Quanta 450 microscope

The same specimens which were analyzed via micro-CT were characterized by means of scanning electron microscopy (SEM; Quanta 450 Microscope, FEI, USA) in high vacuum mode. The scaffolds were cut into 2 mm-thick discs (perpendicular to the long axis of the cylinder) prior to SEM analysis. The resulting sections were coated with a thin layer of gold in an ion sputter (Emitech K550X, Quorum Technologies, UK). The pore size dimensions were measured by means of ImageJ software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2015). The manual mode of the ImageJ analyzer was used for the measurement of the average diameter of the pores. At least 40 pores were assessed at each of five SEM micrographs (mag. 100×) for each scaffold type. Randomly selected pores were analyzed for both long and short pore axis. All parameters of SEM and micro-CT methods are summarized in Table 2.Table 2

Summary of methods and parameters applied in the evaluation of pore size

Characterization of the scaffolds
Method	Parameter	Abbreviation
SEM	Average diameter of the pores	SEM
μCT-2D	Mean thickness	MT
	Major diameter	MD
	Biggest inner circle diameter	BICD
	Area-equivalent circle diameter	AECD
μCT-3D	Sphere-fitting algorithm	3D

The chemical composition of the bone mineral in the explanted rat femurs was verified by means of energy dispersive X-ray spectroscopy (EDS) using an EDS SDD EDAX Apollo (EDAX Genesis system, Mahwah, NJ, USA) detector on a scanning electron microscope (SEM; Quanta 450 Microscope, FEI, Hillsboro, OR, USA) in the high vacuum mode. The histological thin sections were carbon-coated on a K550X (Quorum Technologies, Kent, UK) sputter coater in an argon atmosphere prior to the analysis. The concentrations of elemental Ca and P and the Ca/P molar ratio were determined aimed at the assessment of changes in the bone mineral Ca/P weight ratio that reflected inflammatory processes. Kourkoumelis et al. [48 (link)] have used the EDS method for the evaluation of the Ca/P ratio at different sites in normal and osteoporotic rabbit bones. They proved that EDS provides a suitable analytical method for the in vitro quantification of the Ca/P ratio, that it demonstrates a high enough degree of precision for the taking of semiquantitative measurements and that it allows for a better statistical significance for the Ca/P ratio than does error-prone simple composition assessment. Ten measurements were carried out at different locations on the surface of the bone for each of the eight histological thin sections and for each group of implants.

The microstructure of sponges was characterized by SEM (Quanta 450 Microscope, FEI, Hillsboro, Oregon, USA) in high vacuum mode. Prior to microscopy, the cross-sections of sponges were covered by a thin layer of gold (Emitech K550X ion Sputter, Quorum Technologies, United Kingdom).