Flow perfusion maintains ex vivo bone viability: a novel model for bone biology research

Encased in lacunae, osteocytes receive nutrition and biomechanical signals through the lacunocanalicular system. We have developed a novel flow-perfusion bioreactor designed to support lacunocanalicular fluid flow. We hypothesize that ex vivo fluid flow can maintain endochondral bone viability and, ultimately, serve as a novel model to study bone biology in vitro. Sprague-Dawley rat femurs were harvested, stripped of soft tissue, loaded into a custom-designed bioreactor and perfused with osteogenic culture medium. After 14 days of flow-perfusion or static culture, the bones were harvested, fixed, decalcified, embedded, sectioned and stained with haematoxylin and eosin. Fresh long bone samples were similarly processed for comparison. Osteocyte viability and function were also evaluated, using thiazolyl blue tetrazolium bromide (MTT), fluorospectrophotometric DNA quantification, alkaline phosphatase (ALP) colorimetric assay and fluorochrome labelling of mineralizing surfaces. All samples remained free of infection throughout the study period. After 14 days of flow perfusion, histological analysis showed normal-appearing bony architecture, with 72% of lacunae being osteocyte-filled compared with 93% in freshly harvested samples and only 36% in static samples. MTT staining and assay confirmed osteocyte viability in the flow-perfusion samples as well as in fresh samples. DNA quantification demonstrated DNA to be preserved in flow-perfused samples when compared with freshly harvested samples. ALP activity in flow-perfusion explants was upregulated compared with fresh and static samples. Fluorochrome-labelled mineralizing surfaces were seen throughout the explanted flow-perfused samples. This is the first demonstration that flow perfusion provides adequate chemotransportation to explanted murine endochondal bones.