2.1. Materials
Gelatin (from cold-water fish skin), guar gum, sodium periodate (NaIO4), ethylene glycol, tetrazolium bromide (MTT), were obtained from Sigma-Aldrich. Trypsin–EDTA, fetal bovine serum (FBS), RPMI 1640 medium, and penicillin streptomycin (Pen/Strep) were supplied from Gibco. BNC was purchased from nano novin polymer Co. (Mazandaran Sci. & Tech. Park, IRAN). Honey was collected from Bahraseman village, Jiroft, Iran. The Mueller-Hinton agar and dimethyl sulfoxide (DMSO) were obtained from Merck Chemicals Co (Germany). Fibroblast cells (NIH 3T3) were purchased from the institute Pasture (Iran). Pseudomonas aeruginosa (P. aeruginosa, Gram-positive) and Staphylococcus aureus (S. aureus, Gram-negative) bacteria were obtained from Persian Type Culture Collection (PTCC). All other solvents and reagents were of reagent grade.
2.2. Synthesis of aldehyde-modified Guar gum (AD-GG)
AD-GG was synthesized according to the previous study (1). Firstly, GG solution (0.25 wt%) was prepared by dissolving 0.25 gram GG in 100 ml deionized water at room temperature under stirring. After that, GG solution was oxidized by the addition of 3 mL sodium periodate (0.1 g/mL) solution and the reaction was continued at this condition in the dark place. After 3 hours, the reaction was finished by the addition of 150 μL ethylene glycol to eliminate the excess periodate. Finally, the obtained AD-GG solution was dialyzed with deionized water for 3 days using dialysis membrane (MWCO: 12–14000).
2.3. Preparation of bacterial nanocellulose (BNC) reinforced gelatin-AD-GG films (BNC/Ge/AD-GG)
The Ge powder was dissolved in deionized water at 50°c under stirring to obtain 4% w/v solution. Also, ethylene glycol, as a plasticizer was added to the Ge solution (40% dry weight of the Ge). BNC water dispersions was prepared at 50% 25%, and 12.5% concentration from dilutions of the original source of BNC suspension in deionized water under sonication. Then, BNC suspensions with the predetermined amounts (0.5 ml from the original source and each prepared dispersion of BNC suspension) were separately added to the Ge solution and stirred at 40 °C for 15 min. Finally, the films were prepared by blending the BNC reinforced Ge and AD-GG solutions with the ratio of 1:2 respectively (with total volume of 15 ml) and were poured into petri dishes to dry at the room temperature for 48 hours. Table 1 shows the formulations of the prepared different sample films.
Table 1: Different formulations for the preparation of sample films.
Film sample
|
Ratio of Ge solution
|
Ratio of AD-GG solution
|
BNC suspensions
|
A
B
C
D
E
|
1
1
1
1
1
|
2
2
2
2
2
|
-
0.5 ml (original source)
0.5 ml (50% dispersion)
0.5 ml (25% dispersion)
0.5 ml (12.5% dispersion)
|
|
2.4. Preparation of honey loaded BNC reinforced gelatin-AD-GG films (H/BNC/Ge/AD-GG)
The honey loaded films were prepared according to the previous section; various concentrations of honey based on the % w/v of the film volume (5, 10, and 15) were added to the BNC reinforced Ge and AD-GG blended solution. Based on the mechanical properties of the developed BNC/Ge/AD-GG films, sample D was chosen for the preparation of the H/BNC/Ge/AD-GG films. The H/BNC/Ge/AD-GG films were named based on their honey concentrations: D5, D10, and D15.
2.5. Characterization of the fabricated films
2.5.1. Fourier transform infrared Spectrometer (FTIR)
The samples chemical structures were determined using FTIR spectroscopy (Bruker-Tensor 27) at the wavenumber range of 400-4000 cm-1.
2.5.2. Thermogravimetric analysis (TGA)
To assess the thermal stability and decomposition temperature of the samples, TGA was carried out by TGA instrument (LINSEIS SPA PT 1600 device, Germany) from 25 °C to 700 °C, under a nitrogen atmosphere flow.
2.5.3. Mechanical behaviors of the fabricated films
The mechanical behaviors of the fabricated films were investigated by tensile analyzer (SANTAM Machine, Iran) according to the guidelines established by ASTM. Briefly, the films were cut into the dumbbell-shaped (with 5.4×3 mm sizes) and placed between two grips; the test speed was set at 10 mm/min.
2.5.4. Morphology of the fabricated films
The surface morphology of the obtained films was assessed by scanning electron microscopy (SEM, TESCAN, MIRA3). For this purpose, the films were sputter-coated with a gold layer and the images were captured.
2.5.5. Swelling behavior of the fabricated films
The swelling behavior of the prepared films were evaluated by incubation of them in PBS at 37 ° C. All the dried films were weighted and immersed in PBS (pH: 7.4). After 24 h, the swelled films were taken out from the PBS, their excess surface liquid was removed by filter paper, and weighted. The swelling ratio of the samples were calculated by the following equation, where Wd and Ws represent the initial dry and final swelled weight of the films, respectively:
2.5.6. Degradation of the fabricated films
To investigate the in vitro degradation behavior of the fabricated films, their weight loss was assessed after predetermined times incubation in PBS at 37 ° C. For this purpose, the films were weighted and incubated in PBS (pH: 7.4). At the predetermined times, the samples removed from the PBS, air-dried, and weighed. The degradation percentage of the various films was calculated from the following equation: where Wi and Wf represents the initial dry weight and final weight of the sample at the predetermined time.
2.5.7. Antibacterial study
The antibacterial capacity of the fabricated films was evaluated by disk diffusion manner on agar plate against Staphylococcus aureus (S. aureus as gram-positive) and Escherichia coli (E. coli as gram-negative) bacteria. Briefly, the optimal film and the film containing honey were cut into the circular pieces with 10 mm diameter, UV light sterilized, placed to Muller Hinton Agar, which was cultured with 106 CFU/mL of bacterial suspension, and incubated overnight at 37 ° C. The bacterial growth inhibition was assessed by diameters of the inhibition zones.
2.5.8. Cell viability and proliferation assays
To consider the biocompatibility of the films, MTT assay was carried out using NIH-3T3 fibroblast cells (Yavari Maroufi and Ghorbani 2021). NIH-3T3 fibroblast cells were cultured in RPMI-1640 medium containing 10% FBS and 1% Pen/Strep at 37 °C with 5% CO2. After adequate cellular confluency, the biocompatibility evaluations were fulfilled. In brief, the fabricated films were cut into the small pieces and sterilized under UV light for 1 h. The films were placed into the cell culture plate and the cell suspension with density of 5000 cells were added to each well. At the predetermined times (1, 3, and 5 days after seeding), the medium was taken out, 3 mg/ mL of MTT solution (in PBS) was added to each well, and incubated for 4 h at 37 °C. The MTT solution was removed and DMSO added to dissolve formazan crystals formed inside cells. Finally, the optical density (OD) of the produced violet formazan crystals was determined at 570 nm using spectrophotometer plate reader (Awareness Technologies Stat Fax 2100 Microplate Reader).
2.5.9. Cell attachment study by SEM
For evaluating the NIH-3T3 fibroblast cells attachment on the fabricated films, the fabricated samples were cut into the small pieces and sterilized under UV light. Cells were cultured on each film according to the previous section. After 3 days of cell seeding, following process was used to fix cells on the films: firstly, the medium was removed, films containing cells were washed with PBS, fixed with glutaraldehyde solution (4%), dehydrated by serial ethanol solutions (50%, 60%, 70%, 80%, 90%, and 100%), and finally cells were visualized by SEM imaging.
2.5.10. Statistical analysis
Values were reported as mean ± SD. Statistical differences (P < 0.05) were detected by One-way variance (ANOVA) test by using GraphPad Prism software.