Preparation and Characterization of Freeze-Dried and Electrospun Scaffolds from Chitosan, Gelatin, and Monetite Nanoparticles for Bone Tissue Engineering

Abstract

Chitosan, gelatin, and monetite nanoparticles-based composite scaffolds with tailored structures and properties have great potential for bone regeneration. Herein, we aimed to improve the physicochemical, osteogenic, and mechanical properties of the 3D composites scaffold by the addition of dihydrogen calcium phosphate anhydrous (DCPA, monetite) nanoparticles into the polymer matrix with variation in composition in the developed scaffolds. Scaffolds were fabricated from the viscous slurry containing chitosan, gelatin, and synthesized nano DCPA particles using the freeze-drying and electrospinning method. DCPA nano-sized particles were synthesized using calcium carbonate and phosphoric acid in a water-ethanol medium. The novelty in this thesis lies in the fact that nano dicalcium phosphate anhydrous can enhance the bone regeneration potential of chitosan/gelatin-based freeze-dried and electrospun scaffolds through its chemical and structural modification in vitro to ideally mimic bone apatite. In this case, monetite was used as a bioactive ceramic phase to explore its efficacy in exhibiting both osteoconductive and osteoinductive effects in vitro as well. Moreover, monetite possesses higher solubility than tri-calcium phosphate, octacalcium phosphate, and calcium hydroxyapatite in aqueous solutions at physiological pH and is considered to exhibit a high in vivo resorbability in comparison to other phosphate ceramics. No comparative study has yet been carried out to investigate the mechanical and biological performance of chitosan/gelatin electrospun and freeze-dried scaffold containing monetite as reinforced nano particulate in the microporous/macroporous structure of scaffolds. XRD pattern revealed phase pure DCPA in synthesized nanopowder. Macroporous scaffolds were fabricated by the addition of nano-sized DCPA particles to the extent of 10-20 wt% of total polymer concentration into pure chitosan, pure gelatin, and chitosan-gelatin solution with solid loading varying between 2 to 2.5 wt.%. The prepared scaffold showed significantly high interconnected porosity with pore size varying between 90-390 micrometers. With the addition of DCPA nanoparticles, the average pore size, porosity, swelling, and rate of biodegradation of the prepared scaffolds decreased. With an increase in nano ceramic phase content from 10 wt.% to 20 wt.% of total polymer concentration, the compressive strength of the scaffold increased. Scaffold containing 20 wt.% DCPA revealed the highest average compressive strength of 1.93 MPa for CS-M20, 2.43 MPa for GM20, and 2.15 MPa for CGD20. Higher cellular activities were observed in DCPA-containing scaffold as compared to pure gelatin, pure chitosan and chitosan-gelatin (CG) scaffold suggesting the fact that nano DCPA incorporation into the scaffold stimulated superior osteoblast attachment and proliferation as evident from MTT assay and scanning electron microscopic (SEM) investigation of pre-osteoblast cultured scaffolds. A higher degree of lamellipodia and pseudopodia extensions and superior spreading behavior of osteoblasts were observed in FESEM images of MG-63 cultured DCPA-containing scaffolds. The results demonstrated that both mechanical strength and osteogenic properties of gelatin-chitosan scaffold could be improved by the addition of monetite nanoparticles into it. Separately, fibrous scaffolds were prepared from a combination of pristine chitosan, pristine gelatin, and chitosan-gelatin solution in TFA/DCM and an aqueous suspension of DCPA nanoparticles up to 7 wt% of total polymer concentration using electrospinning technique. The electrospun scaffold with an average fiber diameter varying between 70 to 380 nm was successfully prepared at 20 kV, 15 cm of distance between collector and needle tip from a suspension containing up to 7 wt% of DCPA in total polymer concentration. With the incorporation of monetite nanoparticles, the average fiber diameter, porosity, swelling, and rate of biodegradation of the nanofibrous scaffolds decreased. The results demonstrated that both physiochemical properties and mechanical strength of pure gelatin, pure chitosan, and chitosan-gelatin scaffold could be improved by the addition of monetite nanoparticles into it. Electrospun nanofibers containing 7 wt% monetite exhibited the highest average tensile strength of 12.2 MPa for CH-DCPA7, 18.8 MPa for GD7, and 14.34 MPa for CGM7. Higher cellular activities were observed in monetite-containing nanofibers as compared to pure gelatin, pure chitosan and chitosan-gelatin (CG) nanofibers suggesting the fact that nano monetite addition into the scaffold stimulated superior osteoblast attachment and proliferation as evident from MTT assay and scanning electron microscopic (SEM) investigation of pre-osteoblast cultured scaffolds. A higher degree of lamellipodia and pseudopodia extensions and superior spreading behavior of osteoblasts were observed in SEM images of MG-63 cultured monetite containing scaffolds. The results demonstrated that both mechanical strength and osteogenic properties of pristine chitosan, pristine gelatin, and chitosan-gelatin matrix could be improved by the addition of monetite nanoparticles into it.