Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways.

The emerging field of in situ tissue engineering (TE) of load bearing tissues places high demands on the implanted scaffolds, as these scaffolds should provide mechanical stability immediately upon implantation. The new class of synthetic supramolecular biomaterial polymers, which contain non-covalent interactions between the polymer chains, thereby forming complex 3D structures by self assembly. Here, we have aimed to map the degradation characteristics of promising (supramolecular) materials, by using a combination of in vitro tests. The selected biomaterials were all polycaprolactones (PCLs), either conventional and unmodified PCL, or PCL with supramolecular hydrogen bonding moieties (either 2-ureido-[1H]-pyrimidin-4-one or bis-urea units) incorporated into the backbone. As these materials are elastomeric, they are suitable candidates for cardiovascular TE applications. Electrospun scaffold strips of these materials were incubated with solutions containing enzymes that catalyze hydrolysis, or solutions containing oxidative species. At several time points, chemical, morphological, and mechanical properties were investigated. It was demonstrated that conventional and supramolecular PCL-based polymers respond differently to enzyme-accelerated hydrolytic or oxidative degradation, depending on the morphological and chemical composition of the material. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the investigated supramolecular materials, while, in contrast, the latter materials are more susceptible to oxidative degradation. Given the observed degradation pathways of the examined materials, we are able to tailor degradation characteristics by combining selected PCL backbones with additional supramolecular moieties. The presented combination of in vitro test methods can be employed to screen, limit, and select biomaterials for pre-clinical in vivo studies targeted to different clinical applications.

Beam intensity modulation to reduce the field sizes for conformal irradiation of lung tumors: a dosimetric study.

PURPOSE: In conformal radiotherapy of lung tumors, penumbra broadening in lung tissue necessitates the use of larger field sizes to achieve the same target coverage as in a homogeneous environment. In an idealized model configuration, some fundamental aspects of field size reduction were investigated, both for the static situation and for a moving tumor, while maintaining the dose homogeneity in the target volume by employing a simple beam-intensity modulation technique. METHODS AND MATERIALS: An inhomogeneous phantom, consisting of polystyrene, cork, and polystyrene layers, with a 6 x 6 x 6 cm3 polystyrene cube inside the cork representing the tumor, was used to simulate a lung cancer treatment. Film dosimetry experiments were performed for an AP-PA irradiation technique with 8-MV or 18-MV beams. Dose distributions were compared for large square fields, small square fields, and intensity-modulated fields in which additional segments increase the dose at the edge of the field. The effect of target motion was studied by measuring the dose distribution for the solid cube, displaced with respect to the beams. RESULTS: For the 18-MV beam, the field sizes required to establish a sufficient target coverage are larger than for the 8-MV beam. For each beam energy, the mean dose in cork can significantly be reduced (at least a factor of 1.6) by decreasing the field size with 2 cm, while keeping the mean target dose constant. Target dose inhomogeneity for these smaller fields is limited if the additional edge segments are applied for 8% of the number of monitor units given with the open fields. The target dose distribution averaged over a motion cycle is hardly affected if the target edge does not approach the field edge to within 3 mm. CONCLUSIONS: For lung cancer treatment, a beam energy of 8 MV is more suitable than 18 MV. The mean lung dose can be significantly reduced by decreasing the field sizes of conformal fields. The smaller fields result in the same biological effect to the tumor if the mean target dose is kept constant. Intensity modulation can be employed to maintain the same target dose homogeneity for these smaller fields. As long as the target (with a 3 mm margin) stays within the field portal, application of a margin for target motion is not necessary.

Planning, computer optimization, and dosimetric verification of a segmented irradiation technique for prostate cancer.

PURPOSE: To develop and verify a multisegment technique for prostate irradiation that results in better sparing of the rectal wall compared to a conventional three-field technique, for patients with a concave-shaped planning target volume (PTV) overlapping the rectal wall. METHODS AND MATERIALS: Five patients have been selected with various degrees of overlap between PTV and rectal wall. The planned dose to the ICRU reference point is 78 Gy. The new technique consists of five beams, each having an open segment covering the entire PTV and several smaller segments in which the rectum is shielded. Segment weights are computer-optimized using an algorithm based on simulated annealing. The score function to be minimized consists of dose-volume constraints for PTV, rectal wall, and femoral heads. The resulting dose distribution is verified for each patient by using point measurements and line scans made with an ionization chamber in a water tank and by using film in a cylindrical polystyrene phantom. RESULTS: The final number of segments in the five-field technique ranges from 7 to 9 after optimization. Compared to the standard three-field technique, the maximum dose to the rectal wall decreases by approximately 3 Gy for patients with a large overlap and 1 Gy for patients with no overlap, resulting in a reduction of the normal tissue complication probability (NTCP) by a factor of 1.3 and 1.2, respectively. The mean dose to the PTV is the same for the two techniques, but the dose distribution is slightly less homogeneous with the five-field technique (Average standard deviation of five patients is 1.1 Gy and 1.7 Gy for the three-field and five-field technique, respectively). Ionization chamber measurements show that in the PTV, the calculated dose is in general within 1% of the measured dose. Outside the PTV, systematic dose deviations of up to 3% exist. Film measurements show that for the complete treatment, the position of the isodose lines in sagittal and coronal planes is calculated fairly accurately, the maximum distance between measured and calculated isodoses being 4 mm. CONCLUSIONS: We developed a relatively simple multisegment "step-and-shoot" technique that can be delivered within an acceptable time frame at the treatment machine (Extra time needed is approximately 3 minutes). The technique results in better sparing of the rectal wall compared to the conventional three-field technique. The technique can be planned and optimized relatively easily using automated procedures and a predefined score function. Dose calculation is accurate and can be verified for each patient individually.

Image cytometry characterization of ploidy level, proliferative activity and chromatin pattern in 50 nasal polyps.

A computer-assisted microscope analysis of Feulgen-stained nuclei was carried out on a series of 50 nasal polyps in order to try to identify specific biological subgroups. The present series of 50 nasal polyps includes single polyps both associated (n=9) and unassociated (n=9) with allergy and diffuse polyposis both associated (n=7) and unassociated (n=9) with allergy, cystic fibrosis (n=9) and ASA (aspirin-sinusitis-asthma) related polyposis (n=7). The computer-assisted microscope analysis provides 36 quantitative variables which include 1 variable describing proliferative activity, 9 describing the nuclear desoxyribonucleic acid distribution (DNA ploidy level) and 26 describing nucleus morphology, i.e. its size and chromatin pattern. The results show that the methodology proposed here enabled four major groups of nasal polyps to be identified, i.e. diffuse polyposis associated with allergy, cystic fibrosis-related polyposis, single polyps both associated and unassociated either with allergy and a fourth group including diffuse polyposis not associated with allergy and ASA-related polyposis. These four groups of nasal polyps differed markedly in their morphonuclear characteristics, but not in the proliferative activity- and DNA ploidy-related variables.

Resistance of androgen-mediated aggressive behavior in mice to flutamide, an antiandrogen.

Flutamide (FTA), an anti-androgenic compound, inhibited the effects of methyltestosterone (MT) on the weight of the ventral prostate, seminal vesicles and levator ani in male castrate mice. Castration prevented the development of aggressive behavior in mice isolated for 3 weeks. While chronic administration of MT to castrate isolated mice returned the incidence of fighting behavior to control values, chronic administration of FTA + MTdid not significantly reduce the incidence of fighting as compared to castrate + MT values. These results suggest that the mechanism for androgen stimulation of secondary sex organ weight may differ from that involved in the development and maintenance of aggression resulting from isolation.

A reanalysis of liver cancer incidence in Danish patients administered thorotrast using a two-mutation carcinogenesis model.

In recent years, a two-mutation carcinogenesis (TMC) model has been used to analyze epidemiological data and estimate the radiation risks at low doses for the organs affected. Here the TMC model was used to reanalyze the liver cancer incidence in the Danish population in general and in patients administered Thorotrast, and to estimate the radiation risks for the liver. The data for 807 patients for whom sufficient data on the injected volumes of Thorotrast were available were used in this reanalysis. These data were combined with data on liver cancer incidence in the Danish population as the baseline or background incidence. Because males and females show different baseline liver cancer incidences, separate fits were made for males and females. The fits showed that the radiation effect could be ascribed entirely to the radiation dependence of the first mutation rate of the TMC model, which was higher for females than for males. The second mutation rate was not significantly dependent on dose. The radiation risks for the liver were calculated on the basis of the model parameters. These risks for lifetime exposures are about the same for males and females and are between a factor of 2 and 10 higher than current estimates. The discrepancy between the model results and previous risk estimates probably arises because the model calculations give more complete lifetime radiation risk estimates. For short-term exposures of the liver to ionizing radiation, the maximum radiation-induced excess liver cancer risk per unit dose applies to exposures at the age of about 10; exposures at ages above 35 have a radiation effect of less than approximately 15% of this maximum.

Dosimetric verification of the 95% isodose surface for a conformal irradiation technique.

In the treatment planning of conformal radiotherapy, field shapes are often designed in such a way that a high-value isodose surface fully encompasses the target volume. Therefore, knowledge about the accuracy with which the treatment planning system calculates the position of that isodose surface is essential to prevent field shapes which are either too large or too small. To determine this accuracy for a conformal multi-field technique, the dose in the high-dose region must be measured with a high spatial resolution. A method is presented to reconstruct and evaluate the experimental high-dose region from a set of water phantom scans. This method, which assesses combined dose profiles for multi-field irradiation techniques, can be used for the commissioning and/or quality assurance of a 3-D treatment planning system. For a specific conformal technique, the measured and calculated 95% isodose positions along lines in several directions have been compared. It is shown that different dose values of single beam profiles determine the resulting 95% isodose position, which is important to recognize for quality assurance of treatment planning calculations. It is further found that the uncertainty in the calculated 95% isodose surface can be described by a standard deviation in dose value, which relates to a positional uncertainty through the local dose gradient. Thus the confidence region of the calculated 95% isodose can be indicated in the treatment plan by plotting isodoses at the 95% level plus and minus its standard deviation. Such a procedure is recommended instead of plotting the 95% isodose with a constant width. In addition, restrictions for the cumulative dose-volume histogram of acceptable treatment plans can be formulated, based on the sensitivity of the actual target coverage on the uncertainty with which the prescribed isodose surface is calculated.

Quality assurance of the dose delivered by small radiation segments.

The use of intensity modulation with multiple static fields has been suggested by many authors as a way to achieve highly conformal fields in radiotherapy. However, quality assurance of linear accelerators is generally done only for beam segments of 100 MU or higher, and by measuring beam profiles once the beam has stabilized. We propose a set of measurements to check the stability of dose delivery in small segments, and present measured data from three radiotherapy centres. The dose delivered per monitor unit, MU, was measured for various numbers of MU segments. The field flatness and symmetry were measured using either photographic films that are subsequently scanned by a densitometer, or by using a diode array. We performed the set of measurements at the three radiotherapy centres on a set of five different Philips SL accelerators with energies of 6 MV, 8 MV, 10 MV and 18 MV. The dose per monitor unit over the range of 1 to 100 MU was found to be accurate to within +/-5% of the nominal dose per monitor unit as defined for the delivery of 100 MU for all the energies. For four out of the five accelerators the dose per monitor unit over the same range was even found to be accurate to within +/-2%. The flatness and symmetry were in some cases found to be larger for small segments by a maximum of 9% of the flatness/symmetry for large segments. The result of this study provides the dosimetric evidence that the delivery of small segment doses as top-up fields for beam intensity modulation is feasible. However, it should be stressed that linear accelerators have different characteristics for the delivery of small segments, hence this type of measurement should be performed for each machine before the delivery of small dose segments is approved. In some cases it may be advisable to use a low pulse repetition frequency (PRF) to obtain more accurate dose delivery of small segments.

Histochemical study of the epithelia of nasal polyps by biotinylated lectins and neoglycoprotein. A comparison with the normal human respiratory epithelium.

Formalin-fixed, paraffin-embedded sections from human nasal polyps and the normal respiratory epithelium were glycohistochemically investigated. Three biotinylated lectins were used: peanut (Arachis hypogaea) agglutinin (PNA) which, binds to terminal galactose (beta 1-3) N-acetylgalactosamine residues that can be unmasked by a neuraminidase digestion; wheat germ (Triticum vulgare) agglutinin (WGA), which binds to N-acetylglucosamine and N-acetylneuraminic acids; and gorse seed (Ulex europaeus) agglutinin (UEA-1), which binds to L-fucose. In addition, the presence of accessible galactose (beta 1-3) N-acetylgalactosamine (T-antigen) glycan receptors (endolectins) was also assessed. The avidin-biotin-peroxidase complex (ABC) and diaminobenzidine (DAB) were used as chromogen. The ciliated cells of the normal respiratory epithelium and those of the pseudostratified epithelium of nasal polyps possess similar glycohistochemical characteristics suggesting no major alterations on the level of lectin-reactive carbohydrate epitopes as indicators of cellular glycosylations. Notably, this parameter can respond sensitively to changes in cell differentiation or activation. The basal and mucus-secreting cells in the two epithelia display different reactivity patterns emphasizing the presence of dissimilar sugar residues. Similarly, the dysplasia reflecting squamous epithelium of nasal polyps shows a distinct staining behaviour, indicative for disparate glycoconjugate display. Thus, quantitative differences in the lectin-selective staining of various cell types are detectable. The expression of T-antigen-bearing neoglycoprotein binding is weak and similar in both the normal epithelium and the pseudostratified epithelium lining nasal polyps. Only the most superficial cells of the squamous epithelium disclose a moderate labelling with this probe. These results indicate that further studies in this field are warranted, employing neoglycoproteins and also endolectins from human tissues to correlate glycobiological properties of the epithelium of the conducting airways and its diseased forms with functional features.

Lower radiation weighting factor for radon indicated in mechanistic modelling of human lung cancer.

A two-mutation carcinogenesis (TMC) model was fitted to the age-dependent lung cancer incidence in a cohort of Dutch Hodgkin patients treated with radiotherapy. Employing the results of previous TMC analyses of lung cancer due to smoking (by British doctors) and due to exposure to radon (for Colorado miners) a model fit was obtained with an estimate for the low LET radiation effect at the cellular level. This allows risk calculations for lung cancer from low LET radiation. The excess absolute risks are in tune with the values reported in the literature, the excess relative risks differ among the exposed groups. Comparing the cellular radiation coefficients for radon and for low LET radiation leads to an estimated radiation weighting factor for radon of 3 (0.1-6).

Implications of the analysis of epidemiological data using a two-mutation carcinogenesis model for radiation risks.

A two-mutation carcinogenesis (TMC) model is used as a bridge between cellular radiation biological effects and the incidence of cancer. This model has been applied to several sets of experimental animal and epidemiological data. In this paper the advantage of the model and the implications for radiation risks at low doses are discussed with respect to the age and dose dependence of cancer incidence and the effect of age at exposure on radiation risk; the link between the radiation effect and background cancer incidence and the transfer of radiation risk across different population groups; the implications of acute and protracted radiation exposures for risks at low doses and the dose-effect relationship for radium induced bone cancer.

Analysis of epidemiological cohort data on smoking effects and lung cancer with a multi-stage cancer model.

A stochastic two-stage cancer model is used to analyse the relation between lung cancer and cigarette smoking. The model contains the main rate-limiting stages of carcinogenesis, which include initiation, promotion (clonal expansion of initiated cells), malignant transformation and a lag time for tumour formation. Various data sets were used to test the model. These include the data of a large prospective collaborative project carried out in 10 different European countries, the European Prospective Investigation into Cancer and Nutrition (EPIC). This new data set has not been modelled before. The model is also tested on other published data from CPS-II (Cancer Prevention Study II) of the American Cancer Society and the British doctors' study. The analyses indicate that the EPIC data are best described with smoking dependence on the rates of malignant transformation and clonal expansion. With increasing smoking rates, saturation effects in the two exposure rate-dependent model parameters were observed. The results find confirmation in the biological literature, where both mutational effects and promotional effects of cigarette smoke are documented.

Calculation of the 1995 lung cancer incidence in The Netherlands and Sweden caused by smoking and radon: risk implications for radon.

A two-mutation carcinogenesis model was used to calculate the expected lung cancer incidence caused by both smoking and exposure to radon in two populations, i.e. those of the Netherlands and Sweden. The model parameters were taken from a previous analysis of lung cancer in smokers and uranium miners and the model was applied to the two populations taking into account the smoking habits and exposure to radon. For both countries, the smoking histories and indoor radon exposure data for the period 1910-1995 were reconstructed and used in the calculations. Compared with the number of lung cancer cases observed in 1995 among both males and females in the two countries, the calculations show that between 72% and 94% of the registered lung cancer cases may be attributable to the combined effects of radon and smoking. In the Netherlands, a portion of about 4% and in Sweden, a portion of about 20% of the lung cancer cases (at ages 0-80 years) may be attributable to radon exposure, the numbers for males being slightly lower than for females. In the Netherlands, the proportions of lung cancers attributable to smoking are 91% for males and 71% for females; in Sweden, the figures are 70% and 56%, respectively. The risk from radon exposure is dependent on gender and cigarette smoking: the excess absolute risk for continuous exposure to 100 Bq m-3 ranges between 0.003 and 0.006 and compares well with current estimates, e.g. 0.0043 of the International Commission on Radiological Protection (ICRP). The excess relative risk for continuous exposure to 100 Bq m-3 shows a larger variation, ranging generally between 0.1 for smokers and 1.0 for non-smokers. The results support the assumption that exposure to (indoor) radon, even at a level as low as background radiation, causes lung cancer proportional to the dose and is consistent with risk factors derived from the miners data.

An analysis of bone and head sinus cancers in radium dial painters using a two-mutation carcinogenesis model.

Bone and head sinus cancer incidence after ingestion of 226Ra and 228Ra by radium dial painters is analysed using a two-mutation clonal expansion model for radiation carcinogenesis, taking into account the retention and radiation patterns of these nuclides in the body. The best fit is obtained for compact bone retention and efficient diffusion of 222Rn to the bone cavities and radiation action on both mutation rates of the cancer model, as found in a similar analysis of bone sarcomas after 226Ra injection in beagles. The model parameters of the best fit are consistent with cellular radiobiological data and a previous analysis of lung cancer in uranium miners. Due to the low background incidence of bone and head sinus cancer, the resulting dose-effect relationships for these cancers are linear-quadratic with radium ingestion and alpha radiation dose. These results do not support a threshold dose concept, but the risks at low doses calculated by the model come out to about a factor 10 lower than using a linear extrapolation of the data to low doses, a procedure currently applied by ICRP and EPA. Furthermore, the model results indicate radiation risks at low doses to be related with background cancer incidence between relative and absolute radiation risk projections. The results, which are dependent on the model assumptions, might be more generally applicable for bone seekers and will therefore need further study to arrive at better radiation risk estimations.

Selective adhesion and impaired adhesive properties of transformed cells.

Quantitative studies on the adhesive properties of transformed cells have yielded inconclusive and sometimes contradictory results. The present investigation has examined adhesive interactions between normal human fibroblasts, established as well as virus-transformed animal cell lines, and human tumour-derived cell lines by the cell-cell layer binding assay. The results of these investigations indicate that adhesive selectivity can be observed between normal human fibroblasts and 2 human tumour-derived cell lines, providing an in vitro system to study cell surface components involved in cellular interactions between normal and malignant cells. In addition it is demonstrated that cell layers of transformed cells form a poorly adhesive substratum for both trypsinized normal and transformed cells. Furthermore, it is confirmed that the adhesive properties of transformed cells, including adhesive selectivity, are affected by the dissociation procedure (trypsin or EDTA). In view of the observations made by other investigators, the present results suggest that transformed cells display adhesive properties which can be quantitatively and reproducibly measured but which are modulated by the dissociation procedure as well as by the configuration in which the cells are at the time of the assay.

Growth and surface properties of dispase dissociated human fibroblasts.

The dissociation of human fibroblast cultures with the bacterial neutral protease (Dispase II) was monitored by viability and growth measurement and was compared to the effect of trypsin and EDTA. Cell suspensions with high viability and excellent growth were obtained after 10 min incubation at 37 degrees C in 4 U/ml dispase in 0.02% EDTA. A two to three-fold increase in mitotic index occurred in the cultures within 48 h after dispase dissociation. The initial rate of aggregation was comparable to that of trypsin or EDTA dissociated cells, but attachment to a substratum and agglutination by Wheat Germ Agglutinin were markedly enhanced. The results indicate that dispase-EDTA provides a valuable alternative to the enzymatic dissociation with trypsin. Moreover, it is an additional tool for the dissociation of cultured cells and for the study of the surface properties of single cells.

A consistent two-mutation model of lung cancer for different data sets of radon-exposed rats.

A two-mutation carcinogenesis model, formulated in terms of biologically motivated equations for mutation and expansion steps, has been applied in a mechanistic modelling of the lung cancer incidence in two large data sets of rats exposed to radon, both separately and jointly. Results indicate that (1) the equations employed are able to provide an accurate description of the separate data sets, (2) the parameters in the equations take on similar values for both data sets, and (3) it is possible to construct a consistent and well-fitting solution for the joint data set. It proved not to be necessary to take into account the effect of uranium ore dust, administered to part of the data or the different rat strains of the data sets. The joint solution provides a firm basis to investigate the effects of exposure, exposure rate and age at exposure on cumulative incidence, excess relative risk and excess absolute risk. For the same total exposure, cumulative incidence reaches a maximum for exposure rates between 1 and 10 WLM per day. The so-called inverse-exposure-rate effect acts for higher exposure rates. The influence of age at exposure, however, seems to be even more pronounced. Exposure at a young age leads to considerably higher incidences than exposure at a later age. Parameters derived in this study compare fairly well with those derived for uranium miners, suggesting that a consistent model description for the induction of lung cancer by radon in rats and humans may be possible.