Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering

doi:10.1007/s11706-013-0211-y

Front Mater Sci

2013, Vol. 7

Issue (3) : 269-284 https://doi.org/10.1007/s11706-013-0211-y

RESEARCH ARTICLE

Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering

Min-Dan WANG¹(

), Peng ZHAI¹, David J. SCHREYER^1,2, Ruo-Shi ZHENG³, Xiao-Dan SUN³, Fu-Zhai CUI³, Xiong-Biao CHEN^1,4

1. Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Dr., Saskatoon, SK S7N5A9, Canada; 2. Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK S7N5E5, Canada; 3. School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China; 4. Department of Mechanical Engineering, University of Saskatchewan, 57 Campus Dr., Saskatoon, SK S7N5A9, Canada

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Abstract

Artificial tissue engineering scaffolds can potentially provide support and guidance for the regrowth of severed axons following nerve injury. In this study, a hybrid biomaterial composed of alginate and hyaluronic acid (HA) was synthesized and characterized in terms of its suitability for covalent modification, biocompatibility for living Schwann cells and feasibility to construct three dimensional (3D) scaffolds. Carbodiimide mediated amide formation for the purpose of covalent crosslinking of the HA was carried out in the presence of calcium ions that ionically crosslink alginate. Amide formation was found to be dependent on the concentrations of carbodiimide and calcium chloride. The double-crosslinked composite hydrogels display biocompatibility that is comparable to simple HA hydrogels, allowing for Schwann cell survival and growth. No significant difference was found between composite hydrogels made from different ratios of alginate and HA. A 3D BioPlotter^TM rapid prototyping system was used to fabricate 3D scaffolds. The result indicated that combining HA with alginate facilitated the fabrication process and that 3D scaffolds with porous inner structure can be fabricated from the composite hydrogels, but not from HA alone. This information provides a basis for continuing in vitro and in vivo tests of the suitability of alginate/HA hydrogel as a biomaterial to create living cell scaffolds to support nerve regeneration.

Keywords hyaluronic acid (HA) alginate hydrogel scaffold nerve injury tissue engineering

Corresponding Author(s): WANG Min-Dan,Email:miw554@mail.usask.ca

Issue Date: 05 September 2013

Cite this article:

Min-Dan WANG,Peng ZHAI,Ruo-Shi ZHENG, et al. Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering[J]. Front Mater Sci, 2013, 7(3): 269-284.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-013-0211-y
https://academic.hep.com.cn/foms/EN/Y2013/V7/I3/269

Tab.1 Conditions used to prepare samples for the crosslinking of HA

Tab.2 Conditions used to prepare different samples for the crosslinking of alginate

Tab.3 Conditions used to prepare samples for the crosslinking of alginate/HA

Fig.1 FTIR spectra of HA reactions. Reaction of HA with EDC at pH 4 led to new characteristic peaks of ester bonds at 1708 cm, and amide bonds at 1660 cm (red dash line) and 1550 cm (blue dash line), with the remaining peak for carboxyl groups at 1600 cm (black dash line). Reaction of ADH with HA in the presence of EDC resulted in more amide bonds (red and blue dash lines), fewer ester bonds, and a decrease in carboxyl groups (black dash line), indicating that all the carboxyl groups were transformed into ester bonds, particularly amide bonds.

Fig.2 FTIR spectra of alginate reactions under dispensing. Reaction of 1.5% alginate with ADH in presence of CaCl did not change the spectra, while further addition of EDC led to the characteristic peaks for amide bonds at 1660 and 1550 cm (red and blue dash lines, respectively). Reaction of alginate, ADH with EDC also resulted in the amide formation despite that there were carboxyl groups remaining (black dash line).

Fig.3 FTIR spectra of 1.5% alginate/0.375% HA reactions under stirring or dispensing conditions. There was amide formation when EDC was added to the alginate/HA mixture and the reaction efficiency was increased when ADH was further incorporated. Also, the covalent modification was made to the mixture of alginate, HA and ADH when it was immersed within the CaCl and EDC solution. Black dash line indicates the characteristic peak at 1600 cm for carboxylate anions, and red and blue dash lines represent the characteristic peaks at 1660 and 1550 cm for amide bonds.

Fig.4 Effect of CaClconcentration on the covalent crosslinking of 1.5% alginate/0.375% with ADH. With the increase of the concentration of CaCl, there seemed to be fewer amide bonds, as indicated by the characteristic peaks at 1660 cm (red dash line), and 1550 cm (blue dash line). Higher concentration of CaCl masked the FTIR spectrum, rendering some peaks of interest indistinguishable. The black dash line shows the characteristic peak at 1600 cm for carboxylate anions.

Fig.5 Effect of EDC concentration on the covalent crosslinking of 1.5% alginate/0.375% with ADH. There was more amide formation when EDC was added at a higher concentration. The black dash line indicates the characteristic peak at 1600 cm for carboxylate anions; the red and blue dash lines representes the peaks at 1660 and 1550 cm for amide bonds.

Fig.6 Schwann cell morphology after 2 days’ culture on control PLL-coated culture plate, uncrosslinked HA films, 20%-80% alginate/HA double-crosslinked hydrogels, and crosslinked HA hydrogels.

Fig.7 MTT reduction assay signifying the number of Schwann cells on control and double-crosslinked alginate/HA hydrogel films. Percentages indicate different volume ratios of 3% alginate and 0.75% HA. Error bars represent the standard deviation. ANOVA shows that there is significant difference between groups (<0.0001). Asterisk indicates significant difference from control (Tukey’s test, <0.0001). Substrates containing various ratios of alginate and HA all had significantly decreased Schwann cell viability but there was no significant difference found between the different alginate/HA composite substrates.

Fig.8 MTT result of Schwann cells after 2 days’ culture on the ionically-crosslinked and double-crosslinked alginate/HA hydrogels. Error bars represent the standard deviation; **<0.001, ***<0.0001.

Fig.9 An example of the multilayer scaffolds fabricated from 1.5% alginate/0.75% HA using the 3D Bioplotter system. Ionic crosslinking of alginate by calcium ions occurred upon dispensing, covalent crosslinking of polymers by EDC/ADH reaction could occur after fabrication when the scaffolds are immersed within the crosslinker solution.

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