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1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles
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This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.
Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., Grova, M. M., et al. 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).
Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.
1. Cell Isolation & Expansion
2. Scaffold Preparation
3. Cell Seeding
4. Creation of Calvarial Defects and In Vivo Implantation
5. Quantification of Osteoid Formation
6. Representative Results
Adipose tissue has the potential to serve a vital role in the generation of progenitor cells for clinical application. Adipose tissue has a unique advantage in that there is a readily available supply that can be harvested in a relatively simple procedure that involves minimal morbidity and mortality. Once the tissue is harvested and collected, our protocol is outlined in Figure 2. Adipose-derived stromal cells are isolated through a series of washing steps, collagenase digestion, and centrifugation Once the cells are plated in culture, they can be expanded, placed into various differentiation protocols in vitro, or placed directly in vivo.
The creation of the 4 mm critical-size calvarial defect provides a easily accessible and reproducible in vivo model to test the osteogenic differentiation ability of our adipose-derived stromal cells. Through the use of MicroCT, we are able to follow the formation of skeletal tissue in vivo and track the progress of our interventions (Figure 3). The critical-size calvarial defect will not heal within the lifetime of the animal and we see roughly 90% of the defect remain patent around 8 weeks. The scaffold itself (see discussion) has osteogenic inductive properties and has shown the ability to induce some bony regeneration. The typical results of scaffold placement without cells shows that around a one-third of the defect will have de novo bone formation at 8 weeks. With the augmentation of adipose-derived stromal cells, fully two-thirds or more of the defect will show osseous regeneration at around 8 weeks although there is variability between each animal and surgery. The formation of skeletal tissue can be quantified through histology and typical results show increased osteoid formation in samples with ASCs through Aniline Blue and Pentachrome staining (Figure 3C). In addition, we show that the implanted human cells contribute to the underlying de novo osseous formation through the use of GFP-labeled hASCs that show staining in vivo at 2 weeks near the area of de novo bone formation in the calvarial defect (Figure 3D). In addition, we use immunohistochemistry with human specific antibody to show human cell survival and contribution in the area of the defect (Figure 3E).
Figure 1. A - The lipoaspirate has two layers. The top layer contains the adipocytes and the majority of the processes cellular material while the bottom layer contains the saline used during the lipoaspiration procedure. B - the creation of the calvarial defect through a midline incision over the pericranium to isolate the right parietal bone, subsequent creation of a 4 mm critical sized calvarial defect without disruption of the underlying dura mater.
Figure 2. Overview of the harvesting and application of lipoaspirate from the isolation of adipose-derived stromal cells to their expansion, differentiation and use in vitro and in vivo.
Figure 3. A -MicroCT showing in vivo healing of the critical size calvarial defect with the application of ASCs through a hydroxyapatitie scaffold (Bottom Row). Controls include no scaffold and defect (Top row) and defect with placement of the scaffold without cells (Middle Row) B - Quantification of osseous healing from the MicroCT showing significantly increased healing in the ASC group. C - Histology showing increased osteoid formation of the ASC group (bottom row) through Aniline Blue and Pentachrome staining. For Aniline Blue, the osteoid stains dark blue and for Pentachrome, the osteoid stains yellow. D - hASCS labeled with GFP were seeded onto a scaffold and placed into a calvarial defect and sacrificed at 2 weeks. Staining was done with a GFP labeled antibody to show the human cells contributing to regeneration in the area of the defect. E - Human nuclear antigen immunohistochemistry showing prevalence of human cells in the area of the defect at 2 weeks. Click here to view larger figure.
Since the isolation of adipose-derived stromal cells2 from lipoaspirate, these cells have been differentiated into a wide variety of cellular lineages. Adipose tissue is from mesodermal origins and therefore, multipotent adipose-derived stromal cells will likely be most effective with application towards a mesodermal lineage. The ability to generate skeletal tissue is especially critical given the shortage of donor sites for an autograft and the inherent limitations of synthetic material including infection, rejection, and breakdown over time3. Adipose-derived stromal cells offer a potential autogenous multipotent cell line with a relatively large yield4 per surgical harvest.
During the processing of the stromal vascular fraction and adipocyte tissue, it is important to maintain excellent sterile technique. There is a large risk of contamination of the primary culture given the bulk nature of the lipoaspirate combined with the multiple steps of washing, digestion, and centrifugation with steps outside the sterile cell culture hood Once the pellet is plated onto the dish, the majority of the visible cells will be eyrthrocytes. These can be washed off with sterile PBS once the adipose-derived stromal cells have adhered to the plate or red blood cell lysis buffer can be added. It is important that the cells are relatively confluent on the initial plating step in order to ensure proper growth and differentiation.
There is no consensus on how to exactly define adipose-derived stromal cells. While there have been studies trying to define these population of cells based on cell surface markers through flow cytometry, finding a defined set or markers has been difficult. This is likely due to variability in the patient population and the phenotypic drift that these cells experience in vitro under different cell culture environments and variability in passage numbers. The International Society for Cellular Therapy stated that at the minimum, a mesenchymal stem cell must have certain characteristics including the expression of CD105, CD90, CD73, while lacking CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-DR5. The original definition of the multi-potent nature of these cells came from their ability to differentiate into cells of mesodermal lineages in vitro and these protocols are readily available.
The critical-sized calvarial defect model has been validated as model to study skeletal regeneration using both murine adipose-derived stromal cells6 and human-derived adipose-derived stromal cells1. The creation of a 4 mm defect in the right parietal calvaria of a mouse will not heal in the lifetime of the animal. Therefore, any healing seen is due to the treatment placed within the calvarial defect. During the surgical process, it is especially important to not injure the underlying dura mater. The dura mater itself can stimulate osteogenesis and any injury can significantly hamper the healing process to the calvarial defect7.
The use f the scaffold as a delivery mechanism for stem cells is especially important in the world of skeletal engineering. The function of skeletal tissue is uniquely related to the three-dimensional structure of the macro-environment. The proper scaffold can not only aid in stem cell delivery in vivo, but also augment osteogenesis through the enhanced osteogenic properties of the scaffold itself. Our laboratory incorporates hydroxyapatite, a calcium derivative, onto the PLGA backbone due to the osteoinductive and osteoconductive properties of this material. However, there are many other options for scaffold creation that can have strong osteoinductive and osteoconductive properties8, 9. In addition, the scaffold can also be used to deliver powerful adjuvants such as small molecules10, 11 and vectors to knock down or upregulate gene targets12, 13.
No conflicts of interest declared.
We would like to thank Dr. George Commons and Dr. Dean Vistnes for their support and collaboration of our research. This work is supported by National Institute of Dental and Craniofacial Research grants 1 R21 DE019274-01, R01EB009689 and RC2 DE020771- 02, the Oak Foundation and Hagey Laboratory for Pediatric Regenerative Medicine to M.T.L. Dr. Hyun is supported by the Saint Joseph Mercy Hospital GME.
|Hank's Balanced Salt Solution||Cellgro||21-023-CV|
|Cell Strainer 100 μm||BD Falcon||352360|
|Steri-top 500 ml .22 μm filter||Millipore||SCGPT05RE|
|Z500 Brushless MicromotorsUM50C||NSK||NSKZ500|
|Circular Knife 4.0 mm||Xemax Surgical||CK40|
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