Biomedical subjects

K Klaushofer

Publications and source records attributed to K Klaushofer.

At least 55 records · Page 3Linked to original sources

PubMed1997

Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs.

Fluoride stimulates bone formation, whereas bisphosphonates reduce bone resorption. In clinical trials, both treatments increase bone density, although sodium fluoride (NaF) increases and alendronate (bisphosphonate, ALN) decreases bone turnover. In a comparative study using minipigs an inverse correlation has been reported between bone turnover and elastic modulus. Small-angle X-ray scattering (SAXS) measurements of these bones revealed no structural deterioration of the collagen/mineral composite at the nanometer range for ALN-treated vertebra, whereas a slight increase of the average thickness of the mineral crystals as well as changes of the structure of the collagen/mineral composite were found in the bones of NaF-treated animals. In this study we used quantitative backscattered electron imaging (qBSE) to investigate the cancellous bones from ribs of minipigs treated with vehicle, NaF, or ALN. This method provides information on the local mineral concentration in the micrometer range. Mineralization spectra were obtained from each treatment group, and statistically significant differences between ALN and controls were found for the peak position, the peak height, the peak width, and the average calcium (Ca) concentration of the mineral distribution. The results reveal that the cancellous bone matrix was more uniformly mineralized after ALN treatment. The reduced bone turnover induced by ALN, documented histomorphometrically could be at the origin of this phenomenon. No significant differences were detected between NaF and control. Together with the earlier SAXS data these results may explain in part the increase in bone density and the improvement of biomechanical properties observed after ALN treatment in animals and in osteoporotic patients.

Alendronate↗

PubMed1996

Bone mineralization in an osteogenesis imperfecta mouse model studied by small-angle x-ray scattering.

We have studied the size and orientation of mineral crystals in cortical bone of oim/oim mice, which are known to produce only alpha 1(I) collagen homotrimers and which may serve as a model for human osteogenesis imperfecta. Long bones (femur and tibia) from young (5 wk old) oim/oim mice and from unaffected heterozygous counterparts were investigated by small-angle x-ray scattering (SAXS), which is sensitive to structures smaller than 50 nm. Mineral crystals were compared in terms of their thickness and their alignment with respect to the long bone axis. While electron microscopic tomography has recently shown the existence of large mineral blocks (with all dimensions typically exceeding 50 nm) in mineralized tendons of oim/oim mice, SAXS revealed a family of thin, possibly needle-like, crystals in cortical bone. These crystals were similar in shape to those observed previously in normal mice, but they were thinner and less well aligned in oim/oim mice relative to heterozygotes. Moreover, the crystal thickness and their alignment with the bone axis were more variable in oim/oim bone, with a close correlation (r = 0.94, P < 0.001) between the two parameters. The presence of smaller crystals with more variable alignment in corticalis of oim/oim mice may contribute to the brittleness of their bone, similar to that of human osteogenesis imperfecta.

Animals↗

PubMed1996

Effects of sodium fluoride and alendronate on the bone mineral in minipigs: a small-angle X-ray scattering and backscattered electron imaging study.

Sodium fluoride (NaF), which stimulates bone formation, and bisphosphonates, which reduce bone resorption, are both used in the treatment of osteoporosis, and are binding to bone mineral. In this study, using small-angle X-ray scattering and backscattered electron imaging, we analyzed the bone mineral in the vertebrae of minipigs treated with fluoride, with the bisphosphonate alendronate (ALN), or with vehicle. All specimens were investigated blindly. A slight increase in the average thickness of the mineral crystals as well as changes in the structure of the mineral/collagen composite were found in the case of fluoride-treated animals. No differences were found between ALN-treated animals and controls. The changes produced by fluoride are in the same direction as seen in bones from patients treated with NaF, albeit much smaller. They also correlate quantitatively with the reduction in biomechanical properties of bone in fluoride-treated minipigs found in an earlier study with the same animals. These findings suggest that small changes in the structure of the mineral/collagen composite in bone may considerably affect its biomechanical properties. It also emphasizes the delicate balance between the increase of bone mass and deterioration of bone material properties for the effect of fluoride on the biomechanical properties of bone.

Alendronate↗

PubMed1996

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1).

We investigated the effects of thyroid hormone treatment on morphology, growth behaviour, and cytoskeletal structures of long-term cultured MC3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T3-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T3-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending across the entire cytoplasm. The actin pattern of T3-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T3 treatment. We conclude from these data that the known specific cellular responses to T3 treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.

3T3 Cells↗

PubMed1996

Prostacyclin (PGI2): a potential mediator of c-fos expression induced by hydrostatic pressure in osteoblastic cells.

Application of compressive forces to osteoblastic cells is known to cause specific cellular responses. We report that hydrostatic pressure increased c-fos mRNA expression in MC3T3-E1 cells after 15, 30 and 60 min. This effect was absent when 5 x 10(-7) mol L-1 indomethacin, an inhibitor of prostaglandin synthesis, was present in the culture medium during pressurization. Using radioimmunoassay, a significant increase in the concentrations of 6-keto-PGF1 alpha, the stable conversion product of prostacyclin (PGI2), in the conditioned medium of pressurized cells, was measured after 60 min. In contrast, PGE2 levels were not significantly changed and we therefore assume that under these experimental conditions PGE2 is not responsible for the transduction of the hydrostatic force. However, we also found that PGE2 has the capacity to induce c-fos mRNA in MC3T3-E1 cells. Furthermore, we show for the first time that the stable prostacyclin analogue, Iloprost-Trometamol (Ilomedin), is a potent activator of c-fos gene transcription. Our data suggest that prostacyclin is a likely candidate in mediating the effect of hydrostatic compressive stress on bone cells by regulating the level of c-fos mRNA, a member of the activator protein (AP-1) complex and potent regulator of osteoblastic proliferation and differentiation.

3T3 Cells↗

PubMed1996

Thyroid hormone and retinoic acid induce the synthesis of insulin-like growth factor-binding protein-4 in mouse osteoblastic cells.

Thyroid hormone (T3) is a known regulator of the transcription rate of specific genes. By subtractive hybridization of T2-treated osteoblastic cells, differentially expressed messenger RNAs (mRNAs) were enriched in the form of double stranded complementary DNA (cDNA) fragments. Sequencing of a differentially expressed cDNA that detects a 2.6-kilobase mRNA in Northern blots revealed to homology in the EMBL-Genebank data bases. A mouse genomic library was screened, and the isolated genomic DNA was identified as part of the insulin-like growth factor-binding protein-4 (IGFBP-4) gene including the 3'-untranslated region to which the cloned cDNA fragment was mapped by sequencing. We observed an up-regulation of the 2.6-kilobase IGFBP-4 mRNA transcript in the presence of T3 or retinoic acid. The induction of the IGFBP-4 transcript persisted up to 48 h. This response was inhibited by cycloheximide as well as actinomycin D. Long term induction studies revealed that the T3 effect is present during the complete culture period, with a constant rise in IGFBP-4 mRNA levels until 14 days. Under these culture conditions, the DNA content of MC3T3-E1 cells were significantly reduced by T3 and retinoic acid, indicating the repressive effect of both hormones on cell growth. Western immunoblots showed that the transcriptional induction is consequently transduced to increased IGFBP-4 levels in the conditioned medium of T3-treated cells. Our data show that thyroid hormone and retinoic acid stimulate transcription of IGFBP-4 mRNA in osteoblasts, resulting in increased IGFBP-4 secretion into the medium. IGFBP-4, a known inhibitor of cellular proliferation, might contribute to the antiproliferative effect of T3 and retinoic acid on osteoblasts.

Animals↗

PubMed1996

The cellular protooncogenes c-fos and egr-1 are regulated by prostacyclin in rodent osteoblasts and fibroblasts.

PGs are local regulators of various cellular functions. They exert their effects via specific PG receptor subtypes. Induction of c-fos gene expression has been described for arachidonic acid and its metabolite PGE2. We demonstrate that another very short half-lifed prostanoid metabolite, namely prostacyclin (PGI2), is a regulator of immediate-early genes. PGI2 transiently induced the growth-associated immediate-early genes c-fos and egr-1 in osteoblastic as well as fibroblastic cell lines. Furthermore, we showed that PGI2 dose dependently stimulated new DNA synthesis in the osteoblastic cell line MC3T3-E1. Although PGI2 is known to be a potent inducer of cyclooxygenases, we showed that this pathway is not necessary for protooncogene induction by PGI2. Our data indicate a direct effect of PGI2 on immediate-early gene expression, which does not depend on the synthesis of other prostanoids. Intracellular signal transduction mechanisms were studied with the protein kinase inhibitor H-7, a potent inhibitor of PGI2-induced c-fos expression. Experiments with phorbol esters revealed that protein kinase C activity is not obligatory for the effect of PGI2 on c-fos expression. We conclude from these results that PGI2, a rapidly inactivated prostanoid, has a major impact on cellular oncogene expression and growth in mesenchymally derived cells.

3T3 Cells↗

PubMed1996

Bone mineralization as studied by small-angle x-ray scattering.

Small-angle x-ray scattering (SAXS) can provide information on mean size, predominant orientation and typical shape of mineral crystals in bone. In this paper, recent developments of this technique for application in bone research are reviewed. Then the structure of the collagen/mineral composite in bone, as determined by SAXS, is compared for a number of species. The thickness of the mineral crystals was found to increase with age up to a value around 3 to 4 nanometers in adult animals, depending on the species. The SAXS results also suggest the existence of needle-shaped mineral crystals in mouse or rat bone and more plate-shaped crystals in other tissues like adult human bone or mineralized turkey leg tendon.

Animals↗

PubMed1995

The regulatory role of thyroid hormones in bone cell growth and differentiation.

The clinical effects of thyroid hormones on bone in hypo- and hyperthyroidism are well known but their fundamental role in the regulation of bone remodeling is still poorly understood. In this review the current literature is summarized and experimental data from our laboratory are presented. The direct stimulation of bone resorption by thyroid hormones in organ culture, which in part is mediated by prostaglandins and TGF-beta, and the effect of different agents thereon are reviewed. More recent data concerning thyroid hormone action in the osteoblastic cell line MC3T3E1, are summarized. From their effect on proliferation and alkaline phosphatase activity, we conclude that thyroid hormones accelerate osteoblastic differentiation. The regulation of the transcriptional expression of certain genes by nuclear T3 receptors and their effect on osteoblastic target genes like IGF-I are reviewed. In addition a novel role of triiodothyronine as inhibitor of growth factor induced transcriptional expression of regulatory genes (c-fos, c-jun) is suggested.

Alkaline Phosphatase↗

PubMed1995

A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities.

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium K alpha-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from 0 to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases.

Bone Density↗

PubMed1994

Thyroid hormones increase insulin-like growth factor mRNA levels in the clonal osteoblastic cell line MC3T3-E1.

Thyroid hormones are known to affect skeletal growth and maturation by influencing both bone resorption and bone formation. Their exact mechanism of action, however, is still unknown. Local factors such as prostaglandins, TGF-beta or IGF-I were suggested to mediate their effects. Thyroid hormones were reported to stimulate expression of IGF-I mRNA in liver and kidney and to increase IGF-I release from bone organ cultures and osteoblast-like cells. Therefore we studied the effect of thyroid hormones on IGF-I mRNA expression in MC3T3-E1 cells. The cells were grown in culture for 5 to 7 days and treated with triiodothyronine (10(-11)-10(-6) M) and thyroxin (10(-6) M) for 1-24 h. Cellular mRNA was isolated and subjected to Northern hybridization. The amount of IGF-I mRNA, which is already expressed in this cell line under control conditions, was markedly enhanced by T3 and T4. This effect was found to be dose-dependent with a maximum at 10(-7) M and could already be seen after 3 h increasing up to 24 h. Our findings indicate that IGF-I expression in osteoblasts is directly regulated by thyroid hormones. We conclude that IGF-I expression belongs to the phenotypic characteristics of mature osteoblasts, and that thyroid hormones play an important role in differentiation of MC3T3-E1 cells along the osteoblastic lineage.

Base Sequence↗

PubMed1994

Abnormal bone mineralization after fluoride treatment in osteoporosis: a small-angle x-ray-scattering study.

Sodium fluoride treatment of osteoporosis is known to stimulate bone formation and to increase bone mass, but recent clinical trials failed to prove its antifracture effectiveness. The formation of bone with abnormal structure and, therefore, increased fragility is discussed as a possible explanation. Until now, however, exact information on the mineral structure of osteoporotic bone after fluoride treatment has been lacking. Bone biopsies were taken from three patients with postmenopausal osteoporosis before and after fluoride treatment (60 mg NaF/day for 1-2 years), from one patient with iatrogenic fluorosis, as well as from three normal controls. The mineral in these samples was investigated by a combination of backscattered electron imaging and small-angle x-ray scattering. Depending on the total dose of fluoride, an increasing amount of new bone is laid down on the surface of preexisting trabeculae. Its mineral structure is identical to that of heavy fluorosis and is characterized by the presence of additional large crystals, presumably located outside the collagen fibrils. These large crystals, which are not present in the controls or in osteoporotic bone before fluoride treatment, contribute to increase the mineral density without significantly improving the biomechanical properties of the bone. The possible success of fluoride treatment depends not only on the amount of newly formed bone but also on the rate of bone turnover. Indeed, as soon as significant amounts of fluoride are present, bone turnover leads to the replacement of old (normal) bone by new (pathologically mineralized) bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

[Pathophysiology of fracture healing].

This article briefly summarizes our present knowledge on regulation of proliferation, differentiation and function of bone cells (osteoblasts, osteoclasts) by hormones (1,25-dihydroxyvitamin D3, parathyroid hormone, thyroid hormone, sex steroids, glucocorticoids and calcitonin), cytokines (IL-1, IL-4, IL-6, IL-11 und IFN-gamma) and growth factors (IGF-I, TGF-beta). Interaction of these factors in "basic multicellular units" acting locally on bone surfaces is thought to result in tight coupling of bone formation and resorption in bone-remodelling processes. The significance of the latter in different phases of fracture healing, which proceeds from mesenchymal cell proliferation through callus formation to calcification and bone modelling, is emphasized.

Animals↗

PubMed1993

Collagen packing and mineralization. An x-ray scattering investigation of turkey leg tendon.

Several recent results are suggesting that the collagen packing in mineralized tissues is much less regular than in the case of other nonmineralizing collagen, e.g., rat tail tendon. To clarify this question we have investigated the molecular arrangement in mineralized and unmineralized turkey leg tendon as a model for the collagen of mineralized tissues. Using a combination of diffuse x-ray scattering and computer simulation, it could be shown quantitatively that, although the collagen fibril structure is periodic in the axial direction, it is similar to a two-dimensional fluid in the lateral plane. This has important consequences for the understanding of the mineralization process, which is also discussed.

Animals↗

PubMed1992

Mineral crystals in calcified tissues: a comparative study by SAXS.

The shape, the typical orientation, and the average size of mineral crystals in different types of mineralized tissues were investigated by means of small-angle x-ray scattering (SAXS). To rule out eventual artifacts due to sample preparation, four different standard preparation techniques were used and a comparison showed that the SAXS results were identical for all four methods. In mineralized turkey leg tendon, a frequently used model system for bone, the crystals were found to be typically plate-like with a thickness of the order of 2 nm. This stands in contrast to the case of bone (calvaria, femur, and iliac crest) from mouse, rat, and dog, where mainly needle-like crystals were found. The thickness of these crystals ranged from 3 to 4 nm but was remarkably constant for different bones of a given animal. The preferred orientation of the needle-like crystals was along the main axis of the femur and within the surface of the calvaria (for mouse, rat, and dog). The mineral plates in turkey leg tendon were located inside the hole zone and oriented along the fibril axis. Finally, no periodic arrangement of the crystals inside the hole zone of the collagen could be found.

Animals↗

PubMed1991

On the role of cyclic AMP as a mediator of bone resorption: gamma-interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones.

The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin. gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Inhibitory effects of gamma-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones.

The effects of mouse recombinant gamma-interferon (gamma-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of 45Ca from prelabelled bones as a parameter of bone resorption. In addition, the effects of gamma-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mumol/l) totally abolished bradykinin (1 mumol/l) induced 45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 mumol/l). gamma-IFN (1000 U/ml) almost totally inhibited 45Ca release stimulated by bradykinin (1 mumol/l), but the inhibitory effect could only be partially overcome by PGE2. gamma-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 mumol/l) on 45Ca release. The stimulatory effects of PGE2 (1 mumol/l) on radioactive calcium mobilization was partially inhibited by gamma-IFN (1000 U/ml), whereas indomethacin (1 mumol/l) was without effect. The inhibitory effect of gamma-IFN on 45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3-30 U/ml. Comparative dose-response curves showed that gamma-IFN was most potent as inhibitor of bradykinin induced 45Ca release. Bradykinin (1 mumol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 mumol/l). gamma-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Nucleation and growth of mineral crystals in bone studied by small-angle X-ray scattering.

The mechanism of calcification in bone and related tissues is a matter of current interest. The mean size and the arrangement of the mineral crystals are important parameters difficult to obtain by electron microscopy. Furthermore, most studies have been carried out on poorly calcified model systems or chemically treated samples. In the work presented here, native bone was studied as a function of age by a quantitative small-angle X-ray scattering method (SAXS). Bone samples (calvariae and ulnae) from rats and mice were investigated. Measurements were performed on native bone immediately after dissection for samples up to 1 mm thick. The size, shape, and predominant orientation of the mineral crystals in bone were obtained for embryonal, young, and adult animals. The results indicate that the mineral nucleates as thin layers of calcium phosphate within the hole zone of the collagen fibrils. The mineral nuclei subsequently grow in thickness to about 3 nm, which corresponds to maximum space available in these holes.

Animals↗