Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

Wheat germ agglutinin as a counterstain for immunofluorescence studies of equine hoof lamellae.

Equine laminitis is a common, painful, debilitating condition of the hoof that is a leading cause of disability in horses, often necessitating euthanasia. The equine hoof represents an extreme evolutionary adaptation of an epidermal structure homologous to the human or murine nail units. Immunohistochemistry is frequently utilized in the study of the pathophysiology of laminitis. The complex, multilayered, extensively interdigitated epidermal-dermal lamellar interface renders precise interpretation of immunofluorescence localization difficult, especially when effective technique and reagents render non-reactive tissues completely dark. Fluorescent-conjugated wheat germ agglutinin (WGA) selectively labels dermal extracellular matrix fibres and epidermal cell membranes in tissue sections of horse hoof lamellae, is compatible with indirect immunofluorescence and augments interpretation of indirect immunofluorescence antigen localization. The current report details the use of WGA as a rapid, simple, economical counterstain for immunofluorescence studies of the equine hoof and may have application to other complex epidermal tissue structures.

Three-dimensional processing deformation of three denture base materials.

STATEMENT OF PROBLEM: Heat-polymerized polymethyl methacrylate denture bases deform during and after polymerization, and this deformation may affect the clinical performance of complete dentures. PURPOSE: The purpose of this study was to investigate the processing deformation of 3 denture base materials on a standardized anatomic model by using a contact scanner and surface matching software. MATERIAL AND METHODS: Maxillary reproductions of a definitive cast were digitized by using a contact scanner. The casts were allocated to 4 groups, depending on the denture base material: compression molded (heat polymerized, polymethyl methacrylate based); injection molded (heat polymerized, polymethyl methacrylate based); manually adapted and light-polymerized (urethane dimethacrylate based); and manually adapted, compression molded, and light-polymerized (urethane dimethacrylate-based). The intaglio surfaces of denture bases fabricated on each replicate cast also were digitized by using the contact scanner. Surface-matching software was used to measure dimensional changes between each cast and its corresponding denture base. The Kruskal-Wallis analysis of variance based on ranks was used to assess differences in contraction, expansion, and overall change among groups. The Mann-Whitney U test was performed to determine differences among individual groups. Statistical significance was inferred when P<.01 to compensate for multiple group comparisons. RESULTS: Statistically significant differences in processing deformations were observed among polymethyl methacrylate-based resins and urethane dimethacrylate-based resin. No differences were observed between compression molding and injection molding or between manual adaptation alone and the combination of manual adaptation and compression molding. CONCLUSIONS: Urethane dimethacrylate-based resin showed greater processing deformation compared with polymethyl methacrylate-based resins. Compression molding and injection molding techniques produced similar results for the polymethyl methacrylate-based resins. The methodology used showed patterns of deformation that were too complex to be accurately analyzed by linear measurements.

The fatigue life of a cobalt-chromium alloy after laser welding.

The aim of this study was to investigate the fatigue life of laser welded joints in a commercially available cast cobalt-chromium alloy. Twenty rod shaped specimens (40 mm x 1.5 mm) were cast and sand blasted. Ten specimens were used as controls and the remaining ten were sectioned and repaired using a pulsed Nd: YAG laser welder. All specimens were subjected to fatigue testing (30N - 2Hz) in a controlled environment. A statistically significant difference in median fatigue life was found between as-cast and laser welded specimens (p < 0.001). Consequently, the technique may not be appropriate for repairing cobalt chromium clasps on removable partial dentures. Scanning electron microscopy indicated the presence of cracks, pores and constriction of the outer surface in the welded specimens despite 70% penetration of the weld.

An investigation into the effects of metal primer and surface topography on the tensile bond strength between cobalt chromium alloy and composite resin.

This study examined the influence of surface preparation and metal primer on the tensile bond strength between cobalt chromium alloy and composite resin. The bond strength between 168 cobalt chromium alloy dumb-bells with one of three test surfaces (beaded, machined or sandblasted) to composite resin were tested. Half of each group were treated with metal primer. The weakest bond strength was produced by the unprimed machined surface, many specimens failing before testing. The metal primer increased the bond strengths of all groups tested. The greatest bond strengths were achieved with the primed beaded and sandblasted surfaces. Within the limits of the study it has been shown that the surface preparation of the cobalt-chromium alloy did influence tensile bond strengths with composite resin and Metal Primer II increased the tensile bond strengths for all groups tested. The sandblasted surface treated with Metal Primer II is recommended for the bonding of composite resin to cobalt chromium alloy.

Characterization of the porcine testis-expressed gene 11 (Tex11).

Testis expressed gene 11 (Tex11) is essential for meiosis and male fertility in the mouse. Currently, little is known about the control of spermatogenesis in non-rodent animal models such as pigs. Here, we characterized the sequence and expression profile of the porcine Tex11 gene. We showed that the porcine Tex11 is an X-linked gene that is exclusively expressed in germ cells in the adult pig testis. The expression of porcine Tex11 is correlated with the onset of meiosis and the expression pattern is highly conserved between the Tex11 homologs in pig and mouse. The DNA sequence analysis and the estimated molecular weight also suggested a high level of homology across species. As the mouse Tex11 proved to be essential for male fertility, the important biological function during meiosis is likely conserved in the porcine Tex11.

Immediate replacement complete dentures: pitfalls of ignoring traditional teaching and established practice.

A thirty-year-old female had immediate replacement dentures made by a general practitioner and her teeth extracted and the dentures fitted under general anaesthetic by her local oral surgeon three months previously. Anterior and posterior teeth had been extracted and no attempt had been made to smooth or shape the ridges. Both dentures were constructed with gum fitted anterior teeth without labial flanges. This treatment differs from traditional immediate replacement complete denture teaching. Traditionally, when possible the posterior teeth would have been extracted first and then once there had been a period of healing the immediate replacement complete dentures would have been made replacing the remaining anterior teeth. A trans-septal alveolotomy would have been performed, which would reduce the labial undercut on the edentulous ridge so that the denture could have a labial flange which would enable a border seal to be established to enhance retention but would not displace the lip. Care would have been taken to ensure that the fit surface of the denture would reflect the change in ridge shape that would follow healing. The divergence of this treatment management from a traditional approach raises important questions. In the past oral surgeons were well versed in pre-prosthetic surgery. As demand for this type of treatment has declined, so has the opportunity for oral surgery trainees, who themselves may have limited experience in prosthetic dentistry, to learn the techniques involved. Teaching of this form of removable prosthetic dentistry has been reduced reflecting the reduced frequency of this presenting condition.

Effect of two different disinfectants on dimensional stability of newer alginate impression materials over five days.

The effect of two different disinfectants on the dimensional stability of two alginate impression materials over five days was investigated. 60 impressions were made under standardised conditions, 30 with each alginate, of which ten were disinfected in each disinfectant and ten were controls. Impressions were stored over a period of five days in a container with 100% humidity. Measurements were made between four points. Two-way analysis of variance indicated no differences in the change from baseline to day 5 between any of the combinations of alginate and disinfectant. Only two of the changes between baseline and day 5 reached statistical significance at the p < 0.01 level. Therefore it can be concluded that within the limitations of this study when newer alginate impression materials are disinfected they remain stable over 5 days.

Tensile strength of laser welded cobalt-chromium alloy with and without an argon atmosphere.

The tensile strength and depth of weld of two cobalt chromium alloys before and after laser welding with and without an argon gas atmosphere were investigated. Using two cobalt chromium alloys, rod shaped specimens (5 cm x 1.5 mm) were cast. Specimens were sand blasted, sectioned and welded with a pulsed Nd: YAG laser welding machine and tested in tension using an Instron universal testing machine. A statistically significant difference in tensile strength was observed between the two alloys. The tensile strength of specimens following laser welding was significantly less than the unwelded controls. Scanning electron microscopy showed that the micro-structure of the cast alloy was altered in the region of the weld. No statistically significant difference was found between specimens welded with or without an argon atmosphere.

Is the bond between acrylic resin denture teeth and denture base resin stronger if they are both made by the same manufacturer?

A previous study suggested that a stronger bond may be achieved between acrylic resin denture base material and acrylic denture teeth when both are made by the same manufacturer. Three denture base acrylic resins from three different manufacturers were bonded to three different acrylic resin denture teeth, one of which was manufactured by each of the manufacturers of the base material. In each group there was a trend that the bond strength achieved between the teeth and base material from the same manufacturer was higher than the unmatched pairs but statistical significance was not achieved.

Dimensional stability of newer alginate impression materials over seven days.

The aim of this study was to determine the dimensional stability of the so called '5 day stable' alginates. Ten specimens each of three alginate materials were prepared using a standardised impression technique and a typodont model. Travelling microscope measurements were recorded for six distances at 24 hour intervals. SEM and EDX analysis was undertaken. Changes in dimension over time and differences between materials were tested using analysis of variance. 95% confidence intervals were calculated for the percentage change for comparison with a target of 1.5% based on the ISO Standard. Statistically significant dimensional changes were observed in some measured distances for all materials, but only six out of fifty four distances measured at 1, 5 and 7 days in three materials showed 95% confidence intervals that included the 1.5% ISO standard. There were no statistically significant differences in the proportional change between the three materials. The three materials showed similar appearance under SEM and similar composition by EDX analysis. It is concluded that all materials demonstrated good dimensional stability over the recommended maximum of 5 days.

Effect of curing cycle on the tensile strength of the bond between heat cured denture base acrylic resin and acrylic resin denture teeth.

The effect of different curing cycles on the tensile strength of the bond between one brand of cross-linked acrylic resin teeth and three heat cured denture base acrylic resins was tested. There were differences in the tensile bond strength between the three heat cured denture base acrylic resins and the three curing cycles used. The bond strength of the acrylic resin denture base material made by the same manufacturer as the cross-linked acrylic resin denture teeth was higher. The bond strength following the short cycle was lowest in all cases, individual differences between curing cycles failed to reach statistical significance.

Effect of GC METALPRIMER II on bond strength of heat-cured acrylic resin to titanium alloy (Ti-6Al-4V) with two different surface treatments.

This study investigated the bond strength of heat-polymerized acrylic resin to titanium alloy using a proprietary bonding agent (GC Metalprimer II). Two surface treatments (sandblasted or roughened with a tungsten carbide bur) were compared for their effect on bond strength with or without thermal cycling. Eighty specimens of heat-polymerized acrylic resin bonded to titanium alloy (Ti-6Al-4V) were prepared: 20 specimens (control) and 60 used a bonding agent. Four-point bend testing was used to record the load at failure. A chemical bond between heat-polymerized resin and alloy was achieved using GC Metalprimer II with both surface treatments. Reduced failure loads were recorded after thermal cycling. The predominant mode of failure was cohesive. GC Metalprimer II was effective in achieving a chemical bond with either the sandblasted or roughened surfaces.

Effect of surfactant on surface hardness of dental stone and investment casts produced from polyvinyl siloxane duplicating materials.

Polyvinylsiloxane duplicating materials are typically treated with a topical surfactant before pouring dental models, but the use of topical surfactants in the dental laboratory may affect the surface hardness of the resultant models. The effect of two different topical surfactants on surface hardness of two dental stones (FujiRock and Dentstone) and one phosphate bonded investment material (Croform WB) produced from polyvinyl siloxane (PVS) dental laboratory duplicating moulds was investigated. Topical surfactants affected the surface hardness of FujiRock, Dentstone and Croform WB investment material. Surface hardness of FujiRock increased with Wax-Mate surfactant. However, surface hardness of Croform WB investment material decreased with both topical surfactants.

Long-term dimensional stability and reproduction of surface detail of four polyvinyl siloxane duplicating materials.

OBJECTIVES: Duplicating materials must routinely accurately reproduce the details of dental casts and thus contribute significantly to the close adaptation and success of a removable prosthesis or fixed indirect restoration. It is important to establish the long-term dimensional stability of polyvinyl siloxane materials (PVS) as they are widely used in dental technology and over the duration of a course of treatment, are stored under dental laboratory conditions. The dimensional stability of four PVS duplicating materials was investigated over a 13-week period. MATERIALS AND METHODS: Forty PVS duplicate moulds of a partially dentate maxillary arch were produced from four materials (Elite double 8, Gemini transparent, C & J pourable silicone and Z Dupe). Three dimensions were measured in triplicate at baseline 1, 5, 9 and 13 weeks using computerised image analysis. Half of the specimens were measured following storage at room temperature (21+/-2 degrees C) and half at 37 degrees C to represent storage in hot climates. Specimens for scanning electron microscopy were prepared by duplicating a rugosity standard. RESULTS: At room temperature two materials Elite Double 8 and Z Dupe showed no statistically significant dimensional change, the other two materials showed a slight increase of dimensions. Specimens stored at 37 degrees C showed greater differences in dimensions with Z Dupe showing statistically significant shrinkage. SEM investigation showed no surface deterioration of two materials. CONCLUSIONS: None of the materials showed a change in dimension greater than 2% and the four PVS materials showed good dimensional stability over the time period of the study.

Tensile bond strength of heat and self-cured acrylic denture base resins to the inner and outer layers of two-layered acrylic resin denture teeth.

The tensile bond strength of the inner and outer layers of two-layered acrylic resin denture teeth to heat cured and self-curing acrylic resin denture base material was tested. The bond between the heat-cured resin and the inner layer of the denture teeth was significantly stronger than the bond between the heat-cured resin and the outer layer of the denture teeth. The bond between both layers of the denture teeth and the self-curing acrylic resin was significantly weaker than with the heat-cured acrylic resin. In situations where excessive reduction of the teeth is required there may be a case for choosing teeth with a homogenous structure.

Dimensional change of heat-cured acrylic resin dentures with three different cooling regimes following a standard curing cycle.

The aim of the study was to compare dimensional changes in poly(methylmethacrylate) complete denture bases resulting from three different cooling regimens following a standard heating cycle. Changes in three separate dimensions were measured on ten dentures within each cooling regimen after curing, and before and after removing the denture from the cast using a computer imaging system. No consistent differences occurred as a result of removing the denture from the cast. The results indicated that there was greater change in dimension of dentures with the quenching cooling method than with either overnight cooling in the water bath or bench cooling. This was particularly evident after removal from the cast after curing (p<0.001). It is concluded that slow cooling results in less dimensional change.

Chairside polishing of heat-cured acrylic resin: an SEM and EDA study.

PURPOSE: This study investigated the surfaces produced on two commonly used heat-cured acrylic resins by two chairside polishing systems compared to conventional lathe polishing. It used energy dispersive analysis (EDA) and scanning electron microscopy (SEM) to investigate the abrasive materials bonded in the silicone points that are marketed for chairside polishing of acrylic resin. MATERIALS AND METHODS: Using the three polishing systems (conventional as control, Shofu acrylic polishing system [AcryPoint], and Kenda Queen silicone polisher), three operators polished specimens using a defined protocol. Specimens were subjected to SEM and confocal microscopy. EDA was undertaken on the silicone polishing points. RESULTS: Conventional polishing produced the superior surface, followed by the surfaces produced by the Shofu system and then Kenda Queen. However, both chairside polishing systems produced smoother surfaces on acrylic resin than the surfaces produced by either steel or tungsten carbide burs. Both the Shofu acrylic polishing system and Kenda Queen silicone polisher contain beads of cerium, which is widely used in industry for polishing of glass. CONCLUSION: Silicone polishing points marketed for polishing of acrylic resin could be used to produce smooth surfaces that have been adjusted with burs where there is no access to a laboratory lathe.

Events in the mould during heat processing of poly(methyl methacrylate).

Recordings of temperature and pressure in the mould have raised questions concerning the flow of poly(methylmethacrylate) during heat processing. Three simple experiments investigated flow of polymerised and partly polymerised poly(methylmethacrylate). Flow was not observed at normal processing temperatures. These results, combined with the results of previous work, suggest that as the mould heats up, thermal expansion of the dough exceeds polymerisation contraction, causing increase in pressure rather than flow. Maximum pressure occurs at maximum temperature. Thereafter, thermal expansion is reduced, polymerisation contraction increases and pressure in the mould drops. When the lowest pressure is reached the poly(methylmethacrylate) is polymerised.

Dimensional change in complete dentures fabricated by injection molding and microwave processing.

STATEMENT OF PROBLEM: Acrylic resin complete dentures undergo dimensional changes during polymerization. Techniques with injection molding and polymerization and microwave polymerization are reported to reduce these changes and thereby improve clinical fit. These dimensional changes need to be quantified. PURPOSE: The purpose of this study was to compare differences in dimensional changes of simulated maxillary complete dentures during polymerization and storage in water after injection molding and conventional polymerization, or microwave polymerization against a control of conventionally packed and polymerized simulated maxillary complete dentures. MATERIAL AND METHODS: Forty identical maxillary denture bases were prepared in dental wax with anatomic teeth. They were invested and the wax eliminated from the molds. Ten specimens each were randomly assigned to 1 of 4 groups. Group 1 was compression molded and conventionally polymerized; group 2 was injection molded and conventionally polymerized (Success); group 3 was injection molded and microwave polymerized (Acron MC); and group 4 was injection molded and microwave polymerized (Microbase). Intermolar width and changes in vertical dimension of occlusion, were determined after polymerization and after storage in water for 28 days. Measurements in triplicate were made between points scribed on the second molar teeth with a traveling microscope (accurate to 0.005 mm). Vertical dimension of occlusion was measured between points scribed on the upper and lower members of an articulator by use of an internal micrometer (accurate to 0.05 mm). Data were analyzed by use of a 1-way analysis of variance with Tukey post-hoc contrasts (P <.05). RESULTS: Polymerization contractions (intermolar widths) for each group were: group 1, -0.24%; group 2, -0.27%; group 3, -0.35%; and group 4, -0.37%. The Microbase specimens had greater shrinkage than conventionally polymerized specimens, but there were no significant differences between the groups. All injection methods had less postpolymerization increase in vertical dimension of occlusion (0.63 to 0.41 mm) than the conventional Trevalon control (0.74 mm), but only group 4 was significantly different (P<.004). After storage in water for 28 days, all specimens increased in vertical dimension of occlusion (0.10% to 0.16%) from polymerization techniques, but there were no significant differences between groups. CONCLUSION: Within the limitations of this study, injection molding resulted in a slightly less increase of vertical dimension of occlusion than conventional polymerization techniques, the difference being significant for Microbase compared with the conventional Trevalon control.