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PubMed2003

Impaction bone grafting with freeze-dried irradiated bone. Part II. Changes in stiffness and compactness of morselized grafts: experiments in cadavers.

In the technique of impaction bone grafting, implant stability depends on the mechanical properties of the impacted morselized grafts. Although the procedure is usually performed with fresh-frozen femoral heads, there is still some concern about their supply and safety. Bone processing is a potential solution, but the mechanical properties of this material during and after impaction need to be determined. We used 6 osteoarthrotic femoral heads to prepare two paired batches of morselized bone. One batch was morselized and frozen. The other batch was chemically treated, morselized, freeze-dried and then gamma-irradiated. We impacted 18 samples from each batch in a contained cylinder. Freeze-dried bone grafts were tested after 30 minutes of rehydration. The changes in the compactness and stiffness of the material were monitored during the impaction. The compaction of the freeze-dried bone was faster than that of their fresh-frozen control. The maximal stiffness reached by both materials was the same (55 MPa), but the freeze-dried grafts required three to four times fewer impactions to achieve that stiffness. After 3, 10 and 50 impactions the freeze-dried bone was stiffer than the fresh-frozen bone. As it is easier to impact, the freeze-dried bone may be mechanically more efficient than the fresh-frozen bone in surgical conditions. Moreover, the processed bone meets the highest safety standards, as regards the risk of disease transmission.

Bone Transplantation↗
PubMed2006

The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying.

In a previous study we have shown that the oligosaccharide inulin can prevent aggregation of poly(ethylene glycol) (PEG) coated plasmid DNA/cationic liposome complexes ("PEGylated lipoplexes") during freeze thawing and freeze drying [Hinrichs et al., 2005. J. Control. Release 103, 465]. By contrast, dextran clearly failed as stabilizer. These results were ascribed to the fact that inulin and PEG are compatible while dextran and PEG are not. In this study the stabilizing capacities of inulin and dextran (of various molecular weights) during freeze thawing and freeze drying of four different types of nanoparticles, each type with different amounts of PEG at their surface, were investigated. Freeze drying and freeze thawing of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleoyl-phosphatidyl-ethanolamine (DOPE) liposomes and egg phosphatidyl choline (EPC)/cholesterol (CHOL) liposomes showed that inulins are excellent stabilizers even for highly PEGylated liposomes while (especially higher molecular weight) dextrans dramatically lost their stabilizing capacity when increasing the degree of PEGylation of the liposomes. The same results were obtained for plasmid DNA/DOTAP/DOPE complexes. Finally, both inulin and dextran could prevent full aggregation of plasmid DNA/polyethylenimine (PEI) complexes independent whether PEI was PEGylated or not. It is concluded that inulins are preferred as stabilizers over dextrans for various types of PEGylated nanoparticles due to their compatibility with PEG.

Cholesterol↗
PubMed1999

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Animals↗
PubMed2002

Freeze-dried homogeneous and heterogeneous bone for sinus augmentation in sheep. Part I: histological findings.

Freeze-dried bone has been reported to possess an osteogenetic potential, i.e. to be osteo-inductive or osteoconductive depending on the grafted site. In this animal study homogeneous demineralized freeze-dried bone from the sheep (s-DFDB) and heterogeneous human demineralized freeze-dried bone (h-DFDB) used for single-stage sinus elevation were examined for their potential to improve bone-to-implant bonding. In 72 sinuses of 36 adult female mountain sheep Schneider's membrane was elevated from the local bone through an extra-oral approach and two cylindrical plasma-flame sprayed titanium implants were inserted in the lateral wall of each sinus. At the same time 18 sinuses were reinforced with s-DFDB, another 18 with h-DFDB and yet another 18 with autogenous cancellous bone from the iliac crest. In 18 animals the subantral space remained ungrafted. At each follow-up date, i.e. at 12, 16 and 26 weeks, 12 animals were re-examined and one randomly selected implant from each sinus was analyzed histologically and histomorphometrically. With both s-DFDB and h-DFDB extensive fragmentation and disintegration were seen side by side with sporadic areas of remineralization. These were mainly located in the vicinity of the local host bone and coalesced over time to be ultimately replaced and consolidated by woven bone. But most of the h-DFDB/s-DFDB particles continued to be embedded in abundant collagenous connective tissue and were surrounded by mononucleated and multinucleated giant cells, ultimately causing extensive resorption. The mean bone-to-implant contact length was 16.4% of the implant surface for s-DFDB, 16.9% for h-DFDB, 32.8% for autogenous bone and 22.2% in ungrafted controls. In the autogenous bone group the mean bone-to-implant contact length progressively increased with increasing contact time. In the control group, by contrast, the mean bone-to-implant contact length initially increased in the first two follow-up periods, but dropped again at 26 weeks. Both in the h-DFDB and the s-DFDB group the percent bone contact area was lower at 16 weeks than at 12 weeks but increased again by 26 weeks. In apical implant sections, s-DFDB produced significantly less bone-to-implant contact than autogenous cancellous bone from the iliac crest. In basal implant sections, both DFDB-grafted groups did significantly less well than the two control groups. The results of this experimental study showed that DFDB homografts and heterografts cannot be recommended alone instead of cancellous autografts from the iliac crest for augmenting the maxillary sinus in single-stage sinus elevations. Longer follow-up times than in the present study may perhaps show better results at the bone-implant interface.

Alveolar Ridge Augmentation↗
PubMed1999

Regeneration of transected spinal cord in young adult rats using freeze-dried alginate gel.

We have recently reported that freeze-dried alginate gel, which was developed in our laboratory, enhanced peripheral nerve regeneration. The purpose of this study was to examine whether alginate gel is capable of promoting nerve regeneration in the severed spinal cord of adult mammals. Using Wistar rats at 30 days of age (P30), the T9-T10 spinal cord was totally resected and alginate gel was implanted across the gap. Forty-five days after surgery myelinated and unmyelinated axons regenerated throughout the gap with remaining alginate gel. The elongated axons established electrophysiologically functional projections across the gap. In conclusion, freeze-dried alginate gel could be a promising material as an artificial nerve guide for repair of injured central nervous system.

Alginates↗
PubMed2001

An evaluation of extraction techniques for arsenic species from freeze-dried apple samples.

The extraction of arsenic from freeze-dried apples and subsequent determination of individual arsenic species by HPLC-ICP-MS is described. Solvent extraction with sonication using various aqueous and aqueous/solvent mixtures was initially evaluated by measuring total arsenic extracted by ICP-MS. A two step procedure using overnight treatment with alpha-amylase enzyme followed by sonication for 6 h with 40:60 acetonitrile-water was found to provide good extraction efficiency. The concentration of arsenic extracted was compared with the concentration of total arsenic in the samples determined using ICP-MS after microwave digestion in order to calculate extraction efficiency. Individual arsenic species in the extracts were measured using HPLC-ICP-MS. The three most abundant arsenic species found were arsenite, arsenate and dimethylarsinic acid. Total arsenic concentrations in the freeze-dried apple samples ranged from 8.2 to 80.9 micrograms kg-1 As, dry mass. By HPLC-ICP-MS, the relative amount of inorganic arsenic in the samples ranged from 73 to 90% of the sum of the arsenic species detected in each sample.

Arsenicals↗
PubMed2001

Low-temperature brewing by freeze-dried immobilized cells on gluten pellets.

A biocatalyst, prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets, was freeze-dried without any protecting medium and used for repeated batch fermentations of wort for each of the temperatures 15, 10, 5, and 0 degrees C. The fermentation time for freeze-dried immobilized cells was about 2-fold that of the corresponding time for wet immobilized cells on gluten pellets, and lower than the corresponding time for freeze-dried free cells, especially at 5 and 0 degrees C. Beers produced by freeze-dried immobilized cells contained alcohol levels in the range of 5.0-5.5% v/v, diacetyl concentrations lower than 0.5 mg/L, polyphenol concentrations lower than 145.5 mg/L, and free cell concentrations lower than 3 g/L. As a result, they had a very good clarity after the end of primary fermentation. The amounts of amyl alcohols were lower than 129.1 mg/L and reduced as the temperature was decreased. Ethyl acetate concentrations were found in the range of 22.1-29.2 mg/L, giving a very good aroma and taste in the produced beers.

Alcohols↗
PubMed1971

Repair of injury in freeze-dried Salmonella anatum.

Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.

Adenosine Triphosphate↗
PubMed2000

Freeze-drying and lyopreservation of diblock and triblock poly(lactic acid)-poly(ethylene oxide) (PLA-PEO) copolymer nanoparticles.

In this study, the formulation and process parameters that determine successful production and long-term stability of freeze-dried poly(lactic acid) (PLA) nanoparticles with "hairy-like" poly(ethylene oxide) (PEO) surfaces were investigated. Nanoparticles with grafted (covalently bound) PEO coatings were produced by the salting-out method from blends of PLA and PLA-PEO diblock or triblock copolymers. PLA nanoparticles with physically adsorbed PEO were also produced. The redispersibility of the nanoparticles after freeze-drying under various conditions was assessed. The surface of the nanoparticles was characterized and classified in terms of "brush" and "loop" conformations. Upon freeze-drying, it appeared that the presence of PEO at the nanoparticle surface could severely impair the redispersibility of the particles, especially in the PEO-grafted systems. This effect was shown to be related to the amount and molecular weight of PEO in the various formulations. In most cases, particle aggregation was prevented by use of trehalose as lyoprotective agent. Increasing the concentration of particles in the suspension to be freeze-dried was shown to induce much less damage to the nanoparticles, and freezing the suspension at a very low temperature (-196 degrees C) was found to further improve the lyoprotective effect. Most of the lyoprotected nanoparticles remained stable for at least 12 weeks at 4 and -25 degrees C. The production and preservation of freeze-dried PLA-PEO diblock and triblock copolymer nanoparticles is feasible under optimized lyoprotective conditions.

Drug Delivery Systems↗
PubMed2001

The effects of freeze-drying on the stability of liposomes to jet nebulization.

Egg phosphatidylcholine liposomes were freeze-dried in the presence and absence of trehalose. The lyophilized liposomes were rehydrated and aerosolized using a Pari LC jet nebulizer. The size of the aerosols generated was determined by laser diffraction, which was also used to determine the size distribution of the liposomes before lyophilization, post-rehydration, in the nebulizer post-aerosolization and those deposited in the two stages of a twin impinger. In the absence of trehalose, large liposomes and vesicle aggregates were produced on rehydration, which were rapidly reduced in size on nebulization. Liposomes with a mean size of 1 or 2.5 microm, freeze-dried with trehalose, had a mean size less than 3 microm following rehydration and exhibited enhanced stability to nebulization. Liposomes of 1 microm before freeze-drying were evenly distributed within aerosols generated by the nebulizer, whilst aerosols generated from 2.5 microm liposomes were fractionated in the twin impinger with the largest liposomes collected in the upper stage.

Cryoprotective Agents↗
PubMed1997

Repopulation of freeze-dried porcine valves with human fibroblasts and endothelial cells.

BACKGROUND AND AIMS OF THE STUDY: There is a need for a replacement cardiac valve constructed from non-immunogenic materials but incorporating living, and preferably autologous, cells. The object of this study was to colonize freeze-dried porcine valve leaflets with human fibroblasts and vascular endothelial cells. METHODS: Porcine pulmonary valve leaflets were freeze-dried to produce a porous matrix having communicating cavities of appropriate dimensions for fibroblast repopulation. Cultured human fibroblasts and vascular endothelial cells that had been cryopreserved by standard methods were added to freeze-dried leaflets. Following culture at 37 degrees C, the leaflets were examined by confocal scanning microscopy and transmission electron microscopy. RESULTS: Mechanical perforation of the leaflet surface permitted colonization of the freeze-dried matrix by fibroblasts; under the conditions we studied, the cell density did not reach physiologic levels but those cells that were present were well attached and metabolically active. Gentle cotton abrasion of the surface of the freeze-dried leaflets provided a suitable substrate for endothelial cell attachment and confluence was achieved in 10 days. Leaflets were perforated, cultured with human fibroblasts for 10 days, then gently rubbed with a cotton bud and cultured for a further 10 days with human endothelial cells. The endothelial cells formed a confluent layer on the surface and viable fibroblasts were present within the substance of the leaflet. CONCLUSION: Although these results are preliminary, they demonstrate the basic feasibility of this approach to the production of xenogeneic valves that contain the patient's own cells.

Animals↗
PubMed1981

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens.

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross-sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.

Animals↗
PubMed1994

Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from being killed by freeze-drying.

Wild-type spores of Bacillus subtilis were resistant to eight cycles of freeze-drying, whereas about 90% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins alpha and beta) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing. This role for alpha/beta-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment.

Bacillus subtilis↗
PubMed1990

Freeze-drying foot-and-mouth disease virus antigens. II. For use in the ELISA.

Live and inactivated preparations of foot-and-mouth disease virus strains 01 BFS 1860 and A22 IRQ 24/64 were freeze-dried in the presence or absence of additive solutions and assessed for their reactivity by ELISA at intervals over a six month storage period at various temperatures and also after reconstitution and subsequent storage with or without glycerination. The type specificity of all antigen preparations was maintained throughout the study period and the potency of antigens, judged by titration in ELISA, remained constant during the freeze-drying procedure and throughout subsequent storage at -20 degrees C and 4 degrees C with or without additives having been made to virus suspensions prior to freeze-drying. This was also the case with antigens reconstituted and stored at either -20 degrees C with glycerol or at 4 degrees C without glycerol. Certain additive solutions were necessary, however, to preserve the activity of antigens stored at the elevated temperature of 37 degrees C. The reactivity of all freeze-dried antigens was not unduly affected in the liquid-phase blocking ELISA using bovine convalescent antisera of each of the seven serotypes of foot-and-mouth disease virus and known negative, non-immune bovine sera. The results suggest that shipment and long-term storage of freeze-dried foot-and-mouth disease virus antigens is possible for use in the ELISA in the absence of refrigeration. This has attractive advantages for reducing both shipment and storage costs of antigens and for the development of ELISA kits for the diagnosis of foot-and-mouth disease virus.

Animals↗
PubMed2006

Effect of additives on the viability of bifidobacteria loaded in alginate poly-l-lysine microparticles during the freeze-drying process.

Bifidobacteria-loaded alginate poly-l-lysine microparticles (bap microparticles) were prepared using an air atomization method and then freeze-dried. The viability of the bap microparticles was investigated as a function of the amount of the bifidobacteria cultures, and the addition of a yeast extract, cryoprotectants, antioxidants and neutralizer. The size of the bap microparticles with and without the bifidobacteria was 84.8 +/- 28.5 microm (mean +/- standard deviation) and 113.1 +/- 38.5 microm, respectively. The surface morphology was slightly ellipsoid and wrinkled regardless of the incorporating bifidobacteria. The viability gradually decreased with increasing freeze-drying time. Free-flowing powdered bap microparticles were obtained at least 12 h after freeze-drying the wetted slurry of bap microparticles. However, the particles tended to aggregate when either lactose or ascorbic acid was added. The addition of a yeast extract, cryoprotectants (glycerol and lactose), antioxidants (NaHSO3 and ascorbic acid) and neutralizer (Mg3(PO4)2) resulted in a significantly higher viability of the bifidobacteria in the bap microparticles after freeze-drying (0.34-1.84 log) compared with the culture alone.

Alginates↗
PubMed1982

Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.

Animals↗
PubMed1989

[Non-crystallinity determination by thermal analysis of freeze-dried cephalothin sodium].

Cephalothin sodium (CET-Na) prepared according to the conventional freeze-drying methods is known to easily develop color during storage. Since amorphous CET-Na has been reported to be markedly unstable, the color development is thought to be due to the presence of traces of CET-Na in an amorphous state observed with scanning electron microscopy. Quantitation by use of the powder X-ray diffractometry of such traces of amorphous CET-Na has proved to be of little use. Thermogravimetry (TG) and differential scanning calorimetry (DSC) have demonstrated that the freeze-dried CET-Na by the conventional methods contains three types of CET-Na: amorphous (unstable phase), quasi-crystalline (metastable phase) and crystalline (stable phase). Pyrolysis initiation temperatures of these three types of CET-Na have been demonstrated to become higher in this order. A new parameter for the evaluation of non-crystallinity of CET-Na has been introduced, in which the ratio is calculated from the data of the total weight loss observed through the pyrolysis of both amorphous and quasi-crystalline CET-Na against the total weight loss of all components during the pyrolysis of sample specimen. The ratio thus calculated is defined as "non-crystallinity". This new parameter has successfully been introduced to establish a good correlation to the degree of increasing color intensity with aging of freeze-dried CET-Na.

Calorimetry, Differential Scanning↗
PubMed2005

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients.

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.

Bifidobacterium↗