Bone-like ceramic scaffolds designed with bioinspired porosity induce a different stem cell response

Abstract

Biomaterial science increasingly seeks more biomimetic scaffolds that functionally augment the native bone tissue. In this paper, a new concept of a structural scaffold design is presented where the physiological multi-scale architecture is fully incorporated in a single-scaffold solution. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) bioceramic scaffolds with different bioinspired porosity, mimicking the spongy and cortical bone tissue, were studied. In vitro experiments, looking at the mesenchymal stem cells behaviour, were conducted in a perfusion bioreactor that mimics the physiological conditions in terms of interstitial fluid flow and associated induced shear stress. All the biomaterials enhanced cell adhesion and cell viability. Cortical bone scaffolds, with an aligned architecture, induced an overexpression of several late stage genes involved in the process of osteogenic differentiation compared to the spongy bone scaffolds. This study reveals the exciting prospect of bioinspired porous designed ceramic scaffolds that combines both cortical and cancellous bone in a single ceramic bone graft. It is prospected that dual core shell scaffold could significantly modulate osteogenic processes, once implanted in patients, rapidly forming mature bone tissue at the tissue interface, followed by subsequent bone maturation in the inner spongy structure.

Fig. 1

SEM micrographs of ceramic scaffolds: (A) HA SS, (B) β-TCP CS, (C) HA DC shell scaffold in the transversal section and (D) cross section of β-TCP DC shell scaffold

Fig. 2

Illustration of the pore and interconnection size measurement procedure for SS samples. Black arrows correspond to pore diameter and red ones to interconnection size

Fig. 3

Illustration of the pore size measurement procedure for CS samples. Black and red arrows correspond to long and short axis of the elliptical pore respectively

Fig. 4

Focus on the interface between the two structures showing the slurry intrusion into SS scaffold during the freeze casting process

Fig. 5

Compressive strength of ceramic scaffolds as a function of the macrostructure (Tag values are the porosity level)

Fig. 6

Cell viability analysed by the Live/Dead assay at day 7. Calcein stains live cells in green, Ethidium homodimer-1 stains dead cells in red. A–D HA samples and (E–H) β-TCP samples. In detail: (A) HA SS upper surface; (B) HA SS inner surface. C HA CS upper surface; (D) HA CS inner surface. E β-TCP SS upper surface; (F) β-TCP SS inner surface. G β-TCP CS upper surface; (H) β-TCP CS inner surface. Scale bars 200 µm

Fig. 7

Analysis of cell morphology and scaffold colonization by phalloidin staining at day 7 (A–D) and at day 28 in bioreactor (E, F). Phalloidin in green stains for actin filaments and DAPI in blue stains for cell nuclei. A HA SS; (B) HA CS. C β-TCP SS; (D) β-TCP CS. E, F β-TCP SS. Scale bars 200 μm

Fig. 8

SEM analysis of cells grown on ceramic scaffolds after 7 days. Yellow asterisks indicate some cells. A, B HA SS; (C) HA CS; (D, E) β-TCP SS; (F) β-TCP CS. Scale bars: (A, D) 100 µm; (B, C, E, F) 50 µm

Fig. 9

Relative quantification (2-ΔΔCt) of ALP, SPARC, BGLAP, SPP1 and COL15A1 expression as markers of osteogenesis differentiation for hADSCs, after 28 days of culture in bioreactor. The graphs report the mean and standard error of the samples with respect to the expression of control (A, HA SS; B β-TCP SS) (*p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001)

Fig. 10

Relative quantification (2-ΔΔCt) of ALP, SPARC, BGLAP, SPP1 and COL15A1 expression as markers of osteogenesis differentiation for hADSCs, after 28 days of culture in bioreactor. The graphs report the mean and standard error of the samples with respect to the expression of control (A, β-TCP CS; B, β-TCP SS) (****p ≤ 0.0001)

Fig. 11

Cell distribution analysis performed by Actin staining (phalloidin in green stains for actin filaments and DAPI in blue stains for cell nuclei), (A) and SEM analysis, (B) on β-TCP dual core shell structure scaffold at day 7. Dashed lines show pores of the internal part of the dual scaffold. Scale bars: (A) 200 µm; (B) 500 µm