Noninvasive real-time monitoring by alamarBlue(®) during in vitro culture of three-dimensional tissue-engineered bone constructs

Abstract

Bone tissue engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and nonfunctional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop noninvasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the nontoxic alamarBlue(®) (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was evaluated not only in static cultures, but also in dynamic cultures in a perfusion bioreactor setup. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a noninvasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence, the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct.

FIG. 1.

(A–C) Designed macrostructure of the Ti scaffolds produced via selective laser melting. (A) Diamond-shaped unit cell. (B) Side and (C) top view of the Ti scaffold. (D) Photographic side image of a produced Ti scaffold. The scale bars represent 1 mm.

FIG. 2.

Peristaltic perfusion bioreactor composed of a medium reservoir, a peristaltic pump, and a bone chamber, housing a tissue engineering (TE) construct. (A) Schematic representation of a single circuit. (B) Photographic image of an assembled perfusion bioreactor with seven parallel circuits.

FIG. 3.

Overall metabolic activity of TE constructs dynamically cultured for 7 days. The values at different incubation times were obtained by integrating the initial alamarBlue^® (AB) assay (10 mL) in the bioreactor. The linear fit of the time points below 11 h is represented. Error bars show the standard deviation of three measurements of the same TE construct.

FIG. 4.

Effect of the incubation time of the initial AB assay on the obtained metabolic activity of (A) statically cultured TE constructs (R² values of linear fit between 0.83 and 0.98) in 1 mL AB solution and (B) dynamically cultured TE constructs (R²>0.99) in 10 mL AB solution. Error bars show the standard deviation of five TE constructs, which have all been averaged over three measurements.

FIG. 5.

Correlation of the metabolic activity with the cellularity of the (A, B) seeded TE constructs (static AB protocol a, 2 h incubation) and TE constructs cultured for (C) 14 or (D) 10 days (static and bioreactor AB protocol b). Error bars in (A), (B), and (D) show the standard deviation of 3 measurements of the same TE construct. Error bars in (C) show the standard deviation of three TE constructs, which have all been averaged over three measurements.

FIG. 6.

Green fluorescent images of the top, side, and bottom views of the live/dead-stained bone TE constructs after (A) 7 or (B) 14 days of dynamic bioreactor culturing, and after (C) 14 days of static culturing. The scale bars represent 1 mm. Color images available online at www.liebertpub.com/tec