The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices

The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium. Objective. This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days. Methods. Dentin beams (1 mm x 2 mm x 6 mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus (E) (3-point bending, 15% strain) the beams were divided into 5 groups (n = 11/group) and incubated at 37 degrees C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA. Results. Both the storage medium and the storage time showed significant effects on E, mass loss and HOP release (p < 0.05). The incubation in CM resulted in relatively rapid and significant (p < 0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower (p < 0.05) than in the control CM medium, the recommended storage medium. Conclusions. The storage solutions used to age resin-dentin bonds should be buffered solutions that contain both calcium and zinc. The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs. (c) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Substantivity of chlorhexidine to human dentin

Objectives. To better comprehend the role of CHX in the preservation of resin-dentin bonds, this study investigated the substantivity of CHX to human dentin. Material and methods. Dentin disks (n = 45) were obtained from the mid-coronal portion of human third molars. One-third of dentin disks were kept mineralized (MD), while the other two-thirds had one of the surfaces partially demineralized with 37% phosphoric acid for 15 s (PDD) or they were totally demineralized with 10% phosphoric acid (TDD). Disks of hydroxyapatite (HA) were also prepared. Specimens were treated with: (1) 10 mu L of distilled water (controls), (2) 10 mu L of 0.2% chlorhexidine diacetate (0.2% CHX) or (3) 10 mu L of 2% chlorhexidine diacetate (2% CHX). Then, they were incubated in 1 mL of PBS (pH 7.4, 37 degrees C). Substantivity was evaluated as a function of the CHX-applied dose after: 0.5 h, 1 h, 3 h, 6 h, 24 h, 168 h (1 week), 672 h (4 weeks) and 1344 h (8 weeks) of incubation. CHX concentration in eluates was spectrophotometrically analyzed at 260 nm. Results. Significant amounts of CHX remained retained in dentin substrates (MD, PPD or TDD), independent on the CHX-applied dose or time of incubation (p < 0.05). High amounts of retained CHX onto HA were observed only for specimens treated with the highest concentration of CHX (2%) (p < 0.05). Conclusion. The outstanding substantivity of CHX to dentin and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin-dentin bonds. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Comparative histomorphometric and tomographic analysis of maxillary sinus floor augmentation in rabbits using autografts and xenografts

Our goal was to evaluate bone neoformation promoted by a bovine xenograft composite (XC) compared with autogenous graft for maxillary sinus augmentation in a rabbit model. The left maxillary sinus of 18 male rabbits was filled with 200 mg of cortical and cancellous autogenous bone and the right sinus was filled with 200 mg of a composite comprised organic and inorganic bovine matrices, pool of bBMPs and collagen. Postoperative implant intervals of 2, 4, and 8 weeks were analyzed. Differences in the bone optical density among the groups and experimental periods were evaluated by computed tomography analysis. The tissue response was evaluated by histomorphometric analysis of the newly formed bone, connective tissue and/or granulation tissue, residual material, and bone marrow. The tomographic analyses showed a maximum optical density in the 4-week period for both groups. Histologically, an inflammatory infiltrate was observed at 2 weeks in the XC group but exclusively around the organic particles of the biomaterial. Regarding to the amount of newly formed bone, no statistical differences (p > 0.05) were observed among the two treatments throughout the implant intervals. However, by the end of the 8 weeks, the quantity of bone marrow was two times greater (p < 0.05) in the control group than in the XC group. In conclusion, the xenograft composite promotes formation of new bone in a similar fashion to autogenous bone and could therefore be considered a biomaterial with potential applications as a bone substitute in maxillary sinus floor augmentation. (C) 2007 Wiley Periodicals, Inc.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 A mu g rhBMP-2 + monoolein; II: 3 A mu g rhBMP-2 + monoolein; III: 7 A mu g rhBMP-2 + monoolein; IV: 1 A mu g rhBMP-2 + poloxamer; V: 3 A mu g rhBMP-2 + poloxamer; VI: 7 A mu g rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 A mu g) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.

Gingival crevicular fluid levels of MMP-8, MMP-9, TIMP-2, and MPO decrease after periodontal therapy

P>Background This study aimed at comparing the levels of matrix metalloproteinase (MMP)-8, tissue Inhibitor of MMPs (TIMP)-1 and TIMP-2, Myeloperoxidase (MPO), and MMP-9 in the gingival crevicular fluid (GCF) of chronic periodontitis (CP) patients and controls at baseline and 3 months after non-surgical therapy. Materials and Methods GCF was collected from one site of 15 control subjects and 27 CP patients. MMP-8, MMP-9, TIMP-1, and TIMP-2 were determined by Enzyme-linked immunoabsorbent assay; different forms of MMP-9, by gelatin zymography; and MPO, colorimetrically. Results At baseline, higher levels of MMP-8, TIMP-2, MPO, and the 87 kDa-MMP-9 were found in patients compared with controls (p < 0.001), and these molecules decreased after therapy (p < 0.03). There were no differences between the groups with respect to the higher molecular forms of MMP-9 (180, 130, 92 kDa) or total MMP-9 at baseline. No differences were observed in TIMP-1 levels. In controls, decreased levels of TIMP-2 and the higher molecular forms of MMP-9 (180, 130, 92 kDa) were found 3 months after therapy compared with baseline (p < 0.01). Conclusions Higher levels of MMP-8, TIMP-2, MPO, and 87 kDa MMP-9 were found in the GCF of patients compared with controls, and these markers decreased 3 months after periodontal therapy.

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.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.

Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males

GH is being used by elite athletes to enhance sporting performance. To examine the hypothesis that exogenous 22-kDa recombinant human GH (rhGH) administration could be detected through suppression of non-22-kDa isoforms of GH, we studied seventeen aerobically trained males (age, 26.9 +/- 1.5 yr) randomized to rhGH or placebo treatment (0.15 IU/kg/day for 1 week). Subjects were studied at rest and in response to exercise (cycle-ergometry at 65% of maximal work capacity for 20 min). Serum was assayed for total GH (Pharmacia IRMA and pituitary GH), 22-kDa GH (2 different 2-site monoclonal immunoassays), non-22-kDa GH (22-kDa GH-exclusion assay), 20-kDa GH, and immunofunctional GH. In the study, 3 h after the last dose of rhGH, total and 22-kDa GH concentrations were elevated, reflecting exogenous 22-kDa GH. Non-22-kDa and 20-kDa GH levels were suppressed. Regression of non-22-kDa or 20-kDa GH against total or 22-kDa GH produced clear separation of treatment groups. In identical exercise studies repeated between 24 and 96 h after cessation of treatment, the magnitude of the responses of all GH isoforms was suppressed (P < 0.01), but the relative proportions were similar to those before treatment. We conclude: 1) supraphysiological doses of rhGH in trained adult males suppressed exercise-stimulated endogenous circulating isoforms of GH for up to 4 days; 2) the dearest separation of treatment groups required the simultaneous presence of high exogenous 22-kDa GH and suppressed 20-kDa or non-22-kDa GH concentrations; and 3) these methods may prove useful in detecting rhGH abuse in athletes.

Three-dimensional orthotropic viscoelastic finite element model of a human ligament

Ligaments undergo finite strain displaying hyperelastic behaviour as the initially tangled fibrils present straighten out, combined with viscoelastic behaviour (strain rate sensitivity). In the present study the anterior cruciate ligament of the human knee joint is modelled in three dimensions to gain an understanding of the stress distribution over the ligament due to motion imposed on the ends, determined from experimental studies. A three dimensional, finite strain material model of ligaments has recently been proposed by Pioletti in Ref. [2]. It is attractive as it separates out elastic stress from that due to the present strain rate and that due to the past history of deformation. However, it treats the ligament as isotropic and incompressible. While the second assumption is reasonable, the first is clearly untrue. In the present study an alternative model of the elastic behaviour due to Bonet and Burton (Ref. [4]) is generalized. Bonet and Burton consider finite strain with constant modulii for the fibres and for the matrix of a transversely isotropic composite. In the present work, the fibre modulus is first made to increase exponentially from zero with an invariant that provides a measure of the stretch in the fibre direction. At 12% strain in the fibre direction, a new reference state is then adopted, after which the material modulus is made constant, as in Bonet and Burton's model. The strain rate dependence can be added, either using Pioletti's isotropic approximation, or by making the effect depend on the strain rate in the fibre direction only. A solid model of a ligament is constructed, based on experimentally measured sections, and the deformation predicted using explicit integration in time. This approach simplifies the coding of the material model, but has a limitation due to the detrimental effect on stability of integration of the substantial damping implied by the nonlinear dependence of stress on strain rate. At present, an artificially high density is being used to provide stability, while the dynamics are being removed from the solution using artificial viscosity. The result is a quasi-static solution incorporating the effect of strain rate. Alternate approaches to material modelling and integration are discussed, that may result in a better model.

Spermatozoal ultrastructure of spiny oysters (Spondylidae, Bivalvia) including a comparison with other bivalves

Sperm ultrastructure in three representative species of the marine bivalve family Spondylidae (spiny or thorny oysters) is examined and compared with available data on other bivalves, especially other families of the subclass Pteriomorphia. Spondylid spermatozoa are of the externally fertilizing aquasperm. type (ect-aquasperm). The acrosomal vesicle is conical with a deep basal invagination extending almost the full length of the vesicle. Vesicle contents are divisible into an inner, highly electron-dense anterior layer and a less dense posterior layer. The anterior layer is folded back on itself posteriorly and exhibits radiating plates (best developed peripherally). The vesicle rests on, and is partially embedded in, an extensive granular deposit of subacrosomal. material at the nuclear apex. This deposit extends partly into acrosomal vesicle invagination and also fills a broad depression in the anterior of the nucleus. No pre-formed axial rod (perforatorium) is present. The nucleus is round-pyriform and its contents coarsely fibrogranular. At the base of the nucleus, four broad depressions partially accommodate the midpiece mitochondria. The midpiece consists the four spherical mitochondria and the proximal and distal centrioles. The centrioles are arranged at approximately 90degrees to each other, and each consists of nine, angularly-oriented, microtubular triplets embedded in a granular matrix. A short, periodically banded rootlet connects the proximal centriole to the nuclear fossa, whereas the distal centriole, which forms the basal body to the flagellar axoneme, is anchored to the plasma membrane by nine terminally forked satellite fibres. Extensive deposits of putative glycogen rosettes surround the centrioles and mitochondria. The flagellum consists of a 9+2 axoneme sheathed by the plasma membrane. Spondylid spermatozoa strongly resemble those of the Pectinidae, further confirming the traditional view (based on comparative anatomy and shell morphology) of a close relationship between the Spondylidae and the Pectinidae. Differences in acrosomal shape and dimensions were noted between the three species examined, indicating potential taxonomic utility for comparative sperm ultrastructure within the Spondylidae.

Neural adaptations to resistance training - Implications for movement control

It has long been believed that resistance training is accompanied by changes within the nervous system that play an important role in the development of strength. Many elements of the nervous system exhibit the potential for adaptation in response to resistance training, including supraspinal centres, descending neural tracts, spinal circuitry and the motor end plate connections between motoneurons and muscle fibres. Yet the specific sites of adaptation along the neuraxis have seldom been identified experimentally, and much of the evidence for neural adaptations following resistance training remains indirect. As a consequence of this current lack of knowledge, there exists uncertainty regarding the manner in which resistance training impacts upon the control and execution of functional movements. We aim to demonstrate that resistance training is likely to cause adaptations to many neural elements that are involved in the control of movement, and is therefore likely to affect movement execution during a wide range of tasks. We review a small number of experiments that provide evidence that resistance training affects the way in which muscles that have been engaged during training are recruited during related movement tasks. The concepts addressed in this article represent an important new approach to research on the effects of resistance training. They are also of considerable practical importance, since most individuals perform resistance training in the expectation that it will enhance their performance in-related functional tasks.

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3

Background and Aims: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. Methods: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic ;stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen a 1 (I) mRNA). Results: Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). α-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. Conclusions: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes. (C) 2001 Blackwell Science Asia Pty Ltd.