Skeletal tissue engineering-from in vitro studies to large animal models

Bone is a tissue with a strong regenerative potential. New strategies for tissue engineering of bone should therefore only focus on defects with a certain size that will not heal spontaneously. In the development of tissue-engineered constructs many variables may play a role, e.g. the source of the cells used, the design and mechanical properties of the scaffold and the concentration and mode of application of growth factor(s).Models for studying new strategies for tissue engineering of bone should be based on the target tissue to be restored. However, in light of the many potential variables, which may also interact if used in combination(s), there is also a large need for relatively simple models in which variables can be tested in a limited number of animals. Moreover, in compromised bone there may be a problem with the load-bearing capacity of the remaining healthy bone. In this light, an important prerequisite for tissue-engineering constructs is that they can be tested in loaded conditions. Particularly, this latter prerequisite is very difficult to achieve. Therefore, in vitro tests for mechanical stability are very useful for evaluating the mechanical consequences of a particular reconstruction procedure prior to the animal experiment. Before a tissue-engineered construct can be introduced into a clinical trial, a final test should be available in a large animal model that is as close and relevant to a particular problematic clinical situation as possible.In the past, a series of models were developed in our laboratory that are very useful for testing tissue-engineered constructs. In this paper, we focus on the use of relatively new simple in vitro and in vivo models for hip revision surgery, segmental bone defect restoration and tumour surgery.     

In Situ Thermal Denaturation of Proteins in Dunning AT-1 Prostate Cancer Cells: Implication for Hyperthermic Cell Injury

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from alpha helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees C min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.     

Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies for inflammatory bowel disease: high prevalence in patients with celiac disease

Both celiac disease and inflammatory bowel disease (IBD) are characterized by chronic diarrhea and the presence of distinct (auto)antibodies. In the present study we wanted to determine the prevalence of serological markers for inflammatory bowel disease, i.e., perinuclear antineutrophil cytoplasmic antibodies (pANCA) and/or anti-Saccharomyces cerevisiae antibodies (ASCA), in 37 patients with biopsy-confirmed celiac disease (Marsh IIIb/c). The majority of the patients was positive for IgA (auto)antibodies typically associated with celiac disease, i.e., antiendomysium antibodies (EMA) (86.5%), antigliadin antibodies (AGA) (73%), and antirecombinant human tissue transglutaminase antibodies (rh-tTGA) (86.5%). Four patients with selective IgA deficiency could be identified by analyzing EMA, AGA, and rh-tTGA for the IgG isotype. The prevalence of pANCA and ASCA, markers that are used for IBD, was unexpectedly high in our cohort of patients with celiac disease: 8 patients were positive for pANCA (IgG) and 16 patients were positive for ASCA (IgG and/or IgA). These results indicate that the presence of pANCA or ASCA in the serum of patients with chronic diarrhea does not exclude celiac disease. A prospective study is required to determine whether pANCA and/or ASCA identify patients at risk for developing secondary autoimmune disease.     

Age, gender and litter-related variation in T-lymphocyte cytokine production in young pigs

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.     

Carbon-11 labelled tyrosine to study tumor metabolism by positron emission tomography (PET)

To measure the rate of protein synthesis in human neoplasms by positron emission tomography, we prepared no carrier added DL-(1-¹¹C)-tyrosine by ¹¹C-carboxylation of the appropriate -lithioisocyanide followed by hydrolysis of the isocyanide function and removal of the protecting methoxy group. The purification, resolution and solvent switch to saline was performed by high performance liquid chromatography (HPLC). DL-(1-¹¹C)-Tyrosine in 0.1 N NaH₂PO₄ buffer was prepared with a radiochemical yield of 8%–16% (EOS, 35 min). The enantiomeric separation and solvent switch to saline were achieved in 5 min and 10 min respectively. Consequently L-(1-¹¹C)-tyrosine in physiological saline was obtained in 2%–4% radiochemical yield. Tumor accumulation in rats with the experimental WALKER 256 carcinosarcoma was observed for both the L- and D-isomer. Using positron emission tomography a tumor/muscle ratio of two was observed for the L-isomer 15 min after injection. The corresponding figure for the D-isomer was 2.5. The first clinical results with DL-(1-¹¹C)-tyrosine show accumulation of radioactivity in meningioma, a primary breast carcinoma and in liver metastases of a colonic carcinoma.     

Who contextualises clinical epidemiological evidence? A political analysis of the problem of evidence-based medicine in the layered Dutch healthcare system

We critically examine the discussion on the role of evidence-based medicine (EBM) in healthcare governance. We take the institutionally layered Dutch healthcare system as our case study. Here, different actors are involved in the regulation, provision and financing of healthcare services. Over the last decades, these actors have related to EBM to inform their actor specific roles. At the same time, EBM has increasingly been problematised. To better understand this problematisation, we organised focus groups and interviews. We noticed that particularly EBM’s reductionist epistemology and its uncritical use by ‘professional others’ are considered problematic. However, our analysis also reveals that something else seems to be at stake. In fact, all the actors involved underwrite EBM’s reductionist epistemology and emphasise that evidence should be contextualised. They however do so in different ways and with different contexts in mind. Moreover, the ways in which some actors contextualise evidence has consequences for the ways in which others can do the same. We therefore emphasise that behind EBM’s scientific problematisation lurks a political issue. A dispute over who should contextualise evidence how, in a layered healthcare system with interdependent actors that cater to both individual patients and the public. We urge public administration scholars and policymakers to open-up the political confrontation between healthcare actors and their sometimes irreconcilable, yet evidence-informed perspectives.     

Toxicity of lead acetate to female rabbits after chronic subcutaneous administration. 1. Biochemical and clinical effects

The effect of chronic subcutaneous administration of lead acetate was studied in female rabbits. The low-dose group (15 animals) received three times a week 0.10–0.20 g/kg body weight and the high-dose group (15 animals) 0.80–1.20 g/kg. The control group received the vehicle only. Concentrations of lead in blood in the low-dose group increased to ca. 400 g/l after 70 days and in the high-dose group to ca. 900 g/l after 110 days. After 7.5 months eight animals of each group were sacrificed. The remaining rabbits were kept for an additional 4 months without treatment. Blood lead concentrations decreased with a half-time of 60–70 days. During exposure the gain in body weight was lower in the high-dose group than in the control group and the low-dose group. The high-dose group developed slight anaemia and low MCV, MCH and MCHC, and basophilic stippling of erythrocytes. These effects disappeared during recovery. ALAD activity in erythrocytes was very low during exposure in both exposed groups and did not reach control values during recovery. During exposure the concentrations of ZPP and ALA-U increased, but only ALA-U returned to normal during recovery. No other effects of lead on the composition of the urine were observed. No effects were observed on plasma urea and creatinine concentrations. In the highdose group the concentration of ALAD in the liver decreased by 30%. During recovery this effect was no longer present. No effects were seen in cytochrome P-450 content or cytochrome P-450-dependent enzyme activities. Lead was mainly stored in bones, but some also in serveral soft tissues. After recovery the concentrations in soft tissues decreased to a variable degree. In the high-dose group the relative weights of heart and liver increased. These effects disappeared during recovery. At 400 g lead/l blood no adverse effects were observed that did occur at the high dose level.Part 2, dealing with the histopathology and (electron) microscopy of the kidneys is in preparation     

Isolation,purification, and biological activity of mono- and dihydroxylated paclitaxel metabolites from human feces

Three metabolites of the cytotoxic drug paclitaxel (Taxol) were isolated and purified from the feces of cancer patients receiving the agent as an intravenous infusion. The procedures involved sample homogenization in water followed by liquid-liquid extraction with diethyl ether and high-performance liquid chromatography (HPLC). Approximately 1–3.5 mg of each metabolite was obtained from 100 g of feces. As judged from the chromatographic traces of analytical HPLC with ultraviolet (UV) detection at 227 nm, the purity of each compound was >97%. On-line photodiode-array detection demonstrated that the UV spectrum of the isolated compounds closely resembles that of the parent drug. Mass spectrometry provided evidence that these metabolites are mono- and dihydroxy-substituted derivatives, namely, 6-hydroxypaclitaxel, 3-p-hydroxypaclitaxel, and 6,3-p-dihydroxypaclitaxel. The two 6-hydroxy-substituted metabolites were shown to have lost their cytotoxicity in in vitro clonogenic assays using the A2780 human ovarian carcinoma and the CC531 rat colon-carcinoma tumor cell lines. In addition, the metabolites showed reduced myelotoxic effects as compared with paclitaxel in an in vitro hemopoietic progenitor toxicity assay. Our procedure for the isolation and purification of paclitaxel metabolites in milligram quantities should be useful for testing the biological activities of these compounds and for the preparation of calibration standards essential for pharmockinetics studies.     

Construction,quality assurance and calibration of spherical isotropic fibre optic light diffusers

Spherical isotropic fibre optic light diffusers are used in photodynamic therapy either as a light source or as a light detector. The construction of light diffusers using different materials is described, viz. an optical method involving local polymerization of a dental fissure sealant, which is referred to as the Henderson method, and a second method using plastic or ceramic pre-fabricated spheres. Quality tests necessary for reliable clinical use are presented for the mechanical strength, output power and isotropy. The maximum pull-off force and blow-off output power for the different kinds of diffusers were determined. The calibration procedures are given for measurement of the output power and wavelength of the light emitted by a diffuser and for measurement of the fluence rate by a light-detecting diffuser, using a compact integrating sphere device. With all types of diffusers described, an isotropy can be obtained of better than ± 20% measured over a 320° angle for spheres as small as 1 mm. Larger ceramic diffusers are particularly suitable for delivering high output powers. A 3-mm-diameter ceramic diffuser mounted on a 600-m-core fibre can emit up to 5 W of continuous wave (CW) visible light in air. Diffusers used for light detection can measure the light fluence rate in tissue with 15% accuracy or better if calibration factors are determined for each individual probe.