An ex vivo mouse mandible culture model to study inflammatory bone disease
Periodontal diseases are a group of chronic inflammatory diseases of the supporting tissues of the teeth. Chronic periodontitis is the major cause of tooth loss in the developed world with approximately 54% of the UK adult population are affected by the condition. The consequences of the disease extend beyond oral health as bacterial-induced periodontal inflammation represents a risk factor for heart attacks, bacterial pneumonia and stroke. Destructive forms of periodontal disease result in degradation of the alveolar bone, thought to result from host mediated reactions to pathogenic factors secreted by the bacteria, such as lipopolysaccharide (LPS). Current systems for understanding periodontal disease require animal models to ensure the inclusion of cell/cell and host/bacterial interactions are present.
Why we funded it
This Project Grant aims to reduce the number of animals needed to study periodontal tissue and disease by developing an ex vivo prototype rodent mandibular organ culture system.
In vivo studies are currently used to investigate pathogenesis of periodontal diseases and for testing novel therapeutic treatments. An in vivo study of periodontal disease with five time points uses approximately five animals per time point and a control, thus significant numbers of animals are required to obtain statistically robust data. In vitro studies using rodent cells can also be used but rodent mandibles contain only a small amount of tissue. For enough viable cells to be obtained to create reproducible assays a study may require between 20 and 25 animals. The use of immortalised cell lines overcomes this, but these cells may not behave in the same way as primary, unmodified cells. In addition, cells may behave differently when cultured in 3D in tissue-like environments rather than in dishes as this is more representative of their natural environment. The prototype ex vivo culture system in this Project Grant aims to obtain multiple slices of whole tissue from each mandible, which would be cultured as separate entities. Obtaining multiple slices from each mandible has the potential to reduce the number of animals required by up to 80% per experiment whilst still providing a representative tissue environment.
This prototype ex vivo mandible model aims to maintain the cellular activity, tissue architecture and physiology of the periodontal ligament and surrounding alveolar bone, whilst supporting the wide population of cells present in the tissue. Initial work will seek to compare tissue culture methods to assess the best method for maintaining cell activity and tissue architecture. The cultures will be assessed for viability, with expression of relevant markers and differentiation factors analysed to identify whether relevant cell types are present in the cultures. To ensure the model mimics the degradation of bone prevalent in destructive periodontal disease, osteoclasts and innate immune cells will be transplanted into the ex vivo model. Cultures will be maintained and activity monitored before the tissue is exposed to pro-inflammatory cytokines to investigate the pathways leading to the pathogenesis of periodontal disease.
The objective of this application is to develop a cost effective, reproducible ex vivo culture model of the mouse mandible whereby all tissue and cells are in situ to investigate inflammatory cell behaviour and how inflammation influences bone metabolism during disease processes such as periodontal disease. This will significantly reduce the amount of in vivo animal experimentation and overcome practical and ethical difficulties associated with current animal models. Following development of the ex vivo model, the culture conditions will be manipulated to support the growth of a wide population of cells (mesenchymal, osteoclastic, innate immune) which may be transplanted to, or exist naturally within, the system. The model will then be used to study the effects of bacterial LPS and inflammatory cytokines on the cells supported in the different culture conditions to investigate the effects on cell behaviour, pro-inflammatory cytokine production and bone metabolism. This study will significantly reduce the amount of unnecessary animal experimentation currently used to study inflammatory cell activity in diseases such as periodontal disease. It will also overcome the many practical and ethical issues associated with current animal models. The results will be essential in advancing our understanding of molecular pathogenic mechanisms associated with inflammatory destructive diseases, including periodontal disease and rheumatoid arthritis (RA). It has strong potential to translate into clinical practice, contributing to the development of novel therapies, underpinning the identification of new diagnostic biomarkers and will have widespread application in the reduction of animal experimentation in a number of human disease states.
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