Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

This protocol allows convenient fabrication of chitosan microgels without toxic solvents that can be used for a variety of tissue engineering and drug delivery applications. This technique does not require special equipment or training and does not require toxic emulsion techniques or solvent rinses, making it highly biocompatible and translatable to a clinical setting. We have applied this technique as a biomaterial strategy for treating growth plate injuries.

However, we believe that this technique will also be useful in other regenerative medicine applications, too. This technique can be applied to other regenerative medicine applications that would benefit from an injectable, biodegradable, biomaterial scaffold system with the potential for sustained drug release. Begin by adding acetic acid and purified chitosan to a 10-milliliter Luer lock syringe to form a 6%weight-by-volume chitosan solution.

Using a female female Luer lock connector, connect two Luer lock syringes, then mix the solution back and forth until the chitosan in has fully dissolved in the ascetic acid. Now add 100 microliters of the genipin solution to the chitosan-containing syringe and mix back and forth between the syringes for 30 seconds, then eject the mixture from the syringe onto a 35-millimeter Petri dish. Cover the Petri dish with paraffin film and incubate it at 37 degrees Celsius overnight in a humidified atmosphere.

Use a spatula to break the hydrogel into smaller pieces, then place a filter of the desired mesh size into the back of a clean 10-milliliter syringe. Transfer the broken gel pieces into the syringe fitted with the filter and add 6 milliliters of double distilled water. Connect the syringe via a Luer lock connector to another clean 10-millimeter syringe.

Force the gel and water mixture through the syringe with the filter to create microgels. After the first filtration, open the syringe containing the filter and add the mixture back into this syringe. Force the mixture through the filter again.

Now, transfer the filtered gel mixture to a 50-milliliter conical tube and add double distilled water to bring the volume to 20 milliliters. Vortex the solution to get a homogenous solution. Centrifuge the microgels at 100 times g for 5 minutes at room temperature.

After centrifugation, remove the upper aqueous phase and resuspend the microgels in 10 milliliters of 70%ethanol, then vortex the microgels and place them under UV light for one hour to sterilize. Now, centrifuge the microgels at 1000 times g for 5 minutes at room temperature. Discard the ethanol and rinse 3 times with double distilled water.

Resuspend the microgel pellets in an equal volume of double distilled water. With the increase in pH, the microgels showed a decrease in swelling as depicted by the change in Feret diameter. Also, the size of the microgel particles depends on the pore size of the filter used.

Number 200 mesh produced small particles, while number 100 mesh gave rise to large particles. The presence of microgels at the injured site promoted cartilage regeneration. Alcian blue hematoxylin staining showed that microgels injected at the injured tissue prevented early bony bar formation and microgels loaded with bioactive agents SDF-1a and TGF-B3 promoted cartilage formation.

It is important to ensure that the wire mesh filter is placed correctly into the syringe so that effective filtering can take place. It is also important to repeat the filtration a few times to ensure a homogenous distribution of microgel size. These microgels can be applied to a wide variety of tissue engineering applications that require a biomaterial scaffold substrate that has the potential for sustained release of therapeutics.