Carbon nanotubes play an important role in the spatial arrangement of calcium deposits in hydrogels for bone regeneration

Abstract

Age related bone diseases such as osteoporosis are considered among the main causes of reduced bone mechanical stability and bone fractures. In order to restore both biological and mechanical function of diseased/fractured bones, novel bioactive scaffolds that mimic the bone structure are constantly under development in tissue engineering applications. Among the possible candidates, chitosan-based thermosensitive hydrogel scaffolds represent ideal systems due to their biocompatibility, biodegradability, enhanced antibacterial properties, promotion of osteoblast formation and ease of injection, which makes them suitable for less invasive surgical procedures. As a main drawback, these chitosan systems present poor mechanical performance that could not support load-bearing applications. In order to produce more mechanically-competent biomaterials, the combined addition of hydroxyapatite and carbon nanotubes (CNTs) is proposed in this study. Specifically, the aim of this work is to develop thermosensitive chitosan hydrogels containing stabilised single-walled and multi-walled CNTs, where their effect on the mechanical/physiochemical properties, calcium deposition patterns and ability to provide a platform for the controlled release of protein drugs was investigated. It was found that the addition of CNTs had a significant effect on the sol-gel transition time and significantly increased the resistance to compression for the hydrogels. Moreover, in vitro calcification studies revealed that CNTs played a major role in the spatial arrangements of newly formed calcium deposits in the composite materials studied, suggesting that they may have a role in the way the repair of fragile and/or fractured bones occurs in vivo.

Fig. 1

a Compressibility of the different formulations before (grey) and after (black) sol–gel transition. Results are reported as mean ± SD (n = 3). One way ANOVA (α = 0.05) on the values after gelation returned P = 0.0131; Tukey’s multiple comparisons test: ^* P < 0.05, compared to chitosan; ^# P < 0.05 compared to CS-HACS. Water sorption and desorption isotherms of the freeze dried hydrogels: b chitosan; c CS-HACS-(NOSC-SWNTs); d CS-HACS-(NOSC-MWNTs). Data are presented as mean ± SD (n = 3)

Fig. 2

SEM of chitosan, CS-HACS-(NOSC-SWNTs) and CS-HACS-(NOSC-MWNTs) freeze dried hydrogels. Arrows indicate coated carbon nanotubes within the hydrogel matrix

Fig. 3

Overlay images of SEM pictures with EDS data: Ca (green), P (red) and co-localisation of Ca/P (yellow) (Color figure online)

Fig. 4

a Quantitative analysis of surface calcium deposition presenting % of surface covered by co-localised Ca and P (from SEM). Data are reported as mean ± SD (n = 3). One-way ANOVA (α = 0.05) P < 0.0001 for both gels containing CNTs; Tukey’s multicomparison test, ***P < 0.001 compared to control at time zero, ^### P < 0.001 compared to a specific sample as indicated in the figure. b Average % salt deposition on different types of hydrogel samples (from microCT). Data are reported as mean ± SD (n = 5). One-way ANOVA (α = 0.05) P < 0.0001 for both gels containing CNTs, Tukey’s multicomparison test, all samples presented P < 0.001 compared to control, and P < 0.001 was obtained for all comparisons between 7 and 14 days. Release profile of c BSA d OVA from (black closed circle) chitosan; (red closed square) CS-HACS-(NOSC-SWNTs); (blue closed triangle) CS-HACS-(NOSC-MWNTs) gels. Data are reported as mean ± SD (n = 3) (Color figure online)

Fig. 5

Representative micro CT slices of different gel samples incubated in SBF for 0, 7 and 14 days