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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 issue1,2,

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 Mate.......

Representative 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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Acetic acidSigmaAldrichAX0073
BD Luer-Lock SyringeFisher Scientific14-823-16E
Büchner FunnelFisher ScientificFB966F100 mm diameter
Chitosan (low molecular weight)SigmaAldrich44886975-80% deacetylation
Dialysis Membrane TubingFisher Scientific08-670-5C3500 MWCO
EthanolSigmaAldrich493538
GenipinSigmaAldrichG4796
Heracell 150i IncubatorThermoFisher50116047
ParafilmFisher Scientific13-374-12
Recombinant human SDF-1aPeprotech300-28A
Recombinant human TGF-b3Peprotech100-36E
Whatman Filter Paper Grade 540SigmaAldrichZ2415478 mm pore size
Whatman Filter Paper Grade 541SigmaAldrichWHA154105522 mm pore size
Whatman Filter paper Grade 542SigmaAldrichWHA15421852.7 mm pore size
Wire Mesh SieveMcMaster-Carr9317T86No. 100 Mesh

References

  1. Mizuta, T., Benson, W. M., Foster, B. K., Morris, L. L. Statistical analysis of the incidence of physeal injuries. Journal of Pediatric Orthopaedics. 7 (5), 518-523 (1987).
  2. Mann, D. C., Rajmaira, S.

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Chitosan MicrogelsGenipinEmulsion freeTissue EngineeringRegenerative MedicineBiomaterial ScaffoldDrug DeliveryGrowth Plate InjuriesBiocompatibleBiodegradableSustained Drug Release

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