The overall goal of this procedure is to fabricate a polyelectrolyte complexation shell. A carrier that can entrap and regulate the release of a wide range of growth factors with a heparin binding domain. This method can answer key questions in orthopedic tissue engineering field for efficient entrapment and delivery dedicate growth factors.
The main advantages of this technique is it does not require usage of organics often. Which is known to affect the bio activities of growth factors. Dissolve 200 milligrams of non irradiated sodium alginate or 400 milligrams of eight megarad irradiated sodium alginate in 10 milliliters of double distilled water.
And shake for one hour. Store the alginate solution at four degrees celsius overnight. Filter the alginate solution with a sterile 0.2 micron syringe filter before alginate microbead fabrication.
Disinfect the electrostatic BE generator and syringe pump with 70%ethanol and place them in a class two biological safety cabinet. Then place a glass basin with a magnetic stir bar inside the BE generator. Set the arm electrode of the BE generator nine centimeters above the basin.
Connect the electrode cable of the BE generator to the neuro screw two of the arm electrode. And pour 80 milliliters of strontium chloride solution into the basin. Next, load five milliliters of 0.2 micron filtered alginate solution into the syringe in rubber tube.
After connecting the rubber tube to the arm electrode, switch on the syringe pump at five milliliters per hour. For two minutes. To expel the air inside the tubing and deliver the alginate solution to the tip of the nozzle.
Then, turn off the syringe pump. To commence microbead generation, set the encapsulator to 5.8 kilovolts. Followed by the syringe pump at an alginate flow rate of five milliliters per hour.
Discard the microbeads generated during the first two minutes as these microbeads tend to be irregularly sized and shaped. Collect subsequent microbeads in the 0.2 molar strontium chloride solution. Turn off the syringe pump followed by the encapsulator.
After pumping the preplanned volume of alginate solution. Repeat this for subsequent batches of microbead fabrication. Store the microbeads in 20 milliliters of 0.2 molar strontium chloride solution at four degrees celsius overnight to complete cross linking and stabilize the gel.
Collect 0.5 milliliters of alginate microbeads with a plastic pipette. And place it on a glass slide. View the microbeads under an optical microscope at 10x magnification.
Take 10 images of the microbeads with a microscope CCD camera. Save the images with a 500 micron scale bar in TIFF format at a resolution of 2048 by 1536. Using the length tools in image J, measure the size of the microbeads.
And the scale bar. Then convert the microbead length from pixels to micrometers. Click on the line tools and draw a line across the middle of the alginate bead.
Click analyze on the menu bar and select measure. A pop up window will appear. Repeat these steps to measure all alginate beads within the image.
Measure the scale bar on the image. Collect the microbeads using the 100 micron nylon strainer. And wash the beads with double distilled water.
Using a spatula, transfer all the microbeads made from 0.1 milliliters of the alginate solution into a two milliliter microcentrifuge tube and cover with gauze to prevent drying. Finally, sterilize the microbeads by auto cleaving using liquid mode or in accordance with manufacturer's specifications. Add 1.5 liters of distilled water to the chamber to prevent beads from drying.
Inside the BSL2 hood, incubate the sterile microbeads with one milliliter of sterilized two milligrams per milliliter protamine solution for one hour at room temperature. Wash the protamine coated microbeads twice with double distilled water. Then spin down, using a benchtop centrifuge at 200 times G for three minutes at room temperature.
After centrifugation, aspirate the water using a syringe. Incubate protamine coated microbeads with one milliliter of sterilized 0.5 milligrams per milliliter heparin solution for 30 minutes. This creates a polyelectrolyte complexation shell with avid affinity for heparin binding growth factors.
After incubation, aspirate away the heprain by using a syringe. And wash off the unbound heparin from the polyelectrolyte complexes by washing twice with double distilled water. Load 13.3 microliters of 1.5 milligram per milliliter bone morphogenetic protein 2 or NELL like molecule 1 solution onto 100 micrograms of the polyelectrolyte complex.
Incubate the polyelectrolyte complex at four degrees celsius under 30 RPM, shaking for 10 hours. Next, immerse the microbeads in one milliliter of phosphate buffered saline, or PBS, at 37 degrees celsius with constant shaking. Collect one milliliter of the supernatant and replace it one milliliter of PBS after one, three, six, 10 and 14 days.
Evaluate the uptake and release efficiency of NELL like molecule one using the CBQCA protein assay method according to the manufacturer's protocol. Alternatively, perform elisa. The bioactivity of NELL like molecule one released from the polyelectrolyte complex is assessed by measuring its ability to increase the expression of alkaline phosphatase in rabbit bone marrow stem cells.
Seed 20, 000 rabbit bone marrow stem cells per well in a 24 well plate. And allow them to grow for one day with one milliliter of dulbecco's modified eagle's medium plus 10%fetal bovine serum at 37 degrees celsius and 5%CO2. After 24 hours, replace the medium with one milliliter of an osteogenic medium for seven days at 37 degrees celsius.
And 5%CO2. Place 300 micrograms of polyelectrolyte complex and polyelectrolyte complex NELL like molecule one inside cell culture inserts to keep the complexes separate from the cells. Place the insert into the 24 well plate to avoid the wash out of polyelectrolyte complex microbeads during the osteogenic medium change.
Once every three days, aspirate one milliliter of the osteogenic medium by placing a needle outside the insert. And replace with one milliliter of fresh osteogenic medium. After seven and 14 days of incubation, determine alkaline phosphatase activity with an alkaline phosphatase assay kit.
In accordance with the kit manufacturer's protocol. Package the polyelectrolyte complexes into the pores of a bioresorbable medical grade polycaprolactone tri calcium phosphate scaffold. Using a sterilized spatula inside a BSL2 chamber.
Add a 1.5 milligram or milliliter solution of bone morphogenetic protein two or NELL like molecule one on to the scaffold packed with polyelectrolyte complex. And incubate overnight at 4 degrees celsius. Alginate microbeads are inspected under a microscope to monitor their size and morphology.
Non irradiated alginate microbeads are spherical and the irradiated counterpart is teardrop shaped. Size distribution is an important factor of growth factor uptake. And reduction in size increases the surface to area ratio.
When measuring alginate microbeads with image J software, an average of 100 microbeads provides a good estimation of the size distribution in each match of microbeads. Since growth factor uptake depends on proper coating technique, the layer thickness of each coating is observed by confocal microsopy. Here, confocal laser scanning microsopy images of alginate microbeads incublated with fluorescent analogs of protamine, heparin, NELL like molecule one, and bone morphogenetic protein two are shown.
Alkaline phosphatase activity is used to measure bioactivity of released growth factor. A 2.2 fold increase in alkaline phosphatase activity was observed after incubation with polyelectrolyte complex NELL like molecule one on day 14. Once mastered, this technique can be done within five hours if it is performed properly.
While performing this procedure, it is important to remember that alginate concentrations affect microbead stability. For irradiated alginates, we required four way percent. For non irradiated counterparts, we required two way percent.
Following this procedures, we can further increase growth factor loading by multi layer coating. This technique allows researchers in tissue engineering field to explore growth factor delivery in other disease models. After watching this video, you are able to fabricate polyelectrolyte complex with narrow size distribution.
