IN VITRO EVALUATION OF CONJUGATED HYALURONIC ACID WITH ASCORBIC ACID

Abstract

Hyaluronic acid (Hyalunan, HA), β-1,4-linked D-glucuronic acid and β-1,3 N-acetyl-D-glucosamine polysaccharide, is a nonsulfated glycosaminoglycan(GAG) conserved in the extracellular matrix (ECM). Due to its biocompatibility, biodegradable properties, HA is widely applied for tissue engineering. However, HA also has defects for tissue engineering such as mechanical properties, difficulty of handling. Thus, it is various modified by chemical reaction to produce HA derivative. HA plays an important role in tissue morphogenesis, proliferation and cell differentiation. Ascorbic acid (AA) has an effect on collagen synthesis and bone mineralization. Ascorbate levels also have a significant effect on osteoblast proliferation and alkaline phosphatase (ALP) expression. However AA is weak to heat and light, thus it is easily degradable. Consequently, we conjugated HA with AA in order to make it more stable and effective. In this study, we prepared HA-AA conjugate and evaluated activity of products in pre-osteoblast.

To produce more effective conjugation, we synthesised HA derivative, HA-N-hydroxysuccinimide, an activated ester of the glucuronic acid moiety. This HA-active ester intermediate is a precursor for drug-polymer conjugates. The degree of substitution was calculated by NMR analysis. The modified HA was dialysed and lyophilised. The yield of conjugation is calculated by Gel Permeation Chromatography (GPC). After the process, HA was conjugated with AA once again as previously mentioned. In this study, the resultant HA-AA conjugate was tested on MC3T3-E1, murine pre-osteoblast cells. We examined cellular viability (cytotoxicity), proliferation and gene expression. The expression of Type 1 collagen was examined by RT-PCR and western blot. Osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP), bone proliferation and differentiation marker were detected by RT-PCR. Alkaline phosphatase assay was also performed. For confirmation on bone mineralization, alizarin red staining and von Kossa staining was performed.

In conclusion, the in vitro data demonstrate that HA-AA conjugate has an important role in bone formation, as it can increase proliferation and osteogenic differentiation of MC3T3-E1 cells. These observations further support the use of in vivo system for tissue engineering applications.