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PubMed1989

A comparison of freeze-dried bone allograft and demineralized freeze-dried bone allograft in human periodontal osseous defects.

This study was conducted to clinically compare freeze-dried bone allograft (FDBA) and demineralized freeze-dried bone allograft (DFDBA). Twenty-two defects (11 intrapatient pairs) in 9 patients were grafted with either DFDBA or FDBA. Evaluations were based on standardized radiographs, presurgical and postsurgical soft tissue measurements using the cemento-enamel junction as a fixed reference point, and osseous measurements at the time of surgery. Grafted sites were re-entered at a minimum of 6 months following placement. A mean osseous repair of 1.7 mm (59%) occurred with DFDBA and 2.4 mm (66%) with FDBA. A mean clinical attachment gain of 1.7 mm was obtained with DFDBA and 2.0 mm with FDBA. Probing depths decreased a mean of 2.00 mm with both DFDBA and FDBA. These findings reveal no significant differences between the two materials in primarily intraosseous defects when evaluated at a minimum 6 months postsurgery.

Adult↗
PubMed2002

A retrospective case series comparing the use of demineralized freeze-dried bone allograft and freeze-dried bone allograft combined with enamel matrix derivative for the treatment of advanced osseous lesions.

BACKGROUND: Combined regenerative approaches have been used for treating advanced osseous lesions around teeth. The aim of combining treatments is to enhance both clinical predictability and regenerative outcome compared to a monotherapeutic approach. This case series from a private practice reports on the clinical efficacy of an enamel matrix derivative (EMD) combined with either demineralized freeze-dried bone allograft (DFDBA) or freeze-dried bone allograft (FDBA) in the treatment of advanced infrabony lesions. The advanced lesions were veneered by a rapidly formed absorbable polymer barrier of poly(DL-lactide) to enhance graft containment. METHODS: A total of 22 consecutive patients, each contributing one infrabony lesion, are reported. After patients completed presurgical preparation, the infrabony lesions were surgically treated with a combined approach that included root surface treatment with citric acid. The two groups differed in their composite graft; one received DFDBA-EMD (n = 10) and the other received FDBA-EMD (n = 12). Patients followed a stringent postoperative protocol and were evaluated 6 months postsurgery. Clinical outcomes were assessed by changes in clinical attachment level (CAL) and probing depth (PD) from pretreatment. Surgical re-entry of several sites was possible in each group. RESULTS: CAL at pretreatment measured 9.2 +/- 1.3 mm and 9.1 +/- 1.9 mm for DFDBA-EMD and FDBA-EMD groups, respectively, with corresponding PD of 8.4 +/- 1.6 mm and 8.9 +/- 2.0 mm for each group. At 6 months post-treatment, CALs were reduced to 4.7 +/- 1.3 mm and 3.8 +/- 1.0 mm for DFDBA-EMD and FDBA-EMD groups, respectively; with corresponding PD decreased to 3.0 +/- 0.8 mm and 3.2 +/- 1.0 mm. Relative improvements in CAL for the DFDBA-EMD and DFDBA-EMD groups were 49.1% +/- 11.0% and 57.3% +/- 9.4%, respectively (P <0.07). CONCLUSIONS: This case series demonstrates the clinical benefits of using a combined therapeutic approach in which a biologic mediator (EMD) was combined with either DFDBA or FDBA. In this limited case series, a trend was observed towards greater improvement in clinical attachment level gain in advanced infrabony defects when EMD was combined with FDBA as compared to DFDBA. Larger prospective controlled clinical trials are needed to determine if differences exist in the relative efficacy of DFDBA versus FDBA in combination with EMD.

Absorbable Implants↗
PubMed2005

Thermoanalytical techniques for the investigation of the freeze drying process and freeze-dried products.

A key challenge facing the pharmaceutical industry is the production of biotechnological drug products such as proteins in a stable form. Freeze-drying is preferred for manufacturing such products because of the low temperatures used. However, the protein may still degrade during the process necessitating the inclusion of a protectant. This review describes the range of thermal analysis techniques that have been used to investigate the properties of formulations to be freeze dried and the resultant products. This approach has allowed insight into the key parameters required for design of formulations and processes that will generate the best possible products.

Biological Products↗
PubMed2004

Influence of freezing and freezing plus acidic calcium sulfate and lactic acid addition on thermal inactivation of Escherichia coli O157:H7 in ground beef.

Undercooked ground beef is a leading vehicle for acquiring Escherichia coli O157:H7 infections through consumption of foods. Studies have been performed to determine the effect of freezing and the combined effect of freezing and addition of a mixture of 20% acidic calcium sulfate (final concentration of 0.4% in ground beef) and 10% lactic acid (final concentration of 0.2% in ground beef) (ACS-LA) on the thermal sensitivity of E. coli O157:H7 in ground beef. Five strains of E. coli O157: H7 were separately inoculated into ground beef and held at 5 degrees C for up to 10 days or -20 degrees C for up to 3 weeks then heated at 57, 60, 62.8, 64.3, and 68.3 degrees C to determine rates of thermal inactivation. Results revealed that D-values (decimal reduction times) at equivalent temperatures for four of five E. coli O157:H7 strains were less in the previously frozen than in the refrigerated ground beef and that strains isolated from ground beef in 1993 and 1994 were generally more sensitive to thermal inactivation than those isolated in 1999 and 2000. Only one strain of E. coli O157:H7 was used to determine the effect of ACS-LA in previously frozen or refrigerated ground beef on rates of thermal inactivation. The addition of ACS-LA to ground beef at 20 ml/kg increased the thermal sensitivity of E. coli O157:H7 in both previously frozen and refrigerated ground beef, with greatest rates of inactivation occurring in previously frozen ground beef containing ACS-LA. D-values at 57 degrees C obtained for E. coli O157:H7 in previously refrigerated and frozen ground beef containing ACS-LA and ACS-LA diluted by half were significantly (P < 0.05) less than those obtained in ground beef with no ACS-LA added. D-values at 60 and 62.8 degrees C were consistently less in ACS-LA treated ground beef, but for most treatments the results were not significantly (P > 0.05) different than the controls. Results revealed that the addition of ACS-LA to ground beef, whether frozen or refrigerated, can reduce the temperature or time required to kill E. coli O157:H7 during heating.

Animals↗
PubMed1992

Interactions of stabilizing additives with proteins during freeze-thawing and freeze-drying.

A wide variety of compounds, including sugars, polyols, amino acids and certain salts, are effective at minimizing protein denaturation during freeze-thawing. In this review we provide evidence that the mechanistic basis for this cryoprotection appears to be the same as that described by Timasheff for solute-induced stabilization in aqueous solution. Namely, the stabilizers are preferentially excluded from the surface of the protein, and this interaction makes it thermodynamically unfavourable for proteins to unfold. In contrast, carbohydrate-induced preservation of labile enzymes during freeze-drying is a fundamentally different process. Using Fourier transform intra-red spectroscopy we have found that hydrogen bonding between the carbohydrate and the dried protein is required for stabilization. Thus, it appears that certain carbohydrates protect dried enzymes because these solutes serve as water substitutes for the dried protein, by satisfying the hydrogen bonding requirement of polar groups on the protein's surface. Finally, we discuss some intriguing findings on the synergistic stabilization of proteins by mixtures of divalent cations and organic solutes, which are yet to be explained.

Cryoprotective Agents↗
PubMed1989

Olfactory and nasal respiratory epithelia, and foliate taste buds visualized with rapid-freeze freeze-substitution and Lowicryl K11M embedding. Ultrastructural and initial cytochemical studies.

Rat olfactory and respiratory epithelia and Rhesus monkey taste buds were studied with rapid-freeze, acetone/0.1% uranyl acetate freeze-substitution and low-temperature Lowicryl K11M embedding, usually in the absence of other chemical fixation and cryoprotection procedures. Ultrastructural features of mucus, cytoplasm, including cytoskeletons, and membranes were better retained than with conventional methods. Some major examples: The mucus of the olfactory epithelium consisted of a single layer; that of the respiratory epithelium had an electron-opaque sol layer surrounding cilia and microvilli below a thin laminated electron-lucent gel layer. Taste-bud pores displayed a foam-like opaque secretory product, resembling the contents of secretory granules within Type I taste-bud cells. The electron-opacity of cytoplasmic matrices sometimes obscured features such as radial spokes of respiratory cilia. Membranes had smooth outlines; those of olfactory receptor cell cilia were more electron-opaque than those of olfactory supporting cell microvilli and respiratory cilia. Membranous monolayers of many respiratory cilia across large arrays often partially split apart, all in the same direction. The space between those monolayers contained an electron-lucent substance. Preliminary cytochemistry on olfactory and nasal respiratory epithelial samples with the lectin Concanavalin A (Con A) and antibodies against olfactory marker- and odorant-binding proteins and, in taste only, the sweet-tasting protein thaumatin, were sufficiently successful to warrant further endeavors.

Acrylic Resins↗
PubMed1984

Freeze-thaw activation of the complement attack phase: II. Comparison of convertase generated C--56 with C--56 generated by freezing and thawing.

The activation of the C-attack phase does not necessarily involve the components of the C5 convertases. C--56 hemolytic activity was generated from the same source of C7 depleted serum by the alternative pathway convertase or by freezing and thawing resp. In contrast to activation by the convertase, biological activities of C5a (chemotaxis, serotonin release) were not detected following activation by freezing. The yields of C--56 hemolytic activities were similar and the properties of the activated products were identical. No difference was found in the molecular weight, in the hydrophobicity or with respect to charge. The two activities were in the absence of C7 stable at 37 degrees C and decayed rapidly in the presence of C7. It is proposed that a conformational change in the tertiary structure of the molecule(s) is the critical event in the formation of an active C--56 complex. In this light the cleavage of C5a from the native molecule by the convertase appears as a side reaction, not by itself essential for activation.

Complement Activating Enzymes↗
PubMed1994

A new versatile system for freeze-substitution, freeze-drying and low temperature embedding of biological specimens.

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 1 Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. Fs and subsequent LTE are carried out in an environment of dry cold nitrogen gas provided by evaporating nitrogen from the dewar. Different capsules and moulds may be used for cryodehydration and LTE. FD of bulk specimens or cryosections takes place in an absolutely clean vacuum provided by a cryosorption pump integrated in the FD apparatus. Most of the H2O molecules from the frozen specimen are trapped by large cold surfaces inside the drying chamber. Due to the low LN2 consumption during FS or FD (3-4 1 LN2/day) both procedures may be carried out for 8-10 days without refilling the dewar. A few representative results show that well frozen biological material is stabilized by prolonged FS or FD at temperatures of about -80 degrees C without user of chemical fixatives like OsO4 in the substitution medium during FS or by OsO4 vapor fixation after FD.

Animals↗
PubMed1993

Scanning electron microscopy of muscle myofibrils after high pressure freezing and freeze-substitution-staining.

A novel approach to study the three dimensional ultrastructure of organelles and cells by means of scanning electron microscopy is described. Muscle myofibrils have been used in the development of the techniques since their structure is well characterized using conventional electron microscopic methods. Myofibrils in rigor buffer (with no cryo-protectants or pressure sealants) were frozen at high pressure (2300 bar) within specially designed chambers. The frozen specimens were then freeze-substituted-stained with methanol containing tungsten and iron salts and finally critical point dried. These methods allowed scanning electron microscopic observations of the organization of individual filaments within whole myofibrils over several sarcomeres. Images obtained showed excellent structural preservation with three dimensional information which is not available with other electron microscopic techniques. Success in these approaches was ascribed to (a) rapid and uniform freezing at high pressure without ice segregation patterns, (b) uniform electro-conductivity of the specimen closely attached to the polished carbon piston/carrier, and (c) good electron emission (secondary and back-scattered) from the metal incorporated into the myofibril structure without additional coating.

Animals↗
PubMed1976

Freezing of rat lymphocytes. III. Freezing of plaque-forming cells and restoration by frozen-thawed normal cells of antibody production in irradiated rats.

The present investigation is an extension of earlier work with dimethyl sulphoxide (DMSO) protected frozen-thawed rat lymphocytes. In the present work it is shown that some 85-90% of the haemolytic plaque-forming cells (PFC) survived the freeze-thaw process. Irradiated rats were restored with fresh and frozen-thawed cells and immunized against sheep red blood cells (SRBC). Evidence is presented that a restrictive control of the PFC response by suppressor cells present in the spleen cell suspension is lost during the freeze-thaw process, giving a higher number of PFC/spleen in recipients of frozen-thawed mixed spleen and lymph node cells than in rats receiving the corresponding fresh preparations.

Animals↗
PubMed1996

[Ultrastructural study on rat precorneal tear film by quick freezing-freeze substitution method].

The ultrastructure of rat precorneal tear film was visualized by the quick freezing-freeze substitution (QF-FS) method and the conventional fixative method under the scanning electron microscope (SEM). In the QF-FS method, the eyeballs of rats were quickly frozen with an isopentane-propane mixture cooled by liquid nitrogen applied directly to the eyes. After enucleating the eyes and fracturing the frozen corneas, the corneas were prepared for SEM observation. In contrast to the conventional fixative method, by which the microvilli of the surface epithelial cells could be visualized clearly, a very thin membrane-like structure was observed to cover the corneal surface in the eyes prepared by the QF-Fs method. Between the membrane-like structure and corneal surface, a homogeneous and fine network-like structure was observed. The results suggest that the structure of the tear film might be different from the one we have believed to consist of three layers until now. The QF-FS method is considered to be useful for the morphological study of the precorneal tear film.

Animals↗
PubMed1991

Advantages of fast-freeze fixation followed by freeze-substitution for the preservation of cell integrity.

The use of fast-freeze fixation (FFF) followed by freeze-substitution (FS) has supplied new data in a variety of domains due to the rapidity of this fixation compared to the slow process of the conventional chemical fixation. FFF completely arrests all the cell's biological reactions, and physically immobilizes most molecules within a few milliseconds. This review deals with the main results we obtained concerning the preservation of organelles particularly sensitive to osmotic shock, the maintenance in situ of soluble or depolymerizable cell components, the visualization of brief transient events, and the opening up of new prospects in the field of immunocytochemical labeling.

Amoeba↗
PubMed1991

Routine cryofixation of plant tissue by propane jet freezing for freeze substitution.

Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.

Cryopreservation↗
PubMed1997

Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing.

The influence of the presence of trehalose or sucrose, the osmolality of the freezing medium, and the cooling rate on success of cryopreservation of bull sperm were investigated. Bull semen was frozen at four different cooling rates varying from 40 to 300 degrees C/min in standard Tris-egg yolk medium and in hypertonic or isotonic media containing 0.2 M trehalose or sucrose. Sperm motility, H33258 exclusion, and acrosome morphology were evaluated. In the hypertonic media the motility was almost completely inhibited, but could partly be restored by returning to isotonic conditions. Medium composition and cooling rate had significant effects, with a strong interaction of medium x cooling rate. The optimal cooling rate was found to be between 76 and 140 degrees C/min. At the highest cooling rate, both the presence of the sugars and the high osmolality of the hypertonic media conferred significant protection against fast-cooling damage, sucrose being significantly better than trehalose. At all other cooling rates the medium differences were less pronounced, but the isotonic sugar media were generally superior to standard medium as to preservation of acrosome intactness and H33258 exclusion. This indicates that the isotonic sugar media could enable some improvement of cryopreservation of bull semen over the currently used Tris-egg yolk medium. Further improvement of sperm survival at the AI station should be possible by implementation of controlled rate freezing, using a cooling rate in the optimal range between 76 and 140 degrees C/min.

Animals↗
PubMed1984

Improved preservation of intramitochondrial granules in rat-incisor odontoblasts by rapid-freezing and freeze-substitution fixation.

Odontoblasts fixed by rapid-freezing and freeze-substitution, compared to cells fixed with aldehyde, exhibited a larger number of microfilaments, a granular material inside the rough endoplasmic reticulum, many electron-dense aggregates in cytoplasmic vesicles and numerous intramitochondrial granules. Although these granules were located in odontoblasts facing a well-mineralized layer of dentine, this did not support the possible existence of a transfer pathway for intracellular calcium which plays some part in mineralization processes.

Animals↗
PubMed1990

Tendon collagen fibrillogenesis is a multistep assembly process as revealed by quick-freezing and freeze-substitution.

The ultrastructure of chick embryo tendons has been examined after quick-freezing by liquid helium and freeze-substitution. Several stages of collagen assemblies were observed: intracellular packing of SLS-like aggregates surrounded by membrane containing areas with a clathrin coat; fine non cross-striated filaments connecting the cell membrane at 1 pole of the cells and collagen fibrils; tufts of filaments directly linked to collagen fibrils. This study reveals that some stages are more constant and abundant than supposed (the intracellular SLS-like aggregates) and that other extracellular assemblies that were hypothesized but usually badly preserved by conventional electron microscopy are clearly captured by the method.

Animals↗
PubMed2001

Effect of freezing on the transmembrane distribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus (Diptera, Xylophagidae).

The present study shows that freezing of freeze-tolerant larvae of the wood fly Xylophagus cinctus caused Na(+), K(+) and Mg(++) to move to electrochemical equilibrium across the cell membranes. Na(+) and Mg(++) moved from the haemolymph into the cells, while K(+) moved the opposite way. The original distribution of ions was restored after the larvae were thawed. The transmembrane fluxes of ions were of the same magnitude in the frozen and thawed larvae. The redistribution of ions in the frozen larvae did not give rise to any apparent change in the volume of cells and haemolymph upon thawing, i.e. the redistribution of solutes appeared to be osmotically neutral.

Journal Article↗