(430u) Combination of Proteins on PHBV Microsphere Scaffold to Regulate Hep3B Cells Activity and Functionality for an in Vitro Model of Liver Tissue Engineering

The use of microspheres as scaffolds is a new idea in tissue engineering, which is fast gaining attention because of the versatility that microspheres offer in guiding cell growth. This versatility includes not just the ease of scaffold assembly into various shapes suitable for different tissue application, but also offers easy and controllable surface modification for enhanced cell-material interaction.

In this study, we aim to show the versatility of microspheres as tissue engineering scaffold by easily modifying the scaffold surfaces in a controllable manner. We have modified the whole surface of PHBV microspheres in a uniform monolayer with single ECM proteins of collagen, fibronectin and laminin by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy (XPS). The densities of grafted proteins were quantified by using micro-BCA kit. With the ultimate aim to mimic the in vivo conditions of the body, we cultured a model liver cell line, Hep3B, either on microspheres of a single protein type or on a mixture of proteins, to prove that various ECM proteins do interact with cells in a synergistic manner to enhance cell activity and functionality. Cell proliferation activity was estimated using MTT method and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays respectively. The results indicated that the combination of three ECM proteins on microsphere surface allowed a significant improvement in proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver cells.

Key words: PHBV; protein; surface conjugation; liver tissue engineering