Francis R. Denys

Effects of phosphoric acid concentration and etch duration on the shear bond strength of an orthodontic bonding resin to enamel: An in vitro study

The purpose of this in vitro study was to determine the effects of phosphoric acid (H3PO4) concentration and duration of etching on the shear bond strength of an orthodontic bonding resin to enamel. Nine bonding procedures, each involving 18 extracted human maxillary permanent canines, were used. Ground enamel surfaces were etched with a 37% H3PO4 solution, a 15% H3PO4 gel, or a 5% H3PO4 solution for 60, 30, and 15 seconds, respectively. Cylinders of an orthodontic bonding resin, Concise, were prepared in a special device. The test specimens were disassembled 15 minutes after preparation and stored in distilled water at 37 degrees C for 24 hours. A shear load was applied to the bonded cylinders at a crosshead speed of 0.02 in.min-1 in an Instron testing machine, and the shear bond strengths were calculated and expressed in MN.m-2. A two-factor analysis of the data showed that the H3PO4 concentration had no significant effect on the shear bond strength, but the duration of etching affected shear bond strength significantly. The enamel aspects of the fractured test specimens were examined microscopically and the percent failure within the bonding resin at the bonding sites estimated. The correlation between shear bond strength and percentage failure within the bonding resin was not significant. The effects of the nine etching procedures on ground and unground enamel surfaces were studied by scanning electron microscopy. The etching procedures produced well-defined etching patterns on both ground and unground enamel surfaces.

Liposomes as Oral Adjuvants

In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for mucosal responses when used as oral vaccines. Evidence was provided that oral administration of antigen in liposomes resulted in an augmented mucosal response, compared to the response obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were further enhanced by the use of lipophilic MDP in the antigen/liposome vaccines. In order to better understand the properties of liposomes important for their functional activities, a rapid and reproducible method employing flow cytometry was described which can be conveniently used for the characterization of liposome preparations. Finally, evidence was presented which further supports the potential of recombinant DNA techniques in developing effective and safe oral vaccines against a variety of infectious diseases.

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicers hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiérys periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.

Tensile bond strengths of orthodontic bonding resins and attachments to etched enamel

The purpose of this in vitro study was to determine the tensile bond strengths (TBS) of several orthodontic bonding systems and orthodontic brackets to enamel surfaces exposed to different etching procedures. The TBS of four bonding systems were determined by a test method developed by Kemper and Kilian. Twelve test specimens were prepared for each procedure. The test system was modified to determine the TBS of orthodontic brackets bonded to enamel surfaces by six bonding procedures and the TBS were evaluated 15 minutes and 24 hours after specimen preparation. The specimens were loaded to failure in an Instron machine operated at a crosshead speed of 0.02 inch/min. The TBS were expressed in MN.m-2. The TBS of the four bonding systems to etched enamel were not significantly different (P = 0.2528). The TBS of bonded brackets determined 24 hours after specimen preparation were not significantly different than the TBS recorded after 15 minutes except for brackets bonded with Lee Cleanse and Bond I to enamel surfaces etched with 15% H3PO4 for 30 seconds. The TBS of brackets to enamel etched with 15% H3PO4 for 30 seconds were not significantly different from the TBS to enamel surfaces etched with 5% H3PO4 for 15 seconds except for brackets bonded with Lee Cleanse and Bond II and tested 24 hours after bonding.

Shear Bond Strength of Light-cured Glass Ionomer to Enamel and Dentin

The shear bond strengths of a light-cured glass-ionomer cement to enamel and dentin were determined with use of extracted human maxillary permanent canines and molars. Bonding sites on the ground, etched enamel and ground dentin surfaces were demarcated by the punching of a hole, 3 mm in diameter, in an adhesive tape. The mixed glass-ionomer cement was transferred to the demarcated site, cured by exposure to visible light for 30 s, and the cement surface treated with Scotchprep Dentin Primer followed by Scotchbond 2 Light Cure Dental Adhesive. The embedded teeth were positioned in an assembly apparatus, and Silux composite was bonded to the glass-ionomer-cement surfaces. The specimens were disassembled after 15 min and subjected to a shear load (in an Instron machine) immediately after disassembly; after storage in water at 37°C for 24 h, without and with temperature cycling; and after storage in water for four weeks, without and with temperature cycling. The shear bond strength of the glass-ionomer cement to etched enamel was in the order of 12 MN. m-2, and to dentin it was 9 MN.m -2. Temperature cycling and duration of storage had no adverse effect on the shear bond strength. The enamel and dentin aspects of fractured test specimens were examined, and the percentage of the bonding area that failed in the cement was estimated. Most of the test specimens failed partly at the enamel and dentin interfaces and within the glass-ionomer cement.

Characterization of liposome suspensions by flow cytometry

Novel approaches to drug delivery and induction of immune responses using liposomes have received much attention in recent years. Liposomes, however, are not a singular entity, but can be produced with a diverse group of phospholipids that form microspheres of different sizes, physical structure, electrochemical characteristics, and most importantly, physiologic properties. The purpose of this study was to establish the usefulness of flow cytometry as a convenient, rapid method for assessing the relative size and uniformity of liposomal preparations. Liposomes were made from phospholipid suspensions by sonication alone, or sonication followed by microemulsification. Forward laser light scatter (FSC) analysis of liposomal preparations by flow cytometry indicated that microemulsification produced homogeneous, small vesicles which were less than 1 micron in diameter, compared to the more heterogeneous sized liposomes generated by sonication alone. Transmission electron micrographs of the liposomal preparations were used to confirm the FSC results and showed that liposomes prepared by microemulsification were homogeneous, unilamellar vesicles which exhibited a mean diameter of 99.8 nm, whereas the sonicated-only preparation was more heterogeneous in size, exhibiting a mean diameter of 154.1 nm. Analysis of various liposome preparations by FSC during a 9 week storage period showed that small vesicles were relatively stable. We conclude that flow cytometry using FSC analysis provides a rapid, reproducible and convenient method to evaluate the relative size, uniformity and stability of liposomes.

Ultrastructural localization of the transferrin receptor and transferrin on marrow cell surfaces

Summary. The monoclonal antibody OKT9 (a known transferrin receptor antibody) and a monoclonal antibody to transferrin (ATfn) were used to localize the transferrin receptor and transferrin on marrow cells. After incubation of cell suspensions with the antibody, the cells were reacted with an affinity purified Fab fragment of goat anti‐mouse IgG conjugated to horseradish peroxidase (GAM‐HRP), which was in turn visualized by reaction with 3,3′‐diaminobenzidine (DAB). Erythroblast cell surfaces stained intensely with OKT9‐GAM‐HRP‐DAB, weaker staining was observed on reticulocyte surfaces, whereas erythrocyte surfaces lacked staining. Staining was present on surface caveolae, which often contained endogenous ferritin particles. Moderate to strong OKT9 staining was observed on less than 10% of presumed lymphoid cells. Monocytes, macrophages, promyelocytes, granulocytes, megakaryocytes and platelets consistently lacked OKT9 staining. ATm‐GAM‐HRP‐DAB staining of erythroid cells was similar to that observed with OKT9 staining; however, in contrast to the latter staining, ATfn stained the surfaces of megakaryocytes, platelets, monocytes and most lymphocytes. Promyelocytes stained weakly, whereas late granulocytes lacked staining. These results indicate that the T9 transferrin receptor (1) is largely confined to erythroid cells in marrow, (2) is diffusely distributed on the surface of early erythroid cells, (3) decreases with cell maturation, and (4) is lost when haemoglobin synthesis is completed. Transferrin appears in a similar distribution on erythroid cell surfaces but also appears to bind to some cell surfaces lacking the T9 receptor.

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

Shear bond strength and quantitative microleakage of a multipurpose dental adhesive system resin bonded to dentin

The shear bond strengths of a dental bonding system used in conjunction with a composite resin bonded to dentin were determined 1 minute after irradiation and after storage in saline at 37 degrees C for 24 hours, 1 week without and with temperature cycling, and 4 weeks without and with temperature cycling. The quantitative microleakage of class V preparations in cementum (dentin) restored with the system was determined by a spectrophotometric dye-recovery method. Excellent shear bond strengths ranging from 13.9 MPa (1 minute) to 19.5 MPa (1 week) were obtained. The shear bond strengths and quantitative microleakage of the multipurpose dental adhesive system compared favorably with the data obtained from other dental bonding systems under similar experimental conditions.

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts, osteoid, and bone matrix

SummaryGlycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicers high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiérys periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1–0.5µm in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.