Comparative investigation of porous nano-hydroxyapaptite/chitosan, nano-zirconia/chitosan and novel nano-calcium zirconate/chitosan composite scaffolds for their potential applications in bone regeneration

Abstract

Zirconium (Zr) based bioceramic nanoparticles, as the filler material to chitosan (CS), for the development of composite scaffolds are less studied compared to hydroxyapatite nanoparticles. This is predominantly due to the biological similarity of nano-hydroxyapatite (nHA; Ca₁₀(PO₄)₆(OH)₂) with bone inorganic component. In this study, we compared the physical and biological properties of CS composite scaffolds hybridized with nHA, nano-zirconia (nZrO; ZrO₂), and nano-calcium zirconate (nCZ; CaZrO₃). For the first time in this study, the properties of CS-nCZ composite scaffolds have been reported. The porous composite scaffolds were developed using the freeze-drying technique. The compressive strength and modulus were in the range of 50-55 KPa and 0.75-0.95 MPa for composite scaffolds, significantly higher (p < 0.05), compared to CS alone scaffolds (28 KPa and 0.25 MPa) and were comparable among CS-nHA, CS-nZrO, and CS-nCZ scaffolds. Peak force quantitative nanomechanical mapping (PFQNM) using an atomic force microscope (AFM) showed that the Young's modulus of composite material was higher compared to only CS (p < 0.001), and the values were similar among the composite materials. One of the major issues in the use of Zr based bioceramic materials in bone tissue regeneration applications is their lower osteoblasts response. This study has shown that CS-nCZ supported higher proliferation of pre-osteoblasts compared to CS-nZrO and the spreading was more similar to that observed in CS-nHA scaffolds. Taken together, results show that the physical and biological properties, studied here, of CS composite with Zr based bio-ceramic was comparable with CS-nHA composite scaffolds and hence show the prospective of CS-nCZ for future bone tissue engineering applications.

Keywords: Atomic force microscopy; Cell proliferation; Chitosan; Composite; Mechanical properties; Nano-bioceramics.

Figure 1

Schematic representation of scaffold fabrication process.

Figure 2

Force-separation curve obtained after nanomechanical mapping by AFM.

Figure 3

SEM images showing the highly porous morphology of scaffold surface (A, C, E, and G) and magnified image showing the surface of scaffold wall (B, D, F, and H). (Scale: 100 μm for scaffold surface and 2 μm for pore wall surface).

Figure 4

Elemental map of composite scaffolds using EDS done to observe the distribution of nanoparticles along their surface. (Scale: 300 μm) (Ca: calcium, P: phosphorous, O: oxygen, Zr: zirconium).

Figure 5

XRD pattern of bulk material powder and composite scaffolds. * represents CS peak)

Figure 6

Water absorption capacity of different scaffolds after 24 h and 48 h. CS only scaffolds absorbed more water and thus swelled more than composite scaffolds. * represents the significant between groups indicated (p<0.05).

Figure 7

Compressive strength and modulus of scaffolds. Compressive property was improved significantly on all CS-composite scaffolds compared to CS only scaffolds. * represents the significant difference than the CS-group.

Figure 8

Nano-mechanical property of scaffold materials determined using AFM in PFQNM. All composite groups had higher Young’s modulus compared to CS only scaffolds. * represents the significant difference than CS group.

Figure 9

Proliferation of OB-6 pre-osteoblasts on CS and CS composite scaffolds at day 10 and 20. The proliferation was better on CS-nHA and CS-nCZ groups. (Scale: 250 μm)

Figure 10

SEM images showing the morphology of pre-osteoblast attached and proliferating along the surface of different scaffolds. (Scale: 20 μm for day 7 and 50 μm for day 14 images).