Overview
Topic status: We're looking for students to study this topic.
Silk fibroin is the main protein component of silkworm cocoons. Silk from the domesticated moth Bombyx mori has a long history of use for medical sutures. More recently, fibroin membranes have been shown to display many of the attributes for a good biomaterial for ocular tissue regeneration; they are biocompatible, degrade over a period of a week to a year and, importantly, for ocular use, have high transparency. Furthermore, they are non-mammalian, which essentially eliminates potential disease transmission. Unfortunately, depending on the cell type, cell attachment to fibroin can be poor. For example, human corneal endothelial cells [1] do not attach well and a coating of attachment binding protein is required such as collagen or laminin. Having to use mammalian proteins for cell attachment not only has a risk of potential disease transmission, but also can cause an unfavourable immunological response in the recipient. An alternative method is to functionalise biomaterials with laboratory-synthesised-peptide sequences that contain known cell binding sites, e.g. RGD sequences. Growth of corneal endothelial cells on a transparent biocompatible membrane has immense clinical value for use in corneal transplantation to prevent blindness. Such membranes also have value in other parts of the eye [2] and for other tissue engineering applications. Rather than amend the silk fibroin it is known that fibroin from the Antheraea pernyi moth cocoons naturally contain RGD sequences. Antheraea pernyi silk fibroin has not been as widely studied as Bombyx mori, even though it is an attractive alternative due to the presence of these sequences and its superior strength [3]. The increase in strength will allow thinner membranes that have improved function and better withstand handling.
Hypothesis/Aims: The hypothesis is that Antheraea pernyi fibroin membranes will be stronger and longer lasting than Bombyx mori, whilst at the same time allow cell growth without the need for contaminating proteins. The aim of the project is to perform a side-by-side comparison of the properties, degradation and growth of corneal endothelial cells between, unmodified Antheraea pernyi and Bombyx mori membranes compared with those have been chemically-modified to have RGD sequences on the membrane surface.
Approaches: Fibroin will be extracted from silk cocoons, dissolved in aqueous solution and cast into thin membranes with and without surface patterning and pores to improve cell growth. RGD sequences will be attached to Bombyx mori membranes using well studied carbodiimide coupling chemistry. Infrared spectroscopy, light transmission, contact angle measurement of hydrophobicity and tensile testing will be undertaken to characterise the membranes. Degradation studies will be done in buffered protease solutions. Cell attachment and proliferation studies will be performed under the direction of Dr Peter Madden at the Queensland Eye Institute. In addition to a human endothelial cell line, human donor eye tissue will be used as a source of endothelial cells. Growth success will be graded using live cell examination and fluorescent staining using image analysis techniques. Scanning and transmission electron microscopy will be used to examine cell structure.
References:
- Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003;22:359-89.
- Chirila T, Barnard Z, Zainuddin, Harkin DG, Schwab IR, Hirst L. Bombyx mori silk fibroin membranes as potential substrata for epithelial constructs used in the management of ocular surface disorders. Tissue Eng Part A. 2008;14:1203-11.
- Zhang Y, Yang H, Shao H, Hu X. Antheraea pernyi silk fiber: a potential resource for artificially biospinning spider dragline silk. J Biomed Biotechnol. 2010;2010:683962.
- Study level
- Honours
- Supervisors
- QUT
- Organisational unit
Science and Engineering Faculty
- Research area
- Contact
- Please contact the supervisor.
Associate Professor Damien Harkin
