Synthesis of new chitosan-glutaraldehyde scaffolds for tissue engineering using Schiff reactions

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Abstract

Hydrogels of chitosan (CS) and glutaraldehyde (GA) were synthesized for tissue engineering applications by using a Schiff reaction. CS was reacted with GA (a cross-linker) at different concentrations, which were expressed as percentage of weight (2, 4, 6, 8 and 10 wt%). An evaluation was made of the effect of the different wt% of GA on the swelling and rheological properties of the hydrogels. The Schiff crosslinking reaction was monitored by UV–vis spectroscopy (550 nm) to determine the reaction kinetics and reaction order at 60 °C. The hydrogel structures were characterized by NMR, FT-IR, HR-MS and SEM, while the degree of cross-linking was examined with TGA-DA. The smaller pores and greatest swelling were found in hydrogels containing 10 wt% of GA. However, only the hydrogels with GA at 2, 4 and 6 wt% displayed viable cells, indicating their in vitro cytocompatibility. The rheological studies showed that the values of the loss and storage modules in the hydrogels increased with a rise in temperature from 30 to 35 and finally 40 °C. Further research is needed to verify the adequacy of these hydrogels as a scaffold for tissue engineering in vivo.

Introduction

The development of appropriate scaffolds for tissue engineering is still one of the most important fields in regenerative medicine. [1] Creating scaffolds with the appropriate physicochemical factors to sustain cell growth and tissue formation allows regenerative medicine to improve, restore or replace the biological functions of damaged tissues and organs [2]. The scaffolds should serve as templates to guide adhesion, proliferation, differentiation and cell maturation. Furthermore, they must provide the cells with a free space for vascularization, penetration and transfer of nutrients, oxygen and waste products. [1,3] In other words, the scaffolds need to have appropriate mechanical properties and porous structures that permit the free diffusion of nutrients and waste. Also, the degradation process rate should be equal to the cellular growth rate. [4,5] Essentially, scaffolds serve as an artificial extracellular matrix to offer structural support for the cells and free space for the flow of growth factors. The extracellular matrix consists of a crosslinked mesh between fibrous proteins and glycosaminoglycans (GAGs, such as heparan sulfate, chondroitin sulfate and keratan sulfate) to form proteoglycans. [6]

Materials used as scaffolds include hydrogels, porous nanostructures and nanofibers. [[7]] Hydrogels are three-dimensional polymer networks able to swell and absorb a large amount of aqueous solution without losing their structure [[8], [9], [10]]. They can retain solvent representing at least 20% of their own weight and swell significantly by absorbing water, followed by shrinking again after de-swelling. However, the process of cross-linking creates an insoluble network [11,12].

Several monomers and crosslinking agents have been employed to synthesize hydrogels with a wide range of chemical compositions, many of which could possibly serve as scaffolds [13,14]. There are several routes for the synthesis of these platforms, including Michael, Click and Schiff reactions. A Michael reaction involves the nucleophilic addition of a carbanion or a nucleophile (e.g., thiols and amines) to create a reaction with an 〈,® unsaturated carbonyl compound [15]. A Click reaction consists of a Cu(I)-catalyzed reaction between azide and terminal acetylene groups to form 1,2,3-triazoles [16]. Finally, a Schiff reaction is generated by the condensation of ketone or aldehyde groups and a primary amine [17]. The synthesis process begins with an intermediate of carbonylamine, which upon dehydration forms a carbon-nitrogen double bond (an imine). These methods present advantages and disadvantages for controlling the formation of the hydrogel structure. For instance, some functional groups may not react during the crosslinking reaction, thus resulting in hydrogels with poor mechanical properties [13].

Hence, it is important to choose the type of reaction that is adequate for each particular polymer. For example, chitosan (CS) has been used for the preparation of hydrogels via the Schiff base reaction [[5], [6], [7]]. CS is a linear heteropolymer of glucosamine and N-acetyl glucosamine residues (Fig. 1] that is obtained by the deacetylation of chitin. This weak base is soluble in acidic solution (pH 6.5) and insoluble in water and organic solvents [[18], [19], [20], [21], [22]]. It is biodegradable, biocompatible and non-toxic and exhibits mucoadhesive properties [23]. In the current study, synthesis was carried out of a series of hydrogels with various concentrations of glutaraldehyde (GA). The content of GA is expressed as its percentage of hydrogel weight (wt%), being 2, 4, 6, 8 and 10 wt%. The reaction conversion of GA was monitored by ultraviolet-visible spectroscopy (Uv–vis) and the reaction order was calculated from the corresponding data. The materials were characterized by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and thermogravimetry (TGA). The degree of cross-linking of the hydrogels was determined by Soxhlet techniques, the rheological properties such as loss and storage modulus were measured, and cell viability was evaluated with a cytotoxicity assay.

Section snippets

Materials

CS (75% deacetylated) and 2,4-dinitrophenylhydrazine (DNP) (99%) were obtained from Sigma-Aldrich (Iceland), 99% acetic acid from J.T. Baker (Mexico), ethanol and dichloromethane from Alveg (Mexico), and 25 wt.% GA from Merck (Germany). Distilled water grade II was used as solvent. The high glucose Dulbecco’s modified Eagle’s medium (DMEM), antibiotic-antimitotic 100X and fetal bovine serum (FBS) were purchased from Biowest (Mexico). Trypsin/EDTA solution and phosphate buffed saline (PBS, pH

Results and discussion

A series of hydrogels of CS and GA were synthesized (at 60 °C) by means of the Schiff base method, obtaining five concentrations of GA (2, 4, 6, 8 and 10 wt%) and therefore a range in the degree of cross-linking in the polymer. The synthesis was carried out in triplicate. Photographs of the resulting hydrogels (Fig. 3) reveal that all were a transparent yellow in color. This color became more intense as the concentration of the crosslinking agent increased. Gels with lower concentrations of GA

Conclusions

Novel hydrogels were synthesized by using Schiff reactions to cross-link CS with GA. Hydrogels having different concentrations of GA were tested to determine the effect on swelling and rheological properties. Examination was made of the degree of crosslinking bond formation (with TGA-DA) and of the presence of residual GA in the uncrossed part (with NMR). The reaction kinetics and reaction order, assessed with UV–vis spectroscopy, showed a pseudo cero order for the reaction. Moreover, the

Acknowledgements

This project was supported by grant from the Consejo Nacional de Ciencia y Tecnología (CONACYT, grant # 743999). The authors would like to acknowledge the help of Dr. Hugo Martínez Gutierrez in carrying out the SEM assay, and of Dr. Daniel Arriéta Baez in performing the HR-MS at the Centro de Nanociencias y Micro-nanotecnologías. Finally, we thank Dr. Jose Antonio Serrato Pérez of the Instituto Nacional de Enfermedades Respiratorias (INER).

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