心肌组织工程生物衍生的注射材料的制备
Summary
准备脱细胞组织注射基质凝胶注入大鼠心肌的方法<em>在体内</em>描述。
Abstract
该协议规定编制的注射细胞外基质（ECM）的心肌组织工程中的应用凝胶的方法。简单地说，是冻干脱细胞组织，精，酶消化，然后带来生理pH值。冻干删除所有从组织的含水量，导致干流脑可分为地面用小磨细粉。铣削后，ECM的粉是用胃蛋白酶消化，以形成一种注射矩阵。调整pH至7.4后，液体基质材料可注射到心肌。以往的定性分析的结果表明，脱细胞心包及心肌组织产生的基质凝胶保留原生的ECM成分，包括不同的蛋白质，肽和粘多糖。由于使用这种材料的组织工程，是体内特性特别有用;在这里，左心室壁内注射执行的方法（LV）的游离壁作为分析宿主反应基质凝胶的手段提出一个小动物模型。获得通过膈肌进入胸腔注射略高于在LV游离壁的心尖。生物衍生支架可以可视化和注射前生物素标记，然后与辣根过氧化物酶标记的neutravidin染色的组织切片，并通过二氨基联苯胺（DAB）染色可视化。注射区域的分析，也可以做组织学和免疫组织化学染色。这样，在先前研究的心包和心肌基质凝胶形成纤维，多孔网络和注射区域内促进血管的形成。
Protocol
1。前处理组织准备使用此协议之前，必须已经脱细胞的组织选择。对于这个例子，新鲜的猪和人类心包样品使用去离子（DI）水和十二烷基硫酸钠（SDS），低渗和高渗漂洗脱细胞。 具体来说，首先在DI水冲洗猪心包30分钟，然后搅拌在1％SDS不断在磷酸盐缓冲液（PBS）为24小时，5小时DI水冲洗。对于人类pericardia，首先在DI水冲洗30分钟，然后搅拌在1％SDS的PBS连续60-65小时，?…
Discussion
这种方法允许代生物衍生，注射用心肌组织工程支架。虽然这些方法最初是为制造和开发在体内试验心肌基质凝胶，并提出与心包基质凝胶中，这种协议可以适应任何组织使用，提供了组织可适当的脱细胞。脱细胞应进行验证使用这些方法之前，基质凝胶中的DNA的存在，可能会造成有害的免疫反应。有多种方式decellularize材料，并有关于这一问题的3，4的书面的几个很好的评价。
…Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国立卫生研究院主任的新的创新奖励计划，美国国立卫生研究院医学研究路线图的一部分，通过授予数量DP2 – 1 – OD004309 – 01，部分支持。 SBS – N。想感谢美国国家科学基金会研究生研究奖学金。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Reagents: | ||||
| Pepsin | Sigma-Aldrich | p6887-1G | Lyophilized | |
| Biotin | Thermo Scientific | 21217 | ||
| Neutravidin-HRP | Thomas Scientific | 21130 | ||
| Equipment: | ||||
| Wiley Mini Mill | Thomas Scientific | 3383L10 | ||
| Labconco Lyophilizer | Labconco, Inc | 7670520 | ||
| Surgical supplies: | ||||
| Betadine | Purdue Products, L.P. | 67618-154-16 | ||
| Lactated Ringers Solution | MWI | 003966 | ||
| KY Jelly | MWI | 28658 | ||
| Lidocaine, 2% | MWI | 17767 | ||
| Buprenorphine hydrochloride | Reckitt Benckiser Healthcare (UK) Ltd. | 12496-0757-1 | ||
| Artificial tear ointment | Fisher | NC9860843 | ||
| Triple antibiotic ointment | Fisher | 19082795 | ||
| Isoflurane | MWI | 60307-120-25 | ||
| Otoscope | MWI | 008699 | ||
| Stop cock | MWI | 006245 | ||
| 3-0 Vicrile suture | MWI | J327H | ||
| 5-0 Proline suture | MWI | s-1173 | ||
| Reverse cutting (RC) needle | Ethicon | 8684G | ||
| Microhemostats | Fine Science Tools | 13013-14 | ||
| Rat tooth microforceps | Fine Science Tools | 11084-07 | ||
| No. 10 scalpel | Fine Science Tools | 10110-01 | ||
| Blunt scissors | Fine Science Tools | 14108-09 | ||
| Sharp, curved scissors | Fine Science Tools | 14085-08 | ||
| Large, serrated forceps | Fine Science Tools | 1106-12 | ||
| PE160 suction tubing | BD | 427430 | ||
| Clippers | MWI | 21608 | ||
| Skin staples/stapler | Ethicon | PRR35 | ||
| General supplies: | ||||
| Stir plates | ||||
| 0.1 M HCl | ||||
| 1 M NaOH | ||||
| 10x PBS | ||||
| 1x PBS | ||||
| 70% Ethanol | ||||
| 0.1 mL syringes | ||||
| 10 mL syringe | ||||
| Q-tips | ||||
| Surgical glue | ||||
| Surgical drape | ||||
| Towel clamps | ||||
| Small hand-held vacuum |
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Tags
Biologically Derived Injectable MaterialsExtracellular Matrix GelLyophilizationEnzymatic DigestionPhysiological PHECM PowderPepsin DigestionInjectable MatrixCharacterization AssaysNative ECM ComponentsProteinsPeptidesGlycosaminoglycansIntramural InjectionLeft Ventricular Free WallSmall Animal ModelBiotin-labelingHorse Radish Peroxidase-conjugated Neutravidin
Citar este artigo
Seif-Naraghi, S., Singelyn, J., DeQuach, J., Schup-Magoffin, P., Christman, K. Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering. J. Vis. Exp. (46), e2109, doi:10.3791/2109 (2010).