Effect of monetite reinforced into the chitosan-based lyophilized 3D scaffolds on physicochemical, mechanical, and osteogenic properties

Chitosan and monetite nanoparticles-based composite scaffold with tailored structures and properties possesses tremendous potential for bone restoration. Here, we aimed to enhance the physicochemical, mechanical, and biological properties of a three-dimensional composite scaffold by the addition of monetite nanoparticles into a chitosan matrix with varying content of monetite in the developed scaffolds. The scaffolds were fabricated from the viscous slurry containing chitosan and nano monetite particles using a free-drying process. Macro-porous scaffolds were fabricated by incorporation of nano-sized monetite particles up to 20 wt% of total chitosan concentration into pure chitosan solution. These are then characterized using an X-ray diffractometer (XRD), FTIR, and field emission electron microscope (FESEM). Results revealed that the chitosan/monetite composite scaffolds showed highly porous architecture with an average pore size varying between 90 and 390 mu m, and high interconnected porosity varying from 77 to 88%, respectively. Scaffold containing 20 wt% monetite (CS-M-20) revealed the highest compressive strength of 1.93 MPa, which is closely matching with that of the lower range of compressive strength in cancellous bone. The incorporation of monetite content into the chitosan matrix significantly diminished (p < 0.05) pore size, and porosity and retarded the rate of biodegradation of the monetite-containing scaffolds as evident from 20 wt% and 38 wt% of weight loss in CSM20 and CSM10 scaffolds after 28 days of incubation in a sterile PBS. Monetite-containing scaffolds showed a higher ability of mineralization as compared to the pristine chitosan (CS-M-0) scaffold, depositing bone-like carbonated hydroxyapatite crystals onto their surface after 14 days of immersion in SBF at 37 degrees C. A higher extent of lamellipodia and filopodia protrusions and superior spreading behavior of osteoblasts were noticed in FESEM images of MG-63 cultured monetite containing scaffolds. MTT assay exhibited significantly higher (p < 0.05) MG-63 cell proliferation in monetite-containing CS-M-20 scaffold than that in pristine chitosan scaffold suggesting the fact that monetite nanoparticles incorporation into the chitosan matrix stimulated superior osteoblast attachment and proliferation. Further, immunocytochemistry analysis of MG-63 cultured onto developed scaffolds revealed higher osteogenic activity and differentiation ability in CS-M-20 scaffold as compared to pristine chitosan scaffold. The obtained results demonstrated that the monetite-containing scaffolds have a great potential for the regeneration of bone tissue defects.