Integra真皮再生模板，请通过Integra生命科学公司提供。资金配套工作来源：（上午十时正15090007），这项工作由CIRM，BMBF的早期平移二奖及FONDAP中心基因组调控既要JTE了部分资金。

1，准备的支架<ol><li>通过使用12毫米活检穿孔产生的支架的样品。 </li><li>引进生物活性分子或细胞进入支架，轻轻挤压它们，用无菌纱布引流支架。然后通过加入160微​​升含有生物活性分子或所关注的细胞的溶液中再水化的支架。仔细检查，通过代谢试验，如MTT法生物活化与细胞的成功。 </li><li>如果需要的话，固定的化合物或细胞可以在支架内测试了通过在纤维蛋白的​​凝血酶溶液或水凝胶递送它们，例如，再经过补液如在步骤1.2中描述。 注意：此步骤可以是有益的，当化合物或细胞不机械地附着到材料。一个也可以控制化合物的释放动力学.. </li><li>制备的钛化网格，这将在每一个实验组和对照组被放置在每个支架下，通过cuttin克出圆形片，约14毫米的直径。 注：钛化网格可以作为划定从伤口床再生的组织所需要的物理边界。 </li></ol> 2，动物<ol><li>在实施之前所提出的模型，请咨询相应的动物保护法，并从当地政府获得许可。在这项工作中，所有实验用小鼠进行的，根据目前德国的动物福利法，并经区政府上巴伐利亚（Regierung冯Oberbayern）。 </li><li>慎选动物和应变。尽管建立了小鼠模型中，它可以转化为其他常见的动物，如大鼠，兔子或猪。 </li><li>如果您正在使用的毛皮动物，剃动物的支架植入前回来。 注意：此工作使用的6至8周龄小鼠介于20g和25克，但是不同年龄和b体重ODY权重也可以使用。 </li></ol> 3，麻醉<ol><li>进行无菌条件下操作。为了维持动物的正常体温下，使用加温垫。 </li><li>之前的吸入麻醉的动物接受Buprenorphin在0.05-0.1 mg / kg体重，皮下每天8小时的剂量。 </li><li>将下吸入性麻醉（异氟烷）的动物或申请同等标准的程序。确认麻醉或者轻轻挤压动物的脚趾或通过皮肤捏。 </li><li>为了防止exsiccosis，在操作开始时注入0.5毫升生理盐水溶液皮下注射。 </li></ol> 4，切除的皮肤<ol><li>将动物俯卧位，并标明鼠标的背面采用了细尖永久性笔（ 图1A）的中线。 </li><li>定义切除区域。将缺陷中Figu所示的区域重新1C。请注意，如果缺陷被放置太尾端，动物更容易去除敷料和支架。 </li><li>创建全面采用10毫米活检穿孔（ 图1B）双边缺陷。冲头应仔细被迫靠着皮肤，并且不能用来完全切透皮肤，但划定切除区域（ 图1C及1E）。 </li><li>轻轻抬离皮肤明显用镊子沿采用精细手术剪（ 图1F）标记的圆圈切割。若出血，仔细压缩用无菌纱布。切除的一步一步的描述如图1所示。 注：该缺陷是用较小的穿孔比所述一个用于产生支架创建，以弥补该缺陷的扩大是由于皮肤的弹性。 </li></ol> 5，支架植入术<ol><li>为了使空间的蒂塔欲使网眼，延长皮肤和下面的组织的分离在2-4毫米（ 图2A）的伤口边界。 </li><li>将钛化网进缺损直接对创面的伤口边缘（ 图2A和B）的。 </li><li>脚手架的地方直接在网格。 </li><li>缝合的支架与相邻的伤口边缘与4至6个单结，留下的边缘略微超过所述支架（ 图2C）。 注意：避免使用可生物降解的缝线。 </li><li>缝合的透明伤口敷料上面的缺陷，以保护所述支架，同时允许监测伤口区域（ 图2D）的。 </li></ol> 6，术后护理<ol><li>让每笼一只老鼠，以避免敷料和支架相互操作。 </li><li>通过观察运动活动，体重，疼痛的迹象，宽容评估每天动物的一般状态在更衣室和自动切割。还监测伤口区域的出血，局部和全身感染的迹象和敷料的位置。 </li><li>为了最大限度地减少了动物的痛苦，每日注射Buprenorphin在0.05-0.1 mg / kg体重或等效镇痛药物的剂量。 </li><li>如果鼠标删除或损坏的伤口敷料，更换或重新连接在麻醉下。 </li></ol> 7安乐死的脚手架和外植<ol><li>在期望的时间点，由戊巴比妥（150毫克/千克）的过量牺牲动物。 </li><li>马克皮肤切除的部位用永久性记号笔，其中应包括支架，并尽可能多的鼠标背部皮肤越好（ 图3A）。 </li><li>沿着切开用剪刀或手术刀的标线在皮肤上。拆下整个皮肤，包括支架和钛化目，通过生硬的准备下面的组织（ 网络gure 3B）。 </li><li>将取出的组织伸出在培养皿上（ 图3C和D） </li></ol> 8，可视化的血管网和定量<ol><li>透： <ol><li>成立了透设备，通过安装培养皿上面的白色光（100瓦标准灯泡）强大的源泉。 </li><li>固定透射以上数码相机，以获得数码照片。 </li><li>将样品在培养皿上的透设备倒挂。 </li><li>采取在微距模式的完整支架的照片，以及正常皮肤的同样大小的区域。在进一步的数字分析TIFF格式存储的图像。 </li><li>除组织进行进一步的生化或组织学分析。 </li></ol></li><li>数字细分和量化： <ol><li>下载并安装VesSeg，工具软件，它可以得到FR负责EE在： <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> 。 </li><li>打开图片与VesSeg - 工具软件。 </li><li>选择覆盖所述支架用于数字分析（图片→选择）的图像的区域。 </li><li>为了提高船舶的对比使用选项“迟滞阈值”（图像→血管增强过滤器→迟滞阈值→顶帽变换→计算血管增强）。 </li><li>进行血管图（图像→血管增强过滤器→迟滞阈值→阈值的边界）的分割。选择所述第一阈值（“船只覆盖”），因此每一个像素，这甚至远程容器状，将被标记为容器。选择所述第二阈值（“背景覆盖”），因此，只有那些像素是特别容器状将被标记为容器。 </li><li>手动检查自动分割建议添加非捕获的船只和消除常见的假阳性的结构，它通常是长而薄，血管样结构，例如毛发或缝线。 </li><li>计算血管的长度和覆盖船（图像→图像统计→二进制图像的统计）的总面积。 注：对于血管长度的计算，血管生成的血管地图减薄到线，一个像素20的宽度。 </li></ol></li><li>根据相同的参数的支架分析天然皮肤区域，分配100％的值的原生组织和涉及该支架到该值。例如，如果白色像素的比例是60％，在正常组织和在该支架30％时，就表示该支架的50％的血管。注意：白色容器覆盖分配给周围的脚手架的正方形的面积。调整白色像素覆盖率consi数dering该圆的面积是显著较小（πX读2）比正方形（4×R 2）。 </li></ol>

<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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+scaffolds+for+skin+wound+healing+and+dermal+reconstruction.">Tissue scaffolds for skin wound healing and dermal reconstruction.</a> Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology. 2 (5), 510-525 (2010).</li><li>Liu, G., Zhang, Y., Liu, B., Sun, J., Li, W., Cui, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+regeneration+in+a+canine+cranial+model+using+allogeneic+adipose+derived+stem+cells+and+coral+scaffold.">Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold.</a> Biomaterials. 34 (11), 2655-2664 (2013).</li><li>Hansson, A., Di Francesco, T., Falson, F., Rousselle, P., Jordan, O., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+nanoparticles+for+directed+tissue+engineering.">Preparation and evaluation of nanoparticles for directed tissue engineering.</a> International Journal of Pharmaceutics. 439 (1-2), 73-80 (2012).</li><li>Sarkar, S. 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. E., 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+role+of+endothelial+progenitor+cells+in+prevascularized+bone+tissue+engineering:+development+of+heterogenous+constructs.">The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogenous constructs.</a> Tissue Engineering Part A. 16 (7), 2355-2367 (2010).</li><li>Wang, L., 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=Osteogenesis+and+angiogenesis+of+tissue-engineered+bone+constructed+by+prevascularized+&#946;-tricalcium+phosphate+scaffold+and+mesenchymal+stem+cells.">Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized &#946;-tricalcium phosphate scaffold and mesenchymal stem cells.</a> Biomaterials. 36, 9452-9461 (2010).</li><li>Cuadra, 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=Functional+results+of+burned+hands+treated+with+Integra.">Functional results of burned hands treated with Integra.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 65 (2), 228-234 (2012).</li><li>Wilcke, I., 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=VEGF(165)+and+bFGF+protein-based+therapy+in+a+slow+release+system+to+improve+angiogenesis+in+a+bioartificial+dermal+substitute+in+vitro+and+in+vivo.">VEGF(165) and bFGF protein-based therapy in a slow release system to improve angiogenesis in a bioartificial dermal substitute in vitro and in vivo.</a> Langenbecks Arch Surg. 392 (3), 305-314 (2007).</li><li>Condurache, A., Aach, T., Grzybowsky, S., Machens, H. 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. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nature Medicine. 9 (6), 713-725 (2003).</li><li>Bergeron, L., Tang, M., Morris, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+vascular+injection+techniques+for+the+study+of+perforator+flaps.">A review of vascular injection techniques for the study of perforator flaps.</a> Plastic and Reconstructive Surgery. 117, 2050-2057 (2006).</li><li>Schlatter, P., K&#246;nig, M. F., Karlsson, L. M., Burri, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+intussusceptive+capillary+growth+in+the+chorioallantoic+membrane+(CAM)+of+the+chicken+embryo.">Quantitative study of intussusceptive capillary growth in the chorioallantoic membrane (CAM) of the chicken embryo.</a> Microvascular Research. 54 (1), 65-73 (1997).</li><li>Lehr, H. A., Leunig, M., Menger, M. D., Nolte, D., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+skinfold+chamber+technique+for+intravital+microscopy+in+nude+mice.">Dorsal skinfold chamber technique for intravital microscopy in nude mice.</a> American Journal of Pathology. 143 (4), 1055-1062 (1993).</li><li>Menger, M. D., J&#228;ger, S., Walter, P., Hammersen, F., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+studies+on+the+microvasculature+of+transplanted+islets+of+Langerhans+in+vivo.">A novel technique for studies on the microvasculature of transplanted islets of Langerhans in vivo.</a> International journal of microcirculation, clinical and experimental. 9 (1), 103-117 (1990).</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=Three-dimensional+spheroids+of+adipose-derived+mesenchymal+stem+cells+are+potent+initiators+of+blood+vessel+formation+in+porous+polyurethane+scaffolds.">Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds.</a> Acta Biomaterials. 9 (6), 6876-6884 (2013).</li><li>Ega&#241;a, J. T., 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=Use+of+human+mesenchymal+cells+to+improve+vascularization+in+a+mouse+model+for+scaffold-based+dermal+regeneration.">Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.</a> Tissue Eng Part A. 15 (5), 1191-1200 (2009).</li><li>Condurache, A., Aach, T. <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+in+angiograms+using+hysteresis+thresholding.">Vessel segmentation in angiograms using hysteresis thresholding.</a> Proceedings of the Ninth IAPR Conference on Machine Vision Applications. , 269-272 (2005).</li><li>Ega&#241;a, J. T., 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=Ex+vivo+method+to+visualize+and+quantify+vascular+networks+in+native+and+tissue+engineered+skin.">Ex vivo method to visualize and quantify vascular networks in native and tissue engineered skin.</a> Langenbecks Archives of Surgery. 394, 349-356 (2009).</li></ol>

感兴趣冲突声明： 所有作者：无财务披露： 没有一个作家有财务利益的任何产品，设备，或者在这个手稿中提到的药物。

有必要建立在改善血流灌注组织工程构建，这需要新的可靠的方法发展到生物材料研究中的血管形成过程中的成功做法。用于制造支架的血管的体外可见常见的方法包括使用显微镜，它提供了高分辨率的工具。在大多数情况下，虽然，这种方法仅限于较小的组织区域的分析，而且往往是昂贵的和费时的。此外，它通常包括复杂的标签和染色的方法，其中血液灌注容器不能从非功能性的人<sup...

在最近的几十年中，组织工程开辟了新的治疗选择与人体自身的细胞替换1组织缺损。为了支持组织再生的生理过程，支架被设计成可生物降解的结构，即提供了一种方案，其中从伤口床的细胞能生长和恢复的缺陷2,3。 血管化不足被认为是主要的障碍，这阻碍了生物人工支架4的临床突破。与细胞的向内生长，营养物质和氧的增加，材料的血管化的需求变得至关重要。因此不足或延迟血管可导致组织工程产品5中央坏死。此外，血管提供免疫感受态细胞并去除代谢残留在再生区。高感染率和低再生只部分的血流灌注不足，在组织工程中所观察到的结果，其目的都是为了通过增加支架6,7的血管被避免。 几种策略，目的是改善血管聚焦在生物材料本身和支架的微观结构中的关键作用。有深入的研究努力，从修理转向愈合过程中再生 ，从而（重新）产生的组织与最接近的生理特性，以一个开发新的方法来恢复8,9。到研究和评估与问候他们的再生潜能的生物材料包括胶原蛋白，纤维蛋白，壳聚糖和海藻酸钠10,11。这些生物材料可以使用，组合为骨干，建立使用不同的策略，新的支架，如脱细胞组织，自组装，快速成型和静电12。为了ENHANCE人体自身的再生能力，支架可生物活化。重组血管生成生长掺入因子13或基因载体等因素后14编码已经显示出改善支架的血管。利用干细胞已被广泛证明是一种很有前途的战略，以改善血管化，其中的间充质基质细胞和内皮祖细胞获得了最多的关注15,16。其他方法试图建立包含移植前17预制的血管网络结构。尽管支架设计密集的努力和他们的生物活性，没有战略已经在临床上显著水平提高血管，并与大规模烧伤除真皮替代的，翻译生物工程材料进入临床常规只发生欲言又止18 。 之一的原因的血管人工组织结构仍然是一个未解决的问题，是评价新技术在体内的方法取得成功的难度。虽然在体外实验可提供支架的血管形成潜力的重要的见解，合适的动物模型是必需的，研究的关键参数，如材料的生物相容性，该处理的特别重要的安全性和有效性，并且，该组织的血管构建。因此，可靠的工具来可视化和量化的血管网络， 在体内是必需的。 在本研究中，我们提出了一种简单而可靠的方法，它允许取出的支架内的血管网络的可视化和量化。这个方法是基于组织透和数字分割。由于该方法是非侵入性的，它可以使靶材料的进一步分子和组织学分析。

<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

血管化不足被认为是限制性的组织工程化构建体的临床成功的主要因素之一。为了评价新的策略，其目的是提高血管化的，可靠的方法是必需的，以使得在生长的新血管进入生物人工支架可见和量化的结果。在过去的几年中，我们的团队已经推出了全层皮肤缺损模型，使血管的直接可视化通过透，并提供量化，通过数字化分割的可能性。在此模式中，一个外科地在小鼠背部创建完整皮肤缺陷并将它们替换为所测试的材料。分子或感兴趣的细胞也可以在这些材料掺入以研究它们的潜在影响。经过自己选择的观察时间，材料植进行评估。双侧伤口提供进行内部比较次的可能在减少个体之间的工件，以及减少所需的研究的动物的数量的。相较于其他方法，我们的方法提供了一种简单，可靠和具有成本效益的分析。我们已经实现了这个模式作为常规工具测试不同的生物材料和生物活化方法的血管时，执行高解析度的筛选。

一个可靠的双边全皮肤缺损所用的小鼠（ 图1），其中所述皮肤可以由生物材料进行研究（ 图2）来代替被创建。在这里，没有严重的并发症是中或手术操作后，无论是宏观的感染或异物反应的迹象观察。在极少数情况下，支架被当鼠标中删除丢失。伤口收缩从未被观察到（ 图3）。组织透允许明确的高达30微米的宽度的血管结构的可视化，整个组织样品，其?...

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

血管是关键，成功的组织工程方法。因此，可靠的技术是必需的，以评估组织构建体的血管网络的发展。在这里，我们提出一个简单和具有成本效益的方法来可视化和量化血管在体内 。

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

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

a-full-skin-defect-model-to-evaluate-vascularization-biomaterials

Research

JoVE Journal

Bioengineering

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

12.1K 次观看.  Technische Universität München. 血管是关键，成功的组织工程方法。因此，可靠的技术是必需的，以评估组织构建体的血管网络的发展。在这里，我们提出一个简单和具有成本效益的方法来可视化和量化血管在体内 。 

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

一种意志的运动反应的及时性和准确性，以振动触觉刺激评价方法

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

一种用于生物技术相关金黄色葡萄球菌感染活体可视化和内膜分析的斑马鱼胚胎模型

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...