Image assessment of MSCT and CBCT scans for rapid maxillary expansion: a pilot study

Objectives: The accuracy of cone-beam technique, cone-beam computed tomography (CBCT), compared with that of the multislice spiral CT (MSCT), for image-based linear measurements of midpalatal suture was assessed.

Material and Methods: Two measurements were performed by one investigator on the dry skull by using one digital caliper and in the axial cuts by using software. A 2D object-based image registration process was applied to determine the best affine transformation that maps a 2D input image (CBCT) in a reference image (MSCT).

Results: The value of the intraclass correlation coefficient was approximately 0.9%. The results suggest that differences between two scanners did not exist (observation 1, p = 0.964 and observation 2, p = 0.795). With regard to the dry skull and the image, the significance probabilities equaled zero (observation 1, p = 0.002 and observation 2, p = 0.004), therefore, indicating significant differences.

Conclusion: Measurements acquired in the images were similar and these findings contribute to stimulate the use of CBCT for evaluation of the maxillary expansion procedure.     

Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 °C or 80 °C and precipitation of the polymer with two volumes of ethanol.     

Heat and mass transfer scale-up issues during freeze-drying, III: Control and characterization of dryer differences via operational qualification tests

The objective of this research was to estimate differences in heat and mass transfer between freeze dryers due to inherent design characteristics using data obtained from sublimation tests. This study also aimed to provide guidelines for convenient scale-up of the freeze-drying process. Data obtained from sublimation tests performed on laboratory-scale, pilot, and production freeze dryers were used to evaluate various heat and mass transfer parameters: nonuniformity in shelf surface temperatures, resistance of pipe, refrigeration system, and condenser. Emissivity measurements of relevant surfaces such as the chamber wall and the freeze dryer door were taken to evaluate the impact of atypical radiation heat transfer during scale-up. “Hot” and “cold” spots were identified on the shelf surface of different freeze dryers, and the impact of variation in shelf surface temperatures on the primary drying time and the product temperature during primary drying was studied. Calculations performed using emissivity measurements on different freeze dryers suggest that a front vial in the laboratory lyophilizer received 1.8 times more heat than a front vial in a manufacturing freeze dryer operating at a shelf temperature of −25°C and a chamber pressure of 150 mTorr during primary drying. Therefore, front vials in the laboratory are much more atypical than front vials in manufacturing. Steady-state heat and mass transfer equations were used to study a combination of different scaleup issues pertinent during lyophilization cycles commonly used for the freeze-drying of pharmaceuticals.     

The Use of ToF-SIMS for Analysis of Bioorganic Samples

The technique for the preparation of biological tissue sections developed for Electron Probe Microanalysis has been adapted for ToF-SIMS analysis of mouse GV stage oocytes. GV-oocyte sample preparation involves the following steps: plunge freezing, freeze drying, impregnation in an embedding medium, and section cutting. Molecule-specific images of the distribution of molecules in a single oocyte have been obtained with the described technique and ToF-SIMS analysis. The ToF-SIMS analysis data show that the efficient lateral image resolution is approximately 1 μm. Hence, ToF-SIMS enables us to study the distribution of chemical substances in relation to the morphological data obtained by scanning electron microscopy or conventional light microscopy.     

Drying techniques for the visualisation of agarose‐based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub‐micron level. Achieving suitable drying conditions is especially important with agarose‐based chromatography resins, as over‐drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under‐drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect?/d _w ≈85 µm and Capto? Adhere/d _w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose‐based chromatography media.     

Freezing and freeze-drying of the bacterium Rahnella aquatilis BNM 0523: study of protecting agents, rehydration media and freezing temperatures

Aims: The aim of the present study was to evaluate and compare freezing and freeze‐drying treatments for conserving Rahnella aquatilis (BNM 0523) with the goal to achieve an adequate commercial formulation of this biocontrol agent. Methods and Results: The effect of several protective agents, rehydration media and freezing temperatures on the viability and functional activity of the R. aquatilis was investigated. The storage stability at 3 months and 4 years was determined by checking the viability of the cells and their biocontrol capability against Botrytis cinerea by measuring the percentage of reduction of disease severity on apple. The best results were obtained by the freeze‐drying of the cells using a mixture of skimmed nonfat milk 10%, yeast extract 0·5% and glucose 1% as the protecting and rehydrating medium, and a quickly freezing (?70°C) before the freeze‐drying. In this case, the viability of the cells after 4 years was 98%, and their antagonistic ability showed a little decrease with respect fresh cells. Conclusions: The studies showed that R. aquatilis was resistant to freezing and freeze‐drying when it was used a mixture of cryoprotectants and that it was possible to obtain inoculums with high viability and good effectiveness for reduction of decay caused by B. cinerea. Significance and Impact of the study: This study is probably the first report about the resistance of R. aquatilis to freezing and freeze‐drying treatments and shows that these operations could be useful for obtaining a commercial formulation of this biocontrol agent.     

Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

Liposomal hydrogel formulation for transdermal delivery of pirfenidone

Context: Pirfenidone (PFD) is an anti-fibrotic and anti-inflammatory agent indicated for the treatment of idiopathic pulmonary fibrosis (IPF). The current oral administration of PFD has several limitations including first pass metabolism and gastrointestinal irritation.

Objective: The aim of this study is to investigate the feasibility of transdermal delivery of PFD using liposomal carrier system.

Materials and methods: PFD-loaded liposomes were prepared using soy phosphatidylcholine (SPC) and sodium cholate (SC). Encapsulation efficiency (EE) of PFD in liposomes was optimized using different preparation techniques including thin film hydration (TFH) method, direct injection method (DIM) and drug encapsulation using freeze–thaw cycles. In vitro drug release study was performed using dialysis membrane method. The skin permeation studies were performed using excised porcine ear skin model in a Franz diffusion cell apparatus.

Results and discussion: The average particle size and zeta-potential of liposomes were 191?±?4.1?nm and ?40.4?±?4.5?mV, respectively. The liposomes prepared by TFH followed by 10 freeze–thaw cycles showed the greatest EE of 22.7?±?0.63%. The optimized liposome formulation was incorporated in hydroxypropyl methyl cellulose (HPMC) hydrogel containing different permeation enhancers including oleic acid (OA), isopropyl myristate (IPM) and propylene glycol (PG). PFD-loaded liposomes incorporated in hydrogel containing OA and IPM showed the greatest flux of 10.9?±?1.04?μg/cm²/h across skin, which was 5-fold greater compared with free PFD. The cumulative amount of PFD permeated was 344?±?28.8?μg/cm² with a lag time of 2.3?±?1.3?h.

Conclusion: The hydrogel formulation containing PFD-loaded liposomes can be developed as a potential transdermal delivery system.     

Influence of the Drying Technique of Silica Gels on the Enzymatic Activity of Encapsulated Lipase

Recent studies by Reetz et al. (Reetz, M.T., Zonta, A. and Simpelkamp, J. (1996a) Biotechnology and Bioengineering, 49, 527-534) have shown that the catalytic activity of lipase encapsulated in sol-gel materials, in esterification reactions, depends on many parameters such as the presence of hydrophobic groups grafted on the gel network and of an organic component in the gel network. In the present study, we have examined the effect of the gel pore texture which can be modified by varying the drying technique, for a given silica precursor composition and hydrolysis-condensation procedure. For a given mixture of two silane precursors, propyltri-methoxysilane and tetramethoxysilane, we compared the effects of the presence or absence of an organic component such as polyvinyl alcohol, in combination with drying either by evaporation which leads to the formation of xerogels, or by supercritical drying in CO₂ which leads to the formation of aerogels. For this last technique, the exchange of liquid is also an important step and its effect on the enzyme activity has been examined. The gel pore texture was characterized by nitrogen absorption according to the Brunauer Emmett and Teller method. The catalytic activities of the materials were compared in the esterification of lauric acid by 1-octanol.     

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre‐freeze‐drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25‐dose vials, 50‐dose‐vials and 125‐dose vials). Each freeze‐dried product was measured directly after freeze‐drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700–1600 cm^?1 (amide I band), 1600–1500 cm^?1 (amide II band) and 1200–1350 cm^?1 (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze‐dried samples prepared using different virus medium volumes, containing different doses and using different pre‐freeze‐drying sample treatments in the amide III region. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1107–1118, 2015     

Texture analysis of the 3D collagen network and automatic classification of the physiology of articular cartilage

A close relationship has been found between the 3D collagen structure and physiological condition of articular cartilage (AC). Studying the 3D collagen network in AC offers a way to determine the condition of the cartilage. However, traditional qualitative studies are time consuming and subjective. This study aims to develop a computer vision-based classifier to automatically determine the condition of AC tissue based on the structural characteristics of the collagen network. Texture analysis was applied to quantitatively characterise the 3D collagen structure in normal (International Cartilage Repair Society, ICRS, grade 0), aged (ICRS grade 1) and osteoarthritic cartilages (ICRS grade 2). Principle component techniques and linear discriminant analysis were then used to classify the microstructural characteristics of the 3D collagen meshwork and the condition of the AC. The 3D collagen meshwork in the three physiological condition groups displayed distinctive characteristics. Texture analysis indicated a significant difference in the mean texture parameters of the 3D collagen network between groups. The principle component and linear discriminant analysis of the texture data allowed for the development of a classifier for identifying the physiological status of the AC with an expected prediction error of 4.23%. An automatic image analysis classifier has been developed to predict the physiological condition of AC (from ICRS grade 0 to 2) based on texture data from the 3D collagen network in the tissue.     

Non-uniform strain distribution within rat cartilaginous growth plate under uniaxial compression

Growth plates are highly inhomogeneous in morphology and composition. Mechanical loading can modulate longitudinal bone growth, though the mechanisms underlying this mechanobiology are poorly understood. The proximal tibial growth plates of six rats were tested in vitro under uniaxial compression to 5% strain, and confocal microscopy was used to track and capture images of fluorescently labeled cell nuclei with increasing applied strains. The local strain patterns through the growth plate thickness were quantified using texture correlation analysis. The technique of texture correlation analysis was first validated by comparing theoretical simulated strain maps generated from numerically distorted images. The texture correlation algorithm was sensitive to the grid size superimposed on the original image, but remained insensitive to parameters related to the size of the final image mask, which was searched by the correlation algorithm for each grid point of the original image. Within the growth plate, experimental strain distributions were non-uniform in all six specimens. Growth plates were mostly under compression strains. The strain distributions differed among the histomorphological zones of the growth plate, which was most obvious in specimens with regular growth plate shape: higher compressive strains (4-8 times higher than the applied 5% strain) were located mainly in regions overlapping the reserve and hypertrophic zones with lower compressive strains in the proliferative zone. This study documents the non-uniform mechanical behavior of growth plate across its three histological zones when exposed to compression. Further investigation is required to establish the significance of non-uniform strain fields during growth in vivo.     

Development of a digital image analysis method for real-time estimation of chlorophyll content in micropropagated potato plants

The present work describes a digital image analysis method based on leaf color analysis to estimate chlorophyll content of leaves of micropropagated potato plantlets. For estimation of chlorophyll content, a simple leaf digital analysis procedure using a simple digital still camera was applied in parallel to a SPAD chlorophyll content meter. RGB features were extracted from the image and correlated with the SPAD values. None of the mean brightness parameters (RGB) were correlated with the actual chlorophyll content following simple correlation studies. However, a correlation between the chromaticity co-ordinates ‘r’, ‘b’ and chlorophyll content was observed, while co-ordinate ‘g’ was not significantly correlated with chlorophyll content. Linear regression and artificial neural networks (ANN) were applied for correlating the mean brightness (RGB) and mean brightness ratio (rgb) features to chlorophyll content of plantlet leaves determined through a SPAD meter. The chlorophyll content as determined by the SPAD meter was significantly correlated (RMSE = 3.97 and 3.59, respectively, for linear and ANN models) to the rgb values of leaf image analysis. Both the models indicate successful prediction of chlorophyll content of leaves of micropropagated plants with high correlation. The developed RGB-based digital image analysis has the advantage over conventional subjective methods for being objective, fast, non-invasive, and inexpensive. The system could be utilized for real-time estimation of chlorophyll content and subsequent analysis of photosynthetic and hyperhydric status of the micropropagated plants for better ex vitro survival.     

1H NMR investigation on the role of sorbitol for the survival of Lactobacillus paracasei ssp. paracasei in vacuum‐dried preparations

Aims: The aim of this work was to study the effect of sorbitol as protective agent on the survival of the probiotic strain Lactobacillus paracasei ssp. paracasei (F19) after vacuum drying. Methods and Results: The survival was studied after different drying times and for various concentrations of sorbitol by plate count method. Furthermore, time domain ¹H NMR studies on dehydrated suspensions of Lact. paracasei ssp. paracasei were performed to study the proton mobility in the dried samples. From the obtained signal, T2 relaxation times of single components and fractions with different proton mobility were determined. It was found out that the survival is increased by the presence of a minimum amount of sorbitol that is dependent on drying time. Furthermore, it is shown that the protective effect can only be observed below a critical water content of c. 20%. Nuclear magnetic resonance (NMR) results indicate a transition of sorbitol from liquid to solid like behaviour during drying. The onset of the transition coincides with the critical water content found for a protective effect. Conclusions: The data suggest that sorbitol protons are incorporated into the dried cells of Lact. paracasei ssp. paracasei (F19) below the critical water content and therefore leading to an enhanced survival. Significance and Impact of the Study: The results help to better understand the underlying mechanism of protection of Lact. paracasei ssp. paracasei using sorbitol and to establish vacuum drying as potential alternative drying technique to standard freeze drying.     

The 3-D model: Comparison of parameters obtained from and by simulating different tableting machines

The aim of this study is to apply 3-D modeling to data obtained from different tableting machines and for different compression wheels on a linear rotary tableting machine replicator. A new analysis technique to interpret these data by 3-D parameter plots is presented. Tablets were produced on an instrumented eccentric tableting machine and on a linear rotary tableting machine replicator. The materials used were dicalcium phosphate dihydrate (DCPD), spray-dried lactose, microcrystalline cellulose (MCC), hydroxypropyl methylcellulose (HPMC), and theophylline monohydrate. Tableting was performed to different maximum relative densities (ρ _{rel, max}). Force, time and displacement were recorded during compaction. The 3-D data plots were prepared using pressure, normalized time, and porosity according to Heckel. A twisted plane was fitted to these data according to the 3-D modeling technique. The resulting parameters were analyzed in a 3-D parameter plot. The results show that the 3-D modeling technique can be applied to compaction cycles from different tableting machines as different as eccentric and rotary tableting machines (simulated). The relation of the data to each other is the same even when the absolute values are different. This is also true for different compression wheels used on the linear rotary tableting machine replicator. By using compression wheels of different sizes on this simulator, mainly time plasticity changes. By using bigger compression wheels for simulation, the materials deform slower at lower densification and they deform faster at higher densification. For brittle materials, the stages of higher densification are influenced; for plastically deforming materials, the stages of lower and higher densification can be influenced.     

Analysis of the impact of indole-3-acetic acid (IAA) on gene expression during leaf senescence in Arabidopsis thaliana

Leaf senescence is an important developmental process for the plant life cycle. It is controlled by endogenous and environmental factors and can be positively or negatively affected by plant growth regulators. It is characterised by major and significant changes in the patterns of gene expression. Auxin, especially indole-3-acetic acid (IAA), is a plant growth hormone that affects plant growth and development. The effect of IAA on leaf senescence is still unclear. In this study, we performed microarray analysis to investigate the role of IAA on gene expression during senescence in Arabidopsis thaliana. We sprayed IAA on plants at 3 different time points (27, 31 or 35 days after sowing). Following spraying, PSII activity of the eighth leaf was evaluated daily by measurement of chlorophyll fluorescence parameters. Our results show that PSII activity decreased following IAA application and the IAA treatment triggered different gene expression responses in leaves of different ages.

     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Aims: The effects of different freeze‐drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated. Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze‐drying. The subsequent survival rates during storage and rehydration media of freeze‐dried biocontrol strains were also examined. Conclusions: The results indicated that cellobiose (5%) and d ‐galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze‐drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months. Significance and Impact of the study: This study showed that freeze‐drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale‐up possibilities and formulation development.