Standard Operation Procedures for Fabricating Porous Microspheres: .... at 80oC, and white powder of resin /SiO2 composite microspheres were obtained,.
SOHEYL HABIBIMAJD
(summary)
THESIS PROJECT
Standard Operation Procedures for Fabricating Porous Microspheres: 1) Spray drying1: Materials: 1. Crystalline hydroxyapatite (HA) 2. Gelatin 3. Distilled water Equipment and apparatus: 1. 2. 3. 4. 5. 6. 7.
GPW120-II small-type spray dryer Magnetic Stirring S-570 scanning electron microscope (SEM) D/max-cB X-ray diffractometer (XRD) Nicolet Nexus-470 Fourier transform infrared (FTIR) spectroscope LS13320 Laser Diffraction Particle Size Analyzer SA-3100 Surface Area Analyzer
Procedure: First, using analytical grade Ca (NO3)2.4H2O and (NH4)2HPO4, HA slurry was prepared using precipitation method, and this slurry were putted in room temperature overnight. Second, distilled water was added to dilute the precipitates to an extend after removing supernatant. Third, Gelatin was dissolved into distilled water at 40oC using magnetic stirrer, and then added to slurry. Fourth, HA/gelatin slurry was spray dried via GPW120-II small-type spray dryer, atomized at a pressure of 15 L/min and a flow rate of 400 mL/h, with inlet temperature of the nozzle being 170oC , and outlet temperature of nozzle being 100oC. Fifth, the product was calcined at 700uC and 1000uC for 1 h. Evaluation: S-570 scanning electron microscope (SEM): To examine the morphology of the spray dried HA microspheres. D/max-cB X-ray diffractometer (XRD): Phase analysis was carried out with a scan speed of 4o/min between 10o and 60o 2Ɵ angle, operated at 40 kV and 100 mA. Nicolet Nexus-470 Fourier transform infrared (FTIR) spectroscope: To characterize the functional groups of the micro- spheres. LS13320 Laser Diffraction Particle Size Analyzer: To measure the particle size distribution and mean particle size, and distilled water was employed as diffuse medium. SA-3100 Surface Area Analyzer: To determine specific surface area by the Brunauer-Emmett-Teller (BET), after being degassed at 200uC for 2 h and using nitrogen as the absorbent.
SOHEYL HABIBIMAJD
(summary)
2) W/O/W type double emulsion solvent evaporation
THESIS PROJECT
method2:
Materials: 1. 2. 3. 4.
Ethyl cellulose (EC) (7 cps) Dichloromethane Polyvinyl alcohol, Kuraray POVALTM PVA-220, Kuraray Co.,Ltd.(Tokyo, Japan) gold-palladium at 15 mA
Equipment and apparatus: 1. Homogenizer (T18 Digital ULTRA-TURRAX®, IKA Works GmbH & Co. KG, Staufen im Breisgau, Germany) 2. Freeze Dryer - FDU-2200 (EYELA USA, New York, USA) 3. 75- μm and 53-μm sieves 4. Scanning electron microscopy (SEM) system (JSM- 5500LV, JOEL Ltd., Tokyo, Japan) 5. Sputtering device (Quick Auto Coater JFC-1500, JOEL Ltd.) 6. Laser diffraction particle size analyzer (SALD-2200, Shimadzu Co. Ltd., Kyoto, Japan 7. Surface area analyzer (FlowSorb 2310, Shimadzu Co., Ltd.) Procedure: First, oil phase was obtained by dissolving EC (1.0 gram) in dichloromethane (14 mL). Second, using a homogenizer a 0.5% PVA solution (1 mL) was emulsified into the oil phase for 30 s at 25,000 rpm at room temperature. Third, The obtained W/O emulsion was mixed in a 0.5% PVA solution (20 mL) using the homogenizer for 5 min at 4,000 rpm at room temperature. Fourth, in order to remove the dichloromethane, the resultant emulsion was added to 400 mL of water all at once and stirred at 400 rpm at room temperature for 3 h, and then the hardened EC microspheres were washed. Fifth, using 75- μm and 53-μm sieves the aggregated EC microspheres were removed, and the nonaggregated ones were collected by filtration with a 20-μm sieve. Sixth, the EC microspheres were redispersed in deionized water and lyophilized. Evaluation: Scanning electron microscopy (SEM) system (JSM- 5500LV, JOEL Ltd., Tokyo, Japan) : Using Sputtering device gold-palladium at 15 mA for 3 min were deposited on the prepared EC porous microspheres and observed using a scanning electron microscopy (SEM) system. laser diffraction particle size analyzer (SALD-2200, Shimadzu Co. Ltd., Kyoto, Japan: The sizes of the fabricated EC microspheres were determined. Surface area analyzer (FlowSorb 2310, Shimadzu Co., Ltd.): The specific surface areas of the fabricated EC microspheres were determined using gas adsorption method.
SOHEYL HABIBIMAJD
3) Polymerization
(summary)
THESIS PROJECT
method3:
Materials: 1. 2. 3. 4. 5. 6. 7.
Ethyl alcohol Ammonium hydroxide Distilled water Tetraethyl orthosilicate Urea HCl Formaldehyde
Equipment and apparatus: 1. 2. 3. 4.
Water bath Malvern Zetasizer NanoZS Instrument Scanning electron microscopy (SEM) system Philips XL-30SEM Surface Area & Pore Size Analyzer system (NOVA 2200e)
Procedure: First, Silica sol. was prepared via mixing 60 mL EtOH with 1.5mL ammonium hydroxide and 0.5ml distilled water were stirred at 30 °C water bath, and after half hour, 5g TEOS was rapidly added into the mixture and continued stirring for 8h at 30 °C water bath. Second, 25ml of prepared silica sol diluted with 25ml distilled water and heated to 30oC using water bath. Third, 1.0g urea was added and dissolved by stirring, and 3mol/L HCl also added to adjust the pH 1. Fourth, rapidly 1.85ml formaldehyde was added into the solution and stirred vigorously for about 3 minutes until the solution whitened. Fifth, Maintained at 30 °C water bath for 1h, and then, added large amount of distilled water to end the reaction and kept overnight and precipitated. Sixth, dried overnight at 80oC, and white powder of resin /SiO2 composite microspheres were obtained, and by calcination, resin was removed from the microspheres. Evaluation: Malvern Zetasizer NanoZS Instrument: at 25 °C silica colloidal particle diameter were measured. Scanning electron microscopy (SEM) system Philips XL-30SEM: The prepared microspheres were observed. Surface Area & Pore Size Analyzer system (NOVA 2200e): showed N2 sorption isotherms of the porous silica microspheres, which gives the physical and chemical properties of the porous silica microspheres.
SOHEYL HABIBIMAJD
(summary)
THESIS PROJECT
4) Combination of phase inversion emulsion technique with thermally induced phase separation (TIPS) method4: Materials: 1. 2. 3. 4. 5.
Poly(L-lactic acid) (PLLA) with an inherent viscosity of 1.6 dL/g (from Jinan Daigang Biological Co., China) Glycerol Dioxane (Diox) Obtained from Tianjin Kemiou Chemical Reagent Co., China Tetrahydrofuran (THF) Liquid nitrogen
Equipment and apparatus: 1. Centrifuge 2. Scanning electron microscopy (SEM) (S-3200N, Hitachi, Japan) 3. Belsorp-Mini adsorption measuring apparatus (Bel Japan, Japan) Procedure: First, PLLA was firstly dissolved in 10 mL organic solvents (dioxane, THF or dioxane/THF mixtures). Second, 30 mL of glycerol was heated to 50oC and then added into the PLLA solution dropwise while stirring, and then poured into liquid nitrogen. Third, to exchange the organic solvents, the solidified mixtures were mixed with ten-folds of ice water. Fourth, the PLLA microspheres were washed three times with deionized water and collected by centrifuge and lyophilized. Evaluation: Scanning electron microscopy (SEM) (S-3200N, Hitachi, Japan): (Morphological Characterization) By gold coating of 30s, the morphology was observed by SEM using an accelerating voltage of 15 kV. Belsorp-Mini adsorption measuring apparatus (Bel Japan, Japan): (Surface Area Measurement) after evacuating samples at 258C for 10 h. Surface area of scaffolds was calculated from BET plot of adsorption/desorption isotherm using adsorption points in the P/P0 range of 0.1 to 0.3 (BELSORP-mini analysis software). Microsphere Size Analysis: The size distribution of microspheres were determined and averaged by measuring 100 microspheres from the SEM images using the image analysis software (Nano Measurer 1.2 software). All data were expressed as the mean 6standard deviation. Density and Porosity: The microspheres were filled up into a crucible which had been known volume. The microspheres were then well packed and leveled. The density was calculated.
SOHEYL HABIBIMAJD
5) Seed swelling
(summary)
THESIS PROJECT
method5:
Materials: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Poly(methyl methacrylate) (PMMA) Distilled water Sodium dodecyl sulfate (SDS, 99%) Dibutylphthalate (DBP, 99%) Toluene (99.5%) Methyl methacrylate (MMA, 99%) Ethyleneglycol dimethacrylate (EGDMA, 99%) Benzoylperoxide (BPO, 95%) Polyvinyl alcohol (PVA-124, degree of polymerization 2400, degree of hydrolys is 98%) Tetrahydrofuran (THF, 99%) Ethanol (99.5%)
Equipment and apparatus: 1. Ultrasonic cell crushing apparatus (B1LON92-II, Shanghai Bilang Instrument Co., Ltd., Shanghai, China) 2. Scanning electron microscopy (SEM, JEOL JSM-6309LV, Japan Electronics Co., Ltd., Tokyo, Japan) 3. Three-necked flask 4. Centrifuge Procedure: First, 0.26 g PMMA seeds were dispersed in 10 mL distilled water in a three-necked flask. Second, an emulsion was added which contained 20 mL distilled water, 0.075 g SDS, 1 mL DBP and 1 mL toluene, which the emulsion was formed by sonication at 200 W for 1 h using an ultrasonic. Third, the mixture was putted into stirring at 250 rpm at room temperature for 24 hours. Fourth, another emulsion which was made by sonication of 200 W for 1 h containing 30 mL distilled water, 0.075 g SDS, 0.6 mL MMA, 0.6 mL EGDMA and 0.10 g BPO was added to the three-necked flask and stirred at 250 rpm at room temperature for 24h more and 3.5 mL of PVA aqueous solution with a concentration of 10 wt% was added. Fifth, at stirring rate of 300 rpm at 70 °C for 24 h the polymerization on the swollen seeds were carried out. Sixth, the obtained particles were centrifuged and washed with ethanol three times extracted with THF at 60 °C for 12 h to completely remove the linear polymer and swelling agent and finally particles were centrifuged and washed with ethanol three times. Evaluation: The surface morphology: The characterization of the prepared PMMA seed microspheres and porous PMMA microspheres were carried out using scanning electron microscopy.
SOHEYL HABIBIMAJD
6) Sintering
(summary)
THESIS PROJECT
method6:
Materials: 1. Hydroxyapatite 2. Chitosan (degree of deacetylation, 86.98%; viscosity average molecular weight, 6.96105 Da; intrinsic viscosity, 5.77mPas) 3. Acetic acid (Analytical grade) 4. Liquid paraffin (heavy; viscosity 0.009Pas at 30C and light; viscosity 0.0018Pas at 30C) 5. Sorbitan mono- oleate (Span 80) 6. Glutaraldehyde (25% aqueous solution) 7. Acetone (Analytical grade) 8. Petroleum ether (Analytical grade) Equipment and apparatus: 1. 2. 3. 4.
1000ml reaction flask Halfmoon paddle stirrer Optical microscope (Labophot, Nikon, Japan) Scanning electron microscope (SEM) (S 2400, Hitachi, Japan)
Procedure: First, HA powders 20g together with 40g of 2% (w/v) chitosan solution in 2% (v/v) acetic acid were mixed well. Second, HA/chitosan slurry was dispersed into mixture of heavy and light liquid paraffin and 0.4mg of span 80 as stabilizing agent in a 1000ml reaction flask as dispersion medium by stirring at 400r.p.m. with a half-moon paddle stirrer and stirred for 5mins. Third, q.s. amount of glutaraldehyde were added to harden the spheres obtained. Fourth, after 30min spheres were filtered, washed with acetone and petroleum ether, and dried at 60oC for 3h. Fifth, in order to burn off the chitosan, these chitosan-bonded, spheres were heated initially at 500C for 1h and finally sintered at 1100C for 1h. Sixth, after washing thoroughly the partially burned off chitosan using distilled water, they were heated at 200C for 24h. Evaluation: Microscopy: Spheres were observed and photographed using a scanning electron microscope (SEM) (S 2400, Hitachi, Japan) and an optical microscope (Labophot, Nikon, Japan).
SOHEYL HABIBIMAJD
(summary)
THESIS PROJECT
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2.
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Nie T, He M, Ge M, Xu J, Ma H. Fabrication and structural regulation of PLLA porous microspheres via phase inversion emulsion and thermally induced phase separation techniques. J Appl Polym Sci. 2017;134(22). doi:10.1002/app.44885
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