2.3. Printed parts characterization
Mechanical properties: mechanical properties, including compressive strength and modulus, were evaluated according to ASTM D695 using a universal testing machine (SANTAM STM-20, Santam Co., Iran), with a load cell capacity of 2KN. Cross-head displacement speed was adjusted are 1 mm/min for all tests. All tests were conducted on five samples, to achieve confident results.
The effect of post curing time was evaluated in six levels (0, 1, 3, 6, 12, and 18 hours) on neat resin samples to find the optimum post curing time given in
Table 2.
Two parameters were selected as variables in this research, including weight percentages of GO (0, 0.5, 1, and 1.5 wt%) and heat treatment time (0, 6, 12, and 18 hour).
Table 3 lists a total of 16 experiment runs conducted in this study.
FTIR: Infrared spectroscopic analysis was carried out to determine the creation of crosslinks between GO and PLLA after heat treatment, utilizing FTIR instrument, model L1280127 from Perkin Elmer, US. FTIR spectra in the range of 450–4000 cm-1 were recorded at room temperature. The resolution was 32 cm−1 and the number of scans was eight.
Scaffold morphology: The pore size was measured by Dino-Lite Digital Microscope (Dino-Light Premier, AnMo Electronics, Taiwan) with magnifications of 150x. For measuring porosity of scaffold, the gravimetric method was performed, and calculated using following equation [
30]:
where
Ve and
Vs are the actual volumes of ethanol (soaked into the scaffold) and scaffold.
We1 is the weight of initial ethanol in the vessel and
We2 is the weight of ethanol after removal of the sample (e.g., first immersed and then removed assuring that the scaffold is completely soaked with ethanol
(. To ensure complete penetration of alcohol into all pores of the scaffolds, the samples, while immersed in ethanol, were placed in a vacuum chamber.
Ws is the weight of the scaffold.
ρe and
ρs are the ethanol and scaffold densities, respectively.
MTT assay: for cytotoxicity evaluation of PLLA and PLLA/GO resins, scaffolds were printed with selected GO contents (0, 0.5, 1, and 1.5 wt.%) and MTT assay were conducted on the extraction of the samples. To prepare the extractions, samples were washed with PBS for 24 h, then soaked in diluted ethanol (70 vol.%) for 30 minutes, followed by sterilizing under UV light for 10 minutes. Finally, all samples were placed into a 5 ml culture medium with serum and incubated at 37°C for 24 hours; the final solution was then considered as 100% scaffold extraction. To evaluate the toxicity of the scaffolds, 10,000 mouse fibroblasts cells (L-929, ATCC® CRL-6364™) were cultured in a 96-well micro plate that was filled with DMEM (10 v/v% fetal bovine serum, 100 μg 𝑚𝑙−1 of streptomycin, and 100 𝑈 𝑚𝑙−1 penicillin) at 37 ° C and 5% carbon dioxide. The culture medium was exchanged with 100 ml of scaffold extraction and 5 v/v% FBS at 24, 72, and 168 hours. After cell culture, 20 μl of MTT solution (5 mgr / mlit in FBS medium) was added to each well and the culture wells were incubated for 2 hours. Isopropanol, of 100 μl in amount,was used to dissolve Formazan crystals. After complete dissolution of the crystals, optical density (OD) of each well was measured by BioTek ELX 800, manufactured by BioTek instrument Co., at 570 nm with 670 𝑛𝑚 background subtraction.
Hydrophilicity: The hydrophilicity of the scaffolds was evaluated via calculating the contact angle utilizing a Dino-Lite Digital Microscope (Dino-Light Premier, AnMo Electronics, Taiwan) with a magnifications of 150x . For this purpose, 5 drops of distilled water in the size of 10 microliter were applied on the surface of samples with a diameter of 6 mm, and the image of the first moment the droplet hit the surface of the sample was captured. The contact angle of the droplet was measured using Dino Capture 2.0 software according to Equation 1 and assuming that the water droplets were in the form of spheres.
where θ, w, and h are contact angle, width, and height of the water droplet, respectively.
Bio-degradation behavior: The prepared samples were washed in an ethylene alcohol solution containing 30% distilled water for 12 hrs and let dry for 24 hrs at room temperature, then sterilized by UV light for 15 minutes. The weight of samples was measured utilizing a digital scale with an accuracy of 0.001 g. Next, each sample was immersed in PBS solution with pH of 7.45 and kept in a shaking incubator with a linear movement (Hastaran Teb Co, Iran) for three months with examining interval periods of 1, 4, and 8 weeks). At each time interval, the predetermined samples were dried for 24 hrs at room temperature, then their weights were measured to find weight loss according to the following equation:
where
is the initial weight of samples and
is the weight of samples after degradation. The medium was changed every two weeks and the pH level of the extracted medium was measured using a pH meter (Model 220, Denver Instruments, USA). Compression test was conducted on all samples to evaluate the effect of degradation on the mechanical properties.
Statistical analyzes: The 2-sample t-test (Minitab 16 software) was used for statistical analyses. The mean and ± standard deviation were used to present all experimental data and a p-value<0.05 was considered significant.