# Revealing the True Morphological Structure of Macroporous Soft Hydrogels for Tissue Engineering
## 3 Results and Discussion
### Salt Porogen Features  
The measurement of the distribution of pore sizes after the salt particles were washed shows a higher abundance of smaller pores than the porogen fractionation when a lower sieve size was used. These fine pores below the \"sieving limit\" were remarkable, especially in hydrogels prepared with a porogen in the size ranges of 30-50 and 50-90 \(\upmu\)m, as indicated in Table 2 and Figure 1d. The distribution of the sizes of solid porogen evaluated by SEM shows that the fractionated salt contained mostly irregular particles with an aspect ratio close to 1. Besides larger particles that fit the sieving ranges, all the porogen fractions contained some amount of very fine particles of sizes approximately below 10 \(\upmu\)m held in larger clusters. These clusters were probably formed during sieving (or even existed before), but could be dispersed in the polymerization mixture during the gel preparation. The formation of clusters was most likely caused by environmental humidity, allowing the salt particles to connect by moisture, perhaps with some contribution of electrostatic effects. Thus, in each porogen size fraction, a certain amount of a \"fine dust\" was present, causing in the hydrogel the formation of pores below the sieve size range; see Figure 3.  
Figure 2: Comparison of the structure of sample set 3 (50–90 \(\upmu\)m) image created by LSCM performed with unfrozen gel (**a**) and the SEM of gel previously frozen to \(-195\)\({}^{\circ}\)C (**c**) with salt crystal as porogen (**b**). Selected single pores are highlighted in red.  
Particles below the sieve lower size limit are present in all the salt fractions. In order to relate the porous morphology of gels with the porogen structure, a semi-qualitative evaluation of the porogen particles was performed. The size distribution for each batch was evaluated based on the SEM images of particles using the ImageJ software, with several tens of particles counted in each image of the three images for each size range. The porogen size distribution revealed a low number of two larger fractions in the ranges 0-30 and 30-50 \(\upmu\)m, while the small particles 5-15 \(\upmu\)m originating from clusters were always present, even in the batches sieved through higher sieve sizes; see Figure 4. The porogen fraction at 50-90 \(\upmu\)m contained predominantly particles from 5 to 70 \(\upmu\)m in size. The fractions above represent counts of a pore of respective size (black bars in Figure 4). However, when the distribution of sizes was recalculated to the volume distribution (grey bars in Figure 4), one can see that the most significant volume fraction in the system comprises particles of sizes corresponding to sieve ranges. The volumes were calculated for equivalent spheres of the diameter obtained from the SEM images (c.f., Table 3), with values of the most represented particle size volume-wise and the corresponding volume fractions. When incorporated into the gels and washed away, these particles should form the corresponding volume fraction of pores and pore channels.  
Figure 3: SEM microimages of NaCl particles that serve as a porogen: (**a**) 0–30, (**b**) 30–50, and (**c**) 50–90 \(\upmu\)m. Image (**d**) is a lower magnification image of the 50–90 \(\upmu\)m porogen.