Integra dermal regeneration template was kindly provided by Integra LifeSciences Corporation. Sources of funds supporting the work: This work was partially financed by the CIRM-BMBF Early Translational II Award and the FONDAP Center for Genome regulation (Nr. 15090007) both to JTE.

1. Preparation of Scaffolds
<ol>
	<li>Generate samples of the scaffolds by using 12 mm biopsy punches.</li>
	<li>To introduce bioactive molecules or cells into the scaffold, drain the scaffolds by gently squeezing them with a sterile gauze. Then rehydrate the scaffolds by adding 160 &#181;l of a solution containing the bioactive molecules or cells of interest. Double-check the success of bioactivation with cells through metabolic assays such as MTT Assays.</li>
	<li>If necessary, fix the compounds or cells to be tested...

<ol>
	<li>Rahaman, M. N., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+composite+tissue+constructs+for+regenerative+medicine.">Stem cell-based composite tissue constructs for regenerative medicine.</a> Biotechnology and Bioengineering. 91 (3), 261-284 (2005).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature Biotechnology. 23, 47-55 (2005).</li><li>Machens, H. G., Berger, A. C., Mailaender, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioartificial+skin.">Bioartificial skin.</a> Cells Tissues Organs. 167, 88-94 (2000).</li><li>Priya, S. G., Jungvid, H., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering+for+tissue+repair+and+regeneration.">Skin tissue engineering for tissue repair and regeneration.</a> Tissue Engineering Part B: Reviews. 14, 105-118 (2008).</li><li>Papavasiliou, G., Cheng, M. H., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+vascularization+of+polymer+scaffolds.">Strategies for vascularization of polymer scaffolds.</a> Journal of Investigative Medicine. 58 (7), 838-844 (2010).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+tissue+engineering:+breathing+life+into+constructed+tissue+substitutes.">Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.</a> Tissue Engineering. 12, 2093-2104 (2006).</li><li>Zhong, S. P., Zhang, Y. Z., Lim, C. 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D., Farrugia, B. L., Dargaville, T. R., Dhara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-collagen+scaffolds+with+nano/microfibrous+architecture+for+skin+tissue+engineering.">Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.</a> Journal of Biomedical Materials Research Part A. 18, (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+knitted+mesh-reinforced+collagen-chitosan+hybrid+scaffold+in+the+one-step+repair+of+full-thickness+skin+defects+in+rats.">The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats.</a> Acta Biomaterials. 9 (8), 7822-7832 (2013).</li><li>Rizzi, S. C., Upton, Z., Bott, K., Dargaville, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+dermal+wound+healing:+biomedical+device+approaches.">Recent advances in dermal wound healing: biomedical device approaches.</a> Expert Review of Medical Devices. 1, 143-154 (2010).</li><li>des Rieux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+systems+delivering+VEGF+to+promote+angiogenesis+for+tissue+engineering.">3D systems delivering VEGF to promote angiogenesis for tissue engineering.</a> Journal of Controlled Release. 150, 272-278 (2011).</li><li>Reckhenrich, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivation+of+dermal+scaffolds+with+a+non-viral+copolymer-protected+gene+vector.">Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.</a> Biomaterials. 32, 1996-2003 (2011).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Key+Regulatory+Roles+of+the+PI3K/Akt+Signaling+Pathway+in+the+Functionalities+of+Mesenchymal+Stem+Cells+and+Applications+in+Tissue+Regeneration.">The Key Regulatory Roles of the PI3K/Akt Signaling Pathway in the Functionalities of Mesenchymal Stem Cells and Applications in Tissue Regeneration.</a> Tissue Engineering Part B Rev. 19, 516-528 (2013).</li><li>Fedorovich, N. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+and+analysis+in+laboratory+skin+transplant+micro-angiograms.">Vessel segmentation and analysis in laboratory skin transplant micro-angiograms.</a> Proceedings of the Eighteenth IEEE Symposium on Computer-Based Medical Systems. , 21-26 (2005).</li><li>Danner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+human+sweat+gland-derived+stem+cells+for+enhancing+vascularization+during+dermal+regeneration.">The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.</a> Journal of Investigative Dermatology. 132 (6), 1707-1716 (2012).</li><li>Shaterian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+Time+Analysis+of+the+Kinetics+of+Angiogenesis+and+Vascular+Permeability+in+an+Animal+Model+of+Wound+Healing.">Real Time Analysis of the Kinetics of Angiogenesis and Vascular Permeability in an Animal Model of Wound Healing.</a> Burns. 35 (6), 811-817 (2009).</li><li>McDonald, D. 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Conflict of interest statement:
All authors: None
Financial Disclosures:
None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

There is a need to establish successful approaches in improving blood perfusion in tissue engineered constructs, which demands the development of new reliable methods to study the vascularization processes within the biomaterials. Common methods for making scaffold vascularization ex vivo visible include the use of microscopy, which provides a high-resolution tool. In most cases, though, this method is limited to the analysis of small tissue areas and tends to be expensive and time consuming. Moreover, it often ...

In the recent decades, tissue engineering has opened up a new therapeutic option to replace tissue defects with the body&#8217;s own cells1. In order to support the physiological process of tissue regeneration, scaffolds are designed as a biodegradable structure, that provides a scenario where cells from the wound bed can grow and restore the defect2,3.
Insufficient vascularization is considered to be the main obstacle, which holds back the clinical breakthrough of bioartificial scaffolds4. With the ingrowth of cells, the demand for nutrients and oxygen increases and vascularization of the material becomes essential. Insufficient or delayed vascularization can therefore lead to central necrosis of tissue-engineered products5. In addition, blood vessels provide immune competent cells and remove the metabolic residues in the regenerating area. High infection rates and low regeneration are only some of the consequences of insufficient blood perfusion observed in tissue engineering, which are aimed to be averted by increasing the vascularization of the scaffolds6,7.
Several strategies that aim at improving vascularization focus on the key role of the biomaterial itself and the microstructure of the scaffold. There are intensive research efforts to develop new approaches in shifting the healing process from repair to regeneration, thereby (re)generating a tissue with the closest physiological properties to the one to be restored8,9. Biomaterials that were studied and evaluated with regards to their regenerative potential included collagen, fibrin, chitosan and alginate10,11. These biomaterials can be used and combined as a backbone for building new scaffolds using different strategies such as tissue decellularization, self-assembly, rapid prototyping and electrospinning12. In order to enhance the body&#8217;s own regenerative capacity, scaffolds can be bioactivated. The incorporation of recombinant angiogenic growth factors13 or gene vectors encoding for such factors14 has shown to improve vascularization of the scaffold. The use of stem cells has been widely shown to be a promising strategy to improve vascularization, where mesenchymal stromal cells and endothelial progenitor cells have gained the most attention15,16. Other approaches attempt to build constructs that contain prefabricated vessel networks prior to transplantation17. Despite intensive efforts in scaffold design and their bio-activation, no strategy has improved vascularization at a clinically significant level and, with the exception of dermal replacements in massive burn injuries, the translation of bioengineered materials into the clinical routine is only taking place hesitantly18.
One of the reasons why vascularization of artificial tissue constructs is still an unsolved problem, is the difficulty to evaluate the success of new technologies in in vivo approaches. Although in vitro experiments may provide important insights of the vascularization potential of scaffolds, appropriate animal models are required to study key parameters such as the biocompatibility of the material, the safety and efficacy of the treatment and, of particular importance, the vascularization of the tissue construct. Therefore, reliable tools to visualize and quantify blood vessel networks in vivo are essential.
In this study we present a simple and reliable method that allows the visualization and quantification of the vascular network inside explanted scaffolds. This method is based on tissue transillumination and digital segmentation. Since this method is non-invasive, it allows further molecular and histological analyses of the target material.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:431px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a full skin defect model to evaluate vascularization of biomaterials in vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A reliable bilateral full skin defect can be created in the mouse (Figure&#160;1) where the skin can be replaced by a biomaterial under study (Figure&#160;2). Here, no major complications are observed during or after the operative procedure, neither macroscopic signs of infection or foreign body reaction. In rare cases, a scaffold gets lost when a mouse removes it. Wound contraction was never observed (Figure&#160;3). Tissue transillumination allowed clear visualization ...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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12.1K vues.  Technische Universität München.  La vascularisation est la clé pour les approches en ingénierie tissulaire succès. Par conséquent, des technologies fiables sont nécessaires pour évaluer le développement des réseaux vasculaires dans les tissus-constructions. Nous présentons ici une méthode simple et efficace de visualiser et de quantifier la vascularisation in vivo. 