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Hildner F.,Ludwig Boltzmann Research Institute | Hildner F.,Red Cross | Peterbauer A.,Red Cross | Wolbank S.,Ludwig Boltzmann Research Institute | And 7 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2010

In this study, we investigated the influence of transforming growth factor beta 3 (TGF-β3), bone morphogenetic protein 6 (BMP-6) and basic fibroblast growth factor (FGF-2) on chondrogenesis in adipose derived stem cells (ASC). Cells were isolated from liposuction material, expanded and subjected to chondrogenic differentiation. Micromass pellets were cultured in chondrogenic medium containing 10 ng/mL TGF-β3 which was additionally supplemented with 10 ng/mL BMP-6, 10 ng/mL FGF-2 or a combination of both. We quantitatively evaluated the cartilage specific gene expression after 14 days of culture. The end point measurements on day 35 included glycosaminoglycan (GAG) quantification, histological staining for chondrogenic markers, and transmission electron microscopy (TEM). In comparison to cultures induced with TGF-β3/FGF-2, the presence of TGF-β3/BMP-6 demonstrated strong induction of collagen type II, collagen type IX and aggrecan mRNA expression. This was corroborated by quanti-fication and histological staining for GAGs and immunohistological staining for collagen II. However, when a combination of BMP-6 and FGF-2 in addition to TGF-β3 was added, FGF-2 counteracted BMP-6, as indicated by reduced marker gene expression and weak to absent staining for GAGs. In conclusion, this study demonstrates that BMP-6 combined with TGF-β3 is a potent inducer of chondrogenesis in human ASC. In contrast, FGF-2 does not contribute to differentiation, but rather suppresses the chondrogenic potential of BMP-6. © 2010 Wiley Periodicals, Inc.

Rohringer S.,Ludwig Boltzmann Institute for Experimental and Clinical Traumatology | Hofbauer P.,Ludwig Boltzmann Institute for Experimental and Clinical Traumatology | Schneider K.H.,Ludwig Boltzmann Institute for Experimental and Clinical Traumatology | Husa A.-M.,Ludwig Boltzmann Institute for Experimental and Clinical Traumatology | And 6 more authors.
Angiogenesis | Year: 2014

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering. © 2014 Springer Science+Business Media Dordrecht.

Wolbank S.,Ludwig Boltzmann Research Institute | Pichler V.,Ludwig Boltzmann Research Institute | Ferguson J.C.,Ludwig Boltzmann Research Institute | Meinl A.,Austrian Cluster for Tissue Regeneration | And 4 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2015

Fibrin-based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid-phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non-invasive molecular imaging systems enable real-time tracking of processes in the living animal. In this study, a non-invasive fluorescence based imaging system was applied to follow and quantify site-specific degradation of fibrin sealant. To enable non-invasive tracking of fibrin in vivo, fibrin-matrix was labelled by incorporation of a fluorophore-conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore-labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. © 2014 John Wiley & Sons, Ltd.

Agis H.,Medical University of Vienna | Hueber L.,Austrian Cluster for Tissue Regeneration | Hueber L.,Medical University of Vienna | Pour Sadeghian N.,Austrian Cluster for Tissue Regeneration | And 6 more authors.
Archives of Oral Biology | Year: 2014

Objective Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. Materials Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and 3[H]thymidine incorporation, respectively. Results We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1 h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192 h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. Conclusions In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration. © 2014 Published by Elsevier Ltd.

Agis H.,Medical University of Vienna | Schrockmair S.,Austrian Cluster for Tissue Regeneration | Schrockmair S.,Medical University of Vienna | Skorianz C.,Austrian Cluster for Tissue Regeneration | And 7 more authors.
Journal of Orthopaedic Research | Year: 2013

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet-released supernatant (PRS), serum containing PRS (SC-PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and 3[H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase-of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP+ multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC-PRS and serum decreased the number and activity of TRAP+ multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase-positive colonies while SC-PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis. Copyright © 2013 Orthopaedic Research Society.

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