A confocal system for biomaterials and tissue engineering

Many diseases and loss of tissue due to trauma can be treated using novel surgical procedures involving the implantation of natural or synthetic materials. These materials can be highly porous tissue engineering scaffolds which promote the in-growth and regeneration of tissue once implanted and in some cases can be pre-loaded with the patients own cells prior to implantation. We are currently involved in a large collaboration within the University of Manchester to develop new and improved biomaterials and tissue engineering scaffolds for the regeneration of a variety of tissues and organs such as bone, cartilage, intervertebral disc, skin, ligament, tendon and liver. We are also engaged in nerve repair and the controlled differentiation of stem cells. In order to further the understanding of cellular interactions with our novel scaffolds we are hoping to acquire a confocal laser scanning microscope system which is optimised for use with biomaterials and tissue engineering scaffolds. A confocal system allows cells and the biomolecules they synthesis, labelled with fluorescent markers, to be imaged within 3D. The microscope takes optical sections through cells and tissues (and scaffolds) to enable imaging of specific markers such as proteins, sugars and cell surface receptors where other microscopy techniques cannot. This will allow us to better understand the requirements for regeneration of diseased or damaged tissues and organs via tissue engineering.

We propose to develop a confocal microscope system optimised for biomaterials and tissue engineering research. The system will take advantage of Leica's acousto optical beam splitter for the acquisition of high quality fluorescence images on often autofluorescent materials and scaffolds. The combination of spectral detection and AOBS enables such autofluorescent samples to be successfully imaged. Long working distance lenses are incorporated to allow for cells on surfaces and within scaffolds with highly irregular surfaces and porosity to be imaged. An inverted microscope sytem allows for the elimination of artifactual depth effects on samples that are irregular or sloped. Opaque and metallic materials will be imaged using such a confocal system where conventional fluorescence microscopy is inadequate. The system will be used for a variety of multidisciplinary research projects. Imaging cell colonisation and matrix formation and architecture within novel scaffolds for intervertebral disc tissue engineering will be enabled through acquisition of this equipment. PDLLA/bioactive glass scaffolds have been developed but due to the porosity and autofluorescence of the scaffolds, imaging of such biomarkers has been unsuccessful. We are currently developing a wide variety of novel self-assembling peptide hydrogels for 3-D culture of a variety of cells and for tissue engineering applications. Currently we have shown cell viability and proliferation of fibroblasts, keratinocytes and chondrocytes but now we need to use confocal imaging to determine matrix formation and the development of defined fibroblast/keratinocyte cell layers. We are also developing novel nanomaterials and nanocomposites for controlled cell responses and as implant coatings. Confocal microscopy will allow us to image cell interactions and matrix formation and aid in the development of materials and scaffolds that mimic tissue at the nanoscale.