Dual Porosity Protein-based Scaffolds with Enhanced Cell Infiltration and Proliferation

Abstract

3D dual porosity protein-based scaffolds have been developed using the combination of foaming and freeze-drying. The suggested approach leads to the production of large, highly porous scaffolds with negligible shrinkage and deformation compared to the conventional freeze-drying method. Scanning electron microscopy, standard histological processing and mercury intrusion porosimetry confirmed the formation of a dual network in the form of big primary pores (243 ± 14 µm) embracing smaller secondary pores (42 ± 3 µm) opened onto their surface, resembling a vascular network. High interconnectivity of the pores, confirmed by micro-CT, is shown to improve diffusion kinetics and support a relatively uniform distribution of isolated human dental pulp stem cells within the scaffold compared to conventional scaffolds. Dual network scaffolds indicate more than three times as high cell proliferation capability as conventional scaffolds in 14 days.

Figure 1

Scanning electron microscope images of scaffolds made of Gelatin 5% w/v foamed at 0 (A1), 500 (B1) or 1500 (C1) rpm, 10% w/v foamed at 0 (A2), 500 (B2) or 1500 (C2) rpm, 15% w/v foamed at 0 (A3), 500 (B3) or 1500 (C3) rpm.

Figure 2

Effect of agitation speed and gelatin concentration on the porosity (A,B) and modulus (C,D) of the conventional and foam scaffolds. The outcome of freeze-drying in terms of deformation and shrinkage can be seen for medium size (E) and large size samples (F).

Figure 3

The light microscope images of the cross-sections of conventional (A) and dual network (B) scaffolds prepared and stained using standard histology/tissue processing protocols. The diffusion of fluorescein sodium salt as the model substance into the conventional and dual network scaffolds after 0 (C1), 0.5 (C2) and 1.5 (C3) h immersion in 10 µg/ml aqueous solution. Micro-CT 3D image of the dual network scaffold (D). Pore size distribution profile of the dual network scaffold (E1), % total pore volume and cumulative surface area as a function of pore size (E2) as well as pore volume, surface area and cumulative surface area for different ranges of pore size (E3) obtained by mercury intrusion porosimetry. The SEM images of primary pores (F1), porous wall of a primary pore (F2) and the opening of the secondary pores (F3) of the dual network scaffold.

Figure 4

Isolation of DPSCs was confirmed by flow cytometric analysis (A); for the selected population (A1), the expression of CD44 (A2), CD90 (A3), CD34 (A4) and CD45 (A5) was investigated. The isolated cells exhibited alternatively osteogenic (A6) or adipogenic (A7) differentiation. SEM images revealed DPSCs adhesion to the dual network scaffold (B) at low (B1) and high (B2) magnification. H&E staining corroborated uniform distribution of the cells (blue arrows) all over the dual network scaffold (C1) while revealing superficial growth (dashed box) on the conventional ones (C2). Immunohistochemical staining of the dual network scaffolds (D). The cell proliferation rate reported as the cell number normalized to the initial number of cells (N/N₀) on dual network and conventional scaffolds as well as tissue culture plate (E).