TY - JOUR
T1 - A 300 μm Organotypic Bone Slice Culture Model for Temporal Investigation of Endochondral Osteogenesis
AU - Srinivasaiah, Sriveena
AU - Musumeci, Giuseppe
AU - Mohan, Tamilselvan
AU - Castrogiovanni, Paola
AU - Absenger-Novak, Markus
AU - Zefferer, Ulrike
AU - Mostofi, Sepideh
AU - Bonyadi Rad, Ehsan
AU - Grün, Nicole Gabriele
AU - Weinberg, Annelie Martina
AU - Schafer, Ute
PY - 2019/4/1
Y1 - 2019/4/1
N2 - 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 μm 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 μm 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.
AB - 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 μm 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 μm 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.
KW - bone
KW - bone morphogenesis
KW - bone slice model
KW - cartilage
KW - endochondral ossification
KW - organotypic bone culture
UR - http://www.scopus.com/inward/record.url?scp=85064567804&partnerID=8YFLogxK
U2 - 10.1089/ten.tec.2018.0368
DO - 10.1089/ten.tec.2018.0368
M3 - Article
C2 - 30834810
AN - SCOPUS:85064567804
SN - 1937-3384
VL - 25
SP - 197
EP - 212
JO - Tissue Engineering / C
JF - Tissue Engineering / C
IS - 4
ER -