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Timeline Challenge (https://timelinechallenge.hudsonalpha.org) engages students in a Claim-Evidence-Reasoning (CER) task while they investigate critical advances in agriculture and biotechnology. This freely available digital resource scaffolds students’ investigation of biotechnology by presenting a current and ongoing agricultural problem. The Timeline Challenge utilizes the Progress of Science biotechnology timeline as the backdrop for a series of agriculture-based challenges, such as producing meatier chickens, non-browning apples, or perennial wheat. This web-based module allows students to choose a specific biotech tool to address the challenge and then compare their plan to ongoing modern research related to the challenge. Along the way, students are presented with a real-world complication to encourage thinking about the problem from multiple perspectives.

Life science classrooms often emphasize the exception to the rule when it comes to teaching genetics, focusing heavily on rare single-gene and Mendelian traits. By contrast, the vast majority of human traits and diseases are caused by more complicated interactions between genetic and environmental factors. Research indicates that students have a deterministic view of genetics, generalize Mendelian inheritance patterns to all traits, and have unrealistic expectations of genetic technologies. The challenge lies in how to help students analyze complex disease risk with a lack of curriculum materials. Providing open access to both content resources and an engaging storyline can be achieved using a “serious game” model. “Touching Triton” was developed as a serious game in which students are asked to analyze data from a medical record, family history, and genomic report in order to develop an overall lifetime risk estimate of six common, complex diseases. Evaluation of student performance shows significant learning gains in key content areas along with a high level of engagement.

Recent advances in research and biotechnology are making genetics and genomics increasingly relevant to the lives and health of the general public. For the public to make informed healthcare and public policy decisions relating to genetic information, there is a need for increased genetic literacy. Biotech 101 is a free, short-course for the local community introducing participants to topics in genetics, genomics, and biotechnology, created at the HudsonAlpha Institute for Biotechnology. This study evaluated the effectiveness of Biotech 101 in increasing the genetic literacy of program participants through pre-and-post surveys. Genetic literacy was measured through increases in self-perceived knowledge for each content area covered through the course and the self-reported impact the course had on various aspects of participants’ lives. Three hundred ninety-two individuals attended Biotech 101 during the first three course offerings. Participants reported a significant increase in self-perceived knowledge for each content area (p

Craniosynostosis is a heterogeneous disorder characterized by premature fusion of the skull bone sutures. To evaluate the pathogenesis of premature cranial suture ossification in craniosynostosis, we have evaluated the histologic indices of bone formation and the characteristics of osteoblastic cells derived from normal and affected cranial sutures in 47 infants and children, aged 3-18 months, with nonsyndromic craniosynostosis. The histomorphometric analysis of normal and fused sutures showed an age-related decline in the extent of endosteal bone surface covered with osteoid and osteoblasts during postnatal suture ossification. Bone formation was 20-50% higher at 3-6 months of age in fused sutures compared with normal sutures in the same patients. Cells derived from normal and fused sutures displayed characteristics of the osteoblast phenotype in culture. Analysis of [3H]thymidine incorporation into DNA from 1-14 days of culture showed an age-related decrease in osteoblastic cell growth in both normal and affected sutures. The proliferation of osteoblastic cells isolated from fused sutures was similar at all ages to that of cells isolated from normal sutures in the same patients. In contrast, alkaline phosphatase activity and osteocalcin production by osteoblastic cells cultured in basal conditions and after stimulation with 1,25-dihydroxyvitamin D (1,25[OH]2D3), were 53-74% higher in fused sutures compared with cells isolated from normal sutures in the same patients. The results indicate that bone formation activity at the suture site is locally increased in craniosynostosis, and this disorder is associated with increased in vitro parameters of osteoblastic cell differentiation, suggesting that an increased maturation of osteoblastic cells at the site of the suture leads to the premature ossification in nonsyndromic craniosynostosis.

Adjuvant polyarthritis (AP) in rats is known to result in extensive bone loss. This study investigates the mechanisms responsible for the early trabecular osteopenia evaluated at a single point in time--2 weeks after adjuvant injection--in the hindpaw of female Lewis rats using biochemical and histomorphometric methods. At this early point in time, the inflammation was generalized (inflammatory score, 20; albumin/globulin, -80% versus control). Histomorphometric analysis of the noninjected femur showed that the trabecular bone volume was significantly decreased (-28% versus control) in both proximal and distal parts, and the femur growth rate was unaffected. The trabecular osteopenia was associated with a 90% decrease in osteoid surface and a concomitant thinning (-19%) of the trabeculae. Both the double-fluorescence-labeled surface and the osteoblast surface were also markedly decreased (-75%). In addition, the mineral apposition rate was reduced (-50%) and the bone formation rate was decreased by as much as 90%. The trabecular bone volume was decreased in relation with the extent of double-fluorescence labeling (r = 0.38, p = 0.03) and bone formation rate (r = 0.42, p = 0.01), suggesting that the generalized osteopenia resulted from the reduced bone formation. This was associated with a 26% reduction in plasma osteocalcin. Neither the osteoclast surface nor the number of osteoclasts was consistently affected. However, urinary hydroxyproline was increased by 100-200%, which likely reflected the cartilage and bone destruction at the site of injection. The present data show that the early extensive osteopenia observed 2 weeks after AP induction in rats results from defective bone formation with unchanged bone resorption. The role of cytokines in such an inhibitory effect on bone formation remains to be determined.

SUMMARY Three different inclusion levels of a moisture-mold inhibitor mixture (0, 1, and 2%) were added to corn- and soybean-based diets at the mixer as top-dressing additions, and diets were processed at a commercial and a pilot milling facility. Diets were then evaluated according to feed manufacturing variables, pellet quality, broiler performance, and TMEn. Moisturemold inhibitor additions resulted in an increase in moisture percentages directly after manufacture. After a 2-d storage period, no differences in moisture percentage were detected in diets manufactured at the pilot mill; however, this was not true for diets manufactured commercially. Pellet durability was increased with moisture-mold inhibitor inclusion for the feed processed at the commercial mill, but not for the feed processed at the pilot mill. Relative electrical energy use decreased numerically with increasing levels of moisture-mold inhibitor added to feed processed at the pilot mill. The 1 and 2% diets manufactured commercially resulted in improvements in BW, live weight gain, and FCR, but not the diets manufactured at the pilot mill. There were no differences in TMEn values among treatments, regardless of the manufacturing location. These findings indicate that moisture-mold inhibitor addition has the potential to improve broiler performance and decrease production costs, but improvement depends on the milling technique.

SUMMARY The use of subtherapeutic levels of antibiotics in poultry feed improves performance and morbidity in broiler chickens. However, consumer pressure related to the potential development of antibiotic-resistant bacteria has resulted in the development of nonantibiotic feed additives that may also improve broiler performance. Essential oils, organic acids, and phytogenic compounds enhance production of gastric secretions, stimulate blood circulation, and reduce levels of pathogenic bacteria. The objective of the current study was to assess the use of Biostrong 505+ and Biostrong 510 as natural growth promoters in broiler chickens. Assessment was based on the performance and carcass quality of broilers fed either a maximum-yield or a least-cost commercial broiler diet. The maximum-yield diet improved broiler performance and carcass quality. Biostrong 505+ can be used to improve feed conversion and breast yield when incorporated into diets devoid of antibiotics. This improvement in feed conversion and breast yield was accentuated when Biostrong 505+ was used in conjunction with a maximumyield diet. Biostrong 510 improved feed conversion when used in poultry diets containing antibiotics.

Feed currently constitutes 60 to 65% of the total cost of broiler production. Pellets are the primary feed form for commercially reared broilers. Understanding how to optimize pellet quality through precision thermo-mechanical processing may impact broiler performance and nutrient availability, and thus cost of production. Steam conditioning represents a manipulable thermo-mechanical processing variable. Corn-soybean meal-based diets were conditioned with 1 of 4 steam pressure × temperature combinations: 138 kPa at 82.2°C, 138 kPa at 93.3°C, 552 kPa at 82.2°C, or 552 kPa at 93.3°C. High steam pressure and temperature conditioning were shown to increase pellet quality. Three additional diets were prepared: unprocessed mash, the 552 kPa/93.3°C diet reground to mash, and a 50:50 combination of pellets and mash produced from the 552 kPa/93.3°C treatment to simulate a high fine percentage diet. All diets were fed to Cobb 500 broilers from 21 to 38 d, and nutrient availability was determined with Single Comb White Leghorn roosters. Broilers fed pellets conditioned with high steam temperature demonstrated decreased feed intake and feed conversion ratio. Amino acid and energy availability were not affected by variations in steam conditioning.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:01:27Z No. of bitstreams: 1 S0103-97332008000200002.pdf: 156750 bytes, checksum: b4be8725fa80d6d7a71b633ad76d14d8 (MD5) Made available in DSpace on 2013-08-22T19:01:27Z (GMT). No. of bitstreams: 1 S0103-97332008000200002.pdf: 156750 bytes, checksum: b4be8725fa80d6d7a71b633ad76d14d8 (MD5) Previous issue date: 2008-06-01 Made available in DSpace on 2013-09-30T20:01:28Z (GMT). No. of bitstreams: 2 S0103-97332008000200002.pdf: 156750 bytes, checksum: b4be8725fa80d6d7a71b633ad76d14d8 (MD5) S0103-97332008000200002.pdf.txt: 31860 bytes, checksum: 2718bea668b50288d09bc848b080a2a0 (MD5) Previous issue date: 2008-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:27:40Z No. of bitstreams: 2 S0103-97332008000200002.pdf: 156750 bytes, checksum: b4be8725fa80d6d7a71b633ad76d14d8 (MD5) S0103-97332008000200002.pdf.txt: 31860 bytes, checksum: 2718bea668b50288d09bc848b080a2a0 (MD5) Made available in DSpace on 2014-05-20T13:27:40Z (GMT). No. of bitstreams: 2 S0103-97332008000200002.pdf: 156750 bytes, checksum: b4be8725fa80d6d7a71b633ad76d14d8 (MD5) S0103-97332008000200002.pdf.txt: 31860 bytes, checksum: 2718bea668b50288d09bc848b080a2a0 (MD5) Previous issue date: 2008-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) We study the self-energy of a model with a tri-linear coupling among spinless fields on a manifold with one spatial dimension compactified. The model is considered to be composed by a quantized scalar field interacting with a classical scalar one. In order to improve the compactification we take the quantum field satisfying quasi periodic boundary conditions, which interpolates continuously the periodic and anti-periodic conditions, whilst the background field satisfies periodic boundary conditions. All the calculations are performed in Euclidean coordinates. Universidade Federal de Itajubá ICE UNESP Departamento de Física e Química UNESP Departamento de Física e Química

Poultry diets are formulated based on requirements of birds reared without access to pasture. Consumer demand for organic and free-range poultry has resulted in an increase in the use of certified organic feed. Organic poultry may have the opportunity to utilize nutrients found in forage. The addition of exogenous enzymes and manipulation of cecal microbial populations may affect the utilization of forage nutrients. The objectives of this study were as follows: 1) to determine AMEn and TMEn of organic grower diets with or without nonstarch polysaccharide enzyme supplementation and compare these values to a conventional grower diet, 2) to determine AMEn, TMEn, and true amino acid digestibility (TAAD) of forage samples with or without nonstarch polysaccharide enzyme supplementation, and 3) to determine if cecum modification affects forage AMEn, TMEn, and TAAD. The experiment was conducted using cecectomized, intact, and intact grass-fed roosters as models. Enzyme supplementation increased AMEn and TMEn values for organic feeds but not forage. Bird type had no effect on AMEn and TMEn for any treatment. The TAAD of forage was not significantly affected by enzyme supplementation. However, trends in bird type effect were observed for several amino acids.

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.

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.

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.

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.

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.

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.

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.

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.

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 afte r4ho fculture, 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 hydroxy- apatite compared to plastic after 15 and 28 days of culture, as evaluated by DNA synthesis or cell number. HT cells cul- tured on both substrates produced an abundant extracellu- lar 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, ( 3 H)-proline in- corporation into matrix proteins and the mean thickness of matrix layers were 52% and 26% lower, respectively, on hy- droxyapatite compared to plastic after 4 weeks of culture, indicating that the total collagenous matrix synthesized by HT cells was lower on hydroxyapatite. However, ( 3 H)- 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 plas- tic. 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. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 508-516, 1997.

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.

Aldosterone significantly enhanced the proliferation of osteoblastic cells from rat calvaria, and this effect was inhibited by RU 26752 and ZK 91587, two antagonists specific to the mineralocorticoid receptor (MCR). In addition, aldosterone inhibited the activity of alkaline phosphatase, a marker of the osteoblastic phenotype, and this effect was also reversed by RU 26752. Cytoplasmic staining of MCR was observed in rat calvaria osteoblasts incubated with a specific polyclonal antiserum raised against rat kidney MCR. This anti-MCR immunoglobulin G immunoprecipitated and macroaggregated the MCR-[3H]RU 26752 complex in osteoblastic cytosol. A single 98-kDa band was observed when osteoblastic cytosol was analyzed by Western blotting with anti-MCR serum. The 98-kDa band was also obtained after autoradiography of irradiated osteoblastic cytosol-[3H]R 5020 complex, and this was abolished in the presence of RU 26752. A p26MR probe, specific to COOH-terminal end of MCR, hybridized with the predicted product after amplification of total cell RNA by polymerase chain reaction technique. Furthermore, hybridization of poly(A)+ mRNA from at calvaria osteoblastic cells with the p26MR probe revealed a major band of approximately 4.2 kb. Collectively, our studies demonstrate the existence of a functional MCR in rat calvaria osteoblasts.

Low doses of strontium and fluoride were shown to increase bone formation and trabecular bone density in rodents. To assess whether strontium or fluoride affect the quality of the mineral at doses known to increase bone density, we have determined the effects of low doses of strontium and fluoride on bone formation and bone mineral characteristics in rats. Adult rats were given strontium alone (0.20%), fluoride alone (1 mg/kg per day), or the combined treatment for 8 weeks. Strontium levels in serum and femur were similar in groups treated with strontium alone or in combination, being about 5% of calcium levels. Biochemical and neutron activation analyses in femur showed that calcium and magnesium contents did not differ in the four groups of rats, suggesting that strontium was incorporated in the apatite lattice of the bone mineral in the strontium-treated rats. The mineralized bone volume was significantly increased by 17 % in the strontium-treated group, by 20% in the fluoride-treated group, and by 19% in rats given with the combined treatment. This was associated with increased osteoid surface, osteoblast surface, and double tetracycline labeled surfaces in the strontium-treated and fluoride-treated groups, showing that the number of bone forming sites was increased. However, the mineral apposition rate, the osteoid thickness, and the mineralization lag time were similar in controls and treated groups, reflecting the lack of deleterious effects of low doses of strontium and fluoride on bone mineralization. The density fractionation analysis measured in the femur also showed that neither strontium, nor fluoride at the low doses used, significantly altered the mineralization profile. The results indicate that treatment with low doses of strontium or fluoride increase the number of bone forming sites and vertebral bone volume in rats, but does not have detectable adverse effects on the mineral profile, bone mineral chemistry or bone matrix mineralization.

We investigated the effect of recombinant human transforming growth factor beta 2 (rhTGF-beta 2) administration on trabecular bone loss induced by unloading in rats. Hind limb suspension for 14 d inhibited bone formation and induced osteopenia as shown by decreased bone volume, calcium and protein contents in long bone metaphysis. Systemic infusion of rhTFG-beta 2 (2 micrograms/kg per day) maintained normal bone formation rate, and prevented the decrease in bone volume, bone mineral content, trabecular thickness and number induced by unloading. In vitro analysis of tibial marrow stromal cells showed that rhTGF-beta 2 infusion in unloaded rats increased the proliferation of osteoblast precursor cells, but did not affect alkaline phosphatase activity or osteocalcin production. Northern blot analysis of RNA extracted from the femoral metaphysis showed that rhTGF-beta 2 infusion in unloaded rats increased steady-state levels of type I collagen mRNA but not alkaline phosphatase mRNA levels. rhTGF-beta 2 infusion at the dose used had no effect on metaphyseal bone volume and formation, osteoblast proliferation or collagen expression in control rats. The results show that systemic administration of rhTGF-beta 2 enhances osteoblast precursor cell proliferation and type I collagen expression by osteoblasts, and prevents the impaired bone formation and osteopenia induced by unloading.

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of β-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by fivefold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by ( 3 H)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation. The clonal C1 cells represent therefore, an original model of osteoblast differentiation where the high frequency and synchrony of nodule mineralization make it possible to follow the expression of bone-related genes along the kinetics of osteogenesis in vitro.

The effects of skeletal unloading on osteoblastic cells were evaluated in tail-suspended rats. Hindlimb elevation for 14 days induced osteopenia, decreased histomorphometric indexes of bone formation in tibial metaphysis, and reduced plasma osteocalcin and alkaline phosphatase (ALP) levels compared with controls. The in vitro proliferation of osteoblastic cells isolated from the endosteal bone surface of suspended tibias was decreased by 42 and 31% at 2 and 4 days of culture, respectively, compared with controls, as shown by [3H]thymidine labeling and cell number. The proliferation of ALP-positive marrow stromal cells was also decreased by 20–24% at 1 and 2 days of culture. However, ALP activity in bone-derived cells and marrow stromal cells was not different in unloaded and control rats, and the number of bone cells synthesizing osteocalcin, osteonectin, and type I or type III collagen was identical in the two groups. The results indicate that the inhibition of bone formation induced by skeletal unloading is related to a decreased proliferation of putative osteoblast precursor cells present along the endosteal bone surface and in the marrow stroma.

To determine the osteoblastic dysfunction that may be involved in the pathophysiology of osteoporosis in men we have compared histomorphometric indices of bone formation with in vitro characteristics of osteoblastic cells isolated from the trabecular bone surface in 23 untreated men with eugonadal osteoporosis. In most patients (n = 14), trabecular bone loss resulted from decreased bone formation evidenced by a lower than normal osteoblast surface, double tetracycline labeled surface, bone formation rate, and mean wall thickness. In these patients, DNA synthesis by cultured osteoblastic cells was altered. The peak of [3H]thymidine incorporation into DNA, the maximal DNA synthesis, and the area under the curve of cell proliferation were lower than the values in normal bone cells from age-matched controls. Parameters of bone cell growth were decreased in correlation with the extent of actively bone forming surfaces. By contrast, in patients (n = 9) with normal histomorphometric indices of bone formation, bone cell proliferation in vitro was not different from normal. Parameters of osteoblastic differentiation in vitro such as osteocalcin production and alkaline phosphatase activity were normal in the two groups of patients. This study shows that the trabecular bone loss resulting from defective bone formation in eugonadal osteoporotic men is associated with a lower than normal proliferative capacity of osteoblastic cells lining the trabecular bone surface.

The Green function for a spin-1/2 charged particle in the presence of an external plane wave electromagnetic field is calculated by algebraic techniques in terms of the free-particle Green function.

We investigate the analytic properties of finite-temperature self-energies of bosons interacting with fermions at one-loop order. A simple boson-fermion model was chosen due to its interesting features of having two distinct couplings of bosons with fermions. This leads to a quite different analytic behavior of the bosons self-energies as the external momentum $K^{\mu}=(k^{0},\vec{k})$ approaches zero in the two possible limits. It is shown that the plasmon and Debye masses are consistently obtained at the pole of the corrected propagator even when the self-energy is analytic at the origin in the frequency-momentum space., Comment: 11 pages, no figures, revtex, typos corrected, two references included, to be published in Phys. Rev. D

Summary 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 μg/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

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.

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.

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.

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.

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.

Silicon is known to ensure an essential role in the formation of cross-links between collagen and proteoglycans during bone growth. In this study, we have evaluated the short-term effects of a preventive treatment with silanol, a soluble organic silicon (Si), on trabecular bone in mature ovariectomized rats. Three-month-old rats were sham-operated (sham) or were ovariectomized (OVX) and treated with 10 micrograms/kg/day of 17 beta estradiol (E2), or with 0.1 mg Si/kg/day or 1.0 mg Si/kg/day of silanol for 1 month. Plasma alkaline phosphatase and osteocalcin levels were increased by 50% in OVX rats compared with sham rats and were corrected by E2 but not by silanol treatment. The trabecular bone volume measured at the tibial metaphysis was decreased by 48%, and histomorphometric indices of bone resorption and formation were increased in OVX rats compared with sham, and these parameters were corrected by E2 treatment. Treatment of OVX rats with silanol decreased the osteoclast surface by 31% and the number of osteoclasts by 20%. The mineral apposition rate, the bone formation rate, and the osteoblast surface at the tibia metaphyseal area were increased by 30% at the higher dose of silanol compared with OVX rats. In contrast, silanol treatment had no effect on the periosteal apposition rate. The reduction of the metaphyseal bone resorption and the increased bone formation induced by silanol resulted in a slight improvement of the trabecular bone volume (+14%) compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the role of cytokines produced by osteoblasts in the pathophysiology of bone lesions in postmenopausal osteoporosis (PMOP), we have determined by RIA and immunoradiometric assays the levels of prostaglandin E2 (PGE2), interleukin-1 beta (IL-1), tumor necrosis factor-alpha (TNF alpha), and IL-6 released by cultured bone surface-derived osteoblastic (OB) cells isolated from 24 untreated PMOP women with high, low, or normal bone turnover on bone biopsy. OB cells isolated from patients with high bone formation had a 2-fold increased proliferation rate in vitro compared to OB cells from patients with normal or low bone formation or OB cells from age-matched controls. The spontaneous in vitro production per cell protein of PGE2, IL-1, and TNF alpha, but not of IL-6, was 2- to 3-fold lower in rapidly proliferating OB cells isolated from PMOP patients with high bone formation compared to OB cells from patients with normal or low proliferation or control cells. Treatment with 10 nmol/L 1,25-dihydroxyvitamin D (48 h) increased PGE2 levels to normal values in OB cells with a high proliferation rate, but decreased PGE2 production in cells with low proliferation and in control cells, suggesting that the release of PGE2 was dependent on the stage of maturation of OB cells. Significant correlations were found between IL-1 and TNF alpha (r = 0.87; P < 0.001), IL-1 and PGE2 (r = 0.46; P < 0.05), IL-6 and IL-1 (r = 0.39; P < 0.05), and IL-6 and TNF alpha (r = 0.49; P < 0.05), suggesting that the production of these cytokines was under reciprocal control. The results indicate that the in vitro production of PGE2, IL-1, and TNF alpha, but not IL-6, by OB cells isolated from patients with PMOP is related to the proliferation rate of these cells and the rate of bone formation. The variable production of cytokines by OB cells may contribute to the histological heterogeneity of bone formation in PMOP.

Trabecular bone loss in estrogen deficiency is associated with enhanced bone resorption with a smaller increase in bone formation. We previously reported that low doses of strontium can increase trabecular bone volume in rodents by affecting bone resorption and formation. In this study we determined the effect of a new divalent strontium salt (S12911) on bone loss induced by E2 deficiency. Sprague-Dawley female rats (230 g, n = 15-25 per group) were sham operated or ovariectomized (OVX) and treated with 17 beta-estradiol (E2, 10 micrograms/kg/day, sc) or S12911 by gavage at the dose of 77, 154, or 308 mg/kg/day or the vehicle. Treatment for 60 days with S12911 resulted in a dose-dependent increase in plasma, urine, and bone strontium concentrations without any deleterious effect on total or skeletal growth. OVX rats were osteopenic compared to sham rats as shown by decreased femoral dry bone weight and mineral content measured on bone ash and by DXA. Treatment of OVX rats with S12911 prevented bone loss as bone ash and bone mineral content were restored to the values in sham rats. Trabecular bone volume measured by histomorphometry on the tibial metaphysis was decreased by 46% in OVX rats and was corrected by E2. Treatment of OVX rats with S12911 increased the trabecular bone volume by 30-36%. Histomorphometric indices of bone resorption (osteoclast surface and number) were increased in OVX rats and were reduced by S12911 to the levels in sham rats.(ABSTRACT TRUNCATED AT 250 WORDS)

We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by ( 3 H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48–72 hours). In both species, the effect of PDGF (25ng/ml) was lower than the mitogenic effect of 10%FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH) 2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH) 2 D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.

The effects of mouse epidermal growth factor (EGF) on bone formation and resorption were examined in male mice. EGF administration (2-200 ng.g-1.day-1 ip for 7 days) induced a dose-dependent rise in plasma EGF levels that remained within physiological range. Histomorphometric analysis of caudal vertebrae showed that EGF (20 and 200 ng.g-1.day-1) reduced the endosteal matrix and mineral appositional rates after 5 days of treatment as measured by double [3H]proline labeling and double tetracycline labeling, respectively. This effect was transitory and was not observed after 7 days of EGF administration. EGF administered for 7 days induced a dose-dependent increase in the periosteal osteoblastic and tetracycline double-labeled surfaces. At high dosage (200 ng.g-1.day-1) EGF administration increased the osteoclastic surface and the number of acid phosphatase-stained osteoclasts, although plasma calcium remained normal. The results show that EGF administration at physiological doses induces distinct effects on endosteal and periosteal bone formation and that the effects are dependent on EGF dosage and duration of treatment. This study indicates that EGF at physiological dosage stimulates periosteal bone formation and increases endosteal bone resorption in the growing mouse.

MC-903 is a novel vitamin D analogue which has been shown to promote epidermal cell differentiation but is 100 times less active than 1,25 dihydroxyvitamin D 3 (1,25(OH) 2 D) in causing hypercalcemia. In order to determine the activity of this compound on bone cells, we have compared the effects of MC-903 and 1,25 dihydroxyvitamin D 3 (1,25(OH) 2 D) on parameters of cell proliferation and differentiation in cultured normal human osteoblastic cells derived by migration from trabecular bone fragments. Dose response curves showed that MC-903 was 10 to 100 times less effective than 1,25(OH) 2 D in stimulating the synthesis of the osteoblast specific protein osteocalcin by human bone cells depending on the basal osteocalcin production. In cells showing high basal osteocalcin synthesis, 1,25(OH) 2 D (10 −8 M) was 2- to 3-fold more potent than MC-903 (10 −8 M) in inducing osteocalcin from 48 to 96 h of treatment. The greater activity of 1,25(OH) 2 D over MC-903 was observed in human bone cell cultures with elevated basal osteocalcin levels, indicating that the response to 1,25(OH) 2 D but not to MC-903 was amplified in cells with the higher osteoblastic characteristics. The effects of MC-903 and 1,25(OH) 2 D on alkaline phosphatase activity were not markedly different. Transforming Growth Factor-β (TGFβ) (0.5 ng/mL, 48 h) was found to completely suppress the osteocalcin synthesis induced by 1,25(OH) 2 D (10 −8 and 10 −9 M), whereas the MC-903-induced osteocalcin synthesis was not affected, suggesting a negative interaction between TGFβ and 1,25(OH) 2 D but not MC-903 on osteocalcin synthesis. In contrast, the stimulatory effect of TGFβ (0.5 ng/mL, 48 h) on alkaline phosphatase activity in quiescent human osteoblastic cells was suppressed by both MC-903 and 1,25(OH) 2 D. The two vitamin D derivatives induced similar effects on DNA synthesis in human bone cells and did not affect the stimulation of [ 3 H] thymidine incorporation into DNA induced by TGFβ (0.5 ng/mL). The results show that 1,25(OH) 2 D and MC-903 exert equipotent effects on DNA synthesis and alkaline phosphatase activity but distinct effects on the synthesis of the bone specific protein osteocalcin. The finding that MC-903 has less potent effects than 1,25(OH) 2 D on a specific osteoblastic function further indicates that this compound may be useful in the treatment of psoriatic lesions.