TY - JOUR
T1 - Osteoblast migration into type I collagen gel and differentiation to osteocyte-like cells within a self-produced mineralized matrix
T2 - A novel system for analyzing differentiation from osteoblast to osteocyte
AU - Uchihashi, Kazuyoshi
AU - Aoki, Shigehisa
AU - Matsunobu, Aki
AU - Toda, Shuji
N1 - Funding Information:
This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research No. 22791387 , 24791553 (to KU), 23591050 (to ST), and by personal grants from Koike Hospital, Sasebo Central Hospital and Yamada Clinics (to ST). We thank Prof. M. Mawatari for helpful suggestions, and Messrs. H. Ideguchi, F. Mutoh, S. Nakahara, and Mrs. M. Nishida for their excellent technical assistance.
PY - 2013/1
Y1 - 2013/1
N2 - Osteoblasts are believed to differentiate into osteocytes, becoming embedded in bone, or to undergo apoptosis after the bone formation phase. The regulation of this terminal differentiation seems to be critical for bone homeostasis. However the mechanism remains unclear and there is no assay system currently available to analyze this process. To address this issue, we developed a new model in which osteoblasts are cultured on a type I collagen gel layer with osteogenic supplements β-glycerophosphate and ascorbic acid. Cellular behavior was analyzed by electron microscopy, immunohistochemistry and real-time RT-PCR. Osteoblasts gradually migrated into the gel, produced collagen fibrils, and differentiated to osteocytic cells with bone lacunae- and canaliculi-like mineralization. Osteocalcin, DMP-1 and SOST protein expression was mainly expressed in the migrated cells within the mid-layer of the gel. Osteoblastic (ALP and osteocalcin) and osteocytic (PHEX, DMP-1 and SOST) mRNA expression was significantly increased compared with those of the cells cultured on plastic dishes alone after 21. days. The number of TUNEL-positive apoptotic cells gradually increased, reaching a maximum at 28. days. The cells were distributed at the surface and in the mid-layer of the gel at 7. days and after 14. days of culture, respectively. These data indicate that our model reproduces transition from osteoblasts to osteocytes, suggesting the following: 1) migration of osteoblasts into collagen gel may play a critical role in osteocytic differentiation; and 2) spatiotemporal gene expression and apoptosis may be involved in the terminal differentiation of osteoblasts. Our model will make it possible to study the mechanism of transition from osteoblast to osteocyte, and both cell type-related diseases including osteoporosis and osteonecrosis.
AB - Osteoblasts are believed to differentiate into osteocytes, becoming embedded in bone, or to undergo apoptosis after the bone formation phase. The regulation of this terminal differentiation seems to be critical for bone homeostasis. However the mechanism remains unclear and there is no assay system currently available to analyze this process. To address this issue, we developed a new model in which osteoblasts are cultured on a type I collagen gel layer with osteogenic supplements β-glycerophosphate and ascorbic acid. Cellular behavior was analyzed by electron microscopy, immunohistochemistry and real-time RT-PCR. Osteoblasts gradually migrated into the gel, produced collagen fibrils, and differentiated to osteocytic cells with bone lacunae- and canaliculi-like mineralization. Osteocalcin, DMP-1 and SOST protein expression was mainly expressed in the migrated cells within the mid-layer of the gel. Osteoblastic (ALP and osteocalcin) and osteocytic (PHEX, DMP-1 and SOST) mRNA expression was significantly increased compared with those of the cells cultured on plastic dishes alone after 21. days. The number of TUNEL-positive apoptotic cells gradually increased, reaching a maximum at 28. days. The cells were distributed at the surface and in the mid-layer of the gel at 7. days and after 14. days of culture, respectively. These data indicate that our model reproduces transition from osteoblasts to osteocytes, suggesting the following: 1) migration of osteoblasts into collagen gel may play a critical role in osteocytic differentiation; and 2) spatiotemporal gene expression and apoptosis may be involved in the terminal differentiation of osteoblasts. Our model will make it possible to study the mechanism of transition from osteoblast to osteocyte, and both cell type-related diseases including osteoporosis and osteonecrosis.
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U2 - 10.1016/j.bone.2012.09.001
DO - 10.1016/j.bone.2012.09.001
M3 - Article
C2 - 22985890
AN - SCOPUS:84867236635
SN - 8756-3282
VL - 52
SP - 102
EP - 110
JO - Bone
JF - Bone
IS - 1
ER -
