Abstract
The purpose of this study was to synthesize S-protected thiolated hyaluronic acid (HA) and to evaluate its potential for 3D cell culture scaffold. S-protected thiolated HA was synthesized by the covalent attachment of N-acetyl-S-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)thio)cysteine hydrazide ligand to the HA. Hydrogels were characterized for texture, swelling behavior and rheological properties. Furthermore, the potential of S-protected thiolated HA hydrogels as a scaffold for tissue engineering was evaluated by cell proliferation studies with Caco-2 and NIH 3T3 cells. It showed enhanced cohesion upon addition of N-acetyl cysteine (NAC). Dynamic viscosity of S-protected thiolated HA hydrogel was increased up to 19.5-fold by addition of NAC and 10.1-fold after mixing with mucus. Furthermore, Caco-2 and NIH 3T3 cells encapsulated into hydrogels proliferated in-vitro. As this novel S-protected thiolated HA is stable towards oxidation and forms highly cohesive gels when getting into contact with endogenous thiols due to disulfide-crosslinking, it is a promising tool for 3D cell culture scaffold.
Highlights
Hyaluronic acid (HA), a non-sulfated, glycosaminoglycan (GAG) is found in the extracellular matrix (ECM) of many soft connective tissues (Prestwich, 2011; Shu, Liu, Luo, Roberts, & Prestwich, 2002)
Hyaluronic acid sodium salt, succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-acetyl- L-cysteine (NAC), tetrahydrofuran anhydrous (THF), ethyl acetate, dichloromethane (DCM), methanol, hydrazine (NH2–NH2), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC), Nhydroxysuccinimide (NHS), N,N-dimethylformamide anhydrous (DMF), sodium chloride (NaCl), L-cysteine hydrochloride hydrate, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s reagent), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), trisaminomethane hydrochloride (Tris HCl), sodium borohydride (NaBH4), potassium phosphate dibasic (K2HPO4), hydrochloric acid (HCl), silica gel, dialysis tubing MWCO 3.5 kDa, resazurin (7-hydroxy-3H-phenoxazin-3-one sodium salt), minimum essential medium eagle (MEM) and TritonTM X-100 were all purchased from Merck, Austria
The cell culture medium was made of MEM powder 9.66 g/L, 2.2 g/L sodium bicarbonate, phenol red, 2 mM L-glutamine, 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin solution. 100 mM phosphate-buffered saline (PBS) and FBS were purchased from Invitrogen, Austria
Summary
Hyaluronic acid (HA), a non-sulfated, glycosaminoglycan (GAG) is found in the extracellular matrix (ECM) of many soft connective tissues (Prestwich, 2011; Shu, Liu, Luo, Roberts, & Prestwich, 2002). HA is ubiquitous in the body with many beneficial properties such as hydrophilicity and viscoelasticity (He, Zhao, Yin, Tang, & Yin, 2009) Due to these characteristics, HA has become an important building block for new biomaterials with utility in tissue engineering and regenerative medicine (Bian et al, 2016; Burdick & Prestwich, 2011). A lot of methods such as Michael-type addition reaction (Jin et al, 2010), Schiff-base reaction (Li et al, 2014), photo polymerization (Gramlich, Kim, & Burdick, 2013; Lee & Park, 2009), thiol-ene reaction (Yu et al, 2014) and oxidizing reaction of tyramine (Burdick & Prestwich, 2011) are employed to achieve that goal These methods show potential, there are still a lot of shortcomings such as poor gelation efficiency, uncontrollable gelation processes (Shoham et al, 2013) and safety issues (Lin & Stern, 2001). Disulfide-cross-linked HA hydrogels have been introduced in regenerative medicine as they are easy to synthesize with convenient gelation properties and minor safety concerns due to biodegradability (Lee et al, 2010)
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