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We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulindependent diabetes mellitns) patients and agematched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly reduced in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Three dimensional scaffolds microfabricated using pressure-assisted microsyringe (PAM) with controlled geometry and porous membranes obtained using salt leaching were both tested with three different cell types to identify an optimal microstructural architecture for tissue engineering. MG63 (osteoblast-like cells) were used as models of mesenchymal bone tissue and human endothelial cells and NCTC2544 (keratinocytes) represented two epithelial tissues. Both porosity and stiffness of PLGA structures were measured, and cell morphology and cytoskeletal organization analyzed using SEM and actin labeling. The results show that overall the PAM scaffolds, which have a repeated and regular microstructure, are more biocompatible than the random pore salt-leached membranes, and that surface morphology as well as substrate stiffness modulates cell behavior.

Highly crystalline chitin nanofibrils were isolated from crustacean chitin and characterized by X-ray diffraction and FT-infrared spectrometry. A novel formulation including chitin nanofibrils, chitosan glycolate, and chlorhexidine was manufactured in three presentations: spray, gel, and gauze. The latter included non-woven dibutyryl chitin as a biocompatible support. The products were tested in murine wound models, with phytostimuline-medicated wounds as controls, in two series of experiments, one of which included a concomitant laser treatment capable to activate cells. The gauze consistently induced better epithelial differentiation and keratinization, and better reorganization of the basal lamina. The angiogenetic response induced by the gauze was indicative of satisfactory vascular trophism irrespective of the laser treatment. Immunohistochemical observations showed that the actin cytoskeleton of dermal and epidermal cells was well formed. The gauze was used to treat 75 patients hospitalized for a variety of traumatic wounds with good results in all cases. A single dressing could be kept on site for at least four days, and the healing took place within periods of time similar to those reported for traditional dressings under comparable conditions; in no case secondary infections developed. In conclusion, the spray could be used as a first-aid tool on bleeding abrasions; the gel enhanced physiological repair and was recommended for areas with thin epidermal layers, and the gauze was found to be superior to other dressings insofar as no scar remained. A laser treatment could optionally be applied. The biochemical significance of the chitin/chitosan products was found to accompany their commercial viability in terms of time of healing and clinical labor costs.

Recent advances in process chemistry have made it possible to make chitosan and chitin nanofibril materials more flexible and useful for the development of new biorelated products. In this study, the effectiveness of three chitin nanofibril/chitosan glycolate-based preparations, a spray (Chit-A), a gel (Chit-B), and a gauze (Chit-C), in healing cutaneous lesions are assessed macroscopically and by light microscopy immunohistochemistry. These evaluations are compared to the results obtained using a laser co-treatment. The wound repair provided by the three preparations is clearly evident even without the synergistic effect of the laser co-treatment. These results confirm the effectiveness of chitin nanofibril/chitosan glycolate-based products in restoring subcutaneous architecture. The spray seems to be most effective in healing superficial lesions, including extensive ones; the gel is more effective in repairing shallow lesions as well as an aesthetic factor while the gauze is effective in slow-healing dermo-epidermal wounds.

Cells with a dendritic morphology and/or expression of dendritic cell (DC) markers have been repeatedly described in several human tumors, but the distribution and density of melanoma-associated DCs have not yet been reported. The aim of the present study is to analyze the density and topographical distribution of melanoma-associated DCs and their relation with CD3+, CD4+ and CD8+ T lymphocytes in forty cases of cutaneous human melanoma. In melanocytic tumours different pools of DCs were recognised in the epidermis and in the dermis, particularly in intimate relation with lymphocyte clusters inside the melanocytic proliferation, and more often at the edges of tumours. The number of Langerin-positive DCs showed an inverse correlation with tumour depth (correlation coefficient r= −0.59, P=0.0001) and was significantly lower in thick melanomas compared to thin and intermediate ones (P+ lymphocytes was inversely correlated to the depth of infiltration (r=−0.596, P

Background Merkel cell carcinoma (MCC) is a rare, aggressive cancer of the skin that mainly affects elderly patients. Because of its rarity, there is no established treatment or proven markers to guide therapy or prognosis. Immunohistochemical expression of apoptosis proteins is considered a useful marker of both malignancy and tumour progression. Apoptosis plays a fundamental role in skin homeostasis, and apoptotic cells have been detected in normal and diseased skin. Chemokines posses a wide range of biological activities and CXCR4 is expressed in some cancer cells, where it plays an efficient role in metastasis formation. Objective To identify immunohistochemical parameters that can help clinicians select the most suitable therapy for skin MCC. Design Antibodies against ki67, bcl-2, p53, survivin, p16 and CXCR4 were tested to assess the usefulness of these antigens as indices of proliferation potential and predictors of prognosis. Methods Immunohistochemical detection of apoptosis inhibitors and CXCR4 was performed on tissue from 12 patients with primary MCC. After excision of the primary lesion, five survived and had no metastases, and seven experienced local recurrence or lymph node metastases. Results Expression of ki67 and survivin was increased in patients with local recurrence or metastasis (retrospectively classified as ‘poor prognosis’) compared with those with a ‘good prognosis’, and bcl-2 expression was significantly greater (P = 0.003). P53 and p16 immunostaining was moderate in both groups. A positive correlation was observed between survivin and mutant p53 in the poor prognosis group (r = 0.593, P = 0.033; regression coefficient). High values of p53 were measured in patients with high levels of survivin and vice versa. CXCR4 was not detected at all. Conclusions Our results show strong MCC cell apoptosis inhibition and a high cell proliferation capacity. The positive correlation between survivin and p53 may be a predictor of MCC spread via the lymphatic network. Absent CXCR4 expression may reflect a less aggressive form, with less efficient development of distant and non-organ-selective metastasis formation.

Objective To evaluate Cdc42 expression in eutopic and ectopic endometrial tissue in patients with adenomyosis and ovarian endometriotic cysts compared with patients without endometriosis. Design Experimental retrospective study. Setting University hospital. Patient(s) Twenty-four patients with adenomyosis, 19 with ovarian endometriomata, and 9 with fibroids or benign ovarian cysts. Intervention(s) Hysterectomy and bilateral oophorectomy. Main Outcome Measure(s) Immunostaining for Cdc42 of eutopic and ectopic endometrial tissues. Result(s) In eutopic endometrium of patients with adenomyosis and with fibroids or benign ovarian cysts, the intensity of Cdc42 immunostaining was weaker, especially in the specialized stromal cells, compared with cases with ovarian endometriosis (χ 2 test, P =.003). Expression of Cdc42 in eutopic endometrium showed a trend to be higher in the secretory than in the proliferative phase and in patients with ovarian endometriotic cysts compared with patients with adenomyosis (unpaired t test, P =.005), especially in the proliferative phase. Conclusion(s) An abnormally high expression of Cdc42 in eutopic endometrium in the secretory phase may contribute to the development of ovarian endometriosis, but it does not seem to be involved in the pathogenesis of adenomyosis.

The doping of the apatite with carbonate or/and Mg ions in biologically-like amounts (6 and 1 wt.%, respectively) was performed. Chemico-physical characterizations and cell culture tests were carried out onto the synthetic Mg- and Mg,CO 3 -substituted (∼30–40 nm particle size) powders in comparison with stoichiometric HA (∼160 nm particle size) to determine as mesenchymal stem cells (MSCs) can directly use the mineral microenvironment to stimulate their own proliferation and differentiation activities. At the same time the growth of human osteoblast like cells (MG-63) was evaluated to determine the compatibility of the synthetic doped apatites for bone substitution. Cell morphology analysis by SEM as well as MTT and ALP tests were performed. The peculiar chemico-physical properties of the doped (Mg- and Mg,CO 3 -substituted) materials improved the behaviours of MSC and MG-63 cells in term of adhesion, proliferation and metabolic activation compared to stoichiometric HA.

Conventional renal cell carcinoma (RCC) is characterized by rich neovascularization and shows a fine vascular network around tumor cells. Nephron sparing surgery has been established as a method of choice or necessity for localized tumors. We investigated the importance of microvessel density (MVD), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (Flk-1) immunohistochemical expression in a large series of small conventional clear cell renal carcinomas treated with partial nephrectomy and assessed the prognostic value of their expression in terms of patients survival at long-term followup.A total of 48 patients with a mean age +/- SD of 58.2 +/- 9.5 years who had conventional single RCC were considered. Median tumor diameter was 2.92 +/- 0.82 cm (range 1.3 to 5). Disease was grades 1 to 4 in 15, 29, 2 and 2 patients, respectively. Median followup was 92.9 months (range 17 to 186).Four patients (3.9%) had died of metastatic renal cancer at a median followup of 23.5 months, of whom 1 had a grade 2, 1 had a grade 3 and 2 had grade 4 RCC. Patients with MVD expression higher than the median (44.4 vessels per mm) did not show a significant difference in survival compared to patients with MVD expression lower than the median. Patients with VEGF expression higher than 25% in the histological specimen showed worse survival than patients with VEGF expression lower than 25%. Different Flk-1 expression did not determine a significant difference in survival. On univariate analysis of patient survival in relation to the different considered factors Fuhrman grading was the most important factor for survival.Our study shows that recurrence and death are possible even in patients with small renal tumors. MVD, VEGF and Flk-1 expression do not depend on tumor size in pT1a RCC. Therefore, to date Fuhrman grading appears to be the only factor predictive of survival even in small RCC. Thus, Fuhrman grading is predictive of mortality. While VEGF is not predictive of survival as a single parameter, based on its percent of expression (lower or higher than 25%) it can determine 2 groups that are different from the prognostic point of view.

In this in vitro investigation of a heterogeneous range of bio-functional and modulatory performances of mesenchymal stem cells (MSCs) grown on two plasma-deposited acrylic acid (pdAA) coatings, which differed in chemical characteristics, a view of their potential utility as hybrid scaffolds for bone tissue engineering was obtained. The in vitro behavior of MSCs was compared to that of MG-63 cells, an osteoblast-like cell line that is commonly used to test biocompatibility of materials intended for bone-tissue interface. The coatings exerted a greater stimulus on MSCs and on MG-63 cells in comparison with control cultures. Both studied coatings exhibited satisfactory compatibility and modulatory effects on MSCs, thus they may be suitable for use in 2D or 3D scaffolds for bone tissue reconstruction.

Rapid prototyping, automatic image processing (computer-aided design (CAD)) and computer-aided manufacturing techniques are opening new and interesting prospects for medical devices and tissue engineering, especially for hard tissues such as bone. The development of a bone high-resolution scaffold prototype using these techniques is described. The results testify to the fidelity existing between microtomographic reconstruction and CAD. Furthermore, stereolithographic manufacturing of this scaffold, which possesses a high degree of similarity to the starting model as monitored by morphological evaluations (mean diameter 569 +/- 147 microm), represents a promising result for regenerative medicine applications.

Electroactive polymers such as polypyrrole (PPy) are receiving particular interest because they can combine inherent biological properties that can specifically trigger desired cellular responses for many biological applications. The aim of the present work was to evaluate more efficient solutions, achieved by different oxidisations of tosylate-doped PPy films in different chemico-physical conditions, in view of their long-term biomedical applications. The conductivity and the release of the tosylate ion were assessed. Morpho-functional studies were also performed. A gradual increase in impedance of the oxidised tosylate-doped PPy films soaked in MEM and a reduced conductivity after over-oxidation was found. Scanning electron microscopy (SEM) analysis showed the presence of a hierarchical organisation of the PPy films and a partially different morphology between oxidised and over-oxidised films. Cell behaviour was also randomly affected in relation to the oxidative state of the studied Ppy films. On the basis of the results, growth of these tosylate-doped Ppy films directly in the sol–gel matrix or on the surface of metal alloys could be hypothesised in order to improve bioactive materials of potential use in modern regenerative medicine. However, in view of their in vivo application, over-oxidation must be considered as it may represent the degraded material that is likely to form under a biological oxidising environment.

Water safety is an important quality objective, and special attention needs to be devoted to the pipes in which it runs. Rubber is one of the most widely used materials for drinking-water pipes. The various types of rubber are mixes often with vulcanising agents added to improve mechanical properties. The present study was conducted to evaluate and compare the behaviour of three currently used elastomer prototype mixes devised for use as water pipes before and after an artificial ageing procedure. Contact surface with the water was determined and “quality”-tested using SEM, AFM, FT-IR and biological (biocompatibility and bacterial adhesion) analyses. Differences in rubber composition between the native mixes were shown using FT-IR. Photo-ageing induced degradation of the surface film and in some cases gave rise to alkene derivatives with a small carbonyl component. Comparison of chemical, morphological and biological data before and after photo-ageing highlights the risk to which pipes are exposed over time even in the absence of specific chemico-physical insults.

Summary Background Alopecia areata (AA) is a relatively common inflammatory form of nonscarring hair loss of unknown pathogenesis, but possibly of autoimmune origin. Topical immunotherapy, using a potent contact allergen such as diphencyprone (DPC), is currently considered the most effective mode of treatment. However, the way in which DPC operates on hair follicles in AA still remains to be elucidated. Vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around hair follicles, and several studies provide evidence that apoptosis is a central element in the regulation of hair follicle and vascular regression. The cutaneous lymphocyte-associated antigen (CLA) and the skin-associated chemokine CCL27 highlight an important role for epithelial cells in controlling homeostatic lymphocyte trafficking. Objectives To determine the expression pattern of VEGF, factor (F)VIII, survivin, p16, CD4, CD8, CLA and CCL27 in alopecic skin before and after treatment with DPC. Methods Immunohistochemical staining methods were applied to skin biopsy specimens obtained from alopecic areas of 14 patients before and after DPC treatment and from five healthy subjects. Sections were incubated with monoclonal antibodies against VEGF, FVIII, survivin, p16, CCL27, CLA, CD4 and CD8, and their immunohistochemical expression was evaluated by light microscopy. Results The intensity of VEGF staining in alopecic human hair follicles was significantly lower than in healthy scalp tissue. FVIII immunostaining showed a significantly reduced development of the microvasculature in AA in comparison with healthy scalp tissue. After DPC therapy, cells of alopecic hair follicles showed a significant increase of VEGF immunopositivity, and the number of capillary vessels expressing FVIII was markedly increased in comparison with untreated scalp tissue. The increase in microvessels was associated with strong survivin expression in endothelial cells after treatment. All alopecic specimens showed expression of p16 in the hair follicle outer root sheath (ORS), with a significant increase after therapy. After treatment we observed a significantly decreased number of CD4+ cells and an increase of CD8+ cells (CD4/CD8 ratio 0·85) in alopecic skin compared with untreated scalp tissue (CD4/CD8 ratio 3·45). Most of the T lymphocytes found in inflammatory skin lesions expressed CLA antigen and after therapy we observed a significantly higher CLA positivity in hair follicles (50% or more) in comparison with untreated alopecic scalp tissue. Alopecic patients showed a CCL27 immunopositivity significantly lower than in normal scalp tissue. After DPC therapy the labelling intensity for CCL27 showed a significant increase both in the ORS and in the inner root sheath; similarly, in the basal interfollicular keratinocytes we observed a moderate increase in CCL27 expression. Conclusions Topical immunotherapy exerts an important role in angiogenesis, upregulating VEGF in human hair follicle keratinocytes and upregulating survivin to preserve endothelial cell viability. Moreover, it considerably alters the peribulbar CD4/CD8 ratio, restoring a condition close to normal scalp skin. Our study could contribute to explaining some aspects of AA pathogenesis that are still unknown and aid understanding of how DPC could act in this complex disease.

A new wound dressing made of gelatin and Ag, was evaluated for the management of tissue reconstitution in rats. The gelatin–based dressing, with metabolic stimulation properties that favored the recovery of the skin was made of stiff sheets to supply the repairingtissue with mechanical support. The gelatin sheets enhanced the development of collagen fibers and subsequently cell proliferation at the level of stromal and vascular elements. A protocol based on the utilization of natural biomaterials in the presence of low concentrations of a biocidal was proposed which could control microbiocidal activities principally in burned and debilitated patients.

BACKGROUND Vascular endothelial growth factor (VEGF), an endothelial cell mitogen, plays a hierarchical role in regulating physiologic and pathologic angiogenesis. Moreover, the transformation from noninvasive to invasive carcinomas is accompanied by focal disruption and discontinuity of the basement membrane. Several groups of proteases have been implicated in tumor cell invasion, including the 72-kDa gelatinase A/Type IV collagenase (matrix metalloproteinase 2 [MMP-2]) and the 92-kDa gelatinase B/Type IV collagenase (MMP-9). METHODS The authors assessed the immunohistochemical expression of VEGF and metalloproteinases MMP-2 and MMP-9 in paraffin embedded biopsy specimens of malignant melanomas (18 invasive melanomas and 10 in situ melanomas); dysplastic nevi with architectural disorder and cytologic atypia of melanocytes; Spitz nevi; and compound or predominantly intradermal, ordinary, benign melanocytic nevi. RESULTS Strong cytoplasmic staining for VEGF was observed in melanoma cells in as many as 77% of primary invasive melanomas, whereas only 25% of the in situ melanomas exhibited a detectable immunoreactivity for VEGF. It is interesting to note that no immunoreactivity was shown by any nevi; Spitz nevi, in particular, showed negative immunoreactivity to VEGF. Invasive melanomas and in situ melanomas displayed coexpression of MMP-2 and MMP-9, although to a variable extent. In particular, high MMP-2 staining was observed in 14 of 18 invasive melanomas; moreover, strong MMP-2 expression also was observed in 60% of in situ melanomas, whereas the residual 40% of those melanomas showed a moderate level of positivity. CONCLUSIONS On the basis of the current data showing that malignant melanocytic tumors displayed strong VEGF expression, whereas benign melanocytic proliferations showed no immunoreactivity for VEGF, VEGF also may be used as a discriminating factor to distinguish malignant melanoma from lesions of uncertain histology. Cancer 2002;95:1963–70. © 2002 American Cancer Society. DOI 10.1002/cncr.10888

Oxychitin–chitosan complexes prepared from crustacean chitin and oxypullulan–chitosan complexes prepared from three different preparations of Aureobasidium pullulans pullulan were contacted with solutions of egg white lysozyme, Carica papaya papain, wheat germ lipase, Clostridium histolyticum collagenase, porcine pancreas α-amylase, barley malt α-amylase, and sweet potato α-amylase at nearly neutral pH values and 25 and 37°C, for at least three days. The reducing capacity of the solutions in contact with the complex, due to oligomer release, was measured with the aid of ferricyanide and expressed as net absorbance vs time. The oxychitin–chitosan complex was degraded by lysozyme, lipase and papain. The other enzymes were ineffective. Histological evidence indicated that the oxychitin–chitosan complex tested as a bone prosthesis coating in an animal model, was biochemically active and biodegradable, therefore capable to promote osteoconduction and greater bone formation at the bone-prosthesis interface, with no adverse effect on mineralization. Pullulans of different origins were susceptible to enzymatic hydrolysis, particularly so with animal amylase, nevertheless the chitosan complexes obtained from the corresponding oxypullulans were not degraded over the three-day observation period.

Plates of Ti-6Al-4V alloy were plasma-sprayed with hydroxyapatite and with bioactive glass. Chitosan acetate solution was then used to deposit a chitosan film upon the plasma-sprayed layers, which was further reacted with 6-oxychitin to form a polyelectrolyte complex. The latter was optionally contacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 4°C for 2 h to form amide bonds between the two polysaccharides. Uniform flat surfaces exempt from fractures were observed with an electron microscope. The chitosan film was also submitted to acetylation with acetic anhydride in methanol to obtain a chitin film. In all cases the modified chitosan films were insoluble. The results are useful for the preparation of prosthetic articles possessing an external organic coating capable to promote colonization by cells, osteogenesis and osteointegration.

Background: Metalloproteinases (MMPs) are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. Aims: In order to investigate the role of MMPs in cutaneous oncogenesis we studied the expression of MMP-2 and MMP-9 in 34 cases of epidermal preinvasive neoplastic lesions and invasive carcinomas. We also studied their relationship with the expression of tissue inhibitors of MMPs and with proliferative activity and p53 expression in neoplastic epithelial cells. Results: MMP-9 was found to be focally expressed by neoplastic epithelial cells at the infiltrative edges in microinvasive carcinomas and in dyskeratotic foci in Bowen’s disease and widely invasive carcinomas. Gradation of Mib-1 positivity and p53 expression was found with increasing abnormality in the spectrum of malignancy. Conclusions: Our results seem to suggest the involvement of MMPs in microinvasive carcinomas, which show also low proliferative activity and p53 expression, whether other factors seem to be more important in widely invasive carcinomas.

The osteointegration of Hydroxyapatite (HA), Titanium (Ti-6Al-4V: Ti), Zirconia (ZrO2), Alumina (Al2O3) and 2 biological glasses (AP40 and RKKP) was comparatively investigated in normal and osteopenic rats by means of histomorphometry. Thirty-six Sprague Dawley female rats were left intact (Group C) while 36 were ovariectomized (Group OVX). Group C and OVX were further divided into 6 subgroups. After 16 weeks all animals were submitted to the femoral implant of nails made of the above-mentioned materials. Eight weeks after implantation the animals were euthanized, the femurs were harvested for histomorphometric analysis. The data showed that: (1) all the tested materials were biocompatible in vitro; (2) no significant differences existed in Affinity Index (AI) of Group C; and (3) results from paired comparison applied to the AI showed significant differences among the Groups C and OVX. The AI did not significantly change among intact groups, while it significantly decreased when some materials were implanted in OVX subgroups (AP40, ZrO2 and Ti-6Al-4V: \({p < 0.0005}\), \({p < 0.05}\) and \({p < 0.01}\)). It is confirmed that bone mineral density is a strong predictor of the osteointegration of an orthopedic implant and that the use of pathological animal models is necessary to completely characterize biomaterials.

Surgical lesions in rat condylus were treated with N,N-dicarboxymethyl chitosan and 6-oxychitin sodium salt. Morphological data indicate that the best osteoarchitectural reconstruction was promoted by 6-oxychitin, even though healing was slower compared to N,N-dicarboxymethyl chitosan. Complete healing was obtained with N,N-dicarboxymethyl chitosan within three weeks.

The present in vitro and in vivo comparison of three bioactive (HA, AP40, RKKP) and three bioinert (Ti6-Al4-V, Al2O3, ZrO2) materials was undertaken to identify which of them provide(s) the most suitable coating for prostheses implanted in patients with altered metabolic status. The experimental design included in vitro tests with human osteoblasts and morphological observations by scanning electron microscopy. For the in vivo evaluation, the materials were implanted in the femoral condyle of ovariectomised and intact female rats, and two months after surgery an X-ray microanalytical study was performed. The in vitro study showed good biocompatibility with all materials. Microanalysis evidenced a similar behaviour with all materials except the two biological glasses. The differences in Ca and P content observed between intact and ovariectomised rats can be explained by the intrinsic capability of biological glasses to undergo surface modifications in the presence of alterations of the bone metabolism. Thus, their use seems to be indicated in recipients with osteoporotic pathologies.

Preliminary results on the modifications induced by the growth of a Chinese Hamster Ovary (CHO) cell line on the surface of a CORD 1014 ceramic cordierite are reported. Results proved that cells strongly modify the crystallography and the chemical composition of the surface and near surface regions of the cordierite.

This study aimed to assess platelets as a possible model for screening the accumulation of mitochondrial DNA mutations, particularly during normal ageing. For this purpose we isolated platelets from young and old donors selected by lack of systemic and haematological diseases. We studied the accumulation of a particular deletion (4977-bp deletion) that usually accumulates in an age-related manner in different post-mitotic tissues, such as brain, heart and skeletal muscle, and in some non-post-mitotic tissues (skin, liver). Using different primers, we failed to detect this particular species of deletion in platelets both from young and old individuals. However, we cannot exclude the presence of other species of deletions or point mutations affecting the mitochondrial DNA in platelets during the aging process.

The application of bioactive ceramic coatings to prostheses confers strength to a material (ceramic or biological glass) that exerts beneficial effects on bone-tissue growth but that itself lacks the toughness and stability required of an implant device. The rate of bioactivity is related to the chemical reactivity of the material and causes interface dissolution, precipitation and ion-exchange reactions. Ceramics may differ in sintering temperature and thus exhibit differences in their in vitro dissolution features and in vivo performance. To test these effects, in vitro and in vivo studies were carried out on two biocompatible biological glasses and a ceramic of proven bioactivity in view of their potential utilization as covering materials. In addition, a modified chitosan was adsorbed on the surface of a series of hydroxyapatite (HA) samples. Human fibroblasts and/or osteoblasts were used for the in vitro tests, and normal (INT) and osteoporotic (OVX) rats, normal rabbits and sheep for the in vivo studies. Similar chemical changes were observed in both glasses, suggesting that these materials underwent modifications directly dependent on their biological environment. The in vivo tests point to the possibility of improving the bioactivity of ceramic substrates with chitosan. However, the different behaviour of the materials in vitro and in vivo suggests that these tests should be conducted in parallel.

This study aimed to assess the biocompatibility of two cordierite ceramics (DF and Cord 1014), with similar chemical composition and different porosity, as a potential support for cell growth in a continuous-flow, solid-bed reactor. The Chinese hamster ovary (CHO) cell line transfected with HBV-DHFR recombinant plasmid was seeded on cordierite or polystyrene dishes and evaluated for cell growth and production of recombinant hepatitis B surface antigen. Proliferation of the CHO cells, in terms of cell number, was generally similar in polystyrene and Cord 1014 and always lower in DF. Flow cytometric analysis showed no difference in cell cycle distribution for cells grown on different supports, and showed a two-fold increase in percentage of debris for cells grown on DF than for those grown on Cord 1014 and polystyrene culture dishes. Moreover, the morphology of cells grown on Cord 1014 did not change during the experiment, and cells were well spread and organized. Finally, total recombinant hepatitis B surface antigen production was higher on Cord 1014 than on polystyrene and DF samples. Such evidence suggests that Cord 1014 could be a promising support for growing cells in a continuous-flow, solid-bed reactor.

Ultrastructural difractometric and chemical evaluations of calciumpartially stabilized zirconium (Ca-PSZ) implants were performed in an invivo study on animals in order to evaluate its biological behaviour. Thechemical–morphological investigations demonstrated the presence of anosteogenetic activity at the bone–biomaterial interface. Thenew-osteogenesis was preceded by the formation of a loose connective tissuearound the implants. This mesenchymal-type tissue without a capsularorganization, allowing modulation of the mechanical forces to which the implantis subject, could be considered a positive event in the osteogenetic process andnot a sign of future failure of the implant. Finally, microanalyticalinvestigations carried out on non-implanted and implanted Ca-PSZ tools suggestedthat the surface of this ceramic material does not undergo modification once ithas been inserted in the biological environment (12 months).

The aim of this study was to characterize some phenotypic expressions of fibroblasts from the human oral mucosa. Gingival and lower forearm fibroblasts from young (20–30 years) and elderly (> 60 years) subjects, were analyzed. Gingival fibroblasts were taken from donors with (P) and without (NP) periodontal disease, while skin biopsies were taken from healthy subjects. Cell proliferation was assessed by evaluating the cell multiplication coefficient (C.M.C.). The proliferation potential of gingival fibroblasts from elderly individuals with and without periodontopathy did not differ from that of young subjects in the same condition but differed significantly in the skin samples. Enzyme neutral endopeptidase (EC 3.4.24.11) (NEP) activity, studied as a possible marker of cell ageing, showed an age-related increase in human skin fibroblasts but not consistently in gingival fibroblasts from individuals with or without periodontal disease. Cell area and substrate adhesion were evaluated by morphometric analysis. There were no significant differences between elderly P and NP subjects, while significant differences were observed between young and elderly P subjects. In conclusion, proliferative capacity and NEP activity in gingival fibroblasts did not appear to be age-related, probably because their microenvironment is continually moistened by saliva, which continues to contain growth factors, notably EGF, even into senescence. Tissue reaction and repair are important clinical and therapeutic implications.

The aim of the study was to analyze the relationship between lymph nodal involvement and regional and/or distant recurrences in locally advanced squamous cervical carcinomas, and also evaluate tumor 72-kDa metalloproteinase, as a biologic parameter useful for understanding the mechanisms of disease relapse and prognosis. In particular, 72-kDa metalloproteinase is an enzyme that specifically cleaves type IV collagen and seems to play a critical role in tumor invasion and metastatic dissemination.The medical records of 62 patients with FIGO (International Federation of Gynecology and Obstetrics) stage Ib and IIa squamous cervical carcinoma who underwent primary radical surgery with systematic pelvic and paraaortic lymphadenectomy and then were routinely followed were recruited from our series of 76 consecutive cases and reviewed. Fifty-four patients with complete clinicopathologic information were considered eligible for the study. All recurrences were defined as histologically and/or cytologically documented disease, following a minimum 3-month disease-free interval. Sites of recurrences were classified as distant, or regional to the pelvis. Immunostaining with 72-kDa metalloproteinase was performed on serial sections of tumors using avidin-biotin complex technique. Affinity-purified rabbit anti-72-kDa metalloproteinase antibody was used. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells (72-kDa metalloproteinase index).After a median follow-up of 38 months (range 9-71 months), 11 patients recurred with a 20% overall incidence. Seven patients (64%) recurred regionally, with side-wall infiltration in 2 cases, and 4 patients (36%) recurred distantly. By Cox hazard multivariate analysis, lymph nodal status was significantly related to disease-free survival (P = 0.01); in particular, all the patients with side-wall or distant recurrences had lymph nodal involvement. A significant relationship was also observed between tumor 72-kDA metalloproteinase immunostaining and disease-free survival (P = 0.02). The 72-kDA metalloproteinase index was significantly higher in patients who recurred than in patients with disease-free follow-up (P0.001); in particular, the highest values were detected in patients who recurred distantly. A relationship between 72-kDa metalloproteinase staining and nodal status was observed (P0.001).In conclusion, nodal status and the 72-kDa metalloproteinase index were two independent prognostic parameters, significantly related to recurrence risk and pattern of recurrence in locally advanced cervical carcinoma. Although they are independent prognostic parameters, a relationship between nodal involvement and 72-kDa metalloproteinase was observed. A model of tumor recurrence in which intrinsic tumor factors exert their negative influence directly or by contributing to the development of nodal metastases seems possible.

A platelet (PLT) function modification has been reported in normal pregnancies compared with the nonpregnant condition and it has been hypothesized to play a central role in the pathogenesis of pregnancy-induced hypertension (PIH). The aims of the present study were (i) to evaluate the lipid composition, fluidity at different depths, transport functions, and ultrastructural features of the PLT membrane in PIH and (ii) to ascertain whether similar modifications may be determined by the state of pregnancy in comparison with the nonpregnant condition. The platelets of healthy pregnant women (HPW) showed: (i) an increase in Ca2+ ATPase activity, (ii) a decreased fluidity of the deeper site of the membrane, (iii) a reduced cholesterol concentration, with an increased ratio between unsaturated and saturated fatty acids; (iv) a decreased intramembranous particles (IMP) distribution factor (DF) of the plasma membrane E face in comparison with healthy nonpregnant women. When comparing women affected by PIH with HPW, we observed (i) reduced Na+/K+ ATPase activity and enhanced Ca2+ ATPase activity and intraplatelet calcium concentrations, (ii) an increased membrane fluidity, (iii) an increased cholesterol concentration and ratio between unsaturated and saturated fatty acids, (iv) a reduction of the IMP number and the DF. Pregnancy produces a deep modification of the platelet plasma membrane, and PIH produces a more pronounced alteration of the maternal platelets, which can be responsible for the observed modifications in placental blood flow and in the fetomaternal exchange.

In the present study, we investigated changes of p53 expression and the cell proliferation index detected with MIB 1 in tumors before and after neoadjuvant combination chemotherapy with respect to the outcome of the disease. Our aim was to define more appropriately the significance of chemotherapy in locally advanced cervical carcinoma.Our study included 17 women with locally advanced squamous cervical carcinoma who had been admitted to the Institute of Gynecology and Obstetrics Ancona University, between January 1990 and December 1994. The patients received neoadjuvant combination chemotherapy consisting of three cycles of cisplatin (80 mg/m2) and bleomycin (30 mg/m2). After chemotherapy, radical surgery was performed. Bioptic specimens were obtained from cervical tumors before and after chemotherapy and processed for immunohistochemical staining with a monoclonal antibody against p53 and with the monoclonal antibody MIB 1.Thirteen patients (76.5%) showed a clinical response (4 complete and 9 partial), while of the remaining 4 cases (23.5%) 3 had no change and 1 showed progression after neoadjuvant combination chemotherapy. A significant relationship was observed between the overexpression of p53 and the sensitivity to chemotherapy; responder patients showed a higher frequency of p53 positive cells than non-responders (P = 0.03). No significant relationship with MIB 1 index was observed. Both expression of p53 protein (P0.001) and reaction with MIB 1 (P = 0.003) significantly decreased after chemotherapy. The decrease in expression of p53 protein and staining with antibody MIB 1 was particularly evident in patients who responded to chemotherapy.In tumors, p53 protein and index of proliferating cells as determined with MIB 1 showed a significant modulation after treatment, suggesting an association with sensitivity to chemotherapy. However, the limited number of our series of patients does not permit a statement on the clinical implication of expression of p53 and cell proliferation in patients undergoing neoadjuvant combination chemotherapy for locally advanced cervical carcinoma.

The reactive and reparative capacity of injured tissue when treated with either Pulsed Electromagnetic Fields (PEMFs) or with immunomodulator drugs (Prostaglandin E2 and Thymopentin) was evaluated. A morphological and quantitative evaluation was made of the interactions and the interferences on the structural stages of tissue repair processes. When PEMFs were used the time required for new tissue to completely cover the wound was 26 days after surgery, instead of 30 days for control conditions or for treatment with PGE2 or Thymopentin. PEMFs showed accelerated healing process. Both PGE2 and Thymopentin should be considered, in association with other drugs, in an attempt to strengthen the various immunomodulative phases of the reconstitution of injured tissue.

The purpose of this study was to evaluate the biological significance of Ki67 antigen expression in serous ovarian tumors, through the analysis of MIB 1 monoclonal antibody reactivity in cystoadenomas, borderline tumors, and invasive cystoadenocarcinomas; the correlation between this index of cell proliferation and clinicopathologic parameters (FIGO stage and grade, and disease-free survival) was also investigated in invasive cystoadenocarcinomas. Fifty-four patients with serous ovarian tumors, treated at the Institute of Gynecologic and Obstetrics, Ancona University, Italy, were used as study population; 10 women had serous cystoadenoma, 16 women had serous borderline tumor, and 28 women had invasive cystoadenocarcinoma. The expression of primary tumor proliferation related to Ki67 antigen was immunohistochemically evaluated by monoclonal MIB 1 antibody (Immunotech, Marseille Cedex, France) on microwave oven-processed formalin-fixed paraffin-embedded tissue. Compared to cystoadenomas and borderline tumors, the tissular Ki67 antigen immunostaining was significantly higher in cystoadenocarcinomas, with the highest values in architectural grade 2 and 3 neoplasms (P < 0.001). Within the cystoadenocarcinomas, a relationship was observed between the measured proliferation index and disease FIGO stage, but it was not significant (P = 0.92). However, patients who recurred and/or had disease progression presented a primitive neoplasm with significantly higher expression of Ki67 antigen than that of patients with disease-free survival (P = 0.01). A significant relationship was observed between the Ki67 index and disease-free survival, independent of histologic grade and stage, evaluated by Cox hazards analysis (P = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: The study examined the effects of salt-induced hypertension on vascular contractile responses during pregnancy and the mechanisms of the effects.Methods: Aortic rings from pregnant Wistar rats, fed for 6 weeks on diets containing 0.3% (control) and 8.0% (test) sodium chloride were contracted by phenylephrine, 5-hydroxytryptamine, and potassium chloride, in the presence and absence of either endothelium or 10−6 M indomethacin. Contractile responses to calcium chloride were also assessed.Results: High salt intake increased the systolic blood pressure of the rats. Rings from the high-salt-fed rats showed enhanced reactivity to phenylephrine, but not to potassium chloride and 5-hydroxytryptamine. Indomethacin treatment decreased the contractions of rings from the test rats to phenylephrine, but did not significantly affect the responses of rings from the control rats. Removal of endothelium resulted in similar increase in the contractile responses of rings from both groups of rats to phenylephrine ...

The present study reports on a biological model based on fibroblast proliferation applied to 3 different types of flat-plate dialysis membrane, in order to ascertain whether the artificial materials currently used in hemodialysis cause in vitro cellular proliferation. The study plan we followed involved plate membrane isolation from non-used dialyzers and used dialyzers, observed through scanning electron microscopy (SEM) both before and after testing with human fibroblasts by means of cell culture. Fibroblast growth was assessed by phase contrast light microscopy examination and cytometric DNA content evaluation. Our investigations proved that the artificial materials we considered interact with fibroblast cultures. Noticeable proliferative response was observed both after contact with unused material and on mediation by the protein layer absorbed on the membrane surface at the end of dialysis sessions. In this last case fibroblast proliferative activity appeared higher than that observed with unused membranes, showing that the soluble molecules entrapped in the protein layer appeared able to exert a biological activity even in in vitro tests

A novel modified chitosan carrying covalently linked imidazole groups (average molecular weight 700000, degree of substitution 0.28, degree of acetylation 0.08) was used to stimulate bone formation in an animal model. Lesions (7mm diameter) were surgically made in the femoral condyle of sheep and treated with the modified chitosan. Within 40 d after surgery, the neoformed tissue occluded the surgical hole and assumed a trabecular structure in the peripheral area of the lesion, while looking like a mineralization nodule in the central part in association with a fibrous component. In the control, no sign of osteoinduction or reparative process was observed and bone marrow was rich in adipocytes. Biomaterials (1994) 15, (13) 1075–1081

The use of heterologous collagen in skin repair processes has proved to be an efficacious approach for the reconstitution of injured tissue. A morphological and quantitative evaluation of the reactive and reparative ca pacity of injured tissue treated either with conventional medications or with heterologous collagen (Condress) in the form of sponges was carried out. We have analyzed experimental surgical lesions on an animal model and evalu ated the repair processes of the lesions, including the excision of the cutaneous and subcutaneous tissue. The heterologous collagen sponges provided biologi cal support to obtain healed skin of better quality. Tissue lesions healed more rapidly, thus reducing the risk of recurrence. This experimental study provides morphological and structural data for further investigation.

In order to achieve the most satisfactory results in tissue expansion, a cream containing human recombinant epidermal growth factor (EGF) was applied daily to the expanding skin. The capacity of EGF to stimulate tissue repair has been evaluated on expanded skin flaps, comparing surgical procedures carried out with and without EGF. The present clinical, morphological, immunohistochemical and morphometric results demonstrate that EGF used together with the tissue expander may improve skin repair after mechanical stretching. In EGF-expanded skin flaps, clinical results show a reduction in teleangectasiae and histological evidence of a more regular texture in the dermal tissue, together with a significant thickening of the epidermal malpighian layer. Without EGF some signs of immaturity, such as abundance of fibronectin, persist for longer. EGF seems to be a helpful support to improve the tissue expansion process.

Surgical wounds from wisdom tooth avulsions were medicated with freeze-dried methylpyrrolidinone chitosan, a gel-forming resorbable biopolymer obtained from crab chitosan by chemical modification. Methylpyrrolidinone chitosan promoted osteoconduction and the space left after avulsion was filled with newly formed bone tissue, which conferred desirable mechanical and physiological characteristics to the healed would site. Morphological evidence obtained from biopsies confirmed the radiographic data. Methylpyrrolidinone chitosan was progressively depolymerized by lysozyme and was no longer detected 6 months after surgery. None of the 10 patients reported adverse effects over one year of observation.

The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications. By using both 5-nitroxystearate and 16-nitroxystearate as spin labels, we observed an initial increase in membrane order over 0-24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36-48 h might be related to the process of cellular fusion. The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.

Donor areas on patients undergoing plastic surgery were treated with epidermal growth factor (EGF) to stimulate tissue regeneration. Mor phometric analyses showed that cutaneous explants, treated with EGF, pre sented a higher number of microvessels ( p < 0.01) and mesenchimal cells ( p < 0.01), than untreated areas, while EGF did not improve epidermal thick ening. By electron microscopy, better histoarchitectural maturation of cellular and extracellular dermal compartments was observed after EGF application. This work provides evidence that EGF does stimulate skin repair by promoting the formation of mature cutaneous tissue.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morphofunctional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

Psoriasis is a chronic skin disease, characterized by epidermal hyperplasia, inflammation, angiogenesis and vascular remodelling. An immunohistochemical study on fifteen cryosections of psoriatic skin was performed using antibodies against VEGF, HIF1-alpha, CD34, Factor VIII, MMP-2, MMP-9, TIMP-1 and TIMP-2. Psoriatic skin showed a diffuse VEGF positive staining (13.15+/-6.6), while no expression was observed in normal epidermis. No or faint HIF-1alpha immunostaining was detected in healthy skin, while in psoriatic skin HIF-1alpha was diffusely expressed. A positive correlation between HIF-1alpha and VEGF was reported in psoriatic skin (r= 0.644; p=0.010). In psoriatic sections CD34 expression was significantly higher in respect to control skin (19.15+/-12.61 vs 3.0+/-0.23; p= 0.04), factor VIII immunostaining also demonstrated a significant increased development of the microvasculature in comparison with healthy skin (18.39+/-8.16 vs 7.4+/-0.20; p= 0.033). Total MMP-2 expression of healthy skin (30+/-2.26) was significantly lower in respect to the MMP-2 psoriatic skin (71.5+/-4.13; p= 0.0001) and a positive correlation was observed between VEGF and MMP-2 in psoriatic patients (r= 0.688; p= 0.046). In psoriatic skin MMP-9 expression was significantly increased in comparison to control skin (31+/-3.3 vs 8+/-6.1; p=0.007). All cases of psoriatic skin tissue showed that TIMP-2 and TIMP-1 expression statistically decreased in psoriatic skin (respectively 11+/-1.2 and 12+/-1.5) in comparison with healthy skin (respectively 15+/-3.2 and 53+/-3.8; p=0.0001). In conclusion, we observed that VEGF overexpression correlated with HIF-1alpha and MMP-2 expression, underlining the role of VEGF in psoriasis as a key factor in the link between inflammation and angiogenesis.

The present study was undertaken in order to assess the efficacy of a commercial product containing calcium and silicon (Osteosil-Calcium®) on cell metabolism. MG-63 osteblast-like cells were cultured in the presence of three different drug concentrations (10, 5 and 2.5 μg/mL). Either serum-free culture and standard culture with serum were investigated. Morpho-functional tests (MTT and ALP), scanning electron microscopy (SEM), microanalysis (EDAX) and time-lapse video microscopy were performed. Cell actin cytoskeletal modification with fluorescence phalloidin staining was also tested. Our data show the in vitro functional efficacy of Osteosil-Calcium® on MG63 cell viability and ALP production. This study demonstrates its positive effect on the metabolism of the single cell and suggests wider uses of this drug in health protection and or in Regenerative Medicine therapies which are currently applied to the elderly

The aim of the present study was to evaluate microvessel density (MVD) in the cellular layers of ovarian endometriomata, with particular interest in the relationship with VEGF and survivin expressions by endothelial cells and with the diameter of the cysts.MVD and VEGF and survivin endothelial cell expressions were evaluated in 26 ovarian endometriotic cysts and correlated with the cyst diameter.The mean MVD was higher in the inner specialized stroma of ectopic endometrium than in the outer fibrous capsule, but only in the fibrous capsule was MVD correlated with endothelial cell VEGF and survivin expressions as well as with the cyst diameter.The diameter of ovarian endometriotic cysts seems to be related to the angiogenic process involving the outer fibrous capsule, and not the inner specialized stroma of ectopic endometrium, since only in the capsule are vessels stimulated to proliferate by VEGF and protected from apoptosis by survivin, and their density is correlated to cyst diameter.

Keratinocytes and fibroblasts have been grown onto a series of triblock copolymers based on 1,5-dioxepan-2-one (DXO) and L-lactide (LLA). The molar ratio of DXO and LLA were varied in the copolymers. This resulted in different degrees of hydrophilicity, which in turn influenced the cell growth. On these surfaces, the morphological appearance of the cells with their cell movements and growth were investigated by means of scanning electron microscopy, time-lapse videomicroscopy and immunohistochemistry. All results clearly showed that the keratinocytes and fibroblasts adhered best to the most hydrophilic copolymers. A majority of the keratinocytes seeded on the most hydrophilic copolymer also presented a polarized morphology indicating a migration tendency. The cell growth onto these materials are interesting since a possible application for these unique materials is as polymeric membranes for guided cutaneous and/or periodontal tissue generation.

Aim of this study was to investigate using immunohistochemistry techniques the interrelation between T immunoreactive cells and the expression of CCR10 and its ligand CCL27 in 59 cutaneous melanocytic lesions. In malignant melanomas, T lymphocyte density was significantly decreased from thin melanomas to intermediate and thick ones (P < 0.0005). CCR10 expression was found both in benign and malignant lesions and it was directly correlated with the Breslow depth (P = 0.0298) and inversely with T lymphocyte density (P = 0.0231). Moreover, cases with positive sentinel lymph node tended to have a higher CCR10 expression compared to cases with negative sentinel lymph node (P = 0.0281). When CCR10 and CCL27 expression were evaluated together, CCR10-/CCL27-melanomas tended to have a higher mean density of CD3+ and CD8+ lymphocytes. Our results suggest that in human melanomas CCR10 and CCL27 may act to increase the ability of neoplastic cells to grow, invade tissue, disseminate to lymph nodes and to escape the host immune response.