Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells.

It has been reported that PTH exerts bone-forming effects in vivo when administered intermittently. In the present study, the anabolic effects of PTH(1-34) on osteoblast differentiation were examined in vitro. Osteoblastic cells isolated from newborn rat calvaria were cyclically treated with PTH(1-34) for the first few hours of each 48-h incubation cycle. When osteoblastic cells were intermittently exposed to PTH only for the first hour of each 48-h incubation cycle and cultured for the remainder of the cycle without the hormone, osteoblast differentiation was inhibited by suppressing alkaline phosphatase activity, bone nodule formation, and mRNA expression of alkaline phosphatase, osteocalcin, and PTH/PTHrP receptor. Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and Ca2+/PKC demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, the intermittent exposure to PTH for the first 6 h of each 48-h cycle stimulated osteoblast differentiation. Both cAMP/ PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. Continuous exposure to PTH during the 48-h incubation cycle strongly inhibited osteoblast differentiation. Although both cAMP/PKA and Ca2+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. A neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin mRNA induced by the 6-h intermittent exposure. The inhibitory effect induced by the 1-h intermittent exposure was not affected by anti-IGF-I antibody. These results suggest that PTH has diverse effects on osteoblast differentiation depending on the exposure time in vitro mediated through different signal transduction systems. These in vitro findings explain at least in part the in vivo action of PTH that varies with the mode of administration.

Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.

A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.

Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation.

We investigated the role of Sonic hedgehog (SHH) in osteoblast differentiation and bone formation. The numbers of ALP-positive cells in the mouse fibroblastic cell line C3H10T1/2 and the mouse osteoblastic cell line MC3T3-E1 were increased by co-culture with chicken fibroblasts transfected with chicken Shh cDNA encoding amino-terminal peptide (Shh-N). The conditioned medium of Shh-N-RCAS-transfected chicken fibroblast cultures also significantly increased ALP activity in both C3H10T1/2 and MC3T3-E1 cells. Intramuscular transplantation of Shh-N-RCAS-transfected chicken fibroblasts into athymic mice induced ectopic bone formation. These results indicate that SHH induces osteoblast differentiation and ectopic bone formation.

Effects of ionizing radiation on proliferation and differentiation of osteoblast-like cells.

Diagnostic radiation for immediate post-surgical assessment of osseointegrated dental implants has been discouraged, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. To assess this possibility, we investigated the effects of ionizing radiation on proliferation and differentiation of osteoblasts using osteoblast-like cells isolated from the calvariae of newborn rats (ROB) and a clonal osteoblastic cell line (MC3T3-E1). The cells were exposed on day 3 to a single dose of x-rays at either 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40-mGy dose from a diagnostic chest x-ray machine. The effects of radiation on cell growth and alkaline-phosphatase-specific (ALP) activity were evaluated at three-day intervals after irradiation up to day 12 in ROB cells, and evaluated at day 12 in MC3T3-E1 cells. At the culture end-point, the effects on formation of bone-like nodules were also evaluated in both ROB and MC3T3-E1 cells. Exposure of 4000 mGy differentially affected the two cell types. It inhibited cell growth and alkaline phosphatase activity, and inhibited DNA content in MC3T3-E1 cells. This irradiation also strongly inhibited the formation of bone-like nodules in ROB cells. On the other hand, exposure of 40-, 100-, and 400-mGy (Linac) and 40-mGy (diagnostic quality) irradiation induced no significant changes in cell growth, alkaline phosphatase activity, and formation of bone-like nodules in ROB cells. These doses also induced no significant changes in DNA content and ALP activity in MC3T3-E1 cells. These results indicate that ionizing radiation at a single dose of up to 400 mGy induces no significant changes in cell growth and differentiation of osteoblast-like cells, at least in vitro. Higher radiation doses (4000 mGy) may exert different effects on cell proliferation and cell differentiation of osteoblasts, depending on the cell types affected. Thus, diagnostic radiation seems to have less effect on proliferation and differentiation of osteoblasts.

Cloning of rat type I receptor cDNA for bone morphogenetic protein-2 and bone morphogenetic protein-4, and the localization compared with that of the ligands.

A rat homologue cDNA of mouse (Koenig et al. [1994] Mol. Cell Biol. 14:5961-5974; Suzuki et al. [1994] Proc. Natl. Acad. Sci. USA 91: 10255-10259) and human (ten Dijke et al. [1994] J. Biol. Chem. 269:16985-16988) type I receptors for BMP-2 and BMP-4 was cloned. Tissue distribution of the receptor mRNA was studied by in situ hybridization using rats at embryonic days 9, 13, 15, and 18 as well as 1- and 5-day-old postnatal rats. In the rats at embryonic days 9, 13, and 15, the receptor mRNA was diffusely expressed over the embryonic bodies. At embryonic day 18, the receptor mRNA expression was high in the hair and whisker follicles, tooth bud, cartilage, bone, digestive organs, lung, kidney, heart, and meninges. The receptor mRNA was expressed over a much wider area than those of the ligands in many organs. In the lung and digestive organs, the receptor mRNA was diffusely expressed and most highly expressed in the bronchial epithelium and muscle layer, respectively, in both of which mRNA expression of the ligands was undetectable. The receptor mRNA was highly expressed in the meninges, although neither of the ligands was expressed in or near this region. These results suggest that this receptor participates in both mesoderm formation in early embryogenesis and differentiation of mesodermal cells during maturation of organs, and further suggest the presence of another factor(s) that binds the type I receptor.

Changes in biological activity of bone cells in ovariectomized rats revealed by in situ hybridization.

Twelve-week-old female rats were ovariectomized (OVX) and compared with sham-operated control rats at 3, 5, 7, 10, 14, 30, and 60 days postoperation with respect to the expression of type I collagen and osteopontin mRNAs, as well as bone structure and the number of osteoclasts. The trabecular number and separation were significantly decreased and increased, respectively, in the metaphyseal trabecular bones of OVX rat femurs. The number of osteoclasts was significantly increased in the same region of OVX rats at 3 and 5 days postoperation. Type I collagen mRNA was expressed in osteoblasts, and osteopontin mRNA was expressed in some osteoclasts, in mononuclear cells on the bone resorption surface, and in osteocytes near the resorption surface. In the metaphyseal trabecular bone, type I collagen and osteopontin mRNA expression levels in individual cells was initially increased in OVX rats from 7 to 10 days postoperation, and this was sustained for 60 days. The number of osteopontin mRNA-expressing osteocytes was also significantly increased at 10 days postoperation, which lasted until 60 days. In the epiphysis, an increase in type I collagen mRNA expression was initially observed in OVX rats at 14 days postoperation, which lasted until 60 days. The number of osteopontin mRNA-expressing osteocytes was virtually identical until 30 days postoperation in the epiphysis. These findings indicated that the biological activities of osteoblasts and osteocytes are stimulated in bones of the OVX rat and that the response for OVX differs between the metaphysis and epiphysis. Furthermore, the number of osteopontin mRNA-expressing osteocytes was increased only in bones that tended to be resorbed after OVX. This indicates that some osteocytes were stimulated to express osteopontin mRNA by estrogen deficiency and suggests that these osteopontin mRNA-expressing osteocytes may be involved in regulation of bone metabolism.

Autocrine-paracrine effects of overexpression of hepatocyte growth factor gene on growth of endothelial cells.

Although hepatocyte growth factor (HGF) is synthesized in vascular cells, it is not known whether locally synthesized HGF acts similarly to exogenously added HGF. Therefore, we transfected cultured cells with human HGF vector and examined the effects on growth of vascular cells. Endothelial cells (EC) transfected with HGF vector synthesized and secreted high levels of HGF, and also showed significantly higher number. Addition of conditioned medium from vascular smooth muscle cells (VSMC) or EC transfected with HGF vector to nontransfected EC resulted in a significant increase in cell number, which was abolished by anti-HGF antibody. Co-culture of HGF-transfected VSMC with EC showed that HGF released from VSMC or EC stimulated EC growth. These results demonstrate that endogenously produced HGF by transfection of human HGF vector can exert autocrine and paracrine stimulatory effects on EC growth, but not VSMC growth, suggesting the role of local HGF system in cardiovascular disease.

Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6.

The effects of bone morphogenetic protein-2 (BMP-2) on osteoblastic differentiation of bone marrow stromal cells were investigated using two bone marrow stromal cell lines, ST2 and MC3T3-G2/PA6 (PA6). BMP-2 stimulated ALP activity and induced parathyroid hormone (PTH)-dependent production of cAMP in both ST2 and PA6 cells, but these effects were more apparent in ST2 cells than in PA6 cells. BMP-2 induced the production of osteocalcin in ST2 cells, but not in PA6 cells. BMP-4 and BMP-6 stimulated ALP activity in ST2 cells, but the effect of BMP-6 was less marked than that of BMP-2 and BMP-4. BMP-4 induced PTH-dependent cAMP production of cAMP in ST2 cells, but BMP-6 did not. When ST2 cells were transplanted into the peritoneal cavities of athymic mice with BMP-2 in diffusion chambers, these cells generated mineralized bone in the chambers. These results indicate that BMPs induce the differentiation of bone marrow stromal cells into osteoblasts. However, the effects differ among the BMPs and among the types of cell exposed to these proteins.

An estrogen deficiency caused by ovariectomy increases plasma levels of systemic factors that stimulate proliferation and differentiation of osteoblasts in rats.

To investigate the pathogenesis of accelerated bone formation in estrogen deficiency, diffusion chambers containing osteoblast-like cells isolated from newborn rat calvariae were transplanted into the peritoneal cavity of sham-operated (sham), ovariectomized (OVX) rats, and OVX rats with supplement of 17 beta-estradiol (OVX + E2). Bone formation in the diffusion chambers transplanted into OVX rats was more accelerated than that transplanted into sham rats and OVX + E2 rats. Osteoblast-like cells cultured with the sera isolated from OVX rats exhibited higher levels of the DNA content in the culture wells, alkaline phosphatase activity, messenger RNA expression for alkaline phosphatase and osteocalcin, calcium content in the cell layer, and formation of bone-like nodules than those exposed to the sera from sham rats and OVX + E2 rats. Antibody against IGF-I almost completely inhibited the increase in DNA contents induced by the sera isolated from OVX rats but partially inhibited alkaline phosphatase activity. Adding IGF-I to the sera isolated from sham rats increased the DNA content to the same extent as that induced by the supplement with the sera from OVX rats but did not increase alkaline phosphatase activity appreciably. Addition of various concentrations of 17 beta-estradiol, interleukin (IL)-1, and IL-6 to the sera isolated from sham rats did not increase the DNA content or alkaline phosphatase activity in the osteoblast-like cells. These results indicate that some systemic factor(s) other than IGF-I, IL-1, and IL-6 may be responsible for the stimulative effect on osteoblast differentiation in the pathogenesis of the accelerated bone formation induced by estrogen deficiency in rats.

Ultrastructure of the rat periodontal ligament as observed with quick-freeze, deep-etch and replica methods: arrangement of collagen and related structures.

The ultrastructure of the periodontal ligament of rat molars was examined with the quick-freeze, deep-etch replica methods. It was mainly composed of elongated fibroblast-like cells and 40- to 50-nm-wide collagen fibrils that are arranged parallel to one another to form fibers approximately 1 micron in width. Collagen fibrils are composed of 10-nm-wide substructures that may run helically against the long axis of the fibril. Numerous rod-like structures ('rods') approximately 10 nm in width are present around the collagen fibrils. Individual or groups of rods span spaces between neighboring collagen fibrils to interconnect them. The surfaces of the fibroblast-like cells are also connected to the nearest collagen fibrils through the rods. In place, strands with a thickness similar to that of the rods were seen self-assembled into irregular meshwork structures. The treatment of the tissue with 10% sodium hydroxide for up to 5 days removed most of these rods and strands, thus exposing a three-dimensional arrangement of collagen fibrils that is often not fully visualized in untreated tissues. With histochemical staining of thinly sectioned tissues using Alcian blue, these rods and strands were positively stained, and thus they were demonstrated to be composed of proteoglycans. The ultrastructural arrangement of the periodontal ligament, observed in this study as a delicate interaction of collagen and proteoglycan components, is likely to play a significant role in the transmission of occlusal forces applied to the tissue and in the dissipation of mechanical shock.

Microgravity generated by space flight has little effect on the growth and development of chick embryonic bone.

Seven days' space flight of fertilized chicken eggs pre- incubated for 7 and 10 days on earth caused no differences in the morphology of osteoblasts, osteoclasts, and osteocytes of humerus and tibia from those of control embryos. Bone-resorbing and -forming activities of the femur were not different between control and flight groups. As a consequence, calcium and phosphorus contents of the femora between control and flight groups were not changed. Alkaline phosphatase activity of 3 different regions (resting cartilage, growth cartilage, and cortical bone) of tibia showed no significant difference between control and flight groups. No significant difference of gene expressions of hepatocyte growth factor and receptors of fibroblast growth factor was observed in perichondrium, trabecula, and skeletal muscles and tendons of hind limbs between control and flight groups. Unlike the results of previous space flight experiments in which young growing mammals were used, these morphological and biochemical results indicate that microgravity has little effect on bone metabolism of the chick embryo.

Age-related reduction in bone matrix protein mRNA expression in rat bone tissues: application of histomorphometry to in situ hybridization.

Age-related changes in the biological activity of osteoblastic cells have been studied extensively using histomorphometry, especially in relation to osteoporosis. Nevertheless, the changes occurring in the biological activity of individual osteoblastic cells are not sufficiently clarified by this technique. In the present study, age-related changes in the expression of bone matrix protein mRNAs in individual osteoblastic cells were analyzed in vivo by in situ hybridization using undecalcified bone sections. In the femurs of 8-week-old male rats, strong expression of type I collagen and osteocalcin mRNAs was detected in cuboidal osteoblasts on the bone formation surface. Osteopontin mRNA was detected in some of the mononuclear cells and osteoclasts on the bone resorption surface, and also in osteocytes. In the femurs of 60-week-old and 100-week-old male rats, expression of these bone matrix protein mRNAs was markedly decreased. Histomorphometrical analysis of 8-week-old and 60-week-old rats indicated that both the activity and number of osteoblasts expressing type I collagen mRNA, as well as the number of osteoclasts, were reduced in these tissues in older animals. These results indicate age-related reductions in both biological activity and numbers of osteoblasts.

Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow.

In order to examine the mechanism of the anabolic effect of parathyroid hormone (PTH) on bone formation, human PTH(1-34) [hPTH(1-34)] (30 micrograms/kg) was injected subcutaneously to 9-week-old rats 5 times a week for 1 or 3 weeks. Trabecular bone volume (BV/TV) in the tibial metaphysis was not significantly different between the PTH- and vehicle-treated groups, but the parameters related to bone formation, including osteoid surface (OS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were significantly increased as early as 1 week after PTH treatment. And the parameters related to bone resorption including eroded surface (ES/BS) and osteoclast number (N.Oc/BS) were also significantly increased as early as 1 week after PTH treatment. Treatment with PTH for 1 week induced no significant increase in bone mineral density at the femoral metaphysis, whereas the same treatment for 3 weeks induced a significant increase. When bone marrow cells isolated from femora and tibiae of either PTH- or vehicle-treated rats were cultured at a high density (2 x 10(7) cells/one well of 24-multiwell plate), cellular alkaline phosphatase (ALP) activity was significantly increased in the cells isolated from PTH-treated rats compared with vehicle-treated rats. When bone marrow cells were cultured at a low density (4 x 10(6) cells/a one well of 6-multiwell plate) to generate colonies (colony forming unit-fibroblastic, CFU-F), PTH induced apparent increases in both the total number of CFU-F and the number of ALP-positive CFU-F.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization.

The expression of the mRNAs for osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP) was studied by in situ hybridization during the healing process of an experimental fracture in adult rat femora. At day 1 postoperatively, ON mRNA was detected in the proliferating periosteum. At day 3, ON, OPN, and OC mRNAs were detected in woven bone. From day 5, MGP and ON mRNAs were detected in the immature chondrocytes. From day 7, ON, OPN, and OC mRNAs were detected in the osteoblastic cells in newly formed endosteal trabecular bone. OPN mRNA was also detected in some of the osteocytes in trabecular bone. From day 14, OPN and MGP mRNAs were detected in newly formed periosteal hypertrophic chondrocytes, and the ON, OPN, and OC mRNAs were detected in osteoblastic cells in newly formed periosteal trabecular bone. Although the cell types that expressed each mRNA in fractured bones were similar to those in embryonic bones, the time course of these mRNA expression in fractured bones was different from that in embryonic bones. We considered that this system is useful to investigate the phenotypic change in osteogenic and chondrogenic lineage cells that appears during fracture healing at the molecular level.

Limitation of panoramic radiography in diagnosing adenomatoid odontogenic tumors.

Adenomatoid odontogenic tumor is frequently not diagnosed preoperatively even when radiographs are available. This study evaluates radiographic findings with special emphasis on the characteristic radiopacities of adenomatoid odontogenic tumor. In correlating radiologic findings with pathologic findings, differences in diagnostic accuracy between intraoral periapical and panoramic radiographs were found. The intraoral periapical radiograph allows perception of the radiopacities in adenomatoid odontogenic tumor as discrete foci having a flocculent pattern within radiolucency even with minimal calcified deposits. Panoramic radiography was often unable to demonstrate radiopacities in adenomatoid odontogenic tumor when the calcification was minimal. The result indicates that intraoral radiographs may be essential for correct radiographic interpretation of an adenomatoid odontogenic tumor in the presence of minimal quantities of calcified deposits.

Growth and differentiation properties of normal and transformed human keratinocytes in organotypic culture.

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air-liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three-dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV-transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

The role of gravity in chick embryogenesis.

Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle 'Endeavour' and further incubated for 7 days under microgravity. Twenty out of thirty eggs (9/10 ten-day-old; 10/10 seven-day-old; 1/10 zero-day-old) were recovered alive after landing. The only living embryo of the zero-day-old group died 24 days after launch, and was comparable to a 16-day-old embryo. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.

Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta.

Osteopontin is a phosphorylated, sialic acid-rich, noncollagenous bone matrix protein containing the Arg-Gly-Asp-Ser amino acid sequence responsible for cell adhesion. The protein strongly binds to hydroxyapatite and play an important role in calcification. Expression of osteopontin mRNA was analyzed in human aortic atherosclerotic lesion by Northern blot hybridization, as well as by in situ hybridization. The expression of osteopontin mRNA was detected in 24 out of 25 samples of aorta obtained from 17 autopsy cases, but not in one normal aortic sample. The magnitude of expression was proportional to the stage of atherosclerosis. In situ hybridization revealed that the cells expressing osteopontin mRNA were detected in the wall surrounding atheroma and closely associated with calcification. They were morphologically identified as foam cells and immunohistologically positive with HHF35, appearing to be derived from smooth muscle cells. These findings have suggested that smooth muscle cell-derived foam cells express osteopontin mRNA and play an important role in calcification of the atherosclerotic lesions.

1 alpha,25-dihydroxyvitamin D3 regulates in vivo production of the third component of complement (C3) in bone.

We previously reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] specifically stimulates production of the third component of complement (C3) by murine osteoblastic cells and marrow-derived stromal cells (ST2) in vitro. In the present study we examined tissue-specific production of C3 in vivo in vitamin D-deficient mice, some of which received supplemental 1 alpha,25-(OH)2D3. Western blot analysis indicated that the C3 protein band in bone was undetectable in vitamin D-deficient mice, but became distinct 48 h after 1 alpha,25-(OH)2D3 administration. The mRNA expression of C3 in bone was also undetectable in vitamin D-deficient mice and appeared as early as 24 h after 1 alpha,25-(OH)2D3 administration. mRNA expression apparently preceded the appearance of C3 protein. In contrast, there was no significant difference in the expression of hepatic C3 mRNA among normal mice fed laboratory chow and vitamin D-deficient mice with and without 1 alpha,25-(OH)2D3 administration. The serum concentration of C3 in vitamin D-deficient mice was almost identical to that in normal mice and was unchanged after 1 alpha,25-(OH)2D3 administration. 1 alpha,25-(OH)2D3 receptor (VDR) mRNAs were detected in the kidney and intestine, whereas no appreciable mRNA expression of VDR occurred in the liver. Osteopontin mRNA was expressed in response to 1 alpha,25-(OH)2D3 in the kidney, but not in the intestine. Immunohistochemical studies showed that in normal mice, the C3 protein was located mainly in the periosteal regions of calvaria and on the surfaces of bone trabeculae in the tibial metaphyses. These results demonstrate that 1 alpha,25-(OH)2D3 tissue-specifically regulates in vivo production of C3 in bone. The production of bone C3 cannot be attributed to the presence of VDR alone, and we speculate that other tissue-specific factors are required.

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.