|Articles about Biomaterials|
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| Glucose reactions with acid and base catalysts in hot compressed water at 473 K
Watanabe, M., Y. Aizawa, et al. (2005), Carbohydr Res 340(12): 1925-30.
Abstract: The effects of the homogeneous catalysts (H(2)SO(4) and NaOH) and heterogeneous catalysts (TiO(2) and ZrO(2)) on glucose reactions were examined in hot compressed water (473 K) by a batch-type reactor. From the homogeneous catalyst studies, we confirmed that the acid catalyst promoted dehydration, while isomerization of glucose to fructose was catalyzed by alkali. Anatase TiO(2) was found to act as an acid catalyst to promote formation of 5-hydroxymethylfuraldehyde (HMF). Zirconia (ZrO(2)) was a base catalyst to promote the isomerization of glucose. The effects of the additives were also confirmed through fructose reactions.
| Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase
Hussain, F., D. J. Birch, et al. (2005), Anal Biochem 339(1): 137-43.
Abstract: In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation=295 nm, emission=330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd=0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd=12.5 mM). The glucose response range was increased further (to 120 mM, Kd=57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.
| Glucose-responsive UV polymerised dextran-concanavalin A acrylic derivatised mixtures for closed-loop insulin delivery
Tanna, S., M. Joan Taylor, et al. (2006), Biomaterials 27(8): 1586-97.
Abstract: A novel UV polymerised glucose-responsive mixture containing concanavalin A (con A) and dextran was synthesised and characterised as a "smart" biomaterial to form the basis of a closed-loop delivery device. Dextran and con A precursors were modified with acrylic side groups and then UV polymerised to produce covalently bonded mixtures which were examined by FTIR. The viscoelastic properties of these polymerised mixtures containing glucose concentrations between 0% and 5% w/w were also examined using oscillatory rheometry within the linear viscoelastic range across a frequency range of 0.01-50Hz. As the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1Hz was observed. Increasing the mixture irradiation time produced viscosity profiles at higher values throughout the glucose concentration range. The subsequent testing of such formulations in in vitro diffusion experiments revealed that the leaching of the mixture components is formulation dependent and is restricted significantly in the covalently bonded mixtures. Insulin delivery in response to glucose in the physiologically relevant glucose concentration range was demonstrated using the novel polymerised mixture at 37 degrees C. The performance of this covalently cross-linked glucose-responsive biomaterial has been improved in terms of increased mixture stability with reduced component leaching. This could, therefore be used as the basis of the design of a closed-loop drug delivery device for therapeutic agents used for the management of diabetes mellitus.
| Glutardialdehyde induced fluorescence technique (GIFT): a new method for the imaging of platelet adhesion on biomaterials
Frank, R. D., H. Dresbach, et al. (2000), J Biomed Mater Res 52(2): 374-81.
Abstract: One of the major limitations of biomaterials used in medicine is the adhesion and subsequent activation of platelets upon contact with blood. The development of new or modified materials necessitates adequate methods for the detection and quantification of platelet/material interactions. These interactions are commonly investigated by means of scanning electron microscopy (SEM), radioisotope and immunological techniques, or by quantification of released platelet contents. Given the lack of a simple, rapid, and inexpensive assay, we developed a novel method for the accurate assessment of platelet adhesion after contact with foreign surfaces, which enables quantitative measurements as well as imaging of the platelet shape change, and which omits conventional or immunological staining and time-consuming preparative steps. The glutardialdehyde induced fluorescence technique (GIFT) uses the epifluorescence of glutardialdehyde-fixed platelets detected by fluorescence microscopy and is suitable for opaque and transparent materials. Combined with computer-aided image analysis, numbers of adherent platelets, platelet-covered surface, and average platelet spread area can be determined as markers of surface thrombogenicity. To validate the technique, four materials of different thrombogenicity [polypropylene (PP), poly(D,L-lactide) (PDLLA), 2-hydroxyethyl-methacrylate-grafted PDLLA (PDLLA-HEMA), and heparin-coupled PDLLA-HEMA] were investigated by GIFT and SEM. We found concordant results with SEM and GIFT with the following ranking of thrombogenicity: PP > PDLLA > PDLLA-HEMA > or = PDLLA-HEMA-heparin. GIFT significantly discriminated between the investigated materials. The surface modifications led to improved thromboresistance with reduced platelet adhesion and shape change. The main advantages of GIFT as compared with SEM are: no vacuum-drying or dehydration, less time-consuming procedure, fixation and fluorescence "staining" in one step, and suitability for computer-aided image analysis allowing quantitative assessment of platelet adhesion as well as imaging of the platelet shape change with high-contrast images. In conclusion, GIFT is a valid, rapid, and simple method for the quantitative determination of platelet/material interactions intended for the evaluation of thrombogenicity of biomaterials surfaces.
| Glycocalyx production and adherence of Staphylococcus to biomaterials
Tsai, C. L., T. K. Liu, et al. (1992), Acta Med Okayama 46(1): 11-6.
Abstract: Glycocalyx is suggested to play an important role in the pathogenesis of biomaterial-centered infection. Using an accurate and sensitive method to quantify glycocalyx and bacterial adherence, we have demonstrated that the producer of the most glycocalyx also exhibited the highest adherence index, whereas low producers exhibited the least (p less than 0.01). Additionally, at various concentrations the high producer had the greater tendency to adhere and grow on stainless steel wires and tubes (p less than 0.001). The adherence index, referred as the ratio of tritiated thymidine uptake on wires to colony forming units (CFU), was also the highest in high producers. The adherence index increased as the glycocalyx index increased. It was suggested that glycocalyx production enhanced the adherence of Staphylococcus epidermidis to biomaterials and caused persistent and intractable infections. In short, the glycocalyx index and the adherence index can be reliable indices of biomaterial-centered infection.
| Glycolide copolymer staple-line reinforcement reduces staple site bleeding during laparoscopic gastric bypass: a prospective randomized trial
Nguyen, N. T., M. Longoria, et al. (2005), Arch Surg 140(8): 773-8.
Abstract: HYPOTHESIS: The use of staple-line reinforcement sleeves during laparoscopic gastric bypass reduces staple-line bleeding, which may translate into a reduction in the rate of gastrointestinal hemorrhage. DESIGN: Prospective randomized trial. SETTING: University hospital. PATIENTS AND INTERVENTIONS: Thirty-four patients undergoing laparoscopic gastric bypass were randomly assigned to receive either no reinforcement (control group, n = 17) or reinforcement of the staple line with glycolic copolymer sleeves (treatment group, n = 17). MAIN OUTCOME MEASURES: Demographic data, the number of stapler loads used, the number of staple-line bleeding sites, the amount of blood loss, the length of time required to obtain hemostasis of the staple lines, operative time, intraoperative and postoperative complications, and serial hemoglobin levels. RESULTS: The mean number of stapler loads used was similar between groups. The mean number of staple-line bleeding sites was significantly fewer in the treatment group for division of gastric tissue (0.4 vs 2.5 bleeding sites), jejunal tissue (0.1 vs 0.6 bleeding site), and mesenteric tissue (0 vs 0.8 bleeding site). The mean blood loss was lower in the treatment group (84 vs 129 mL). Staple misfire occurred in 1 (0.7%) of 143 stapler loads used in the treatment group compared with 0 (0%) of 138 stapler loads used in the control group. The time to obtain staple-line hemostasis was shorter in the treatment group (1.2 vs 10.1 minutes). The total operative time was similar between groups. There was no mortality or postoperative leaks. One patient in the control group had postoperative gastrointestinal hemorrhage requiring blood transfusion and reoperation. There was no significant difference in the mean hemoglobin level between groups on the first postoperative day. CONCLUSIONS: The use of glycolide copolymer staple-line reinforcement sleeves in patients undergoing laparoscopic gastric bypass is safe and significantly reduces staple-line bleeding sites and may reduce the incidence of gastrointestinal hemorrhage.
| Glycosaminoglycan mimetic biomaterials. 4. Synthesis of sulfated lactose-based glycopolymers that exhibit anticoagulant activity
Sun, X. L., D. Grande, et al. (2002), Biomacromolecules 3(5): 1065-70.
Abstract: Cyanoxyl persistent radicals can be used as chain-growth moderators of the statistical copolymerization of a variety of monomers. We report herein the preparation of fully sulfated lactose-based glycopolymers by cyanoxyl (OC[triple bond]N)-mediated free-radical polymerization of acrylamide derivatized glycomonomers in good yield (60-80%) and low polydispersity (1.1 < M(w)/M(n) < 1.6). Prolongation of the activated partial thromboplastin time (aPTT) was observed, and structure-activity relationships were defined. Specifically, the anticoagulant effect varied in response to both polymer molecular weight and the density of pendant sulfated lactose units. Nonetheless, measured thrombin times were only modestly prolonged suggesting that the observed anticoagulant effect is not primarily related to direct thrombin inhibition.
| Glycosaminoglycan-mimetic biomaterials. 3. Glycopolymers prepared from alkene-derivatized mono- and disaccharide-based glycomonomers
Baskaran, S., D. Grande, et al. (2002), Bioconjug Chem 13(6): 1309-13.
Abstract: Mono- and disaccharide-containing glycopolymers were synthesized by two different free-radical processes, and their ability to act as heparan sulfate glycomimetics in promoting the binding of Fibroblast Growth Factor-2 (FGF-2) to its receptor (FGFR-1) was evaluated using an in vitro cell-based assay. Cyanoxyl (*OC triple bond N)-mediated polymerization of acrylamide with alkene-derivatized mono- and disaccharides including sulfated or nonsulfated N-acetyl-D-glucosamine is described. The results of this approach are compared to those obtained via the classical ammonium peroxodisulfate (APS)/N,N,N',N'-tetramethylethylenediamine (TMEDA) initiating system and confirm the capacity of cyanoxyl-mediated polymerization to generate a variety of glycopolymers with high saccharide contents and low polydispersity indexes. In vitro assays demonstrate that specific glycopolymers can potentiate FGF-2/FGFR-1 binding interactions.
| Gold nanoshell bioconjugates for molecular imaging in living cells
Loo, C., L. Hirsch, et al. (2005), Opt Lett 30(9): 1012-4.
Abstract: Advances in scattering-based optical imaging technologies offer a new approach to noninvasive point-of-care detection, diagnosis, and monitoring of cancer. Emerging photonics technologies provide a cost-effective means to image tissue in vivo with high resolution in real time. Advancing the clinical potential of these imaging strategies requires the development of optical contrast agents targeted to specific molecular signatures of disease. We describe the use of a novel class of contrast agents based on nanoshell bioconjugates for molecular imaging in living cells. Nanoshells offer significant advantages over conventional imaging probes including continuous and broad wavelength tunability, far greater scattering and absorption coefficients, increased chemical stability, and improved biocompatibility. We show that nanoshell bioconjugates can be used to effectively target and image human epidermal growth factor receptor 2 (HER2), a clinically relevant biomarker, in live human breast carcinoma cells.
| Gold nanotubes by template-directed synthesis
Barreca, D., A. Gasparotto, et al. (2005), J Nanosci Nanotechnol 5(6): 994-8.
Abstract: Gold nanotubes were prepared by radiofrequency (RF)-sputtering through a template-directed synthesis in porous alumina substrates. The resulting composite material was subsequently treated in acidic or alkaline aqueous solutions in order to selectively remove the membrane, thus resulting in the obtainment of self-supporting Au nanotubules. The adopted strategy allows the preparation of both composites and free-standing metal nanostructures with an aspect ratio tunable as a function of the synthesis conditions and the membrane pore size.
| Grafting of gelatin on electrospun poly(caprolactone) nanofibers to improve endothelial cell spreading and proliferation and to control cell Orientation
Ma, Z., W. He, et al. (2005), Tissue Eng 11(7-8): 1149-58.
Abstract: We modified the surface of electrospun poly(caprolactone) (PCL) nanofibers to improve their compatibility with endothelial cells (ECs) and to show the potential application of PCL nanofibers as a blood vessel tissue-engineering scaffold. Nonwoven PCL nanofibers (PCL NF) and aligned PCL nanofibers (APCL NF) were fabricated by electrospinning technology. To graft gelatin on the nanofiber surface, PCL nanofibers were first treated with air plasma to introduce -COOH groups on the surface, followed by covalent grafting of gelatin molecules, using water-soluble carbodiimide as the coupling agent. The chemical change in the material surface during surface modification was confirmed by X-ray photoelectron spectroscopy and quantified by colorimetric methods. ECs were cultured to evaluate the cytocompatibility of surface-modified PCL NF and APCL NF. Gelatin grafting can obviously enhance EC spreading and proliferation compared with the original material. Moreover, gelatin-grafted APCL NF readily orients ECs along the fibers whereas unmodified APCL NF does not. Immunostaining micrographs showed that ECs cultured on gelatin-grafted PCL NF were able to maintain the expression of three characteristic markers: platelet-endothelial cell adhesion molecule 1 (PECAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). The surface-modified PCL nanofibrous material is a potential candidate material in blood vessel tissue engineering.
| Grafting reaction of poly(D,L)lactic acid with maleic anhydride and hexanediamine to introduce more reactive groups in its bulk
Pan, J., Y. Wang, et al. (2005), J Biomed Mater Res B Appl Biomater 74(1): 476-80.
Abstract: Bioactivity of biomaterials was a new requirement, especially in tissue engineering and drug delivery. As a traditional used biomaterial, polylactide (PLA) had no bioactivity, of course, and it still had few reactive groups to introduce some bioactive molecules in its bulk. Here, we want to introduce carboxyl groups and amino groups in the side chain of PLA to get more reactive groups for incorporating bioactive molecular later and to maintain the structure of main chain to keep its biodegradability, and to settle the acidity of PLA during hydrolysis at the same time. It was performed as follows: first, maleic anhydride was covalently grafted onto the side chain of PLA by a free radical reaction at 100 degrees C for 20 h with BPO as the initiator. Then, by amidation with a maleic anhydride group on PLA at room temperature, hexanediamine was incorporated. The resulting polymers have been characterized via GPC, (13)C NMR, DSC, and TGA. The graft ratio was tested by titration. The pH changes during hydrolysis in 0.1 M PBS with pH 7.4 of PLA, MPLA, and HPLA were investigated. All the results showed that this research has grafted maleic anhydride and then hexanediamine in the bulk of PLA. The molecular weight degradation during reaction was less than 20%. The graft ratios of were 2.68, 2.36, and 1.86%, respectively in 5, 10, and 20% raw MA in MPLA; and the anhydride groups grafted in MPLA can completely react with hexanediamine at room temperature. The pH value of HPLA remained neutral within 12 weeks' hydrolysis compared with the resulted acidity of PLA and MPLA.
| Granulocyte colony-stimulating factor for the treatment of biomaterial-associated staphylococcal infections in-vitro
Schlobe, A., N. Schnitzler, et al. (2003), Urol Res 30(6): 394-8.
Abstract: Staphylococcal infections are a common and severe complication after the implantation of a prosthesis. We developed an in-vitro model for biomaterial-associated infections and studied the effects of human recombinant granulocyte colony-stimulating factor (rhuG-CSF; filgrastime) on the eradication of bacteria from the surface of biomaterial. Latex beads (25 micro m) were incubated with 10(7) colony forming units of either a slime producing (DSM 3269) or non-slime producing strain (ATCC 14990) of Staphylococcus epidermidis. Infected particles were consecutively confronted with effector cells, derived from heparinized whole blood samples taken from healthy volunteers, after stimulation with rhuG-CSF (5,000 IU/ml, 10,000 IU/ml). Control blood specimens were not stimulated or conditioned with normal saline. The results indicate that stimulation with rhuG-CSF induced an increased rate of phagocytosis and lead to a more rapid reduction of adhering bacteria from the surface of the beads. Therefore, the in-vitro data suggest that patients with prosthesis infection may profit from an additional treatment with rhuG-CSF.
| Granulocyte-macrophage colony-stimulating factor (GM-CSF)-coated implants and their potential for reducing biomaterial-associated infection in neutropenic hosts
Rozalska, B., B. Sadowska, et al. (1998), Zentralbl Bakteriol 288(2): 237-51.
Abstract: The incidence of infections associated with the use of medical biomaterials is high for skin-penetrating devices, when microbes of the normal skin flora like coagulase-positive and coagulase-negative staphylococci dominate as causative organisms. The most serious ones are infections in immunocompromised individuals. A mouse model of subcutaneous staphylococcal infection yielding abscesses in cyclophosphamide-induced neutropenic mice implanted with heparinized polyethylene (H-PE) was used. The present study addresses the question of the effects of implant modification with recombinant granulocyte-macrophage stimulating factor (rGM-CSF) on the course of infection. Our findings demonstrate that such modification reduces the proliferation of bacteria within the abscess and as a consequence limits the dissemination of bacteria from the local infection induced in the neutropenic host.
| Growth and reactions of SiOx/Si nanostructures on surface-templated molecule corrals
Liu, Y., Z. Zhang, et al. (2005), Langmuir 21(19): 8883-91.
Abstract: Surface-templated nanostructures on the highly oriented pyrolytic graphite (HOPG) basal plane were created by controlled Cs+- or Ga+)ion bombardment, followed by subsequent oxidation at high temperature, forming molecule corrals. The corrals were then used for template growth of SiOx/Si nanostructures. We demonstrate here that, for SiOx/Si nanostructures formed in controlled molecule corrals, the amount of silicon deposited on the surface is directly correlated with the corral density, making it possible to generate patterned SiOx/Si nanostructures on HOPG. Since the size, depth, position, and surface density of the nanostructures can be controlled on the HOPG, it is possible to produce surfaces with patterned or gradient functionalities for applications in fields such as biosensors, microelectronics, and biomaterials (e.g., neuron pathfinding). If desired, the SiOx structures can be reduced in size by etching in dilute HF, and further oxidation of the nanostructures is slow enough to provide plenty of time to functionalize them using ambient and solution reactions and to perform surface analysis. Organosilane monolayers on surface-templated SiOx/Si nanostructures were examined by X-ray photoelectron spectroscopy, time-of-flight secondary ion mas spectrometry, and atomic force microscopy. Silanes with long alkyl chains such as n-octadecyltrichlorosilane (C18) were found to both react on SiOx/Si nanostructures and to condense on the HOPG basal plane. Shorter-chain silanes, such as 11-bromoundicyltrimethoxysilane (C11) and 3-mercaptopropyltrimethoxysilane (C3) were found to react preferentially with SiOx/Si nanostructures, not HOPG. The SiOx/Si nanostructures were also found to be stable toward multiple chemical reactions. Selective modification of SiOx/Si nanostructures on the HOPG basal plane is thus achievable.
| Growth at high pH increases Enterococcus faecalis adhesion to collagen
Kayaoglu, G., H. Erten, et al. (2005), Int Endod J 38(6): 389-96.
Abstract: AIM: To evaluate the effect of growth at pH levels from 7.1 to 9.5 on the adherence of Enterococcus faecalis to bovine serum albumin (BSA) and collagen type I. METHODOLOGY: Enterococcus faecalis strain A197A was grown in broth of adjusted pHs varying between 7.1 and 9.5. Aliquots of bacterial suspensions were added to wells coated either with BSA or with collagen type I. Bacteria adhering to the surfaces were stained with crystal violet. Spectrophotometric measurements of the dissolved stain were used to assess the number of bacteria adhering to the surfaces. The data obtained were analysed using the Kolmogorov-Smirnov test, Levene's test and Student's t-test, with alpha = 0.05 as the level for statistical significance. RESULTS: The adhesion of E. faecalis to BSA-coated surfaces decreased inversely with alkalinity of the growth medium. The pH 7.1-grown bacteria bound to BSA significantly more than the other BSA groups. On the contrary, the adhesion to collagen type I-coated surfaces of bacteria grown at pH 8.0 and 8.5 was significantly greater than for those grown at pH 7.1. CONCLUSIONS: A minor increase in pH up to 8.5, which may be a consequence of insufficient treatment with alkaline medicaments such as calcium hydroxide, increases the collagen-binding ability of E. faecalis, in vitro. This can be a critical mechanism by which E. faecalis predominates in persistent endodontic infections.
| Growth factor delivery for bone tissue repair: an update
Varkey, M., S. A. Gittens, et al. (2004), Expert Opin Drug Deliv 1(1): 19-36.
Abstract: Growth factors (GFs) are endogenous proteins capable of acting on cell-surface receptors and directing cellular activities involved in the regeneration of new bone tissue. The specific actions and long-term effects of GFs on bone-forming cells have resulted in exploration of their potential for clinical bone repair. The concerted efforts have led to the recent approval of two GFs, bone morphogenetic protein-2 and osteogenic protein-1, for clinical bone repair, and human parathryroid hormone (1-34) for augmentation of systemic bone mass. This review provides a selective summary of recent (2001-2004) attempts for GF delivery in bone tissue regeneration. First, a summary of non-human primate studies involving local regeneration and repair is provided, with special emphasis on the range of biomaterials used for GF delivery. Next, efforts to administer GFs for systemic augmentation of bone tissue are summarised. Finally, an alternative means of GF delivery, namely the delivery of genes coding for osteogenic proteins, rather than the delivery of the proteins, is summarised from rodent models. To conclude, future avenues of research considered promising to enhance the clinical application of GFs are discussed.
| Growth hormone stimulates the degradation of calcium phosphate biomaterial by human monocytes macrophages in vitro
Guicheux, J., S. Kimakhe, et al. (1998), J Biomed Mater Res 40(1): 79-85.
Abstract: This study investigated the effects of human growth hormone (hGH) on the monocyte/macrophage lineage, the first cell population involved in degradation of calcium phosphate ceramic after in vivo implantation. Monocytes isolated from human blood were cultured on biphasic calcium pellets (200 mg) for 8 days in the presence of lipopolysaccharides (LPS, 0.5 microgram/mL), hGH (10 and 50 ng/mL), or an association of LPS with hGH (10 and 50 ng/mL). Unlike LPS, hGH significantly decreased (about 25%) the total number of lacunae formed by monocytes. However, hGH induced the formation of lacunae with a greater surface area (about a 90% increase) as compared to the control. Finally, intense upmodulation (about a 250% increase) of lacuna surface area was observed in the presence of both soluble factors, suggesting that hGH and LPS act synergistically. In view of the development of a drug delivery system for hGH bone release, this study shows that hGH not only stimulates bone cells implicated in the synthesis of the extracellular matrix but also those involved in the early degradation of calcium phosphate biomaterial.
| Growth in the biomaterials market: the nature of growth factors
Williams, D. (1998), Med Device Technol 9(7): 6-11.
Abstract: An increasing number of medical devices are demonstrating better performance if they are used in association with growth factors. This is especially so in the area of tissue regeneration. This article describes the nature of these molecules and discusses some of the issues associated with their incorporation into medical devices.
| Growth of embryonic renal parenchyme at the interphase of a polyester artificial interstitium
Minuth, W. W., L. Denk, et al. (2005), Biomaterials 26(33): 6588-98.
Abstract: The construction of an artificial kidney module by tissue engineering or the application of cell-based therapies for the treatment of renal failure requires exact information regarding the cellbiological mechanisms of parenchyme development in combination with different kinds of biomaterials. To learn more about these processes tissue cultures are frequently used experimental tools. However, apart from experiments with early kidney anlagen there is a lack of suitable in-vitro models regarding the generation and long-term maintenance of renal tubules. In the present paper we like to demonstrate an advanced culture technique, which allows to generate tubular elements derived from renal stem cells. For the growth of tubules it is essential to fine-tune the interface between the embryonic tissue and the dead fluid space within a perfusion culture container by offering a polyester artificial interstitium. Culture was performed in IMDM supplemented with hormones and growth factors but using serum-free conditions over 14 days. Formation of tissue was then analysed by immunohistochemistry and two-dimensional (2D) electrophoresis. Culture in pure IMDM leads to a complete loss of tissue formation. In contrast, application of aldosterone (A) induces the development of numerous polarised tubules. Surprisingly, addition of epidermal growth factor (EGF), a cocktail of insulin, transferrin and selenium (ITS), retinoic acid (RA), cholecalciferol (VitD3) or bovine pituitary extract (BPT) does not further improve development of tubules, but leads to intensive cell clustering and a decrease of tubule formation. 2D Western blots of developing tissue probed with soybean agglutinin (SBA) reveal a unique pattern of newly detected proteins. It is found that growth factors do not support but abolish protein spots upregulated by aldosterone. It remains to be investigated, which cellbiological effect stimulates the embryonic cells to develop tubules in competition to cell clusters at the interphase of an artificial interstitium.
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