Atomic level structure of Extracellular Matrix (ECM): spectroscopic approaches to the systems biology of intact tissue

The extracellular matrix (ECM) is a network of solid protein and other substances which supports the cells that make up our various tissues. The maintenance of healthy tissues into older age has huge implications for the economy and our quality of life. Moreover, the design of tissue replacements is of vital importance when tissues wear out or become damaged, and the ECM is critical to the faithful integration of these replacements into the host tissue. This project will develop methods for growing ECMs from cells in the laboratory that on molecular and nano-lengthscales look as far as possible like the ECM found in bone. These ECMs may then be used to grow bone tissue in the laboratory for grafting purposes and for further basic science research into the detailed structure of bone and its reaction to drugs and other chemicals, for instance. In order to do this, we will first develop ways of examining the molecular structure of the ECM using a form of spectroscopy, solid-state nuclear magnetic resonance (SSNMR). To test these methods, we will use molecules which resemble those found in the ECM of bone, but which can be synthesised by normal chemical reactions in the laboratory. We will then use various cell culturing methods to grow ECMs from a several different cell types that are capable of producing bone-like material, supporting the cells on different materials and under a variety of different conditions in order to investigate what features are necessary to produce an ECM which most closely resembles that in bone, as judged by the SSNMR methods we have developed in the first part of the project and electron microscopy to examine the nanostructure of the material. Finally we will apply the SSNMR methods we have developed to study bone into which we have introduced NMR-active isotopes as 'reporters' of molecular structure in order to show that the material we have produced using cell culture represents a valid model of the real material.

The ECM is of paramount importance as a cell scaffold, for cell signalling and for cellular nutrition. Equally important is its material role in giving strength and structural integrity to tissues. The maintenance of healthy tissues into older age has huge implications for the economy and our quality of life. Concurrently, the design of biocompatible tissue prostheses is of vital importance in the replacement or repair of degenerated or traumatized tissue, and the ECM is critical to the faithful integration of these devices into the host tissue. The purpose of this application is twofold: first to develop a solid-state NMR toolkit for determining the molecular-level structure of the ECM, focussing particularly on that in bone and the interactions between its various components and then to use this to examine how ECM structure depends on the differentiation and type of the osteogenic cells producing the matrix. Different cell scaffolds and culture conditions will be used to produce cell populations with different differentiation and specifically, different levels of expression of the various proteins important in ECM production. Correlating the resulting ECM molecular structure (via NMR) and nanostructure (via electron microscopy) with the details of cell differentiation and protein expression will yield essential information on the structural relevance of key components in the mechanism of ECM production.