Isolamento rápido de BMPR-IB + adiposo-derivados de células estromais para uso em um Defeito Modelo Cura calota craniana
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
Os principais defeitos ósseos resultantes de lesão, infecção ou cancro invasivo tem um impacto significativo sobre a recuperação e a qualidade de vida de um paciente. Existem técnicas para preencher esses defeitos com osso saudável de outras partes do próprio corpo do paciente, mas essa transferência leva seu próprio morbidade e risco de complicações 1, 2, 3. Além disso, alguns defeitos são tão grandes ou complexos que óssea de doadores suficiente não está disponível para preencher o defeito. Dispositivos protéticos são uma opção potencial para preenchimento de defeitos ósseos mas estes estão associados com várias desvantagens, incluindo o risco de infecção, falha de hardware, e reação de corpo estranho 4.
Por estas razões, há um grande interesse na possibilidade de engenharia substitutos ósseos biológicos utilizando as próprias células 5 de um paciente. células estromais derivadas de tecido adiposo (ASCs)tem potencial para esta aplicação, porque eles estão disponíveis em abundância no próprio tecido de gordura do paciente e eles têm demonstrado a capacidade de curar defeitos ósseos, gerando um novo tecido ósseo 6, 7. ASC são uma população diversa de células e vários estudos têm mostrado que a selecção de marcadores específicos da superfície celular pode produzir populações de células com actividade osteogénica aumentada de 8, 9. Seleccionar ASC com o maior potencial osteogénico iria aumentar a probabilidade de que um andaime semeadas com estas células poderia regenerar um grande defeito ósseo.
Sinalização proteína morfogenética do osso (BMP) é crítico para a regulação de diferenciação do osso e formação 10 e do tipo de receptor de BMP IB (BMPR-IB) é conhecida por ser importante para a osteogénese em 11 ASC. Recentemente, mostrámos que a expressão de BMPR-IB pode be usado para selecionar para ASC com reforçada actividade osteogénica 12. Aqui demonstramos um protocolo para o isolamento de BMPR-IB-expressando ASC de gordura humana, seguido de um ensaio da sua actividade osteogénica utilizando um modelo in vivo de calvária defeito.
Protocol
Representative Results
Discussion
Passos críticos dentro do Protocolo
Durante a colheita da ASC, o passo crítico é a digestão adequada de gordura com colagenase. digestão inadequada resultará em um baixo rendimento da ASC. Durante FACS triagem de células BMPR-IB +, é importante definir cuidadosamente o portão para a positividade. Definindo portas muito frouxamente pode resultar em populações classificadas que não são puros. Durante a confecção do defeito calvária, é crítico para perfurar o defeito através do …
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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