BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型
Summary
Adipose-derived stromal cells may be useful for engineering new tissue from a patient’s own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.
Abstract
Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient’s own body. The ability to grow bone de novo using a patient’s own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient’s own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.
Introduction
从损伤，感染，或浸润癌造成重大的骨缺损对患者的康复和生活质量有显著的影响。技术存在从患者自身的身体别处填充健康骨这些缺陷，但这种传输携带它自己的发病率和并发症1，2，3的风险。此外，一些缺陷是如此之大的或复杂的足够的供体骨不可用于填充缺损。假体装置是用于填充骨缺损的潜在的选择，但这些都与若干缺点，包括感染风险，硬件故障，以及异物反应4相关联。
由于这些原因，有在工程使用患者自身的细胞5生物骨替代品的可能性极大的兴趣。脂肪衍生的基质细胞（ASCs）具有潜在的这种应用，因为它们是在患者自身的脂肪组织大量获得，他们已经证明通过产生新的骨组织6,7愈合的骨缺损的能力。携带者的细胞和几个研究的多样化的人口已经表明，选择特定的细胞表面标志物可产生具有增强成骨活性8,9细胞群。最高的成骨能力选择的ASC将增加的可能性，这些细胞接种支架可以再生一个大的骨缺损。
骨形态发生蛋白（BMP）信令是用于调节骨分化和形成10和BMP受体类型IB（BMPR-IB）已知是用于在携带者11成骨重要的关键。最近，我们已经表明BMPR-IB的表达可以b使用E要选择具有增强成骨活性12携带者。在这里，我们证明了的BMPR-IB-表达来自人脂肪的ASCs接着用体内颅骨缺损模型中的他们的成骨活性的测定中隔离的协议。
Protocol
Representative Results
Discussion
该议定书中的关键步骤
携带者的收获过程中，关键的一步是脂肪用胶原酶消化充分。不足消化将产生的ASC的低产率。在BMPR-IB +细胞的FACS分选，仔细定义大门的积极性是非常重要的。定义门太松可能会导致排序人群是不纯洁的。在创建过程中的颅骨缺损的，关键是通过头骨的骨钻缺陷，但可以不前进到硬脑膜。这将导致大量出血和脑，这将需要的动物实施安乐死的曝光。
<p cla…Disclosures
The authors have nothing to disclose.
Acknowledgements
C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.
Materials
| 100 micron cell strainer | Falcon | 352360 | |
| 15 blade scalpel | Miltex | 4-515 | |
| 24 well plate | Corning | 3524 | |
| 40 micron cell strainer | Falcon | 352340 | |
| 50 mL conical centrifuge tubes | Falcon | 352098 | |
| 6-0 Ethilon nylon suture, 18", P-3 needle, | Ethicon | 1698G | |
| Anti-BMPR-IB primary antibody | R&D systems | FAB5051A | |
| BioGel PI surgical gloves | Mölnlycke Health Care | ALA42675Z | |
| Buprenorphine SR | ZooPharm | ||
| Castro-Viejo needle driver | Fine Science Tools | 12565-14 | |
| CD1 nude mouse | Charles River | 086 | |
| Collagenase Type II powder | Gibco | 17101-015 | |
| DMEM medium | Gibco | 10564-011 | |
| Drill: Circular knife 4.0 mm | Xemax Surgical | CK40 | |
| Drill: Z500 Brushless Micromotor | NSK | NSKZ500 | |
| FBS | Gicbo | 10437-077 | |
| Fisherbrand Absorbent Underpads, 20" x 24" | Fisher Scientific | 14-206-62 | |
| Fisherbrand Sterile cotton gauze pad, 4" x 4" | Fisher Scientific | 22-415-469 | |
| Heating pad | Kent Scientific | DCT-20 | |
| Hyclone 199/EBSS medium | GE Life Sciences | SH30253.01 | |
| Isothesia isoflurane | Henry Schein | 050033 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Paraffin film (Parafilm) | Bemis | PM996 | |
| PBS | Gibco | 10010-023 | |
| Pen-Strep | Gibco | 15140-122 | |
| PLGA scaffolds | Proprietary Formulation | ||
| Poloxamer 188, 10% | Sigma | P5556-100ML | |
| Polylined Sterile Field, 18" x 24" | Busse Hospital Disposables | 696 | Cut a rectangular hole of the appropriate size |
| Polysucrose Solution: Histopaque 1119 | Sigma | 11191 | |
| Povidone Iodine Prep Solution | Medline | MDS093944H | |
| Puralube petrolatum ophthalmic ointment, 1/8 oz. tube | Dechra Veterinary Products | ||
| RBC lysis buffer | Sigma | 11814389001 | |
| Webcol alcohol prep swabs | Covidien | 6818 |
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