Templated Assembly of Collagen Fibers Directs Cell Growth in 2D and 3D.

Collagen is widely used in tissue engineering and regenerative medicine, with many examples of collagen-based biomaterials emerging in recent years. While there are numerous methods available for forming collagen scaffolds from isolated collagen, existing biomaterial processing techniques are unable to efficiently align collagen at the microstructural level, which is important for providing appropriate cell recognition and mechanical properties. Although some attention has shifted to development of fiber-based collagen biomaterials, existing techniques for producing and aligning collagen fibers are not appropriate for large-scale fiber manufacturing. Here, we report a novel biomaterial fabrication approach capable of efficiently generating collagen fibers of appropriate sizes using a viscous solution of dextran as a dissolvable template. We demonstrate that myoblasts readily attach and align along 2D collagen fiber networks created by this process. Furthermore, encapsulation of collagen fibers with myoblasts into non-cell-adherent hydrogels promotes aligned growth of cells and supports their differentiation. The ease-of-production and versatility of this technique will support future development of advanced in vitro tissue models and materials for regenerative medicine.

Synergistic effects of growth and differentiation factor-5 (GDF-5) and insulin on expanded chondrocytes in a 3-D environment.

OBJECTIVE: To investigate the effects of growth and differentiation factor-5 (GDF-5) alone or in combination with insulin on engineered cartilage from primary or expanded chondrocytes during 3-dimensional in vitro culture. DESIGN: Juvenile bovine chondrocytes were seeded either as primary or as expanded (passage 2) cells onto polyglycolic acid fiber meshes and cultured for 3 weeks in vitro. Additionally, adult human chondrocytes were grown in pellet culture after expansion (passage 2). The culture medium was supplemented either with GDF-5 in varying concentrations or insulin alone, or with combinations thereof. RESULTS: For primary chondrocytes, the combination of GDF-5 and insulin led to increased proliferation and construct weight, as compared to either factor alone, however, the production of glycosaminoglycans (GAG) and collagen per cell were not affected. With expanded bovine chondrocytes, the use of GDF-5 or insulin alone led to only very small constructs with no type II collagen detectable. However, the combination of GDF-5 (0.01 or 0.1 microg/ml) and insulin (2.5 microg/ml) yielded cartilaginous constructs and, in contrast to the primary cells, the observed redifferentiating effects were elicited on the cellular level independent of proliferation (increased production of GAG and collagen per cell, clear shift in collagen subtype expression with type II collagen observed throughout the construct). The synergistic redifferentiating effects of the GDF-5/insulin combination were confirmed with expanded adult human cells, also exhibiting a clear shift in collagen subtype expression on the mRNA and protein level. CONCLUSIONS: In combination with insulin, GDF-5 appears to enable the redifferentiation of expanded chondrocytes and the concurrent generation of cartilaginous constructs. The demonstration of these synergistic effects also for adult human chondrocytes supports the clinical relevance of the findings.

Lipidic implants for controlled release of bioactive insulin: effects on cartilage engineered in vitro.

Controlled release systems for growth factors and morphogens are potentially powerful tools for the engineering or the treatment of living tissues. However, due to possible instabilities of the protein during manufacture, storage, and release, in the development of new release systems it is paramount to investigate into the maintenance of bioactivity of the protein. Within this study, recently developed protein releasing lipid matrix cylinders of 2 mm diameter and 2 mm height made from glycerol tripalmitate were manufactured in a compression process without further additives. Insulin in different concentrations (0.2%, 1%, and 2%) served as model protein. The bioactivity of the protein released from the matrices was investigated in a long-term cartilage engineering culture for up to four weeks; additionally, the release profiles were determined using ELISA. Insulin released from the matrices increased the wet weights of the cartilaginous cell-polymer constructs (up to 3.2-fold), the amount of GAG and collagen in the constructs (up to 2.4-fold and 3.2-fold, respectively) and the GAG and collagen content per cell (1.8-fold and 2.5-fold, respectively), compared to the control. The dose-dependent effects on tissue development correlated well with release profiles from the matrices with different insulin loading. In conclusion, the lipid matrices, preserving the bioactivity of incorporated and released protein, are suggested as a suitable carrier system for use in tissue engineering or for the localized treatment of tissues with highly potent protein drugs such as used in the therapy of brain cancer or neurodegenerative CNS diseases.

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes: further evidence using 5-HT moduline, polyclonal 5-HT1B receptor antibodies and 5-HT1B receptor knock-out mice.

In previous paper based on classical pharmacological tools, we identified a Gi protein-coupled presynaptic 5-hydroxytryptamine (5-HT) 1B receptor causing inhibition of dopamine (DA) release in rat striatal synaptosomes. It was the aim of the present study to further explore this receptor, using 5-HT moduline, a polyclonal antibody directed against 5-HT1B receptors and 5-HT1B receptor knock-out mice. Preincubation of rat striatal synaptosomes with 5-HT moduline (0.1, 1, or 10 microM) significantly reduced the inhibitory effect of CP93,129, a selective rat 5-HT1B receptor agonist, on K+-evoked overflow of [3H]DA in a non-competitive manner: 5-HT moduline did not modify the IC50 of CP93,129, but concentration-dependently reduced the maximal inhibitory effect. Preincubation of rat striatal synaptosomes with a specific polyclonal 5-HT1B receptor antibody also resulted in a significant attenuation of the inhibitory effect of CP93,129 on K+-evoked overflow of [3H]DA. In female 129/Sv wild-type mice, CP93,129 and 5-carboxyamidotryptamine maleate (5-CT), a non-selective 5-HT1B receptor agonist, inhibited the K+-evoked [3H]DA overflow in a concentration-dependent manner. Sumatriptan, a selective rat 5-HT1D receptor agonist, did not modify the overflow of [3H]DA. SB224289, a selective 5-HT1B receptor antagonist, abolished the inhibitory effects of CP93,129 and 5-CT. The inhibitory effects of CP93,129 and 5-CT were absent in synaptosomes from 5-HT1B receptor knockout mice. No compensatory inhibition effect in mutant mice was observed using sumatriptan. In conclusion, the results show that a non-competitive antagonist of the 5-HT1B receptor concentration-dependently decreases the maximal inhibitory effect of a 5-HT1B receptor agonist on the synaptosomal K+-evoked release of [3H]DA in striatum. Moreover, a specific antibody raised against the receptor and particularly directed against a region of the receptor protein involved in signal transduction, namely the coupling with the G-protein, also antagonizes the inhibitory effect of the stimulation of 5-HT1B receptor on the release of [3H]DA. Ultimately the disruption of 5-HT1B receptor gene in 5-HT1B knock-out mice leads to a total suppression of the effect of 5-HT1B receptor agonists on [3H]DA release. These observations further support our previous observations using selective agonists/antagonists, indicating that 5-HT1B receptors control the release of neuronal DA as presynaptic heteroreceptors.

Differential sensitivity of 5-HT1B auto and heteroreceptors.

The effect of the native and rodent-selective 5-HT1B receptor agonists (5-hydroxytryptamine (5-HT) and CP93,129) on the K+-evoked overflows of [3H]5-HT, [3H]dopamine (DA) and [3H]acetylcholine (ACh) was studied in synaptosome preparations obtained from rat brain striatum or hippocampus loaded with radiolabeled neurotransmitter. The aim of the study was to compare the different potencies of the specific 5-HT1B receptor agonists to stimulate the auto and heteroreceptors and to modulate the different neurotransmitter release. Results show that under the same experimental conditions, 5-HT and CP93,129 exhibited significantly higher potencies in inhibiting the K+-evoked overflow of [3H]5-HT from synaptosomes of rat striatum (IC50=2.0+/-1.8 nM and 20.5+/-3.1 nM, respectively) than in inhibiting the K+-evoked overflow of [3H]DA from synaptosomes of the same cerebral region (IC50= 0.8+/-0.2 microM and 1.8+/-0.4 microM, respectively), or [3H]ACh from synaptosomes of hippocampus (IC50=1.7+/-0.8 microM for CP93,129). The inhibitory effects of the 5-HT1B receptor agonists on [3H] K+-overflows were antagonized by the selective 5-HT1B receptor antagonist (SB224289), further indicating that the observed effects were 5-HT1B receptor specific. Sumatriptan, a selective r5-HT1D receptor agonist, did not show any significant effect on the K+-overflow of [3H]5-HT in the range of concentrations (10(-10) to 10(-6) M), and did not affect the K+ overflow of [3H]DA or [3H]ACh at concentrations (10(-9) to 10(-4) M), which exclude the involvement of 5-HT1D receptors. These inhibitory effects of the 5-HT1B receptor agonists were highly attenuated by pertussis toxin in the three systems studied, suggesting the involvement of Gi/Go-proteins in the transduction mechanism pathway of the receptor generated signal. In conclusion, these results suggest that 5-HT1B heteroreceptors located on dopaminergic and cholinergic terminals exhibit a lower sensitivity to 5-HT1B receptor agonist and antagonist than do 5-HT1B autoreceptors. The observed difference in functional sensitivities of 5-HT1B auto- and heteroreceptors may represent important consequences in the physiological control of the release of serotonin versus that of other neurotransmitters.

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes.

The effect of the selective r5-HT1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K(+)-evoked overflow of [3H]dopamine was studied in rat striatal synaptosomes loaded with [3H]dopamine. The aim of the study was to investigate the participation of 5-HT1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca2(+)-dependent, tetrodotoxin-resistant K(+)-evoked overflow of [3H]dopamine was inhibited by CP93,129 (0.01-100 microM) in a concentration-dependent manner (IC50=1.8 microM; maximal inhibition by 35.5% of control). [+/-]8-OH-DPAT, a 5-HT(1A) receptor agonist, [+/-]DOI, a 5-HT2 receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT3 receptor agonist, at concentrations ranging from 0.01 microM to 100 microM did not show any significant effect. Neither ketanserin (1 microM and 5 microM), a selective 5-HT2/5-HT1D receptor antagonist, nor ondansetron (1 microM), a 5-HT3 receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT1B receptor antagonists, in contrast, used at a concentration of 1 microM, antagonized the inhibitory effect of CP93,129 (3 microM and 10 microM). SB224289, a selective 5-HT1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K(i) value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K(+)-evoked release of dopamine is regulated by the presynaptic 5-HT1B heteroreceptors.

Mapping of 5-HT-moduline binding sites in guinea-pig brain by film and digital autoradiography.

5-HT-moduline is a cerebral tetrapeptide [Leu-Ser-Ala-Leu] that was recently isolated from bovine brain tissue and shown to interact specifically with 5-HT1B receptors, particularly in rodents. The pharmacological properties of 5-HT1B receptors in rodents are different from those in other species. In order to better understand the role of this peptide in non-rodent species, we determined the distribution of 5-HT-moduline binding sites in guinea-pig brain using both the film autoradiography and digital autoradiography with a newly developed high resolution beta-imaging techniques. We found that 5-HT-moduline binding sites were expressed in various brain regions. Quantitative analysis showed that densities of binding sites were similar to those observed previously in rat brain. Regions with the highest labelling included cortex, septum, hippocampus and some regions of basal ganglia. Our results extend previous data and show that 5-HT-moduline interacts with the two forms of 5-HT1B receptors that are distinct pharmacologically. By this interaction, 5-HT-moduline may play an important role in regulating the functional activity of 5-HT1B receptors, thereby contributes to the pathophysiology of serotonergic transmission.

Sub-chronic cold stress reduces 5-HT1A receptor responsiveness in the old but not in the young rat.

The inhibitory effect of the prototypical 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-n-propyl amino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity, has been examined as an index of the functional activity of 5-HT1A receptors in the hippocampus of young (3 months) and old (18 months) rats exposed during 24 h or 5 days to cold. In both young and old rats exposed to cold stress during 24 h, there was a reduction in the potency (EC50) and/or the maximal inhibitory effect (Emax) of 8-OH-DPAT in reducing forskolin-induced cAMP accumulation. The properties of the hippocampal 5-HT1A sites labelled by [3H]8-OH-DPAT were not affected by these stressful conditions. Moreover, while the sensitivity of 5-HT1A receptors to 8-OH-DPAT in young rats returned to control values after 5 days of cold exposure, old rats still exhibited a significant desensitization of 5-HT1A receptors as compared to naive animals. These results point out the capacity of young but not of old rats to adapt to the aversive effects of a subchronic stressor.

Stress-induced 5-HT1A receptor desensitization: protective effects of Ginkgo biloba extract (EGb 761).

The effects of sub-chronic cold stress on the functioning of hippocampal 5-HT1A receptors in old isolated rats and the possible protective effects of Ginkgo biloba extract (EGb 761) were investigated. Cold exposure during five days, produced a significant reduction of the inhibitory effect of 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity. In contrast, neither the affinity nor the density of hippocampal [3H]8-OH-DPAT binding sites were affected indicating that the reduced sensitivity of 5-HT1A receptors induced by stress is probably due to a modification of their coupling mechanisms to adenylyl cyclase. The stress-induced desensitization of 5-HT1A receptors was prevented by the administration of EGb 761 (50 mg/kg per os/14 days). These results clearly indicate that 5-HT1A receptors are desensitized by stress and point out the reduced capacity of old rats to cope with the adverse effects of a chronic stressor. EGb 761 appears to restore the age-related decreased capacity to adapt to a chronic stressor.

Evaluation of a rapid fluorogenic method for the detection of Escherichia coli in dairy products.

A rapid fluorogenic medium was evaluated for the detection of Escherichia coli in dairy products. The medium was capable of detecting Esch. coli after 7.5 h incubation at 41.5 degrees C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-beta-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers of Esch. coli detected on the two media. MUG-7 medium had a specificity of 98.6% and the small number of organisms giving a false positive reaction were identified as Klebsiella pneumoniae. The incidence of false negative results was approximately 2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.

A rapid fluorogenic method for the detection of Escherichia coli by the production of beta-glucuronidase.

A medium containing the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide was developed for the isolation and identification of Escherichia coli within 7.5 h and was based on the detection of beta-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41.5 degrees C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6.1% and false-positives were 3.7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.

Regional distribution of 3-O-methyl-DOPA in the cortex and subcortical structures in the rat: effect of aging.

Dihydroxyphenylalanine (DOPA) and its metabolite 3-O-methyl-DOPA (3-OMDOPA) have been identified as normal constituents in blood, CSF and brain in human and several animal species. In the present study, we report results of 3-OMDOPA measurements in seven cortical areas, hippocampus and striatum of young (3-month-old), middle aged (10-month-old) and aged (27-month-old) rats. 3-OMDOPA was identified in all the areas considered. Regional differences were observed, with the highest levels being found in the hippocampus and the somatomotor and pyriform cortex. A marked increase in the level of 3-OMDOPA was observed in all the cortical areas and in the hippocampus of aged rats. In contrast, this level remained unchanged in the striatum. These modifications were compared to those observed in the levels of dopamine (DA) in the same areas.

Bacteria and food poisoning organisms in milk.

Individual milk samples of 50 goats, 50 ewes and 50 cows were examined for the total viable count, coliform bacteria, staphylococci and salmonellae. Growth of enterotoxin A producing Staphylococcus aureus NCTC 10652 in the milk of the three animal species was also studied. The average total count was 1.9 X 10(7) cells/ml for cow's, 7.7 X 10(6) for goat's and 2.7 X 10(6) for ewe's milk with micrococci staphylococci, rods and streptococci being the predominant organisms in the three milks, respectively. Goat's milk contained the lowest numbers of coliforms and ewe's milk the highest numbers. Staphylococcus aureus could not be detected in goat's milk, whilst 16 and 26% of the ewe's and cow's milk samples contained 100 and 80 cells/ml, respectively. Out of 39 coagulase positive staphylococci, 27 were thermonuclease positive, 18 produced lecithinase and 15 fermented mannitol. Red blood cells of sheep origin were much more resistant to lysis by ewe's strains compared to bovine strains. Growth curves of Staphylococcus aureus were nearly linear at 17 degrees C but exponential at 31 degrees C without lag phase. Hazardous numbers of about 10(6) cells/ml were readily reached at 31 degrees C after 6 h and at 17 degrees C after 18 h. Salmonellae could not be detected in any of the samples examined. Out of 19 enterobacteria suspected to be salmonellae 11 proved to be Proteus and 8 Citrobacter.