Histological Outcomes on the Development of New Space-making Devices for Maxillary Sinus Floor Augmentation

Background: Previous studies have pointed out that the mere elevation of the maxillary sinus membrane promotes bone formation without the use of augmentation materials. Purpose: This experimental study aimed at evaluating if the two-stage procedure for sinus floor augmentation could benefit from the use of a space-making device in order to increase the bone volume to enable later implant installation with good primary stability. Materials and Methods: Six male tufted capuchin primates (Cebus apella) were subjected to extraction of the three premolars and the first molar on both sides of the maxilla to create an edentulous area. The sinuses were opened using the lateral bone-wall window technique, and the membrane was elevated. One resorbable space-making device was inserted in each maxillary sinus, and the bone window was returned in place. The animals were euthanatized after 6 months, and biopsy blocks containing the whole maxillary sinus and surrounding soft tissues were prepared for ground sections. Results: The histological examination of the specimens showed bone formation in contact with both the schneiderian membrane and the device in most cases even when the device was displaced. The process of bone formation indicates that this technique is potentially useful for two-stage sinus floor augmentation. The lack of stabilization of the device within the sinus demands further improvement of space-makers for predictable bone augmentation. Conclusions: It is concluded that (1) the device used in this study did not trigger any important inflammatory reaction; (2) when the sinus membrane was elevated, bone formation was a constant finding; and (3) an ideal space-making device should be stable and elevate the membrane to ensure a maintained connection between the membrane and the secluded space.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

A Clinical Study of 406 Sinus Augmentations With 100% Anorganic Bovine Bone

Background: The aim of the present study is to evaluate the use of anorganic bovine bone (ABB) associated with a collagen membrane (CM) for a sinus graft by means of clinical, histologic, and radiographic parameters in cases with bone availability <= 7 mm. A preliminary evaluation consisted of a clinical examination, computed tomography (CT), and a panoramic x-ray. Methods: Ninety-two patients requiring bilateral sinus grafts and 222 requiring unilateral procedures (total: 406 sinuses) participated in this study. A total of 1,025 implants were placed in the grafted sinuses. A total of 118 implants were placed simultaneously with the sinus graft (one stage), and 907 implants were placed in a subsequent surgery (two stages), 6 to 12 months after the graft was performed. In seven cases, a biopsy was harvested for histomorphometric analysis. Recall appointments were scheduled every 6 months, and panoramic and periapical x-rays were required every year for 3 years. Results: Among 1,025 implants, 19 were lost (survival rate: 98.1%). The difference in survival rates for implants placed in native bone: <= 3 mm (98.1%), >3 to <= 5 mm (98.6%), and >5 to <= 7 mm (97.0%) was not statistically significant (P = 0.3408). The survival rates for implants with rough and machined surfaces (98.6% and 97.0%, respectively) were not statistically significant (P = 0.0840). The histomorphometric analysis showed new bone formation (39.0% +/- 12%), marrow space (52.9% +/- 9.3%), and residual ABB (8% +/- 2.7%). Conclusion: Our results indicated that 1,025 implants placed in sinuses grafted exclusively with ABB combined with CM led to an excellent and predictable survival rate of 98.1%. J Periodontol 2009;80:1920-1927.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Vimentin Expression and Myofibroblast Infiltration Are Early Markers of Renal Dysfunction in Kidney Transplantation: An Early Stage of Chronic Allograft Dysfunction?

Introduction. The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up. Methods. Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function. Results. Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 +/- 8.5 months. The mean Cr at biopsy time was 1.44 +/- 0.33 mg/dL, and after the follow-up it was 1.29 +/- 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively. Conclusion. Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.

Functional and morphologic evaluation of kidney proximal tubuli and correlation with renal allograft prognosis

P>Renal transplant patients with stable graft function and proximal tubular dysfunction (PTD) have an increased risk for chronic allograft nephropathy (CAN). In this study, we investigated the histologic pattern associated with PTD and its correlation with graft outcome. Forty-nine transplant patients with stable graft function were submitted to a biopsy. Simultaneously, urinary retinol-binding protein (uRBP) was measured and creatinine clearance was also determined. Banff`s score and semi-quantitative histologic analyses were performed to assess tubulointerstitial alterations. Patients were followed for 24.0 +/- 7.8 months. At biopsy time, mean serum creatinine was 1.43 +/- 0.33 mg/dl. Twelve patients (24.5%) had uRBP >= 1 mg/l, indicating PTD and 67% of biopsies had some degree of tubulointerstitial injury. At the end of the study period, 18 (36.7%) patients had lost renal function. uRBP levels were not associated with morphologic findings of interstitial fibrosis and tubular atrophy (IF/TA), interstitial fibrosis measured by Sirius red or tubulointerstitial damage. However, in multivariate analysis, the only variable associated with the loss of renal function was uRBP level >= 1 mg/l, determining a risk of 5.290 of loss of renal function (P = 0.003). Renal transplant patients who present PTD have functional alteration, which is not associated with morphologic alteration. This functional alteration is associated to progressive decrease in renal function.

A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.

Bayesian Estimation for Performance Measures of Two Diagnostic Tests in the Presence of Verification Bias

Sensitivity and specificity are measures that allow us to evaluate the performance of a diagnostic test. In practice, it is common to have situations where a proportion of selected individuals cannot have the real state of the disease verified, since the verification could be an invasive procedure, as occurs with biopsy. This happens, as a special case, in the diagnosis of prostate cancer, or in any other situation related to risks, that is, not practicable, nor ethical, or in situations with high cost. For this case, it is common to use diagnostic tests based only on the information of verified individuals. This procedure can lead to biased results or workup bias. In this paper, we introduce a Bayesian approach to estimate the sensitivity and the specificity for two diagnostic tests considering verified and unverified individuals, a result that generalizes the usual situation based on only one diagnostic test.

Noninvasive Evaluation of Oral Lesions Using Depth-sensitive Optical Spectroscopy

BACKGROUND: Optical spectroscopy is a noninvasive technique with potential applications for diagnosis of oral dysplasia and early cancer. In this study, we evaluated the diagnostic performance of a depth-sensitive optical spectroscopy (DSOS) system for distinguishing dysplasia and carcinoma from non-neoplastic oral mucosa. METHODS: Patients with oral lesions and volunteers without any oral abnormalities were recruited to participate. Autofluorescence and diffuse reflectance spectra of selected oral sites were measured using the DSOS system. A total of 424 oral sites in 124 subjects were measured and analyzed, including 154 sites in 60 patients with oral lesions and 270 sites in 64 normal volunteers. Measured optical spectra were used to develop computer-based algorithms to identify the presence of dysplasia or cancer. Sensitivity and specificity were calculated using a gold standard of histopathology for patient sites and clinical impression for normal volunteer sites. RESULTS: Differences in oral spectra were observed in: (1) neoplastic versus nonneoplastic sites, (2) keratinized versus nonkeratinized tissue, and (3) shallow versus deep depths within oral tissue. Algorithms based on spectra from 310 nonkeratinized anatomic sites (buccal, tongue, floor of mouth, and lip) yielded an area under the receiver operating characteristic curve of 0.96 in the training set and 0.93 in the validation set. CONCLUSIONS: The ability to selectively target epithelial and shallow stromal depth regions appeared to be diagnostically useful. For nonkeratinized oral sites, the sensitivity and specificity of this objective diagnostic technique were comparable to that of clinical diagnosis by expert observers. Thus, DSOS has potential to augment oral cancer screening efforts in community settings. Cancer 2009;115:1669-79. (C) 2009 American Cancer Society.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Crystallization and Preliminary Crystallographic Analysis of Laminarinase from Rhodothermus marinus: A Case of Pseudomerohedral Twinning

Thermophilic endo-1,3(4)-beta-glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75%. Diffraction data were collected to a resolution of 1.95 angstrom and resulted in a dataset with an overall R-merge of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

Microbiopsies of Surface Dental Enamel as a Tool to Measure Body Lead Burden

Lead (Pb) poisoning is preventable but continues to be a public health problem in several countries. Measuring Pb in the surface dental enamel (SDE) using microbiopsies is a rapid, safe, and painless procedure. There are different protocols to perform these microbiopsies, but the reliability of dental enamel lead levels (DELL) determination is dependent upon biopsy depth (BD). It is established that DELL decrease from the outermost superficial layer to the inner layer of dental enamel. The aim of this study was to determine DELL obtained by two different microbiopsy techniques on SDE termed protocol I and protocol II. Two consecutive enamel layers were removed from the same subject group (n = 138) for both protocols. Protocol I consisted of a biopsied site with a diameter of 4 mm after the application of 10 l HCl for 35 s. Protocol II involved a biopsied site of 1.6 mm diameter after application of 5 l HCl for 20 s. The results demonstrated that there were no significant differences for BD and DELL between homologous teeth using protocol I. However, there was a significant difference between DELL in the first and second layers using both protocols. Further, the BD in protocol II overestimated DELL values. In conclusion, SDE analyzed by microbiopsy is a reliable biomarker in protocol I, but the chemical method to calculate BD in protocol II appeared to be inadequate for measurement of DELL. Thus, DELL could not be compared among studies that used different methodologies for SDE microbiopsies.

Preparation and characterization of colloidal Ni(OH)(2)/bentonite composites

A conductive and electrochemically active composite material has been prepared by the combination of bentonite and nickel hydroxide precursor sol. This material exhibits the characteristic intercalation properties of the clay component and the electrochemical and optical properties of nickel hydroxide. The clay particles seem to induce the aggregation of nickel hydroxide, leading to the formation of a layer of alpha-Ni(OH)(2) exhibiting needle like morphology. The composite forms stable films and has been conveniently used for the preparation of modified electrodes exhibiting intercalation and electrochemical properties, thus providing an interesting material for the development of amperometric sensors. (C) 2008 Elsevier Ltd. All rights reserved.

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal trarisduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.