What makes osteoblasts

Although transgenic mice were born normally, they quickly started to display bone fractures as early as 1 wk after birth , and within 3 wk most of them suffered from bone fracture. Bone fractures were found most frequently in tibiae, fibulae, calcanei, and femurs, whereas fractures in upper limbs and ribs were found also, indicating that the skeleton of transgenic mice was extremely fragile. Teeth of transgenic mice were also fragile and sometimes broken. The phenotype of teeth will be described elsewhere in detail.

We generated seven F 0 transgenic mice with fractures. Although we succeeded in establishing a line from one of them, the other F 0 mice were affected too severely to be bred and were analyzed directly. Although these F 0 mice suffered from severe fractures Fig. The transgene expression of the line was about five times higher than endogenous Cbfa1 expression Fig.

The body weight of transgenic Tg mice became progressively lower than that of wild-type WT littermates during development and reached nearly half of the normal average value at 4 wk of age Tg, male 9. The fragility of bones and teeth seemed to cause the growth retardation. The serum concentration of calcium was similar between wild-type and transgenic mice at 6 wk of age WT, 9.

Radiological analysis showed that the whole skeleton of transgenic mice was proportionally shorter, and their bone was generally more radiolucent Fig. The fracture healing was observed in tibiae, fibulae, and calcanei, and cortices were thinner Fig. The histological appearance of tibiae clearly showed osteopenia in the transgenic mice Fig.

The cortical bone of transgenic mice was porous and thinner at both 3 and 6 wk of age. The analysis by peripheral quantitative computed tomography pQCT also showed thinner cortical bone and enlarged marrow cavity in 3-mo-old transgenic mice Fig. The trabecular bone was decreased at 3 wk of age but not at 6 wk of age in transgenic mice Fig.

This was also demonstrated by pQCT analyses in 3-mo-old mice, which showed similar amounts of mineral content in the trabecular region WT, 0. Maintenance of trabecular bone and thinner porous cortical bone were observed also in histological analyses of 3- and 8-mo-old transgenic mice unpublished data.

Osteocytes in bone increase during growth. At 6 wk of age, both trabecular and cortical bone contained many osteocytes in wild-type mice but had few osteocytes in transgenic mice Fig. The drastic decrease of osteocytes was observed until 1 yr of age Fig. The decrease of osteocytes in transgenic mice is not due to cell death, since empty lacunae, which represent the death of osteocytes, were absent in bone.

Numerous tartrate-resistant acid phosphatase TRAP -positive osteoclasts were observed on the surface of trabecular bone but not in cortical bone of wild-type mice, whereas they were observed not only on trabecular bone but also on cortical bone, especially in the cavities in transgenic mice Fig. Polarization microscopy revealed that cortical bone of transgenic mice at 3 mo of age was composed mainly of immature bone, described as woven bone, in which collagen fibers ran in all directions, instead of mature lamellar bone, which has highly ordered parallel collagen fibers as seen in wild-type mice Fig.

The composition of bone matrix was also different. Osteopontin protein was restricted to trabecular bone in wild-type mice, whereas it was distributed widely and extensively in the whole bone, including cortical bone, in transgenic mice Fig. The cause of osteopenia was examined in detail by histomorphometrical analyses at 3 and 6 wk of age. Although the number of osteoblasts in transgenic mice was more than twice that in wild-type mice at both 3 and 6 wk of age, matrix production by osteoblasts was impaired severely in transgenic mice as shown by the decreased thickness of newly deposited matrix osteoid thickness; O.

Th Fig. Osteocytes were decreased dramatically in transgenic mice, with their number in cortical bone about one-tenth of that in wild-type mice Fig. S and eroded surface ES Fig. Therefore, the maintenance of trabecular bone volume in 6-wk-old transgenic mice seems to be due to decreased osteolytic activity. To perform kinetic analyses of bone formation and mineralization, calcein was injected twice at a 3-d interval in 3-wk-old mice and a 7-d interval in 6-wk-old mice.

Calcein injections formed two consecutive labels in most parts of 3- and 6-wk-old wild-type bone Fig. In contrast, transgenic bone showed only a single band of labeling or dual lines with decreased distance between them, and these bands were interrupted frequently by engraved cavities Fig. Trabecular dynamic histomorphometry was performed on the longitudinal sections of 3- and 6-wk-old bone. In transgenic mice, the mean distances between the two labels were short at both 3 and 6 wk of age Fig.

Cortical dynamic histomorphometry was performed on cross sections of 3-wk-old bone. In transgenic mice, the mean distances between the two labels in both periosteum and endosteum were short Fig. Further, pQCT analysis showed that bone mineral density was decreased in the cortical bone of transgenic mice Fig.

These data indicate that osteoblast function was impaired in both matrix production and mineralization in transgenic mice. Because osteoblasts were increased but their function was impaired severely in transgenic mice, osteoblastic markers were investigated by in situ hybridization using probes of type I collagen , osteopontin , and osteocalcin.

Mice at birth, 2 wk, 4 wk, 6 wk, 3 mo, and 8 mo of age were examined Fig. In wild-type mice at birth, most bone was covered by type I collagen—positive and osteopontin-positive cells, but the number of osteocalcin-positive cells was very low Fig.

In transgenic mice at birth, osteopontin was expressed strongly in type I collagen—positive cells, but osteocalcin was barely detectable Fig. In wild-type mice, osteopontin-positive cells decreased gradually and osteocalcin-positive cells increased gradually during development, with osteocalcin-positive cells widely distributed at 3 mo of age Fig. However, in transgenic mice osteopontin-positive cells always covered most of the trabecular and cortical bone, but osteocalcin expression was much weaker than in their wild-type counterparts Fig.

Even in 8-mo-old transgenic mice, most bone was covered by osteopontin-positive cells, whereas only a small area of bone was covered by osteopontin-positive cells in the wild-type mice unpublished data. Since osteopontin expression is detected earlier than osteocalcin expression in osteoblast differentiation Yoon et al.

In wild-type mice, the expression pattern of Cbfa1 was similar to that of osteopontin but different from that of osteocalcin at any age, although some overlaps of Cbfa1 and osteocalcin expression were observed, especially at younger ages. It indicates that a major population of Cbfa1 highly positive cells consists of less mature osteoblasts.

In transgenic mice, endogenous Cbfa1-positive cells were increased, and the level of the expression seemed to be upregulated. Expression of the genes related to bone matrix proteins, including type I collagen , osteopontin , bone sialoprotein , osteocalcin , and matrix metalloproteinase MMP 13 , is considered to be regulated by Cbfa1 Yamaguchi et al.

Alkaline phosphatase ALP is considered to be upregulated during osteoblast differentiation Stein et al. The expression of osteopontin and bone sialoprotein was increased but that of ALP , osteocalcin , and MMP13 was decreased in transgenic mice. Cbfa1 transgenic mice showed severe osteopenia and suffered from multiple fractures. Although trabecular bone was conserved, cortical bone was reduced severely.

Cortical bone, which is composed of immature bone with a few osteocytes, was invaded by osteoclasts. Enlarged bone marrow cavity also implied the osteolysis of cortical bone by osteoclasts. However, osteoclastogenesis was not accelerated in transgenic mice. Osteoblast number was increased from an early developmental stage, but osteoblast function was impaired in both matrix production and mineralization.

Osteoblast maturation was inhibited at a late stage, and less mature osteoblasts accumulated to form the impaired bone in adult mice. Therefore, failure in the terminal differentiation of osteoblasts resulted in osteopenia and fragility in transgenic mice, demonstrating that Cbfa1 inhibits osteoblast differentiation at a late stage.

Previous in vitro and in vivo data suggested that Cbfa1 plays an important role in maturation of osteoblasts. However, our data showed that the late stage of osteoblast maturation was inhibited in transgenic mice as indicated by the accumulation of osteopontin-positive cells and the decrease of highly osteocalcin-positive cells, osteocytes, and ALP and osteocalcin expression.

The accumulation of less mature osteoblasts in transgenic mice seemed to be caused not only by the maturational blockage of osteoblasts but also by acceleration of osteoblast differentiation at an early stage of cell development because osteoblasts were increased in number at neonatal stage Fig. Furthermore, the proliferation and apoptosis of less mature osteoblasts in transgenic mice have to be considered, although their analyses in vitro were unsuccessful because of the loss of the transgene expression in primary culture of calvaria-derived cells unpublished data as previously described Krebsbach et al.

ALP activity is detected at an early stage of osteoblast differentiation and continues to increase during osteoblast maturation until the mineralization phase Stein et al. Although the difference in ALP expression level between transgenic and wild-type mice was not apparent at birth, it became evident during development Fig.

This suggests that overexpression of Cbfa1 blocks osteoblast maturation at a certain stage in vivo. Since osteocalcin expression is restricted to mature osteoblasts and odontoblasts, it is a convenient marker for fully differentiated osteoblasts Mark et al. These findings suggested that Cbfa1 is the most important factor for osteoblast-specific osteocalcin expression in vitro. However, a major population of Cbfa1 highly positive cells consisted of less mature osteoblasts in wild-type mice, and overexpression of Cbfa1 failed to upregulate osteocalcin expression in vivo Figs.

These data indicate that other factors, which are induced at a late stage of osteoblast differentiation, are required for the regulation of osteocalcin expression or that some factors suppress osteocalcin expression at an immature stage of osteoblast differentiation in vivo. Further, Runx1 is known to interact with the corepressor mSin3A Lutterbach et al. Thus, these repressors may play an important role in the transcriptional regulation of osteocalcin by repressing Cbfa1-dependent activation at an early stage of osteoblast differentiation.

Overexpression of DN-Cbfa1 at a late stage of osteoblast differentiation caused a decrease in the bone formation rate and decreased expression of the genes encoding main bone matrix proteins and resulted in osteopenia Ducy et al. Surprisingly, our transgenic mice overexpressing Cbfa1 at both early and late stages of osteoblast differentiation also showed an osteopenic phenotype, although the mechanism for osteopenia was different. The two kinds of transgenic mice had a common feature because Cbfa1 function was suppressed in fully differentiated osteoblasts of DN- Cbfa1 transgenic mice, and the fully differentiated osteoblasts were diminished in our Cbfa1 transgenic mice.

Therefore, both transgenic mice lacked at least Cbfa1-dependent function of fully differentiated osteoblasts, which finally caused osteopenia in both transgenic mice. It indicates that Cbfa1 inhibits osteoblast differentiation at a late stage, but some level of Cbfa1 is required for the expression of the genes encoding main bone matrix proteins. It also indicates that the transcriptional regulation of bone matrix genes by Cbfa1 is dependent on the maturational stage of osteoblasts as discussed about osteocalcin expression in the previous paragraph.

It was suggested that osteoprogenitor cells have more potential to support osteoclast development than more differentiated cells Manolagas, Therefore, the decrease of RANKL may reflect the relative decrease of osteoprogenitor cells because Cbfa1 seemed to accelerate the early stage of osteoblast differentiation Fig. In transgenic mice, cortical bone mass but not trabecular bone mass was reduced severely without acceleration of osteoclastogenesis.

However, the enlarged bone marrow cavity and the presence of numerous osteoclasts in cortical bone showed that cortical bone loss was a result of osteolytic activity. This seemed to be caused by the immature composition of cortical bone, which contains abundant osteopontin with the small cell attachment motif Arg-Gly-Asp recognized by integrins and promotes the attachment of osteoclasts to the extracellular matrix Fig.

The expression of bone sialoprotein, which also has the Arg-Gly-Asp motif, was increased in Northern blot analysis Fig.

The drastic decrease of osteocytes that is a unique phenotype of the transgenic mice was caused by the inhibition of osteoblast maturation. Osteocytes are spaced regularly throughout the bone and communicate with each other and with osteoblasts and bone marrow stromal cells using their processes.

Although the exact function of osteocytes remains unknown, they are considered to work as mechanosensors Nijweide et al. Thus, the fragility of transgenic bone might be caused by the near absence of osteocytes, causing an inability to detect mechanical stress and microfractures. Overexpression of Cbfa1 in osteoblasts increased osteoblast number but inhibited their terminal maturation, resulting in accumulation of less mature osteoblasts and osteopenia.

Therefore, in an attempt to increase bone mass by Cbfa1 intermittent induction of Cbfa1 in osteogenic cells might permit a periodic increase of immature osteoblasts and their maturation. This concept might be related to the anabolic action on bone mass induced by the intermittent administration of parathyroid hormone Dempster et al. We demonstrated using Cbfa1 transgenic mice that Cbfa1 negatively regulates osteoblast differentiation at a late stage of osteoblast development.

However, Cbfa1 seems to regulate osteoblast differentiation positively at an early stage. These opposite functions of Cbfa1, depending on the maturational stage of osteoblasts, may play an important role in the regulation of bone mass.

Since Cbfa1 is an essential factor for osteoblast differentiation, many factors and substances that have an effect on bone mass will influence Cbfa1 expression or activation. Thus, our findings are expected to be of great benefit to future trials to increase bone mass. A DNA fragment containing the 2. The fragments from the final construct, including the 2. Transgene integration and expression were identified by Southern and Northern blot analyses, respectively, using the whole length cDNA of type II Cbfa1 as a probe.

A transgenic line was maintained on a B6C3H F 1 background. To confirm the activity of the promoter used in this study, we also subcloned the DNA fragment covering the 2. Transgenic mice and their wild-type littermates were anesthetized and subjected to x-ray exposure in a micro-FX Fuji Film, Inc. Long bones were dissected from killed mice and exposed to x-rays.

Voxel size was 0. The contour of the total bone was determined automatically by the pQCT software algorithm. The cortical and trabecular parameters were obtained at the diaphysis and 2 mm from distal epiphysis, respectively.

For histological analyses, mice were killed at birth, 2 wk, 3 wk, 4 wk, 6 wk, 3 mo, 8 mo, and 1 yr of age. For the assessment of dynamic histomorphometric indices, mice were injected twice with calcein at a dose of 0. The 3-wk group received dual injections at 6 and 3 d before sacrifice, and the 6-wk group received them at 8 and 1 d before sacrifice.

Long bones were fixed with ethanol, and the undecalcified bones were embedded in glycolmethacrylate. Nomenclature, symbols, and units used are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research Parfitt et al. Some of the sections were stained with TRAP. To examine collagen fiber deposition, sections from tibiae were stained with hematoxylin and eosin and observed under polarized light Bucay et al.

To examine osteoblast markers, sections of tibiae at different ages were also used for in situ hybridization using probes for type I collagen , osteopontin , osteocalcin , and Cbfa1 as described previously Inada et al.

Localization of the first antibody was visualized by incubation with biotinylated F ab 2 fragments of swine anti—rabbit IgG antibody Dako at room temperature for 40 min and then treated with the ABC reagents Vector Laboratories. Finally, sections were stained by DAB substrate and counterstained with methyl green.

Total RNA was extracted from long bones without fracture from newborn and 4- and wk-old transgenic and wild-type mice by lithium chloride. We thank M. Iwamoto for critical discussions, B.

Murayama Kureha Chemical Industry for bone histomorphometrical analysis, K. Hiraiwa for maintaining mouse colonies, and R. Ohkawa for secretarial assistance. Generation of transgenic mice. C Longitudinal section of forelimb. Staining is observed specifically in osteoblasts around the diaphysis and in immature osteoblastic cells around the metaphysis.

Sections were counterstained with eosin. D Northern blot hybridized with Cbfa1 probe. E Northern blot analysis comparing the transgene and endogenous Cbfa1 levels of expression in long bones of newborn Cbfa1 transgenic mice. Radiological analysis. A—C X-ray analysis of 6-wk-old Cbfa1 transgenic mice from the established line.

Whole skeletons of transgenic mice are proportionally shorter and more radiolucent A. The transgenic mouse suffered from fractures in the tibia, fibula, and calcanei, and fracture healing is observed in these regions A and B.

The radiolucency is caused mainly by thinner cortices because metaphyseal trabeculation is similarly observed in femurs C. D Radiograph of 9-wk-old F 0 transgenic mice. Whole skeletons are also more radiolucent, and fracture healing is observed in tibiae, fibulae, and calcanei in both lower limbs. WT, wild-type mouse; Tg, transgenic mouse. Histological appearance of transgenic bone. Cortical bone in transgenic mice is thin and porous at both 3 and 6 wk of age.

Transgenic mice show reduced trabeculation at 3 wk of age but not at 6 wk of age. Undecalcified sections were stained with toluidine blue. Bar, 1 mm. Diaphyses of femurs from wild-type and transgenic female mice at 3 mo of age were analyzed by pQCT. Note the increased width of the marrow cavity and the concomitant reduction in cortical thickness in transgenic mice.

Mineral densities are shown as different colors according to the standard mineral density gradients. C—F Cortical thickness C , endosteal circumference D , periosteal circumference E , and bone mineral density F were measured for wild-type white bars and transgenic black bars mice.

Bars, 1 mm. Decreased osteocytes, osteoclast invasion, collagen structure, and osteopontin deposition in cortical bone of transgenic mice.

Note that osteocytes are diminished greatly in transgenic mice. C and D Polarized microscopy of cortical bone in tibias of wild-type C and transgenic D mice at 3 mo of age. Cortical bone in transgenic mice shows the woven pattern instead of the lamellar collagen deposition seen in wild-type mice. E and F Immunohistochemical analysis in cortical bone of wild-type E and transgenic F mice at 3 mo of age using antiosteopontin antibody.

Osteopontin is deposited heavily in cortical bone of transgenic mice. Note that osteocytes are spaced regularly in wild-type bone, but a few osteocytic cells are spaced irregularly in transgenic bone. Bone volume, matrix deposition, and cell parameters in Cbfa1 transgenic mice. Th B , osteoblast number N. Ob C , osteoblast surface Ob.

S D , osteocyte number N. Ot E , osteoclast number N. Oc F , osteoclast surface Oc. S G , and eroded surface ES H are compared between wild-type white bars and transgenic mice black bars at 3 and 6 wk of age. The analyses were done using proximal parts of tibiae at secondary spongiosa except osteocyte number, which was counted at cortical bone of diaphysis. Bone formation in trabecular and cortical bone.

To assess osteoblast function in mineralization, wild-type and transgenic mice were double labeled with calcein at 3 and 6 wk of age. A and B Fluorescent micrographs of the two labeled mineralization fronts in femurs of wild-type A and transgenic B mice at 3 wk of age. The distances between the double labeling in transgenic mice are much less than those of wild-type mice.

Christina Vrahnas, Natalie A. Overview Fingerprint. Abstract The cells of the osteoblast lineage osteoblasts, osteocytes, and bone lining cells have multiple stage-specific functions in the skeleton, including formation of the bone matrix. Leder, Marc N. Link to publication in Scopus. Together they form a unique fingerprint. View full fingerprint. Wein Eds. Contemporary Endocrinology. Humana Press Inc.. Later, usually after birth, secondary ossification centers form in the epiphyses.

Ossification in the epiphyses is similar to that in the diaphysis except that the spongy bone is retained instead of being broken down to form a medullary cavity. When secondary ossification is complete, the hyaline cartilage is totally replaced by bone except in two areas. A region of hyaline cartilage remains over the surface of the epiphysis as the articular cartilage and another area of cartilage remains between the epiphysis and diaphysis.

This is the epiphyseal plate or growth region. Bones grow in length at the epiphyseal plate by a process that is similar to endochondral ossification. The cartilage in the region of the epiphyseal plate next to the epiphysis continues to grow by mitosis. The chondrocytes, in the region next to the diaphysis, age and degenerate. Osteoblasts move in and ossify the matrix to form bone.

This process continues throughout childhood and the adolescent years until the cartilage growth slows and finally stops. When cartilage growth ceases, usually in the early twenties, the epiphyseal plate completely ossifies so that only a thin epiphyseal line remains and the bones can no longer grow in length.

Bone growth is under the influence of growth hormone from the anterior pituitary gland and sex hormones from the ovaries and testes. Even though bones stop growing in length in early adulthood, they can continue to increase in thickness or diameter throughout life in response to stress from increased muscle activity or to weight.

The increase in diameter is called appositional growth.