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ABSTRACT: The aim of the present in vitro study has been to investigate the effects of a enriched platelet derived growth factors on proliferation and migration of human endothelial and mesenchymal stem cells and on osteogenic differentiation of stem cells. Platelet rich plasma has been produced, yielding a four time higher number of thrombocytes than normal plasma. Degranulation of platelets has been performed by means of calcium and thrombin. Plasma has served as a control, whereas plasma in combination with calcium and thrombin was used to distinguish the difference between calcium and/or thrombin mediated effects and growth factor induced effects on the cells. The observed enhanced proliferation and migration of endothelial cells towards the platelet derived growth factors was driven by the plasma component of these preparations. However PDGF solely stimulated the migration and proliferation of mesenchymal stem cells. The increased osteogenic differentiation of growth factor treated mesenchymal stem cells was mostly driven by the high level of calcium used for the platelets degranulation. In summery, the different components of platelet derived growth factors work together to influence human endothelial and mesenchymal stem cells. This is of special clinically interest regarding the stimulation of bone healing in orthopaedic and traumatic surgery.
European journal of medical research 08/2004; 9(7):337-44. · 1.13 Impact Factor
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ABSTRACT: Proliferation and differentiation of wild-type, BMP-2 and BMP-4 transfected cells of C3H10T1/2, a mouse mesenchymal stem cell line that can differentiate into chondrocytes, were studied under monolayer (2D-) and encapsulation (3D-) conditions. Cells were encapsulated in a novel class of alginate. The alginate was of clinical grade (CG) because of complete removal of mitogenic and cytotoxic contaminants by chemical means. Compared to commercial alginates used so far for encapsulation it was characterized by ultra-high viscosity (UHV; viscosity of a 0.1% w/v solution of about 20 cP). In contrast to monolayer cultures, proliferation of cells was prevented when the cells were encapsulated in UHV/CG alginate at the same suspension density. As revealed by immunohistochemistry and quantitative RT-PCR, transfected and wild-type monolayer cells showed synthesis of type I collagen after transfer into differentiation medium, while culture in an alginate scaffold resulted in an upregulation of type II collagen and other hyaline cartilage proteins. BMP-4 transfected cells produced considerably more type II collagen than BMP-2 transfected and wild-type cells. BMP-4 transfected cells were also characterized by type I collagen production up to Day 10 and exhibited transient alkaline phosphatase activity levels that were much higher than the peak values observed for the other two cell lines. The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondroprogenitor-like cell.
Biomaterials 06/2002; 23(9):2003-13. · 7.40 Impact Factor
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U Zimmermann,
F Thürmer, A Jork,
M Weber,
S Mimietz,
M Hillgärtner,
F Brunnenmeier,
H Zimmermann,
I Westphal,
G Fuhr,
U Nöth,
A Haase,
A Steinert,
C Hendrich
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ABSTRACT: In the light of results of clinical trials with immunoisolated human parathyroid tissue Ba2+-alginate capsules were developed that meet the requirements for long-term immunoisolated transplantation of (allogeneic and xenogeneic) cells and tissue fragments. Biocompatibility of the capsules was achieved by subjecting high-M alginate extracted from freshly collected brown algae to a simple purification protocol that removes quantitatively mitogenic and cytotoxic impurities without degradation of the alginate polymers. The final ultra-high-viscosity, clinical-grade (UHV/CG) product did not evoke any (significant) foreign body reaction in BB rats or in baboons. Similarly, the very sensitive pERK assay did not reveal any mitogenic impurities. Encapsulated cells also exhibited excellent secretory properties under in vitro conditions. Despite biocompatible material, pericapsular fibrosis is also induced by imperfect capsule surfaces that can favor cell attachment and migration under the release of material traces. This material can interact with free end monomers of the alginate polymers under formation of mitogenic advanced glycation products. Smooth surfaces, and thus topographical biocompatibility of the capsules (visualized by atomic force microscopy), can be generated by appropriate crosslinking of the UHV/CG-alginate with Ba2+ and simultaneous suppression of capsule swelling by incorporation of proteins and/or perfluorocarbons (i.e., medically approved compounds with high oxygen capacity). Perfluorocarbon-loaded alginate capsules allow long-term non-invasive monitoring of the location and the oxygen supply of the transplants by using 19F-MRI. Transplantation studies in rats demonstrated that these capsules were functional over a period of more than two years.
Annals of the New York Academy of Sciences 12/2001; 944:199-215. · 3.15 Impact Factor
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ABSTRACT: 19F nuclear magnetic resonance imaging (MRI) can be used as a non-invasive tool to simultaneously determine the location, the integrity and the oxygen supply of Ba2+-alginate implants. This requires that the beads (implants) are pre-loaded with the perfluorocarbon compound F-44E. Implantation of solid 19F-labelled beads into the peritoneum, below the kidney capsule or into the muscle of Wistar WU rats demonstrated that these beads could be detected by 19F-MRI for up to 18 months after implantation. This indicated that F-44E is not considerably released from the beads during implantation. The signal to noise ratio of liquid-core beads was higher by a factor of 4 than the signal to noise ratio of solid beads, but liquid-core beads were more fragile and also too large for implantation under the kidney capsule and into the intramuscular tissue. Quantitative 2-dimensional 19F-T1 maps (resolution 0.5 x 0.5 mm) could be deduced from 19F-MRI measurements. These T1-maps correlated to the local pO2-values. The partial oxygen pressure estimated in F-44E-loaded Ba2+-alginate beads showed that the oxygen supply inside the beads was very poor when they were implanted below the kidney capsule or into the peritoneal cavity. These low pO2-values obtained for the renal subcapsular site and the peritoneum may explain the failure of previous immunoisolated islet transplantation studies using these locations.
Artificial Cells Blood Substitutes and Biotechnology 04/2000; 28(2):129-46. · 0.98 Impact Factor
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A Jork,
F Thürmer,
H Cramer,
G Zimmermann,
P Gessner,
K Hämel,
G Hofmann,
B Kuttler,
H J Hahn,
O Josimovic-Alasevic,
K G Fritsch,
U Zimmermann
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ABSTRACT: A simple procedure is described for the extraction and purification of alginate from the inner stipes of the kelp Laminaria pallida. Alginate yield was about 10-15% of the dry mass, with a 70:30 mannuronic/guluronic acid ratio. Analysis of the purified alginate revealed a low polyphenol content while proteins were below detection level. The purified alginate was highly viscous, with 10-15 mPa s and 281 mPa s for a 0.1% and 0.5% solution, respectively, indicating a very high molecular mass (larger than 250 kDa). Bead formation occurred in the presence of divalent cations, but also in the presence of artificial serum (FCSIII) without added divalent cations. The biocompatibility of the alginate was tested with the in vitro mice lymphocyte test as well as by implantation of Ba2+ cross-linked beads beneath the kidney capsule of BB/OK rats. There was no evidence for significant mitogenic activity or fibrotic reaction. Biocompatibility of the alginate was also demonstrated by the encapsulation of human chondrocytes into Ca2+ cross-linked alginate beads. Immobilized chondrocytes grew and remained functional (i.e. they produced collagen).
Applied Microbiology and Biotechnology 03/2000; 53(2):224-9. · 3.42 Impact Factor
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M. Hillgärtner,
H. Zimmermann,
S. Mimietz, A. Jork,
F. Thürmer,
H. Schneider,
U. Nöth,
C. Hasse,
A. Haase,
G. Fuhr,
M. Rothmund,
U. Zimmermann
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ABSTRACT: Transplantation studies with immunoisolated foreign cells / tissues (encapsulated in Ba2+-cross-linked alginate) show that several obligatory requirements have to be met before this immunoisolation technique can be used for routine clinical trials. We present chemical procedures and technical developments which could address and solve the current problems and limitations of the microencapsulation technique. Large-scale production of highly purified (biocompatible) alginate is possible and this material does not evoke any foreign body reactions under implantation conditions.Atomic force microscopy (AFM) studies suggest that geometric inhomogeneities may lead to immunological reactivity of the cross-linked alginate. Material traces released from fibroblasts, macrophages and / or lymphocytes migrating over surfaces can also be studied by AFM and may initiate the primary foreign body reactions.Long-term stability of the alginate beads can be improved by incorporating proteins or (medically approved) perfluorocarbons during the cross-linking process and by subsequent treatment with 6 mM sulphate. Long-term stability and local oxygen supply can be monitored in vivo using non-invasive 19F-nuclear magnetic resonance imaging (MRI) of perfluorocarbon-loaded alginate beads.These improvements and developments allow clinical trials with allogenic (and xenogenic) tissue by alginate immunoisolation.Immunisolierung von Transplantaten durch Einschluß in 19F-markierte Alginatgele: Herstellung, Biokompatibilität, Stabilität und Langzeit-Überprüfung der FunktionalitätTransplantationsstudien mit fremden, immunisolierten Zellen bzw. Geweben (die in eine mit Ba2+-Ionen vernetzte Alginatkapsel eingeschlossen waren) haben gezeigt, daß zahlreiche zwingende Anforderungen erfüllt sein müssen, bevor eine routinemäßige Anwendung dieser Immunisolierungstechnik in der Klinik durchgeführt werden kann. Chemische Verfahren und technische Entwicklungen werden vorgestellt, die die bestehenden Probleme und Beschränkungen der Mikroverkapselungstechnik aufzeigen bzw. lösen. Eine halb-technische Produktion von hochgereinigtem (biokompatiblem) Alginat ist möglich. Der Nachweis wird erbracht, daß dieses Material unter Implantationsbedingungen keine Fremdkörperreaktion hervorruft. Untersuchungen mit dem Rasterkraftmikroskop (AFM) weisen darauf hin, daß eine mögliche immunologische Reaktivität der Oberfläche der Alginatkapseln durch geometrische Inhomogenitäten hervorgerufen werden könnte. Die zellulären Prozesse, die die primären Fremdkörperreaktionen hervorrufen könnten (d. h. die Absonderung von Materialspuren durch Fibroblasten, Makrophagen und / oder Lymphozyten während ihrer Wanderung auf Oberflächen), können ebenso mit Hilfe der Rasterkraftmikroskopie aufgeklärt werden. Die Langzeitstabilität der Alginatkapseln kann durch den Einschluß von Proteinen oder von (medizinisch zugelassenen) Perfluorcarbonverbindungen während des Vernetzungsprozesses und die anschließende Behandlung der Kapseln mit 6 mM Sulfat verbessert werden. Der Implantationsort und die Langzeitstabilität von mit Perfluorcarbon beladenen Alginatkapseln sowie ihre Versorgung mit Sauerstoff können unter Verwendung der nicht-invasiven 19F-Kernspinresonanzbildgebungstechnik (NMR) überprüft werden.Die genannten Verbesserungen bzw. Entwicklungen erlauben klinische Untersuchungen mit allogenem (bzw. xenogenem) Gewebematerial, nachdem es in Alginat immunisoliert wurde.
Materialwissenschaft und Werkstofftechnik 02/2000; 30(12):783 - 792. · 0.54 Impact Factor
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ABSTRACT: Semipermeable hydrogels formed with a biocompatible alginate solution and Ba(2+) ions protect encapsulated cells and tissues from a foreign immune system. For the viability and metabolic activity of the encapsulated materials, a sufficient oxygen supply inside the capsules is necessary. Quantitative (19)F-MRI was performed on perfluorocarbon-loaded alginate capsules implanted into the peritoneal cavity, the musculus quadriceps femoris, and beneath the kidney capsule of rats, in order to determine in vivo the partial oxygen pressure (pO(2)) inside the capsules at these implantation sites. The temporal behavior of the pO(2) values was observed for at least 3 months. The most stable values over time were observed in the kidney, where inter-rat pO(2) differences were considerable. In the muscle, the values were very high directly after implantation and decreased to nearly zero after 2 weeks. In the peritoneal cavity, values changed randomly over a wide range between different rats and over time. Magn Reson Med 42:1039-1047, 1999.
Magnetic Resonance in Medicine 01/2000; 42(6):1039-47. · 2.96 Impact Factor
