In this dissertation, two geometrically different materials were nanoengineered for application in regenerative medicine. The first material used was titanium and the second was halloysite-PCL scaffold. The present study investigated anodized titanium, as surface modified nanoporous substrate. Uniform nanopores of 78nm diameter were produced by optimizing the weight % of HF, applying the voltage and using two different molar concentration of H2SO4. Nanoporous titanium showed higher cell proliferation and differentiation compared to smooth titanium.
The fabrication of the halloysite-PCL scaffold was optimized by using different wt % of halloysite in the PCL-chloroform mixture. The location of the halloysite in the halloysite-PCL scaffold was finalized by use of SEM and FTIC labeling of the halloysite. 2 wt% halloysites in the PCL-chloroform mixture gave the best architecture. Up to 7 wt% halloysites produced a fair quality of the halloysite-PCL scaffold. The Halloysite-PCL scaffold showed a higher cell differentiation and proliferation compared to the PCL scaffold.
The effect of different drug loaded halloysite-PCL scaffolds was observed on bacteria. Results show that the halloysite-PCL scaffolds were able to load and release sufficient drugs to kill the bacteria. There for the halloysite-PCL scaffold can be used as a drug loaded band aid for different applications in the future.
Several studies can be done in the future to further build upon the obtained results. Future studies need to be done to advance this development such as:
Use of local anesthetic drug, anti-inflammatory drugs, growth factors and other drugs for encapsulation and sustained release from halloysite.
Check the effect of the growth factor loaded halloysite-PCL scaffold on osteoblasts.
Application of layer by layer assembly, using various polymers on the drug-loaded halloysite-PCL scaffold as an additional step for controlled for drug diffusion.
Elaboration of drug loading study of halloysite for application in cosmetics.
Investigate the growth of osteoblasts on nanoporus titanium loaded with growth factors.
Try a combination of nanopours titanium and layer by layer technique.
Nanoporous titanium with surface functionalization with bioactive coating such as collagen should be tested.
Check the hierarchical hybrid micro/nano-textured titanium surface for osteoblast growth.
Complete aMEM (alpha minimum essential medium), fetal bovine serum and antibiotic (AB)
Complete aMEM (alpha minimum essential medium) is prepared by mixing 90% aMEM with 2mM L-glutamine and 1 mM sodium pyruvate without ribonucleosides and deoxyribonucleosides, 10% fetal bovine serum and 1% antibiotics.
Cryovials containing osteoblasts, 75% alcohol, centrifuge tubes, complete aMEM and tissue culture flasks.
A liquid nitrogen tank contains different containers labeled with varying colours. Cryovials of different cell lines are placed in different containers. The log book is checked before taking out cryovials from the liquid nitrogen. When removing cryovials from the liquid nitrogen, protective goggles and gloves need to be worn. After removing from the cry vials cap need to be loosened to release liquid nitrogen trapped in the threads of vial. Re-tight the cap and using a float lower half of the vials was placed in water at room temperature to thaw. Sterilize the exposed area of the vial with 75% alcohol before using it. Transfer the suspension to the centrifuge tube from the vial so that nothing will be left in the cryovial. Add complete aMEM to the desired concentration. Centrifuge it. Discard the supernatant, resuspend the pellet in complete aMEM. Plate the desired number of cells on TCP or a tissue culture flask. Ensure the uniform distribution of cells in the TCP or the flask to avoid uneven clumps of cells. Incubate the cells at 37°C, 5% CO2 and 95% air in a humidified cell culture incubator.
70% ethanol, cryovial floating membrane or thawing cells, culture dishes, pipetting aids, sterile pipettes, disposable centrifuge tubes, pipette tips, flask and funnel , hemacytometer, light microscope, 37ºC incubator. Hanks buffer saline solution (HBSS), trypsin, complete media aMEM. Note: reagents can be used for up to two months after preparation.
Laminar hood was cleaned with 70% alcohol. The laminar hood was turn on for 30 minutes so that the laminar hood will be sterilized. Then the cell culture works was started. aMEM was taken out from the refrigerator and equilibrate to 37ºC. Sterilization of the bottle surface was done with 70% alcohol in sterile hood. Cryopreserved vial of cells was taken out from the liquid nitrogen storage tank. Osteoblasts were obtained from ATCC (7F2 CRL-12557).
Proper protection for the eyes and hands was used. The cryovial of the cells was Kept in a 37ºC water bath for thawing. Once it was thaw; it was wiped with 70% alcohol. Cells in the cryovial were transferred to the centrifuge tube, containing 5ml of the complete aMEM. It was centrifuged at 1.2 rpm for 7 to 10 minutes. The supernatant was discarded because it contains cell freazing media. The pellet was resuspend in 5 ml of complete aMEM. It was transfer into culture dishes. Cells in one cryovial can be plated into one or two 100 mm culture dishes. Add some more medium if needed. The culture was incubated at 37ºC, 5% CO2 and 95% humidified air.
Check for the presence of microbial contamination every day. Using a microscope, check for the confluency of the culture every day. Once the cells become confluent, subcultivate them to keep the cells actively growing.
Pre-warn HBSS, 1X Trypsin and Complete aMEM medium to room temperature.Take out and discard the medium using sterile pipette from the culture dish. Wash the cell monolayer two times with 5-10 ml of HBSS. This process of washing with HBSS removes traces of fetal bovine serum. Fetal bovine serum interferes with trpsin. HBSS washing is followed by, addition 3-5 ml of 1 X trypsin and incubate for 5 minutes at room temperature. Check for the detachment of the cells under the microscope. When the cells start detaching, take out all the media with a sterile transfer pipette and transfer it to a centrifuge tube. Centrifuge at 1.2 rpm for 10 minutes. Discard the supernatant because it contains Trypsin. Resuspend the pellet in 5 ml of complete aMEM. Transfer it into culture dishes. Add some more medium if needed. Incubate the culture at 37ºC, 5% CO2 and 95% humidified air.
Extra osteoblast cells, sterile cryovials, liquid nitrogen and cell freezing medium.
Extra osteoblast cells were stored in cryovials in liquid nitrogen for long-term use. The temperature of the liquid nitrogen was maintained at -80ºC. Extra Osteoblasts were collected in a centrifuge tube. They were counted for cell numbers. They were centrifuge at 1.2 rpm for 7 to 10 minutes. The supernatant of complete aMEM was discarded. The cells were resuspended in cell freezing medium (complete aMEM + 10 % DMSO) at a concentration of about 106 – 107 cells/ml. The cryovials were filled with 1 – 1.5 ml of cells suspended in a freezing medium. The cell count, passage, source of cells, viability and confluence were labeled on the cryovials. Before the cryovials were stored at -80°C, they were initially stored for about 6 hours at 4°C and then for 24 hours at -20°C. The cyovials with its details (type of cell line, passage number, date and number of vials) were recorded in a cell-freezing logbook.
A hemocytometer is used for counting the osteoblast cells per unit volume of a suspension. Cover slips used for the counting chambers are thicker than the usual microscopic cover slips. The cover slip is kept over the counting surface then cell suspension is dropped into one of the V-shaped wells. Due to this, the area under the cover slip fills by capillary action. Once the counting chamber is filled, then the counting grid is focused at a low power. The counting chamber has 9 large squares (Figure F.1). Each square has an area of 1mm2 and the depth of 0.1 mm.
The osteoblast cell suspension was dilute enough so that the cells do not overlap each other and uniformly distribute on the counting chamber. The central large square was selected to perform the count.
Description: 2
Figure F.1 Cell counting chamber (Picture credit goes to Nexcelom Bioscience)
Osteoblasts were cultured in 12 well plates. Cells were detached from the plate by trypsinization and transferred to 15ml tubes. They were centrifuged at 1200 rpm for 5 minutes.The supernatant was discarded and the pellet was resuspended in 300 µl of the cell lysis solution. It was kept on ice for 15 minutes. It was then centrifuged at 3200 rpm for 20 minutes at 4 ºC. The supernatant was collected and stored at-70 ºC until needed for further use.
Picogreen assay was used to obtain measurement on the rate of cell proliferation, which was determined by quantifying ds DNA content produced by dividing cells.
Lambda DNA standard, picogreen dsDNA reagent, 20X TE buffer, 1X TE buffer, DNase-free distilled water, plastic container, aluminum foil, samples and fluorescence plate reader.
Kit contains lambda DNA standard, picogreen dsDNA reagent and 20X TE buffer. 1X TE buffer: 20X TE buffer was diluted 20 fold by adding DNase-free distilled water to prepare 1X TE buffer. Picogreen working reagent: Picogreen stock solution was diluted 200 fold with the 1X TE buffer solutions. This process of dilution was done in a plastic container as the solution adsorbs to glass surfaces. This plastic container was covered with aluminum foil and kept in a dark place as the solution is photodegradable. DNA standers were prepared from DNA stock by diluting it in an appropriate amount of 1X TE buffer. For standards, 100 µl is mixed with 100 µl of picogreen working reagent. For samples, 100 µl of cell lysate sample is mixed with 100 µl of picogreen reagent. Incubate in dark at room temperature for 2-5 minutes. Then from each tubes 200 µl will be transferred in 96 black well plates to avoid photodegradation. As fluorescence plate reader was used to detect fluorescence emitted by the samples at an excitation wavelength of 480nm and emission wavelength of 520nm.
50 µl (0.05ml) of each sample was pipetted into the appropriate test tube. 1.5 ml of the Coomassie® Plus Reagent was added to each well and mixed. The plate was incubated for 10 minutes at room temperature. Measure the absorbance at or near 595 nm with a plate reader.
Alizarin red-S, sodium phosphate dibasic, sodium phosphate monobasic, cetyl pyridinium chloride, distilled water and spectrophotometer.
40mM Alizarin Red solution was prepared by adding 2g of Alizarin Red-S to 100ml of distilled water. 10mM Sodium Phosphate solution was prepared by adding two different solutions. Solution A, consisting of 138g of Sodium Phosphate Dibasic in 100ml of distilled water and, solution B consisting of142g of Sodium Phosphate Monobasic to 100ml distilled water. 42.3ml of Solution A and 47.7ml of Solution B, all together this makes 100ml of 10mM sodium phosphate solution. To this, 10% cetyl pyridinium chloride was added to prepare the destaining solution. The scaffolds were then stained with alizarin red for 10min. All the excess stain was removed and the scaffolds are destained with 10% cetyl pyridinium chloride in 10mM sodium phosphate solution. This was measured on a spectrophotometer at 410nm.
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