Ferric chloride (FeCl3) and Genipin were utilized as cross-linkers to create two types of nanocomposite hydrogels through physical and covalent cross-linking methods, respectively. The hydrogels were composed of unmodified Gum Arabic (GA), Chitosan (Ch), and natural nano-Hydroxyapatite (nHA) using an acrylic acid solvent. Both the natural nHA and the FeCl3 vs. genipin cross-linked GA/Ch/nHA nano-composite hydrogels were prepared and characterized using various in vitro and in vivo analysis techniques. The use of FeCl3 and genipin cross-linkers resulted in the formation of novel hydrogels with compressive strengths of (15.43-22.20 MPa), which are comparable to those of natural cortical bone. In vivo evaluation was conducted by creating calvarial defects (6 mm) in Sprague-Dawley male rats. The results showed the formation of new, full-thickness bone at the implantation sites in all groups, as evidenced by digital planar tomography and histological staining with Hematoxylin and Eosin stain (H & E). Additionally, the use of genipin as a cross-linker positively affected the hydrogel's hydrophilicity and porosity. These findings justify further investigation into the potential of these nanocomposite hydrogels for bone regeneration applications.
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