Elsevier

Carbohydrate Polymers

Volume 102, 15 February 2014, Pages 8-11
Carbohydrate Polymers

Short communication
Lyophilization as a novel approach for preparation of water resistant HA fiber membranes by crosslinked with EDC

https://doi.org/10.1016/j.carbpol.2013.10.063Get rights and content

Highlights

  • The HA fiber mats have been prepared via a facile approach – lyophilization.

  • The water-resistance property has been enhanced by cross-linked via EDC.

  • The degradation rate could be controlled by the cross-linking time with EDC.

  • The degradation rate was rapidly in PBS because of the strong ionic environments.

Abstract

The hyaluronic acid (HA) fibers scaffold with an extracellular matrix mimic structure has been prepared via lyophilization. The morphology of the HA fibers varying the concentration from 0.05 wt% to 0.15 wt% was characterized by scanning electron microscope (SEM). The diameter of HA fibers increased and the morphology changed from fiber to sheet-like structure as the concentration of HA solution increased. To enhance the water-resistance, the pure HA fiber membranes were chemical cross-linked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and confirmed by fourier transform infrared (FT-IR) spectra. In vitro degradation behavior of cross-linked HA fiber membranes in both of water and PBS solutions was investigated, the physical properties were also studied by differential scanning calorimetry (DSC) and thermogravimetry (TG). The results showed that the bonding water capacity increased after crosslinking, and indicated that the crosslinked HA fibers could be used as scaffold in tissue engineering.

Introduction

Tissue engineering using three-dimensional (3D) porous scaffolds provided support for the tissue regeneration (Collins & Birkinshaw, 2013). The 3D substrate serves as a template for tissue regeneration. The ideal scaffolds should have an appropriate surface chemistry and microstructures to facilitate cellular attachment, proliferation and differentiation (Ma, Kotaki, Inai, & Ramakrishna, 2005). The fabrication of the applicable scaffolds was the core task of tissue engineering. The high surface area and interconnected 3D pore system were considered as the key factor for scaffolds due to facilitating cell attachment and permitting the diffusion of nutrients. Thus, various techniques, including solvent casting/salt leaching, phase separation, emulsion freezing/drying (Kim et al., 2008, Lam et al., 1994, Naznin and Wang, 2012, Pakavadee and Masahiro, 2012) have been developed for the preparation of foam-like scaffolds with the highly porous structure. Recently, the fabrication of bio-mimic architecture to mimic the native extracellular matrix (ECM), which provided an intricate fibers web for cell adhesion, has been driven an increasing interest.

The fibers were suitable for tissue engineering scaffold, due to the continuous structure, high porosity, high surface-to-volume ratio and natural ECM mimic morphological which is the most important characteristic (Li et al., 2006a, Li et al., 2006b). Various strategies, such as self-assembly (Zhang & Eisenberg, 1998) and electrospinninng (Ji et al., 2006), have been developed to mimic the architecture of the natural ECM. Electro spinning fibers have been extensively applied in the tissue engineering because of their closely mimic of the EMC architecture (Rajesh & Dhirendra, 2006). However, some limitations of electrospinnability in natural macromolecules, such as gelatin, chitosan and hyaluronic acid (Li et al., 2006a, Li et al., 2006b, Ohkawa et al., 2004), restricted its application in natural materials fibers.

In order to improve the water-resistance properties, hyaluronic acid (HA), found ubiquitously in the ECM of virtually all mammalian connective tissues (Collins and Birkinshaw, 2008, Laurent et al., 1995), has been served as a potential tissue engineering scaffold material cross-linked with DVS, epoxide, formaldehyde, glutaraldehyde or genipin because of the excellent water-hold properties to mimic the microenvironment of cell regeneration (Burns et al., 1996, Eun et al., 2008, Tan et al., 2011Tomihata and Ikada, 1997, Zhao, 2000).

Lyophilization is a simple and versatile route for the preparation of 3D monolithic structures using ice as the template (Collins and Birkinshaw, 2011, Qian and Zhang, 2011). An aqueous solution is frozen following with sublimation of the ice to template the 3D architecture of ice crystals. It is a flexible technology that can be applied to a range of materials such as natural macromolecule, polyelectrolyte and polymer colloids to prepare the 3D scaffold. Versatile complex ordered structures have been fabricated via lyophilization by controlling the freezing parameters. However, few articles have been reported to fabricate fiber membranes via lyophilization.

Herein, HA fibers were prepared by the lyophilization with the HA solutions and subsequently cross-linked with EDC, and then the water-resistance property was enhanced for the application of tissue engineering. The influence of the concentration ranging from 0.05 wt% to 0.15 wt% on the morphology of the HA fibers has been investigated. Both in water and PBS solutions, in vitro degradation behavior of cross-linked HA fiber membranes were also investigated. It was expected that the cross-linked fiber membranes might have potential applications in tissue engineering.

Section snippets

Materials

Hyaluronic acid (HA, MW = 2,000,000) was purchased from Freda Biopharm. Co., Ltd. (Shandong, China).1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Aladdin (Shanghai, China). Ethanol (AR) was purchased from Richjoint Chem. Co., Ltd. (Shanghai, China). The deionized water was prepared by UPT ultrapure water polishing system.

Preparation of HA fiber membranes

0.05 wt%, 0.15 wt% and 0.3 wt% HA solutions were prepared by dissolving HA in deionized water by gentle agitation in a 250 mL container at

Results and discussion

The fiber membranes were prepared by freezing HA solutions at different concentrations 0.05 wt%, 0.15 wt%, and 0.3 wt%, then cross-linked with EDC for 24 h. The SEM images of HA fibers were shown in Fig. 1. The HA solution frozen in liquid nitrogen with 0.15 wt% concentration showed uniform fiber structure (Fig. 1b), most of which had ribbon-like structure. The morphology of the fibers changed according to the concentration of the solutions. At the concentration of 0.05 wt%, it showed fiber and

Conclusion

In this study, HA fiber membranes were prepared by the lyophilization technique. The cross-linked HA fiber membranes by EDC showed improvement in the stability in PBS and water. SEM showed that the diameter of the fibers increased with the increase of concentration of HA solutions. FT-IR results confirmed the formation of ester bond and indicated that the cross-linking reactions occurred and the cross-linked fiber membranes were more stable in PBS and water. DSC and TGA results confirmed that

Acknowledgements

We would like to thank the Project Supported by National Natural Science Foundation of China (Grant No.201304005) and Jiangsu Provincial Natural Science Foundation (Grant No. BK20131145).

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