Note on the use of different approaches to determine the pore sizes of tissue engineering scaffolds: what do we measure?

Abstract

Background: Collagen-based scaffolds provide a promising option for the treatment of bone defects. One of the key parameters of such scaffolds consists of porosity, including pore size. However, to date, no agreement has been found with respect to the methodology for pore size evaluation. Since the determination of the exact pore size value is not possible, the comparison of the various methods applied is complicated. Hence, this study focuses on the comparison of two widely-used methods for the characterization of porosity-scanning electron microscopy (SEM) and micro-computed tomography (micro-CT).

Methods: 7 types of collagen-based composite scaffold models were prepared by means of lyophilization and collagen cross-linking. Micro-CT analysis was performed in 3D and in 2D (pore size parameters were: major diameter, mean thickness, biggest inner circle diameter and area-equivalent circle diameter). Afterwards, pore sizes were analyzed in the same specimens by an image analysis of SEM microphotographs. The results were statistically evaluated. The comparison of the various approaches to the evaluation of pore size was based on coefficients of variance and the semi-quantitative assessment of selected qualities (e.g. the potential for direct 3D analysis, whole specimen analysis, non-destructivity).

Results: The pore size values differed significantly with respect to the parameters applied. Median values of pore size values were ranging from 20 to 490 µm. The SEM values were approximately 3 times higher than micro-CT 3D values for each specimen. The Mean thickness was the most advantageous micro-CT 2D approach. Coefficient of variance revealed no differences among pore size parameters (except major diameter). The semi-quantitative comparison approach presented pore size parameters in descending order with regard to the advantages thereof as follows: (1) micro-CT 3D, (2) mean thickness and SEM, (3) biggest inner circle diameter, major diameter and area equivalent circle diameter.

Conclusion: The results indicated that micro-CT 3D evaluation provides the most beneficial overall approach. Micro-CT 2D analysis (mean thickness) is advantageous in terms of its time efficacy. SEM is still considered as gold standard for its widespread use and high resolution. However, exact comparison of pore size analysis in scaffold materials remains a challenge.

Keywords: Bone regeneration; Micro-CT; Pore size; Porosity; SEM; Scaffold.

Fig. 1

Representative images of the composite scaffold (RT MID specimen): a micro-CT 2D section, b SEM (×100), c micro-CT 3D image; section area is presented in red color at one side. Scale bars = 400 µm

Fig. 2

Micro-CT 3D visualization of selected specimens: a RT MID, b ORIG, c 37 MIN. The right halves depict the scaffold matrix, the left halves combine the scaffold matrix with color-coded pore size values. Scale bar (white) = 3 mm, the color-coded scale bar is shown on the right. The 3D dimensional structure must be considered when evaluating these images. In the ORIG (b) specimen red pores seem to be much smaller than red pores and even green pores in RT MID and 37 MIN. This is caused by the sectioning of the marginal part of a larger pore and can easily be misunderstood

Fig. 3

Sphere-fitting algorithm in 3D pore size evaluation and the effect of noise voxels inside a pore. a 2D image presenting a section through the fitted spheres inside the pores (the color is dependent on sphere diameter; the pore walls can be seen in black), b color-coded 3D pore size visualization of a segmented pore in 3D combined with a scaffold matrix visualization (white), c artificially added noise pixels are shown by the red arrow; changes in calculated pore size are apparent. The white scale bar = 500 µm; the color-coded scale bar for b and c is shown on the right; the color-coded scale bar for a is not shown

Fig. 4

Structural porosity of differently cross-linked scaffolds expressed by means of differing 2D and 3D parameters. Open circle denotes pairs without statistically significant differences (Kruskal–Wallis, Bonferroni procedure, 0.05)

Fig. 5

Structural porosity of differently cross-linked scaffolds expressed by means of differing 2D and 3D parameters. Statistically significant differences are evident between each of the 6 different porosity parameters of each scaffold before and after the differing cross-linking procedures except for those pairs denoted by open circle (Kruskal–Wallis, Bonferroni procedure, 0.05)

Fig. 6

Illustration of varying results provided by micro-CT 2D pore size analysis. Pores (in gray) of 3 differing shapes (a, b, c) were evaluated by means of 4 micro-CT 2D parameters (MT—mean thickness, MD—major diameter, BICD—biggest inner circle diameter, AECD—area-equivalent circle diameter) and their values are presented in panels below the images (in mm). The results tend to differ with increasing shape irregularity. Scale bar = 0.2 mm

Fig. 7

Coefficient of variation for applied method of pore size analysis. * Denotes statistically significant differences (p ≤ 0.05)