Cartilage Tissue Engineering via Characterization and Application of Carboxymethyl Chitosan-Based Bioink

International researchers continue the trend in exploring natural biomaterials for bioprinting, detailing their findings in the recently published ‘Characterization and Application of Carboxymethyl Chitosan-Based Bioink in Cartilage Tissue Engineering.’

Examining chitosan as an ingredient for bioink in cartilage tissue engineering, the authors realize previous challenges in using printable inks overall—along with difficulty in sustaining cells in the lab environment. Such material has been featured in 4D printing studies, along with experimentation in bioprinting with chitosan-gelatin hydrogels.

Chemical crosslinking has also been used by many research teams, employing chemicals like glutaraldehyde, formaldehyde, and carbodiimide; however, many such agents are high in toxicity, leading to negative reactions. Because chitosan is a natural polysaccharide, it is being used more often in bioprinting applications.

Schematic diagram of hydrogel preparation and printing. (a) First step: chitosan reacting with EDTA, unreacted carboxyl groups (green) take part in the next step. (b) Second step: additional chitosan is added to the solution and crosslinked with CaCl2 solution after printing to form hydrogel. (c) Hydrogel printing method.

For this study, the researchers focused on tissue engineering of cartilage, seeking ways to regenerate cells:

“The characteristics of chitosan are similar to those of hyaluronic acid and glycosaminoglycans which are distributed extensively in native cartilage, and the degraded products of chitosan are involved in chondrification,” stated the researchers. “However, the weak mechanical property of pristine chitosan limited its further utilization in cartilage regeneration, and the poor water solubility hinders the large-scale use.”

To overcome hurdles for the development of materials with chitosan, the authors developed ink with ‘enhanced mechanical properties,’ allowing them to print hydrogel templates for cartilage bioprinting. Relying on carboxymethyl chitosan, hydrogels were suitably complemented.

Bioink was created via both pneumatic and piston-driven methods (Hkable 3D):

“In order to maintain the continuity of printed hydrogel line and prevent clogging at the extruder, the diameter of the needle used for 3D printing in this work was 0.5 mm, the air pressure was controlled by an affiliated precise regulator and set at 110 psi, and the travel speed of the extruder was set to 300 mm/min.”

Printed samples with different chitosan : modified chitosan (CE) ratios. Images on the left, from top to bottom, show highly viscous bioink resulting in a discontinuous print, highly viscous bioink printed using a large diameter needle resulting in an inaccurate print, and low-viscous bioink incapable of holding its shape after printing. Image on the right shows an accurate printed structure with a chitosan : CE ratio of 90 : 10.

Four bioink samples were evaluated in the study, compared as CE powder weight was kept the same for all but the amount of added chitosan was varied. Experimentation revealed that greater amounts of CE caused higher storage and loss modulus, as it proved also to be the main factor in strength enhancement.

(a) Storage and (b) loss modulus of chitosan/CE hydrogel. Four Chitosan/CE conjugate ratios tested. (c) Storage modulus (G′) and loss modulus (G″) of the bioink as a function of crosslinking time. Solid lines represent 45 min of crosslinking, and dashed lines represent 30 min of crosslinking. CaCl2 (1 M) solution is used as the crosslinking agent

Effect of crosslinker concentration on gel retraction and appearance. Images of hydrogel discs crosslinked with (a) 0.1 M, (b) 0.5 M, (c) 1 M, and (d) 2 M CaCl2 solution. Top images in each set represent gel precursor before the final crosslinking, and bottom images represent the resulting gel after crosslinking. The chitosan/CE conjugate ratio of the samples shown is 90 : 10, and the crosslinking time is 45 min for all samples.

(a) Live/dead staining of chondrocytes. (b) Flow cytometry result of cell viability in the control group. (c) Flow cytometry result of cell viability in the hydrogel mesh group. (d) Quantification of cell viability in both groups. Scale bar = 100 μm.

Overall, the bioink showed stability and mechanical properties required for both fast gelation and precision in bioprinting.

“According to the rheology and mechanical testing results, the bioink viscoelastic properties and mechanical strength are tunable by adjustment of the proportions of the components which provides a platform to expand the application of the bioink in tissue engineering,” concluded the authors.

“Furthermore, cell studies with chondrocytes show that the bioink is biocompatible, and it supports cell proliferation as well as helps cells to retain their chondrogenic phenotype. Our results illustrate that the developed bioink has the potential to be adopted for 3D bioprinting of scaffolds for tissue engineering.”

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