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Transparency in a fibrin and fibrin-agarose corneal stroma substitute generated by tissue engineering.

Purpose: To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within. Methods: The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka–Munk equations. Results: The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka–Munk scattering coefficient of the fibrin–agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin–agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin–agarose than for fibrin scaffolds, especially for short wavelengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development. Conclusions: The transparency, scattering, and absorption of both fibrin and fibrin–agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use.

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin‐agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin‐agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow‐up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis. Copyright

ELECTRON PROBE X-RAY MICROANALYSIS OF CULTURED EPITHELIAL TUMOUR CELLS WITH SCANNING ELECTRON MICROSCOPY

Three methods have been used to prepare cultured cells for electron probe X-ray microanalysis (EPXMA): (1) analysis at the subcellular level of freeze-dried ultrathin cryosections with scanning transmission electron microscopy (STEM); (2) analysis at the cellular level of whole freeze-dried cells with STEM; and (3) analysis at the cellular level of whole freeze-dried cells with scanning electron microscopy (SEM) (for a review see: Wroblewski and Roomans, 1984; Wroblewski and Wroblewski, 1993; Warley, 1994). However, EPXMA of whole freeze-dried cells with SEM has some disadvantages. This method requires that cultured cells be adapted to growth on a thick substrate such as plastic or glass coverslips (Zierold and Schafer, 1988), graphite discs (Abraham et al., 1985; Larsson et al., 1986) and microcarrier beads (Hall et al., 1992). In addition, a suitable washing procedure to remove the extracellular medium must be found because the deposition of culture medium on the cell surface after freezing and freeze-drying may interfere with X-ray spectra from the cell. Moreover, it is difficult to perform absolute quantitative analyses of elemental content, since the substrate may contribute to the background, decreasing the peak-to-background (P/B) ratio (Roomans, 1981). We present here a simple method to study the intracellular concentrations of elements in whole cultured epithelial tumour cells by EPXMA with scanning electron microscopy.

UV absorbance of a bioengineered corneal stroma substitute in the 240-400 nm range.

Purpose: To determine the UV absorbance of a bioengineered human corneal stroma construct based on fibrin and fibrin-agarose scaffolds in the 240-400 nm range. Methods: Three types of artificial substitutes of the human corneal stroma were developed by tissue engineering using fibrin and fibrin with 0.1% and 0.2% agarose scaffolds with human keratocytes immersed within. After 28 days of culture, the UV absorbance of each sample was determined using a spectrophotometer. The thickness of corneal stroma samples was determined by light microscope. Results: For all the 3 types of corneal stroma substitutes studied, the range of the UV absorbance values was similar to that of the native human corneal stroma, although the fibrin with 0.1% agarose stroma substitute had the best UV filtering properties. The higher UV absorbance of the artificial substitute of the human corneal stroma was in the UV-B and -A ranges, suggesting that these artificial tissues could have potential clinical usefulness and proper UV light-absorption capabilities. Conclusion: Our data suggest that the bioengineered human corneal substitute of fibrin with 0.1% agarose is an effective absorber of harmful UV radiation and could therefore be potentially useful.

Changes in elemental concentrations in K562 target cells after conjugation with human lymphocytes studied by X‐ray microanalysis.

There is considerable interest in determining the mechanisms by which natural killer (NK) cells are able to kill tumor cells. Morphological evidence supports the suggestion that target cell death may occur by either apoptosis or necrosis (I). There have been no studies which describe changes in elemental concentrations in NK cell-mediated cytotoxicity, which would support the mechanism of cell death. Electron probe X-ray microanalysis (EPXMA) should provide the ideal technique for undertaking such studies, since it is the only method which allows elemental analysis of a known (morphologically defined) cell type (2).

An in vitro biocompatibility study of conventional and resin-modified glass ionomer cements.

PURPOSE To evaluate the biocompatibility of a glass-ionomer (GIC) and a resin-modified glass-ionomer cement (RM-GIC), cell viability was examined in a model of human gingival fibroblasts using morphological, biochemical, and ionic patterns by means of phase contrast microscopy, lactate dehydrogenase (LDH) release, and quantitative x-ray microanalysis (EPXMA). MATERIALS AND METHODS The GIC Ketac-Molar Easymix (3M ESPE) and the RM-GIC Vitrebond (3M ESPE) were compared in human gingival fibroblasts exposed to the cements for 72 h. As controls, fibroblasts cultured with DMEM culture medium (negative control) and with 1% triton × (positive control) were used. RESULTS Light microscopic findings showed greater morphological alterations in cells exposed to RM-GIC than to GIC. The relative percentage of LDH released from the cells to the supernatant was significantly higher in RMGIC cultures than in the control. Quantitative x-ray microanalysis showed that cultures exposed to RM-GIC were characterized by an increase in intracellular Na and a decrease in intracellular Cl and K. These changes in ion composition were significant compared to control and GIC cultures. CONCLUSION The three indicators of cellular biocompatibility after 72 h of exposure showed that RM-GIC led to more marked alterations than GIC in human gingival fibroblasts.

Retroperitoneal schwannoma. A complex surgical treatment of a tumor with uncertain behavior.

Abstract Background : Retroperitoneal schwannoma is a rare nerve sheath tumor; the surgical removal of this tumor is sometimes compromised by its location. The aim of this study is to analyze our experience with the diagnosis and treatment of this type of tumor. Method : We present our experience between 1999 and 2011 in the diagnosis and treatment of retroperitoneal schwannoma. During that time, we diagnosed and treated five female patients (four adults and one infant) with the condition. The tumors appeared sporadically and were not associated with neurofibromatosis or other syndromes. Diagnosis was performed by computed tomography (CT) imaging in four cases and by magnetic resonance imaging (MRI) in one case. Results : All patients underwent surgical treatment and complete resection of the lesion. An open resection was performed in four cases, and in the most recent case, the excision was conducted laparoscopically. In all of the cases, the histological diagnosis was retroperitoneal schwannoma, and in one case, there was a melanocytic variant that was not associated with Carney syndrome. At the time of this report, there has been no evidence of recurrence. Conclusion : Retroperitoneal schwannoma is a tumor that is difficult to diagnose with imaging techniques, and because of its localization, the tumor is difficult to remove surgically.

Histological and immunohistochemical study of an unusual type of gallbladder duplication

Gallbladder duplication is a rare congenital anomaly, with an incidence of 1 in 3,800 autopsies. The correct diagnosis and treatment of this type of entity is important in clinical practice, because it may cause some clinical and surgical problems. In this report, we present the clinical case of a 28-year-old female with abdominal pain. Ultrasound of the upper abdomen showed a distended gallbladder with the presence of a septum that could suggest a congenital anomaly of the extrahepatic biliary system. During surgery, a distended and inflamed gallbladder with a lithiasis was found. In addition, a complete septum and double cystic duct were observed. The gross and histopathological evaluation of the surgical specimen allowed us to confirm the diagnosis of a Y- shaped type gallbladder duplication according to Boydens classification. In conclusion, in presence of an atypical imaging of the gallbladder, diagnosis of this group of congenital anomalies should be considered in order to adequately plan surgical intervention if necessary.

An immunohistochemical study of cytokeratins distribution of the human adult male and female urethra.

Surgical treatment of diseases affecting long urethral areas represents a challenge in urology. Recent developments of tissue-engineered urethral substitutes represent a hope for patients. However finding an ideal tissue source for urethral reconstruction first requires proper understanding of the native human urethra physiology and a deep knowledge of the histological and molecular features of the native human urethra. Here we present a comprehensive characterization of male and female urethra by histological, histochemical and immunohistochemical methods with a panel of 15 antibodies. The results demonstrated that the histology of the male and female urethra depend on the area where the sample is taken along its length. Proximal areas of male and female urethra have differential expression of the epithelial basal and suprabasal layer markers CK14 and CK10 which distinguished the prostatic/membranous and proximal female urethra from the bulbar/penile and distal female areas of the urethra. The distal male (penile) and female may be further divided by the distinct expression pattern of CK19. On the other hand, the expression of CK5/6 and CK19 also make a distinction of the proximal and distal female urethra. These results should facilitate a more informed selection of donor graft tissues for urethral replacement. Besides, novel bioengineered urethral tissue approaches should take into account the characterization of the different areas of the urethra presented in this work.

Electron microprobe analysis in periodontal guided tissue regeneration.

Electron microprobe analysis was used to determine the evolution of Ca, P and S in regenerated tissue surrounding incisors roots after periodontal treatment with guided tissue regeneration. Our results, which showed increased Ca and P, and decreased S are discussed in relation to the process of mineralization electron probe microanalysis with potentially provided an accurate means of assessing the degree of mineralization in extremely small tissue samples.