Tom Flanagan

Publications (17) View all

• Article: Non-Destructive Analysis of Extracellular Matrix Development in Cardiovascular Tissue-Engineered Constructs.

  M Tuemen, D V A Nguyen, J Raffius, T C Flanagan, M Dietrich, J Frese, T Schmitz-Rode, S Jockenhoevel
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  ABSTRACT: In the field of tissue engineering, there is an increasing demand for non-destructive methods to quantify the synthesis of extracellular matrix (ECM) components such as collagens, elastin or sulphated glycosaminoglycans (sGAGs) in vitro as a quality control before clinical use. In this study, procollagen I carboxyterminal peptide (PICP), procollagen III aminoterminal peptide (PIIINP), tropoelastin and sGAGs are investigated for their potential use as non-destructive markers in culture medium of statically cultivated cell-seeded fibrin gels. Measurement of PICP as marker for type I collagen synthesis, and PIIINP as marker of type III collagen turnover, correlated well with the hydroxyproline content of the fibrin gels, with a Pearson correlation coefficient of 0.98 and 0.97, respectively. The measurement of tropoelastin as marker of elastin synthesis correlated with the amount of elastin retained in fibrin gels with a Pearson correlation coefficient of 0.99. sGAGs were retained in fibrin gels, but were not detectable in culture medium at any time of measurement. In conclusion, this study demonstrates the potential of PICP and tropoelastin as non-destructive culture medium markers for collagen and elastin synthesis. To our knowledge, this is the first study in cardiovascular tissue engineering investigating the whole of here proposed biomarkers of ECM synthesis to monitor the maturation process of developing tissue non-invasively, but for comprehensive assessment of ECM development, these biomarkers need to be investigated in further studies, employing dynamic cultivation conditions and more complex tissue constructs.
  Annals of biomedical engineering 01/2013; · 2.41 Impact Factor
• Article: Tissue Engineering: Selecting the Optimal Fixative for Immunohistochemistry.

  Sabine Koch, Nadine Stappenbeck, Christian G Cornelissen, Thomas Cormac Flanagan, Petra Mela, Jörg Sachweh, Benita Hermanns-Sachweh, Stefan Jockenhoevel
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  ABSTRACT: Background: In the immunohistochemical analysis of tissue-engineered structures, aggressive treatments for fixation and antigen retrieval can impair the quality of specimen staining and visualization. Hypothesis: We hypothesized that the adequate choice of fixative and antigen-retrieval method might improve the quality of immunohistochemical staining. Methods: Tissue-engineered vascular grafts were fixed using formalin, Carnoy's, or HOPE(®) fixative. Antigen retrieval was performed where necessary and samples from each group were stained using hematoxylin and eosin to assess overall tissue preservation. For a set of proteins relevant to cardiovascular tissue development, immunohistochemical staining was applied to formalin-, Carnoy's-, and HOPE-fixed specimens to allow a comparative analysis. Results: In tissue-engineered constructs, antigen retrieval methods necessary after formalin fixation led to significant destruction of the overall tissue structure. Carnoy's fixation resulted in good overall tissue preservation and adequate results for immunohistochemical staining of alpha-smooth muscle actin (α-SMA), vimentin, type I collagen, elastin, and laminin. HOPE fixative led to a loosened tissue structure and a swollen appearance but showed adequate results for staining against type III collagen and elastin. Formalin fixation without antigen retrieval led to inadequate visualization of α-SMA, vimentin, type I- and type III collagen, and laminin. Conclusion: Based on the present study, we recommend that Carnoy's fixative is employed for the preservation of tissue-engineered constructs to allow immunohistochemical analysis of type I- and type III collagen, elastin, laminin, α-SMA, and vimentin. However, it is clear that the technique requires optimization based on the particular tissue engineering application.
  Tissue Engineering Part C Methods 06/2012; · 4.64 Impact Factor
• Article: The BioStent: Novel Concept for a Viable Stent Structure.

  Stefan Weinandy, Lisanne Rongen, Fabian Schreiber, Christian Cornelissen, Thomas Cormac Flanagan, Andreas Mahnken, Thomas Gries, Thomas Schmitz-Rode, Stefan Jockenhoevel
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  ABSTRACT: Objectives: Percutaneous stenting of occluded peripheral vessels is a well-established technique in clinical practice. Unfortunately, the patency rates of small-caliber vessels after stenting remain unsatisfactory. The aim of the BioStent concept is to overcome in-stent restenosis by excluding the diseased vessel segment entirely from the blood stream, in addition to providing an intact endothelial cell layer. Design: The concept combines the principles of vascular tissue engineering with a self-expanding stent: casting of the stent within a cellularized fibrin gel structure, followed by bioreactor conditioning, allows complete integration of the stent within engineered tissue. Materials and Methods: Small-caliber BioStents (Ø=6 mm; n=4) were produced by casting a nitinol stent within a thin fibrin/vascular smooth muscle cell (vSMC) mixture, followed by luminal endothelial cell seeding, and conditioning of the BioStent within a bioreactor system. The potential remodeling of the fibrin component into tissue was analyzed using routine histological methods. Scanning electron microscopy was used to assess the luminal endothelial cell coverage following the conditioning phase and crimping of the stent. Results: The BioStent was shown to be noncytotoxic, with no significant effect on cell proliferation. Gross and microscopic analysis revealed complete integration of the nitinol component within a viable tissue structure. Hematoxylin and eosin staining revealed a homogenous distribution of vSMCs throughout the thickness of the BioStent, while a smooth, confluent luminal endothelial cell lining was evident and not significantly affected by the crimping/release process. Conclusions: The BioStent concept is a platform technology offering a novel opportunity to generate a viable, self-expanding stent structure with a functional endothelial cell lining. This platform technology can be transferred to different applications depending on the luminal cell lining required.
  Tissue Engineering Part A 04/2012; 18(17-18):1818-26. · 4.64 Impact Factor
• Article: Influence of platelet-derived growth factor-AB on tissue development in autologous platelet-rich plasma gels.

  Simone Wirz, Maren Dietrich, Thomas C Flanagan, Gudrun Bokermann, Wolfgang Wagner, Thomas Schmitz-Rode, Stefan Jockenhoevel
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  ABSTRACT: Fibrin-based scaffolds are widely used in tissue engineering. We postulated that the use of platelet-rich plasma (PRP) in contrast to platelet-poor plasma and pure fibrinogen as the basic material leads to an increased release of autologous platelet-derived growth factor (PDGF)-AB, which may have a consequent positive effect on tissue development. Therefore, we evaluated the release of PDGF-AB during the production process and the course of PDGF release during cultivation of plasma gels with and w/o platelets. The influence of PDGF-AB on the proliferation rate of human umbilical cord artery smooth muscle cells (HUASMCs) was studied using XTT assay. The synthesis of extracellular matrix by HUASMCs in plasma- and fibrin gels was measured using hydroxyproline assay. The use of PRP led to an increase in autologous PDGF-AB release. Further, the platelet-containing plasma gels showed a prolonged release of growth factor during cultivation. Both PRP and platelet-poor plasma gels had a positive effect on the production of collagen. However, PDGF-AB as a supplement in medium and in pure fibrin gel had neither an effect on cell proliferation nor on the collagen synthesis rate. This observation may be due to an absence of PDGF receptors in HUASMCs as determined by flow cytometry. In conclusion, although the prolonged autologous production of PDGF-AB in PRP gels is possible, the enhanced tissue development by HUASMCs within such gels is not PDGF related.
  Tissue Engineering Part A 03/2011; 17(13-14):1891-9. · 4.64 Impact Factor
• Article: Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation.

  Sabine Koch, Thomas C Flanagan, Joerg S Sachweh, Fadwa Tanios, Heike Schnoering, Thorsten Deichmann, Ville Ellä, Minna Kellomäki, Nina Gronloh, Thomas Gries, René Tolba, Thomas Schmitz-Rode, Stefan Jockenhoevel
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  ABSTRACT: There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre ( approximately 5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts (n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.
  Biomaterials 03/2010; 31(17):4731-9. · 7.40 Impact Factor