2.2. Preparation

Figure 1 shows the procedure for producing porous gel. CS and gelatin mixture was first made as follows. A 2 v/v% ethanoic acid and 2 wt% CS solution were heated to 60 °C and simultaneously mixed for twelve hours. CS solution and gelatin solution were blended with a volume ratio of 9:1 and constantly stirred for 4 min at 45 °C. Next, 1 v/v% glutaraldehyde (GA) solution was stirred at 25 °C for 5 min, after which 0.1 wt% of MMT, ZnO, or TiO₂ was added to the CS/gelatin mixture, respectively, followed by 0.02 g of sodium bicarbonate. The mixtures were heated to 55 °C and mixed for 3 min, and then 5 mL of GA solution was incorporated and mixed for 1 min at 55 °C using magnetic stirrers. The final mixtures were kept still at 55 °C on the mixer for another 4 min. The denotations and specifications of samples are listed in Table 1 and Table 2. The final mixtures were then placed in a microwave oven (NN-SM33H, PANASONIC, Fukushima, Osaka, Japan) with an output of 800 W for one minute, and the optimal porous gel was determined accordingly.

Processing flow chart of CS/gel/mineral porous gel and schematic of self-made mold for bone scaffolds.

Denotation and specification of CS/gel/mineral porous gel.

Denotation and specification of CS/Gel/MMT/PCL bone scaffolds.

Figure 1 shows the procedure for the production of bone scaffolds. The flaky polycaprolactone with a melting point between 50 °C and 64 °C was heated until it became a liquid state. Next, a small amount of Vaseline was smeared over the hollow self-made cylinder mold to decrease the possible difficulty of removing the sample afterward. The liquid was then infused into the mold and not removed until it became solid. The hollow polycaprolactone cylinders were then combined with porous gel, thereby forming the bone scaffolds. Refer to Table 1 and Table 2 for sample definitions and specifications of CS/gel/mineral porous gel and CS/Gel/MMT/PCL bone scaffolds.

Bone scaffolds were photographed using a stereo microscope (SMZ-10A, Nikon Instruments Inc., Chiyoda, Tokyo, Japan) with an adjusted focal length and with the aim of ToupView software (Hangzhou ToupTek Photonics, Xihu, Hangzhou, China) in order to acquire images with intensified pixels.

ToupView software (Hangzhou ToupTek Photonics, China) was used to measure the pore size, determining the maximal, minimal, and major pore sizes.

The swelling property test was conducted based on a modified method according to the study by Ngadaonye et al. [20,25]. Samples were immersed in deionized water at 25 °C and then weighed with an interval of one hour until samples reached the swelling equilibrium status. Three samples for each specification were used.

where W_w is the sample weight when wet and W_d is the sample weight when dry for a period of time (t).

The compressive strength of bone scaffolds was measured using a universal testing machine as specified in ASTM D6641M-09, thereby examining whether they were compatible with the strength required by the impaired bones. The compressive rate was 1.3 mm/min, the distance between two compressive clamps was adjusted to 1.2 cm, and cylinder samples had a diameter of 3.7 cm and a thickness of 1.2 cm. The compressive clamps compress the sample to 50% of its thickness and then undo it.

This measurement was designed by the authors. First, 7.5 mL of ethanol and 2.5 g of a black dye were blended to form a coloring solution. The coloring solution was dripped over the surface of samples and left for the evaporation of ethanol. A stereo microscope was used to photograph the surface and cutting section of the samples in order to observe the permeation level, examining whether the samples demonstrate interconnected pores.

This test was conducted according to the method proposed by Wu et al. [23,26]. The cell invasion and proliferation were dependent on the porous micropore structure of the stents, which was thus used to measure the porosity in this study. Samples were trimmed into a specified size and immersed in pure water. The height that the water climbs was recorded and computed to have the sample volume and is referred to as Vv. The apparent volume was referred to as V_t which includes the solid parts and voids. The porosity can be computed using the equation as follows.

where Ø is the porosity, is the apparent volume (including solid part and pores), and V_v is the volume of pores.

All quantitative data are presented in mean standard deviation using one-way statistical analysis (one-way ANOVA) using THE SPSS Statistics 17 with * p < 0.05 and ** p < 0.01 indicating the significance level.