Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Influence of matrix composition on polymerization stress development of experimental composites

Objective. Stress development at the tooth/restoration interface is one of the most important reasons for failure of adhesive restorations. The aim of this study was to evaluate the influence of BisGMA/TEGDMA (B/T) and UDMA/TEGDMA (U/T) ratios on polymerization stress (PS) and on the variables related to its development: degree of conversion (DC), polymerization maximum rate (Rp(max)), volumetric shrinkage (VS), elastic modulus (E), stress relaxation (SR) and viscosity of experimental composites. Method. Composites were formulated containing B/T or U/T in mol% ratios of 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3 and 8: 2, and 15 wt% of fumed silica. PS was determined with a universal testing machine. VS was measured with a linometer. E and SR were obtained in three-point bending. DC and Rp(max) were determined by real time NIR spectroscopy and viscosity was measured in viscometer. Data were submitted to one-way ANOVA, Tukey test (alpha = 0.05%) and regression analyses. Results. PS, VS, E and DC decreased and viscosity and Rp(max) increased with base monomer content in both series. PS showed strong correlation with VS, DC and viscosity. PS, VS and DC were higher and viscosity was lower for UDMA-based materials. Significance. Reduced viscosity, kinetics parameters and molecular characteristics led UDMA-based composites to elevated conversion and relatively lower PS at lower TEGDMA contents, compared to B/T composites. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Al(2)O(3)/GdAlO(3)fiber for dental porcelain reinforcement

The aim of this study was to test the hypothesis that the addition of continuous or milled GdAlO(3)/Al(2)O(3) fibers to a dental porcelain increases its mechanical properties. Porcelain bars without reinforcement (control) were compared to those reinforced with long fibers (30 vol%). Also, disk specimens reinforced with milled fibers were produced by adding 0 (control), 5 or 10 vol% of particles. The reinforcement with continuous fibers resulted in significant increase in the uniaxial flexural strength from 91.5 to 217.4 MPa. The addition of varied amounts of milled fibers to the porcelain did not significantly affect its biaxial flexural strength compared to the control group. SEM analysis showed that the interface between the continuous fiber and the porcelain was free of defects. On the other hand, it was possible to note the presence of cracks surrounding the milled fiber/porcelain interface. In conclusion, the reinforcement of the porcelain with continuous fibers resulted in an efficient mechanism to increase its mechanical properties; however the addition of milled fibers had no significant effect on the material because the porcelain was not able to wet the ceramic particles during the firing cycle. (C) 2008 Elsevier Ltd. All rights reserved.

Activity of Matrix Metalloproteinases in Bovine versus Human Dentine

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 +/- 2.2, 14.6 +/- 2.0, 9.7 +/- 1.2 and 12.4 +/- 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 +/- 2.0, 15.3 +/- 1.3, 15.4 +/- 1.3 and 15.5 +/- 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9. Copyright (C) 2011 S. Karger AG, Basel

Effect of 2% Chlorhexidine Digluconate on the Bond Strength to Normal Versus Caries-affected Dentin

This study evaluated the effect of 2% chlorhexidine digluconate (CHX) used as a therapeutic primer on the long-term bond strengths of two etch-and-rinse adhesives to normal (ND) and caries-affected (CAD) dentin. Forty extracted human molars with coronal carious lesions, surrounded by normal dentin, were selected for this study. The flat surfaces of two types of dentin (ND and CAD) were prepared with a water-cooled high-speed diamond disc, then acidetched, rinsed and air-dried. In the control groups, the dentin was re-hydrated with distilled water, blot-dried and bonded with a three-step (Scotchbond Multi-Purpose-MP) or two-step (Single Bond 2-SB) etch-and-rinse adhesive. In the experimental groups, the dentin was rehydrated with 2% CHX (60 seconds), blot-dried and bonded with the same adhesives. Resin composite build-ups were made. The specimens were prepared for microtensile bond testing in accordance with the non-trimming technique, then tested either immediately or after six-months storage in artificial saliva. The data were analyzed by ANOVA/Bonferroni tests (alpha=0.05). CHX did not affect the immediate bond strength to ND or CAD (p>0.05). CHX treatment significantly lowered the loss of bond strength after six months as seen in the control bonds for ND (p<0.05), but it did not alter the bond strength of CAD (p>0.05). The application of NIP on CHX-treated ND or CAD produced bonds that did not change over six months of storage.

Ascorbate-induced osteoblast differentiation recruits distinct MMP-inhibitors: RECK and TIMP-2

The bone formation executed by osteoblasts represents an interesting research field both for basic and applied investigations. The goal of this work was to evaluate the molecular mechanisms involved during osteoblast differentiation in vitro. Accordingly, we demonstrated that, during the osteoblastic differentiation, TIMP-2 and RECK presented differential expressions, where RECK expression was downregulated from the 14th day in contrast with an increase in TIMP-2. Concomitantly, our results showed a temporal regulation of two major signaling cascades during osteoblast differentiation: proliferation cascades in which RECK, PI3 K, and GSK-3 beta play a pivotal role and latter, differentiation cascades with participation of Ras, Rho, Rac-1, PKC alpha/beta, and TIMP-2. Furthermore, we observed that phosphorylation level of paxillin was downregulated while FAK(125) remained unchangeable, but active during extracellular matrix (ECM) remodeling. Concluding, our results provide evidences that RECK and TIMP-2 are involved in the control of ECM remodeling in distinct phases of osteoblast differentiation by modulating MMP activities and a multitude of signaling proteins governs these events.

Long-term effects of developmental exposure to di-n-butyl-phthalate (DBP) on rat prostate: Proliferative and inflammatory disorders and a possible role of androgens

In the present study we evaluated the toxic effects on the male adult rat prostate of DBP exposure during fetal and lactational periods, because although many studies have addressed the influence of phthalates on the male reproductive system, only a few have discussed their possible effects on prostate development. Pregnant females were distributed into two experimental groups: Control (C) and Treated (T). The females of the T group received DBP (100 mg/kg, by gavage) from gestation day 12 to postnatal day 21, while C rats received the vehicle (corn oil). In adulthood (90 days old), the animals were euthanized. The serum and testicular testosterone levels were measured. Ventral prostate was removed and weighed. Distal segment fragments of the ventral prostate were fixed and processed for histochemistry and immunohistochemistry to detect androgen receptor (AR) and Ki67 antigens. Protein extraction from ventral prostate fragments was performed for AR immunoblotting and Gelatin zymography for MMP-2 and MMP-9 (MMP, metalloproteinase). Stereological and histopathological analyses were also performed. Serum and testicular testosterone levels and prostate weight were comparable between groups. In the T group the relative proportions (%) of epithelial (C=32.86; T=42.04*) and stromal (C=21.61; T=27.88*) compartments were increased, while the luminal compartment was decreased (C=45.54; T=30.08*), *p < 0.05. In T, disseminated inflammatory infiltrate in the stroma, associated or not with epithelial dysplasia and PIN (Prostatic Intraepithelial Neoplasia), was observed. Increases in AR expression, proliferation index and metalloproteinase 9 (MMP-9) activity were noted in T animals. In some T animals, collagen fibrils accumulated adjacent to the epithelium. As far as we are aware, this is the first report in the literature showing that phthalates could play a role in proliferative and inflammatory disorders of the rat prostate. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

The Bone Formation Capabilities of the Anorganic Bone Matrix-Synthetic Cell-Binding Peptide 15 Grafts in an Animal Periodontal Model: A Histologic and Histomorphometric Study in Dogs

Background: The aim of this study is to verify the regenerative potential of particulate anorganic bone matrix synthetic peptide-15 (ABM-P-15) in class III furcation defects associated or not with expanded polytetrafluoroethylene membranes. Methods: Class III furcation defects were produced in the mandibular premolars (P2, P3, and P4) of six dogs and filled with impression material. The membranes and the bone grafts were inserted into P3 and P4, which were randomized to form the test and control groups, respectively; P2 was the negative control group. The animals were sacrificed 3 months post-treatment. Results: Histologically, the complete closure of class III furcation defects was not observed in any of the groups. Partial periodontal regeneration with similar morphologic characteristics among the groups was observed, however, through the formation of new cementum, periodontal ligament, and bone above the notch. Histologic analysis showed granules from the bone graft surrounded by immature bone matrix and encircled by newly formed tissue in the test group. The new bone formation area found in the negative control group was 2.28 +/- 2.49 mm(2) and in the test group it was 6.52 +/- 5.69 mm(2), which showed statistically significant differences for these groups considering this parameter (Friedman test P <0.05). There was no statistically significant difference among the negative control, control, and test groups for the other parameters. Conclusions: The regenerative potential of ABM-P-15 was demonstrated through new bone formation circumscribing and above the graft particles. The new bone also was accompanied by the formation of new cementum and periodontal ligament fibers. J Periodontol 2010;81:594-603.

Acellular Dermal Matrix Graft for Gingival Augmentation: A Preliminary Clinical, Histologic, and Ultrastructural Evaluation

Background: The aim of this study was to evaluate clinically, histologically, and ultrastructurally the integration process of the acellular dermal matrix used to increase the band of keratinized tissue while achieving gingival inflammation control. Methods: Ten patients exhibiting a mucogingival problem with bands of keratinized tissue <= 1 mm and gingival inflammation of the related teeth were included in the study. The surgical procedures were performed to augment the gingival tissue using acellular dermal matrix. Clinical measurements were assessed at baseline and after 3 months. A specimen of the allograft and surrounding tissues was obtained immediately before the surgery and 4 minutes and 1, 2, 3, 4, 6, and 10 weeks after grafting. Results: Clinically, a gain of keratinized tissue of 2.92 +/- 0.65 mm was observed after 3 months. Histologically and ultrastructurally, many macrophages were observed phagocytosing preexisting collagen fibers in the first weeks. From week 2 on, fibroblasts synthesizing new collagen, epithelial cells colonizing the graft surface, and revascularization were noticed. After 6 weeks it was difficult to find the acellular dermal matrix preexisting collagen fibers. This process of substitution was completed after 10 weeks, when the reepithelialization of the entire graft throughout a well-structured basement membrane was achieved. Conclusion: The acellular dermal matrix graft seemed to be an easily handled material for use in keratinized tissue augmentation that, in humans, was substituted and completely reepithelialized in 10 weeks according to histologic and ultrastructural results. J Periodontol 2009;80:253-259.

Comparison between two surgical techniques for root coverage with an acellular dermal matrix graft

Aim: The aim of this randomized, controlled, clinical study was to compare two surgical techniques with the acellular dermal matrix graft (ADMG) to evaluate which technique could provide better root coverage. Material and Methods: Fifteen patients with bilateral Miller Class I gingival recession areas were selected. In each patient, one recession area was randomly assigned to the control group, while the contra-lateral recession area was assigned to the test group. The ADMG was used in both groups. The control group was treated with a broader flap and vertical-releasing incisions, and the test group was treated with the proposed surgical technique, without releasing incisions. The clinical parameters evaluated before the surgeries and after 12 months were: gingival recession height, probing depth, relative clinical attachment level and the width and thickness of keratinized tissue. Results: There were no statistically significant differences between the groups for all parameters at baseline. After 12 months, there was a statistically significant reduction in recession height in both groups, and there was no statistically significant difference between the techniques with regard to root coverage. Conclusions: Both surgical techniques provided significant reduction in gingival recession height after 12 months, and similar results in relation to root coverage.

In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

Ridge Preservation With Acellular Dermal Matrix and Anorganic Bone Matrix Cell-Binding Peptide P-15 After Tooth Extraction in Humans

Background: Preventing ridge collapse with the extraction of maxillary anterior teeth is vital to an esthetic restorative result. Several regenerative techniques are available and are used for socket preservation. The aim of this study is to analyze by clinical parameters the use of acellular dermal matrix (ADM) and anorganic bovine bone matrix (ABM) with synthetic cell-binding peptide P-15 to preserve alveolar bone after tooth extraction. Methods: Eighteen patients in need of extraction of maxillary anterior teeth were selected and randomly assigned to the test group (ADM plus ABM/P-15) or the control group (ADM only). Clinical measurements were recorded initially and at 6 months after ridge-preservation procedures. Results: In the clinical measurements (external vertical palatal measurement [EVPM], external vertical buccal measurement [EVBM], and alveolar horizontal measurement [AHM]) the statistical analysis showed no difference between test and control groups initially and at 6 months. The intragroup analysis, after 6 months, showed a statistically significant reduction in the measurements for both groups. In the comparison between the two groups, the differences in the test group were as follows: EVPM = 0.83 +/- 1.53 mm; EVBM = 1.20 +/- 2.02 mm; and AHM = 2.53 +/- 1.81 mm. The differences in the control group were as follows: EVPM = 0.87 +/- 1.13 mm; EVBM = 1.50 +/- 1.15 mm; and AHM = 3.40 +/- 1.39 mm. The differences in EVPM and EVBM were not statistically significant; however, in horizontal measurement (AHM), there was a statistically significant difference (P<0.05). Conclusion: The results of this study show that ADM used as membrane associated with ABM/P-15 can be used to reduce buccal-palatal dimensions compared to ADM alone for preservation of the alveolar ridge after extraction of anterior maxillary teeth. J Periodontol 2011;82:72-79.

Importance of estimating matrix quality for modeling species distribution in complex tropical landscapes: a test with Atlantic forest small mammals

Information to guide decision making is especially urgent in human dominated landscapes in the tropics, where urban and agricultural frontiers are still expanding in an unplanned manner. Nevertheless, most studies that have investigated the influence of landscape structure on species distribution have not considered the heterogeneity of altered habitats of the matrix, which is usually high in human dominated landscapes. Using the distribution of small mammals in forest remnants and in the four main altered habitats in an Atlantic forest landscape, we investigated 1) how explanatory power of models describing species distribution in forest remnants varies between landscape structure variables that do or do not incorporate matrix quality and 2) the importance of spatial scale for analyzing the influence of landscape structure. We used standardized sampling in remnants and altered habitats to generate two indices of habitat quality, corresponding to the abundance and to the occurrence of small mammals. For each remnant, we calculated habitat quantity and connectivity in different spatial scales, considering or not the quality of surrounding habitats. The incorporation of matrix quality increased model explanatory power across all spatial scales for half the species that occurred in the matrix, but only when taking into account the distance between habitat patches (connectivity). These connectivity models were also less affected by spatial scale than habitat quantity models. The few consistent responses to the variation in spatial scales indicate that despite their small size, small mammals perceive landscape features at large spatial scales. Matrix quality index corresponding to species occurrence presented a better or similar performance compared to that of species abundance. Results indicate the importance of the matrix for the dynamics of fragmented landscapes and suggest that relatively simple indices can improve our understanding of species distribution, and could be applied in modeling, monitoring and managing complex tropical landscapes.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.