Quantitative stereological analysis of the highly porous hydroxyapatite scaffolds using X-ray CM and SEM

Background: Material properties of the scaffolds as well as their microstructure are vital in determining in vivo cellular response. Three-dimensional (3D), highly porous scaffolds are used in tissue engineering to provide a suitable microenvironment and to support regeneration of bone. Both pore sizes and their architecture, in particular interconnection density, impact functionality of scaffold during its biomedical applications.

Objective: In this paper a comparative study of the microstructure of highly porous hydroxyapatite scaffolds produced via gelcasting of foamed slurries and replication of polyurethane sponge were carried out.

Methods: Quantitative stereological analysis of the microstructure was conducted using transmission X-ray computed microtomography (μCT) and scanning electron microscopy (SEM). Application of the X-ray microtomography allowed obtaining the 2D cross-sectional images of examined samples, and then the 3D reflection of individual samples.

Results: In our studies we proved that the distribution of pores in HAp bioceramics can be controlled by selection of the manufacturing method. In the case of material produced by the gelcasting method, the porosity of the samples was about ∼78 vol.%, while for the method of replication of the porous organic matrix it was higher ∼84 vol.%. Application of gelcasting method resulted in bioceramics with the macropores ranging from 95 μm to 158 μm (the modal value of 120 μm). Furthermore, micropores of size 34 μm-60 μm - so called "windows", were observed on spherical macropores surfaces. In the case of replication of polyurethane sponge only macropores from 295 μm to 337 μm (the modal value of 300 μm) were obtained. Application of μCT and SEM give more information than classical mercury intrusion porosimetry in studies of porous bioceramics. Developed materials met the criteria for porous bone substitutes.

Conclusions: The results of quantitative description of microstructure allowed determining the differences between porous hydroxyapatite bioceramics obtained via replication of porous organic matrix and gelcasting of foamed slurry. The stereological analysis demonstrated, that bioceramics prepared via gelling of foamed slurry has a lower pore size and grains (1.1-1.9 μm) than the material obtained by the method of replication of polyurethane sponge (2.1-2.3 μm). Based on morphological analysis the porosity of tested materials was determined. In the case of material produce by the gelcasting, porosity of the samples was about ∼78 vol.%, while for method of replication of the porous organic matrix the porosity was higher and constituted ∼84 vol.%. Furthermore, evaluated materials varied in porosity and the pore size distribution. It was stated that the method of gelcasting resulted in hydroxyapatite bioceramics with the macropores diameter (95-158 μm), micropores so called "windows" (34-60 μm) - observed on spherical macropores walls and micropores of size 0.6 μm-1.3 μm, which were visible in sintered areas. When the method of replication of polyurethane sponge was applied only macropores from 295 μm to 337 μm were obtained. The comparable values of shape factors such as elongation, curvature of pours boundary and convexity, confirmed that macropores in both studied series had similar shape.