Abstract It is not known how bone proteins appear in the matrix before and after calcification during embryon- ic osteogenesis. The present study was designed to in- vestigate expressions of the five major bone extracellular matrix proteins – i.e. type I collagen, osteonectin, osteo- pontin, bone sialoprotein and osteocalcin – during osteo- genesis in rat embryonic mandibles immunohistochemi- cally, and their involvement in calcification demonstrat- ed by von Kossa staining. Wistar rat embryos 14 to 18 days post coitum were used. Osteogenesis was not seen in 14-day rat embryonic mandibles. Type I collagen was localized in the uncalcifed bone matrix in 15-day mandi- bles, where no other bone proteins showed immunoreac- tivity. Osteonectin, osteopontin, bone sialoprotein and osteocalcin appeared almost simultaneously in the calci- fied bone matrix of 16-day mandibles and accumulated continuously in 18-day mandibles. The present study suggested that type I collagen constitutes the basic framework of the bone matrix upon which the noncollag- enous proteins are oriented to lead to calcification, whereas the noncollagenous proteins are deposited si- multaneously by osteoblasts and are involved in calcifi- cation cooperatively. Key words Bone · Calcification · Type I collagen · Noncollagenous proteins · Immunohistochemistry Introduction The bone matrix is composed of organic and mineral components. Type I collagen is a primary gene product of osteoblasts during bone matrix formation (Aubin and Liu 1996) and comprises 85% to 90% of the total organ- ic bone matrix (Termine and Robey 1996). The most abundant noncollagenous protein produced by osteo- blasts is osteonectin, a phosphorylated glycoprotein that has high affinity for binding ionic calcium and physio- logic hydroxyapatite (Termine and Robey 1996). Osteo- pontin is a phosphorylated glycoprotein secreted by os- teoblasts and has been suggested to occur at an early stage during bone development and to facilitate attach- ment of osteoblasts to the extracellular matrix (Butler 1989; Butler et al. 1996). Bone sialoprotein (BSP) is an osteoblast-derived heavily glycosylated protein of the bone matrix expressed at late stages of differentiation (Bianco et al. 1991) and its appearance is tightly corre- lated with the appearance of mineral (Bianco et al. 1993). Osteocalcin is a γ-carboxy glutamic acid-contain- ing protein that has been considered the latest of expres- sion markers in mature osteoblasts (Aubin and Liu 1996). Distribution of the bone matrix proteins in embryonic and infant bones has been reported using immunohisto- chemistry for osteopontin and ostecalcin in rats (Mark et al. 1988), osteocalcin in human (Ohta et al. 1989), osteo- pontin and osteonectin in pigs (Chen et al. 1991) and os- teopontin and BSP in rats (Pinero et al. 1995). The gene expression of the bone matrix components in embryos and infants has been reported for BSP in rats (Chen et al. 1992), osteonectin,osteopontin and osteocalcin in mice (Nakase et al. 1994), and osteonectin, osteopontin, BSP and osteocalcin in mice (Sommer et al. 1996). However, it is not known how the gene products of the major five bone proteins, i.e. type I collagen, osteonectin, osteopon- tin, BSP and osteocalcin are sequentially deposited and accumulated extracellularly during embryonic osteogen- esis in a single animal model and how the maturation of the bone organic matrix is involved in calcification. Y. Sasano ( ✉ ) · J.-X. Zhu · M. Kagayama Second Department of Oral Anatomy, Tohoku University School of Dentistry, Sendai 980-8575, Japan Tel.: 022-717-8287, Fax: 022-263-9867 S. Kamakura Department of Maxillofacial Surgery I, Tohoku University School of Dentistry, Sendai 980-8575, Japan S. Kusunoki LSL Co., Ltd., Tokyo 178-0061, Japan I. Mizoguchi Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu-Ishikari, Hokkaido 061-0293, Japan Anat Embryol (2000) 202:31–37 © Springer-Verlag 2000 ORIGINAL ARTICLE Yasuyuki Sasano · Jing-Xu Zhu · Shinji Kamakura Shinichiro Kusunoki · Itaru Mizoguchi Manabu Kagayama Expression of major bone extracellular matrix proteins during embryonic osteogenesis in rat mandibles Accepted: 21 December 1999