Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

The overall goal of this methodology is to assess both the adhesive properties and cellular biocompatibility of the proposed hydrogel composite for intervertebral disc tissue engineering of the nucleus pulposus. This method can help answer key questions in the field of tissue engineering, like how to design and prepare thermal reversible hydrogels with bioadhesive properties for cell encapsulation. The main advantage of this technique is that we are able to demonstrate in-vitro studies with an injectable, thermally-sensitive composite to evaluate its use as a potential replacement for the nucleus pulposus.

In this current study, we evaluate a novel hydrogel scaffold by performing both tensile mechanical tests, and cellular viability assays. Demonstrating the biocompatibility assays will be Emily Schmidt, Mark Dittmer, Edward Goldschmidt, and Frantzeska Giginis, students from the departments of Biological Sciences and Biochemistry. Prior to the synthesis of the bioadhesive hydrogel, purify NIPAAm monomer and mCS, as described in the text protocol.

Then, co-dissolve 10 grams of purified NIPAAm and 2.209 grams of mCS in 232 milliliters of deionized water. Purge the solution of oxygen by using inert nitrogen gas. Maintain a vigorous, yet low gas flow rate, to prevent the solution from bubbling over for 15 minutes.

Continue purging the solution with nitrogen gas and add 0.976 milliliters of TEMED. Next, initiate the polymerization reaction by mixing 97.6 milligrams of ammonium persulfate. Mix the solution for approximately 20 seconds, and quickly seal the solution to prevent any air from entering the vessel.

Allow the solution to polymerize under fluorescent light for 24 hours before processing the sample into a fine powder, as described in the text protocol. Create an oil and water mixture by combining 100 milliliters of canola oil, one milliliters TWEEN 20, and 20 milliliters of 2%of alginate. Emulsify the mixture for 10 minutes with a homogenizer at 15, 000 RPM.

Additionally, mix with a magnetic stir bar at 200 RPM to create large microparticles, or 2, 000 RPM to create small microparticles. Aspirate the 2%calcium chloride solution using a syringe with an 18-gauge needle tip. Slowly add the calcium chloride drop-wise into the emulsion.

After allowing the microparticles to cross-link for 10 minutes, evenly distribute the batch of microparticles into capped 50 milliliter conical tubes. Centrifuge at 1, 400 times G for two minutes to remove the initial layer of canola oil before washing and lyophilizing the microparticles as described in the text protocol. Use the resulting freeze-dried polymer powder to create a 5%weight volume PNIPPAm graft CS solution by first dissolving 50 milligrams of hydrogel powder in one milliliter of 1X phosphate buffered saline, or PBS.

Mix the solution using a vortexer before chilling in a refrigerator at four degrees Celsius for 24 hours. Once the viscous solution forms, create a homogenous composite by adding 25 or 50 milligrams of freeze-dried alginate microparticles to the hydrogel solution and vortexing. Store the composite in a refrigerator at four degrees Celsius for later use.

Prior to performing tensile tests, extract cartilage substrate from a porcine ear, as detailed in the text protocol. Then, attach the force gauge to the arm of the test stand. Place a hotplate on top of the force stand, and set the temperature to 50 degrees Celsius.

Then, affix a one centimeter by one centimeter piece of cartilage to the stainless steel block with cyanoacrylate glue and a pair of tweezers. Glue an additional piece of cartilage to the Delrin cylinder, and attach the cylinder to the force gauge. It is very important that both cartilage surfaces face each other and contact the hydrogel.

Place the plexiglass water bath on top of the hotplate, and insert the stainless steel block with the cartilage substrate inside the bath. Lower the arm of the test stand, and align both cartilage substrates with one another. Then, raise the arm, leaving enough room to apply the hydrogel.

With a positive displacement pipette, dispense 200 microliters of the bioadhesive hydrogel to the bottom piece of cartilage. Lower the arm of the force stand at one milliliter per minute until the hydrogel contacts the upper piece of cartilage, and exerts a preload force of 0.001 newtons. Pour the 37-degrees Celsius saline solution into the bath until the volume reaches the designated water level.

Check the temperature of the solution with a thermocouple. After allowing the hydrogel to set for a total of five minutes, raise the arm at a speed of two millimeters per minute. The test is completed once the bioadhesive detaches from the cartilage substrate, or when a significant drop in force occurs, signaling failure.

Collect the recorded data, and raise the arm of the test stand. Remove the Delrin cylinder from the force gauge. Pour the saline solution into its previous container, and reheat to 37 degrees Celsius.

Calculate the stresses, as described in the text protocol. Before performing the Live/Dead assay with PNIPAAm graft CS, prepare human embryonic kidney, or HEK 293 cells, as detailed in the text protocol. Create positive control monolayers by pipetting multiple 300-microliter volumes of the cell mixture into a 24-well plate to generate four to six replicate samples.

Maintain the same cell concentration by re-suspending the cells in approximately two milliliters of PNIPAAm graft CS for each replicate sample. Pipette the polymer cell mixture up and down using a serological pipette until homogenous. If seeding with alginate microparticles, transfer two milliliters of the PNIPAAm graft CS cell suspension into a 35-millimeter culture dish, and introduce 200 milligrams of particles into the mixture.

Pipette the cell mixture up and down using a serological pipette until homogenous. Then, with a serological pipette, dispense multiple 300-microliters volumes of the polymer cell mixture into each of the wells for killed control, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles to generate four to six replicate samples. Incubate the plate at 37 degrees Celsius for 10 to 15 minutes, and allow the PNIPAAm graft CS to transition from a liquid to a gel.

The polymer forms an opaque white disc. Position a slide warmer in the hood, and set the temperature to 37 degrees Celsius. Use the slide warmer to maintain well plate temperature, and pipette 600 microliters of pre-warmed media into each well.

To further prevent the transitioning of the polymer to a liquid state, position a lamp with a fluorescent bulb above the dish to warm up the air above it. Incubate the cells for five days at 37 degrees Celsius with 5%carbon dioxide. After five days have elapsed, remove the media from the wells of the monolayer, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles.

Wash all of the wells thoroughly, but gently, with PBS, and then, remove the PBS. Allow the hydrogel discs containing alginate microparticles to liquefy at room temperature, and add two milliliters of 50 millimolar sodium citrate to each of the wells. Remove the media from the killed cell wells.

Allow the hydrogel discs to liquefy at room temperature, and add 300 microliters of 70%methanol to each well before incubating the plate for 45 minutes at 37 degrees Celsius. Remove the sodium citrate and methanol solutions from the wells, and pipette each of the hydrogel suspensions into individual microcentrifuge tubes. Centrifuge the tubes at 2, 000 times G for 10 minutes to separate the cells from the hydrogel suspension.

Carefully remove the suspension by pipetting the solution from the conical tube, and leaving behind the cell pellet. Re-suspend the pellet in 300 microliters of PBS, and transfer to the original well plate. Next, add 300 microliters of the Live/Dead dye mix to each of the wells.

Wrap the well plate in aluminum foil, and incubate for 45 minutes on a rocker at room temperature before imaging all of the samples under an inverted fluorescence microscope with a 10X objective. Representative tensile strength results for both PNIPAAm graft CS, and PNIPAAm graft CS with varying alginate microparticle formulations are shown here. The tensile strength of PNIPAAm graft CS quadrupled with the addition of 50 or 75 milligrams per milliliter of alginate microparticles.

As expected, PNIPAAm graft CS contracts and shrinks in volume above its lower critical solution temperature. Introducing microparticles within the hydrogel imparts a swelling effect over a seven-day period in PBS. HEK 293 cells encapsulated in both PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles showed active cellular fluorescence using Live/Dead dye after a period of five days.

These results can be compared to the positive and negative control wells. In parallel, a quantitative assay was used to confirm the Live/Dead imaging, which showed no significant differences in cellular viability between PNIPAAm graft CS, PNIPAAm graft CS with alginate microparticles, and the control monolayer. A hydrogel composite consisting of thermally-sensitive poly(N-isopropylacrylamide)grafted with chondroitin sulfate and alginate microparticles was created for nucleus pulposus replacement.

While attempting this procedure, it's important to remember that the composite is temperature-sensitive, and will liquefy below its lower critical solution temperature. Once mastered, tensile mechanical tests can be performed with ease as long as careful precision is practiced. Swelling studies reveal a hydrogel's ability to retain water.

However, these tests must mimic the native in-vivo environment. Cellular viability of the thermally-sensitive gels can be confirmed and assessed by a combination of fluorescence-marking and metabolic-producing reagents. After watching this video, you should have a good understanding of how thermally-sensitive injectables are applied in intervertebral discs, tissue engineering, and how composites may enhance certain material properties.