Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits.

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.

Neoplastic disorders of prostate glands in the light of synchrotron radiation and multivariate statistical analysis.

The prostate gland is the most common site of neoplastic disorders in men. The pathogenesis of inflammatory cells, prostatic intraepithelial neoplasia (PIN) lesions, and prostate cancer is still under investigation. Inflammatory cells by producing free radicals are considered as major and universal contributors to cancerogenesis. PIN is regarded as a precursor lesion to prostate cancer or a marker signaling the vulnerability of the epithelium to neoplastic transformation [1]. Differentiation markers that are frequently changed in early invasive carcinoma are also changed in PIN lesions. In this study, prostate tissue samples obtained during surgical operation and classified as various disease states (inflammation, PIN lesions, and cancer) were examined. The samples were measured by means of microbeam synchrotron-radiation-induced X-ray emission (micro-SRIXE). Special attention was paid to examine the relationship between the earlier-mentioned disorders and changes in relative concentrations of S, K, Ca, Fe, Cu, and Zn. Applying the image-processing program ImageJ enabled us to select the areas of interest from two-dimensional maps of various prostate samples according to the histopathologist's evaluation. Detailed analysis of micro-SRIXE spectra based on multivariate methods shows significant differences between elemental concentrations in inflammatory cells, PIN lesions, and cancerous tissues, which confirms that this method can be used to distinguish various pathological states in prostate tissues. Information obtained in this way may provide better understanding of the biochemistry of unhealthy prostate tissues, thus opening the way to find new medicines/treatments to prevent or slow down some harmful intracellular processes.

Correlation of concentrations of selected trace elements with Gleason grade of prostate tissues.

The causes of prostate cancer are still obscure but some evidence indicates that there is a close connection between several trace elements and processes which may lead to malignant cells. In our study the microbeam synchrotron radiation X-ray fluorescence emission (micro-SRIXE) technique was applied for quantitative analysis of selected elements. For the first time, we correlate the concentrations of Mn, Fe, Cu, and Zn with the clinical stage of the prostate cancer at the time of operation (described by Gleason grade). Serial sections of prostate tissues were collected from patients undergoing radical prostatectomy. One section, stained with hematoxylin and eosin, was prepared for histopathological analysis; a second, adjacent unstained section was used in micro-SRIXE experiments. All experiments were performed at beamline L at HASYLAB, DESY, Germany. Our results seem to be valuable in light of the determination of the changes in the concentrations of trace elements as a potential diagnostic marker and their etiological involvement in the different stages of prostate diseases.

Development of galanin-containing nerve fibres in rat tibia.

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non-neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin-containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin-immunoreactive (GAL-IR) nerve fibres. The earliest, sparse GAL-IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL-IR fibres were also seen in the bone marrow cavity and in the region of the inter-condylar eminence of the knee joint. Intramedullary GAL-IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL-IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin-containing nerve fibres resemble the development of substance P- and CGRP-expressing nerve fibres, thus suggesting their sensory origin.

Inhibition of five lipoxygenase activating protein (FLAP) by MK-886 decreases atherosclerosis in apoE/LDLR-double knockout mice.

BACKGROUND: Recent reports point to an important role of leukotrienes in atherogenesis. Leukotrienes are produced by 5-lipoxygenase co-operating with five lipoxygenase activating protein (FLAP). We hypothesized that MK-886, an inhibitor of FLAP, could attenuate the development of atherosclerosis in the atherogenic apolipoprotein E/low density lipoprotein receptor (apoE/LDLR) double knockout (DKO) mouse model. MATERIALS AND METHODS: Female apoE/LDLR-DKO mice at the age of 8 weeks were put on Western diet. The experimental group (n = 10) received the same diet as the control group (n = 10), but mixed with MK-886 (Merck, Rahway, NJ) at a dose of 4 microg per 100 mg of body-weight per day. At age 6 months the mice were sacrificed under anaesthesia. RESULTS: Measured by the en face method, the percentage of area occupied by lesions in aortas in the control group was 25.15 +/- 2.9%, whereas in the MK-886-treated group it was 11.16 +/- 0.7% (P < 0.05). Lesion area measured by cross-section of aortic roots was 455,494 +/- 29,564 microm(2) in the control group versus 263,042 +/- 20,736 microm(2) in the MK-886-treated group (P < 0.05). The MK-886 did not change the plasma cholesterol lipoprotein profile as compared with the control mice. Finally, we show that MK-886 may increase plaque stability by decreasing the macrophage content as well as increasing the collagen and smooth-muscle cell content. CONCLUSIONS: Our results show for the first time that inhibition of FLAP by MK-886 reduces development of atherosclerosis in gene-targeted apoE/LDLR-DKO mice.

One-dimensional iterative algorithm for three-dimensional point-spread function engineering.

We present a new method with which to binarize pupil filters designed to control the three-dimensional irradiance distribution in the focal volume of an optical system. The method is based on a one-dimensional iterative algorithm, which results in efficient use of computation time and in simple, easy to fabricate binary filters. An acceptable degree of resemblance between the point-spread function of the annular binary filter and that of its gray-tone counterpart is obtained.

Development of the innervation of long bones: expression of the growth-associated protein 43.

It has been known from clinical and experimental observations that the peripheral nervous system is involved in the development of long bones. Expression of growth-associated protein 43 (GAP-43/B-50) was found in axonal growth cones during embryonic and postnatal ontogeny as well as in regenerating axons after nerve injury. The aim of the present study was to examine the occurrence of growing nerve fibers in rat tibia from gestational day 16 (GD 16) to postnatal day 28 (PD28). An indirect immunoenzymatic reaction using antibodies raised against GAP-43 was applied to detect outgrowing nerve fibers penetrating into the developing bone. On GD 16 and GD 17 no GAP-43-immunoreactive (IR) fibers were observed in the close vicinity of bone rudiments. On GD19 GAP-43-IR fibers were scarcely present within the periosteum of the central portion of the diaphysis. In the perichondrium surrounding the proximal epiphysis, nerve fibers were first detected around birth. From PD1 onward, numerous fibers were seen in the fibrous buds of the perichondrium at the epi-metaphyseal junction (Ranvier's grooves), some of them being adjacent to the blood vessels. Nerve fibers penetrating into the bone and located in the bone marrow, predominantly associated with blood vessels, were first observed on GD21 and their number increased with further development. They were initially located in the central portion of the diaphysis and later extended towards the metaphyses. On PD4 an increased number of GAP-43-IR fibers appeared in the perichondrium of proximal and distal epiphyses. In the fibrous strands penetrating into the epiphyses and in the secondary ossification centers, nerve fibers were first observed on PD10. From PD14 onward the pattern of tibial innervation remained unchanged but the intensity of GAP-43 immunostaining visibly decreased. The present study demonstrates that developing long bones of rat hindlimbs are supplied by growing nerve fibers immunoreactive for GAP-43 from GD 19 onward. Time and location of their appearance were at least partially correlated with known events taking place during long bone development, e.g. formation of primary and secondary ossification centers. Decreased expression of GAP-43 immunoreactivity in later developmental stages is believed to reflect nerve fiber maturation.

[Calcification of allogenic aortic heart valves]. / Mineralizacja allogennych zastawek aortalnych serca.

The material used for the studies consisted of allogenic aortic valves (AAV) collected from 14 individuals. The necessity of AAV replacement arose from growing circulation insufficiency and AAV dysfunction. The aim the study was the determination of the elemental composition and crystallographic structure of the inorganic deposits in AAV. Moreover, the results of the physicochemical investigations were correlated with clinical data (age of the patient, time between valve replacement surgeries, endomyocarditis, number of infections during last 12 months, arterial hypertension and disturbance of the lipid balance) and with echocardiographic examinations (cusp mineralization and perforation, vegetation, systolic and diastolic dimensions of the left ventricle, maximal and average gradient through allograft valve as well as range of the recoil wave to left ventricle). It was found that mineralization of the AAV cusps was a time-dependent process and took place predominantly at the surface of the cusp. The elemental composition and crystallographic data revealed that the inorganic deposits in AAV were composed of hydroxyapatite crystals. However, the presence of other calcium salts was also found. The development of the mineralization process in AAV does not correlate with endomyocarditis, arterial hypertension and the disturbance of the lipid balance. Probably, endomyocarditis and arterial hypertension induce the pathologic alternations of AAV independently from the mineralization process. The echocardiographic estimations of the pathomorphologic changes of the aortic valve cups are not always consistent with the results of the physicochemical studies.

Optical-digital method of local histogram calculation by threshold decomposition.

A theorem for calculating the local histograms of a gray-scale input image by means of convolution of input-image binary slices with a binary kernel is presented and proved. The calculation of the local histograms of a gray-scale image for all resolution cells and its arbitrary neighborhoods is optically implemented in a shadow-casting correlator. The choice of different rank-order values from the local histograms can lead to a wide spectrum of nonlinear filtration algorithms.

Calcification of the aortic wall in hypercalcemic rabbits.

The mineralization process was investigated in the aortic wall of hypercalcemic rabbits. The elevated calcium level in serum was induced by intramuscular injection of vitamin D3. The animals were killed at different times of the experiment (max. 246 d). The freeze-dried tissue homogenates were used for elemental composition studies by means of proton induced X-ray emission (PIXE) and atomic absorption spectroscopy. The structural information was obtained from infrared (IR) and X-ray diffraction (XRD) spectra. Moreover, the ascending part of the aortic arch was separated and used for micro-PIXE (PIXE in combination with proton microprobe) and histochemical examinations. It was found that hypercalcemia (blood serum Ca content elevated by about 20%) induced calcification of the aortic wall. The mineral phase within the aortic wall consisted of Ca-P salts. The Ca/P ratio continuously increased during the experiment and approached 2 after 246 d of the vitamin D3 treatment. The IR and XRD studies made possible the identification of the complex phase composition of the samples. The hydroxyapatite crystals were detected after 196 days, however, in earlier phases of the experiment, amorphous calcium phosphate, dicalcium phosphate dihydrate and octacalcium phosphate were also observed. On the basis of the data obtained, the mechanism of the precipitation and growth of inorganic deposits in the tunica media of the aortic wall was discussed.

Physicochemical characterization of the inorganic phases in the aortic wall of young individuals.

The autopsy samples of human aortic wall were investigated to determine the structure of the inorganic deposits formed at the onset of the mineralization process. The studies were concentrated on the material for which histochemical staining of randomly selected sections did not reveal the presence of minerals. The highly mineralized human media samples as well as broad spectrum of model compounds were investigated for comparative purposes. It was found that the inorganic phase at the onset of tunica media mineralization is composed of a few compounds including octacalcium phosphate as the predominant Ca-P compound, and a Mg-compound, probably magnesium carbonate or double/triple salts containing magnesium carbonate.

Calcification of aortic wall in cholesterol-fed rabbits.

Development of the mineralization process in the course of atherogenesis was studied using the cholesterol-fed rabbit model. The aorta samples were investigated by means of proton and electron microprobes, infrared spectroscopy and X-ray diffraction as well as selected histochemical staining. Blood serum was analysed every 2 weeks to determine the content of cholesterol, triglycerides, inorganic phosphorus, ionized calcium, elemental composition as well as activity of alkaline phosphatase. It was found that the administered diet did not disturb the calcium and phosphorus homeostasis. Histochemical findings confirmed the formation of lipid-rich lesions blocking the lumen of the vessel. The dystrophic calcification was observed only in the atheroma, while in the tunica media a slight mineralization similar to that found in controls was observed after 210 days of the diet. In the atheroma the only phase detected was a defective hydroxyapatite. The perfection of the crystals, as well as the diameter of the deposits, increased during the course of the diet reaching about 2 microns after 210 days. The crystals were not contaminated with carbonate groups regardless of the duration of the diet.

Developing an artificial fallopian tube: successful in vitro trials in mice.

This report describes the design and testing of an artificial fallopian tube for the treatment of tubal infertility. Within the device, mouse eggs incubated with sperm were fertilized and a microinfusion pump was used to transport the fertilized ova through the tube. Normal offspring resulted from transfer of the developing embryos into pseudopregnant recipients. These results provide encouraging evidence that an artificial fallopian tube warrants further investigation as a potential alternative to in vitro fertilization.

Intramembranaceous ossification analyses by a proton microprobe.

The proton induced X-ray emission method in combination with a proton microprobe was applied to study the intramembranaceous ossification. As material sections of mouse embryo skulls from the 17th and 19th day of gestation were used. The morphology of the sample was examined by routine histochemical procedure performed on the sections adjacent to that irradiated by the proton microprobe. The measurements were made in line scan and raster scan mode. The concentrations of P, S, Cl, K, Ca, Fe and Zn were determined at each irradiated point. The average element concentrations were calculated for four parts of each section (bone, cartilage, mesenchymal tissue close to the bone and mesenchymal tissue in other places). The distributions of Ca and P (less markedly than Ca) concentrations almost exclusively correlate with localization of the bone while S, Cl and K concentrations show preference to the cartilage. The amount of inorganic material in flat bones of the 17-day embryo amounts to 14% of the dry mass. The material is characterized by a Ca/P ratio of about 1.6. In the embryo 2 days older the amount of the inorganic phase is practically the same (15%) while the Ca/P ratio approaches 2. This suggests the presence of the precursor phase in the flat bone calcification. It is possible that octacalcium phosphate (Ca/P ratio equals to 1.72) is formed at the onset of the flat bone mineralization which transforms rapidly (in 2 days) to a more stable mineral (defective hydroxyapatite).

Peroxidase activity in peritoneal macrophages of patients with terminal renal failure treated by intermittent peritoneal dialysis.

Peroxidase activity was assessed cytochemically in macrophages from peritoneal dialysate of 50 patients with terminal renal failure treated by intermittent peritoneal dialysis. In 22 patients cells were harvested for the second time after development of peritonitis as the complication of the treatment. The obtained results were compared with those of the control group consisting of 30 patients with normal renal function. Patients treated with peritoneal dialysis showed both in complication-free period and during the course of peritonitis significantly higher peroxidase activity in macrophages as revealed by the semiquantitative score test. In the course of peritonitis, peroxidase activity in macrophages was significantly lower than in complication-free period. Electron-microscopic cytochemistry demonstrated that in macrophages obtained from both control and dialyzed patients the peroxidase activity was located exclusively in cytoplasmic granules, suggesting a similarity of the studied cells and exudative macrophages.

Artery wall calcification: correlation of atherosclerosis with mineralization.

Popliteal arteries from 14 individuals (17-85 y old) were investigated. The concentrations and localizations of P, S, Cl, K, Ca, Fe, Cu, Zn and Br were measured by means of PIXE and micro-PIXE methods. The presence of PO4(3-) and CO3(2-) groups was assessed using the IR technique. The amount of P and Ca increased with age approaching at places 9% and 20% and mineral deposits were detected in tunica media. At the same time an increase in the Ca/P ratio and in the crystallinity of deposits was observed. The samples from old individuals also contained more CO3(2-) groups. The concentrations and localization of Zn and Br showed artery wall layer-dependent changes. In some places of the artery wall, minerals were also found in young persons. They were not correlated with places of blood vessel branching.

Changes in activity of selected lysosomal enzymes in peritoneal macrophages of renal failure patients on peritoneal dialysis.

Activity of acid phosphatase (AP), beta-glucuronidase (GR), N-acetyl-beta-D-glucosaminidase (GZ), and peroxidase (P) was assessed using a semiquantitative cytochemical method in peritoneal macrophages of 30 patients with end-stage renal failure treated by intermittent peritoneal dialysis and of 30 control patients with normal renal function. The dialysed patients showed a significantly higher activity of GR and P at the beginning of the treatment as compared with the respective activities observed in the control group and a further significant rise of these activities after 4 months of dialysis. Activity of AP at the beginning of the treatment was insignificantly lower than in the control group and the difference became significant at the end of the investigated period. There was no significant difference between the dialysed patients and the control group in the activity of GZ assessed at the beginning of the dialytic treatment and after 4 months of dialysis. A significant decrease in that activity was, however, observed in the course of dialysis.

The analysis of mineral deposits and proteoglycans content in the cartilage of mouse trachea using PIXE in combination with proton microprobe.

The tracheal cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and quantitative measurements of P, S, Cl, K, Ca, Fe, and Zn concentrations as well as direct and indirect assessment of glycosaminoglycans by measurement of S content and measurement of bound colloidal iron at pH = 1.8 has been performed. Adjacent sections were stained with the Hale method in Müller modifications and the v. Koss method for sulphated mucins and inorganic deposits respectively. It has been found that hyaline cartilage in trachea contains mineral deposits and that P + Ca amounts up to 22% of cartilage dry mass. The Ca/P ratio approaches 2 what indicates hydroxyapatite type crystals. The cartilage contains substantial amounts of S reflecting the presence of sulphate groups. It was found that the cartilage binds also colloidal iron at low pH. There is a good correlation between places with high amount of bound colloidal iron assessed by PIXE and places showing strong Prussian Blue staining. The Fe/S ratio was, however, much lower in the cartilage than in other tissues what indicates that the colloidal iron method does not give quantitative results. There were no regions showing substantial decrease in Fe/S ratio which we found previously as typical for degenerating and calcifying growth plate cartilage. This may be connected with a relatively low degree of calcification degree of the tracheal cartilage.