Crocin-Loaded 3D-Printed Chitosan/Collagen/nano-Hydroxyapatite Scaffolds for Rabbit Calvarial Defect Repair: In Vitro and In Vivo Evaluation

Abstract

Crocin (Cro), a bioactive compound derived from saffron, has shown potential in bone healing by regulating osteoclast activity, promoting osteoblast differentiation, and supporting bone regeneration. This study evaluated a 3D-printed scaffold composed of chitosan (Cs), collagen (Col), and nano-hydroxyapatite (nHA), loaded with Cro, for bone tissue engineering (BTE). Using an advanced three-dimensional (3D) printing process, scaffolds containing 25 and 50 µM Cro were fabricated and assessed for structural, biological, in vitro, and in vivo properties. It was hypothesized that Cs/Col/nHA scaffolds loaded with Cro would exhibit enhanced bioactivity and improved suitability for BTE. The results demonstrated that Cro loading was associated with enhanced scaffold structural integrity, interconnected porous architecture, and thermal stability, with measured porosity values reaching approximately 90%. Differential scanning calorimetry (DSC) confirmed successful Cro loading and favorable interactions within the scaffold matrix. Cell studies demonstrated time and concentration dependent responses of human adipose-derived mesenchymal stem cells (hADSCs), with preserved cell attachment and morphology across all groups and the Cs/Col/nHA/Cro50 scaffold showing the most favorable response. Histopathological evaluation following implantation showed acceptable tissue compatibility, with no major adverse tissue responses observed. Masson’s trichrome staining revealed greater collagen deposition in the Cro-loaded scaffolds, particularly at 25 µM Cro, providing preliminary evidence of enhanced extracellular matrix (ECM) formation and regenerative potential. In vivo, preliminary observations from this pilot animal study suggested a potential improvement in cranial defect repair following Cro loading. These findings highlight the potential of Cro-loaded Cs/Col/nHA scaffolds as customizable matrix for bone repair and support the need for further comprehensive investigation.