3D conductive nanocomposite scaffold for bone tissue engineering

Abstract

Bone healing can be significantly expedited by applying electrical stimuli in the injured region. Therefore, a three-dimensional (3D) ceramic conductive tissue engineering scaffold for large bone defects that can locally deliver the electrical stimuli is highly desired. In the present study, 3D conductive scaffolds were prepared by employing a biocompatible conductive polymer, ie, poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) (PEDOT:PSS), in the optimized nanocomposite of gelatin and bioactive glass. For in vitro analysis, adult human mesenchymal stem cells were seeded in the scaffolds. Material characterizations using hydrogen-1 nuclear magnetic resonance, in vitro degradation, as well as thermal and mechanical analysis showed that incorporation of PEDOT:PSS increased the physiochemical stability of the composite, resulting in improved mechanical properties and biodegradation resistance. The outcomes indicate that PEDOT:PSS and polypeptide chains have close interaction, most likely by forming salt bridges between arginine side chains and sulfonate groups. The morphology of the scaffolds and cultured human mesenchymal stem cells were observed and analyzed via scanning electron microscope, micro-computed tomography, and confocal fluorescent microscope. Increasing the concentration of the conductive polymer in the scaffold enhanced the cell viability, indicating the improved microstructure of the scaffolds or boosted electrical signaling among cells. These results show that these conductive scaffolds are not only structurally more favorable for bone tissue engineering, but also can be a step forward in combining the tissue engineering techniques with the method of enhancing the bone healing by electrical stimuli.

Keywords: PEDOT:PSS; bioactive glass nanoparticles; bone scaffold; conductive polymers; conductive scaffold; gelatin.

Figure 1

Schematic diagram of the preparation of three-dimensional scaffolds with the freeze-drying method. Notes: 0 P, 0.1 P, and 0.3 P represent the scaffolds prepared from 0%, 0.1%, and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles. Abbreviations: 3D, three dimensional; BaG, bioactive glass nanoparticles; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Gel, gelatin; NHS, N-hydroxysuccinimide; PEDOT:PSS, poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate).

Figure 2

(A) Chemical structure of poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate). (B) The Fourier transform infrared spectroscopy spectra of noncrosslinked gelatin, 0 P, and 0.3 P scaffolds. More intense peaks of amide bonds in the gelatin spectra can be related to crosslinking of carboxyl and amine groups. The presence of bioactive glass nanoparticles is indicated by the silicon peaks. Notes: 0 P and 0.3 P represent the scaffolds prepared from 0% and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 3

¹H high-resolution magic angle spinning nuclear magnetic resonance spectra of 0 P (blue) and 0.3 P (red) scaffolds soaked in heavy water (D₂O). Peak assignments for gelatin follow Rose and Gross.⁶² Silicone resonance is due to disc and O-rings in the magic angle spinning rotor. Gelatin resonances have lower amplitude for the 0.3 P scaffold compared to the 0 P scaffold. The peak height of arginine δ significantly decreased with the addition of poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate), suggesting that poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) molecules are in close interaction with arginine side chains of gelatin. Notes: 0 P and 0.3 P represent the scaffolds prepared from 0% and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 4

The X-ray diffraction pattern of the 0.3 P scaffold. No diffraction peak in the spectra indicates an amorphous structure of bioactive glass nanoparticles in the composition. Notes: 0.3 P represents the scaffolds prepared from 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 5

Scanning electron micrograph of the (A) 0 P, (B) 0.1 P, and (C) 0.3 P scaffolds, and the (D) 0.3 P scaffold at high magnification. (E) Micro-computed tomography image of the 0.3 P scaffold and (F) overall view of the scaffolds. The interconnected network of spherical pores with an average size of 100–200 μm provides a good environment for cell migration and facilitates the diffusion of nutrients. Notes: 0 P, 0.1 P, and 0.3 P represent the scaffolds prepared from 0%, 0.1%, and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 6

(A) Cytoplasmic content of human mesenchymal stem cells on the 0 P, 0.1 P, and 0.3 P scaffolds in comparison to tissue culture plastic (control sample) shows that the number of viable cells increases by increasing the poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) concentration in the composition of scaffolds. The values are mean ± standard deviation (number of samples =3). (B) Scanning electron microscopy and (C) confocal fluorescent microscopy images of a cell on the 0.3 P scaffold. (D) Scanning electron microscopy and (E) confocal fluorescent image of cell distribution on the 0.3 P scaffold. Enhanced cell attachment is observed for the conductive scaffolds. Notes: 0 P, 0.1 P, and 0.3 P represent the scaffolds prepared from 0%, 0.1%, and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles. Abbreviation: con, control.

Figure 7

(A) Thermal gravimetric analysis and (B) differential scanning calorimetry curves of the 0 P and 0.3 P scaffolds. Four thermal stages can be observed in the differential scanning calorimetry plot (water loss, decomposition, pyrolysis, and oxidative degradation). The location of the peaks shows a slight shift to the right for the 0.3 P scaffold. Notes: 0 P and 0.3 P represent the scaffolds prepared from 0% and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 8

(A) Swelling and (B) in vitro enzyme degradation analysis of the 0 P, 0.1 P, and 0.3 P scaffolds. Although the presence of poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) did not change the swelling behavior significantly, it dramatically influenced the biodegradation resistance of scaffolds. Notes: 0 P, 0.1 P, and 0.3 P represent the scaffolds prepared from 0%, 0.1%, and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.

Figure 9

The electrical conductivity of scaffolds is increased by the addition of poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate). A 70% increase in conductivity was measured in scaffolds with 0.3% poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate). Notes: 0 P, 0.1 P, and 0.3 P represent the scaffolds prepared from 0%, 0.1%, and 0.3% (w/w) poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) in the mixture of 10% (w/v) gelatin and 30% (w/v) bioactive glass nanoparticles.