Biomimetic dual layered keratin/ hydroxyapatite scaffolds for alveolar bone regeneration

Abstract A new biomimetic dual layered keratin/hydroxyapatite scaffold was designed using the iterative freeze-drying technique. The physical, structural, and chemical properties of the keratin/hydroxyapatite (HA) scaffolds were studied using several analytical techniques. The developed multilayered, interconnected, porous keratin scaffold with different HA content in the outer and inner layer, mimics the inherent gradient structure of alveolar bone. The FT-IR analysis confirmed that the secondary structure of keratin was not destroyed with the successful incorporation of hydroxyapatite particles during the process of scaffold fabrication. X-ray diffraction analysis showed that the keratin and hydroxyapatite retained their crystalline structures in the prepared keratin/HA scaffolds. SEM studies showed an interconnected porous architecture of the prepared scaffolds with seamless integration between the upper and lower layers. EDX analysis showed that the inorganic phases of both the upper and lower layers of the keratin/HA scaffold mainly consist of calcium and phosphorous ions. EDX mapping confirmed the even distribution of calcium (Ca) and phosphorous (P) in both the upper and lower layers of the scaffold. The prepared scaffolds showed a gradual degradation during the 28-day incubation period in PBS. The incorporation of HA improved the mechanical properties of keratin/HA scaffolds. The keratin/HA scaffolds exhibited superior mechanical properties in terms of Young’s modulus and compressive strength in comparison to pure keratin scaffolds. The biocompatibility studies suggested that both keratin and keratin/HA scaffolds were cytocompatible, in terms of cell viability and proliferation. Furthermore, it showed that both the tested materials can be served as an ideal substrate for the differentiation of Saos-2 cells, leading to mineralization of the extracellular matrix. In summary, the development of a non-toxic green method was investigated for keratin extraction to fabricate keratin/HA scaffolds and our results showed great potential for the use of these scaffolds to regenerate alveolar bone due to their structural similarity and excellent in vitro biocompatibility.