The overall goal of this procedure is to visualize blood vessels in tissue engineered constructs. This is accomplished by creating a standardized full skin defect on the backs of mice. Next, a sample of the tissue engineered material is implanted into the skin defect.
Then at the end of the observation time, the material is implanted and placed in a trans illumination device. Finally, high resolution photos of the vascular network are acquired with a standard digital camera. Ultimately, the vessel network is analyzed by digital segmentation and quantification using the Vest egg tool software.
The main advantage of this technique over existing methods like microscopy, computer tomography, or magnetic resonance imaging, is that allows time and cause effective visualization and quantification of vascular processes within biomaterials focusing all new functional vessels. This method can provide insight into vascularization of dermal replacement materials. It can also be applied to other model organisms or to study vascularization in other organs.
To begin, use 12 millimeter biopsy punches to generate samples of scaffolds, such as from bovine, collagen based dermal replacement materials cut 14 millimeter diameter round tetin mesh pieces for placing under each scaffold. After anesthetizing a mouse, checking the depth of anesthesia with a toe pinch and removing the back fur according to the text protocol. Place the animal in a prone position and use a fine tip permanent pen to mark the midline of the mouse's back.
Define an excision area that is not too far coddly to prevent the animal from removing the scaffold. Using a 10 millimeter biopsy punch, create round bilateral defects by carefully forcing the punch against the skin. To delineate the excision area with forceps, gently lift off the marked skin and use surgical scissors to incise along the marked circle.
If bleeding occurs, use sterile gauze to carefully compress the area to make space for the Titanic mesh. Extend the separation of skin and underlying tissue at the wound's border and additional two to four millimeters. Place the mesh into the defect directly on the wound bed and under the wound edges.
Then place the scaffolds directly over the mesh. Use four to six single knots to suture the scaffold, so the adjacent wound edges leaving the edges slightly over the scaffold. Then suture a transparent wound dressing above the defects to protect the scaffold while allowing monitoring of the wound area.
Evaluat, evaluate the general state of the animal daily by monitoring motor activity, body weight, signs of pain, tolerance to the dressing and auto mutilation. Also, monitor the wound area for bleeding local and systemic signs of infection and the position of the dressing. After euthanizing the animal at the desired time point.
According to the text protocol, use a permanent marker to mark the excision site that includes the scaffolds. Use a pair of scissors or a scalpel to incise the skin along the marked lines through a blunt excision, detach the entire skin, including the scaffolds and mesh from the underlying tissue, placed a tissue stretched out upside down into a Petri dish. To visualize the sample, place it over a trans illumination device.
In macro mode, take pictures of the complete scaffolds and equally sized areas of normal skin and store the images in a TIFF format for further digital analysis. After downloading the ves EQ tool software, open an image and select a region of interest for analysis by pressing the backspace key. Then press invert.
To visualize the vascular network in white, choose image vessel enhancement filter hysteresis thresholding top hat transformation, and calculate vessel enhancement in all previous steps. Scroll to zoom in and out the picture. Segment the vessel map by selecting the first threshold or vessel coverage so that every pixel that is even remotely vessel like will be labeled as a vessel.
Press calculate. To preview your selection, select the second threshold or background coverage so that only those pixels that are particularly vessel like will be labeled as vessels. To calculate the length of the vessels and the overall area covered by them, click on image, image statistics and binary image statistics.
Vessel length is best estimated by pre thinning the vascular network to one pixel width. For this, go to image morphological filters and skeletonizing before the calculation. The ratio of vessel area to vessel length gives a value for the vessel size in the region of interest.
If necessary, eliminate artifacts by selecting only the vessels of interest. By clicking under the selection mode, press backspace to copy the selected vessels into a new image. Finally, analyze the native skin area under the same parameters as a scaffold, assigning a value of 100%to the native tissue and relate the scaffold to that value.
For example, if the percentage of white pixels is 30%in the normal tissue and 15%in the scaffold, this represents a 50%vascularization of the total area of the scaffold. Please note that the values correspond to the whole picture area. Therefore, it is necessary to correct it to the area of the region selected as shown here.
Two weeks after implantation tissue, trans illumination allowed clear visualization of vascular structures of up to 30 micrometers in width in a whole tissue sample that included both native tissue and implanted scaffolds. Using digital segmentation to analyze the explanted tissue vascularization levels of 62.28 plus or minus 8.6%were observed in the scaffold compared to the native skin. While attempting this procedure, it's important to remember to create reproducible full skin defects and fix the biomaterial reliably to the wound.
Following this procedure, expanded tissue can be used for further analysis such as histology or RNA and protein expression.
