In vivo Regeneration of Mineralized Bone Tissue in Anisotropic Biomimetic Sponges

Abstract

In the last two decades, alginate scaffolds have been variously studied as extracellular matrix analogs for tissue engineering. However, relevant evidence is still lacking concerning their ability to mimic the microenvironment of hierarchical tissues such as bone. Hence, an increasing amount of attention has recently been devoted to the fabrication of macro/microporous sponges with pore anisotropy able to more accurately replicate the cell niche structure as a trigger for bioactive functionalities. This paper presents an in vivo study of alginate sponges with anisotropic microporous domains (MAS) formed by ionic crosslinking in the presence of different fractions (30 or 50% v) of hydroxyapatite (HA). In comparison with unloaded sponges (MAS0), we demonstrated that HA confers peculiar physical and biological properties to the sponge, depending upon the inorganic fraction used, enabling the sponge to bio-mimetically support the regeneration of newly formed bone. Scanning electron microscopy analysis showed a preferential orientation of pores, ascribable to the physical constraints exerted by HA particles during the pore network formation. Energy dispersive spectroscopy (EDS) and X-Ray diffraction (XRD) confirmed a chemical affinity of HA with the native mineral phase of the bone. In vitro studies via WST-1 assay showed good adhesion and proliferation of human Dental Pulp-Mesenchymal Stem Cells (hDP-MSC) that increased in the presence of the bioactive HA signals. Moreover, in vivo studies via micro-CT and histological analyses of a bone model (e.g., a rat calvaria defect) confirmed that the maximum osteogenic response after 90 days was achieved with MAS30, which supported good regeneration of the calvaria defect without any evidence of inflammatory reaction. Hence, all of the results suggested that MAS is a promising scaffold for supporting the regeneration of hard tissues in different body compartments.

Keywords: alginate; anisotropic structure; hard tissues; hydroxyapatite; in vivo models; microporous sponges.

FIGURE 1

Pore morphology of alginate porous sponges as a function of the HA volume fraction. (A) SEM images of sponge cross-sections (scale bar: 1 mm). Pores originated by the development of crystal domains with preferential orientation (see red arrows). This was determined by the presence of rigid HA phases that constrain the formation of pores to make them anisotropic. (B) SEM images of the pore surface to evaluate HA distribution. In the red square, EDS analysis and mapping for MAS30: the results confirmed the homogeneous elemental distribution along a portion of the surface -carbon (green), calcium (red), and phosphorus (blue).

FIGURE 2

In vitro biological response of hDP-MSC to MAS0, MAS30, and MAS50. (A) Cell adhesion at 4 and 24 h and (B) cell viability after 2, 4, 6, and 8 days of cell culture.

FIGURE 3

(A) Sequence of micro-CT images of sponges after 8, 30, 60, and 90 days in the in vivo calvaria defect model. (A) No treatment (NT), (B) MAS0, (C) MAS30, and (D) MAS50. (E) Measurement of bone mineral density (BMD) after 8, 30, 60, and 90 days in the calvaria defect model. (F) Percentage of in vivo total bone regeneration after 90 days in the calvaria defect model.

FIGURE 4

Photomicrographs of histological sections stained with H&E. The images are representative of the different groups at 90 days. (A,B) No treatment (NT). (C,D) MAS0. (E,F) MAS30. (G,H) MAS50. All images are shown at 20× and 40×, respectively. The asterisks correspond to the MAS scaffolds.