Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

The overall goal of this procedure is to provide controlled biomolecule delivery with spatial control to mimic the cellular niche from hydrophilic or hydrophobic polymeric composites scaffolds. This is accomplished by first creating a sacrificial framework of desired polymers to provide the spatial arrangement of the biomolecule releasing fibers called interfacial poly electrolyte, or IPC fibers. The second step is to incorporate IPC fibers containing the targeted hydrophilic biomolecule on a sacrificial frame.

Next, the IPC fibers on frame constructs are embedded onto a larger scaffold using a micro pattern substrate as a casting mold incorporating topographical cues on the substrate. The final step is to allow full assimilation of the fibers on frame, construct, and solidification of the polymers to create un patterned or micro patterned substrates. Ultimately, standard assays can be used to show the release profiles and bioactivity of the target molecules.

The main advantage of this technique offer other existing method like microencapsulation that the incorporation of hydrophilic biomolecule into a wide range of hydrophilic or hydrophobic polymer scaffold is now possible to be done in a simple incorporation and fabrication process. This method can help answer key questions in the drug delivery field, including multiple regulated biochemical release biochemical gradient, and this synergistic effect of topographical cues and sustained biochemical release on cell behavior. Visual demonstration of the poly electrolyte fiber formation is critical as the steps are difficult to learn.

The speed concentration and the purity of the poly electrolyte solution are all critical in the stable formation of IPC fibers To begin mix proteins, growth factors, or other biomolecules of interest into 10 to 20 microliter aliquots of the poly electrolyte solutions as described in the accompanying text protocol. Next place, small 10 to 20 microliter droplets of chitosan and alginate on a stable flat surface that is covered with paraform. The droplets of chitosan and alginate should be placed in close proximity, but not in contact with each other.

Lightly dip one tip of a pair of forceps into the kites sand droplet and the other tip into the alginate droplet. Then bring the droplets of poly electrolytes together by pinching the forceps. When the droplets come into contact with each other, slowly pull the forceps vertically to draw the interfacial poly electrolyte complexation fiber, known as the IPC fiber from the interface of the two droplets.

Carefully place the end of the drawn IPC fiber onto a collector consisting of a flat polymeric scaffold affixed on a rotating mandrel. Rotate the mandrel at a fixed speed of 10 millimeters per second to allow formation of uniform and bead less IPC fibers. Increasing the speed of drawing the IPC fibers will form beads, which will cause a burst release of incorporated biochemical and premature fiber termination.

In agreement with the observation from liao et al, and KO et al. When the fiber terminates dilute the remaining liquid with 500 microliters of PBS, measure the concentration of the remaining protein or growth factor content in the residue. In order to determine the incorporation efficiency of the biomolecules, weigh out three grams of Poland polysaccharide and add it to 15 milliliters of distilled deionized water.

In order to create a 20%aqueous solution, mix the Poland solution overnight to ensure homogeneity the next day. Pour 15 milliliters of the Poland solution into a 10 centimeter diameter tissue culture polystyrene dish. Dry the solution overnight at 37 degrees Celsius once dry, cut the films into seven millimeter by seven millimeter square sacrificial Poland frames.

Next, create a 30%solution of the polysaccharides Poland and dextrin in distilled deionized water in a three to one ratio. Mix overnight to ensure homogeneity slowly add sodium bicarbonate to the polysaccharide solution to achieve a final concentration of 20%Mix the solution overnight and store the final polysaccharide solution at four degrees Celsius until it is needed. Affix the sacrificial Poland frame in a desired orientation to the rotating mandl using an alligator clip and some plastic coated adhesive tape.

Rotate the mandl with the affixed frame at a constant speed of 10 millimeters per second. Then begin to draw the IPC fibers as shown in the previous section. However, this time, attach the drawn end of the IPC fibers onto the rotating Poland frame.

Wait for the end of the IPC fiber drawing afterwards. Dry the fibers on frame construct overnight at room temperature. Next, release the construct from the rotating mandl by removing it from the alligator clip.

Place the construct in a micro centrifuge tube cap. Then place five grams of the Poland Dextrin solution into a beaker and begin stirring it at 60 RPMs. Using a stir plate, add 500 microliters of an 11%sodium trim phosphate solution, and 500 microliters of 10 molar sodium hydroxide.

To crosslink the solution. Continue mixing the solution for one to two minutes and then pour the viscous polysaccharide solution onto the fibers on frame construct to fully embed the IPC fibers. Incubate the construct at 60 degrees Celsius for 30 minutes to form a chemically cross-linked composite scaffold to induce pore formation in the composite scaffold.

Submerge the whole scaffold into a solution of 20%acetic acid for 20 minutes. Then wash the scaffold three times in PBS for five minutes while shaking at 100 RPMs. To remove any remaining unreactive reagents, remove the excess PBS and immediately freeze the composite scaffolds at minus 80 degrees Celsius overnight.

Next, lyophilize the scaffolds for at least 24 hours before they're used in any controlled release or bioactivity assays. Start by creating a pristine and patterned PDMS substrate with a desired topography using standard soft lithographic methods. Next, prepare both the sacrificial frame of PCL for IPC fiber collection and the patterned PCL base by first dissolving 0.9%PCL in di chloro methane.

For every one square centimeter area of the PDMS film casting mold drop 500 microliters of the 0.9%PCL solution onto the mold. Allow the solvent to fully evaporate in the fume hood, and then repeat the process of casting 0.9%PCL to thicken the film. Next, prepare the sacrificial frame by placing 500 microliters at a time of the 0.9%PCL solution onto a separate pristine PDMS substrate.

To create a pristine PCL base cast multiple layers of PCL to obtain a scaffold with the desired thickness. Allow all of the chloro methane solvent to fully evaporate in the fume hood when the desired thickness has been reached. Remove the dried PCL film from the pristine PDMS substrate and punch a hole in the PCL frame using a PSAP sized puncher.

Next, mount the PCL frame on the collection mandrel and begin drawing the IPC fiber onto the frame as previously described. When the IPC fiber drawing ends, remove the fiber on frame construct and allow it to dry overnight at 25 degrees Celsius. Place the fiber on frame construct on top of the patterned PCL base and add the 0.9%PCL solution on the fiber on frame construct multiple times to get the desired thickness and to ensure that the IPC fibers are fully embedded.

Bovine serum albumin or BSA released from the Poland dextrin IPD composite showed near linear kinetics with an initial attenuated burst release followed by a concomitant steady state. After two months, the BSA achieved a total release of 97%In contrast, standalone IPC fibers exhibited a rapid release of 80%of the BSA within four hours. Using this same composite vascular growth factor was also able to be sustainably released from the fibers using human umbilical vein endothelial cells as a test platform.

The released growth factor was found to be bioactive following its release at each time.Point. In this example, nerve growth factor was loaded into the fibers over the course of 18 days. Approximately 80%of the loaded nerve growth factor was released from the P-C-L-I-P-C composite in a sustained linear release using a PC 12 neurite outgrowth assay.

The amount of bioactive nerve growth factor released from the scaffold and into the media at each time point had a similar effect on neurite outgrowth as adding 30 nanograms per milliliter of nerve growth factor to plain media. P-C-L-I-P-C composite scaffolds that contain both topography and controlled nerve growth factor release may have a synergistic effect on cellular behavior. Human mesenchymal stem cells grown to nano pattern composite scaffolds showed a higher level of neuronal differentiation compared to cells exposed to either topography or growth factor alone.

While attempting this procedure, it's important to remember to mix the desired hydrophilic molecule into the poly electrolyte solution of the same net charge to incorporate it into the fibers. After watching this video, you should have a good understanding of how to create composite poly scaffold with the capacity for control release of bar molecules. The large gain in this video should allow you to create pulmonary scaffold with the capacity of sustained release of growth factor, release of multiple growth factor, and to create a gradient of biochemical cues that will recapitulate the physiological niche.

Don't forget that working with acidic acid, sodium hydroxide and chlor methane can be extremely hazardous and precautions such as the use of personal protective equipment and the film hood should always be taken when performing this procedure.