2. Materials and Methods:
The keratin was extracted from Chicken feather which were collected from the local market of Raipur (21.14°N 81.38°E) Chhattisgarh, India. The 91% food grade alginate (sodium salt form) was purchased from Loba Chemie. The β-mercatpoethanol (mol. wt. 78.13) from LOBA Chemie, sodium dodecyl sulfate (SDS) (mol. wt. 288.38) and urea (mol. wt. 60.06) was purchased from Merck, and ethanol was purchased from Sigma-Aldrich. Antimicrobial strain of gram negative bacteria Escherichia coli (E. coli) [MTCC 118] and gram positive bacteria Bacillus subtilis (B. subtilis) [MTCC 121] was purchased from MTCC Chandigarh, India.
2.1. Fabrication of Porous Scaffold from Keratin/Alginate binary blend:
The extraction of keratin from Chicken feather was done by using method, described by Yamauchi et al. 14. The homogenized stock solution of keratin 7% (w/v) and alginate 4% (w/v) were prepared and store at ambient temperature. The keratin and alginate samples were characterized by FTIR before using it as blend material. The blend solution was prepared using the optimum parameter, obtained by the CCD model of RSM. The freeze-extraction method as described by the Wang et al. 12, were used to fabricate the scaffold. 2.2. Compatibility study of keratin and alginate: The compatibility/miscibility between polymer of keratin and alginate was confirmed using “X-ray Diffractometer” (XRD) and “Fourier Transform Infrared Spectroscopy” (FTIR). The XRD (X’pert PRO, PANalytical) was equipped with a Cu Kα radiation source (λ = 0.154 nm) set at 45 KV and 30 mA. The samples were scanned at a step size of 4°, between the grade ranges (2θ) of 5° to 40°. The intensity pattern of keratin, alginate and keratin/alginate blend was analyzed to check compatibility between them. The FTIR spectra of keratin, alginate and keratin/alginate blend were recorded on ALPHA FTIR spectrometer (BRUKER). The spectra were recorded with a spectrums 4000-400 cm-1 and 12 scans per sample. 2.3. Determination of the Apparent Porosity of Scaffold: The apparent porosity (% AP) of the scaffold was measured by using the Archimedes principle. Apparent porosity is a calculation of open pore volume as a percentage of bulk volume15. The calculation of % AP was performed by applying the Dry, Soaked and Suspended weights of the scaffolds in the following equation: 2.4. Optimization of Parameters by Response Surface Methodology: Response surface methodology was used to optimize three independent parameters for improvement % AP of scaffold prepared from a …show more content…
2.4.1. Statistical Analyses:
All the statistical analyses were done by using Design Expert© 9.0 (Stat-Ease, USA) software package program. Total 20 experimental studies were run to prepare porous scaffold and for each run % AP was calculated manually by use of Archimedes principle. The observed response values (% AP) then put into CCD model and generate a response surface image (Figure 2). After that, the numerical optimization was done by maintaining all parameter values “in range” and response goal was set to “maximize” mode. The optimum parameter which was predicted by the RSM model was then validated experimentally in triplicates.
2.5. Scanning Electron Microscopy analysis of scaffold:
The scaffold was examined under scanning electron microscope (SEM) (ZEISS EVO 18) under acceleration voltage of 10 kV. The uncoated sample was used for SEM
The keratin was extracted from Chicken feather which were collected from the local market of Raipur (21.14°N 81.38°E) Chhattisgarh, India. The 91% food grade alginate (sodium salt form) was purchased from Loba Chemie. The β-mercatpoethanol (mol. wt. 78.13) from LOBA Chemie, sodium dodecyl sulfate (SDS) (mol. wt. 288.38) and urea (mol. wt. 60.06) was purchased from Merck, and ethanol was purchased from Sigma-Aldrich. Antimicrobial strain of gram negative bacteria Escherichia coli (E. coli) [MTCC 118] and gram positive bacteria Bacillus subtilis (B. subtilis) [MTCC 121] was purchased from MTCC Chandigarh, India.
2.1. Fabrication of Porous Scaffold from Keratin/Alginate binary blend:
The extraction of keratin from Chicken feather was done by using method, described by Yamauchi et al. 14. The homogenized stock solution of keratin 7% (w/v) and alginate 4% (w/v) were prepared and store at ambient temperature. The keratin and alginate samples were characterized by FTIR before using it as blend material. The blend solution was prepared using the optimum parameter, obtained by the CCD model of RSM. The freeze-extraction method as described by the Wang et al. 12, were used to fabricate the scaffold. 2.2. Compatibility study of keratin and alginate: The compatibility/miscibility between polymer of keratin and alginate was confirmed using “X-ray Diffractometer” (XRD) and “Fourier Transform Infrared Spectroscopy” (FTIR). The XRD (X’pert PRO, PANalytical) was equipped with a Cu Kα radiation source (λ = 0.154 nm) set at 45 KV and 30 mA. The samples were scanned at a step size of 4°, between the grade ranges (2θ) of 5° to 40°. The intensity pattern of keratin, alginate and keratin/alginate blend was analyzed to check compatibility between them. The FTIR spectra of keratin, alginate and keratin/alginate blend were recorded on ALPHA FTIR spectrometer (BRUKER). The spectra were recorded with a spectrums 4000-400 cm-1 and 12 scans per sample. 2.3. Determination of the Apparent Porosity of Scaffold: The apparent porosity (% AP) of the scaffold was measured by using the Archimedes principle. Apparent porosity is a calculation of open pore volume as a percentage of bulk volume15. The calculation of % AP was performed by applying the Dry, Soaked and Suspended weights of the scaffolds in the following equation: 2.4. Optimization of Parameters by Response Surface Methodology: Response surface methodology was used to optimize three independent parameters for improvement % AP of scaffold prepared from a …show more content…
2.4.1. Statistical Analyses:
All the statistical analyses were done by using Design Expert© 9.0 (Stat-Ease, USA) software package program. Total 20 experimental studies were run to prepare porous scaffold and for each run % AP was calculated manually by use of Archimedes principle. The observed response values (% AP) then put into CCD model and generate a response surface image (Figure 2). After that, the numerical optimization was done by maintaining all parameter values “in range” and response goal was set to “maximize” mode. The optimum parameter which was predicted by the RSM model was then validated experimentally in triplicates.
2.5. Scanning Electron Microscopy analysis of scaffold:
The scaffold was examined under scanning electron microscope (SEM) (ZEISS EVO 18) under acceleration voltage of 10 kV. The uncoated sample was used for SEM