Evaluation of bioactive extracellular soluble Hemarina (M101) derived scaffolds for neovascularization in periodontal tissue regeneration

Periodontitis leads to progressive destruction of periodontal tissues, where low oxygen levels and inadequate vascularization limit regenerative healing. Hemarina-101 (M101), a marine-sourced extracellular hemoglobin with high oxygen-binding and antioxidant potential, may overcome these barriers. This study investigated the ability of M101-incorporated chitosan–alginate scaffolds to support angiogenesis and cell compatibility for potential use in periodontal regeneration. Chitosan–alginate scaffolds containing 10 % w/w M101 were fabricated and compared with plain scaffolds, untreated controls, and vascular endothelial growth factor (VEGF)-treated groups. Human periodontal ligament cells (PDLCs) and endothelial cells (EA.hy926) were cultured on the scaffolds. Cell viability and proliferation were analyzed using MTT assays and live/dead staining. Expression of angiogenic genes (VEGF-A, ANGPT1, CD31, HIF-1α) was quantified by qPCR. In vivo-like angiogenic responses were assessed using the chorioallantoic membrane (CAM) assay. Statistical significance was set at p < 0.05. Scaffolds with M101 showed significantly greater cell viability and proliferation than plain scaffolds ( p < 0.05), comparable to VEGF-treated controls. Live/dead staining confirmed high densities of viable cells on M101 scaffolds. Gene expression analysis revealed notable upregulation of VEGF-A, ANGPT1, CD31, and HIF-1α in the M101 group, approaching levels seen with VEGF treatment. The CAM assay demonstrated dense, radially organized vessel networks forming around M101 scaffolds, indicating a strong pro-angiogenic effect. M101-incorporated scaffolds enhanced endothelial cell growth, angiogenic gene activation, and neovascularization compared with plain scaffolds. These findings support the potential of M101-based biomaterials as promising candidates for periodontal tissue regeneration, meriting further preclinical and clinical validation.