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

Summary

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo, and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Abstract

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

Introduction

The growth plate, also known as the physis, is the cartilage structure located at the end of long bones that mediates growth in children. If the growth plate becomes injured, repair tissue known as a "bony bar" can form, which interrupts normal growth and can cause growth defects or angular deformities. Epidemiological data have shown that 15%-30% of all childhood skeletal injuries are related to the growth plate. Bony bar formation occurs in up to 30% of these injuries, making growth plate injuries and their associated treatment a significant clinical manifestation issue^1,2,3,4. When bony bar formation occurs, the most common treatment avenue involves resectioning the bony bar and inserting an interpositional material, such as silicon or adipose tissue⁵. However, patients that undergo bony bar resection surgery often have a poor prognosis for full recovery, as there is currently no treatment that can fully repair an injured growth plate^6,7,8. In light of these shortcomings, there is a critical need for effective strategies for treating growth plate injuries, both in preventing the formation of a bony bar and regenerating healthy physeal cartilage tissue.

Hydrogel microparticles, or microgels, have recently gained interest as injectable scaffolds that can provide sustained release of therapeutics⁹. Due to their high tunability and biocompatibility, microgels are also well-suited for bioactive factor or cell encapsulation. Microgels can be made of various materials, ranging from synthetic polymers, such as polyethylene glycol (PEG), to natural polymers like alginate or chitosan^10,11,12. Chitosan has been shown to have several beneficial effects for tissue engineering, such as its ability to destabilize the outer membrane of gram-negative bacteria, thereby offering inherent antimicrobial activity^13,14. Additionally, chitosan is cost-effective, cell-interactive, and easily modified using its amine-containing structure. Chitosan-based microgels promise a biomaterial strategy for drug delivery and material signaling that can promote tissue regeneration while preventing bacterial infection. However, chitosan microgels are often fabricated with a wide range of techniques that require special equipment, emulsion techniques, or cytotoxic solvent rinses. For example, some studies have fabricated chitosan microgels with emulsion-based methods, but these protocols require solvent rinses and cytotoxic crosslinkers, potentially negating their translation to clinical settings^15,16. Other studies have used microfluidics or electrospray approaches to fabricate chitosan microgels, which require special equipment, preparation, and training^17,18. Chitosan microgels are also commonly made with a dropwise process of crosslinker into chitosan solution; however, this method is highly dependent on solution viscosity, polymer concentration, and flow rate, making it difficult to control the size and dispersity of the microgels^19,20. Conversely, the method for microgel fabrication described herein requires no specialist equipment or solvent rinses, making these microgels viable for fabrication in nearly any lab or setting. Therefore, these microgels represent highly relevant biomaterials for a quick, cost-effective, and easy-to-produce drug delivery vehicle for many applications.

By modulating a microgel's composition and material characteristics, researchers can gain precise control over the cellular microenvironment, thus directing cell behavior in a material-dependent manner. Microgels can be employed on their own or combined with bulk biomaterial systems to impart specific functionalities, such as the extended release of bioactive factors or precise special signaling for native or exogenous cells. Biomaterials and microgels have emerged as attractive treatment avenues for growth plate injuries. Significant effort has been dedicated to developing alginate and chitosan-based biomaterials to treat growth plate injuries^{21,22,23,24,25}. Due to the dynamic temporal nature of growth plate ossification and bone elongation, the mechanism of bony bar formation is not fully understood. Therefore, several animal models have been developed to better elucidate the mechanisms of endochondral ossification and bony bar formation, such as in rats, rabbits, and sheep^26,27,28. One such model is a rat growth plate injury model, which uses a drill-hole defect in the rat tibia to produce a bony bar in a predictable and reproducible manner and mimics human injuries across all three zones of the growth plate^29,30. Several biomaterial-based strategies for treating growth plate injuries have been tested using this model. Additionally, two different methods for fabricating chitosan microgels have been developed, which can be used as an injectable biomaterial system that releases therapeutics in a sustained manner^10,31. These microgels have been employed in a rat physeal injury model, and they showed improved cartilage regeneration³¹ when releasing SDF-1a and TGF-b3. The techniques provided in this protocol describe methods developed to fabricate these chitosan microgels, which can then be employed in a wide variety of tissue engineering applications. For example, recent studies have used thermo- or magento-responsive chitosan microgels for controlled oncological drug delivery applications^32,33.

Protocol

All animal procedures were approved by the University of Colorado Denver Institutional Animal Care and Use Committee. 6-week old male Sprague-Dawley rats were used for the present study. The rat growth plate injury model was created following a previously published report³⁰.

1. Preparation of the chitosan polymer

1. Obtain purified and lyophilized low molecular weight (LMW) chitosan from commercially available sources (see Table of Materials).
2. Add 495 mL of double-distilled water (ddH₂O) and a stir bar to a 1 L beaker. Add 5 g of chitosan (Step 1.1.) and mix well.
   NOTE: Chitosan is only sparingly soluble in an aqueous solution at physiological pH, so the chitosan will not dissolve easily at this step.
3. Add 5 mL of glacial acetic acid to the above-prepared chitosan solution.
4. Stir covered at 300 rpm for 18 h with the beaker set in a water bath held at 50 °C.
5. Using a Büchner flask and funnel, filter the chitosan solution through decreasing sizes of filter paper: 22 µm, 8 µm, and 2.7 µm (see Table of Materials).
6. Add the filtered chitosan solution to cellulose dialysis tubing (see Table of Materials) and allow to dialyze in ddH₂O at room temperature for 4 days, changing the ddH₂O every day.
   NOTE: Use ultrapure ddH₂O water for the last change.
7. Transfer the dialyzed chitosan solution to a beaker and adjust the pH to 8.0 using 1 M NaOH.
8. Aliquot the chitosan into centrifuge tubes and centrifuge at 4000 x g for 5 min at room temperature.
9. Decant the supernatant to a waste stream and resuspend the chitosan in ddH₂O, repeating 2x.
10. Freeze and then lyophilize the chitosan pellet.
    1. Each day, remove the lyophilized product and record the mass.
       ​NOTE: When the mass of the lyophilized product is no longer changing, the product is fully dried and can be stored at -20 °C until ready for use.

2. Fabrication of chitosan hydrogel

1. Add 2 mL of 6% acetic acid and 120 mg of purified chitosan (Step 1) to a 10 mL Luer-lock syringe to form a 6% w/v chitosan solution.
2. Connect the Luer-lock syringe to another identical syringe using a female-female Luer-lock connector and mix the solution back and forth for 30 s, or until the chitosan has fully dissolved in the acetic acid.
3. Before crosslinking, add any therapeutic or bioactive agent to the chitosan solution (if needed). For the present study, 200 ng of SDF-1a and TGF-b3 (see Table of Materials) were added to the microgels.
   NOTE: SDF-1a and TGF-b3 are bioactive agents relevant to growth plate tissue regeneration. SDF-1a promotes migration of mesenchymal stem cells to the defect site, and TGF-b3 serves as a chondrogenic factor to induce differentiation of these stem cells down the chondrogenic lineage³¹. 
   NOTE: Mix the chitosan again between the syringes to fully incorporate the therapeutic.
4. Prepare 100 mM of stock crosslinker solution of genipin (see Table of Materials) in 100% ethanol.
5. Add 100 µL of the prepared genipin solution (Step 2.4.) to the chitosan-containing syringe, and again mix back and forth between syringes for 30 s.
6. Extrude the mixture from the syringe onto a 35 mm Petri dish, cover it with paraffin film, and incubate it at 37 °C overnight in a humidified atmosphere.
   NOTE: The solution will turn dark blue, indicating that the crosslinking reaction between chitosan and genipin has occurred, leading to the formation of chitosan microgel.
7. Filter the prepared chitosan microgel following the steps below.
   1. Gently break the hydrogel into smaller pieces using a spatula.
      NOTE: The pieces should be small enough to be transferred to the back of a 10 mL syringe, ~1-2 cm in diameter.
   2. Place a filter of desired mesh size into the back of a clean 10 mL syringe.
      NOTE: The typical size range for microgels is between 50-200 µm.
   3. Transfer the broken gel pieces into the syringe fitted with the filter and add 6 mL of ddH₂O.
      ​NOTE: The chitosan gel will swell significantly in the aqueous medium, so a large change in the gel volume is expected.
   4. Connect the syringe via a Luer-lock connector to another clean 10 mL syringe.
   5. Force the gel + water mixture through the syringe with the filter to create microgels with a specified maximum diameter.
      1. After the first filtration, open the back of the syringe containing the filter and extrude the mixture back into this syringe.
      2. Replace the back of the syringe and force the mixture through the filter again.
      3. Repeat the filtration 5-6x or until there is little resistance through the filter.
8. Rinse and purify the filtered microgels.
   1. Transfer the filtered gel mixture to a 50 mL conical tube, bring the total volume up to 20 mL with ddH₂O, and then vortex the mixture to ensure homogeneous dispersion.
   2. Centrifuge the microgels at 100 x g for 5 min at room temperature and decant the upper aqueous phase.
   3. Resuspend the microgels in 10 mL of 70% ethanol, vortex, and place under UV light for 1 h to sterilize.
   4. Centrifuge the microgels at 1,000 x g for 5 min at room temperature, discard the ethanol, and rinse 3x with ddH₂O.

3. Preparation of microgels for in vitro or in vivo applications

NOTE: For the present study, cartilage regeneration in growth plate injuries was studied in a rat model. For details, see reference³¹.

1. Resuspend the microgel pellets 1:1 in ddH₂O. Microgels can be stored for up to 1 month suspended in ddH₂O at 4 °C. If a bioactive agent is used, the microgels must be used immediately.
2. Create the injury site in the animal following a previously published report³⁰.
3. Flush the injury site with saline and either keep the animal untreated (for control study) or inject the chitosan microgels only or the microgels loaded with the bioactive agents (Step 3.2.).
4. Close the wound in the animal and administer post-surgical analgesics³⁰.
5. At days 7 or 28 post-surgery, euthanize the rat by CO₂ overdose, excise the limbs and perform histology to assess the tissue repair at the injury site³¹.

Results

Successful fabrication of chitosan microgels relies on the crosslinking reaction between genipin and chitosan, specifically involving the amines on the chitosan polymer chains. In contrast to other microgel fabrication techniques, this method does not require emulsions or solvent rinses and can be quickly and easily conducted with inexpensive equipment. A hallmark indicator for successful microgel fabrication is the distinct color change from off-white to dark blue after the chitosan and genipin have been mixed. The cros...

Discussion

Microgels have been widely researched in recent years due to their high level of applicability for various purposes, such as drug delivery or cell encapsulation⁹. The ease of manufacturing of micro-scale biomaterial constructs is of significant relevance in tissue engineering, as it allows researchers to develop hydrogel-based strategies at a specific size and time scale. However, most methods for fabricating chitosan microgels require expensive equipment and reagents, emulsions, or cytotoxic solv...

Materials

Name	Company	Catalog Number	Comments
Acetic acid	SigmaAldrich	AX0073
BD Luer-Lock Syringe	Fisher Scientific	14-823-16E
Büchner Funnel	Fisher Scientific	FB966F	100 mm diameter
Chitosan (low molecular weight)	SigmaAldrich	448869	75-80% deacetylation
Dialysis Membrane Tubing	Fisher Scientific	08-670-5C	3500 MWCO
Ethanol	SigmaAldrich	493538
Genipin	SigmaAldrich	G4796
Heracell 150i Incubator	ThermoFisher	50116047
Parafilm	Fisher Scientific	13-374-12
Recombinant human SDF-1a	Peprotech	300-28A
Recombinant human TGF-b3	Peprotech	100-36E
Whatman Filter Paper Grade 540	SigmaAldrich	Z241547	8 mm pore size
Whatman Filter Paper Grade 541	SigmaAldrich	WHA1541055	22 mm pore size
Whatman Filter paper Grade 542	SigmaAldrich	WHA1542185	2.7 mm pore size
Wire Mesh Sieve	McMaster-Carr	9317T86	No. 100 Mesh