Newborn Rat Calvaria Research Articles

Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite

Microspheres comprised of hydroxyapatite particles, dispersed in reconstituted fibrous collagen, were prepared and characterized. The hydroxyapatite particles distributed evenly throughout the collagen matrix of the microsphere. Diameters of the reconstituted collagen fibers ranged from 30 to 90 nm, and exhibited a regular banding pattern with cross-striation of 50–60 nm under transmission electron microscope, suggesting that the reconstitution of collagen was not hindered by the hydroxyapatite particulates. When osteoblast cells isolated from newborn rat calvaria were seeded and cultured on the microspheres, the cell density increased from 2×10 4 to 3.2×10 4 cells/cm 2 in 8 days. Von Kossa staining exhibited spotty accumulation of mineral deposits on microspheres indicating matrix mineralization of the cultured cells. Analyses by electron microscopy and confocal microscopy showed that the osteoblast cells spread and attached to the microsphere via focal adhesion, while F-actin and DNA staining demonstrated the presence of stress fibers; moreover, mitotic cells could be observed. Together, these results indicate that osteoblast cells are capable of proliferating, differentiating and mineralizing in the matrix of the microspheres, and suggest that the microspheric composite is a potential grafting material for future clinical applications.

Early expression of bone matrix proteins in osteogenic cell cultures.

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.

Distinct anabolic response of osteoblast to low-intensity pulsed ultrasound.

Low-intensity pulsed ultrasound, a form of mechanical energy transmitted as high-frequency acoustical pressure waves, provides noninvasive therapeutic treatment for accelerating fracture repair and distraction osteogenesis. Relatively young osteoblasts respond to ultrasound by transiently upregulating message levels of immediate-early genes as well as that of osteocalcin and insulin-like growth factor I (IGF-I). Osteocytes derived from newborn rat tibia and calvaria responded to a lesser extent only in c-fos and cyclooxygenase-2 (COX-2) messages. Compared with the stretched osteocytes, which use stretch-activated and parathyroid hormone (PTH)-potentiated Ca2+ influx as an entry route to the protein kinase A (PKA) signal transduction pathways, there was no evidence of Ca2+ internalization by any of the cells tested on exposure to the ultrasound. On the other hand, inhibitors of p38 mitogen-activated protein kinase (MAPK) and upstream phosphoinositide 3-kinase (PI3K) blocked COX-2 and osteocalcin upregulation by the ultrasound-exposed ST2, murine bone marrow-derived cells. This is distinct from the aforementioned osteocytic response to low-frequency stretching and implies the involvement of integrins. Our findings suggested that accelerated fracture repair and distraction osteogenesis by the low-intensity pulsed ultrasound depend, at least in part, on the stimulation of osteoblastic cells at relatively early stages of osteogenic lineage. Bone is under control of multiple regulatory mechanisms so that diverse physical forces can be reflected to the microenvironment of each cell, in turn, to the entire bone.

Parathyroid hormone-stimulated resorption in calvaria cultured in serum-free medium is enhanced by the calcium-mobilizing activity of 1,25-Dihydroxyvitamin D 3

1,25(OH) 2D 3 enhances parathyroid hormone (PTH)-induced Ca 2+ signaling in osteoblasts by activating plasma membrane voltage-sensitive Ca 2+ channels (VSCCs). The ability of 1,25(OH) 2D 3 or the VSCC-activating analog AT (25-hydroxy-16-ene-23-yne-D 3) to enhance parathyroid hormone-stimulated 45Ca 2+ release from cultured new-born rat calvaria was measured. Analog BT (1,24-dihydroxy-22-ene-24-cyclopropyl-D 3), that does not mobilize Ca 2+, also was tested along with PTH. Control experiments were performed with and without PTH and with and without serum. Individual calvaria labeled in utero with 45Ca 2+ were cultivated in serum-free medium on filters at the medium/air interface of 24-well culture plates and 45Ca 2+ release followed over 72 h. The results demonstrated that 1,25(OH) 2D 3 and the Ca 2+-mobilizing analog, AT, but not the nuclear receptor-binding analog, BT, enhanced PTH-stimulated 45Ca 2+ release under serum-free conditions. This enhancement effect of the seco-steroids was not evident in the presence of 10% fetal calf serum. The effect of analog AT was faster than that of 1,25(OH) 2D 3. Nitrendipine, a specific L-type VSCC blocker, attenuated enhancement by vitamin D compounds, indicating that the high-threshold L-type VSCC is a molecular transducer of costimulation. These results emphasize the synergy between the calcitropic hormones 1,25(OH) 2D 3 and PTH in cultures containing osteoblasts and osteoclasts, and suggest that the Ca 2+-mobilizing activity of 1,25(OH) 2D 3 enhances Ca 2+ release from bone.

The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair

We investigated the effects of transplantation of osteoblastic cells with a bone morphogenetic protein (BMP)/carrier complex on bone repair by in vitro and in vivo experiments. Poly- d, l-lactic- co-glycolic acid/gelatin sponge (PGS) was used as a carrier for cell transplantation. In the in vitro experiments, three cell types, C3H10T1/2 cells, MC3T3-E1 cells, and primary osteoblastic cells, isolated from newborn rat calvariae (ROB cells), were cultured for 2 weeks on PGS alone or PGS containing BMP-2 (PGS/BMP). C3H10T1/2 cells cultured on PGS/BMP expressed several markers related to differentiation of both osteoblasts and chondrocytes, such as alkaline phosphatase (ALP) activity and mRNAs for osteocalcin and aggrecan, whereas the cells cultured on PGS alone expressed no such markers. MC3T3-E1 cells cultured on PGS/BMP exhibited a more ALP-positive cells than those cultured on PGS alone. PGS/BMP promoted ROB cell differentiation into both osteoblasts and chondrocytes. In the in vivo experiments, we transplanted ROB cells, which had been cultured on PGS alone or PGS/BMP in vitro for 2 weeks, into bone defects created in rat calvariae. Transplantation of ROB cells cultured on PGS alone generated little new bone. Transplantation of ROB cells cultured on PGS, which absorbed a low dose (10 ng) of rhBMP-2,; induced significantly higher bone mineral content than PGS/BMP alone, although application of a high dose (1 μg) of rhBMP-2 induced no difference in bone mineral content between transplantation of PGS/BMP with or without ROB cells. These results show that transplantation of osteoblastic cells after induction of osteoblast maturation in vitro by cultivation on PGS/BMP is a potent technique for cell therapy of bone repair.

Alveolar mononuclear cells can develop into multinucleated osteoclasts: An in vitro cell culture model

Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro. To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days. On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts. Bone resorption characteristics were observed both with light and scanning electron microscopy. When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells. Resorption pits were seen in the 7–14 days long-term cultures. These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D3. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 52, 142–147, 2000.

Alveolar mononuclear cells can develop into multinucleated osteoclasts: an in vitro cell culture model.

Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro. To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days. On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts. Bone resorption characteristics were observed both with light and scanning electron microscopy. When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells. Resorption pits were seen in the 7-14 days long-term cultures. These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D(3).

Regulation of cell‐cell communication in rat bone cells: The effect of phorbol esters

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pHi) and cytosolic calcium ([Ca+2]i) in this process. The results show that TPA (5 × 10−8 M) caused a complete inhibition of intercellular communication within 40–60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pHi. However, neither an increase of pHi alone nor exposure to TPA, under conditions preventing pHi-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pHi-shift itself rather than on the absolute value of pHi. In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pHi and the increase of intracellular calcium are involved in repression of intercellular communication by TPA. J. Cell. Biochem. 74:349–356, 1999. © 1999 Wiley-Liss, Inc.

Regulation of cell-cell communication in rat bone cells: The effect of phorbol esters

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pH(i)) and cytosolic calcium ([Ca(+2)](i)) in this process. The results show that TPA (5 x 10(-)(8) M) caused a complete inhibition of intercellular communication within 40-60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pH(i). However, neither an increase of pH(i) alone nor exposure to TPA, under conditions preventing pH(i)-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pH(i)-shift itself rather than on the absolute value of pH(i). In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pH(i) and the increase of intracellular calcium are involved in repression of intercellular communication by TPA.

Differential expression of fibroblast growth factor receptor-1, -2, and -3 and syndecan-1, -2, and -4 in neonatal rat mandibular condyle and calvaria during osteogenic differentiation in vitro

Fibroblast growth factors (FGFs) play important roles in the control of skeletal cell growth and differentiation. To identify the mechanisms of regulation of FGF actions during chondrogenesis and osteogenesis, we investigated, by immunohistochemistry, the spatiotemporal expression of the high-affinity FGF receptors (FGFR-1, -2, and -3) and coreceptors (syndecans-1, -2, and -4) in newborn rat condyle and calvaria during chondrogenesis and osteogenesis in vitro. During chondrogenesis at 4 days of culture, condyle chondrocytes showed weak FGFR-1, FGFR-2, and syndecan-1 immunoreactivity; stronger syndecan-2 expression; and marked FGFR-3 and syndecan-4 immunolabeling. At a later stage (i.e., 9 days of culture), FGFR-1, -2, and -3 were coexpressed with syndecan-4 in chondrocytes. Condyle progenitor cells located in the condyle perichondrium initially expressed strong syndecan-2 and -4 and weak syndecan-1 labeling, whereas no FGFR was detectable. When these cells differentiated into osteoblasts, they expressed syndecan-2 and -4 coincidently with FGFR-1, -2, and -3 at 9 days of culture. In newborn rat calvaria, syndecan-1, -2, and -4 were coexpressed mainly with FGFR-1 and -2 in osteoblasts. In the two models, treatment with FGF-2 (100 ng/mL) at 4–9 days of culture increased cell growth and decreased glycosaminoglycan or collagen synthesis, respectively, suggesting interactions of FGF-2 with distinct FGFRs and syndecans during chondrogenesis and osteogenesis. The coincident or distinct spatiotemporal expression pattern of FGFRs and syndecans in chondrocytes, progenitor cells, and osteoblasts represents a dynamic mechanism by which FGF effects on skeletal cells may be controlled in a coordinate manner during cartilage and bone formation in vitro.

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8.

Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell's osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.

Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15–16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway. Anat. Rec. 252:554–567, 1998. © 1998 Wiley-Liss, Inc.

Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone

Osteoprogenitors present in cell populations derived from fetal or newborn rat and mouse calvaria differentiate in long term culture and form osteoblastic bone-forming colonies (bone nodules). Previous reports have indicated considerable differences between bone cell populations derived from these two species with regard to their proliferation in response to glucocorticoids. In the present investigation, we have focused on proliferation and differentiation of osteoprogenitor cells in these bone cell populations and evaluated the effect of corticosterone, the principal glucocorticoid of both mouse and rat. Cells were isolated by sequential collagenase digestion from calvaria of newborn (2–5 days) CD-1 mice [mouse calvariae (MC) cells] and term fetal Wistar rats [rat calvaria (RC) cells] and cultured for up to 25 days in α-minimal essential medium containing 10% fetal bovine serum (FBS), antibiotics, 50 μg/mL ascorbic acid, and 8–10 mmol/L β-glycerophosphate. In agreement with previous observations by us and others, corticosterone increased cell growth in RC cell cultures, but inhibited cell growth in MC cultures. In RC cell cultures, corticosterone (1–1000 nmol/L) increased the nodule number in a dose-dependent manner ( p < 0.001 for all concentrations above 3 nmol/L) with a maximal effect at 300 and 1000 nmol/L (threefold increase over control). In MC cells, on the other hand, corticosterone (0.3–1000 nmol/L) increased the nodule number only at 30 nmol/L (50%, p < 0.01) but inhibited nodule formation by 33% ( p < 0.001) at 1000 nmol/L. In both RC and MC cultures a linear relationship was found between the number of cells plated and number of nodules formed. When cultures were treated with cortisol (30–300 nmol/L), similar effects were observed; the number of nodules dose dependently increased in RC cell cultures and dose dependently decreased in MC cell cultures. Significantly, however, the inactive glucocorticoid cortisone also increased bone nodule formation in RC cell cultures and decreased bone nodule formation in MC cell cultures. Carbenoxolone, which blocks 11 β hydroxysteroid dehydrogenase and thus prevents conversion of cortisone to cortisol, partially inhibited the cortisone-induced effects on bone nodule formation in both RC and MC cell cultures, indicating that both RC and MC cells can convert inactive glucocorticoids to active metabolites. In conclusion, our results show that the glucocorticoids corticosterone and cortisol inhibit proliferation and differentiation of osteoprogenitors in MC cell cultures but stimulate these processes in rat-derived osteoprogenitors.

Changes in Osteoblast Phenotype During Differentiation of Enzymatically Isolated Rat Calvaria Cells

Osteoblasts enzymatically isolated from newborn rat calvariae show various phenotypes including formation of mineralized bone nodules in culture. We investigated the temporal changes in osteoblast phenotype in these cells up to day 20 in culture. These cells formed unmineralized nodules by day 5. Mineralization was observed at the center of nodules by day 10, and nodules became larger on day 15. The nodules were surrounded by numerous alkaline phosphatase (ALP)-positive cells. ALP activity gradually increased by day 20. Parathyroid hormone (PTH) responsiveness increased with time in culture. Osteoblasts produced no osteocalcin by day 10, but its synthesis was detected from day 15. These cells expressed substantial levels of ALP and PTH/PTHrP receptor mRNAs as early as day 5 in culture, but very weak expression of osteocalcin mRNA on day 5. The levels of expression of these transcripts increased with time in culture. In situ hybridization demonstrated that PTH/PTHrP receptor and osteocalcin mRNAs were strongly expressed in nodules, but the former appeared much earlier than the latter. BMP-2 and BMP-4 mRNAs also appeared in the cells forming nodules. Immunohistochemical analysis demonstrated that cells expressing either BMP-2/4 or their receptors (BMPR-IA, BMPR-IB, and BMPR-II) preferentially appeared in nodules. These observations suggested that BMPs play an important role in the formation of mineralized bone nodules in an autocrine and/or paracrine fashion in these cells. The present study confirmed that osteoblasts enzymatically isolated from newborn rat calvariae are a useful tool for studying the differentiation process of osteoblasts.

Osteogenesis of Electrically Stimulated Bone Cells Mediated in Part by Calcium Ions

Culture selected and expanded osteoblastic cells may be able to be reintroduced into massive skeletal defects to accelerate cell mediated regeneration of skeletal tissues, especially in bone ingrowth in total joint replacement, fracture healing, and osteoporosis. In vitro osteogenic cell culture is a useful model in studying the mechanism of bone metabolism under direct current stimulation. In this study, an osteoblastlike cell line was isolated from newborn rat calvaria. The osteogenic processes of the in vitro cultured cell line were studied by cytochemical, electron microscopic, and energy dispersive x-ray analysis techniques that resembled those observed in membrane bone ossification centers in vivo. Direct current stimulation of 100 microA/cm2 accelerated greatly the proliferation and calcification of the in vitro cultured cells. Intracellular free calcium ion metabolism was measured with an Adherent Cell Analysis and Sorting Machine. Under direct current stimulation, intracellular free calcium ion concentration increased an average of 2.3 times of the original level, which may play a key role in regulating osteogenesis and bone metabolism.

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.

Ultrastructure and cytochemical detection of alkaline phosphatase in long-term cultures of osteoblast-like cells from rat calvaria.

Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation.

Effects and fate of human IGF-binding protein-5 in rat osteoblast cultures.

Osteoblasts prepared from calvaria of newborn rats produce insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP), IGFBP-5 was discovered in bone extracts. However, we could not detect IGFBP-5 in the medium of newborn rat osteoblasts, although we found mRNA expression. To find an explanation for this discrepancy and to learn more about the physiological role of IGFBP-5 in these cells, we studied the biological activity and the fate of recombinant human (rh) IGFBP-5 in comparison to rhIGFBP-3. IGFBP-5 but not IGFBP-3 stimulated thymidine incorporation into DNA both in the absence and presence of IGF-I. However, IGFBP-5 did not enhance uridine incorporation into RNA and glucose incorporation into glycogen. 125I-rhIGFBP-5 but not 125I-rhIGFBP-3 rapidly disappeared from the culture medium consistent with the observation that endogenous (rat) IGFBP-3 but not IGFBP-5 accumulated in the medium. However, intact 125I-labeled or unlabeled rhIGFBP-5 was associated with the cell-layer matrix, whereas IGFBP-5 fragments appeared in the medium. Trapping of IGFBP-5 in the cell layer matrix may enhance local availability of IGF.

Immediate early-gene induction in rat osteoblastic cells after mechanical deformation

Previous studies have reported changes in proliferation, second-messenger generation and activation of various cellular processes when osteoblasts have been mechanically stimulated. Recent evidence suggests that mechanical loading of long bones induces immediate early-gene expression. Immediate early genets, such as Egr-1, are genes that control cell proliferation, are involved in signal transduction, and share properties of transcription factors. The purpose of this study was to examine how mechanical deformation of osteoblasts affects cellular proliferation and Egr-1 mRNA induction. Osteoblasts were isolated from collagenase digestion of newborn rat calvariae, cultured in Petri dishes with flexible bottoms and then constantly stretched, producing an increase of 3 or 7% in surface area. A mechanical stretch of 7% for 0.5 or 24 h resulted in a doubling of [ 3H]thymidine incorporation, while 50 nM of epidermal growth factor resulted in a 4-fold increase. A time-course experiment showed that a 7% stretch induced Egr-1 mRNA as early as 15 min, reaching maximum levels by 60 min and returning to baseline by 120 min. Epidermal growth factor at 50 nM for 60 min resulted in a 3.8-fold Egr-1 mRNA induction. A mechanical stretch of 3% for 30 min also produced an Egr-1 mRNA induction. No induction of Egr-1 mRNA was seen in osteoblasts that were exposed to conditioned media from deformed cells. It is concluded that the immediate early gene, Egr-1, may be directly involved in the signal-transduction pathway of mechanical stimuli in osteoblasts.