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Culture Period (culture + period)
Kinds of Culture Period Selected AbstractsLong-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture mediumDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Yasuto Fukui Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3,4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro. [source] Retinoic acid increases the length and volume density of ducts in the rat embryonic pancreasDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2003Carene Erasmus In this study, the role of all -trans retinoic acid (RA) on the proliferation of rat embryonic pancreas ducts and on the proportion of insulin cells was investigated. All- trans RA (10,6 m) was added to Ham's F12. ITS serum-free medium in which 12.5 day rat dorsal pancreatic buds were cultured on Matrigel. Control explants were cultured on Matrigel in Ham's F12. ITS alone or in Ham's F12. ITS containing ethanol (the diluent for RA). After a 7 day culture period, explants were incubated with bromodeoxyuridine (BrdU) for assessment of cell proliferation. Explants were processed for both morphometry and immunocytochemistry. The length density and volume density of the pancreatic ducts were assessed using an image analysis system. Cells positive for insulin, BrdU and glucagon were localized on adjacent serial sections. RA treatment caused a statistically significant increase in the volume density (P < 0.007) and length density (P < 0.008) of the ducts, as well as a 1.2-fold increase (P < 0.0001) in the proportion of insulin to glucagon cells, compared to both control groups. Few insulin cells were BrdU positive, indicating that cells had a low proliferation rate. The increased proportion of insulin cells may relate to the increased volume density and length density of the ducts in RA-treated explants. It is suggested that RA stimulated the production of additional progenitor cells and not proliferation of existing insulin cells. [source] Inverse relationship between seizure expression and extrasynaptic NMDAR function following chronic NMDAR inhibitionEPILEPSIA, Issue 2010Suzanne B. Bausch Summary We showed previously that electrographic seizures involving dentate granule cells in organotypic hippocampal slice cultures were dramatically reduced following chronic treatment with the NR2B-selective antagonist, Ro25,6981, but were increased following chronic treatment with the high-affinity competitive antagonist, D(-)-2-amino-5-phosphonopentanoic acid (D-APV). To begin to investigate the potential mechanisms underlying the differential effects of N -methyl- d -aspartate receptor (NMDAR) antagonists on seizures, electrophysiologic experiments were conducted in dentate granule cells in hippocampal slice cultures treated for the entire 17,21 day culture period with vehicle, Ro25,6981 or D-APV. Initial experiments revealed a lack of an association between miniature excitatory postsynaptic current (mEPSC) measures and seizures suggesting that shifts in mEPSC were unlikely to account for the differential effects of D-APV and Ro25,6981 on seizures. However, the amplitude of tonic NMDAR-mediated currents was reduced in cultures treated chronically with D-APV and dramatically enhanced in cultures treated chronically with Ro25,6981. Because tonic NMDAR currents are mediated primarily by extrasynaptic NMDAR, these data show an inverse relationship between changes in extrasynaptic NMDAR function and alterations in seizure expression. [source] Dedifferentiation of intrinsic response properties of motoneurons in organotypic cultures of the spinal cord of the adult turtleEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2000Jean-François Perrier Abstract Explant cultures from the spinal cord of adult turtles were established and used to study the sensitivity of the intrinsic response properties of motoneurons to the changes in connectivity and milieu imposed by isolation in culture. Transverse sections 700 ,m thick were explanted on cover slips and maintained in roller-tube cultures in medium containing serum and the growth factors brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT3), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF). The gross morphology of acute sections was maintained after 4 weeks in culture. Cell bodies of motoneurons remained stainable in fixed cultures with an antibody against choline acetyltransferase (ChAT) throughout the culture period. During culture, motoneurons maintained stable resting membrane potentials and were contacted by functional synapses. The ability to generate action potentials was also preserved as was delayed inward rectification and generation of calcium spikes in the presence of tetra-ethyl ammonium (TEA). In response to depolarization, however, motoneurons presented strong outward rectification, and only 41% of the cells recorded from maintained the ability to fire repetitively. By the second week in culture, a fraction of motoneurons displayed fast and slow transient outward rectification and low-threshold calcium spikes, features not seen in turtle motoneurons in acute slices. On the other hand, properties mediated by L-type Ca2+ channels disappeared during the first few days in culture. Our observations show that the phenotypical intrinsic response properties of mature spinal motoneurons are modified in explant cultures. The properties acquired resemble the properties in juvenile motoneurons in several species of terrestrial vertebrates. [source] Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: An approach to tissue engineeringJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2007Wah W. Thein-Han Abstract Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007 [source] Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactorJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2002Vassilios I. Sikavitsas Abstract The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague,Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L -lactic- co -glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 62: 136,148, 2002 [source] A Dominant Negative Cadherin Inhibits Osteoblast Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Su-Li Cheng Abstract We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCad,C) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCad,C, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally,45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCad,C-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and ,-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCad,C cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells. [source] Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat modelJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2006U. Kneser Abstract Introduction: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. Methods: Discs of PBCB (Tutobone®) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. Results: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. Discussion: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects. [source] Generation of a scaffold free cartilage-like implant from a small amount of starting materialJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2006M. J. Stoddart Abstract Introduction: An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. Methods: Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFG,1 and dexamethasone was investigated. Results: During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95±5ng/ng compared with 37.3±1.8ng/ng in the controls (P <0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P < 0.001). IGF1 also induced a increase of DNA content 1383±314ng/bead compared to 512±19ng/bead in the controls (P < 0.001). The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4±13.14ng/ng GAG/DNA and 42.93±10.23ng/ng collagen II. Discussion: We believe alginate released chondrocytes provide a real alternative to artificial scaffolds. [source] Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitroJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 3 2001Yu-Chao Chang Abstract Background, aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. Method: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 ,g/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. Results: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 ,g/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p<0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 ,g/ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p<0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 ,g/ml and 100 ,g/ml, arecoline depleted about 18% and 56% of thiols (p<0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 ,g/ml, arecoline suppressed the growth of PDLF by about 33%, and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. Conclusion: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone. Zusammenfassung Zielsetzung: Das Kauen von Betelnüssen gehört zum Alltag von ungefähr 200 Millionen Menschen. Betelnußkauer weisen eine höhere Prävalenz von Parodontalerkrankungen auf als Personen, die keine Betelnüsse konsumieren. In dieser Studie sollte der pathobiologische Effekt des Arekolins, das die Hauptkomponente des Betelnußalkaloides darstellt, auf menschliche Desmodontalfibroblasten (PDLF) in vitro untersuchen. Material und Methoden: Zellvitalität, Proliferationsrate, Proteinsynthese und zelluläre Thiolspiegel wurden genutzt, um zu untersuchen, welche Auswirkungen eine Exposition der PDLF gegenüber Arekolinspiegeln von 0 bis 200 ,g/ml hat. Zusätzlich wurde Nikotin beigefügt, um festzustellen wie das Nikotin den Effekt des Arekolins beeinflußt. Ergebnisse: Arekolin hemmt die Zellproliferation signifikant in dosisabhängiger Weise. Bei Konzentrationen von 10 und 30 ,g/ml unterdrückt Arekolin das Wachstum der PDLF um 20% bzw. 50% (p<0.05). Arekolin unterdrückt ebenfalls dosisabhängig die Proteinsynthese während der 24-stündigen Kultivierungsperiode. Ein Arekolinspiegel von 100 ,g/ml reduzierte die Proteinsynthese auf 50% im Vergleich zur unbehandelten Kontrollkultur (p<0.05). Auch die intrazellulären Thiolspiegel wurden dosisabhängig reduziert. Bei Konzentrationen von 25 und 100 ,g/ml wurden die Thiolspiegel um 18% bzw. 56% reduziert (p<0.05). Bei einer Konzentration von 60 ,g/ml unterdrückte das Arekolin das PDLF-Wachstum um 33%. Die Zugabe von 5 mM Nikotin verstärkte die durch Arekolin induzierte zytotoxische Wirkung, so daß es zum Zelltot von 66% kam. Schlußfolgerungen: Es scheint, daß Arekolin selbst zu der Schädigung des Parodonts beiträgt, die der Betelnuß zugeschreiben wird. Außerdem deuten die Ergebnisse darauf hin, daß Personen, die Betelnußkauen mit Nikotinkonsum kombinieren, empfindlicher für Schädigungen des Parodonts sind als solche, die nur Betelnüsse kauen. Während der Inaktivierung des Thiols könnte das Arekolin PDLF verletzlicher für andere reaktive Substanzen wie Nikotin machen. Résumé L'habitude de mastiquer de la noix de betel affecte la vie quotidienne de près de 200 millions de personnes. Les mâcheurs de betel présentent une prévalence plus élevée de maladies parodontales. Cette étude examine les effets pathologiques de l'arécoline, un composant majeur des alcaloïdes de la noix de betel, sur des fibroblastes du ligament parodontal humain (PDLF) in vitro. La viabilité cellulaire, la prolifération, la synthèse protéique, et les niveaux cellulaires de thiol ont été utilisés pour observer les effets de l'exposition de PDLF humains à des taux d'arécoline de 0 à 200 ,g/ml. De plus, de la nicotine fut ajouté pour tester la façon dont cela modulait les effets de l'arécoline. L'arécoline inhibait significativement la prolifération cellulaire de façon dose dépendante. A des concentrations de 10 à 30 ,g/ml, l'arécoline supprime la croissance des fibroblastes par 20 et 50% (p<0.05), respectivement. L'arécoline dimunuait également la synthèse des protéines de façon dose dépendante pendant une période de culture de 24 h. Une concentration de 100 ,g/ml d'arécoline inhibit la synthèse protéique à seulement 50% de celle du groupe controle non traité (p<0.05). De plus, l'arécoline réduit les thiols intracellulaires de façon dose dépendante. A des concentrations de 25 ,g/ml et 100 ,g/ml, l'arécoline réduit environ 18 à 56% des thiols, respectivement (p<0.05). Cela suggère que l'arécoline, elle même, peut augmenter la destruction du parodonte en association avec l'utilisation de noix de betel. De plus, l'addition de nicotine entrainait un effet synergique sur la cytotoxicité induite par l'arécoline. A une concentration de 60 ,g/ml, l'arécoline supprimait la croissance des PDLF d'environ 33% et 5 mM de nicotine augmentait cette réponse cytotoxique induite par l'arécoline, jusqu'à entrainer 66% de morts cellulaires. Lors de la réduction des thiols, l'arécoline pourrait rendre les PDLF humains plus vulnérables à des agents réactifs entrant dans la composition des cigarettes. Pris ensemble, les gens qui combinent des habitudes de mastication de noix de betel et de tabagisme, pourrait être plus susceptibles à des dommages parodontaux, que les gens qui utiliserait uniquement la noix de betel, mais sans fumer. [source] Inter-subtype cross-neutralizing antibodies recognize epitopes on cell-associated HIV-1 virionsJOURNAL OF MEDICAL VIROLOGY, Issue 2 2003Helen Donners Abstract HIV-1 infected individuals with cross-neutralizing antibodies against primary HIV-1 isolates belonging to Group M (env A-H) and O, are identified. To investigate the neutralization-kinetics of primary isolates with these antibodies, different neutralization assay conditions are compared. Each set is summarized as a/b/c where a is the time in hours for which antibody is incubated with virus, b is the time in hours allowed for virus to absorb to cells, c is the total culture period in days, from the cells' first exposure to virus, before antigen production (peripheral blood mononuclear cells) or number of fluorescent cells (GHOST) are measured. In HIV-infected individuals, neutralizing antibodies can be detected against a wide range of primary isolates (Group M; A,H and Group O) in PBMC-assays with short incubation phases (1/2/7 or 1/24/7). If cultures are extended (1/2/14 or 1/24/14), however, neutralization can be lost. In kinetic experiments, neutralization can even be seen without pre-incubation (a,=,0 hr). This study shows that neutralization of primary HIV isolates by cross-reactive antibodies can continue after the virus has bound to its target cell. This neutralization, however, is not an all or nothing loss in virus infectivity. Most often it leads only to a reduction in viral replication rates. J. Med. Virol. 69:173,181, 2003. © 2003 Wiley-Liss, Inc. [source] Homeostasis of neuroactive amino acids in cultured cerebellar and neocortical neurons is influenced by environmental cuesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Helle Waagepetersen Abstract Neuronal function is highly influenced by the extracellular environment. To study the effect of the milieu on neurons from cerebellum and neocortex, cells from these brain areas were cultured under different conditions. Two sets of cultures, one neocortical and one cerebellar neurons, were maintained in media containing [U- 13C]glucose for 8 days at initial concentrations of 12 and 28 mM glucose, respectively. Other sets of cultures (8 days in vitro) maintained in a medium containing initially 12 mM glucose were incubated subsequently for 4 hr either by addition of [U- 13C]glucose to the culture medium (final concentration 3 mM) or by changing to fresh medium containing [U- 13C]glucose (3 mM) but without glutamine and fetal calf serum. 13C Nuclear magnetic resonance (NMR) spectra revealed extensive ,-aminobutyric acid (GABA) synthesis in both cultured neocortical and cerebellar neurons after maintenance in medium containing [U- 13C]glucose for 8 days, whereas no aspartate labeling was observed in these spectra. Mass spectrometry analysis, however, revealed high labeling intensity of aspartate, which was equal in the two types of neurons. Addition of [U- 13C]glucose (4 hr) on Day 8 in culture led to a similar extent of labeling of GABA in neocortical and in cerebellar cultures, but the cellular content of GABA was considerably higher in the neocortical neurons. The cellular content of alanine was similar regardless of culture type. Comparing the amount of labeling, however, cerebellar neurons exhibited a higher capacity for alanine synthesis. This is compatible with the fact that cerebellar neurons could ameliorate a low alanine content after culturing in low glucose (12 mM) by a 4-hr incubation in medium containing 3 mM glucose. A low glucose concentration during the culture period and a subsequent medium change were associated with decreases in glutathione and taurine contents. Moreover, glutamate and GABA contents were reduced in cerebellar cultures under either of these conditions. In neocortical neurons, the GABA content was decreased by simultaneous exposure to low glucose and change of medium. These conditions also led to an increase in the aspartate content in both types of cultures, although most pronounced in the neocortical neurons. Further experiments are needed to elucidate these phenomena that underline the impact of extracellular environment on amino acid homeostasis. © 2004 Wiley-Liss, Inc. [source] Ex vivo static tensile loading inhibits MMP-1 expression in rat tail tendon cells through a cytoskeletally based mechanotransduction mechanismJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2004Steven P. Arnoczky Abstract To determine the effect of various degrees of ex vivo static tensile loading on the expression of collagenase (MMP-1) in tendon cells, rat tail tendons were statically loaded in tension at 0.16, 0.77, 1.38 or 2.6 MPa for 24 h. Northern blot analysis was used to assay for mRNA expression of MMP-1 in freshly harvested, 24 h load deprived, and 24 h statically loaded tendons. Western blot analysis was used to assay for pro-MMP-1 and MMP-1 protein expression in fresh and 24 h load deprived tendons. Freshly harvested rat tail tendons demonstrated no evidence of MMP-1 mRNA expression and no evidence of the pro-MMP-1 or MMP-1 protein. Ex vivo load deprivation for 24 h resulted in a marked increase in the mRNA expression of MMP-1 which coincided with a marked increase of both pro-MMP-1 and MMP-1 protein expression. When tendons were subjected to ex vivo static tensile loading during the 24 h culture period, a significant inhibition of this upregulation of MMP-1 mRNA expression was found with increasing ioad (p < 0.05). A strong (r2 = 0.78) and significant (p < 0.001) inverse correlation existed between the level of static tensile load and the expression of MMP-1. Disruption of the actin cytoskeleton with cytochalasin D abolished the inhibitory effect of ex vivo static tensile loading on MMP-1 expression. The results of this study suggest that up-regulation of MMP-1 expression in tendon cells ex vivo can be inhibited by static tensile loading, presumably through a cytoskeletally based mechanotransduction pathway. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Collagen gene expression and mechanical properties of intervertebral disc cell,alginate culturesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2001Anthony E. Baer Cells of the intervertebral disc have a limited capacity for matrix repair that may contribute to the onset and progression of degenerative disc changes. In this study, the biosynthetic capacity of cells isolated from specific regions of the porcine intervertebral disc was evaluated in vitro. Using a competitive reverse transcription-polymerase chain reaction technique, gene expression levels for types I and II collagen were quantified in cells cultured for up to 21 d in a three-dimensional alginate culture system and compared to levels obtained for cells in vivo. The mechanical properties of cell-alginate constructs were measured in compression and shear after periods of culture up to 16 weeks. Cells from the anulus fibrosus expressed the most type I collagen mRNA in vivo and in vitro, while cells from the transition zone expressed the most type II collagen mRNA in vivo and in vitro. Mechanical testing results indicate that a mechanically functional matrix did not form at any time during the culture period; rather, decreases of up to 50% were observed in the compressive and shear moduli of the cell,alginate constructs compared to alginate with no cells. Together with results of prior studies, these results suggest that intervertebral disc cells maintain characteristics of their phenotype when cultured in alginate, but the molecules they synthesize are not able to form a mechanically functional matrix in vitro. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Signal transduction pathways involved in the stimulation of tissue type plasminogen activator by interleukin-1, and Porphyromonas gingivalis in human osteosarcoma cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2006Yu-Chao Chang Background:, Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. Objectives:, The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1, (IL-1,) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Methods:, IL-1, and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. Results:, Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1, and P. gingivalis during a 2-day culture period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1, or P. gingivalis -stimulated t-PA production, respectively (p < 0.05). Conclusions:, Our findings demonstrated that IL-1, and P. gingivalis enhance t-PA production in human osteosarcoma cells, and that the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA production and/or activity and may therefore be valuable therapeutics in t-PA-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis. [source] Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of tissue non-specific alkaline phosphatase activityJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2000M. T. M. Van Der Pauw The capacity of the periodontal ligament to alter its structure and mass in response to mechanical loading has long been recognized. However, the mechanism by which periodontal cells can detect physical forces and respond to them is largely unknown. Besides transmission of forces via cell-matrix or cell-cell interactions, the strain-derived flow of interstitial fluid through the periodontal ligament may mechanically activate the periodontal cells, as well as ensure transport of cell signaling molecules, nutrients and waste products. Mechanosensory cells, such as endothelial and bone cells, are reported to respond to a flow of fluid with stimulated prostaglandin E2(PGE2) and nitric oxide production. Therefore, we examined the PGE2 and nitric oxide response of human periodontal ligament and gingival fibroblasts to pulsating fluid flow and assessed the expression of tissue non-specific alkaline phosphatase activity. Periodontal ligament and gingival fibroblasts were subjected to a pulsating fluid flow (0.7±0.02 Pa, 5 Hz) for 60 min. PGE2 and nitric oxide concentrations were determined in the conditioned medium after 5, 10, 30 and 60 min of flowing. After fluid flow the cells were cultured for another 60 min without mechanical stress. Periodontal ligament fibroblasts, but not gingival fibroblasts, responded to fluid flow with significantly elevated release of nitric oxide and decreased expression of tissue non-specific alkaline phosphatase activity. In both periodontal ligament and gingival fibroblasts, PGE2 production was significantly increased after 60 min of flowing. Periodontal ligament fibroblasts, but not gingival fibroblasts, produced significantly higher levels of PGE2 during the postflow culture period. We conclude that human periodontal ligament fibroblasts are more responsive to pulsating fluid flow than gingival fibroblasts. The similarity of the early nitric oxide and PGE2 responses to fluid flow in periodontal fibroblasts with bone cells and endothelial cells suggests that these three cell types possess a similar sensor system for fluid shear stress. [source] Effects of Two Densities of Caged Monosex Nile Tilapia, Oreochromis niloticus, on Water Quality, Phytoplankton Populations, and Production When Polycultured with Macrobrachium rosenbergii in Temperate PondsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2007Jason J. Danaher The effects of different densities of caged Nile tilapia, Oreochromis niloticus, on water quality, phytoplankton populations, prawn, and total pond production were evaluated in freshwater prawn, Macrobrachium rosenbergii, production ponds. The experiment consisted of three treatments with three 0.04-ha replicates each. All ponds were stocked with graded, nursed juvenile prawn (0.9 ± 0.6 g) at 69,000/ha. Control (CTL) ponds contained only prawns. Low-density polyculture (LDP) ponds also contained two cages (1 m3; 100 fish/cage) of monosex male tilapia (115.6 ± 22 g), and high-density polyculture (HDP) ponds had four cages. Total culture period was 106 d for tilapia and 114 d for prawn. Overall mean afternoon pH level was significantly lower (P , 0.05) in polyculture ponds than in CTL ponds but did not differ (P > 0.05) between LDP and HDP. Phytoplankton biovolume was reduced in polyculture treatments. Tilapia in the LDP treatment had significantly higher (P , 0.05) harvest weights than in the HDP treatment. Prawn weights were higher (P , 0.05) in polyculture than prawn monoculture. These data indicate that a caged tilapia/freshwater prawn polyculture system may provide pH control while maximizing pond resources in temperate areas. [source] Production Characteristics, Water Quality, and Costs of Producing Channel Catfish Ictalurus punctatus at Different Stocking Densities in Single-batch ProductionJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2006Brent E. Southworth Channel catfish Ictalurus punctatus farming is the largest component of aquaculture in the USA. Culture technologies have evolved over time, and little recent work has been conducted on the effects of stocking density on production characteristics and water quality. Twelve 0.1-ha ponds were stocked with 13- to 15-cm fingerlings (16 g) at either 8600, 17,300, 26,000, or 34,600 fish/ha in single-batch culture with three replicates per treatment. Fish were fed daily to apparent satiation with a 32% floating commercial catfish feed. Nitrite-N, nitrate-N, total ammonia nitrogen (TAN), total nitrogen, total phosphorus, chemical oxygen demand (COD), Secchi disk visibility, chlorophyll a, chloride, total alkalinity, total hardness, pH, temperature, and dissolved oxygen (DO) were monitored. Ponds were harvested after a 201-d culture period (March 26, 2003 to October 13, 2003). Net yield increased significantly (P < 0.05) as stocking density increased, reaching an average of 9026 kg/ha at the highest density. Growth and marketable yield (>0.57 kg) decreased with increasing stocking density. Survival was not significantly different among densities. Mean and maximum daily feeding rates increased with density, but feed conversion ratios did not differ significantly among treatments (overall average of 1.42), despite the fact that at the higher stocking densities, the feeding rates sometimes exceeded 112 kg/ha per d (100 lb/ac per d). Morning DO concentrations fell below 3 mg/L only once in a 34,600 fish/ha pond. Concentrations of chlorophyll a, COD, nitrite-N, and TAN increased nominally with increasing feed quantities but did not reach levels considered problematic even at the highest stocking densities. Breakeven prices were lowest for the highest stocking density even after accounting for the additional time and growth required for submarketable fish to reach market size. While total costs were higher for the higher density treatments, the relatively higher yields more than compensated for higher costs. [source] Growth of Stocker Channel Catfish to Large Market Size in Single-Batch CultureJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2004Bartholomew W. Green Catfish farmers increasingly are producing fish larger than the traditional size of 0.45-0.57 kg/fish in order to meet processing plant requirements for larger fish. Production of larger channel catfish Ictalurus punctatus in multiple-batch culture has been investigated in a few studies, but the impact of understocked fingerlings on growth of carry-over fish is unknown. The present study was conducted to quantify growth, feed conversion ratio, net daily yield, and net and total yield of stocker channel catfish grown in single-batch, one-season culture to mean individual weights of 0.60, 0.72, 0.91, or 1.17 kg/fish. Channel catfish (mean weight = 0.26 kg/fish) were stocked into 12 0.1-ha ponds at 11,115 fish/ha. Fish were fed a 32% crude protein floating extruded feed once daily to apparent satiation. When the average weight of the fish population reached the target weight, three randomly selected ponds were harvested. Fish growth was linear in all treatments. Growth rates were similar for fish grown to 0.60, 0.72, and 0.91 kg/fish, and significantly lower (P < 0.05) than for fish grown to 1.17 kg. Variation in individual fish weight increased linearly with increased duration of culture period. Feed conversion ratio averaged 1.9 and did not differ significantly among treatments. The percentage of the fish population at harvest that fell within the 0.57 to 2.04 kg-size range preferred by processing plants increased from 56.6 to 98-5% as the mean weight at harvest increased from 0.60 to 1.17 kg/fish. [source] Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineeringJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2 2010Gail C. Donegan Abstract Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space. Copyright © 2009 John Wiley & Sons, Ltd. [source] Monocyte-derived dendritic cells from HCV-infected patients transduced with an adenovirus expressing NS3 are functional when stimulated with the TLR3 ligand poly(I:C)JOURNAL OF VIRAL HEPATITIS, Issue 11 2008I. Echeverría Summary., Dendritic cells (DC) transfected with an adenovirus encoding hepatitis C virus (HCV) NS3 protein (AdNS3) induce potent antiviral immune responses when used to immunize mice. However, in HCV infected patients, controversial results have been reported regarding the functional properties of monocyte-derived DC (MoDC), a cell population commonly used in DC vaccination protocols. Thus, with the aim of future vaccination studies we decided to characterize MoDC from HCV patients transfected with AdNS3 and stimulated with the TLR3 ligand poly(I:C). Phenotypic and functional properties of these cells were compared with those from MoDC obtained from uninfected individuals. PCR analysis showed that HCV RNA was negative in MoDC from patients after the culture period. Also, phenotypic analysis of these cells showed lower expression of CD80, CD86, and CD40, but similar expression of HLA-DR molecules as compared to MoDC from uninfected individuals. Functional assays of MoDC obtained from patients and controls showed a similar ability to activate allogeneic lymphocytes or to produce IL-12 and IL-10, although lower IFN-, levels were produced by cells from HCV patients after poly(I:C) stimulation. Moreover, both groups of MoDC induced similar profiles of IFN-, and IL-5 after stimulation of allogeneic T-cells. Finally, migration assays did not reveal any difference in their ability to respond to CCL21 chemokine. In conclusion, MoDC from HCV patients are functional after transduction with AdNS3 and stimulation with poly(I:C). These findings suggest that these cells may be useful for therapeutic vaccination in chronic HCV infection. [source] Limnology and culture-based fisheries in non-perennial reservoirs in Sri LankaLAKES & RESERVOIRS: RESEARCH AND MANAGEMENT, Issue 3 2005U. Asanka D. Jayasinghe Abstract This study was carried out to investigate the possibility of using the limnological characteristics of non-perennial reservoirs in Sri Lanka for the future management of culture-based fisheries. Forty-five reservoirs were randomly selected to study their limnology, out of which 32 were stocked with fish fingerlings of Chinese and Indian carps, tilapia and freshwater prawn at stocking densities ranging from 218,4372 fingerlings ha,1. Of these, 23 reservoirs were harvested at the end of the culture period (6,10 months). Thirteen limnological parameters were measured during the water retention period of each of the 45 reservoirs between November 2001 and January 2004. The mean values of the limnological parameters were used to ordinate the reservoirs through principal component analysis. Ordination showed a productivity gradient among reservoirs where Secchi disc depth, total phosphorus, chlorophyll- a, inorganic turbidity and organic turbidity were identified as key factors. The total fish yield of culture-based fisheries was positively correlated to the scores of the first principal component axis. This study reveals that it is possible to classify non-perennial reservoirs in Sri Lanka based on the above limnological parameters in order to develop culture-based fisheries and that they could be applicable in comparable water bodies elsewhere in the tropics. [source] In vitro analysis of cryopreserved alginate,poly- l -lysine,alginate-microencapsulated human hepatocytesLIVER INTERNATIONAL, Issue 4 2010Hualian Hang Abstract Background: The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. Methods: Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 °C for 12,24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate,poly- l -lysine,alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at ,80 °C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed. Results: Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. Conclusions: The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 °C for 12,24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans. [source] Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liverLIVER TRANSPLANTATION, Issue 4 2007Paul P.C. Poyck The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading. Liver Transpl 13:589,598, 2007. © 2007 AASLD. [source] Cryopreservation of primary human hepatocytes: The benefit of trehalose as an additional cryoprotective agentLIVER TRANSPLANTATION, Issue 1 2007Ekaterina Katenz Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 ± 13 vs. 46.9 ± 11%, P < 0.01) and plating efficiency (41.5 ± 18 vs. 17.6 ± 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation. Liver Transpl, 2007. © 2006 AASLD. [source] Inhibition of nifedipine-induced proliferation of cultured human gingival fibroblasts by Saiko, a Chinese herbal medicinePHYTOTHERAPY RESEARCH, Issue 8 2006Toshimi Hattori Abstract Saiko is predominantly contained in Saireito, a Chinese herbal medicine. The present study was conducted to determine whether or not Saiko is involved in the inhibition by Saireito of nifedipine-induced proliferation and collagen synthesis in gingival fibroblasts. Nifedipine (10 µm) significantly enhanced the proliferation starting on day 5 of the culture period. When added together with nifedipine, Saiko at concentrations of 0.05%,0.2% (w/v) dose-dependently inhibited the nifedipine-induced proliferation, and at the highest concentration tested (0.2%), Saiko inhibited the nifedipine-induced proliferation by about 40%. Moreover, Saiko (0.2%) also inhibited the normal proliferation at days 11 and 14. Sole application of nifedipine (10 µm) augmented the release of bFGF, and Saiko concentration-dependently reduced the level of bFGF in the nifedipine-containing culture medium. Nifedipine (10 µm) increased the production of type I collagen to almost twice that of the control (normal medium), and Saiko at concentrations above 0.1% significantly reduced the nifedipineinduced production of collagen. In conclusion, the present findings demonstrate that Saiko inhibited the nifedipine-induced proliferation of gingival fibroblasts by reducing the release of bFGF and that Saiko is involved in the Saireito-induced inhibition of nifedipine-stimulated proliferation and collagen synthesis in gingival fibroblasts. Copyright © 2006 John Wiley & Sons, Ltd. [source] Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering,THE LARYNGOSCOPE, Issue 1 2010Jennifer L. Long MD Abstract Objectives/Hypothesis: One potential treatment option for severe vocal fold scarring is to replace the vocal fold cover layer with a tissue-engineered structure containing autologous cells. As a first step toward that goal, we sought to develop a three-dimensional cell-populated matrix resembling the vocal fold layers of lamina propria and epithelium. Study Design: Basic science investigation. Methods: Adipose-derived stem cells were cultured in fibrin hydrogels with various growth factors. At the end of the culture period, matrices were sectioned and labeled with immunomarkers to identify cell phenotype. Results: Adipose-derived stem cells survived, attached, and populated three-dimensional fibrin matrices. Under select conditions, a superficial layer of cells expressing epithelial marker proteins overlay a deeper mesenchymal cell layer. Conclusions: A three-dimensional structure of fibrin and adipose-derived stem cells was created as a prototype vocal fold replacement. Two segregated cell phenotypes occurred, producing a bilayered structure resembling epithelium over lamina propria. This preliminary work demonstrates the feasibility of tissue engineering to produce structures for vocal fold replacement. Laryngoscope, 2010 [source] Dietary protein level and natural food management in the culture of blue (Litopenaeus stylirostris) and white shrimp (Litopenaeus vannamei) in microcosmsAQUACULTURE NUTRITION, Issue 3 2003L.R. Martinez-Cordova Abstract The effect of dietary protein level and natural food management on the production parameters of blue and white shrimp, as well as on water quality, was evaluated in a microcosms system (plastic pools simulating aquaculture ponds). Two experimental trials were carried out in the facilities of DICTUS, University of Sonora, Northwest México. Treatment with low protein diet (LP) consisted of a low protein input (diet with 250 g kg,1 crude protein) through the culture period; treatment with high protein diet (HP) consisted of a high protein input (diet with 400 g kg,1 crude protein) through the trial, and finally treatment VP consisted of an adjustment of protein input (diets with 250, 350 or 400 g kg,1 crude protein), depending on the abundance of biota (zooplankton and benthos) in the system. Each species responded differently to the treatments. For blue shrimp, low protein input resulted in the lowest final body weight (12.9 ± 0.6 g) and biomass (696.0 g pool,1). Survival and feed conversion ratio were similar in the three treatments. For white shrimp, the best growth, biomass and food conversion ratio were obtained in the low protein input treatment. Water quality parameters such as nitrate, ammonia and organic matter during the two trials, were better for LP and VP treatments. White shrimp seems to have lower protein requirements than blue shrimp. For the blue shrimp culture, adjusting protein input according to natural food abundance (zooplankton and benthos) in the system, seems to be advantageous because of the possibility of getting a production similar to that obtained with a high protein input through the farming period, but at lower feed cost, and with a lower environmental impact. It is concluded that a high protein input through the whole farming period is not the best feeding strategy for any of the two species. [source] Chironomid abundance and consumption by juvenile channel catfish in plastic-lined and earthen culture pondsAQUACULTURE RESEARCH, Issue 9 2010Bonnie L Mulligan Abstract In 2004, research was conducted to compare chironomid larvae populations and their use by channel catfish (Ictalurus punctatus) fingerlings in two different culture systems. Over a 4-month culture period, chironomid larvae densities in plastic-lined ponds were significantly less than those in earthen ponds. The consumption of chironomid larvae by channel catfish fingerlings was related to chironomid abundance in earthen ponds. The significance of these findings is the possible relationship among pond type, initial consumption of commercial diets and subsequent survival rates of fingerling channel catfish. [source] Heterosis in fingerlings from a diallel cross between two wild strains of silver perch (Bidyanus bidyanus)AQUACULTURE RESEARCH, Issue 11 2009Jeffrey A Guy Abstract Cross-breeding was investigated as a strategy to improve performance of the Australian native freshwater fish, silver perch (Bidyanus bidyanus Mitchell) through the exploitation of heterosis during the fingerling phase of production. Growth, and mid and best parent heterosis of two wild strains, Cataract Dam (C,× C,) and Murray River (M,× M,) and their reciprocal crosses (C,× M, and M,× C,) were evaluated in cages and ponds through summer, and in tanks in a re-circulating aquaculture system during winter. The M × C cross grew significantly faster than the reciprocal cross and pure strains in cages and tanks, had the lowest coefficients of variation of weight and length and was 20.9% and 16.0% heavier than mid-parent and best-parent average, respectively, when grown in ponds. Differences in growth between the reciprocal crosses were also evident, with C × M expressing significantly less heterosis in cages and tanks. Faster growth of M × C was attributed to greater appetite; however, at sizes approaching 250 g this feeding vigour diminished. The results of this study suggest that use of the M × C cross has the potential to reduce the length of the culture period and lower costs of silver perch production. 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