Multiparticle collision dynamics for diffusion-influenced signaling pathways.

An efficient yet accurate simulation method for modeling diffusion-influenced reaction networks is presented. The method extends existing reactive multiparticle collision dynamics by incorporating species-dependent diffusion coefficients, and developing theoretical expressions for the reactant-dependent diffusion control. This off-lattice particle-based mesoscopic simulation tool is particularly suited for problems in which detailed descriptions of particle trajectories and local reactions are required. Numerical simulations of an intracellular signaling pathway for bacterial chemotaxis are carried out to validate our approach, and to demonstrate its efficiency.

[Are investment activity and backlog in investments risks for university medicine in Germany?]. / Investitionstätigkeit und Investitionsrückstand als Risiko-faktoren für die Hochschulmedizin?

University medicine in Germany requires significantly higher funding and investment because its tasks not only include health care but also research and teaching. However, over recent decades less and less funding compared to the development of the turnover has been available. This trend is due to decreasing public funding. The diminishing funding has caused a major backlog of investment at German university hospitals. The first part of the article summarizes the investments policies at university hospitals and other hospitals. The second part describes the investment needs in university medicine and exposes risk factors for research, education and health care due to the process of investment planning and realization. Goal-oriented solutions are shown to facilitate investments. The third part discusses several risks caused by insufficient investments in university medicine. There are special risks for research, teaching, and the capacity for innovation in university medicine besides economical and medical risks. Some policies and financial strategies to overcome the backlog in investments are presented. After a summary, the article concludes with some practical examples of further measures to ensure sustainable funding.

Tissue factory: conceptual design of a modular system for the in vitro generation of functional tissues.

Tissue factory is a modular system designed to generate artificial tissues under optimal perfusion culture conditions. The microenvironment within the culture containers can be fine-tuned to meet the physiological needs of individual tissues, so that the generation of differentiated three-dimensional tissue constructs becomes possible. An optimal physiological environment is created by modulating a liquid phase as well as an artificial interstitium surrounding the growing construct. An innovative construction principle allows production of tissue culture containers, gas exchangers, and gas expanders at minimal material expenditure. Therefore it will be possible for the first time to produce sterile one-way perfusion culture modules for the generation of artificial tissues. The modules can be used separately as well as in a combined module. The system is designed to provide a possible platform for the standardized production of artificial tissues for future applications in biomedicine.

Controlled respiratory gas delivery to embryonic renal epithelial explants in perfusion culture.

During generation of artificial tissues high levels of oxygen are usually available whereas after implantation into a recipient's body the implant is not vascularized immediately, which leads to low oxygen partial pressures within the implanted tissue. Under these conditions cells will experience an oxygen shortage, contrasting with the abundance of oxygen during culture. It is uncertain whether tissues can be trained to tolerate such an acute hypoxic situation so that nonphysiological stress reactions and tissue necrosis can be avoided. To investigate the effects of varying oxygen levels on embryonic renal tissue in vitro we have been developing a model system combining continuous medium renewal with the ability to control levels of oxygen and carbon dioxide by gas equilibration through gas-permeable tubing. Renal embryonic tissue from neonatal rabbit was cultured in serum-free Iscove's modified Dulbecco's medium at 45, 90, 115, and 160 mmHg oxygen partial pressure for 14 days under continuous medium exchange in such a setup. After a 14-day culture period tissue sections were analyzed by cell biological methods and compared with fresh tissue histology. Surprisingly, embryonic renal explants survive and maintain good morphology for 14 days under all O(2) conditions tested. Expression of cytokeratin 19 within the established epithelium remains unchanged, indicating a structurally intact tissue. However, Na/K-ATPase is clearly downregulated under low O(2) conditions, whereas COX-2 expression increases drastically. An antiparallel effect of decreased O(2) concentrations on glycoprotein expression can be demonstrated with the lectin Dolichos biflorus agglutinin. Scanning electron microscopy reveals oxygen-dependent changes in cellular surface differentiation of developed collecting duct epithelium.

Characterization of micro-fibers at the interface between the renal collecting duct ampulla and the cap condensate.

The development of renal histo-architecture substantially depends on the three-dimensional extension of the collecting duct (CD) ampulla, since under its influence, nephron induction takes place in the surrounding mesenchyme. Recently, micro-fibers were detected by soybean agglutinin (SBA), which line from the basal aspect of each CD ampulla through the mesenchyme towards the organ capsule in embryonic kidney. Their unique distribution suggests that they may play an important role in the control of CD ampulla growth and in forming the renal stem cell niche. A profound analysis of interstitial proteins between the CD ampulla and the nephrogenic mesenchyme is lacking. Consequently, the goal of the current investigation was to colocalize the micro-fibers detected by SBA with interstitial proteins. For this reason a detailed cell biological analysis of extracellular molecules at this site was carried out. Double labeling showed that the micro-fibers do not correspond to known collagens and other extracellular matrix molecules such as agrin, versican or MMP-9. In addition, it could be demonstrated that the micro-fibers do not contain epithelial or mesenchymal cell elements. Furthermore, two-dimensional electrophoresis with subsequent Western blotting yielded two different amino acid sequences (1: GHYADPTSPR; 2: NNGCCSSDYHA) obtained from SBA-labeled protein spots. Both amino acid sequences could not be assigned to known rodent proteins. The findings suggest that the SBA-labeled micro-fibers represent a new type of extracellular structure between the CD ampulla, the mesenchyme and the organ capsule.

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.

[Health care reform 2000--effects on inpatient care]. / Gesundheitsreform 2000--Auswirkungen auf die stationäre Versorgung.

The health reform 2000 in Germany has little impact on the structural deficits of the German health service. The monopolies of hospitals for inpatient and of contract doctors for outpatient care are not changed. The budgeting of hospitals remains. In future there will be a catalog of operations that may only be carried out on an outpatient basis. The duty for quality assurance is specified and intensified. Implementation of innovative medical treatments in hospital will to be done only under strict control of a new countrywide committee. A new DRG-based price system for hospitals will be established.

Human progenitor cells for bone engineering applications.

In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies.

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

[Third level centers in Integrated Health Care: no room, no interest, no needs?]. / Maximalversorger und Integrierte Versorgung: Kein Raum, kein Interesse, kein Bedarf?

Third level health care providers are often highly integrated in the sense that they provide a broad variety of medical specialties. They mostly lack cooperative structures with physicians who are running private practices. By this "isolation", they realize disadvantages in the race for more patients. This is one reason why more university teaching hospitals are growingly interested in contracts for Integrated Health Care. Another field of growing needs for cooperative structures is rehabilitation to ensure achieved therapeutic success especially in highly specialized centers. The paper outlines these growing interests but also formulates preconditions for contracts which should be regarded if university hospitals are to become involved in Integrated Health Care.

[Biomechanical evaluation of biointegrable suture anchors composed of bovine compact bone in a pull-to-failure test in porcine tibial head specimens]. / Biomechanische Untersuchungen zur Fixationsfestigkeit von Fadenankern aus boviner Kompakta (CB-Anker) in porciner Tibiakopfspongiosa im Ausreissversuch.

AIM: Suture anchors of various designs have gained wide acceptance for securing soft tissues to bone. The biointegrable Tutofix CB anchors derived from bovine compact bone are available with diameters of 3 mm (CB3 anchor), 4 mm (CB4 anchor) and 5 mm (CB5 anchor). The CB anchors are push-in anchors and, from the biomechanical standpoint, they are a combination of press-fit and angulation anchors. The purpose of this study was to evaluate the CB anchors for singular pull load-to-failure strength using porcine tibial head specimens as a test model. METHODS: In all specimens, the joint surface was removed by performing a subchondral osteotomy. Axial PQ-CT scans of 12 specimens were obtained to determine the trabecular BMD. The anchors were implanted posteromedially and centrolaterally in the porcine tibial head specimens. After threading the anchors with a steel suture and predrilling of the anchor holes perpendicular to the osteotomy surface they were inserted 4 mm below the osteotomy surface. A universal testing machine applied tensile loads parallel to the axis of insertion at rates of 10 mm/min and 500 mm/min until pull-out failure or anchor breakage and mean anchor fixation strengths were calculated. RESULTS: The fixation strength of the CB anchors was found to be much higher at the dorsomedial implantation site than at the centrolateral implantation site. The CB4 anchors and CB5 anchors provided nearly the same fixation strength at a level much higher than that of the CB3 anchors. Bone mineral density had a strong influence on axial pull-out force of the anchors, especially the CB4 anchors and CB5 anchors. The overall correlation coefficient for bone mineral density with ultimate load-to-failure was 0.869 for the CB4 anchors and 0.716 for the CB5 anchors. Differences in failure strengths were also seen between the low and high extraction rates. With the high extraction rate much higher failure strengths were obtained than with the low extraction rate. The sudden pull eccentrically on the anchors caused a better fixation due to angulation of the anchor within the drill holes. CONCLUSION: In spite of the double worst-case scenario in the testing conditions, the CB anchors provided a high fixation strength in the trabecular bone of porcine tibial head specimens with the CB4 anchors and CB5 anchors being nearly equal and both being superior to the CB3 anchors. Bone mineral density had a strong influence on the axial pull-out force. Our results show that the CB anchors seem to be a reasonable alternative to metal and bioabsorbable suture anchors.

[Impact of the public health reform law on research and education]. / Auswirkungen des Gesundheitsstrukturgesetzes auf Forschung und Lehre.

The statutory health insurance reform law of 1993 had a serious impact on the management of university hospitals. Firstly, the phase of strict budget caps, new in and out patient services could only be introduced when compensated with the elimination of other services. Most universities have mastered this challenge. In the second phase, an entirely new system of hospital fees will be introduced. This will lead to a high degree of uncertainty on the financial situation of the university hospitals. The new regulation on hospital fees only insufficiently takes into consideration tertiary care hospitals. A severe loss of income has to be reckoned with. The funding of current subsidies for medical research, training, construction projects and big-ticket equipment is threatened by the fiscal budget crisis due to the current recession and the heavy financial burdens of the German reunification. Therefore fundamental reforms with regard to the statutory form, the form of ownership, the responsibilities of the university hospital, the organization of management and decision processes, the public service law and investment financing are all unavoidable.

Nephron induction--the epithelial mesenchymal interface revisited.

While more and more humoral factors are being implicated in nephrogenesis, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. Hence we examined this area in the cortex of neonatal rabbit kidneys by scanning and transmission electron microscopy. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme where morphogenic signals are exchanged during nephron induction. Close contact between these two tissues is assumed during nephrogenesis to allow direct cellular contact or diffusion of soluble factors across a short distance. However, our data show the presence a wide cleft around the collecting duct ampulla spatially separating the inducer and the competent mesenchyme during nephron induction. This cleft is filled with a characteristic fibrillar mesh-work.

Partial identification of the mab (CD)Amp1 antigen at the epithelial-mesenchymal interface in the developing kidney.

The nature of the primary functional events of nephron induction is still unknown, making it impossible to completely understand the mechanism of tissue interaction between collecting duct ampulla and the surrounding nephrogenic mesenchyme. Soluble morphogenic substances are known to be exchanged in the process and it is assumed that nephron induction requires close contact between both tissues involved. Contrasting with that assumption our previous investigation revealed a thick fibrous meshwork separating nephron inducer and mesenchyme. Our present investigation focused on the molecular characterization of the mab (CD)Amp1 antigen, which is found only in this meshwork. The protein was shown immunohistochemically to be located exclusively at the embryonic collecting duct ampulla and could be clearly distinguished from other extracellular matrix proteins such as collagen type IV, laminin, reticulin, and fibronectin. Two-dimensional electrophoresis of the soluble form of P(CD)Amp1 showed a molecular weight of 87,000 and an isoelectric point of 4.3-4.4. Results from N-terminal sequencing indicated a partial sequence homology of P(CD)Amp1 to collagen type IV alpha 2-chain precursor but additionally yielded unknown sequences. Thus P(CD)Amp1 is a novel, collagen-related protein, restricted to the fibrous meshwork at the mesenchymal-epithelial interphase, which is the site of primary epithelial-mesenchymal interaction.

High efficacy and minor renal effects of indomethacin treatment during individualized fluid intake in premature infants with patent ductus arteriosus.

AIM: To determine the efficacy and the renal side effects of indomethacin treatment for closure of a patent ductus arteriosus (PDA) in premature infants during an individualized fluid regime that avoids hypovolaemia and subsequent prostaglandin-dependent renal perfusion. METHODS: Observational retrospective analysis of the efficacy of indomethacin in premature infants with PDA treated in a single institution from June 1992 to May 2000. The clinical course and renal effects were analysed in the subgroup of infants born from June 1995 to May 2000. The management of infants at risk and the treatment of infants with PDA followed a standardized protocol that included echocardiographic screening for PDA, indomethacin treatment before congestive failure develops (early symptomatic treatment) and an individualized fluid intake. RESULTS: In total, 412 infants with a gestational age < or = 32 wk were identified. Fifty-six infants with a PDA (14%) were treated with indomethacin [mean birthweight 936 (95% confidence interval 866-1006) g; gestational age 27.3 (26.8-27.9) wk]. Indomethacin treatment was successful in 52 infants (93%). The clinical course and renal effects were analysed in 41 infants. Most infants received three indomethacin doses of 0.2 mg kg(-1) every 12 h. Urine output transiently decreased from 5.6 (4.6-6.4) to 4.6 (3.9-5.3) ml kg(-1) (h(-1). Serum creatinine temporarily increased from 0.90 (0.83-0.98) to 1.06 (0.87-1.24)mg dl(-1). Fluid intake was 158 (148-168) ml kg(-1) d(-1) before indomethacin and decreased to 142 (131-154) ml kg(-1) d(-1). CONCLUSION: Indomethacin is very effective for closure of a PDA, even in very premature infants, and is not associated with clinically significant renal side effects.

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.

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.