修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate
Summary
该协议描述了隔离的脂肪组织的基质细胞从lipoaspirate 4毫米的临界大小的颅骨缺损，以评估骨骼的再生和创造的。
Abstract
颅面骨骼的修复和再生的细胞为基础的方法，利用干细胞通过重新组织形成提供了保证。脂肪基质细胞（ASCs）已被证明是一个丰富的来源，能够进行成骨细胞，软骨细胞，脂肪细胞和成肌细胞分化的多能干细胞。许多研究探讨了这些细胞在体内的成骨潜能细胞输送各种脚手架生物材料的使用。它已被证明，通过利用一个骨传导，以羟基磷灰石涂敷的聚（乳酸 – 共 – 羟基乙酸）（HA-PLGA）支架种子与先进的结构陶瓷，颅骨缺损的关键尺寸，其无法进行自发定义的缺陷，这会愈合的动物的整个生命周期，可有效地显示出强劲的骨再生。这在体内模型演示旨在再生的骨组织的基础上的平移的方法-在蜂窝成分和生物基质。这种方法的模型作为最终的临床应用祖细胞对一个特定的组织缺损的修复。
Protocol
1。细胞分离和扩增所有病人的同意和实验方案进行了审查，并批准由斯坦福大学的机构审查委员会（议定书＃2188，＃9999）。 获取人体皮下脂肪组织从本地/全身麻醉下的的选修lipoaspiration程序。 将有两个在lipoaspirate层（ 图1A）。将上清液包含处理后的多孔材料的绝大部分。底层主要是注射生理盐水。 脂肪衍生的基质细胞，可以从任一层收获，但产量从上?…
Discussion
由于隔离脂肪衍生的基质细胞从lipoaspirate 2，这些细胞已分化成各种各样的细胞谱系。脂肪组织是来自中胚层的起源，因此，多能的脂肪组织衍生的基质细胞将可能是最有效的应用向中胚层谱系。生成骨组织的能力是特别重要的，因为短缺的自体移植的供区和合成材料固有的局限性，包括感染，排斥反应，随着时间的推移和故障3。脂肪间充质干细胞提供了一个潜在的自体多能细胞…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们想感谢乔治共享他们的支持和合作，我们的研究和博士院长Vistnes的。支持这项工作是由美国国立牙科和颅面研究所拨款1 R21 DE019274-01，R01EB009689和RC2 DE020771-02，橡树基金会和Hagey来说小儿再生医学博士炫MTL实验室的支持，圣约瑟夫慈善医院GME的。
Materials
| Name of the reagent: | Company | Catalogue number | Comments (optional) |
| Lipoaspirate Harvest | |||
| PBS | Gibco | 10010-023 | |
| Hank’s Balanced Salt Solution | Cellgro | 21-023-CV | |
| Collagenase | Sigma | C6885-500MG | |
| Cell Strainer 100 μm | BD Falcon | 352360 | |
| Steri-top 500 ml .22 μm filter | Millipore | SCGPT05RE | |
| Calvarial Defect | |||
| Z500 Brushless MicromotorsUM50C | NSK | NSKZ500 | |
| Circular Knife 4.0 mm | Xemax Surgical | CK40 |
References
- Levi, B., James, A. W., Nelson, E. R. Human adipose-derived stromal cells heal critical size mouse calvarial defect. PLoS One. 5, (2010).
- Zuk, P. A., Zhu, M., Ashjia, P. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell. 13, 4279-4295 (2002).
- Keefe, M. S., Keefe, M. A. An evaluation of the effectiveness of different techniques for intraoperative antibiotics into alloplastic implants for use in facial reconstruction. Arch Facial Plastic Surg. 11, 246-251 (2009).
- Mitchell, J. B., McIntosh, K., Zvonic, S. Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 24, 376-385 (2006).
- Dominici, M., Blanc, K. L. e., Mueller, I. Minimal criteria for defining multipotent mesenchymal stroma cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8, 315-317 (2006).
- Cowan, C. M., Shi, Y. Y., Aalami, O. O. Adipose-derived adult stromal cells heal critical-size calvarial defects. Nat Biotechnol. 22, 560-567 (2004).
- Levi, B., Nelson, E. R., Li, S. Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects. Stem Cells. 29, 1241-1255 (2011).
- Phipps, M. C., Clem, W. C., Catledge, S. A. Mesenchymal stem cells responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite. PLoS One. 6, (2011).
- Yuan, H., Zang, Z., Li, Y. Osteoinduction by calcium phosphate biomaterials. J. Mater. Sci. Mater. Med. 9, 723-726 (1998).
- Wei, G., Jun, Q., Giannobile, W. V. The enchancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomaterials. 28, 2087-2096 (2007).
- Li, C., Verpari, C., Jin, H. J. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials. 27, 3115-3124 (2006).
- Zhang, Y., Fan, W., Nothdurft, L. In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for bone morphogenetic protein-7 gene delivery. Tissue Eng Part C Methods. 17, 789-797 (2011).
- Levi, B., Hyun, J. S., Nelson, E. R. Non-integrating knockdown and customized scaffold design enhances human-adipose-derived stem cells in skeletal repair. Stem Cells. 29, 21028-21029 (2011).
Tags
RepairCritical-sized Calvarial DefectAdipose-derived Stromal CellsLipoaspirateCraniofacial Skeletal RepairCell-based ApproachStem CellsOsteogenic PotentialScaffolding BiomaterialsCellular DeliveryOsteoconductiveHydroxyapatite-coated Poly(lactic-co-glycolic Acid)HA-PLGA ScaffoldOsseous RegenerationTranslational ApproachesProgenitor Cell
Citer Cet Article
Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., Grova, M. M., Lee, M., Wan, D. C., Longaker, M. T. Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate. J. Vis. Exp. (68), e4221, doi:10.3791/4221 (2012).