Calcium isotope ratios in blood and urine: A new biomarker for the diagnosis of osteoporosis.

We assessed the potential of Calcium (Ca) isotope fractionation measurements in blood (δ44/42CaBlood) and urine (δ44/42CaUrine) as a new biomarker for the diagnosis of osteoporosis. One hundred post-menopausal women aged 50 to 75 years underwent dual-energy X-ray absorptiometry (DXA), the gold standard for determination of bone mineral density. After exclusion of women with kidney failure and vitamin D deficiency (<25 nmol/l) 80 women remained in the study. Of these women 14 fulfilled the standard diagnostic criteria for osteoporosis based on DXA. Both the δ44/42CaBlood (p < 0.001) and δ44/42CaUrine (p = 0.004) values were significantly different in women with osteoporosis (δ44/42CaBlood: -0.99 ±â€¯0.10‰, δ 44/42CaUrine: +0.10 ±â€¯0.21‰, (Mean ±â€¯one standard deviation (SD), n = 14)) from those without osteoporosis (δ44/42CaBlood: -0.84 ±â€¯0.14‰, δ44/42CaUrine: +0.35 ±â€¯0.33‰, (SD), n = 66). This corresponded to the average Ca concentrations in morning spot urine samples ([Ca]Urine) which were higher (p = 0.041) in those women suffering from osteoporosis ([Ca]Urine-Osteoporosis: 2.58 ±â€¯1.26 mmol/l, (SD), n = 14) than in the control group ([Ca]Urine-Control: 1.96 ±â€¯1.39 mmol/l, (SD), n = 66). However, blood Ca concentrations ([Ca]Blood) were statistically indistinguishable between groups ([Ca]Blood, control: 2.39 ±â€¯0.10 mmol/l (SD), n = 66); osteoporosis group: 2.43 ±â€¯0.10 mmol/l (SD, n = 14) and were also not correlated to their corresponding Ca isotope compositions. The δ44/42CaBlood and δ44/42CaUrine values correlated significantly (p = 0.004 to p = 0.031) with their corresponding DXA data indicating that both Ca isotope ratios are biomarkers for osteoporosis. Furthermore, Ca isotope ratios were significantly correlated to other clinical parameters ([Ca]Urine, ([Ca]Urine/Creatinine)) and biomarkers (CRP, CTX/P1NP) associated with bone mineralization and demineralization. From regression analysis it can be shown that the δ44/42CaBlood values are the best biomarker for osteoporosis and that no other clinical parameters need to be taken into account in order to improve diagnosis. Cut-off values for discrimination of subjects suffering from osteoporosis were - 0.85‰ and 0.16‰ for δ44/42CaBlood and δ44/42CaUrine, respectively. Corresponding sensitivities were 100% for δ44/42CaBlood and ~79% for δ44/42CaUrine. Apparent specificities were ~55% for δ44/42CaBlood and ~71%. The apparent discrepancy in the number of diagnosed cases is reconciled by the different methodological approaches to diagnose osteoporosis. DXA reflects the bone mass density (BMD) of selected bones only (femur and spine) whereas the Ca isotope biomarker reflects bone Ca loss of the whole skeleton. In addition, the close correlation between Ca isotopes and biomarkers of bone demineralization suggest that early changes in bone demineralization are detected by Ca isotope values, long before radiological changes in BMD can manifest on DXA. Further studies are required to independently confirm that Ca isotope measurement provide a sensitive, non-invasive and radiation-free method for the diagnosis of osteoporosis.

Body image in patients with somatoform disorder.

BACKGROUND: Although body-related problems are common in patients with somatoform disorder, research focusing on how patients with somatoform disorder perceive and evaluate their body is scarce. The present study compared differences in body image between patients with somatoform disorder and respondents from a general population sample. It also examined differences within the somatoform disorder group between men and women and between the diagnostic subgroups conversion disorder, pain disorder and undifferentiated somatoform disorder. METHODS: Data were obtained from 657 patients (67.5% female) with somatoform disorder (DSM-IV-TR 300.7, 300.11, 300.81, 300.82) and 761 participants (58.6% female) from the general population. The Dresden Body Image Questionnaire (DBIQ) was used to assess body image in five domains: body acceptance, vitality, physical contact, sexual fulfilment, and self-aggrandizement. Confirmatory factor analysis and analyses of variance were performed. Since differences in age and sex were found between the somatoform disorder sample and the comparison sample, analyses were done with two samples of 560 patients with somatoform disorder and 351 individuals from the comparison sample matched on proportion of men and women and age. RESULTS: Patients scored significantly lower than the comparison sample on all DBIQ domains. Men scored higher than women. Patients with conversion disorder scored significantly higher on vitality and body acceptance than patients with undifferentiated somatoform disorder and pain disorder. CONCLUSIONS: The mostly large differences in body image between patients with somatoform disorder and the comparison sample as well as differences between diagnostic subgroups underline that body image is an important feature in patients with somatoform disorder. The results indicate the usefulness of assessing body image and treating negative body image in patients with somatoform or somatic symptom disorder.

Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering.

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.

Polar application of test substances in an organotypic environment and under continuous medium flow: a new tissue-based test concept for a broad range of applications in pharmacotoxicology.

We have established a new test concept for in vitro pharmacological trials. Our model employs tissue explants to test compounds for toxicity which arises with the metabolic interactions among different cell types. Microsurgical preparation of tissue explants avoids the destruction of the organ-specific tissue architecture. Explants were mounted in tissue carriers to improve nutrition and handling of the sample. To allow for the omission of serum supplementation of the culture medium, explants were cultured under continuous medium flow. Test substances are applied considering the polar architecture of most tissues in vivo, for example, covering the apical aspect of epithelia. In principal, all tissues obtained from any species, including man, can be used in this system. A trial application was performed with vitreous body substitutes, substances used in ophthalmology. One compound had passed cell culture tests, but caused massive blood vessel deterioration in vivo. Using our test system based on the developing renal vessel system, we could confirm, within 24 hours, severe vessel damage which resembles the injury suffered by the rabbit retina. We demonstrate that an improved tissue culture assay is a suitable tool for the detection of toxicity that remained unidentified in cell culture tests.

Modulation of cell differentiation in perfusion culture.

An in vitro model was used to investigate the terminal differentiation mechanisms leading from embryonic to adult renal tissue. For these experiments the capsula fibrosa with adherent embryonic tissue was isolated from neonatal rabbit kidneys. These explants were mounted onto special tissue carriers and cultured in medium containing serum for 24 h. During that time collecting duct (CD) cells grew out and formed a monolayered epithelium covering the whole surface of the explant. The carriers were then transferred to perfusion culture containers to obtain an optimal degree of differentiation. A special type of container allowed us to continuously superfuse the epithelia with individual media on the luminal and basal sides. Using this method it became possible to culture embryonic CD epithelia in a fluid gradient for weeks. The epithelia were superfused with standard Iscove's modified Dulbecco's medium (IMDM) on the basal side, while IMDM containing additional NaCl was used on the luminal side. In controls IMDM was superfused on both the luminal and basal sides. It was found that the degree of differentiation in the CD epithelia is dependent on the influence of fluid gradient exposure. Perfusion culture under isotonic conditions revealed that less than 5% of cells were immunopositive for principal and intercalated cell features, while epithelia cultured in a luminal-basal gradient showed more than 80% positive cells. Immunoreactivity for characteristic markers started to develop after an unexpectedly long latent period of 3-6 days, then increased continuously during the following 5 days and reached a maximum on day 14. After switching back from the gradient to isotonic culture conditions the immunoreactivity for some markers decreased within 5 days, while other characteristic features remained stable. Thus, differentiation was not only under the control of growth factors but was also regulated by the electrolyte environment.

The influence of culture media on embryonic renal collecting duct cell differentiation.

During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove's modified Dulbecco's medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams' medium E, McCoys 5A medium, and Dulbecco's modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.

Existence of a dense reticular meshwork surrounding the nephron inducer in neonatal rabbit kidney.

While more and more humoral factors involved in nephrogenesis are being discovered, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. For that reason we examined this area in the cortex of neonatal rabbit kidneys by scanning electron-microscopical and transmission electron-microscopical techniques. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme, where morphogenic signals are to be exchanged during nephron induction. Close contact between these two tissues involved in nephrogenesis is assumed to allow direct cellular contact or diffusion of soluble factors across a short distance. Our data, however, show the presence of a dense fibrillar meshwork around the collecting duct ampulla, spatially separating the inducer and the competent mesenchyme during nephron induction.

Transitional stages in the development of the rabbit renal collecting duct.

The collecting duct (CD) epithelium of the mammalian kidney is an extraordinary structure with respect to its functional changes during development and its heterogeneous composition when matured. All of the different nephron epithelia of the mammalian kidney consist of one single cell type. In contrast, the differentiated CD is composed of at least three distinct cell types [principal, alpha intercalated-, and beta intercalated cells] that are responsible for the multiple physiological functions of this kidney compartment. During development the function of the CD changes: initially, the CD ampulla serves as an embryonic inducer, while the matured epithelium plays a key role in maintaining the homeostasis of body fluids. At present the process of CD maturation is not well understood. Neither the time course of development nor the morphogenic factors leading to the heterogeneously composed epithelium are known. In the present study the differentiation of the CD epithelium was investigated using newly developed monoclonal antibodies and well-characterized antisera. The morphological changes induced during differentiation were monitored by immunohistochemistry and scanning electron microscopy. The experiments were performed on neonatal and adult rabbit kidneys. Results obtained by light microscopical techniques and scanning electron microscopy revealed that the ampullary tip can be distinguished from the ampullary neck, as well as from the maturing CD. A number of proteins that were not detectable in the ampulla were detected in the neonatal CD and were found at even higher concentrations in the adult CD (PCD8, chloride/bicarbonate exchanger). Other proteins (PCD9) were downregulated during differentiation. For the first time the transient character of the differentiation stage of the neonatal CD could be demonstrated unequivocally. Furthermore, considerable heterogeneity in protein expression patterns (PCD6 and PCD9) was demonstrated within the beta IC cell population of the mature CD.

Communicative styles of mothers interacting with their preschool-age children: a factor analytic study.

The aim of this study was to determine if mothers display identifiably different communicative styles in their interaction with their normally developing two- to five-year-old children. In order to investigate this issue an extensive coding system was developed, which assessed the structural organization and the communicative function of the speech of 71 mothers as they interacted with their children. By means of factor analysis three maternal communicative styles were distinguished: non-intervening, explaining and directing. In the non-intervening style there is no direct pressure from the mother on the child to respond verbally. The explaining mother is primarily concerned with providing information to her child in a way that gives the child little opportunity to take the speaking turn. The directing mother is mainly engaged in directing the child's behaviour by means of verbal control. The internal consistency of the three communicative styles appeared to be both satisfactory and related to relevant child and mother features.

Basic fibroblast growth factor is a morphogenic modulator in kidney vessel development.

During kidney organogenesis the development of renal vessels must be synchronized with the maturation of nephrons and the collecting duct system. Several reports showed that hormones and mitogenic peptides as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) are involved in this regulatory process. It is a known fact that bFGF receptors are expressed by differentiating tubular epithelium and mesenchyme, but little information is available about the function of bFGF in kidney organogenesis. The role of bFGF during kidney development was investigated using an organotypic culture system and immunohistological techniques. Renal cortex explants were prepared from the kidneys of neonatal rabbits with a microsurgical method, retaining the natural tissue composition. The explants were cultured serum free under continuous medium perfusion. Our results indicate a new and unexpected role of bFGF during the differentiation process. When bFGF alone was applied, vessels could no longer be detected. The inhibitory influence of bFGF could be overcome by addition of VEGF or hormones such as retinoic acid and aldosterone/vitamin D3. The combination of these factors with bFGF resulted in the expression of small vessel-like structures. We conclude that bFGF has a morphogenic rather than a mitogenic function during kidney vessel development.

Tissue engineering: generation of differentiated artificial tissues for biomedical applications.

A new field in biomedical science has been established. Cell biologists, engineers, and surgeons now work within a team. Artificial connective, epithelial, or neuronal tissues are being constructed using living cells and different kinds of biomaterials. Numerous companies and laboratories are presenting dynamic developments in this field. Prognoses predict that, at the beginning of the coming century, the industry of tissue engineering will reach the importance of the present genetic technology. An enormous demand for organ and tissue transplants motivates research activities and drives the acquisition of innovative techniques and creative solutions. At the front of this development is the creation of artificial skin for severely burned patients and the generation of artificial cartilage for implantation in articular joint diseases. Future challenges are the construction of liver organoids and the development of an artificial kidney on the basis of cultured cells. In this paper we show strategies, needs, tools, and equipment for tissue engineering. The presupposition for all projects is the induction, development, and maintenance of differentiation within the tissue under in vitro conditions. As experiments in conventional culture dishes continued to fail, new cell and tissue culture methods had to be developed. Tissues are cultured under conditions as close as possible to their natural environment. To optimize adherence or embedding, cells are grown on novel tissue carriers and on individually selected biomatrices or scaffolds. The tissues are subsequently transferred into different types of containers for permanent perfusion with fresh culture medium. This guarantees constant nutrition of the developing tissue and prevents the accumulation of harmful metabolites. An organo-typical environment for epithelial cells, for example, is obtained in gradient containers, which are permanently superfused at the apical and basal sides with different media. Long term experiments result in cultured tissues in a quality thus far unreached.

Electrolyte environment modulates differentiation in embryonic renal collecting duct epithelia.

The influence of electrolytes on the development of renal principal and intercalated collecting duct cells is unknown. Consequently embryonic collecting duct epithelia were exposed to different electrolyte concentrations, and their degree of differentiation was registered by immunohistochemical methods. Embryonic collecting duct epithelia were isolated from neonatal rabbit kidneys and placed on tissue carriers. The apical urine and the basal serum compartments were simulated in a gradient culture container. The two sides of the epithelium were each constantly superfused with medium for 13 days. In controls the medium on both apical and basal side was standard Iscove's modified Dulbecco's Medium (IMDM) with 112 mmol/l Na+ and 85 mmol/l Cl-. In experimental series the NaCl concentration at the basal side of the epithelium was increased up to 137 mmol/l Na+ and 99 mmol/l Cl- as found in the serum of neonatal rabbits. Light microscopy revealed morphologically faultless epithelia following gradient perfusion culture in standard and NaCl-adapted IMDM. The development of principal and intercalated cell features was monitored with the monoclonal antibodies 703, 503, PCD9, and peanut lectin. Cells immunopositive for monoclonal antibody 703, for example, increased from less than 10% in controls to more than 80% in NaCl-adapted IMDM. It is a new finding that the development of collecting duct cell features is influenced by the extracellular electrolyte environment.

Culture of embryonic renal collecting duct epithelia in a gradient container.

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

[Vital long-term preservation of human gingiva in perfusion culture]. / Lebende Langzeitkonservierung von humaner Gingiva in der Perfusionskultur.

Perfusion culture offers the advantage of keeping gingiva alive for a long time as an stable explant according to cell biological parameters. To investigate the suitability of cultured human gingival explants for transplantations the biopsies were put into a newly developed perfusion chamber and cultured for at least 21 days. Gingiva explants were derived from healthy donors undergoing surgical removal of molar teeth. The tissue pieces were cultured without prior proteolytic desintegration or subculture. Immediately after excision a morphological and immunohistochemical analysis of the tissue was carried out and the distribution pattern of cytokeratin and vimentin was examined. Gingival explants cultured for 7, 14 and 21 days in serum-free keratinocyte growth medium in perfusion culture were analyzed in the same way. The morphology of the cultured explant (21 days) was well preserved from stratum basale up to stratum corneum. As proved by immunohistochemical incubation with antibodies to CK 5/6, CK 14 and CK 19, a tissue-specific cytokeratin (CK) expression pattern was maintained during the whole perfusion period. After 7 days of culture vimentin was synthesized in the fibroblast layer and was found in small quantities in each layer of the epithelium. In contrast to conventional cultures, where dissociation of the tissue and a subculture interruption is usually needed for long-term culture, this is not necessary for perfusion cultured tissue. The use of perfusion-cultured gingival explants as autogenous transplants is investigated herein.

Three-dimensional organization of the developing vasculature of the kidney.

Kidney function depends on a well-developed vascular system. Any impairment of the blood supply disturbs the integrity and function of the organ. The differentiation of renal vessels has been investigation for many years, but little is known about the relationship between nephrogenesis and vessel development. In the present work the spatial organization of the differentiating vessels was analyzed in precisely oriented tissue sections and in optical sections acquired by laser scan microscopy. Developing vessels as well as small capillaries were visualized with two endothelium-detecting antibodies. Small vessels running in parallel towards the organ capsule were detected in numerous cortico-medullary-oriented tissue sections. Cross-sections of the nephrogenic zone showed a regularly arranged network, which was composed of cells detected by both monoclonal antibodies. Parts of this network were localized in regions of the nephrogenic zone which have been assumed to be free of vessels or vessel-like structures for a long time. These results were confirmed by the laser-scan-microscopic analysis of complete cortex explants. The extraordinarily regular arrangement of the endothelial network in the nephrogenic zone allowed us to reconstruct the developing vascular system. The results presented here underline the close relationship between nephrogenesis and vessel development.

PCDAmp1, a new antigen at the interface of the embryonic collecting duct epithelium and the nephrogenic mesenchyme.

In the neonatal rabbit kidney nephrogenesis is not yet terminated. The ampullar collecting duct epithelium acts as an inducer that generates the nephron anlagen, however, to date the morphogenic mechanisms involved are unknown. A presupposition for successful nephron induction is the close tissue interaction between the basal aspect of the ampullar collecting duct epithelium and the surrounding mesenchyme. To gain new insights in this area we raised monoclonal antibodies (mabs), to identify specific structures localized at the tissue interface. With the generated mab CDAmp1 we found an intensive immunohistochemical reaction between the basal aspect of the ampullar collecting duct epithelium and the mesenchyme. The label was most concentrated at the ampullar tip and continuously decreased in the shaft region. In the maturing collecting duct of the neonatal kidney and in the adult renal collecting duct no immunohistochemical reaction was found. The binding pattern of mab CDAmp1 is different from that of all known collecting duct cell markers and from antibodies against known basement membrane compounds such as laminin or collagen type IV. Under in vitro conditions immunoreactivity with mab CDAmp1 was obtained using embryonic collecting duct epithelia and perfusion culture. The antigen was present in specimens treated with Iscove's modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum. Omittance of serum or hormonal treatment with aldosterone, insulin or vitamin D3 led to the disappearance of the newly detected antigen, while characteristics of the differentiated collecting duct cells were up-regulated. We conclude that the expression of PCDAmp1 is a characteristic feature of the embryonic parts of the collecting duct epithelium. It may play a pivotal role during nephron induction.

In vitro development and preservation of specific features of collecting duct epithelial cells from embryonic rabbit kidney are regulated by the electrolyte environment.

During kidney development the embryonic collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into functional principal (P) and intercalated (IC) cells. Aldosterone is able to modulate this differentiation. Consequently we investigated whether increased concentrations of extracellular NaCl or Na gluconate may also have an influence on the development of individual CD cell features. Embryonic CD epithelia were isolated from neonatal rabbit kidneys, placed on tissue carriers and cultured in gradient containers, which were constantly perfused with medium for 13 days. Isotonic culture conditions could be mimicked, when on both the luminal and basal side standard Iscove's Modified Dulbecco's Medium (IMDM) was used. In another set of experiments, gradient culture was performed. Standard IMDM was applied on the basal side and IMDM supplemented with 12 mM NaCl and 17 mM Na gluconate on the luminal side. This adaptation of IMDM led to the same Na concentrations as found in the serum of neonatal rabbits. The development of CD cell features was monitored by cellular markers such as the monoclonal antibodies (Mabs) 703 and 503 recognizing P and IC cell features respectively. Epithelia cultured under isotonic conditions showed less than 5% Mab 703- and 503-immunopositive cells. In contrast, epithelia cultured in a luminal-basal medium gradient revealed more than 80% positive cells. Immunoreactivity started to develop after a long lag period of 4 days, then increased continuously during the following 5 days and reached a maximum at day 14. When the medium gradient was then changed to an isotonic environment for another 5 days immunoreactivity for Mab 703 remained stable, while the number of Mab 503-positive cells was found to be decreased to 10%. Thus, the extra-cellular electrolyte environment not only induces but also preserves individual cell features.