The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The overall goal of this methodology is to synthesize hydrogels for tissue engineering applications very easily. This method can help answer key questions in the tissue engineering field, such as the possibility to maintain high cell viability in three dimensional cell culture systems. The main advantage of this technique is that is using quick reaction, we can easily modify hydrogel systems to improve their ability to maintain cell viable.

The implications of this technique extend toward the therapy of spinal cord injury because the cells loaded within the scaffold can rebuild the damaged tissue. For this method can provide significant insight into cell therapies as regenerative medicine and their implication in many methodologies. It can also be applied in several other systems like bone, heart, and cartilage.

To synthesize the RGD-azide derivative, dissolve 50 milligrams of RGD in one milliliter of one molar sodium hydroxide. Then dissolve 24 milligrams of 4-azidobutanoyl chloride in two milliliters of tetrahydrofuran. Add all of the RGD solution into the 4-azidobutanoyl chloride solution at zero degrees Celsius dropwise.

Return to room temperature and stir overnight. Add one milliliter of one molar HCL. Remove the solvent under reduced pressure using a rotary evaporator.

Characterize the obtained product by proton nuclear magnetic resonance spectroscopy, dissolving the sample in deuterium oxide or D2O. To perform the PAA alkine modification, dissolve 200 milligrams of 35%in weight PAA solution in 15 milliliters of distilled water. Add 15.4 milligrams of propargylamine hydrochloride.

Then dissolve 42.8 milligrams of HOBt in 14 milliliters of a one to one ratio of acetonytryle distilled water solution by heating to 50 degrees Celsius. Add all of the HOBt solution to the PAA solution at room temperature. Next, add 53.6 milligrams of EDC to the reaction mixture.

Use one molar HCL to adjust the PH to 5.5 and stir the reaction system overnight at room temperature. Next, dissolve 11.2 grams of sodium chloride in two liters of distilled water. And then add 200 microliters of 37%weight by weight HCL.

Dialyze the solution using a membrane with a molecular weight cutoff of 3.5 kilodaltons. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.

Store the final solution at minus 80 degrees Celsius. Characterize the functionalized polymer by proton nuclear magnetic resonance spectroscopy, dissolving the sample in D2O. To synthesize the PAA-RGD polymer, dissolve 78 milligrams of PAA modified alkine in 10 milliliters of distilled water.

Then dissolve 25 milligrams of the RGD-azide derivative in five milliliters of tetrahydrafuran. Add all of the RGD solution to the polymeric solution. Next, add 2.2 milligrams of copper iodide.

And 2.2 milligrams of sodium ascorbate. Reflux the resulting mixture overnight at 60 degrees Celsius with stirring. Cool the mixture to 25 degrees Celsius.

To dialyze the solution, dissolve 11.2 grams of sodium chloride in two liters of distilled water and add 200 microliters of 37 percent weight by weight HCL. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.

Then place the final solution at minus 80 degrees Celsius. Characterize and store the resulting PAA-RGD polymer as done for its constituents. Blend 40 milligrams of carbomer and 10 milligrams of the functionalized PAA in nine milliters of phosphate-buffered saline at room temperature until complete dissolution.

Add 400 milligrams of PEG to the solution and keep stirring for 45 minutes. Stop the stirring and allow the system to settle for 30 minutes. Then use one molar sodium hydroxide to adjust the PH to 7.4.

To five milliliters of the obtained mixture, add 25 milligrams of agarose powder. Irradiate the system with microwave radiation at 500 watts for 30 seconds to one minute until boiling. And electromagnetically heat up the solution to 80 degrees Celsius.

Leave the mixture exposed to room temperature until its temperature decreases to 50 degrees Celsius. Then add five milliliters of PBS to obtain a solution at a one to one volumetric ratio. Prepare a 12-multiwell plate containing steel cylinders with a diameter of 1.1 centimeters.

Take 500 microliters aliquots from the solution and place them into each steel cylinder. Leave the plate at rest for 45 minutes until complete gelification of the system. Remove the cylinders using stainless steel forceps to obtain the hydrogels.

See the text protocol for characterizing the physicochemical properties of the RGD-functionalized hydrogels. Carbomer 974P, PAA-RGD, and PEG, together with agarose, take part in a statistical random polycondensation after microwave irradiation. Hydroxyl groups contained in PEG and agarose react with carboxyl groups present in Carbomer 947P and PAA-RGD to form ACPEG-RGD hydrogels.

Considering rheological properties, the storage modulus was found to be approximately one order of magnitude higher than the loss modulus, indicating an elastic rather than viscous material. Moreover, it is clearly visible that hydrogel behavior is dominated at low strain values by the elastic modulus. Scanning electron microscopy shows the morphology of an RGD functionalized hydrogel sample and a hydrogel without functionalization.

Both hydrogels revealed that they possess a highly entangled structure of interconnected pores, with some bigger pores containing small pores and some fibular networks on the pore walls. The similar properties indicate that the presence of RGD does not alter the polymer network. After this development, this technique paved the way for research in the field of tissue engineering to explore the stability of these cell culture systems in several other diseases.

After watching this video, you should have a good understanding of how to functionalize hydrogels. Improving their ability to maintain viable stem cells.