Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The overall goal of this experiment is to visualize In Vivo blood vessel formation in three dimensional scaffolds in real time by multi photon microscopy. These measured are key question in bone generating field such as how to neutralize and trick in vivo blood vessel formation in three dimensional scaffolds in real time. The one advantage of this technique is that it enables to simultaneously neutralize CERES, extracellular metrics and surrounding molecular networks in vivo.

To begin, prepare a homogenous solution containing 20 grams of PLGA and 20 milliliters of 1, 4 dioxane. Then, add two grams of powdered nHAP to the solution. Using a magnetic stirrer, mix the suspension vigorously at 1500 rpm at room temperature for 16 hours to form a uniform paste that will be used for printing porous PLGA NHAP scaffolds.

Once printed, freeze dry the scaffolds for 48 hours to completely remove any solvent. And then sterilize them in 75 percent ethanol for one hour. Afterward, lifalyze them once more to obtain neutral, antiseptic material.

Next, add four point five times 10 to the fifth of lentiviral particles to each scaffold and allow the particles to absorb in a humidified incubator maintaining 37 degrees celsius for two hours. Now that lentiviral particle's absorbed on the scaffolds, wash the scaffolds twice with two milliliters of PBS to remove any unbound residues. Snap freeze the washed scaffolds in liquid nitrogen.

And after lifalyzing once again, store them at minus 80 degrees celsius for further studies. Transfer 3d scaffolds previously coated with four point five times ten to the fifth lentiviral particles carrying green fluorescent protein in two point zero milliliter cryogenic vials filled with one milliliter of complete DMEM and incubate them at 37 degrees celsius with shaking for five days. Every twelve hours of incubation, collect one milliliter samples of the media containing lentiviral particles released from the scaffolds, and replace them with one milliliter of fresh media.

To transduce HEK293T cells, first remove the medium from HEK293T cells cultured in a 12 well plate and wash the cells twice with PBS. Then, add one milliliter of the previously collected media to the washed HEK293T cells and incubate the plate for 48 hours at 37 degrees celsius in a humidified incubator. After 48 hours of incubation, measure the number of GFB positive HEK293T cells by flow cytometry to determine the number of bioactive lentiviral particles.

To begin, randomly divide C57 black six mice into three treatment groups:control, PH, and PHp of 14 animals each. Shave the skin on the head of an anesthetized mouse and disinfect the surface with an iodine tincture and then 75 percent alcohol solution. Place the head of the mouse in a stereotactic instrument and size the skin with a scalpel and then using scissors, make a one centimeter linear cut.

Using a sterile cotton swab, scrape the periosteum off the cranial bone to reveal it's surface. Then, on the left side of the skull, create with a trephine bur a four millimeter diameter critical size defect. Transplant the scaffold into the side of the bone defect.

Use ophthalmic forceps to gently cover the surgical site with the periosteum. Afterward, close the wound by applying three stitches per each centimeter of incision. Finally, administer the analgesic buprenorphine to control post-operative pain and keep the animal on a heating pad to maintain it's body temperature until it wakens.

When the mouse regains consciousness, provide it with food and water and monitor it's everyday activity until the end of the experiment. After assembling the multi photon microscopy system for TPEF and SHG imaging, tune the femtosecond titanium sapphire laser to 860 nanometers. To visualize newly formed blood vessels, inject the mouse intravenously with 200 microliters of a 10 milligram per milliliter dextran solution in saline, then, using the stereotactic instrument, immobilize the anesthetized animal on a heating plate to maintain it's body temperature at 37 degrees celsius.

Inject the mouse with 200 microliters of saline intraperitoneally to prevent dehydration. Finally, provide the animal with a three percent mixture of isoflurane in 100 percent oxygen to maintain sufficient anesthesia and analgesia during imaging. To begin imaging, scan a pair of galvo mirrors to create a 512 by 512 micrometer sampling area.

Then, using an NA one point O water immersion objective lens, focus the excitation beam on the sample and collect the back scattered TPEF HSG signal. Set the field of view so that it covers the normal bone area and the edge of the defect in the control group, but only the implanted scaffold when imaging the animal from PH or PHp group. Then, scan five different sites of the defect.

Acquire image stacks every 12 seconds over a two hour imaging period. Presented here is a photograph of the scaffold prepared with the 3d printing technique, along with a micro-CT image showing the overall porosity of the prepared material. The microporous structure of the scaffold is shown in an SEM image acquired at 5000 times magnification.

As shown, HEK293T cells treated with media containing lentiviral particles released from the scaffolds efficiently expressed the green fluorescent protein. As estimated by flow cytometry, the total number of the released particles reached approximately 40 percent. The most robust release effect was observed within the first 24 hours of the study, and gradually decreased over time, confirming that lentiviral particles coated on the scaffolds maintain their biological activity for up to 96 hours.

In vivo analysis of the blood vessels formed within a calvarial bone defect showed that new blood vessels, while undetectable in the control group of mice, were clearly visible in both PH and PHp groups eight weeks after scaffold transplantation. Additionally, while in both PH and PHp groups many collagen deposition sites were detected, only in the PHp group an increasing number of smaller vessels growing into scaffolds could be observed and the overall blood vessel area was significantly different. Once mastered, this technique can be done in two hours if it is performed properly.

While attempting this procedure, it's important to remember to immobilize the head of the mouse to keep it still during scanning to avoid any artifacts. Following this procedure, other methods such as photoacoustic imaging can be performed in order to provide additional data such as deep tissue images. After its development, this technique paved the way for researchers in the field of bone regeneration to investigate angiogenesis in transplants.

After watching this video, you should have a good understanding of how to visualize in vivo plasma formation in 3d scaffolds in real time, and microphoton microscopy.