Journal
TISSUE ENGINEERING PART C-METHODS
Volume 25, Issue 4, Pages 197-212Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/ten.tec.2018.0368
Keywords
bone slice model; organotypic bone culture; bone; cartilage; endochondral ossification; bone morphogenesis
Categories
Funding
- Marie Skodowska-Curie Actions of the European Union's Seventh Framework Programme FP7/2007-2013/under REA grant [289163]
- CAMed (COMET K-Project) - Austrian Federal Ministry of Transport, Innovation and Technology (BMVIT) [871132]
- Austrian Federal Ministry for Digital and Economic Affairs (BMDW)
- Styrian Business Promotion Agency (SFG)
- University Research Grant (Triennial Research Plan 2016-2018), Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, Italy
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Translational studies to elucidate the response of immature bone to biologic and physical stimuli have been held back by the lack of a viable long-term functional bone explant model. This study attempts to bridge this gap between cell culture and animal model studies. In this study, we describe a methodology to derive a 300 mu m organotypic femur slice comprising physiological zones (epiphysis and meta-diaphysis) essential for endochondral bone development. The unique capability of slice culture model incorporating enhanced nutrient access to distinct bone tissue components associated with linear bone growth facilitates the investigation of the orchestrated cellular transition of chondrogenic and osteogenic cells involved in endochondral bone development in an ex vivo setup. Bone slices of 300 mu m were prepared from 4-day-old postnatal rats and were viable in culture up to 21 days. On days 7 and 15, an increase in chondrogenic and osteogenic modulations was confirmed in epiphysis, metaphysis, and diaphysis. An increase in osteocytes, osteoblasts, and hypertrophic cells were found at these time points, as well as a noticeable increased expression of chondrogenic and osteogenic markers (collagen II, Runx2, and osteocalcin) confirmed endochondral progression. Osteoclast-mediated bone resorption was demonstrated on day 15 by tartrate-resistant acid phosphatase staining. Attenuated total reflection infrared spectroscopic analyses, furthermore, confirmed a time-dependent increase in phosphate levels, bone minerals, and hydroxyapatite for 15 days. Our establishment of a bone slice culture model closely mimicking the in vivo cellular transitions and endochondral microenvironment of a mineralizing bone provides a vital new tool for the elucidation of cellular and endochondral mechanisms of bone development, maturation, and growth plate modulations. The presented model has the potential to be utilized in implementation of preclinical, toxicological, and therapeutic investigations.
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