Preparation and cytocompatibility evaluation for hydrosoluble phosphorous acid-derivatized cellulose as tissue engineering scaffold material.

Chemical modification of cellulose by phosphorylation enhances its bioactivity and provides new derivatives and materials with specific end uses. In the present study, cellulose derivatized with phosphorous acid was obtained using the reaction of microcrystalline cellulose with phosphorous acid-urea mixture, in molten state, in comparison with others methods that used different solvents and catalysts. Completely water soluble films with a substitution degree close to one were obtained and characterized by analytical and spectral analysis (FT-IR, (31)P NMR), contact angle, metallographic microscopy and atomic force microscopy (AFM). 31P NMR spectra of derivatized cellulose showed a signal at 2.58 ppm (assigned to P-O-C6) while the doublets at 4.99-5.29 and at 7.38 ppm were assigned to P-O-C2 and P-O-C3, respectively; thus, the formation of monosubstituted phosphorous acid esters of cellulose is advocated. Contact angle measurements showed that the work of adhesion is more important in water than in ethylene glycol, for the phosphorous acid derivatized cellulose. The cytocompatibility of this hydrosoluble derivatized cellulose was tested by direct contact and also by indirect assays on normal human dermal fibroblasts and on osteoblast-like cells (human osteosarcoma). Cell growth on phosphorylated cellulose pellicle and the results from viability assays had shown a good cytocompatibility and lack of toxicity. Phosphorous acid derivatized cellulose would offer a promising biomaterial, useful as scaffolds for new biopolymer composites, and subject for further development as an ionic crosslinker.

Immunohistochemical and transmission electron microscopy study regarding myofibroblasts in fibroinflammatory epulis and giant cell peripheral granuloma.

Fibroblasts represent the main cellular population in the connective tissue; they have a central role in extracellular matrix (ECM) synthesis, degradation and remodeling. These cells may express a substantial heterogeneity regarding their morphology and functions in pathological conditions and during tissue remodeling. Myofibroblasts are a good example for heterogeneity and phenotypical changes. These cells can be morphologically and immunologically defined by the expression of specific cytoskeleton proteins. Myofibroblasts show cytoplasmic actin microfilaments organized as stress fibers and interconnected by gap or adherens junctions. These cells come also in contact with extracellular matrix by focal contacts. Myofibroblasts play fundamental roles in pathologic conditions, even by activation and proliferation or by deletion. Moreover, these cells seem to be involved in formation and repair of the ECM compounds, proliferation and differentiation of the epithelial, vascular or neurogenic elements. The purpose of the present study is to emphasize the presence and distribution of myofibroblasts in the reactive stromal tissue of granulation tumors in the oral area, fibroinflammatory epulis and giant cells peripheral granuloma, by means of immunocytochemical and transmission electron microscopy studies. Both tumor types shown a common characteristic of the presence of reactive inflammatory stromal tissue and myofibroblasts are a common issue.

Correlations between morphological changes induced by curcumin and its biological activities.

Curcumin is a phytochemical polyphenol extracted from turmeric rhizome, with multiple biological activities, intensively studied in various therapeutic areas. Its effects covers a wide range of specialties, from the neuroprotective to the antimetastatic properties, influencing pathologies from cardiovascular, neuronal and oncological fields, as a part of its broad spectrum of action. These effects are explained by antioxidant, anti-inflammatory and anticarcinogenic simultaneous roles of curcumin and its derivatives. In this review, we selected the information about morphological evidences correlated with the biological effects on the following organ systems: the central nervous system (including neurological pathology, such as Parkinson's and Alzheimer's disease), the cardiovascular system (including disorders like atherosclerosis, endothelial dysfunction and drug-induced myotoxicity), multiple forms of cancer, and metabolic syndromes including diabetes. The central point of this review was to target a variety of morphological changes at microscopic level induced by curcumin, using different microscopy techniques.

The ultrastructural features of the premalignant oral lesions.

Premalignant oral lesions are among the most important risk factors for the development of oral squamocellular carcinoma. Recent population studies indicate a significant rise in the prevalence of leukoplakia, erythroplakia/erythroleukoplakia, actinic cheilitis, submucous fibrosis and erosive lichen planus. Since standard histopathological examination has numerous limitations regarding the accurate appreciation of potential malignant transformation, the present study aims to aid these evaluations using the transmission electron microscopy (TEM) technique, which emphasizes ultrastructural changes pertaining to this pathology. Oral mucosa fragments collected from 43 patients that were clinically and histopathologically diagnosed with leukoplakia, erosive actinic cheilitis and erosive lichen planus have been processed through the classic technique for the examination using TEM and were examined using a Philips CM100 transmission electron microscope. The electron microscopy study has confirmed the histopathological diagnosis of the tissue samples examined using photonic microscopy and has furthermore revealed a series of ultrastructural details that on the one hand indicate the tendency for malignant transformation, and on the other reveal characteristic features of tumor development. All the details furnished by TEM complete the overall picture of morphological changes, specific to these lesions, indicating the importance of using these techniques in establishing both a correct diagnosis and prognosis.

The molecular mosaic of the premalignant cutaneous lesions.

In the last three decades, the premalignant cutaneous lesions have represented a milestone for the clinicians and the anatomopathologists given the increased risk of malignant transformation not only in the old but also in the young population. Recent research indicates the fact that, though multiple progresses were recorded in the diagnosis and treatment of the cutaneous squamocellular carcinomas, developed in more than 85% of the cases in premalignant lesions, however the prognosis and survival up to five years did not register significant improvements. For the achievement of the diagnosis with certainty, the histopathological examination, considered until recently the "golden standard", principally based on the TNM criterion, has an increased percentage of subjectivity and it is relatively unsure, being known the fact that two apparently identical tumors answer differently to the same therapy. The variability of the morphological aspects from simple dysplasia to in situ carcinomas and the cancers themselves impose the identification of some cellular and molecular markers typical to the premalignant and malignant cutaneous lesions. In this respect, the knowledge and characterization of the molecular mosaic allow the establishment of some clear criterion for an early diagnosis, corresponding monitoring and adequate treatment.

In vitro evaluation of curcumin effects on breast adenocarcinoma 2D and 3D cell cultures.

UNLABELLED: Breast adenocarcinoma cell line MDA-MB-231, even if it expresses low levels of E-cadherin, still readily form multicellular aggregates of cells, namely spheroids. Curcumin is a diarylheptanoid antitumoral drug while it significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. Curcumin photoactivation may enhance antiapoptotic role against cancer cells. AIM: To evaluate the effect of low curcumin concentrations, ranged from 1.9 to 15 µM, with and without photoactivation, using a manufactured 670 nm LED-matrix. A secondary aim was to evaluate the ideal method to produce easy-to-use tumor cell spheroids, comparing two low adherence plate supports. MATERIALS AND METHODS: Breast adenocarcinoma cell line MDA-MB-231 were cultured according to 2D monolayer and 3D spheroid models then submitted to normal and photoactivated curcumin in micromolar concentrations. MTT assay was used to evaluate cell viability following curcumin application on cells. On 2D cell cultures, curcumin inhibits cell tumor development and proliferation at concentrations of 15 µM, with a viability of 65.7% at 48 hours incubation time. A decreased viability up to 25% for a concentration of 15 µM was recorded following photoactivation and cytotoxic action on breast cancer tumor cell line continued at concentrations of 7.5 and 3.75 µM. Curcumin photoactivation increases pro-apoptotic effects in both 2D and 3D tumor cell culture models and also responsiveness to curcumin is slightly reduced in spheroid-like structures. Thus, 3D tumor cell culture systems appear to be the ideal environment for in vitro assays regarding anticancer drug effects on cell viability.

Morphopathological stigmata in acne fulminans.

Acne fulminans is the most aggressive and destructive form of acne vulgaris, being also known as acne maligna. The onset is acute and systemic involvement is always present. Most commonly, acne fulminans (AF) occurs in male adolescents as a brutal complication of a preexisting mild or moderate acne. The etiology of AF remains incompletely elucidated. The skin lesions are polymorphic, the symptoms and clinical signs vary, and thus the diagnosis is not easy. In making a certain diagnosis of AF, histopathology has a decisive role. In this respect, we will present some of the most suggestive aspects of histopathology, immunohistochemistry and electron microscopy in a 16-year-old patient clinically diagnosed with AF. This patient presented on admission nodular inflammatory and ulcerative necrotic lesions on the face and chest, extremely, accompanied by significant myalgias and arthralgias.

E-cadherin expression in invasive ductal carcinoma associates ultrastructural changes in desmosomes structure.

UNLABELLED: The relationship between E-cadherin presence/absence and the integrity of the desmosomes at the same level becomes important regarding the discrepancies between E-cadherin lack of expression in severe invasive carcinomas and the desmosome activity. PURPOSE: In the present study, we have evaluated the presence of E-cadherin (EC) in 34 cases of metastatic or non-metastatic invasive ductal breast carcinomas (IDC) by immunohistochemistry followed by transmission electron microscopy (TEM) evaluation on samples prepared from paraffin sections. MATERIALS AND METHODS: Our study has analyzed 34 paraffin blocks incoming from 20 cases with documented presence of metastatic invasion and 14 cases without metastases. All samples were processed and stained by classic Hematoxylin-Eosin, immunohistochemistry to detect EC presence and transmission electron microscopy. RESULTS: Our results, even on a small pilot group, emphasize that EC presence is associated with the complete desmosomal integrity in non-metastatic cases, at least for the investigated areas. From the metastatic IDC cases, we have observed reduced EC expression in only three cases and important loss of desmosomal arrangement, mainly at the desmosomal plate level. CONCLUSIONS: This observation can issue the hypothesis that even in IDC cells that express EC, the invasive potential depend not only on EC-dependent junctions but also on the desmosome integrity. Also, correlated relationship between EC expression, potentially explored desmogleins, desmocollin, desmoplakin and plakoglobin expression and TEM ultrastructure can lead to a conclusion about the invasive potential of these malignant cells.

Electron microscopy analysis of skin biopsies in CADASIL disease.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited vascular disorder, non-amyloid and non-atherosclerotic, affecting predominantly the central nervous system. We examined samples of skin biopsies from six patients (men, 43-52-year-old), admitted for treatment in the Neurology Clinic regarding the presence of partial motor impairment on upper and lower right limbs, facial asymmetry and phrasing impairment (three of the patients); These three patients had family history remarkable for early-onset strokes: mother and two brothers deceased by early strokes (40-50-year-old). Skin biopsy samples were fixed in glutaraldehyde and post-fixed in osmium tetroxyde. After dehydration, tissue samples were embedded in Epon. Ultrathin sections were mounted on copper grids and stained with uranyl acetate and lead citrate as usual and examined with a transmission electron microscope Phillips CM100. In all cases ultrastructural study showed granular osmiophilic material (GOM) in extracellular locations, between degenerating smooth muscle cells in dermal arteries or in their indentations. Deposits of GOM varied in size and electron density. Degeneration and loss of smooth muscle cells (SMCs) leads to abnormal enlargement of the space between these cells Ultrastructural analysis in three cases showed chromatin condensation and peripheral aggregation of nuclear material suggesting cells entry to apoptosis. These aspects and the marked destruction of the vascular wall were correlated with MRI findings and the severity of clinical manifestations at these patients. Our study showed that findings of GOM deposits, degeneration and loss of SMCs (probably by apoptosis), cell adhesion elements disturbance are characteristic for CADASIL disease and sufficient for diagnose of certainty. Moreover, electron microscopy analysis of skin biopsies is a useful tool for a differential diagnosis and can be considered as first choice method.

Correlation between E-cadherin abnormal expressions in different types of cancer and the process of metastasis.

Cell-cell adhesion plays a critical role in the establishment and maintenance of cell polarity and cell society. Reduced cell-cell adhesiveness allows cancer cells to disobey the social order, resulting in destruction of the histological structure, the morphological hallmark of malignant tumors. Morbidity in most cancer patients is not due to primary cancer but to metastatic disease. Thus, understanding the progression of tumors to metastatic state and the changes that take place in highly aggressive cells is important in the development of novel approaches to the diagnosis and treatment of progressive malignancies. Cell adhesion molecules are implicated in human carcinogenesis. E-cadherin is a calcium-dependent cell adhesion molecule the intact function of which is crucial for the establishment and maintenance of epithelial tissue polarity and structural integrity. The gene encoding E-cadherin (CDH1, on chromosome 16q22.1) was one of the first to be considered as an invasion-suppressor gene. Mutations in CDH1 occur in diffuse type gastric cancer, lobular breast cancer, and endometrial cancer. In human cancers, partial or complete loss of E-cadherin expression correlates with malignancy. Through immunohistochemical analysis it has been assessed the abnormal expressions of E-cadherin in three types of cancer: gastric carcinoma, lobular breast carcinomas and cutaneous melanoma and the correlation with the multistep process of metastasis.

[Ultrastructural aspects in the development of the human nephron--electron microscopy study]. / Aspecte ultrastructurale în dezvoltarea nefronului uman. Studiu de microscopie electronica.

UNLABELLED: We have looked for electron microscopy aspects tissue fragments of human nephrons harvested from fetuses of 7,5-19 weeks to show ultrastructure aspects of the nephron during development. MATERIAL AND METHODS: We have used 9 fragments from human embryos; Four cases were aged of 7,5, 8, 12 and 17 weeks. The other 5 cases were between 18 and 19 weeks. Tissue fragments were fixed in glutaraldehyde at 4 degrees C and processed by the classic technique. RESULTS: The electron microscopy study shows the evolutive steps of the human nephron (stages I-IV), stages met for the 12-19 weeks embryo. For the cases under this age the meta-nephrogenic blastema was dominant. In the IVth development stage, the cells in the proximal tubule show a higher development degree than the distal segments. CONCLUSION: These observations indicate that during early development the proximal segment is better developed and probably more functional than the distal segments.

[Gelatinases in peri-implantation stage]. / Gelatinazele în perioada periimplantara.

The periimplantation stage in mice is marked around the 5th day after mating by blastocyst presence in the emdometrium. The events that are marking the implantation event are focused on the evolution of the extracellular matrix around the mezenchime cells that undergoes decidualization. The inductive process is guided by gelatinases and the purpose of our study is to demonstrate their presence at this level.

[Electron microscopy study regarding the etiology and the pathogenesis of acne vulgaris]. / Contributii la studiul electronomicroscopic privind etiopatogenia acneei vulgare.

The etiology and the pathogenesis of acne vulgaris are not complete cleared up yet. This work presents the results of the electronomicroscopic examinations effected on lesions of acne vulgaris biopsied from 5 patients with different clinical forms of disease (including acne fulminans). In this study we had in view the investigation and description of the ultrastructural modifications that followed the intervention of the 4 factors incriminated in the etiology and pathogenesis of acne: sebaceous hypersecretion, hyperkeratosis pilosebaceous infundibulum, bacterial colonisation, perifollicular inflammation. The ultrastructural aspects that we had in view underline the role of the 4 etiology and pathogenesis factors of acne vulgaris, confirming the data related by the specialised literature.