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

## TL;DR

This study shows that M101 scaffolds can boost cell growth and blood vessel formation, making them a promising tool for regenerating periodontal tissues.

## Contribution

The study introduces M101-incorporated scaffolds as a novel approach for enhancing periodontal tissue regeneration through improved angiogenesis.

## Key findings

- M101 scaffolds significantly increased cell viability and proliferation compared to plain scaffolds.
- M101 upregulated key angiogenic genes and induced dense vessel networks in the CAM assay.
- M101 scaffolds showed angiogenic effects comparable to VEGF-treated controls.

## Abstract

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.