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
<ol>
	<li>Obtain purified and lyophilized low molecular weight (LMW) chitosan from commercially available sources (see Table of Materials).</li>
	<li>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.</li>
	<li>Add 5 mL of glacial acetic acid to the above-prepared chitosan solution.</li>
	<li>Stir covered at 300 rpm for 18 h with the beaker set in a water bath held at 50 &#176;C.</li>
	<li>Using a B&#252;chner flask and funnel, filter the chitosan solution through decreasing sizes of filter paper: 22 &#181;m, 8 &#181;m, and 2.7 &#181;m (see Table of Materials).</li>
	<li>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.</li>
	<li>Transfer the dialyzed chitosan solution to a beaker and adjust the pH to 8.0 using 1 M NaOH.</li>
	<li>Aliquot the chitosan into centrifuge tubes and centrifuge at 4000 x g for 5 min at room temperature.</li>
	<li>Decant the supernatant to a waste stream and resuspend the chitosan in ddH2O, repeating 2x.</li>
	<li>Freeze and then lyophilize the chitosan pellet.
	<ol>
		<li>Each day, remove the lyophilized product and record the mass. 
		&#8203;NOTE: When the mass of the lyophilized product is no longer changing, the product is fully dried and can be stored at -20 &#176;C until ready for use.</li>
	</ol>
	</li>
</ol>
2. Fabrication of chitosan hydrogel
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.&#160; 
	NOTE: Mix the chitosan again between the syringes to fully incorporate the therapeutic.</li>
	<li>Prepare 100 mM of stock crosslinker solution of genipin (see Table of Materials) in 100% ethanol.</li>
	<li>Add 100 &#181;L of the prepared genipin solution (Step 2.4.) to the chitosan-containing syringe, and again mix back and forth between syringes for 30 s.</li>
	<li>Extrude the mixture from the syringe onto a 35 mm Petri dish, cover it with paraffin film, and incubate it at 37 &#176;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.</li>
	<li>Filter the prepared chitosan microgel following the steps below.
	<ol>
		<li>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.</li>
		<li>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 &#181;m.</li>
		<li>Transfer the broken gel pieces into the syringe fitted with the filter and add 6 mL of ddH2O. 
		&#8203;NOTE: The chitosan gel will swell significantly in the aqueous medium, so a large change in the gel volume is expected.</li>
		<li>Connect the syringe via a Luer-lock connector to another clean 10 mL syringe.</li>
		<li>Force the gel + water mixture through the syringe with the filter to create microgels with a specified maximum diameter.
		<ol>
			<li>After the first filtration, open the back of the syringe containing the filter and extrude the mixture back into this syringe.</li>
			<li>Replace the back of the syringe and force the mixture through the filter again.</li>
			<li>Repeat the filtration 5-6x or until there is little resistance through the filter.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Rinse and purify the filtered microgels.
	<ol>
		<li>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.</li>
		<li>Centrifuge the microgels at 100 x g for 5 min at room temperature and decant the upper aqueous phase.</li>
		<li>Resuspend the microgels in 10 mL of 70% ethanol, vortex, and place under UV light for 1 h to sterilize.</li>
		<li>Centrifuge the microgels at 1,000 x g for 5 min at room temperature, discard the ethanol, and rinse 3x with ddH2O.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Resuspend the microgel pellets 1:1 in ddH2O.&#160;Microgels can be stored for up to 1 month suspended in ddH2O at 4 &#176;C. If a bioactive agent is used, the microgels must be used immediately.</li>
	<li>Create the injury site in the animal following a previously published report30.</li>
	<li>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.).</li>
	<li>Close the wound in the animal and administer post-surgical analgesics30.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Mizuta, T., Benson, W. M., Foster, B. K., Morris, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+analysis+of+the+incidence+of+physeal+injuries.">Statistical analysis of the incidence of physeal injuries.</a> Journal of Pediatric Orthopaedics. 7 (5), 518-523 (1987).</li><li>Mann, D. C., Rajmaira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+physeal+and+nonphyseal+fractures+in+2,650+long-bone+fractures+in+children+aged+0-16+years.">Distribution of physeal and nonphyseal fractures in 2,650 long-bone fractures in children aged 0-16 years.</a> Journal of Pediatric Orthopaedics. 10 (6), 713-716 (1990).</li><li>Eid, A. M., Hafez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+injuries+of+the+distal+femoral+physis.+Retrospective+study+on+151+cases.">Traumatic injuries of the distal femoral physis. Retrospective study on 151 cases.</a> Injury. 33 (3), 251-255 (2002).</li><li>Barmada, A., Gaynor, T., Mubarak, S. 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. M., Abdelgawad, A., Heinrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+growth+arrest:+current+practice+and+future+directions.">Management of growth arrest: current practice and future directions.</a> Journal of Clinical Orthopaedics and Trauma. 9, 58-66 (2018).</li><li>Williamson, R. V., Staheli, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+physeal+growth+arrest:+treatment+by+bridge+resection+and+fat+interposition.">Partial physeal growth arrest: treatment by bridge resection and fat interposition.</a> Journal of Pediatric Orthopedics. 10 (6), 769-776 (1990).</li><li>Escott, B. G., Kelley, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+traumatic+physeal+growth+arrest.">Management of traumatic physeal growth arrest.</a> Orthopaedics and Trauma. 26 (3), 200-211 (2012).</li><li>Newsom, J. P., Payne, K. 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=Microgels:+modular,+tunable+constructs+for+tissue+regeneration.">Microgels: modular, tunable constructs for tissue regeneration.</a> Acta Biomaterialia. 88, 32-41 (2019).</li><li>Riederer, M. S., Requist, B. D., Payne, K. A., Way, J. D., 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=Injectable+and+microporous+scaffold+of+densely-packed,+growth+factor-encapsulating+chitosan+microgels.">Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.</a> Carbohydrate Polymers. 152, 792-801 (2016).</li><li>Xin, S., Wyman, O. M., Alge, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+PEG+microgels+into+porous+cell-instructive+3D+scaffolds+via+thiol-ene+click+chemistry.">Assembly of PEG microgels into porous cell-instructive 3D scaffolds via thiol-ene click chemistry.</a> Advanced Healthcare Materials. 7 (11), 1800160 (2018).</li><li>Kim, P. -. H., 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=Injectable+multifunctional+microgel+encapsulating+outgrowth+endothelial+cells+and+growth+factors+for+enhanced+neovascularization.">Injectable multifunctional microgel encapsulating outgrowth endothelial cells and growth factors for enhanced neovascularization.</a> Journal of Controlled Release. 187, 1-13 (2014).</li><li>Rabea, E. I., Badawy, M. E. -. T., Stevens, C. V., Smagghe, G., Steurbaut, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+as+antimicrobial+agent:+applications+and+mode+of+action.">Chitosan as antimicrobial agent: applications and mode of action.</a> Biomacromolecules. 4 (6), 1457-1465 (2003).</li><li>Sarmento, B., Goycoolea, F. M., Sosnik, A., das Neves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+and+chitosan+derivatives+for+biological+applications:+chemistry+and+functionalization.">Chitosan and chitosan derivatives for biological applications: chemistry and functionalization.</a> International Journal of Carbohydrate Chemistry. 2011, 1 (2011).</li><li>Galdioli Pell&#225;, M. C., 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=Chitosan+hybrid+microgels+for+oral+drug+delivery.">Chitosan hybrid microgels for oral drug delivery.</a> Carbohydrate Polymers. 239, 116236 (2020).</li><li>Echeverria, C., 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=One-pot+synthesis+of+dual-stimuli+responsive+hybrid+PNIPAAm-chitosan+microgels.">One-pot synthesis of dual-stimuli responsive hybrid PNIPAAm-chitosan microgels.</a> Materials &amp; Design. 86, 745-751 (2015).</li><li>Kim, M. Y., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+microgels+embedded+with+catalase+nanozyme-loaded+mesocellular+silica+foam+for+glucose-responsive+drug+delivery.">Chitosan microgels embedded with catalase nanozyme-loaded mesocellular silica foam for glucose-responsive drug delivery.</a> ACS Biomaterials Science &amp; Engineering. 3 (4), 572-578 (2017).</li><li>Mora-Boza, A., 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=Microfluidics+generation+of+chitosan+microgels+containing+glycerylphytate+crosslinker+for+in+situ+human+mesenchymal+stem+cells+encapsulation.">Microfluidics generation of chitosan microgels containing glycerylphytate crosslinker for in situ human mesenchymal stem cells encapsulation.</a> Materials Science and Engineering: C. 120, 111716 (2021).</li><li>Zhang, H., Mardyani, S., Chan, W. C. W., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+biocompatible+chitosan+microgels+for+targeted+pH-mediated+intracellular+release+of+cancer+therapeutics.">Design of biocompatible chitosan microgels for targeted pH-mediated intracellular release of cancer therapeutics.</a> Biomacromolecules. 7 (5), 1568-1572 (2006).</li><li>Huang, P., 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=Effect+of+pH+on+the+mechanical,+interfacial,+and+emulsification+properties+of+chitosan+microgels.">Effect of pH on the mechanical, interfacial, and emulsification properties of chitosan microgels.</a> Food Hydrocolloids. 121, 106972 (2021).</li><li>Fletcher, N. 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=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. (125), e55571 (2017).</li><li>Erickson, C., Stager, M., Riederer, M., Payne, K. A., Krebs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulsion-free+chitosan-genipin+microgels+for+growth+plate+cartilage+regeneration.">Emulsion-free chitosan-genipin microgels for growth plate cartilage regeneration.</a> Journal of Biomaterials Applications. 36 (2), 289-296 (2021).</li><li>Yang, D., 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=Microfluidic+synthesis+of+chitosan-coated+magnetic+alginate+microparticles+for+controlled+and+sustained+drug+delivery.">Microfluidic synthesis of chitosan-coated magnetic alginate microparticles for controlled and sustained drug delivery.</a> International Journal of Biological Macromolecules. 182, 639-647 (2021).</li><li>Marsili, L., Dal Bo, M., Berti, F., Toffoli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+chitosan-grafted-poly(N-vinylcaprolactam)+microgels+via+ionotropic+gelation+for+oncological+applications.">Thermoresponsive chitosan-grafted-poly(N-vinylcaprolactam) microgels via ionotropic gelation for oncological applications.</a> Pharmaceutics. 13 (10), 1654 (2021).</li><li>Muzzarelli, R., El Mehtedi, M., Bottegoni, C., Aquili, A., Gigante, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genipin-crosslinked+chitosan+gels+and+scaffolds+for+tissue+engineering+and+regeneration+of+cartilage+and+bone.">Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regeneration of cartilage and bone.</a> Marine Drugs. 13 (12), 7314-7338 (2015).</li><li>Muzzarelli, R. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genipin-crosslinked+chitosan+hydrogels+as+biomedical+and+pharmaceutical+aids.">Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids.</a> Carbohydrate Polymers. 77 (1), 1-9 (2009).</li><li>Butler, M. F., Ng, Y. -. F., Pudney, P. D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+kinetics+of+the+crosslinking+reaction+between+biopolymers+containing+primary+amine+groups+and+genipin.">Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin.</a> Journal of Polymer Science Part A: Polymer Chemistry. 41 (24), 3941-3953 (2003).</li><li>Marquez-Curtis, L. A., Janowska-Wieczorek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+migration+ability+of+mesenchymal+stromal+cells+by+targeting+the+SDF-1/CXCR4+axis.">Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis.</a> BioMed Research International. 2013, 1-15 (2013).</li><li>Tang, Q. O., 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=TGF-&#946;3:+A+potential+biological+therapy+for+enhancing+chondrogenesis.">TGF-&#946;3: A potential biological therapy for enhancing chondrogenesis.</a> Expert Opinion on Biological Therapy. 9 (6), 689-701 (2009).</li><li>Hogg, R., Turek, M. L., Kaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+particle+shape+in+size+analysis+and+the+evaluation+of+comminution+processes.">The role of particle shape in size analysis and the evaluation of comminution processes.</a> Particulate Science and Technology. 22 (4), 355-366 (2004).</li><li>Raval, N., Maheshwari, R., Kalyane, D., Youngren-Ortiz, S. R., Chougule, M. B., Tekade, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+physicochemical+characterization+of+nanoparticles+in+pharmaceutical+product+development.">Importance of physicochemical characterization of nanoparticles in pharmaceutical product development.</a> Basic Fundamentals of Drug Delivery. , 369-400 (2019).</li></ol>

The authors have nothing to disclose.

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 solvent rinses, which prevents their translation to clinical use15,16,17,18,19,20. These microgels excel in terms of their significant convenience of fabrication, requiring no emulsion techniques or solvent rinses. Additionally, these microgels retain the ideal properties for a tissue-engineered construct, such as pH-dependent swelling and drug loading, tuned degradation behavior, and sustained release of therapeutics.
The most critical step in fabricating these chitosan microgels is the filtration between syringes. These microgels start as bulk hydrogel and are filtered to a specific size range using wire mesh filters. Without filtering, the microgels&#39; applicability, mechanical properties, and drug release characteristics would be significantly different. The filtering step allows for precise control over the size of the hydrogels, and it also allows for high throughput fabrication of microgels that exhibit pH-dependent swelling and sustained release of therapeutics.
A limitation of this process is that the filtering step did not lead to hydrogels with a perfectly spherical shape, which may be an important factor to consider for some applications. For this reason, the characteristic size of the microgels was described using Feret diameter (Figure 3), which is useful for quantifying irregularly-shaped particles39. Although the geometry of the microgels was not a perfect sphere, the average size of the particles was easy to control based on the mesh size of the syringe filter, and, for many applications, having perfectly spherical particles is not necessary. The microgels&#39; polydispersity index (PDI) was quantified using the square-ratio of the standard deviation of Feret diameter to the average Feret diameter obtained from a large population of particles (n = 74). The PDI was calculated as 0.076 using the equation
PDI = (s/D)2
where s is the standard deviation of the average Feret diameter and D is the average Feret diameter40. Due to the filtering done during this process and the use of the Feret diameter for irregularly shaped particles, the polydispersity index of these particles was quite low, to the extent that they could be considered monodisperse.
For future research, several modifications to this protocol could be made to better fit the given research need. For example, only two proteins, SDF1-a and TGF-b3, have been studied for their controlled release with these microgels. Previous work has shown sustained release of these bioactive factors up to ~30 days in vitro. However, other relevant therapeutics, such as nanoparticles, RNA interfering (RNAi) molecules, other biologics, or small-molecule drugs could also be explored to quantify their release rate and efficacy when applied with this chitosan microgel technology. Another variable that could be investigated in the future is changing the size range of the microgels, which is done simply by changing the mesh size of the syringe filter. This could also have a significant impact on the release rate of therapeutics from the microgels, allowing for convenient control over release kinetics without altering the chemistry of crosslinking. Additionally, this protocol can be easily scaled-up using larger syringes and filters or vacuum filtration techniques to produce large quantities of chitosan microgels.

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><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&amp;uuml;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 crosslinking reaction between genipin and amine-containing compounds, such as chitosan or other proteins, has been well-characterized in the literature34. In short, the crosslinking mechanism is considered to be a nucleophilic attack by the amino groups of chitosan, in which genipin acts as a dialdehyde with stable condensation products35. The short chains of stable, condensed genipin act as crosslinking bridges between the chitosan polymers. The crosslinking reaction causes the solution to turn a dark blue, likely due to oxygen radical-induced polymerization and dehydrogenation of intermediate compounds, which follows the ring-opening reaction from nucleophilic attack36.
Once the microgels have been filtered and resuspended in a 1:1 water dilution, they can be easily employed in various biomaterial applications. Work has recently been published using these emulsion-free chitosan microgels to promote cartilage regeneration in growth plate injuries. The microgels were fabricated as described herein and either kept empty or loaded with SDF-1a and TGF-b3, which are bioactive agents that are relevant in growth plate tissue regeneration, with SDF-1a promoting migration of mesenchymal stem cells to the defect site and TGF-b3 serving as a chondrogenic factor to induce differentiation of these stem cells down the chondrogenic lineage37,38. The release rate of the proteins was quantified in vitro via ELISA, and the release of these molecules was sustained over time31. Then, the microgels were injected into a growth plate injury in an in vivo rat model, and the injected microgels prevented early bony bar formation in vivo31. These injectable, cost-effective, and simple-to-produce chitosan microgels could easily be employed in many biomaterial applications.
Although this process for microgel fabrication has been optimized for simple setup and applications, several problems could still arise that researchers should be mindful of. Insufficient mixing of the polymer and crosslinking components is the most likely cause for different results during fabrication. The solid chitosan must be mixed vigorously between the syringes, and the resulting chitosan solution must be entirely homogenous before the genipin crosslinker is added. If the solution is not homogenous, the solid chitosan chunks remaining in the solution will form lumps, and uneven crosslinking will occur, preventing effective filtering and resulting in poly-dispersed microgels with significantly varying diameters. Another important factor to consider during fabrication is avoiding evaporation during the crosslinking period, which must be prevented with paraffin film or other evaporation-trapping techniques. If the chitosan hydrogel dries out, it will not swell during the water rinses, and it will not filter through the syringe. Lastly, the microgels must be suspended in excess water during the filtration process and stored in water at 4 &#176;C when not in use. The microgels are not extrudable or injectable unless suspended in at least a 1:1 dilution of water.
Figure 1 shows a broad overview of the microgel fabrication process. The same process is depicted again in Figure 2, which shows photographs of the process, emphasizing the protocol stages that are difficult to understand from text alone. For example, Figure 2D shows how a wire mesh filter is inserted into the 10 mL syringe. Once fully seated against the top of the syringe, this wire mesh filter allows for quick and convenient filtration of the chitosan microgels without specialist equipment or solvents. Similarly, Figure 2E shows the flow of hydrated chitosan gel through the mesh filter, which is the basis for microgel fabrication. Figure 3 was adapted from our previous publication on these microgels and shows their pH-dependent swelling behavior and the differences in the size of the microgels dependent on the pore size of the mesh filter. Different mesh sizes can be ordered from the manufacturer, which allows for convenient control over the size of the microgels. This precise control over microgel size is highly important when designing drug delivery systems with well-defined therapeutic load release rates. Previous work on microgels also showed that they degrade significantly in the presence of lysozyme at 2-4 weeks31. Finally, Figure 4 shows histology images31 in a rat growth plate injury model treated with the chitosan microgels loaded with SDF-1a and TGF-b3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63857/63857fig01.jpg" /> 
Figure 1: Schematic overview of chitosan microgel fabrication. The figure was created using biorender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63857/63857fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63857/63857fig02.jpg" /> 
Figure 2: Photographs of the microgel fabrication process. (A) Chitosan solution in syringes connected using Luer lock. (B) Extrusion of chitosan gel into a 35 mm Petri dish. (C) Retrieval of chitosan gel after crosslinking color change from off-white to dark blue. (D) Top-down view inside the syringe showing the wire mesh sieve fitted against the nozzle of the syringe. (E) Chitosan gel was pressed through a mesh filter to produce microgels. (F) Microgels were stored in a 1:1 dilution of ddH2O in a conical tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63857/63857fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63857/63857fig03.jpg" /> 
Figure 3: pH-dependent swelling behavior of the microgels. (A) Normal distribution graph of the Feret diameter showing swelling behavior of the microgels in response to pH changes. (B) Fluorescent images of microgels fabricated using No. 200 mesh (upper image: &#60;75 &#181;m sized microgels) and No. 100 mesh (lower image: 75-150 &#181;m sized microgels). The figure is reprinted with permission from reference31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63857/63857fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63857/63857fig04.jpg" /> 
Figure 4: Histology images in a rat growth plate injury model treated with the chitosan microgels loaded with SDF-1a and TGF-b3. 10x histological images showing growth plate repair tissue of intact (A) and (E), untreated (B) and (F), microgel treated (C) and (G), microgel + SDF-1a treated (D) and (H), and microgel + TGF-b3 treated (I) limbs. No day 7 animals were treated with microgel + TGFb3. Alcian blue hematoxylin (ABH) stains the bone orange to red, the fibrous tissue pink, and the cartilage blue. The microgel appears as a dark red fibrous-like tissue. Scale bars = 500 &#181;m. The figure is reprinted with permission from reference31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63857/63857fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

fabrication-size-controlled-emulsion-free-chitosan-genipin-microgels

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.3K Views.  Colorado School of Mines. Dit protocol maakt gemakkelijke fabricage van chitosan microgels mogelijk zonder giftige oplosmiddelen die kunnen worden gebruikt voor een verscheidenheid aan weefselmanipulatie en medicijnafgiftetoepassingen. Deze techniek vereist geen speciale apparatuur of training en vereist geen toxische emulsietechnieken of oplosmiddelspoelingen, waardoor het zeer biocompatibel en vertaalbaar is naar een klinische omgeving. We hebben deze techniek toegepast als een biomateriaalstrategie voor de behandel...

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

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Chemistry

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

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

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

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

Samenvatting

Meer video's verkennen