1[alpha],25-dihydroxyvitamin D(3) enhances annexin II dependent proliferation of osteoblasts.

Cells experience a variety of physiological and non-physiological stresses and consequently have appropriate mechanisms to deal with such deviations from homeostasis. Particularly subject to mechanical stress and shear forces are the cells that make up the bones. Osteoblastic cells can interpret this stress as a stimulus for proliferation; however, the molecular mechanisms underlying this phenomenon are poorly understood. We have identified annexin II as being specifically upregulated in mechanically stressed osteoblasts and found that increased levels of this protein are necessary for 1[alpha],25-dihydroxyvitamin D(3) mediated augmentation of the proliferative response of osteoblasts after mechanical stress. Our data demonstrate a novel interaction between 1[alpha],25-dihydroxyvitamin D(3) and annexin II in the proliferative response of osteoblasts as well as a novel function for annexin II in the stress response. These findings may offer new therapeutic opportunities for conditions that require regenerative osteoblastic activity such as osteoporosis.

Characterization of the increase in [Ca(2+)](i) during hypotonic shock and the involvement of Ca(2+)-activated K(+) channels in the regulatory volume decrease in human osteoblast-like cells.

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca(2+)](i), in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca(2+)](i) from approximately 150 nm up to 1.3 microm. This increase in [Ca(2+)](i) was mainly due to an influx of extracellular Ca(2+) since removing of extracellular Ca(2+) reduced this increase to approximately 250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca(2+)](i) on membrane currents in these cells. An increase in [Ca(2+)](i) revealed two types of Ca(2+)-activated K(+) channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca(2+)](i) buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca(2+)](i) to 10 microm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from -160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than -40 mV from a holding potential of -80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca(2+)](i) elicited through hypotonic solutions. In combination with the volume-regulated Cl(-) conductance in the same cells this K(+) channel seems to play a vital role in volume regulation in human osteoblasts.

[In vitro cell behavior of human osteoblasts after physiological dynamic stretching]. / In-vitro-Zellverhalten humaner Osteoblasten nach physiologischen dynamischen Dehnungen.

The cell activity of human bone derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human bone derived cells were stretched in rectangular silicone dishes with cyclic uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 mustrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human cancellous bone derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25% and 5-32% respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

In vitro effects of dynamic strain on the proliferative and metabolic activity of human osteoblasts.

AIM OF THE STUDY: It has been well shown by human and animal studies that mechanical load is an important regulator of skeletal mass and architecture. However, cellular reactions which adapt bone tissue to the mechanical environment are not definitively determined. For this purpose we studied the cell activity of human bone derived cell cultures after mechanical stimulation by cyclic, uniaxial strain at a magnitude occurring in normal loaded bone tissue. MATERIALS AND METHODS: Human osteoblasts were isolated from cancellous bone biopsies of 5 different donors. Cell seeding was made in DMEM in a density of 10.000 cells/cm(2) on deformable culture dishes for three days prior to initiating cell stretching at 1000 microstrain, 1Hz for 1800 cycles for two subsequent days with an especially developed cell stretching device. 48h after the second stimulation cells were harvested and cell number was determined with a Coulter Counter. Cell bound alkaline phosphatase activity was analyzed in cell lysates by a colorimetric assay, osteocalcin and CICP (procollagen I propeptide) production were analyzed in cell supernatants with ELISAs. Three parallel cultures were tested. STATISTICS: Wilcoxon. RESULTS: In all experiments mechanical stimulation resulted in a significant increase in cell number (10-48%) and CICP release (7-49%). Simultaneously a significant decrease in alkaline phosphatase activity (9-25%) and osteocalcin release (5-32%) could be observed. CONCLUSIONS: The results demonstrate that cyclic strain at physiologic magnitude leads to an increase of early osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased. These new findings confirm in vivo observations about the importance of dynamic strain for bone formation during fracture healing and bone remodeling and could contribute to the optimization of fracture healing.

Clinical evaluation of a more rapid and sensitive Psoriasis Assessment Severity Score (PASS), and its comparison with the classic method of Psoriasis Area and Severity Index (PASI), before and after climatotherapy at the Dead-Sea.

BACKGROUND: The Psoriasis Area and Severity Index (PASI) is used to quantify the extent of the disease, and to evaluate its improvement with treatment. It is considered to be a slow, rough, nonsensitive, and complex tool, with high interobserver variability and low reproducibility. OBJECTIVES AND METHODS: To develop a simpler, more sensitive, and more rapid end-point determination for evaluating the psoriatic condition, and to compare its sensitivity with that of the classic PASI score in psoriatic patients undergoing 4-week climatotherapy at the Dead-Sea (Israel). RESULTS: This study describes a new, rapid, and simple Psoriasis Assessment Severity Score (PASS), whose readings are spread over a longer scale, making the test more sensitive than PASI, and allow better differentiation. CONCLUSIONS: The comparison between the classic PASI and our new PASS emphasizes the weight of the "sensitivity to change" (responsivity) in selecting a better evaluation method for psoriatic patients.

Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity.

The cell activity of human-bone-derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human-bone-derived cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 microstrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human-cancellous bone-derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25 and 5-32%, respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

Complete transposition in a chick embryo demonstrated by scanning electron microscopy.

Chick embryos are frequently used as animal models when researching the developing heart. In the past, every attempt to induce complete transposition (the combination of concordant atrioventricular and discordant ventriculo-arterial connections) failed in chicks, suggesting that it might be impossible to develop a chicken model for this malformation. We demonstrate, to the best of our knowledge, the first well-documented case of complete transposition occurring in the chick.

Effects of mechanical factors on the fracture healing process.

An interdisciplinary study based on animal experiments, cell culture studies, and finite element models is presented. In a sheep model, the influence of the osteotomy gap size and interfragmentary motion on the healing success was investigated. Increasing gap sizes delayed the healing process. Increasing movement stimulated callus formation but not tissue quality. Typical distributions of intramembranous bone, endochondral ossification, and connective tissue in the fracture gap are quantified. The comparison of the mechanical data determined by a finite element model with the histologic images allowed the attribution of certain mechanical conditions to the type of tissue differentiation. Intramembranous bone formation was found for strains smaller than approximately 5% and small hydrostatic pressure (< 0.15 MPa). Strains less than 15% and hydrostatic pressure more than 0.15 MPa stimulated endochondral ossification. Larger strains led to connective tissue. Cell culture studies on the influence of strain on osteoblasts supported these findings. Proliferation and transforming growth factor beta production was increased for strains up to 5% but decreased for larger strains. Osteoblasts under larger strains (> 4%) turned away from the principal strain axis and avoided larger deformations. It is hypothesized that gap size and the amount of strain and hydrostatic pressure along the calcified surface in the fracture gap are the fundamental mechanical factors involved in bone healing.

Embryological observations on the formal pathogenesis of double-outlet left ventricle with a right-ventricular infundibulum.

The term 'double-outlet left ventricle' (DOLV) denotes congenital heart malformations in which the aorta and the pulmonary trunk both arise entirely or predominantly above the morphologically left ventricle. In the past, the formal pathogenesis of DOLV was explained by an abnormal topogenesis of the origin of the great arteries, which could be caused by an excessive leftward shift of the embryonic conotruncus, or by errors in differential conal growth or absorption. However, modern embryological and pathological research is casting doubt on the validity of these concepts. In the present paper we demonstrate a case of DOLV found in a chick fetus. In this heart the main derivatives of the embryonic conotruncus (right-ventricular infundibulum and proximal portions of the great arteries) principally are in the normal position and of normal dimensions. The anomaly leading to DOLV under these conditions is a misalignment of the ventricular septum. The subarterial portion of the ventricular septum above the crista supraventricularis is not oriented in the normal oblique plane between the pulmonary and aortic valve, but is oriented in a frontal plane anterior to the origin of both great arteries. The consequences of this anomaly are the separation of the right-ventricular infundibulum from the origin of both great vessels (DOLV) and a lack of continuity between the malpositioned portion of the ventricular septum (posterior wall of the right-ventricular infundibulum) and a septum dividing the semilunar valve level. The infundibulum of the right ventricle is derived from the upstream portion of the embryonic conotruncus (conus) whereas the semilunar valves and great arteries are derived from its downstream protion (truncus arteriosus) and the aortic sac. Therefore, our findings suggest that the division of the conotruncus is performed by at least two different septa, one dividing the conus and another dividing the truncus arteriosus and aortic sac. The misalignment of the ventricular septum leading to the presented type of DOLV could result from a misalignment of the septal anlagen of the embryonic conus, the conus ridges. Our findings are discussed with respect to human cases of DOLV and with respect to a recent concept on the septation of the embryonic conotruncus.

The formal pathogenesis of isolated common carotid or innominate arteries: the concept of malseptation of the aortic sac.

Deficient connections (= isolation) of the innominate artery or the common carotid artery to the aorta are rare congenital anomalies of the human aortic arch complex that are usually associated with a patent vascular connection between the isolated artery and a pulmonary artery. In the present study we demonstrate chick fetuses with a corresponding anomaly, the isolation of the brachiocephalic artery. In our chick fetuses the left brachiocephalic artery did not arise from the aortic arch, but was connected to the pulmonary trunk proximal (upstream) to the patent left and right ductus arteriosus. These cases are of interest because the presence of a congenital pulmonary-systemic arterial connection proximal (upstream) to the ductus arteriosus cannot be explained by the traditional concept of the morphogenesis of the aortic arch complex. The development of the normal and abnormal branching patterns of the aortic arch arteries is traditionally explained by transformation of the primitive embryonic pharyngeal arch arterial system due to obliteration of some of its vascular segments. Based on this concept, the isolation of an aortic arch artery can be explained by obliteration of vascular segments proximal and distal to this artery, whereas its connection to a pulmonary artery can be explained only by deficient obliteration (persistence) of the distal portion of the right or left sixth pharyngeal arch artery. The connecting "vascular segment" between an isolated aortic arch artery and the pulmonary circulation, therefore, is traditionally interpreted as a patent ductus arteriosus. The formal pathogenesis of congenital pulmonary-systemic arterial connections proximal (upstream) to the ductus arteriosus is discussed. The presented cases of isolation of the brachiocephalic artery are explained by disturbances in the partition of the embryonic aortic sac, possibly due to abnormal development of the "cardiac" neural crest.

Experimental study on the significance of abnormal cardiac looping for the development of cardiovascular anomalies in neural crest-ablated chick embryos.

During normal development, ectomesenchyme from the cardiac neural crest migrates to pharyngeal arches 3, 4, 6 and the developing heart. It participates in the formation of the aorticopulmonary septum and the wall of the great arteries. Removal of the cardiac neural crest resulted in anomalies of the great arteries and in two categories of severe heart defects: (1) outflow septation defects of the persistent truncus arteriosus (PTA) type, (2) alignment defects. It has been hypothesized that PTA occurs if the number of cardiac neural crest cells is reduced below a level critical for complete formation of the aorticopulmonary septum. Alignment defects would be indirect consequences of neural crest defects, possibly caused by altered blood flow in the pharyngeal arch region. We found that these concepts were not in agreement with some experimental facts reported previously, so we considered whether there could be other mechanisms responsible for the heart defects described. To investigate whether mechanical interference with cardiac looping could possibly contribute to the pathogenesis of these anomalies, we removed the entire cardiac neural crest in chick embryos with micro-needles. Postoperative development was checked during cardiac looping and after normal completion of cardiac septation. Our data suggested that abnormal cardiac looping did not contribute to the pathogenesis of the aortic arch artery anomalies and PTA. With respect to the alignment heart defects, we could not elucidate the role of looping anomalies because we did not observe such heart defects. Moreover, PTA occurred only in 28% of survivors. This finding conflicts with previous studies where extensive ablation of the cardiac neural crest has led to a high incidence of PTA (73-100% of survivors). The possible reasons for this discrepancy are discussed. It is shown that the use of different microsurgical techniques (mechanical cutting/microcautery) may be responsible for the different incidence of PTA. We speculate that microcautery hampers a normal complete repair of neural crest defects, possibly by release of abnormally high levels of growth factors.

Embryological observations on the morphogenesis of double-outlet right ventricle with subaortic ventricular septal defect and normal arrangement of the great arteries.

Double-outlet ventricle (DORV) is generally regarded as a congenital heart defect resulting from impaired morphogenesis of either the outflow portion (conotruncus) or the conoventricular flange (crista prima) of the embryonic heart. However, we demonstrate in this study chicken fetal hearts with DORV in which the conotruncal derivatives (great arteries and subpulmonary part of the ventricular septum = conus septum) and the region of the crista prima are normally developed. The anomalies leading to DORV under these conditions are found at the atrioventricular region. The posterior-anterior axis of the tricuspid orifice is not directed to the right anterior but to the left anterior side of the heart. Thereby the posterior connection line between the muscular ventricular septum and conus septum, which usually follows the left margin of the tricuspid orifice, is not connected to the right portion of the conus septum but instead is directed towards the left portion of the conus septum. In consequence of the abnormal connection between the muscular ventricular septum and the conus septum, the interventricular foramen is formed at the left side of the subaortic flow path. The subaortic flow path arises from the right ventricle. These findings show that DORV can result not only from impaired development of the conotruncus or conoventricular flange, but also from abnormal development of the atrioventricular region. We suggest distinguishing between conotruncal, conoventricular (crista prima), and atrioventricular types of DORV.

The role of extracardiac factors in normal and abnormal development of the chick embryo heart: cranial flexure and ventral thoracic wall.

The present study was designed to investigate whether the formation of the cranial flexure is involved in the normal positional changes of the embryonic heart tube that occur during its transformation from the c- to s-shaped loop. For this purpose, the formation of the cranial flexure was locally suppressed in chick embryos by introducing a straight hair into the neural canal. In the experimental embryos, prevention of cranial flexure did not suppress the normal positional changes of the heart tube. However, other anomalies in the looping of the heart tube were frequently observed. These anomalies were caused by alterations in the formation of the ventral thoracic wall, which in turn seemed to be related not to the prevention of the cranial flexure but rather to accidental injuries during the implantation of the hair. In the embryos with abnormal looping of the heart tube, the incidence of delayed/defective septation of the heart was significantly higher than in embryos with normal looping. These results show that in the chick embryo: (1) cranial flexure is not involved in normal positional changes of the heart loop; (2) manipulations at the head region of the embryo can unintentionally result in developmental disorders of the ventral thoracic wall; (3) such disorders can result in congenital heart defects through mechanical interference with normal looping of the embryonic heart. The possible significance of these findings for the evaluation of experimental studies of chick embryos is discussed in the context of anomalies observed after surgical ablation of the premigratory cranial neural crest.

Formation of the cervical flexure: an experimental study on chick embryos.

It has been proposed that the cervical flexure of vertebrate embryos arises from the normal morphogenesis of the heart. This hypothesis is based on experiments in which the heart tube is removed or disrupted in early chick embryos. It has been reported that, in normal atmosphere, these embryos continued normal morphogenesis except for cervical flexure formation. In the present study, we performed similar experiments. In contrast to previous work, however, only one set of our heart-deprived chick embryos was reincubated in normal air. The other sets were reincubated in oxygen-enriched air. Under normoxia, heart removal resulted not only in prevention of the cervical flexure, but also in mesenchymal defects, and in a remarkable hypoplasia of the craniocervical region. Under hyperoxia, heart-deprived embryos developed no severe mesenchymal defects and the growth of the upper body portion was more normal, with the hypoplasia confined to the cranial region. The formation of the cervical flexure was now normalized. These results show that cervical flexure formation is not directly dependent on normal morphogenesis of the heart, but does depend on a sufficient oxygen supply to the cervical region. During early development, the cranio-cervical region of a chick embryo is more sensitive to circulatory failure than the trunk.

Correlation between the embryonic head flexures and cardiac development. An experimental study in chick embryos.

The aim of the present study is to examine whether the formation of the cranial and cervical flexures is involved in the process of cardiac looping, and whether looping anomalies are causally involved in the development of cardiac malformations. For this purpose, the formation of the cranial and cervical flexures was experimentally suppressed in chick embryos by introducing a straight human hair into the neural tube. In the experimental embryos, the absence of the cervical flexure, alone or in combination with a reduced cranial flexure, was always associated with anomalies in the looping of the tubular heart. The convergence of the primary distant venous and arterial ends of the heart, as well as the normal movement of the ventricular region from its original position, cranial and ventral from the cardiac inflow, to its final position caudal to the presumptive atria, was suppressed to an extent related to the degree to which the formation of the flexures was prevented. Positional immaturity of the heart loop (increased distance between its inflow and outflow, and cranio-ventral position of the ventricular region) was associated with incomplete deformations (reduced angulations) of the cardiac wall at the atrioventricular or conoventricular junctional areas. Reduced angulations were associated with the hypoplasia of the anlagen of the cardiac septa at the level of the angulation (av-cushions, conal ridges). Hypoplasia of these anlagen was followed by incomplete or absent fusion of their opposite free edges, which finally resulted in atrioventricular or ventricular septal defects. These results show that the convergence of the venous and arterial ends of the tubular heart and the caudo-dorsal movement of its ventricular region are related to the formation of the cervical flexure, and that the mesenchymal septa of the heart seem to develop in response to deformations of the embryonic heart, which are generated by the process of cardiac looping. Therefore, the positional and morphological changes of the looping heart are regarded as playing a key role in the process of normal and abnormal morphogenesis of the heart.

Developmental aspects of the sinus valves and the sinus venosus septum of the right atrium in human embryos.

In 32 human embryos from 5 to 27 mm of length, stages 13 to 23 (according to the Carnegie system of stages), the contributions of the sinus venosus septum and the right sinus valve of the right atrium to the formation of the Eustachian and Thebesian valve were examined by scanning electron microscopy. The sinus septum takes part in the subdivision of the right sinus valve into the Eustachian and the Thebesian valves. From its first origin the sinus septum forms a septal structure between the orifices of the right hepatic vein (hepatic portion of the inferior caval vein), the precursor of the inferior caval vein, and the left horn of the sinus venosus, the precursor of the coronary sinus. Before the incorporation of the sinus venosus into the right atrium, it has an intra-sinusal position, and extends between the bases of the left and the right sinus valve. During the incorporation of the sinus venosus into the right atrium the sinus septum receives an intra-atrial position, and its positional relationships to the sinus valves and the orifices of the corresponding veins remain unchanged in principle. Due to the connection between the sinus septum and the right sinus valve, after completion of the incorporation of the sinus, the superior portion of the right sinus valve branches y-like into a lateral limb, (i.e. its original inferior portion) and into a medial limb, (i.e. the sinus septum).(ABSTRACT TRUNCATED AT 250 WORDS)

[The orientation of prisms in the dental enamel of human permanent teeth]. / Uber die Ausrichtung der Prismen im Zahnschmelz menschlicher permanenter Zähne.

Enamel of human permanent teeth was sectioned and ground with 2 planes perpendicular to each other extending centrifugally from the dentino-enamel-junction to the crown surface. Prisms were made visible by acid etching before evaluation under the SEM. In the vicinity of the dentino-enamel-junction more prisms were found to be cut longitudinally, while close to the crown surface more prisms were cut transversely. In the perpendicularly ground plane the corresponding prisms were seen to deviate the more from the centrifugal orientation the more close they came to the crown surface. In a geometric model the angle under which the prisms deviate from the centrifugal orientation was calculated in dependence from the distance to the dentino-enamel-junction. The results correspond with the SEM-findings. We conclude that--since the prism diameter is today known to be constant--the form of the enamel mantle is created by a specific orientation of the prisms: They stand perpendicular at the dentino-enamel-junction, and the farther away they run towards the periphery the more they deviate from the perpendicular path. By this increasingly oblique orientation of the prisms the volume increment of the enamel mantle is created. The maximal angle of deviation is found morphologically and mathematically between 60 degrees-70 degrees at the crown surface. This arrangement of the prisms demonstrated by us is now seen to be the reason for erroneous assumptions about an increase of the prism diameter when ground sections were used. Because the prisms are oriented more and more oblique towards the periphery, correspondingly larger effective diameters must be produced while making ground sections tangential to the crown surface.

[Early development of human tooth germs including the surrounding structures]. / Zur Frühentwicklung menschlicher Zahnanlagen unter Einschluss der Umgebung.

It could be shown by means of 3-dimensional reconstructions made from serial sections of human embryos, that the dental lamina of the lower jaw as well as that of the upper jaw is spatially impeded at its distal ends by the presence of the mandible and Meckel's cartilage. This spatial impediment of the odontogenetic epithelium can be seen to be of influence during the formation of the tooth buds.

[The orientation of the enamel prisms at the enamel surface]. / Uber die Richtung der Schmelzprismen an der Schmelzoberfläche.

The angulation that prisms obtain with the surface in human permanent teeth was analysed on broken enamel by means of the scanning electron microscope. In the cervical region the prisms end nearly perpendicular towards the surface, while the angle becomes more and more acute towards the occlusal region of the crown. In the cuspal area the deviation from the perpendicular direction of the prisms approaches 70 degrees. The size discrepancy between the inner and the outer surface of the enamel mantle can be explained by the more or less angulated position of the prisms.