The overall goal of this procedure is to sequentially track and quantify the migration proliferation and differentiation of endogenous osteogenic stem and progenitor cells in bone fracture repair. This is accomplished by first labeling mixo virus, influenza virus resistance, one or MX one positive cells in vivo by poly cynic poly cyto ilic acid injection, and then eliminating the resident MX one positive hematopoietic cells by irradiation. In the second step, wild type bone marrow cells are systematically administered to the irradiated mice.
Next micro fractures are generated in the mouse Cal area by needle drilling. Finally, sequential intra vial imaging of the micro fracture repair is performed. Ultimately, these intravital images can be used to evaluate the dynamics of the osteogenic stem and progenitor cell influx and expansion into the fracture sites.
So the main advantage of this technique is that allows for in vivo repetitive imaging of osteogenic stem and progenitor cells during the course of fracture healing. The implication of this technique extended toward the therapy of fracture healing and osteoporosis. As this method offer a new tool for monitoring endogenous osteogenic stem progenitor cells in bone regeneration and repair.
Visual demonstration of this method is critical as a generation of the microfracture with minimal tissue damage and the repeated in vivo imaging of the fracture injury without inducing severe scar formation are important to the method and technically challenging To label MX one positive cells in vivo begin by injecting 200 microliters of poly acidic poly cyto ilic acid per 20 grams of MX one Cree reporter mouse weight intraperitoneal once every other day for 10 days. Then to eliminate the resident MX one positive hematopoietic cells irradiate the injected mice with a single dose of 9.5 gras. After 24 hours, transplant one times 10 to the sixth wild type bone marrow cells intravenously per mouse.
Four to six weeks later, anesthetize the mouse by intraperitoneal injection with 50 microliters of ketamine xylazine After confirming sedation by toe pinch, clip the scalp pair of the bone marrow cell transplanted mouse, and then sterilize the exposed skin with a 70%alcohol swab. Apply tear gel to prevent corneal dehydration, and then make a transverse incision less than one centimeter long across the scalp, starting from one ear and ending at the other. Next, turn the animal about 60 degrees and continue the incision until it reaches about two to three millimeters from the nose using forceps.
Pull the flap of tissue toward the animal sides to separate the skin, both the frontal bones and the intersection of the sagittal and coronal sutures should now be clearly exposed. Flush the open surface with sterile PBS and then gently wipe away the residual hairs with cotton swabs. Then to generate microfractures on the calver, hold the mouse head with one hand and a 30 gauge needle with the other.
Use the 30 gauge needle tip to gently drill a micro puncture on one side of the frontal bones near the intersection of the sagittal and coronal sutures. To avoid penetrating the brain switch to a bigger gauge and twist the new needle to widen the puncture hole to about 0.5 millimeters in diameter. After generating a second puncture on the contralateral frontal bone, continuously drop PBS into each spot wiping until the bleeding stops.
Then to avoid drying, apply a sufficient enough drop of sterile physiological saline solution onto the skull to fully cover the area to acquire the Vidal images. First turn on the polygon based laser scanner to allow simultaneous multi-channel image acquisition at a video rate of 30 frames per second. Next turn on the femtosecond titanium sapphire laser for multi photon imaging.
Set the power and wavelength to 880 nanometers for second harmonic generation imaging of the calvarial bone at 440 nanometers for GFP and TD tomato excitation, turn on the 491 nanometer and 561 nanometer solid state lasers. And then turn on the photo multiplier tube detectors for each signal a 435 plus or minus 20 nanometer band pass filter for second harmonic generation, A 528 plus or minus 19 nanometer band pass filter for GFP and a 590 plus or minus 20 nanometer band pass for TD tomato. Now, place the animal on an XYZ axis motorized microscope stage on top of an electric heating pad set to 37 degrees Celsius.
Use tape to hold the mouse in position and then apply a drop of warm 2%Methylcellulose based gel onto the skull. To avoid drying, put a cover glass on the imaging area. Then use a 30 x water immersion objective with 0.9 numerical aperture and the XY, Z axis controller to detect the SHG signal from the bones.
To find the surface of the calver, identify a crucial landmark location such as the intersection between the sagittal sutures and coronal sutures, and acquire an image. Mark the XY, Z coordinates of the landmark and then continue to search for the location of injury using the SHG and fluorescent signals as guides. When each region of interest is found, acquire an image of the best focal plane containing the SHG and fluorescent signals from the cells of interest.
Save the XY, Z coordinates and the distance to the intersection of the sagittal and coronal sutures to define their precise location for the next round of imaging. Then to collect 3D cellular and bone structures of the fracture injury. Record images in roughly two to five micron Z stack intervals with about a 100 micron depth from the endo bone surface.
Finally, after imaging both injury sites use multiple drops of sterile saline to remove the 2%Methylcellulose gel from the skull. Now coat the surface with antibiotics and cover the skull with the skin flaps, reclose the scalp with hypoallergenic suturing thread and keep the animal in a warm recovery chamber until it regains sufficient consciousness. After three to five days, repeat the Intravital imaging steps to track the cellular change during the healing of the fracture.
In this representative experiment, two microfractures were generated on the frontal bones of genic MX one tomato osteocalcin, GFP mice after irradiation and bone marrow replacement sequential 3D intra vital imaging of the micro fractures showed a relocation of the tomato positive osteogenic stem and progenitor cells within the site of the fracture at day two, as well as their expansion at day five. Although no GFP positive osteoblasts were detected at this time, on day 12, a subset of osteo progenitors near the fracture surface initiated the differentiation of tomato positive GFP positive osteoblasts as indicated by the arrow, the accumulation of new osteoblasts and new bone formation on day 21 indicates that the migration and proliferation of the osteogenic progenitor cells was a major mechanism for supplying new osteoblasts for healing the fracture. To test whether this method provides a consistent and quantitative output of osteo progenitor numbers during fracture healing, MX one YFP positive osteogenic stem and progenitor cells were tracked for 14 days after injury.
Small numbers of the progenitors were consistently detected at the injury site by three days. The cell numbers continuously increased at day seven, reaching a peak population at day 10 that was sustained at 14 days. The kinetics of the generation of the osteo progenitors was quantified by measuring the YFP signal intensity over time, and correlated with that observed by intra vital microscopy.
The progenitors were first observed on day three, peaked by day 10, and remained at a similarly high intensity through day 14. While attempting this procedure, it's important to remember that skilled suture techniques and minimal bleeding can reduce the formation of scars and lower the background autofluorescence. After watching this video, you should have good understanding of how to image and track the migration proliferation and differentiation of endogenous osteogenic stem progenitor cells in the process of fracture repair.
