Study on βTCP/P(3HB) Scaffolds-Physicochemical Properties and Biological Performance in Low Oxygen Concentration

Abstract

The search for new materials for bone regenerative purposes is still ongoing. Therefore, we present a series of newly constructed composites based on β tricalcium phosphate (βTCP) and poly(3-hydroxybutyrate) bacteria-derived biopolymer (P(3HB)) in the form of 3D scaffolds with different pore sizes. To improve the polymer attachment to the βTCP surface, the etching of ceramic sinters, using citric acid, was applied. As expected, pre-treatment led to the increase in surface roughness and the creation of micropores facilitating polymer adhesion. In this way, the durability and compressive strength of the ceramic-polymer scaffolds were enhanced. It was confirmed that P(3HB) degrades to 3-hydroxybutyric acid, which broadens applications of developed materials in bone tissue engineering as this compound can potentially nourish surrounding tissues and reduce osteoporosis. Moreover, to the best of our knowledge, it is one of the first studies where the impact of βTCP/P(3HB) scaffolds on mesenchymal stem cells (MSCs), cultured in lowered (5%) oxygen concentration, was assessed. It was decided to use a 5% oxygen concentration in the culture to mimic the conditions that would be found in damaged bone in a living organism during regeneration. Scaffolds enabled cell migration and sufficient flow of the culture medium, ensuring high cell viability. Furthermore, in composites with etched βTCP, the MSCs adhesion was facilitated by hydrophilic ceramic protrusions which reduced hydrophobicity. The developed materials are potential candidates for bone tissue regeneration. Nevertheless, to confirm this hypothesis, in vivo studies should be performed.

Keywords: biomaterials; bone tissue engineering; in vitro studies; mesenchymal stem cells; poly(3-hydroxybutyrate); polyhydroxyalkanoates; scaffolds; β tricalcium phosphate.

Figure 1

XRD patterns of: (a) TCP powder before calcination, (b) βTCP powder after calcination, (c) TM scaffold, (d) P(3HB) polymer, (e) composite βTCP/P(3HB) disc.

Figure 2

TG-DSC curves of (a) TS, (b) eTS, (c) TS/P, and (d) eTS/P scaffolds.

Figure 3

Microstructure of the obtained scaffolds.

Figure 4

The average pore size of the scaffolds.

Figure 5

Cross-section of the composite etched βTCP disc covered with P(3HB).

Figure 6

Total (a) and open (b) porosity of the obtained materials.

Figure 7

Water contact angle (a) and surface free energy (b) of the obtained materials.

Figure 8

Compressive strength of the scaffolds. Statistically significant differences were indicated by * p ≤ 0.01.

Figure 9

SEM micrographs of the (a) TM, (b) TM/P, (c) eTM/P scaffolds after compression test and macroscopic view of the compressed (d) TM, (e) TM/P, (f) eTM/P specimens.

Figure 10

The changes in (a) pH of SBF during sample incubation and (b) ionic conductivity around samples incubated in distilled water. Morphology of the P(3HB) on the eTM/P scaffold (c) before and (d) after 60 days of incubation in distilled water. The results of the UHPLC-MS analysis of the sample after 60-day incubation concerning the presence of hydroxy acids (e).

Figure 11

The mean percentage of viable MSC at 7 and 21 DIV. Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval).

Figure 12

Growth of MSC cells on scaffolds. Live cells are green and dead are marked with red dots. Red arrows show cells growing on the edges of the pores and the white arrows show cells growing on the bottom and sides of the pores. Image captured under 10× objective.

Figure 13

Z-stack of MSC cells growing on eT/M scaffold. Photographs were taken at different focal planes along the vertical z-axis (interval 40 µm), showing the location of the cells in the scaffold pore. Red arrows show cells growing on the edges of the pores and white arrows show cells growing on the bottom and walls of the pores.

Figure 14

The average number of MSC cells growing on scaffolds per field of view at (a) 7 and (b) 21 DIV. Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval), Significant digits (*) for p-value * 0.0332, ** 0.0021, *** 0.0002, **** 0.0001. Box plot showing the median (horizontal line), X—mean value, the box covers values from the first to the third quartile and whiskers—total range.

Figure 15

The mean depth (µm) of scaffold penetration (MSC overgrowth) at 7 and 21 DIV. Test: One-way ANOVA with post hoc Bonferroni’s multiple comparisons test, alpha 0.05 (95% confidence interval), Significant digits (*) for p-value * 0.0332, ** 0.0021, *** 0.0002, **** 0.0001, whiskers—standard deviation.