Nanostructured Composite Hydrogels Incorporating Antimicrobial Peptides and Osteogenic Factors for Periodontal Regeneration

This study reports the development of an injectable nanostructured composite hydrogel integrating the antimicrobial peptide LL-37 and osteogenic growth factor BMP-2 for multifunctional periodontal regeneration. The chitosan/gelatin hydrogel, reinforced with 5% nano-hydroxyapatite (nHA), exhibited a highly porous structure (~90% porosity) and increased surface area (21.4 m²/g vs. 6.8 m²/g in blank) with homogeneously dispersed nanoparticles, as confirmed by FTIR, XRD, SEM, TEM, SAED, XPS, and BET analyses. Sustained release was achieved, with LL-37 releasing ~80% and BMP-2 ~60% by day 7, facilitated by nHA binding. In vitro antibacterial assays demonstrated >99% killing efficiency of Porphyromonas gingivalis, corroborated by live/dead staining and SEM evidence of membrane disruption. Cytotoxicity tests confirmed >90% viability of human periodontal ligament stem cells (hPDLSCs), and osteogenic induction assays showed the AMP+BMP hydrogel produced the highest ALP activity and mineralized nodule formation (p < 0.01 vs. controls). Shear strength testing on wet tissue revealed adhesion of 5.12 ± 1.36 kPa for the composite, compared with 3.54 ± 1.08 kPa for blank hydrogels. In a rat periodontitis model, micro-CT analysis at 6 weeks revealed markedly improved bone regeneration in the dual-loaded group, with bone volume fraction reaching 82 ± 5%, CEJ–ABC distance reduced to 0.34 ± 0.08 mm (vs. 0.94 ± 0.11 mm untreated), trabecular number increased to 2.05 ± 0.15 1/mm, and trabecular separation decreased to 0.20 ± 0.03 mm. Histology-based quantification demonstrated extensive new bone bridging (6/8 animals), increased bone area fraction (BA/TA = 66.8 ± 7.5% in AMP+BMP vs 18.7 ± 7.2% No-Tx), reduced osteoclast indices (N.Oc/B.Pm = 0.9 ± 0.2 mm⁻¹; Oc.S/BS = 2.8 ± 0.9%), and higher PDL-like fiber organization scores (median 3 [IQR 2–3]) at 6 weeks. These findings highlight the synergistic antibacterial and osteoinductive efficacy of LL-37/BMP-2/nHA hydrogels as a promising platform for comprehensive periodontal regeneration.