Investigation of calvarial bone regeneration in a rat model using three-dimensional polycaprolactone/carboxymethyl chitosan nano composite scaffolds containing hydroxyapatite nanoparticles along with the icariin and atorvastatin synthesized by the freeze-casting method

Abstract

Although autografts and allografts remain common for bone defect repair, they entail donor-site morbidity, limited availability, and potential immune rejection. The development of tissue engineering has provided a potential solution to overcome these and facilitate effective bone regeneration. Extensive research has confirmed the osteogenic potential of bioactive molecules like Atorvastatin (ATV) and Icariin (ICA). But despite the increasing body of evidence supporting their individual merits, few studies have investigated the synergistic integration of these materials in Nanocomposite scaffolds. A novel three-dimensional scaffold composed of polycaprolactone (PCL), carboxymethyl chitosan (CMCs), and nano-hydroxyapatite (nHA), co-loaded with Icariin and Atorvastatin, and fabricated using the freeze-casting technique, is described. This study aimed to evaluate the scaffold’s effectiveness in promoting calvarial bone regeneration in Wistar rats, contributing to the advancement of biomaterials in bone tissue engineering. Scaffolds containing PCL/CMCs/nHA with 0.1% ICA and 0.1% ATV were fabricated using the freeze-casting method. In vitro assessments were conducted to evaluate the biomechanical and physiological properties of the scaffolds. In vivo experiments involved implanting the scaffolds into calvarial bone defects in six groups of Wistar rats. After 12 weeks, histological analysis was performed to assess bone regeneration, including fibrous tissue formation, bone formation, osteon development, and osteoblast cell numbers and fibroblast cell numbers. After 72 h of incubation, the PCL/CMCs/nHA/ATO/ICA scaffold significantly enhanced cell viability compared to other groups, however, the differences observed between the other groups were not statistically significant. In vivo, results showed significantly greater bone formation, osteon development, and osteoblast numbers in the PCL/CMCs/nHA/ATO/ICA group than in the negative and other groups. The PCL/CMCs/nHA/ATO/ICA scaffold demonstrated superior bone regeneration outcomes, showing comparable performance to autografts in terms of new bone tissue formation, osteon structure, and 72-h cell viability, suggesting its potential as a viable alternative in bone tissue engineering.