Fabrication, characterization, and optimization of a novel copper- incorporated chitosan/gelatin-based scaffold for bone tissue engineering applications

Introduction: Fabricating composite scaffolds with improved physicochemical properties as artificial microenvironments are of great interest in bone tissue engineering. Given advantageous properties of nano-hydroxyapatite/chitosan/gelatin (nHA/Cs/Gel) scaffolds, the present study aimed to synthesize a modified nHA/Cs/Gel biomimetic scaffold with improved features. Methods: Pure and copper (Cu)-substituted nHA was synthesized using the chemical precipitation method under controlled pH and temperature. Pure and Cu-substituted nHA/Cs/Gel scaffolds were fabricated by salt-leaching/freeze-drying method. Physicochemical characteristics of nanoparticles and scaffolds were explored using XRD, FTIR, FE-SEM/EDX, and ICP. Besides, scaffold mechanical strength, degradation, porosity, swelling, biomineralization, and cytocompatibility were assessed. Results: Pure and Cu-substituted nHA were synthesized and characterized with appropriate Cu substitution and improved physical properties. All scaffolds were highly porous (porosity >98%) and Cu incorporation reduced porosity from 99.555 +/- 0.394% to 98.69 +/- 0.80% while enlarged the pore size to more than100 mu m. Cu-substitution improved the scaffold mechanical strength and the best result was observed in nHA.Cu5%/Cs/Gel scaffolds by the compressive strength 88.869 +/- 19.574 MPa. Furthermore, 3% and 5% Cu-substituted nHA enhanced the scaffold structural stability and supported osteoblast spread, adhesion, survival, mineralization, and proliferation. Moreover, longterm and sustainable Cu release from scaffolds was observed within 28 days. Conclusion: Cu-substituted nHA/Cs/Gel scaffolds mimic the porous structure and mechanical strength of cancellous bone, along with prolonged degradation and Cu release, osteoblast attachment, viability, calcium deposition, and proliferation. Taken together, our results indicate the upgraded properties of nHA.Cu5%/Cs/Gel scaffolds for future applications in bone tissue engineering.