NOTE: Samples were obtained from patients who gave informed consent. All protocols were reviewed and approved by the appropriate Stanford University Institutional Review Board. While handling human tissue and cells, always adhere to Biosafety Level 2 (BSL2) precautions, as specified by your institution.
1. Preparation of Reagents
2. Harvesting and Isolation of ASCs
NOTE: Ensure that adequate institutional approvals are in place for using human tissue and for isolating human stem cells. Obtain human abdominal, flank, or thigh subcutaneous fat from a healthy donor undergoing elective liposuction. Keep the fat in a plastic suction canister.
3. FACS Sorting for BMPR-IB Positive Cells and Preparation of Cell-containing Scaffolds
4. Creation of Calvarial Defects and Application of ACS-containing Scaffold
NOTE: Ensure that adequate institutional approvals are in place for the creation of calvarial defects in live mice. This protocol has been approved by the Stanford University Institutional Animal Care and Use Committee.
Micro CT scan done on the day of surgery will clearly show the skull defect. At this time there will be no ingrowth into the 4 mm defect. Subsequent scans are obtained over time to quantify the size of the defect over time compared with the baseline. Defects seeded with BMPR-IB+ cells should demonstrate more rapid closure of the defect when compared with BMPR-IB- and Unsorted cells (Figure 5). In addition, the portion of the skull containing the defect can be decalcified and processed for histology using standard methods 12. Sections stained with Movat's pentachrome stain will reveal greater bone regeneration in the defect treated with BMPR-IB cells compared with the other cell populations (Figure 6).
Figure 1: Lipoaspirate after PBS Wash. A canister of lipoaspirate after PBS is added and the mixture is allowed to settle. The top tissue layer is the adipose tissue which hosts ASCs, while the bottom layer is the aqueous layer, mainly comprised of saline and blood. Please click here to view a larger version of this figure.
Figure 2: Placement of Cell Suspension onto Polysucrose Solution. (a) The cell suspension must be pipetted very gently onto the side of the tube, allowing it to layer on top of the polysucrose solution. (b) This is the correct appearance of a cell suspension layer on top of the polysucrose prior to centrifugation. Please click here to view a larger version of this figure.
Figure 3: Seeding of Cells onto the Scaffold. (a) On the sterile surface of one well of a 24-well cell culture plate, load a dry scaffold with approximately 50 μL of cell suspension. (b) Incubate the scaffold in a cell culture incubator for 30 min to allow cell adhesion. Note: In the figure, a 10-cm plate is used for demonstration purposes. Please click here to view a larger version of this figure.
Figure 4: Creation of the Calvarial Defect. The calvarial defect (arrows) is visible within the open skin incision. Note the intact dural blood vessels (arrowheads), indicating that the drilling has not breached the dura. Scale bar, 5 mm. Please click here to view a larger version of this figure.
Figure 5: Healing of Critical-sized Calvarial Defects with Different ASC Subpopulations. (A) Three-dimensional micro- CT reconstructions were performed for calvarial defects re- paired with unsorted, BMPR-IB(+), or BMPR-IB(-) ASCs. (B) Quantification of healing at 8 weeks demonstrated significantly greater bone regeneration with BMPR-IB(+) ASCs (92%) compared with unsorted and BMPR-IB(-) ASCs (58% and 46%, respectively, **p < 0.01). Significant differences in healing were also seen at 2 weeks (**p < 0.01), 4 weeks (***p < 0.001), and 6 weeks (***p < 0.001). Micro-CT, micro-computed tomography. Reprinted with permission from McArdle et al.12. Error bars represent standard deviation. Please click here to view a larger version of this figure.
Figure 6: Histological Staining of Bone Regenerate. (A) Movat's pentachrome staining of bone regenerate in defects repaired with unsorted, BMPR-IB(+), or BMPR-IB(-) ASCs at 5X magnification using bright field microscopy. Note more robust bone formation in BMPR-IB(+) group compared with BMPR-IB(-) group. The dotted line represents the extent of the defect area. The area within the black rectangle is shown on higher magnification using (B) 10X and (C) 40X bright field microscopy of defect area. Reprinted with permission from McArdle et al.12. Please click here to view a larger version of this figure.
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 |
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.
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.
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.