The overall goal of this experiment is to quantify strain maps of skin during tissue expansion using isogeometric analysis and multi-view stereo. This method can help answer key questions in the field of reconstructive surgery with tissue expansion such as determining regional deformations induced by different expander shapes, sizes and inflation rates. The main advantage of this technique is that it uses computational analysis and flexible 3D imaging techniques.
Thus, it can be used in the operating room without altering the routine procedure. The implications of this technique extend towards therapy because it can be easily translated to the clinical setting to monitor skin deformation maps of human patients undergoing tissue expansion. To begin, prepare and anesthetize Yucatan mini pig for the surgery by shaving its dorsal skin with a razor.
Remove any remaining hair with tape and wipe the skin thoroughly with an isopropyl alcohol to clean it up from contaminants. Next, identify the dorsal midline on the animal's body, and using a pen, mark the dorsal limits of four grids so that they are positioned approximately two centimeters away from the dorsal midline. After marking on the skin the location of the first anterior grid, leave a three centimeter gap and mark the position of the posterior grid.
Finally, confirm that the marked anterior and posterior grid locations are symmetric with respect to the dorsal midline and positioned at the same supracaudal distance. Once the locations of the grid are marked, prepare a paper template of a 10 centimeter by 10 centimeter grid. To do this, heavily outline the grid by coloring the lines repetitively with a ballpoint pen and then trim the template.
Next, thoroughly moisten the desired skin area with isopropyl rubbing alcohol to facilitate the transfer of the grid tracing, and allow the excess alcohol to air dry, which will prevent the ink from leaking. Align the edges of the prepared grid template to the marking on the skin, and apply the grid to the skin with the ink side facing down, then gently press the grid template against the skin to ensure a complete transfer of the ink. After several seconds, slowly remove the grid from the skin and make sure the grid tracing was properly transferred.
Prior to tattooing, cover the grid tracing with petroleum jelly to decrease smearing of the ink, and using a tattooing device, cover the lines of the grid with tattoo ink. Once the first grid is tattooed, transfer to the animal's skin the tracing for the second grid, ensuring that both grids are positioned three centimeters apart from each other. After tattooing the second grid, confirm that both grids are located two centimeters lateral to the dorsal midline.
Once four grids are tattooed, confirm that they are positioned two centimeters away from the dorsal midline and spread apart in three centimeters one more time. To begin the surgery, mark on the skin of the anesthetized pig an incision site, located at the midpoint between two grids, then mark the desired location of the expander so that it is positioned in the upper part of the tattooed grid. Incise the skin with a surgical blade, and using scissors, create a tight subcutaneous tunnel connecting the initial incision with an area where the expander is going to be placed, then bluntly dissect out the pocket within the marked expander placement site.
Next, wash the created tunnel with an antibiotic solution to prevent infection and insert the expander to its subcutaneous pocket. Once the expander is in place, mark the sites of two incisions adjacent to the dorsal midline that will be used for an expander port placement, and make the cuts with the scalpel. Afterward, insert a hemostat through the caudal incision and dissect another subcutaneous tunnel connecting it with the incision previously used for expander placement, then seize the expander's tubing and pull it out through the caudal port incision.
Use the second incision to dissect a pocket for an inflation port placement, and insert the port in the pocket. Create with hemostat another subcutaneous tunnel connecting the two port placement incisions, then seize the port's tubing with the hemostat and pull it out through the same incision where the expander tubing is placed. After trimming both of the tubes, connect them through a metal adapter.
Secure the connection on both sides of the adapter by suturing around the tubing. Pull the tube through the initially made incision so that the tubing system is placed subcutaneously, then introduce a 21 gauge needle attached to a 50 milliliter syringe filled with liquid through the skin to the expander port and remove the air from the expander by pulling the plunger. Once all air is removed, inject to the expander port approximately 10 milliliters of the liquid to confirm that it fills properly.
Finally, close all three incisions by applying surgical sutures, then proceed by marking the incision site between the two grids on the opposite side of the pig's body to place the second expander diagonally in respect to the first expander placement site. After anesthetizing the animal, attach plastic flexible tape measures between the grids using surgical tape, then place the animal on one side to photo document the grid. It is critical to take the photographs from many different angles, making sure that the images are focused and contain primarily the object of interest rather than the background.
This will ensure accurate 3D geometry reconstruction, which is crucial to the analysis. Begin by positioning the camera above the animal but leaning to the caudal side to capture a shot approximately parallel to the ground where the tattooed grids are fully visible and fill the frame, then move in a circular pattern around the animal in an arch from the caudal to the rostral direction, taking photographs along the way and always capturing all of the tattooed grids in frame, with the grids filling the frame. Next, position the camera towards the ventral side to capture a shot angle that is approximately parallel to the ground, and take photographs in an arch from the ventral to dorsal region.
After photographing both sides of the animal, perform the inflation step according to the protocol in use. First, cleanse the skin of the animal around the port with isopropyl alcohol. Next, load a syringe with 0.9%injectable saline and attach it to a 25 gauge butterfly needle.
Next, attach the needle to the port, then inject the saline into the expander. After the injection, photo document the grids again, as before. To make the digital reconstruction, use commercially available software.
In this demonstration, MVS is used. Begin by selecting photo to 3D on the top left corner. Next, click add photos, browse to the location of the images and manually select the 30 photographs corresponding to a single model, then name the model and click create.
It can take several minutes for the model to be created. Next, click dashboard on the right to go back to see representative images of the geometric models. To save a model for analysis, select it at the bottom right corner, click downloads and select obj.
Use opensource software to process the geometric model. Import the file generated from the MVS software, then on the bottom of the 3D view, click on viewport shading and select texture. Look for a tab on the right of the 3D view with the submenu containing shading.
From shading, select shadeless, then right click on a geometry to select it, and on the bottom of a 3D view, choose edit mode to visualize the triangular mesh. Now, one by one, select the nodes of the grid by right clicking and highlighting the point. The coordinates for the point will appear on the tab on the right hand side of the 3D view.
Select and copy those coordinates, then paste them into a text file. Proceed to do this for all the points of the grid to save all 121 coordinates, then follow the instructions in the text protocol to ultimately quantify the deformation from this data. This methodology has been successfully employed to study the deformation induced by different expander geometries.
The variations in regional strains induced by sphere and crescent expanders were focused on. Both devices were filled to the same volume at every time point. From the reconstructed spline surfaces, deformations were calculated using a reference and a deformed grid.
Day zero was considered the reference. Comparing the end of every inflation step to the reference configuration results and the contour plots, the progression of the area change, theta, and the stretching in the two orthogonal directions, lambda, was then analyzed. Ultimately, the spacial variation of the contour plots revealed that skin was stretched more at the center of the expander than at the periphery.
Interestingly, even though both expanders were filled to the same volume, filling the sphere shaped expander induced larger deformations compared to the filling of the crescent shaped expander. After watching this video, you should have a good understanding as to how to quantify deformation maps of skin using multi-view stereo and isogeometric analysis. While attempting this procedure, it's important to photo document every step of the process with tape measures so the geometries can be scaled during analysis.
Following this procedure, other methods, like tissue biopsies, subsequent histochemical analysis, and genome sequencing can all be performed to answer additional questions about the underlying cellular responses to skin stretching.
