IMMUNOREGULATORY PROPERTIES OF SELF-ASSEMBLING PEPTIDES AND HYALURONAN SOFT BIOMATERIALS

Immune cells, particularly macrophages (MΦs), are key orchestrators of musculoskeletal healing, determining outcomes ranging from chronic inflammation to successful regeneration. For severe injuries, rapid-sealant biomaterials can guide nearby and infiltrating monocytes and MΦs toward optimal healing. This study aimed to develop injectable biomaterials for controlling inflammation by investigating how chemical modifications to biomaterials influence MΦ modulation. We focused on self-assembling peptides (SAPs) and hyaluronan (HA), which allow precise control of nanostructure and chemistry to regulate cellular signals. Tyrosine, a versatile amino acid, was incorporated into SAPs and HA to explore its effects on MΦ behaviour. Five β-sheet SAPs were designed: EFEFKFEFK ( EF8 ), Y EFEFKFEFK ( YEF8 ), EFEFKFEFK Y ( EF8Y ), Y EFEFKFEFK Y ( YEF8Y ) and E Y EFKFEFK ( EYF8 ) (F: phenylalanine; E: glutamic acid; K: lysine, Y : tyrosine). 3 The self-assembly and gelation of these peptides were fully characterized using a series of physiochemical and computational methods. For HA, we synthesized tyramine-modified variants (THA) with low (M w = 280 kDa, LMW) and high (M w = 1640 kDa, HMW) molecular weights, confirmed by 1 H-NMR and UV-Vis spectroscopies. 2 The polarization effects of SAPs and THA were studied using THP-1 cell-derived MΦs and PBMC-derived-MΦs, with polarization states evaluated through immunocytochemistry, flow cytometry, gene expression analysis, confocal microscopy, and multiplex ELISAs. Flow cytometry and multiplex ELISA indicated that LMW THA induced a pro-inflammatory M1-like phenotype, while HMW THA led to a mixed M1/M2 profile, suggesting a transitional state ( Fig. 1 ). EF8 promoted an anti-inflammatory M2 phenotype with upregulation of CD105 and CD163, while YEF8 favoured a pro-inflammatory M1 state with increased HLA-DR expression ( Fig. 1 ). The other SAPs ( EF8Y , YEF8Y , EYF8 ) showed minimal inflammatory responses. These effects were linked to the nanostructure differences in SAPs. The results were validated in PBMC-derived-MΦs from human donors, highlighting donor variability in inflammatory responses. This study confirms the critical role of biomaterial chemistry in MΦ modulation. EF8 and YEF8 SAPs demonstrate potential as biomaterials to either promote tissue repair or induce pro-inflammatory responses, respectively. These findings offer valuable insights for designing immunomodulatory biomaterials for spatiotemporal control of inflammation and the induction of regenerative pathways. This work was supported by the AO Foundation, EU Horizon 2020 (no.893099), LEADING HOUSE MENA Research Partnership (RPG-2022-38) and Consolidation (COG-2023-35) grants. For any figures or tables, please contact the authors directly.