TSH prevents bone resorption and with calcitriol synergistically stimulates bone formation in rats with low levels of calciotropic hormones.

Thyroid-stimulating hormone exerts both antiresorptive and anabolic effects on bone remodeling in aged ovariectomized rats and thyroid stimulating hormone-receptor null mice, supported by clinical results demonstrating that low thyroid-stimulating hormone level is associated with increased bone loss. To further explore the effect of thyroid-stimulating hormone on bone metabolism we introduced here a rat model with removed thyroid and parathyroid glands to obtain low serum concentrations of thyroid and parathyroid hormone, calcitonin and 1,25(OH)2D3. Surgery resulted in hypocalcemia, low parathyroid and thyroid hormone, 1,25(OH)2D3, C-telopeptide, and osteocalcin serum level. Intermittent administration of thyroid-stimulating hormone resulted in a further decrease of serum calcium and decreased level of serum C-telopeptide due to the suppression of bone resorption, while in the same animals osteocalcin in serum was higher indicating an increased bone formation rate. A combination of thyroid-stimulating hormone and 1,25(OH)2D3 significantly increased the serum Ca2+, C-telopeptide and serum osteocalcin values. MicroCT analyses of the distal femur and proximal tibia showed that rats treated with 1,25(OH)2D3 alone or in a combination with thyroid-stimulating hormone had an increased trabecular bone volume, and enhanced trabecular bone quality. Biomechanical testing of the trabecular bone showed an increased maximal load for 105% and 235%, respectively, in rats treated with 1,25(OH)2D3 alone, or in a combination with thyroid-stimulating hormone. We suggest that thyroid-stimulating hormone independently of calciotropic hormones suppressed bone resorption and stimulated bone formation, while in combination with 1,25(OH)2D3 acted synergistically on bone formation resulting in an increased bone volume.

Correction to: Analysis and quantification of bone healing after open wedge high tibial osteotomy.

Correction to: Wien Klin Wochenschr 2019 https://doi.org/10.1007/s00508-019-01541-8The original version of this article unfortunately contained a mistake. The presentation of Tab. 4 was incorrect. The corrected table is given below.The original article has been ….

Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.

Talc granulomatosis in the rat: the relationship between osteoblast insufficiency and adjacent bone marrow hyperplasia.

Bone loss in talc granulomatosis is paralleled by hyperplasia of bone marrow in the rat. To test the hypothetical relation between those two phenomena, bone matrix osteoinduction was employed as a model in which bone formation and bone marrow appearance are separated in time. Implantation of demineralized bone matrix to normal rats was followed by three talc injections (one weekly), starting 1 week after matrix implantation. Implants of demineralized bone and [3H]thymidine-labeled tibial metaphyses from talc-injected and normal rats were analyzed histologically and evaluated for alkaline and tartrate-resistant acid phosphatase activity on days 7, 14, 21, and 30 after matrix implantation. Analysis of tibial autoradiographs showed a marked growth arrest and bone marrow hyperplasia in talc-injected rats 7 days after first talc injection. Alkaline phosphatase activity in homogenates of bone implants was low in talc-injected rats on day 14 after implantation. Moreover, histology of the bone implants showed numerical and functional inhibition of osteoblasts on the same day, causing marked growth delay. Bone marrow appeared as late as day 21 after bone matrix implantation. We conclude that hyperplasia of the adjacent bone marrow is not the cause of bone loss in talc granulomatosis, but rather its compensatory consequence.

Molecular background and physiological consequences of altered peripheral serotonin homeostasis in adult rats perinatally treated with tranylcypromine.

Serotonin (5-hydroxytryptamine, 5-HT) is a biologically active molecule present in mammals in the brain and peripheral tissues where it exerts many physiological functions. Developmental exposure to 5-HT-enhancing agents has been reported to induce long-lasting changes in the brain, but the long-term effects of perinatal 5-HT enhancement on 5-HT balance and function in the peripheral compartment have not been explored. Perinatal treatment of rats with monoamine oxidase (MAO) inhibitor tranylcypromine (TCP), leads to persistent imbalance in central (increased 5-HT degradation and decreased 5-HT concentrations in the brain) and peripheral (increased platelet and decreased plasma 5-HT concentrations) 5-HT homeostasis. In this study, we explored the molecular background of peripheral 5-HT imbalance, and its possible consequences on bone remodeling and hematopoiesis. Jejunum, liver and blood samples were collected from TCP- and saline-treated rats on post-natal day 70. Relative mRNA levels for tryptophan hydroxylase 1 (TPH1) and MAO A were analyzed using quantitative RT-PCR, femoral trabecular bone parameters were measured using microcomputed tomography, while peripheral blood cell number was determined by cell counter. TCP-treated rats displayed significant decrease in expression of Tph1, and significant increase in percentage of bone volume, trabecular number, connectivity density, and leukocyte number. In addition, significant negative correlation was observed between relative concentrations of TPH1 mRNA and trabecular bone parameters. Our results: a) show that perinatal exposure to tranylcypromine leads to long-lasting compensatory decrease in Tph1 expression in the peripheral compartment, accompanied with alterations in bone remodeling and hematopoiesis, b) suggest that peripheral and central 5HT compartment use different strategies to compensate for 5-HT imbalances of the same cause, and c) indicate dominant role of peripheral over central 5-HT in the regulation of bone maintenance, as well as possible negative in vivo influence of peripheral 5-HT on leukocyte development and/or sustainment.

Effects of 1 alpha,25- and 24R,25-dihydroxyvitamin D3 on aluminum-induced rickets in growing uremic rats.

Rats were subjected to a two-stage subtotal nephrectomy or sham operation, and treated with aluminum (Al) or both aluminum and vitamin D3 metabolites for 5 weeks with a cumulative dose of 13.6 mg aluminum. Animals were injected with 3H-thymidine and 3H-proline. The following analyses were performed: quantitative histology of tibial metaphyses and cytomorphometric electron microscopy of osteoclasts, quantitative (ICP-spectroscopy) and qualitative determination (histochemical staining) of aluminum within organs, and serum biochemistry (Ca, P, Mg, vitamin D3 metabolites, alkaline phosphatase, urea). The following new facts of the aluminum-related bone disease became evident: (a) Application of aluminum to growing uremic rats induced rickets, whose major epiphyseal growth plate changes were 1 alpha,25(OH)2D3-dependent. Addition of 1 alpha,25(OH)2D3 prevented the formation of rachitic metaphysis, but failed to prevent osteoid accumulation on epiphyseal and metaphyseal trabecular surfaces. Moreover, calcitriol produced hyperosteoidosis and osteosclerosis in the same rats. Aluminum did not alter the function of osteoblasts, while osteoclasts seemed inactivated. (b) The development of rickets was associated with suppressed serum levels of 1,25(OH)2D3, reduced phosphorus level and the high content of aluminum in the bone, kidney, and liver. The addition of 24R,25(OH)2D3 markedly exaggerated the reduction of serum levels of calcitriol. We suggested that aluminum induces rickets in growing uremic rats, which consists of two components: vitamin D refractory osteomalacia and 1 alpha,25(OH)2D3-dependent epiphyseal growth plate changes.

Calcium signaling in endothelin- and platelet-derived growth factor-stimulated chondrocytes.

In bovine articular chondrocytes, endothelin (ET) and platelet-derived growth factor (PDGF) receptors mediate agonist-induced increases in inositol phosphate hydrolysis, cytoplasmic calcium concentration ([Ca2+]i), and mitogenesis. In most cells, ET stimulated nonoscillatory [Ca2+]i elevations with dose-dependent increases in both spike and plateau amplitudes. However, about 15% of cells showed oscillatory Ca2+ responses with a constant frequency and variable shape and duration of spiking. ET-1 and ET-2 were more potent than ET-3 in stimulating [Ca2+]i responses in inhibiting the specific binding of 125I-ET-1 and 125I-ET-3 and in promoting internalization of the receptor-ligand complex, consistent with actions through endothelin ETA receptors. Similar nonoscillatory and oscillatory patterns of Ca2+ responses were observed in PDGF-stimulated cells. In cells showing nonoscillatory Ca2+ responses to ET-1, subsequent stimulation with PDGF was frequently followed by the development of an oscillatory Ca2+ response. Nonoscillatory responses to both agonists were only slightly reduced in Ca(2+)-deficient medium, but the oscillatory responses were critically dependent on Ca2+ entry. Ca2+ spiking was not altered in the presence of the voltage-sensitive Ca2+ channel blocker, nifedipine; also, depolarization of chondrocytes by high K+ did not induce [Ca2+]i responses, confirming that voltage-sensitive calcium channels are not expressed in these cells. At high agonist concentrations, ET- but not PDGF-stimulated cells underwent rapid desensitization. Activation of ETA and PDGF receptors was associated with differential stimulation of thymidine incorporation; ET-1 induced a low-amplitude bell-shaped dose-response curve; PDGF induced a sustained sigmoidal and dose-dependent rise. These data indicate that two distinct types of Ca(2+)-mobilizing receptors initiate similar patterns of [Ca2+]i responses but have different capacities to maintain and reinitiate the Ca2+ signaling, as well as to promote mitogenesis.

Cellular basis of inflammation-induced osteopenia in growing rats.

Local nonosseous inflammation provoked by four subcutaneous talc powder injections induced a marked trabecular bone loss in rats within 7 days. The disturbance included suppression of bone elongation, inefficiency and decreased number of trabecular osteoblasts, decreased osteoprogenitor cell number in tibial metaphyses, and bone marrow expansion. Neither the appearance and function of osteoblasts in the vicinity of the cortical bone nor the number of osteoclasts in the metaphysis were found to be altered. The loss of trabecular bone in granulomatosis was based on a suppression of bone elongation and a failure of osteoblasts to form normal secondary spongiosa.

TGF-beta and basement membrane matrigel stimulate the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria.

Primary cultures of fetal rat calvarial cells contain a spectrum of osteogenic phenotypes including undifferentiated mesenchymal cells, osteoprogenitor cells, and osteoblasts. We recently demonstrated that rat calvarial osteoblast-like cells grown on basement membrane undergo profound morphological changes resembling a canalicular network in bone. In the present study, we examined the effect of reconstituted basement membrane Matrigel on chondroblastic versus osteoblastic differentiation of different cell subpopulations obtained by five consecutive enzymatic digestions of rat calvarial cell populations. We found that the appearance of canalicular cell processes decreased with the later digests. When cells from the fourth and fifth digest were grown on top of Matrigel for 7 days, the majority of the cell aggregates displayed chondrocytic characteristics but none of the cells became hypertrophic. When individual chondroblastic cell aggregates were subsequently transferred from Matrigel to plastic, they started expressing types I and X collagens, alkaline phosphatase, and osteocalcin. Within the next 7 days (days 8-14 of the experiment), the majority of cells increased in size, and at day 17 on plastic (day 24 of the experiment) mineralized bone nodules formed. The chondroblastic differentiation of calvarial cells grown on Matrigel could be inhibited by a specific transforming growth factor-beta 1 (TGF-beta 1) but not by a TGF-beta 2 antibody. Addition of recombinant TGF-beta 1 to similar cultures promoted the appearance of chondroblastic cell aggregates. The cartilage phenotype could not, on the contrary, be promoted by growing the cells on other extracellular matrices such as a collagen I gel. We suggest that TGF-beta 1 in concert with the basement membrane extracellular matrix induces chondroblastic differentiation of rat calvarial osteoprogenitor cells.

Opposite effects of osteogenic protein and transforming growth factor beta on chondrogenesis in cultured long bone rudiments.

Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-beta superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-beta 1 and porcine TGF-beta 1 and beta 2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-beta 1 and beta 2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-beta inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-beta exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-beta have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.

Developing human lung and kidney are major sites for synthesis of bone morphogenetic protein-3 (osteogenin).

Bone morphogenetic protein-3 (BMP-3; osteogenin) is a member of the transforming growth factor-beta superfamily. We used human BMP-3 cDNA probes and a specific BMP-3 polyclonal peptide antibody to analyze BMP-3 expression and synthesis in human fetal and adult tissues. Northern blot hybridization revealed two mRNA species of 7 and 3 KB. High levels of BMP-3 mRNA were found in fetal lungs. By in situ hybridization, the BMP-3 transcripts were found in lung bronchial epithelium, straight collecting kidney tubules, intestinal mucosa, perichondrium, periosteum, and osteoblasts of human embryos of 6-14 weeks' gestation. Cellular BMP-3 immunostaining co-localized with the distribution of RNA. In addition, bone matrix showed intensive BMP-3 staining. These data suggest that although BMP-3 has been isolated from bone matrix, it may have additional regulatory roles in the morphogenesis and/or function of human lung, kidney, and intestine.

Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development.

Osteogenic protein-1 (OP-1; BMP-7) is a member of the bone morphogenetic protein subfamily. Because members of the TGF-beta superfamily have a role in tissue development, the distribution of OP-1 expression in developing human embryos (5-8 gestational weeks) and fetuses (8-14 gestational weeks) and mouse (9.5-17.5 gestational days) fetuses was examined. Northern hybridization with specific OP-1 probes revealed two mRNA species of 4 and 2.2 KB. Highest levels of OP-1 mRNA were found in human fetal kidney and heart between 12-14 weeks of gestation. By in situ hybridization, the OP-1 transcripts were found in various tissues, i.e., the ectodermal epithelium of the mouse fore- and hindlimbs, heart, teeth, intestinal epithelium, perichondrium, hypertrophic chondrocytes, and periosteum/osteoblast layer of developing human bones. In kidneys, transcripts were first detected in the epithelium of the branching uretheric buds, whereas at later stages glomeruli were the major site of OP-1 mRNA accumulation. These data suggest that, although OP-1 has been isolated from bone matrix, it may have additional regulatory roles in the morphogenesis and/or function of the kidney, limb bud, tooth, heart, and intestine.

24R,25-dihydroxyvitamin D3 prevents aluminum-induced alteration of brain gangliosides in uremic rats by keeping the metal within perivascular astrocytes of the blood-brain barrier.

The administration of aluminum (Al) to uremic rats leads to Al accumulation in different brain regions with subsequent alteration of brain gangliosides. Addition of 24R,25-dihydroxyvitamin D3[24R,25-(OH)2D3] did not influence the brain Al content determined by plasma argon emission spectrometry, but prevented the decrease in brain gangliosides. By using electron microscopy and laser microprobe mass analysis, it was demonstrated that in rats given 24R,25-(OH)2D3 together with Al, the metal was mainly kept within perivascular astrocytes of the blood-brain barrier. On the contrary, in rats given Al only, the metal was evenly distributed throughout the brain areas causing extensive demyelination, chromatolysis of nerve cells in some brain regions (hippocampus) and brain edema. Our results could find application in the prevention of Al-induced encephalopathy in patients on hemodialysis.

Bone morphogenetic protein-7 (osteogenic protein-1, OP-1) and tooth development.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Talc granulomatosis in the rat. Involvement of bone in the acute-phase response.

We investigated the dynamics of the acute-phase response (APR) and osteoblast trabecular surface in rats with subcutaneous inflammation provoked by magnesium silicate (talc). The first visible indicator of the APR was a rapid and profound hypozincemia, paralleled by a decrease in metaphyseal trabecular surfaces covered with osteoblasts in long bones. Both the intensity of serum APR and the decrease in osteoblast trabecular surface were directly proportional to the number of granulomas. Alterations in bone metabolism were specific for the inflammation, whereas mild hypozincemia and decrease in mononuclear and increase in polymorphonuclear peripheral white blood cell fractions developed in animals pair-fed with rats bearing two or four granulomas. Rats with talc granulomatosis had high serum ACTH and corticosterone levels, but neither adrenalectomy nor high doses of hydrocortisone could revert bone alterations in talc-injected animals. Glucocorticoids were necessary for the development of hypozincemia and hypercupremia seen in talc granulomatosis, as well as for normal bone metabolism. Inhibition of prostaglandin synthesis had no effect on bone alterations and serum APR in rats bearing talc-induced granulomas. We conclude that the decrease in bone formation constitutes an important aspect of the host acute-phase response in a rat model of talc granulomatosis.

Holographic analysis of the human pelvis.

Twelve fresh human pelves with preserved lumbar spines, hip joints, and ligaments, were tested by double-exposure and sandwich-hologram interferometry. During physiologic loadings (50-300 N), the pelvis moved as a whole downward and backward. Iliac wings exhibited marked undulation, except for the central part, which showed minor deformations. The sacrum moved downward and rotated forward over an axis 5-9 cm below the promontorium. Removal of the sacroiliac interosseous ligaments eliminated all joint movements and caused a tighter contact between articular surfaces. Removal of the sacrotuberous and sacrospinous ligaments had no influence on the pelvic behavior. The magnitudes of deformations as well as their underlying mineral contents were unequally distributed between the two pelvic sides. These results indicate that the sacroiliac interosseous ligaments are the main determinant of sacral movement. Asymmetric load transmittance to the hip joints might be responsible for the mineral content differences between the pelvic sides.

New bone formation in autologous membranous bone transplanted into the anterior abdominal wall.

During a gross anatomy dissection, a piece of the calvarial bone was found between aponeuroses of the anterior abdominal wall in an 82-year-old male cadaver. It corresponded closely in size to the calvarial defect after the neurosurgery performed 5 years before death, showing that very little resorption had occurred over the years. Moreover, 13 percent of the transplant was new bone, indicating that membranous bone can, in the long term, induce new bone formation in humans.

Acute zinc deficiency and trabecular bone loss in rats with talc granulomatosis.

Subcutaneous inflammation induced by magnesium silicate (talc) leads to the suppression of bone elongation, osteoblast insufficiency, and subsequent bone loss in rats. Since bone and immunological changes in talc granulomatosis are similar to those observed in zinc deficiency, we investigated the kinetics of zinc tissue distribution and the effects of zinc supplementation on the development of bone loss in rats with talc-induced inflammation. Decrease in serum zinc concentration was observed between 5 and 15 h in rats with talc granulomatosis. It was paralleled by the accumulation of zinc in the liver and rapid disappearance of osteoblasts from the trabecular bone surfaces. However, talc-injected rats supplemented parenterally and orally with zinc sulfate exhibited a decrease in osteoblast trabecular surface comparable to that of unsupplemented rats bearing granulomas despite normalized serum zinc concentrations. Zinc supplementation slightly increased osteoblast trabecular surface in all supplemented groups, but this effect was not significant. We conclude that zinc is the earliest indicator of the acute-phase response in rats with talc granulomatosis. Although zinc appears to be important for the normal function of bone cells, there is no causative relationship between acute zinc deficiency and decreased osteoblast number and activity in rats with talc granulomatosis.

Distribution of structural and trace elements in human temporal bone.

This study was undertaken to evaluate a systematic analysis of mineral and trace elements of individual functionally determined parts of adult temporal bone. Marked differences were observed in basic structural elements (Ca, P, Mg, and Zn) among different bone regions. The more so, molar Ca/P ratio was significantly different in various regions, being highest in the hammer and vestibular regions. Taxonomic analysis revealed specific differences in the mineral ratio between the two petrous bone regions believed to develop from various embryonal bases. According to results, the observed differences in mineral trace element composition of particular regions of human temporal bone might be explained by their developmental specificities and functional adaptation.

Effects of parathyroidectomy on tissue calcium, phosphorus, magnesium, and copper concentrations in aluminum-loaded uremic rats.

Rats were subjected to a two-stage 5/6 nephrectomy and treated with Al for 2 and 4 wk with a cumulative dose of 4.2 and 8.4 mg of Al, respectively. Other animals were parathyrectomized (PTx) and loaded with 8.4 mg of Al for 4 wk. Total Al, Ca, P, Mg, and Cu contents were analyzed in the liver, kidney, and bone by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that Al given to growing uremic rats significantly increased the content of Al in the liver, kidney, and bone. Moreover, Al treatment increased the liver and kidney Ca levels and decreased the Ca and P values in bone. Previous parathyroidectomy significantly reduced Al accumulation within organs and changes in the Ca and P levels in the bone, liver, and kidney. The result was not influenced by different degrees of renal failure.