Strengthened Anti-Inflammatory Potential of a Purified Antimicrobial Peptide Extract in Leukocyte-Biomaterial Interactions.

Excessive inflammation following implant insertion may impair early healing and predispose to postoperative complications. Since neutrophils constitute the predominant leukocyte population during the initial inflammatory phase, and monocytes subsequently orchestrate tissue resolution and repair, understanding their direct response to biomaterials and bioactive compounds is essential. In the present study, we evaluated the effect of purified bioactive extract combined with biomaterial on neutrophil and monocyte activity in vitro, comparing their modulatory potential with that of non-purified extract. Porcine neutrophils and monocytes were cultured in contact with hydroxyapatite (HA) mixed with purified fractions obtained through sequential chromatographic isolation. Cellular responses were assessed by measuring enzyme release and reactive oxygen/nitrogen species (ROS/RNS) generation. Neutrophils exposed to HA alone displayed a pronounced oxidative response, confirming strong activation. Purified extracts, however, significantly reduced ROS/RNS production (NO: 2.71 ± 0.07 µM vs. HA-only group 5.72 ± 0.10 µM, p < 0.05), whereas non-purified extracts elicited only a partial reduction. Enzyme release followed the same trend, with purified fractions demonstrating a markedly higher inhibitory effect on neutrophil degranulation. Similarly, monocytes exposed to HA exhibited prolonged oxidative stress at 24 h and 48 h, which was markedly downregulated by the purified extract (HA + EX), demonstrating significant reductions in both nitric oxide (NO) and superoxide generation. These findings indicate that purification enhances the bioactivity and specificity of the tested extracts, improving their capacity to modulate excessive neutrophil and monocyte activation. To address this challenge, this study introduces a novel, advanced purification method designed to isolate and refine AMPNE-type particles from the crude extract. Furthermore, we investigated whether the purified particles retain their structural and functional integrity post-purification. By comparing the biological efficacy of the extract before and after purification, we evaluated whether the cleaning process maintains, enhances, or modifies their original bioactive properties, providing crucial insights into the viability of the proposed technology. Our study highlights the potential of purified bioactive molecules to more effectively regulate early inflammatory responses to biomaterials. These results provide a foundation for further research into targeted immunomodulatory strategies aimed at improving implant integration and reducing inflammatory complications.