This project in a collaborative and interdisciplinary venture between Dr Paxton, an expert in tissue engineering, and Dr Murray, an expert in molecular analysis of the neuromuscular system.
There are many regions in the human body where two different tissues join together. For example, in the musculoskeletal system, the soft tissues of tendon and ligament join to the hard tissue of bone though a unique, carefully constructued connecting region, which has an important job in protecting both tissues from damage1. As a functional connection, the microanatomy of the bone-tendon/ligament interface (or enthesis) is important to understand since tendon/ligament injury is common in sports. When a tendon/ligament is ruptured, and surgery required to re-attach the soft tissue to bone, the normal tissue connection structure is lost, and unorganised scar tissue is produced, leaving the surgical interface weak and prone to further injury.
This project aims to apply novel techniques in tissue engineering and 3D cell culture methodologies to develop a reproducible co-culture method whereby the cells from two different musculoskeletal tissues (e.g. bone and tendon) can be grown in close apposition. Then, we aim to apply cutting-edge molecular techniques to investigate what happens when cells from ajoining tissues grow together during development and after injury. By doing so, we will develop a tool whereby future investigations into treatment options can be explored with the ultimate goal of improving patient outcomes following musculoskeletal injury. To accomplish this, we will employ the novel 3D culture methods such as cell sheet technology2 and/or spheriod-based culture3 to create monocultures of bone and tendon cells. Then monocultures will be combined to create co-cultures. These 3D co-cultures will be used to profile the molecular changes occuring when these two cell populations interact. RNA will be extracted from co-cultures at regular time points following co-culture creation, and q-RT-PCR arrays will be used to profile the gene expression of key pathways as they interact. These commercially available arrays contain >80 transcript specific primer sets on a collection of transcripts relating to a given cellular function. For initial analysis, we plan to use arrays focused on extracellular matrix and adhesion molecules.
The key experimental techniques involved in this project are; • 2D culture • 3D culture (cell sheets/spheroids), • Histology, • immunohistochemistry, • q-RT-PCR • analysis of transcriptional data
Training will also be provided in experimental design, data collection, data analysis and presentation skills.
1. Benjamin M., McGonagle D. (2009) Entheses: tendon and ligament attachment sites Scand J Med Sci Sports 19(4):520-7
2. Kobayashi J., Kikuchi A., Aoyagi T., Okano (2019) Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation J Biomed Mater Res A 107(5):955-967.
3. Fennema E., Rivron N., van Blitterswijk C., de Boer J. (2013) Spheroid culture as a tool for creating 3D complex tissues. Trends in Biotechnology 31(2) 108-115