Baaijens, F.P.T.

Bouten, C.V.C.

Post, M.J.

Pullens, R.A.A.

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Vascularization of tissue engineered constructs

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 Vascularization of tissue engineered constructsCitation for published version (APA):Pullens, R. A. A., Bouten, C. V. C., Post, M. J., & Baaijens, F. P. T. (2005). Vascularization of tissue engineeredconstructs. Poster session presented at Mate Poster Award 2005 : 10th Annual Poster Contest.Document status and date:Published: 01/01/2005Document Version:Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers)Please check the document version of this publication:• A submitted manuscript is the version of the article upon submission and before peer-review. There can beimportant differences between the submitted version and the official published version of record. Peopleinterested in the research are advised to contact the author for the final version of the publication, or visit theDOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and pagenumbers.Link to publicationGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal.If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, pleasefollow below link for the End User Agreement:www.tue.nl/taverneTake down policyIf you believe that this document breaches copyright please contact us at:openaccess@tue.nlproviding details and we will investigate your claim.Download date: 04. Oct. 202312/mhjVascularization of tissue engineered constructsRolf A.A. Pullens, Carlijn V.C. Bouten, Mark J. Post, Frank P.T. BaaijensBiomechanics and Tissue Engineering, Soft Tissue Biomechanics & EngineeringIntroductionTissue engineering has advanced a lot over the last years.One of the challenges we’re facing now is creating thickerconstructs. The thickness of a tissue engineered constructis now limited to the diffusion of nutrients to the cells in thecenter. In order to create thicker constructs in which cell via-bility throughout the construct is maintained vascularizationin vitro is necessary [1].It was observed that endothelial cells (EC) seeded on top ofmyofibroblast (MF) seeded PGA/P4HB scaffolds make tubelike structures. Experiments were performed to understandand control the formation of these structures.Material and methodsTwelve rectangular PGA/P4HBscaffolds (20x7x1 mm) wereglued into a wells culture using20% polyurethane solved inTHF (figure 1). The scaffoldswere seeded with human venasaphena MFs using fibrin as a cellcarrier. After 3 days of culturehuman vena saphena ECs wereseeded on top of the construct.Figure 1 PGA/P4HB scaffoldglued in wells plateThe constructs were cultured for 22 days in EGM-2 mediumsupplemented with 20% FBS and growth factors. At day 5, 8,11, 15 and 21 two constructs were cut loose and stained withCell Tracker Orange, staining the cytoplasma of viable cellsand a UEA-1 lectin staining which is a specific for ECs. Differ-ent z-stacks of the EC side of the constructs were made, usinga confocal laser scanning microscope (CLSM). These z-stackswere analyzed for the presence of an EC monolayer and ECtube like structures.Figure 2 Culturing protocolResultsAfter seeding, the ECmonolayers started to grow, resulting in80-100% confluent layers at day 21. At day 11 the first signsof the tube like structures appeared. On day 15 and 21 tubelike structures were seen in most of the samples. They areup to 500 µm long and can be found up to 80 µm below thesurface of the construct. Figure 3 shows a typical example oftube like structures lying under layers of ECs and MFs.The EC tube like structures were detected at different posi-tions: (1) under an EC and a MF layer, (2) under a MF layeronly and (3) on top of a MF layer. Some tube like structuresseem to be connected to the overlying EC monolayer (figure4), indicating that they originate from that layer.Figure 3 A: CLSM image at 0µm with pieces of the EC monolayer (green). B:CLSM image at a depth of -23µmwith the MF layer (red). C: CLSM image at adepth of -80µm with EC tube like structures (green) and fibers of PGA (red).D: Projection of all slices in z direction.Figure 4 Side projection of part of the stack, showing a EC tube like structure(green) that it is connected with the top monolayer of ECs (green).DiscussionThe EC tube like structures were found in most of the con-structs after 15 days. It is hypothesized that the MFs in thecenter of the constructs are experiencing a low oxygen con-centration and are producing substances which trigger theECs to form the tube like structures.Future researchHistological slides will be made of the constructs in whichthe ECs will be stained with a different endothelial specificimmunological staining, in order to visualize the tube likestructures in another way. In order to verify if the MFs in thecenter of the constructs are hypoxic, a hypoxy probe will beused in coming experiments.References:[1] LEVENBERG S, ET AL.: Nat Biotechnology, 2005, 23: 879-884

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