Vascular Wall-Mesenchymal Stem Cells Differentiation on 3D Biodegradable Highly Porous CaSi-DCPD Doped Poly (α-hydroxy) Acids Scaffolds for Bone Regeneration

Abstract

Vascularization is a crucial factor when approaching any engineered tissue. Vascular wall-mesenchymal stem cells are an excellent in vitro model to study vascular remodeling due to their strong angiogenic attitude. This study aimed to demonstrate the angiogenic potential of experimental highly porous scaffolds based on polylactic acid (PLA) or poly-e-caprolactone (PCL) doped with calcium silicates (CaSi) and dicalcium phosphate dihydrate (DCPD), namely PLA-10CaSi-10DCPD and PCL-10CaSi-10DCPD, designed for the regeneration of bone defects. Vascular wall-mesenchymal stem cells (VW-MSCs) derived from pig thoracic aorta were seeded on the scaffolds and the expression of angiogenic markers, i.e. CD90 (mesenchymal stem/stromal cell surface marker), pericyte genes α-SMA (alpha smooth muscle actin), PDGFR-β (platelet-derived growth factor receptor-β), and NG2 (neuron-glial antigen 2) was evaluated. Pure PLA and pure PCL scaffolds and cell culture plastic were used as controls (3D in vitro model vs. 2D in vitro model). The results clearly demonstrated that the vascular wall mesenchymal cells colonized the scaffolds and were metabolically active. Cells, grown in these 3D systems, showed the typical gene expression profile they have in control 2D culture, although with some main quantitative differences. DNA staining and immunofluorescence assay for alpha-tubulin confirmed a cellular presence on both scaffolds. However, VW-MSCs cultured on PLA-10CaSi-10DCPD showed an individual cells growth, whilst on PCL-10CaSi-10DCPD scaffolds VW-MSCs grew in spherical clusters. In conclusion, vascular wall mesenchymal stem cells demonstrated the ability to colonize PLA and PCL scaffolds doped with CaSi-DCPD for new vessels formation and a potential for tissue regeneration.

Keywords: angiogenesis; biobased materials; biodegradable mineral scaffolds; engineered tissue; green biomaterials; green scaffolds; oral bone defects; polylactic acid (PLA), poly-e-caprolactone (PCL); vascular wall–mesenchymal stem cells.

Figure 1

Surface porosity evaluation of PLA (a) and PLA-10CaSi-10DCPD (b) scaffolds on one random area at 500× and 1000× magnifications. Mineral doped formulation revealed a more compact surface, with a lower number of porosities when compared to pure PLA formulation.

Figure 2

Surface porosity evaluation of PCL (a) and PCL-10CaSi-10DCPD (b) scaffolds on one random area at 500× and 1000× magnifications. PCL scaffolds reveal a more regular surface with larger porosities when compared to PLA scaffolds. CaSi and DCPD mineral fillers are widely distributed on the scaffold surface, partially occluding the pores.

Figure 3

Cell seeding efficiency performed through an indirect method. After 24 h from the seeding on the scaffold surface, the unattached cells were quantified and cell seeding efficiency was calculated by the equation: CSE (%) = (1 − cells_u/cells_i) × 100. Data represent the mean ± SD of three independent biological replicates (n = 3) and were analysed using one-way ANOVA followed by the Tukey’s post hoc comparison. No differences were detected among different scaffolds.

Figure 4

Metabolic activity of VW-MSCs seeded on the different scaffolds after 24, 48 and 72 h of culture was evaluated by MTT based assay. The data represented as the mean ± SD of three independent biological replicates (n = 3), were analysed using one-way ANOVA followed by the Tukey’s post hoc comparison. Significant differences among the experimental scaffolds are represented by asterisks (*) (p < 0.05).

Figure 5

Expression of CD90, αSMA, NG2, and PDGFR-β genes in VW-MSCs evaluated after 72 h of culture in the presence of different experimental 3D scaffolds or in 2D standard culture condition (CTR). Data represent mean ± range of relative expression (RE) of three biological replicates (n = 3). Data were analysed using one-way ANOVA followed by the Tukey’s post hoc comparison test. Statistically significant differences among the scaffolds are represented by asterisks (*) (p < 0.05).

Figure 6

DAPI staining for PLA-10CaSi-10DCPD (a) and PCL-10CaSi-10DCPD (b) without cells and in the presence of 72 h cultured cells: PLA-10CaSi-10DCPD (c), PCL-10CaSi-10DCPD (d). Immunofluorescence analysis for alpha-tubulin of 72 h cultured cells on PLA-10CaSi-10DCPD (e) and PCL-10CaSi-10DCPD (f). Scale bar = 50 µm for a–d and 20 µm for e,f, *indicate spherical cluster.