Comprehensive in vitro and in vivo assessment of spongin/hydroxyapatite biohybrid scaffolds for bone regeneration

Abstract

The selection of an appropriate scaffold is vital for successful bone tissue engineering. Biohybrid scaffolds, combining the mechanical tunability of synthetic polymers with the biocompatibility of natural materials, have gained significant attention. In this study, biohybrid scaffolds were developed using spongin isolated from the marine sponge Sarcotragus foetidus (Sf), mimicking natural bone architecture. These scaffolds were functionalized with hydroxyapatite (HAp) and boron-doped HAp (B-HAp) to enhance mechanical and osteoconductive properties. Micro-computed tomography (μCT) revealed interconnected porosity above 70 % in all groups, facilitating nutrient exchange and cell migration. Elemental analysis confirmed the presence of carbon, oxygen, calcium, and phosphorus, essential for bone regeneration. Mechanical testing showed increased Young's modulus values, reaching ∼20 kPa for Sf/HAp and ∼ 22 kPa for Sf/B-HAp, due to the reinforcing coatings. In vitro assays over 21 days showed that the Sf/B-HAp scaffold exhibited the highest mineralization, alkaline phosphatase (ALP) activity, collagen synthesis, and extracellular matrix (ECM) formation, indicating enhanced osteogenic capacity. Additionally, antibacterial tests showed superior activity for the Sf/B-HAp group. In vivo studies conducted using a rat calvarial bone defect model over a 20-week period demonstrated significant bone regeneration across all groups, with the Sf/B-HAp scaffold exhibiting the most pronounced effect. Overall, Sf-based scaffolds supported cell adhesion, proliferation, and differentiation, with the Sf/B-HAp variant offering superior mechanical, osteoconductive, osteoinductive, and antibacterial properties—making it a promising candidate for advanced bone tissue engineering.