An Ex Vivo Bone Culture Model for Investigating Cell Responses

Objectives: The placement of dental implants that extend from the bone to the oral cavity lends these sites prone to bacterial infection and implant failure. The interaction of bacteria with cells and extracellular matrix in bone marrow leads to bone destruction, but is poorly understood; an understanding that could improve current treatment options. Aim: to develop and characterise cell fate within an ex vivo bone marrow culture model with the intention that it can be used to investigate specific cell responses to bacterial infection established in osseous tissues.
Methods: Femoral shafts were dissected from male Wistar rats, which were then split once in cross-section and once in a longitudinal direction. Samples were mounted in semi-solid agar and cultured in a basal αMEM media as Trowell-type cultures at the liquid air interphase for 0-48h. Cells were visualised and counted following haematoxylin and eosin. The presence of apoptotic and necrotic cells were assessed by immuno-staining for lactate dehydrogenase, BAX and cleaved caspase 3 and proliferative activity of cells was assessed using an MTT assay. The persistence of monocytes, macrophages and neutrophils was assessed via immuno-staining for CD14, F4/80, neutrophil elastase and TRAP staining.
Results: During the first 24h of culture approximately 30% cell loss was noted, but remaining cells demonstrated a proliferative activity during the following 24-48h culture. Cell loss was as a result of apoptosis and some necrosis. Mesenchymal cell populations appeared to be maintained, along with the connective tissue matrix architecture. Analysis indicated the loss of haematopoietic cells macrophages and neutrophils.
Conclusions: The data suggests the successful development of a bone marrow culture model which is capable of supporting mesenchymal cells and tissue architecture. This provides the basis for using the model for the specific study of bacterial interplay with these cells within a defined 3D matrix environment.