Elsevier

Carbohydrate Polymers

Volume 202, 15 December 2018, Pages 545-553
Carbohydrate Polymers

Facile strategy involving low-temperature chemical cross-linking to enhance the physical and biological properties of hyaluronic acid hydrogel

https://doi.org/10.1016/j.carbpol.2018.09.014Get rights and content

Highlights

  • The cross-linking and degradation rates of HA were affected by the temperature.

  • Degradation rate of HA was more sensitive to the temperature than the cross-linking rate.

  • Mechanical Properties of the HA were markedly enhanced when it was fabricated at 10 °C.

  • HA hydrogels cross-linked at 10 °C exhibited superior biocompatibility and durability.

Abstract

Here, we present a novel strategy to fabricate hyaluronic acid (HA) hydrogels with excellent physical and biological properties. The cross-linking of HA hydrogel by butanediol diglycidyle ether (BDDE) was characterized under different reaction temperatures, and the resulting physical properties (i.e., the storage modulus and swelling ratio) were measured. The ratio between the cross-linking rate (a strengthening effect) and the hydrolysis rate (a weakening effect) was much greater with lower cross-linking temperatures after sufficient cross-linking time, resulting in a noticeably higher storage modulus. As the cross-linking temperature decreased, the formed HA hydrogel structure became denser with smaller pores. Moreover, the introduction of low-temperature HA cross-linking strategy also resulted in an enhanced several important characteristics of HA hydrogels including its enzymatic resistivity and its ability to elicit a cellular response. These results indicate the performance of HA hydrogels can be markedly enhanced without further additives or modifications, which is expected to contribute to the advancement of applications of HA hydrogels in all industrial fields.

Introduction

Hyaluronic acid (HA) is a high-molecular-weight linear glycosaminoglycan with repeating disaccharide units of d-glucuronic acid and N-acetyl glucosamine. It is one of the most widely used biomedical polymers because of its unique set of characteristics including a high water-retention capacity, viscoelasticity, biodegradability, and excellent biocompatibility (Fakhari & Berkland, 2013; Leach & Schmidt, 2005; Schanté, Zuber, Herlin, & Vandamme, 2011). HA is naturally present in the skin as an integral component of the extracellular matrix; thus, it does not tend to elicit adverse reactions from the body, making it very promising for cosmetic applications (Kenne et al., 2013; Maleki, Kjoniksen, & Nystrom, 2007; Seidlits et al., 2010). One of the largest markets for HA is its use as a subcutaneous or intradermal filler. However, despite these unique advantages, HA cannot be used in its natural state for this application because of its inadequate mechanical properties and rapid degradation in vivo. Its half-life ranges from 2 to 5 min in the bloodstream; even in the epidermis of the skin, the half-life only reaches 1 to 2 days (Maleki et al., 2007; Schanté et al., 2011; Tavsanli & Okay, 2016). Therefore, to provide a long-lasting aesthetic effect, it is necessary to modify its structure to improve its mechanical properties and retard its degradation.

To extend the durability of HA in the body and enhance its mechanical properties, HA is chemically cross-linked to form a hydrogel by incorporating polyfunctional reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, divinyl sulfone, 1,2,7,8-diepoxyoctane (DEO), poly(ethylene glycol) diglycidyl ether (PEGDE), or butanediol diglycidyl ether (BDDE) (Al-Sibani, Al-Harrasi, & Neubert, 2015; Kenne et al., 2013; Maleki et al., 2007; Segura et al., 2005; Tavsanli & Okay, 2016; Yang, Guo, Zang, & Liu, 2015). A number of studies over the past few decades have shown that adding larger amounts of the cross-linker, even in a solid-state cross-linking reaction (Larrañeta et al., 2018), creates a higher level of covalent connections within the polymer network, resulting in a higher stiffness and slower degradation (Choi et al., 2015; Yang, Tan, Cen, & Zhang, 2016). However, these cross-linking agents are innate invariable chemicals with various functional groups, which presents a high risk of cytotoxicity and adverse effects when they are incorporated into living tissues (Choi et al., 2015; Liang, Chang, Liang, Lee, & Sung, 2004; Manchun, Dass, Cheewatanakornkool, & Sriamornsak, 2015). Even BDDE, which is the most widely used cross-linker for HA on the commercial market, should not be used in large amounts to reduce the degradation rate and improve the mechanical properties of HA hydrogels because its residues are potentially cytotoxic (Choi et al., 2015). Therefore, it is highly desirable to find alternative ways to improve the durability and mechanical properties of HA hydrogels with a minimal amount of the cross-linking agent to minimize the adverse effects and attain the best overall treatment outcome.

The experimental parameters of the cross-linking reaction, such as the initial polymer concentration, the molecular weight of the polymer, and the reaction time, can be tuned to enhance the cross-linking density of the polymer network (Collins & Birkinshaw, 2013). In particular, the pH and temperature are considered critical variables that influence the chemical reaction kinetics, and thereby affecting the degree of cross-linking of HA. Temperature increases stimulate the movement of the cross-linker, increasing the rate of cross-linking reaction, while acidic or basic pH conditions activate the cross-linker and facilitates the formation of covalent bonds between HA molecules (Collins & Birkinshaw, 2013; Kim, Potta, Park, & Song, 2017). These variables also substantially affect the degradability of the resulting HA chain. For example, DEO, PEGDE, and BDDE contain epoxide rings on both ends of the main chain; these rings open and form strong ether bonds primarily with the hydroxyl groups on HA under alkaline conditions (De Boulle et al., 2013; Yang et al., 2015). However, even with short-term alkaline treatments, HA is readily degraded through the base-catalyzed hydrolysis on d-glucuronic acid and N-acetyl glucosamine units, including the continuous elimination of end groups (called a “peeling” reaction) and cleavage of glycosidic bonds (Stern, Kogan, Jedrzejas, & Soltes, 2007; Tokita & Okamoto, 1995; Whistler & Bemiller, 1958). These hydrolytic reactions cause scissoring of the HA main chain at multiple locations, producing mixtures of randomly sized oligo- and monosaccharides. These processes make it difficult to optimize the physical properties of HA (Stern et al., 2007).

In this study, we report, for the first time, a new method for forming HA hydrogels that results in a reduced degradation rate as well as enhanced mechanical properties. The proposed process involves controlling the cross-linking temperature and time but does not require additional cross-linking agents or additives. Because the degree of chemical cross-linking and the degradation rate of HA are substantially influenced by temperature changes (Kim et al., 2017; Tavsanli & Okay, 2016), we explored the effects of wide ranges of the cross-linking temperature (from 3 °C to 37 °C) and time (up to 4 weeks) on the physical properties of HA hydrogels. BDDE was chosen as a representative chemical cross-linker for this study because its reaction profile is similar to those of other cross-linkers and have been shown to be safe for in vivo applications because of its low toxicity and biodegradability (Al-Sibani et al., 2015; De Boulle et al., 2013; Yang et al., 2015). The cross-linking treatment was applied under alkaline conditions, and the physical properties of obtained HA hydrogels were evaluated in terms of the rheological behavior and swelling ratio. The storage modulus, G′, was measured to estimate the activation energy for the cross-linking and degradation of the hydrogel. In addition, the biological properties of the HA hydrogels were assessed in terms of the in vitro cellular response, in vitro enzymatic decomposition, and in vivo behavior in an animal model compared with a commercial dermal filler as a control.

Section snippets

Fabrication of HA hydrogel

10% (wt/vol) HA (Bioland, Korea) with a molecular weight ranging from 1.8 to 2.5 MDa was dissolved in 9.8 mL of a 0.2-M NaOH solution at room temperature. Then, 200 μL of the cross-linker, 1,4-butanediol diglycidyl ether (BDDE; Sigma-Aldrich USA), was mixed with HA solution at a final concentration of 2% (vol/vol). The mixture was then distributed into the molds, which were then sealed and incubated at 37 °C, 20 °C, 10 °C, or 3 °C for gelation. The initial G′ value (G′0) of each HA solution was

Analysis of cross-linking conditions

The effects of the cross-linking time and the temperature during cross-linking on the mechanical properties of the resulting HA hydrogels were evaluated by considering the influences of the two main processes occurring simultaneously: degradation and cross-linking. These processes are caused by the alkaline NaOH solution containing the HA and BDDE and are considered to greatly influence the mechanical properties of the HA hydrogel. To quantify the mechanical properties, the ratio of G′ to

Conclusion

The results of this study demonstrated that the properties of the HA hydrogel were markedly enhanced when it was synthesized at a low temperature of 10 °C under alkaline conditions. The HA hydrogels cross-linked at lower temperatures exhibited high mechanical strengths because the ratio between the cross-linking rate and base-catalyzed degradation rate increased at lower temperatures. This novel approach facilitates better cross-linking efficiency, which leads to the formation of denser polymer

Acknowledgments

This research was supported by Basic Science Research Program (2018R1C1B6001003) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Technology Innovation Program (Grant No. 10037915, WPM Biomedical Materials-Implant Materials) funded by the Ministry of Trade, Industry & Energy (MI, Korea), and Seoul National University Research Grant in 2017.

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