How will Mahanarva spectabilis (Hemiptera: Cercopidae) Respond to Global Warming?

The aim of this study was to determine the favorable constant temperature range for Mahanarva spectabilis(Distant) (Hemiptera: Cercopidae) development as well as to generate geographic distribution maps of this insect pest for future climate scenarios. M. spectabilis eggs were reared on two host plants (Brachiaria ruziziensis(Germain and Edvard) and Pennisetum purpureum(Schumach)), with individual plants kept at temperatures of 16, 20, 24, 28, and 32 °C. Nymphal stage duration, nymphal survival, adult longevity, and egg production were recorded for each temperature*host plant combination. Using the favorable temperature ranges for M. spectabilis development, it was possible to generate geographic distribution. Nymphal survival was highest at 24.4 °C, with estimates of 44 and 8% on Pennisetum and Brachiaria, respectively. Nymphal stage duration was greater on Brachiaria than on Pennisetum at 20 and 24 °C but equal at 28 °C. Egg production was higher on Pennisetum at 24 and 28 °C than at 20 °C, and adult longevity on Pennisetum was higher at 28 °C than at 20 °C, whereas adult longevity at 24 °C did not differ from that at 20 and 28 °C. With these results, it was possible to predict a reduction in M. spectabilis densities in most regions of Brazil in future climate scenarios.

Wide localized solutions of the parity-time-symmetric nonautonomous nonlinear Schrödinger equation.

By using canonical transformations we obtain localized (in space) exact solutions of the nonlinear Schrödinger equation (NLSE) with cubic and quintic space and time modulated nonlinearities and in the presence of time-dependent and inhomogeneous external potentials and amplification or absorption (source or drain) coefficients. We obtain a class of wide localized exact solutions of NLSE in the presence of a number of non-Hermitian parity-time (PT)-symmetric external potentials, which are constituted by a mixing of external potentials and source or drain terms. The exact solutions found here can be applied to theoretical studies of ultrashort pulse propagation in optical fibers with focusing and defocusing nonlinearities. We show that, even in the presence of gain or loss terms, stable solutions can be found and that the PT symmetry is an important feature to guarantee the conservation of the average energy of the system.

Wide vector solitons in systems with time- and space-modulated nonlinearities.

In this work we apply point canonical transformations to solve some classes of two coupled nonautonomous nonlinear Schrödinger equations with specific cubic and quintic-time- and space-dependent-nonlinearities. The method applied here allows us to find a class of wide localized (in space) vector soliton solutions of nonautonomous nonlinear Schrödinger equations. The vector solitons found here can be applied to theoretical studies of Bose-condensed atoms in two different internal states and of ultrashort pulse propagation in optical fibers with focusing and defocusing nonlinearities.

Wide localized solitons in systems with time- and space-modulated nonlinearities.

In this work we apply point canonical transformations to solve some classes of nonautonomous, nonlinear Schrödinger equations, namely, those which possess specific cubic and quintic (time- and space-dependent) nonlinearities. In this way we generalize some procedures recently published which resort to an ansatz to the wave function and recover a time- and space-independent nonlinear equation which can be solved explicitly. The method applied here allows us to find wide localized (in space) soliton solutions to the nonautonomous, nonlinear Schrödinger equation. We also generalize the external potential which traps the system and the terms of the nonlinearities.

S12911-2 reduces bone loss induced by short-term immobilization in rats.

Skeletal immobilization induces trabecular bone loss resulting from increased bone resorption and decreased formation. In this study we determined the effect of S12911-2, a compound containing two atoms of stable strontium, on trabecular bone loss induced by short-term immobilization of hind limbs in rats. Male Sprague-Dawley rats were randomly allocated to six groups (n = 25 per group). At 9 weeks of age, five groups of rats had their right hind limb immobilized for 10 days, using a plaster cast, whereas one control group was not immobilized (CT). Four groups of immobilized rats were treated for 10 days with 50, 200, or 800 mg/kg/day of S12911-2 or the vehicle. One group of immobilized rats was pretreated (PT) for 2 weeks with 200 mg/kg/day of S12911-2 and continued treatment during the immobilization period. Immobilization of the right hind limb induced bone loss as shown by decreased ash weight (-12%) and bone mineral density measured by dual energy x-ray absorptiometry of the femur (-9%), and confirmed by decreased trabecular bone volume measured by histomorphometry of the tibial metaphysis (-25%). This effect was unrelated to alteration in long bone length and was associated with increased urinary hydroxyproline excretion (+12%), increased osteoclast surface and number (+27%), decreased mineral apposition rate (-30%), and tetracycline double labeled surface (-17%) in the immobilized tibia. S12911-2 (800 mg/kg/day) partially reduced bone loss, as shown by increased bone mineral density (+4%) and trabecular bone volume (+19%) compared with untreated immobilized rats. Furthermore, S12911-2 (800 mg/kg/day) increased bone density (+5%) in the contralateral nonimmobilized leg. These effects resulted from inhibition of bone resorption, as shown by normalization of urinary hydroxyproline excretion and histomorphometric indices of bone resorption. This study shows that the bone resorption induced by immobilization in rats can be suppressed by treatment with S12911-2, resulting in partial reduction of the bone loss.

Spaceflight inhibits bone formation independent of corticosteroid status in growing rats.

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (-12%; p < 0.05) associated with approximately 50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (-42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.

The Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation induces PKC-independent downregulation of FGFR-2 associated with premature calvaria osteoblast differentiation.

We recently showed that the Apert Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation causes premature osteoblast differentiation and increased subperiosteal calvaria bone matrix formation. To gain further insight into the cellular mechanisms involved in these effects, we examined the effects of the mutation on the expression of FGFRs in relation to cell proliferation and differentiation markers in vivo and in vitro, and we analyzed the underlying signaling pathways in mutant cells. Immunohistochemical analysis of the Apert calvaria suture showed that the Ser252Trp FGFR-2 mutation increased type 1 collagen, osteocalcin, and osteopontin expression in preosteoblasts compared to normal, whereas cell growth was not affected. The premature osteoblast differentiation induced by the mutation was associated with lower than normal FGFR-2 immunolabeling, whereas FGFR-1 and FGFR-3 levels were not decreased. Immunocytochemical analysis in osteoblasts isolated from Apert coronal suture showed that the Ser252Trp mutation induced constitutive downregulation of FGFR-2 in mutant cells. Western blot analysis of FGFRs in immortalized mutant osteoblastic cells confirmed that the mutation induced FGFR-2 downregulation. FGFR-2 mRNA levels were not altered in mutant cells, indicating that FGFR-2 downregulation resulted from receptor internalization rather than from changes in receptor mRNA. The signaling pathway involved in FGFR-2 downregulation was studied using specific inhibitors of FGF signaling molecules. The selective PKC inhibitor calphostin C markedly reduced FGFR-2 protein levels in mutant cells, in contrast to the p38 MAP kinase inhibitor SB 203580 or the Erk 1,2 MAP kinase inhibitor PD-98059, showing that PKC is involved in FGFR-2 regulation, but not in FGFR-2 downregulation in mutant cells. The results indicate that the premature osteoblast differentiation induced by the FGFR-2 Ser252Trp mutation is associated with a PKC-independent downregulation of FGFR-2 in human calvaria cells.

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.

Genomic insertion of the SV-40 large T oncogene in normal adult human trabecular osteoblastic cells induces cell growth without loss of the differentiated phenotype.

In the present study, we established a new adult human trabecular osteoblastic (AHTO) cell line, immortalized by SV-40 Large T (LT) oncogene. From seven proliferative colonies identified, we selected clone 7 with high alkaline phosphatase (ALP) activity for further analysis. AHTO-7 cells were able to grow for at least 8 months and 25 passages, with a doubling time of about 22 hours. Immunocytochemistry staining and RT-PCR analysis indicated that the extended life-span of AHTO-7 cells results in genomic insertion of SV-40 LT oncogene. The cells responded to PTH and PGE2 in terms of cAMP accumulation. The time course study, in the presence of 10(-8) M vitamin D3 (vit D3) showed a marked increase (fourfold) in ALP activity with a peak at day 3. Furthermore, in the presence of ascorbic acid (50 microg/ml) and inorganic phosphate (3 mM), AHTO-7 cells produced abundant calcified extracellular matrix, as examined by the von Kossa staining after 2 weeks of culture. Molecular analysis of mRNAs for phenotypic osteoblast markers at day 15 showed the expression of ALP, osteocalcin (OC), and collagen type I (Col I) mRNAs constitutively. Col I expression was inhibited by vit D3 and dexamethasone treatment. In contrast, treatment with vit D3 induced a marked increase of ALP and OC transcripts. Therefore, the immortalized AHTO-7 cells express osteoblast markers that are induced by calciotropic hormones, and constitute a suitable model for identifying specific osteoblastic genes and their regulation during human osteoblast differentiation.

Calcitonin gene-related peptide (CGRP) increases intracellular free Ca2+ concentrations but not cyclic AMP formation in CGRP receptor-positive osteosarcoma cells (OHS-4).

Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.

Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation.

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.

Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria.

Matrix and cell surface proteoglycans (PGs) may play important roles in the control of cellular actions of heparan-binding growth factors such as fibroblast growth factor (FGF) during chondrogenesis and osteogenesis. In this study, we used 4-methylumbelliferyl-beta-d-xyloside, an inhibitor of PG synthesis, and sodium chlorate, a competitive inhibitor of glycoconjugate sulfation, to determine the functional consequences of alterations of PG metabolism on osteogenesis and on FGF actions in neonatal rat condyle and calvaria in vitro. Biochemical analysis showed that beta-d-xyloside (1 mM) or chlorate (15 mM) treatment for 1-8 days inhibited cellular PG synthesis by 60-80% in condyle and calvaria, as evaluated by [35S]sulfate incorporation. Histochemistry and immunohistochemistry showed that the inhibition of PG synthesis by beta-d-xyloside resulted in reduced incorporation of chondroitin sulfate into cartilage and bone matrix. This was associated with a 75% reduction in cell growth in condyle, determined by DNA synthesis, and in collagenous matrix synthesis in condyle and calvaria, evaluated by tritiated proline incorporation and type I collagen immunohistochemistry. Morphological and quantitative autoradiographic analyses also showed that inhibition of PG synthesis by beta-d-xyloside blocked bone matrix formation by perichondral progenitor cells in condyles and by osteoblasts in calvaria. In addition, alteration of PG metabolism blocked the mitogenic response to rhFGF-2 in calvaria. The data show that functional proteoglycans are essential for osteogenesis and for the growth response to FGF-2 during osteogenic differentiation in vitro.

Isolation and characterization of human clonogenic osteoblast progenitors immunoselected from fetal bone marrow stroma using STRO-1 monoclonal antibody.

Osteoprogenitor cells present in human fetal bone marrow (BM) stroma have not been characterized. We used density gradient centrifugation, aggregation on binding lectin, and enrichment by magnetic activated cell sorting with STRO-1 antibody to isolate STRO-1+ cells from nonadherent human fetal BM stromal cells. Immunoselected STRO-1+ cells were immortalized using SV-40 large T antigen and a clone, F/STRO-1+ A, with weak alkaline phosphatase (ALP) activity was selected. The cloned cells proliferated rapidly but were not tumorigenic. Preconfluent F/STRO-1+ A cells showed immunoreactivity for osteopontin, alpha1(I) procollagen, and parathyroid hormone-related peptide, but not for the late osteoblast differentiation markers, osteocalcin (OC), or bone sialoprotein. However, differentiation of F/STRO-1+ A cells was induced by dexamethasone and 1,25-dihydroxyvitamin D3, as shown by increased ALP activity. In addition, osteogenesis occurred in F/STRO-1+ A cells cultured in three-dimentional aggregates, as assessed morphologically, histologically, and biochemically. Moreover, reverse transcription-polymerase chain reaction analysis showed that OC expression was silent in exponentially growing cells and occurred when cell-cell contacts were established in monolayer and in aggregates, showing induction of mature osteoblast phenotype by cell-cell contacts. Thus, clonal F/STRO-1+ A cells immunoselected from human fetal BM stroma display features of immature osteoprogenitor cells which can differentiate into mature osteogenic cells by cell-cell interactions or inducing agents. The generation by immunoselection of an immortalized clonogenic human fetal BM stroma-derived cell line which behaves like an osteoprogenitor cell provides a novel model system for identifying the signals required for the commitment of osteoprogenitors in the human fetal BM stroma.

Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis.

The effects of fibroblast growth factor-2 on human neonatal calvaria osteoblastic cells are differentiation stage specific.

Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1-100 ng/ml, 2-5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 microg/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1-28 days, or a transient rhFGF-2 treatment for 1-7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8-28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8-28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3-4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor beta2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation.

Effects of BMP-2 on osteoblastic cells and on skeletal growth and bone formation in unloaded rats.

A previous study showed that skeletal unloading induced by hindlimb suspension for 14 days in rats reduces osteoblastic cell proliferation, inhibits skeletal growth and bone formation and induces metaphyseal bone loss. This study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in this model. In vitro analysis showed that rhBMP-2 (25-100 ng/ml, 48-96 h) increased alkaline phosphatase activity, an early marker of osteoblast differentiation, in rat neonatal calvaria cells and adult marrow stromal cells, showing that rhBMP-2 induced the differentiation of osteoblast precursor cells in vitro. In contrast, rhBMP-2 did not increase rat calvaria or marrow stromal cell proliferation. Biochemical and histomorphometric analysis showed that systemic infusion with rhBMP-2 (2 microg/kg/day) in unloaded rats had no significant effect on serum osteocalcin levels and on histomorphometric indices of bone formation. Accordingly, rhBMP-2 infusion did not prevent the decreased skeletal growth, trabecular bone bone volume and bone mineral content induced by unloading. The present data indicate that, although rhBMP-2 stimulates osteoblastic cell differentiation, rhBMP-2 infusion is not effective in increasing bone formation and in preventing trabecular bone loss induced by unloading in rats.

Osteopontin is associated with bioprosthetic heart valve calcification in humans.

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreated porcine valves after implantation in humans. Thirty-eight glutaraldehyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-binding proteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted values, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted values and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these values are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.

Expression of the CT/CGRP gene and its regulation by dibutyryl cyclic adenosine monophosphate in human osteoblastic cells.

There is general agreement that calcitonin (CT) inhibits bone resorption by its effects on osteoclast function. CT was also found to have direct effects on osteoblast-like cells. In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)2, cAMP), a cAMP analog. We first detected by Northern blot hybridization the presence of CT and CGRP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells. In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP). OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry. Stimulation of OHS-4 cells with 1 mM (Bu)2 cAMP induced a significant increase in mRNA levels for CT (x 2.5) and CGRP (x 3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure. The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay. In addition, (Bu)2 cAMP increased OC (x 4) and ALP (x 3) mRNA levels in OHS-4 cells. These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu)2, cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells. The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)2 cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)2 cAMP is associated with enhanced expression of CT and CGRP in human osteoblastic cells.

Proliferation and differentiation of human trabecular osteoblastic cells on hydroxyapatite.

In order to evaluate whether human osteoblastic cells differentiate normally on hydroxyapatite, we have compared the adhesion, proliferation, and differentiation of human trabecular (HT) osteoblastic cells on synthetic-dense hydroxyapatite and on standard plastic culture. We show here that initial HT cell attachment was 4-fold lower on hydroxyapatite than on plastic after 4 h of culture, and that normal cell attachment on hydroxyapatite was restored after 18 h of culture. HT cell proliferation was similar on the two substrates at 2-8 days of culture, but was lower on hydroxyapatite compared to plastic after 15 and 28 days of culture, as evaluated by DNA synthesis or cell number. HT cells cultured on both substrates produced an abundant extracellular matrix which immunostained for Type I collagen. The levels of carboxyterminal propeptide of Type I procollagen (P1CP) in the medium were lower in HT cell cultures on hydroxyapatite than on plastic. In addition, (3H)-proline incorporation into matrix proteins and the mean thickness of matrix layers were 52% and 26% lower, respectively, on hydroxyapatite compared to plastic after 4 weeks of culture, indicating that the total collagenous matrix synthesized by HT cells was lower on hydroxyapatite. However, (3H)-proline and calcium uptake expressed per cell was higher on hydroxyapatite than on plastic. The results show that human osteoblastic cells attach, proliferate, and differentiate on dense hydroxyapatite with a sequence similar to that of plastic. However, the growth of human osteoblastic cells is lower on hydroxyapatite in long-term culture, which results in a reduced amount of extracellular matrix, although matrix production per cell may be increased.