyl cyclase-associated protein 1 (CAP1), to drive macrophage polarization and release inflammatory factors proportional to the DA. Additionally, ACS activated macrophages release large amounts of Chitotriosidase-1 (CHIT1), which degrades ACS scaffolds into smaller fragments recognized by TLR2, further amplifying inflammatory signals via classical pathways. This dual regulatory mechanism mediated by ACS-TLR4-CAP1 and ACS-CHIT1-TLR2 provides new insights into the immune activation induced by ACS scaffolds. These findings identify potential therapeutic targets and offer guidance for optimizing ACS scaffold design to improve tissue regeneration outcomes.