Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

The overall goal of this procedure is to monitor the integration of cranial bone grafts in terms of bone regeneration and neovascularization using multimodal imaging approaches. This method can help answer key questions in the field of regenerative medicine, such as the osteogenesis angiogenesis coupling. The main advantage of this technique is that it provides a high definition 3D determination of the vascular three, and it allows the study of the complete organ demonstrating the procedure will be doron Vic, a PhD student in my lab After euthanizing seven to eight week old Bab sea mouse.

According to the text protocol, use a hair clipper to shave the calvarial region against the direction of hair growth. Apply 70%isopropanol to the carcass, head to disinfect. Then use a number 11 scalpel to cut the scalp skin and remove the periosteum using a dental drill and a 4.5 millimeter inner diameter tine.

Detach a circular fragment of calvarial bone. Transfer the bone fragment to a 100 millimeter plastic dish containing sterile saline. At this point, observe blood and soft tissue attached to the bone fragment with curved tweezers.

Hold one bone fragment at a time and use a cotton tipped applicator to swab it thoroughly. Removing any brain tissue, soft tissue, and blood. Use fine scissors to gently cut away any bone chips that remain attached to the graft so that the graft will have a perfect rounded shape.

Dip each graft once in a 15 milliliter tube containing 70%ethanol and twice in 15 milliliter tubes containing PBS to wash off the ethanol. Freeze the allograft in a minus 80 degrees Celsius freezer in cryo tubes for at least one week after freezing. Ensure the allograft appear transparent working with recipient transgenic mice that express luciferase under the control of the osteocalcin promoter.

After anesthetizing a seven to eight week old female mouse using injectable anesthetics and verifying sedation. Use a hair clipper against the direction of hair growth to shave the calvarial region. Apply a depilatory to remove any fur remnants and use dry gauze to wipe it off after no longer than two minutes.

Then use gauze soaked in saline to wipe down the area. Disinfect the scalp by applying 5%chlorhexidine subcutaneously. Inject five milligrams per kilogram of carprofen diluted in 200 microliters of saline.

Apply eye ointment to prevent dryness and keep the animal on a thermal pad at all times. Then position the animal under the operating binoculars. Next, using fine scissors and tweezers, make a one centimeter long oval cut in the scalp skin with tweezers.

Hold the periosteum and use fine scissors to cut it away with a dental drill and a five millimeter outer diameter tr refine drill the calvarial defect two millimeters anterior to the lambdoid suture to avoid penetration of the dura mater. Drill three fourths of the way into the bone and use a spatula to detach the bone fragment. One critical step in the protocol is the termination of the defect location.

Injury to the labo suture will lead to massive bleeding. Therefore, care should be taken not to disrupt the underlying durometer and the superior sagittal. To implant an auto graft.

Use 10 milliliters of sterile saline in a 10 millimeter plastic plate to wash the bone fragment. Remove blood clots without removing soft tissue to implant an allograft thaw in advance and normalize the sample to room temperature. Then wash the bone fragment with curved tweezers.

Place the bone graft in the defect so it will not touch the defect margins. Then using fibrin gel, glue the graft to the host bone. Suture the skin with four oh Vicryl suture and apply povidone iodine to the surgical cut.

Mark the mouse ear and inject 0.25 milligrams per kilogram anti sedan. To reverse the anesthetic effect of the meato. Carry out postoperative care according to the text protocol.

Prepare heparinized saline by weighing 2000 units of sodium heparin and transferring to a 50 milliliter tube. Add 20 milliliters sterile saline to the tube and shake. Well fill a 20 milliliter lure lock syringe with 15 milliliters of warmed heparinized saline and connect the syringe to a 23 gauge scalp vein set.

Affix the syringe to a syringe pump and set the pump for 10 milliliters at one milliliter per minute. After anesthetizing a mouse according to the text protocol, confirm sedation with a toe pinch and secure the mouse on. Its back to a fixation board wrapped with an absorbent surface liner.

Next, cut the skin from the abdomen to the neck. Cut the abdominal wall up to the xiphoid process. Then cut the rib cage along its lateral aspect, making sure the heart is fully exposed and the heart and lungs are undamaged.

Use a hemostat to retract the sternum. Insert the butterfly needle three millimeters into the left ventricle. Then with three second glue, glue the needle to the heart and chest wall Insertion of the butterfly needle into the left ventricle is a delicate procedure.

Try to avoid insertion into the right ventricle or too deep into the left ventricle because it will penetrate the posterior heart wall. Next, immediately activate the pump. After a minute, dissect the inferior vena cva in order to depressurize the vasculature.

Tilt the board so that fluids flow away from the head. Successful perfusion will result in a clearing of the liver and blood vessels. Now perfused 10 milliliters of 4%formaldehyde.

Avoiding penetration of air bubbles into the vasculature. When perfusion is complete as signified by a stiffening of the tail, use 10 milliliters of heparinized saline to flush out the formaldehyde. Prepare the radio opaque contrast agent according to the manufacturer's instructions.

By mixing the compound diluent and curing agent in a plastic tube, perfuse the animal with the contrast agent at a rate of one milliliter per minute. The coronary intestinal and hepatic arteries should turn yellow when perfusion is complete. Cut the tubing to the butterfly needle and wrap the carcass in paper to prevent leakage of the solution to the calvarial region.

Place the animal at four degrees Celsius overnight to allow polymerization the following day. To dissect the calvarial region, use a scalpel to cut the skin as lateral as possible, avoiding damaging the cranial region of interest. Locate the bone graft and then use fine scissors to cut the cranial bone 10 millimeters away from the graft.

Using a micro CT scanner. Set the field of view to 20.5 millimeters x-ray energy to 55 KVP intensity to 145 microamps a thousand projections per 360 degrees and an integration time of 200 milliseconds. Scan the isolated calvarial bone sample.

Observe the 2D reconstructed slices that were generated by the micro CT software. Make sure to scan the region of interest properly and then locate the calvarial defect. If the perfused vessels are readily identifiable, use 6%trichloroacetic acid diluted in double distilled water.

To decalcify the sample, re-scan the sample using the same parameters as just described. Locate the lambdoid suture in the reconstructed 2D slices. Define a cylindrical volume of interest or VOI two millimeters in height and with a diameter of eight millimeters that is tangential to the suture.

This is the anatomical site of the defect. Next to segment the bone from the soft tissue. Set the lower threshold to 130 and apply a constrained 3D Gaussian Filter to partially suppress noise in the volumes.

Generate a 3D reconstruction. Making sure the VOI is properly positioned using IPL software and the DT object. Peram function generate a thickness map.

This function describes the vessel volume for each vessel diameter carry out imaging and micro computed tomography. According to the text protocol, as shown here, seven days after surgery, CT scanning demonstrated a significantly higher volume of small and medium diameter blood vessels that nourished the entire graft area in mice that had received autographs as opposed to mice received allografts where the vessels seemed to penetrate from outside. In addition, fluorescence imaging showed significantly higher blood perfusion in autograph treated animals.

By day 14, the vascular tree in the allograft reach all of the grafts proximity. Yet vessel volume was significantly lower compared to the Autographt group. This difference was most prominent when vessels with 0.08 to 0.1 millimeter diameters were compared as seen here 28 days after surgery, the vasculature volume returned to baseline seven days after surgery.

The autograft group demonstrated significantly greater bone mineralization as seen by fluorescence imaging using a hydroxyapatite directed probe. Bone formed throughout the graft proximity in this group while it formed only at the defect margins in the allograft group as marked by a ring shape 56 days after surgery, bone volume was significantly higher and graft thickness was significantly greater in animals that received autographs versus allografts Once mastered, this surgical technique can be done in about 25 minutes. Following this procedure, fluorescence imaging using other probs can be performed in order to answer additional questions, for example, osteoclast activities and involvement of immune response.

After watching this video, you should have a good understanding of how to image bone regeneration in the underlying angiogenesis.