Name: fabrication of size-controlled and emulsion-free chitosan-genipin microgels for tissue engineering applications
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Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health under award numbers R03AR068087 and R21AR071585 and by the Boettcher Foundation (#11219) to MDK. CBE was supported by NIH/NCATS Colorado CTSA Grant Number TL1 TR001081.

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
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	<li>Obtain purified and lyophilized low molecular weight (LMW) chitosan from commercially available sources (see Table of Mate...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+physeal+closure+following+distal+tibia+physeal+fractures:+a+new+radiographic+predictor.">Premature physeal closure following distal tibia physeal fractures: a new radiographic predictor.</a> Journal of Pediatric Orthopaedics. 23 (6), 733-739 (2003).</li><li>Shaw, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+approaches+for+the+treatment+of+pediatric+physeal+injuries.">Regenerative medicine approaches for the treatment of pediatric physeal injuries.</a> Tissue Engineering Part B: Reviews. 24 (2), 85-97 (2018).</li><li>Dabash, S., Prabhakar, G., Potter, E., Thabet, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+anti-VEGF+from+injectable+hydrogel+systems+provides+a+prolonged+decrease+of+endothelial+cell+proliferation+and+angiogenesis+in+vitro.">Sustained delivery of anti-VEGF from injectable hydrogel systems provides a prolonged decrease of endothelial cell proliferation and angiogenesis in vitro.</a> RSC Advances. 8 (16), 8999-9005 (2018).</li><li>Fletcher, N. A., Babcock, L. R., Murray, E. A., Krebs, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+delivery+of+antibodies+from+injectable+hydrogels.">Controlled delivery of antibodies from injectable hydrogels.</a> Materials Science and Engineering: C. 59, 801-806 (2016).</li><li>Fletcher, N. A., Von Nieda, E. L., Krebs, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-interactive+alginate-chitosan+biopolymer+systems+with+tunable+mechanics+and+antibody+release+rates.">Cell-interactive alginate-chitosan biopolymer systems with tunable mechanics and antibody release rates.</a> Carbohydrate Polymers. 175, 765-772 (2017).</li><li>Erickson, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+degradation+rate+of+alginate-chitosan+hydrogels+influences+tissue+repair+following+physeal+injury.">In vivo degradation rate of alginate-chitosan hydrogels influences tissue repair following physeal injury.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. , 34580 (2020).</li><li>Erickson, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-VEGF+antibody+delivered+locally+reduces+bony+bar+formation+following+physeal+injury+in+rats.">Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats.</a> Journal of Orthopaedic Research. , 24907 (2020).</li><li>Lee, M. A., Nissen, T. P., Otsuka, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+a+murine+model+to+investigate+the+molecular+process+of+transphyseal+bone+formation.">Utilization of a murine model to investigate the molecular process of transphyseal bone formation.</a> Journal of Pediatric Orthopaedics. 20 (6), 802-806 (2000).</li><li>Planka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+mesenchymal+stem+cells+with+chondrocytes+in+prevention+of+partial+growth+plate+arrest+in+pigs.">Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.</a> Biomedical Papers. 156 (2), 128-134 (2012).</li><li>Yu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+model+of+physeal+injury+for+the+evaluation+of+regenerative+medicine+approaches.">Rabbit model of physeal injury for the evaluation of regenerative medicine approaches.</a> Tissue Engineering Part C: Methods. 25 (12), 701-710 (2019).</li><li>Xian, C. J., Zhou, F. H., McCarty, R. C., Foster, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembranous+ossification+mechanism+for+bone+bridge+formation+at+the+growth+plate+cartilage+injury+site.">Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.</a> Journal of Orthopaedic Research. 22 (2), 417-426 (2004).</li><li>Erickson, C. B., Shaw, N., Hadley-Miller, N., Riederer, M. S., Krebs, M. D., Payne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+tibial+growth+plate+injury+model+to+characterize+repair+mechanisms+and+evaluate+growth+plate+regeneration+strategies.">A rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies.</a> Journal of Visualized Experiments. 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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...

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 &#34;bony bar&#34; 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 issue1,2,3,4. 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 plate6,7,8. 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 chitosan10,11,12.&#160;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 activity13,14. 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 settings15,16. Other studies have used microfluidics or electrospray approaches to fabricate chitosan microgels, which require special equipment, preparation, and training17,18. 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 microgels19,20. 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&#39;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 injuries21,22,23,24,25. Due to the dynamic temporal nature of growth plate ossification and bone elongation, the mechanism of bony bar formation is not fully understood.&#160;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 sheep26,27,28. 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 plate29,30. 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 manner10,31. 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 applications32,33.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>SigmaAldrich</td>
			<td>AX0073</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lock Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>B&#252;chner Funnel</td>
			<td>Fisher Scientific</td>
			<td>FB966F</td>
			<td>100 mm diameter</td>
		</tr>
		<tr>
			<td>Chitosan (low molecular weight)</td>
			<td>SigmaAldrich</td>
			<td>448869</td>
			<td>75-80% deacetylation</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Tubing</td>
			<td>Fisher Scientific</td>
			<td>08-670-5C</td>
			<td>3500 MWCO</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SigmaAldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>SigmaAldrich</td>
			<td>G4796</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i Incubator</td>
			<td>ThermoFisher</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SDF-1a</td>
			<td>Peprotech</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF-b3</td>
			<td>Peprotech</td>
			<td>100-36E</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper Grade 540</td>
			<td>SigmaAldrich</td>
			<td>Z241547</td>
			<td>8 mm pore size</td>
		</tr>
		<tr>
			<td>Whatman Filter Paper Grade 541</td>
			<td>SigmaAldrich</td>
			<td>WHA1541055</td>
			<td>22 mm pore size</td>
		</tr>
		<tr>
			<td>Whatman Filter paper Grade 542</td>
			<td>SigmaAldrich</td>
			<td>WHA1542185</td>
			<td>2.7 mm pore size</td>
		</tr>
		<tr>
			<td>Wire Mesh Sieve</td>
			<td>McMaster-Carr</td>
			<td>9317T86</td>
			<td>No. 100 Mesh</td>
		</tr>
	</tbody>
</table>

fabrication of size-controlled and emulsion-free chitosan-genipin microgels for tissue engineering applications

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.

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...

Watch this Scientific Journal Video about Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications at JoVE.com

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

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,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

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.

Research

JoVE Journal

Bioengineering

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