Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

A Study of the Effect of Oxygen on the Rate of Dissolution of Gold in Cyanide Solutions.

The accepted chemical reactions in the dissolution of gold by cyanide solutions require the presence of gold, cyanide, water, and oxygen. The importance of dissolved oxygen in cyanide solutions as a factor is recognized by those familiar with cyanidation. Manufacturers of cyanidation equipment realize the necessity of oxygen, as shown by the appliances they have developed which are attached to the agitators in order to saturate the cyanide solutions with air.

The Effect of Temperature on the Rate of Dissolution of Gold in Cyanide Solutions which have a Constant Oxygen Content.

Since the development of cyanidation into a highly efficient process for treating gold ores, many papers have been written on its various aspects. Although, there has been much work done on it, the chemistry of the reaction is not yet completely understood.

A Study of the Dissolution of Pyrolusite in Sulphuric Acid

With an eye to the future development of its re­sources, the large deposits of low-grade manganese ore within Montana's boundaries show great promise of developing an important industry. With the pre­sent war demands and the possibility of a future mar­ket it is essential that work expended to utilize this important mineral.

Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.

Influence of enamel matrix derivative on cells at different maturation stages of differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

Periodontal healing after application of enamel matrix derivative in surgical supra/infrabony periodontal defects in rats with streptozotocin-induced diabetes

BACKGROUND AND OBJECTIVE Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.

Effects of buffering properties and undissociated acid concentration on dissolution of dental enamel in relation to pH and acid type

To quantify the relationships between buffering properties and acid erosion and hence improve models of erosive potential of acidic drinks, a pH-stat was used to measure the rate of enamel dissolution in solutions of citric, malic and lactic acids, with pH 2.4-3.6 and with acid concentrations adjusted to give buffer capacities (β) of 2-40 (mmol·l(-1))·pH(-1) for each pH. The corresponding undissociated acid concentrations, [HA], and titratable acidity to pH 5.5 (TA5.5) were calculated. In relation to β, the dissolution rate and the strength of response to β varied with acid type (lactic > malic ≥ citric) and decreased as pH increased. The patterns of variation of the dissolution rate with TA5.5 were qualitatively similar to those for β, except that increasing pH above 2.8 had less effect on dissolution in citric and malic acids and none on dissolution in lactic acid. Variations of the dissolution rate with [HA] showed no systematic dependence on acid type but some dependence on pH. The results suggest that [HA], rather than buffering per se, is a major rate-controlling factor, probably owing to the importance of undissociated acid as a readily diffusible source of H(+) ions in maintaining near-surface dissolution within the softened layer of enamel. TA5.5 was more closely correlated with [HA] than was β, and seems to be the preferred practical measure of buffering. The relationship between [HA] and TA5.5 differs between mono- and polybasic acids, so a separate analysis of products according to predominant acid type could improve multivariate models of erosive potential.

The effect of monoalkyl phosphates and fluoride on dissolution of hydroxyapatite, and interactions with saliva

The aims were to investigate the effect of monoalkyl phosphates (MAPs) and fluoride on dissolution rate of native and saliva-coated hydroxyapatite (HA). Fluoride at 300 mg/l (as NaF) inhibited dissolution of native HA by 12%, while potassium and sodium dodecyl phosphates (PDP, SDP), at 0.1% or higher, inhibited dissolution by 26-34%. MAPs, but not fluoride, also showed persistence of action. MAPs at 0.5% and fluoride at 300 mg/l were then tested separately against HA pre-treated with human saliva for 2 or 18 h. Agents were applied with brushing to half the specimens, and without brushing to the other half. In control (water-treated) specimens, pre-treatment of HA with human saliva reduced dissolution rate on average by 41% (2 h) and 63% (18 h). Brushing did not have a statistically significant effect on dissolution rate of saliva-coated specimens. In brushed specimens, fluoride significantly increased the inhibition due to 2- or 18-hour saliva pre-treatment. It is hypothesised that brushing partially removes the salivary film and allows KOH-soluble calcium fluoride formation at the surfaces of HA particles. Inhibition was reduced by PDP in 2-hour/non-brushed specimens and in 18-hour/brushed specimens. PDP did not affect dissolution rates in the remaining groups and SDP did not affect dissolution rate in any group. Possible reasons for these variable results are discussed. The experiments show that pre-treatment with saliva can significantly modify results of tests on potential anti-erosive agents and it is recommended that saliva pre-treatment should be a routine part of testing such agents.

Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model

Study Design. In vitro study to develop an intervertebral disc degeneration (IDD) organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4 and HTRA1.Objective. This study aimed to develop an in-vitro model of enzyme-mediated IDD to mimic the clinical outcome in humans for investigation of therapeutic treatment options.Summary of Background Data. Bovine IVDs are comparable to human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an IDD model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4 and HTRA1 were injected at a dose of 10μg/ml each. Phosphate buffered saline (PBS) was injected as a control. Discs were cultured for 8 days and loaded diurnally (day 1 to day 4 with 0.4 MPa for 16 h) and left under free swelling condition from day 4 to day 8 to avoid expected artifacts due to dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, glycosaminoglycan (GAG) content, total collagen content, relative gene expression and histological investigation.Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 compared to the day 0 control discs. Disc height was decreased following injection with HTRA1, and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.Conclusion. MMP-3, ADAMTS-4 and HTRA1 neither provoked visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height which positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may therefore be necessary to induce disc degeneration

Characterization of granite matrix porosity and pore-space geometry by in situ and laboratory methods

Most available studies of interconnected matrix porosity of crystalline rocks are based on laboratory investigations; that is, work on samples that have undergone stress relaxation and were affected by drilling and sample preparation. The extrapolation of the results to in situ conditions is therefore associated with considerable uncertainty, and this was the motivation to conduct the ‘in situ Connected Porosity’ experiment at the Grimsel Test Site (Central Swiss Alps). An acrylic resin doped with fluorescent agents was used to impregnate the microporous granitic matrix in situ around an injection borehole, and samples were obtained by overcoring. The 3-D structure of the porespace, represented by microcracks, was studied by U-stage fluorescence microscopy. Petrophysical methods, including the determination of porosity, permeability and P -wave velocity, were also applied. Investigations were conducted both on samples that were impregnated in situ and on non-impregnated samples, so that natural features could be distinguished from artefacts. The investigated deformed granites display complex microcrack populations representing a polyphase deformation at varying conditions. The crack population is dominated by open cleavage cracks in mica and grain boundary cracks. The porosity of non-impregnated samples lies slightly above 1 per cent, which is 2–2.5 times higher than the in situ porosity obtained for impregnated samples. Measurements of seismic velocities (Vp ) on spherical rock samples as a function of confining pressure, spatial direction and water saturation for both non-impregnated and impregnated samples provide further constraints on the distinction between natural and induced crack types. The main conclusions are that (1) an interconnected network of microcracks exists in the whole granitic matrix, irrespective of the distance to ductile and brittle shear zones, and (2) conventional laboratory methods overestimate the matrix porosity. Calculations of contaminant transport through fractured media often rely on matrix diffusion as a retardation mechanism.