Hyaluronate-alginate hybrid hydrogels prepared with various linkers for chondrocyte encapsulation
Introduction
Tissue engineering has become a promising approach to replace damaged tissues or organs (Kim, Shin, & Lim, 2012). A biomimetic scaffold, one of the critical components required in typical tissue engineering approaches, should provide a suitable environment for the attachment, differentiation, and migration of cells (Murphy, O’Brien, Little, & Schindeler, 2013). Among the different types of scaffolds, hydrogel has frequently been used for cell delivery (Tibbitt & Anseth, 2009). Hydrogel forms a three-dimensional structure through its network of hydrophilic polymer chains. In addition, hydrogels can retain large amounts of water, and they have a porous structure (Ahmed, 2015), making them suitable for cell delivery. Hydrogel has often been used as a scaffold for cartilage regeneration due to its composition, mechanical stiffness, and swelling degree, which are particularly similar to those of the extracellular matrix (ECM) in native cartilage (Liu et al., 2017). Articular cartilage damage is caused by abrasion from frequent and unbearable stress and may eventually result in osteoarthritis. Damage to the cartilage tissue itself is difficult to repair due to its avascular and aneural structure (Buckwalter & Mankin, 1997; Newman, 1998).
Natural polymers occur abundantly, have excellent biocompatibility (Dang & Leong, 2006), and have attracted much attention as a scaffolding material in tissue engineering approaches (Malafaya, Silva, & Reis, 2007). Hyaluronate (HA) is a naturally-occurring polysaccharide and the main component of ECM in connective tissues (Jiang, Liang, & Noble, 2007). HA can interact with chondrocytes via surface receptors, such as CD44 (Jin et al., 2010), and is often utilized in hydrogel preparations for cartilage tissue engineering (Kim, Mauck, & Burdick, 2011). HA typically forms hydrogel via chemical cross-linking, and the mechanical properties of that gel are dependent on the type and amount of chemical cross-linking agent. However, chemical cross-linkers that remain in the gel may cause toxic side effects in the body (Hunt, Chen, van Veen, & Bryan, 2014). Alginate (ALG) is also a natural polysaccharide, and it can easily form into hydrogel by ionic cross-linking in the presence of divalent cations such as calcium ions (Lee & Mooney, 2012).
Hydrogels for tissue engineering scaffolds should be designed considering various parameters, including biodegradability, biocompatibility, mechanical stiffness, minimally invasive injectability, and cell-specific interaction (Kim et al., 2011). Controlling the mechanical stiffness of hydrogel is critical in tissue engineering approaches, especially cartilage regeneration, as it may significantly affect cell behavior, such as attachment, migration, and differentiation (Discher, Janmey, & Wang, 2005; Vining & Mooney, 2017). We previously reported that hyaluronate-alginate hybrid (HAH) can be synthesized by the covalent conjugation of ALG to HA using ethylenediamine as a linker, and that HAH hydrogel can be formed using calcium ions without additional cross-linkers. For a same concentration of calcium ions, the mechanical stiffness of HAH hydrogel was dependent on the composition ratio of HA and ALG. HAH hydrogel showed great potential as a scaffold for cartilage regeneration (Park, Woo, & Lee, 2014).
In addition to mechanical stiffness, the cellular interaction of a scaffold is important in tissue engineering. HAH was modified with ligands to enhance both cell-cell and cell-matrix interactions. A peptide with the sequence arginine-glycine-aspartic acid (RGD) is a well-known ligand that interacts with the integrin receptor of cells to allow cell-matrix interactions and promotes cartilage regeneration (Hersel, Dahmen, & Kessler, 2003; Koo, Irvine, Mayes, Lauffenburger, & Griffith, 2002). The histidine-alanine-valine (HAV) peptide is derived from cadherin and is involved in cell-cell interactions (Zhu et al., 2016). Therefore, both HAV and RGD peptides were introduced into HAH to enhance cell-cell and cell-matrix interactions, respectively (An et al., 2018).
In this study, we hypothesized that the type and length of the linkers between HA and ALG in HAH could significantly influence the mechanical properties of HAH hydrogel. Various diamines and dihydrazides were utilized as linkers to synthesize HAH, and its hydrogel containing RGD and HAV peptides was prepared by ionic cross-linking with calcium ions. Various characteristics of HAH hydrogels were investigated, and a mouse chondrogenic cell line (ATDC5) was used to determine whether the mechanical stiffness of peptide-modified HAH hydrogels could regulate chondrogenic differentiation in vitro depending on the linker type.
Section snippets
Materials
Sodium hyaluronate (Mw = 1000 kDa) was purchased from Humedix (Anyang, Korea). Sodium alginate (Mw = 250 kDa, guluronate unit content = 66%) was purchased from FMC Biopolymer (Sandvika, Norway). RGD peptide (G4RGDSP) and HAV peptide (G4SHAVSS) were purchased from Anygen (Seoul, Korea). 1-Ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) was purchased from Proteochem (Hurricane, UT, USA). N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) was purchased from Covachem (Loves Park, IL, USA).
Synthesis and characterization of hyaluronate-alginate hybrid
HAH polymers were synthesized using various bifunctional linkers, including diamine and dihydrazide (Table 1). First, HA was conjugated with either diamine or dihydrazide (HA-linker) via carbodiimide chemistry. Alginate modified with RGD and HAV peptides (ALG-peptide) was next coupled to the HA-linker to synthesize HAH (Fig. S1). HAH-sebacic dihydrazide was not synthesized, due to the extremely low solubility of sebacic dihydrazide in MES buffer solution. More than 96% of amino/hydrazide groups
Conclusions
We demonstrated that the type and length of the linkers used for preparing hyaluronate-alginate hybrid (HAH) could influence the mechanical stiffness of the resultant HAH hydrogel. HAH hydrogel was successfully formed with calcium ions using the gelation feature of the alginate in the hybrid. The mechanical stiffness of HAH hydrogel was significantly influenced by varying the length of the linker type between HA and ALG. As the length of the linker increased, the G' value of the HAH hydrogel
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2016R1A2A2A10005086).
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