This work was supported by NIH grant 1R21EB021590-01A1 to Arun Gosain and Ellen Kuhl.

This protocol involves animal experiments. The protocol was approved by the IRB of Ann and Robert H. Lurie Children&#39;s Hospital of Chicago Research Center Animal Care and Use Committee to guarantee humane treatment of animals. The results for two expansion studies using this protocol have been published elsewhere 16,31.
Execution of this protocol requires a team with complementary expertise. The first part of the protocol describes ...

<ol>
	<li>Gosain, A. K., Zochowski, C. G., Cortes, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinements+of+tissue+expansion+for+pediatric+forehead+reconstruction:+a+13-year+experience.">Refinements of tissue expansion for pediatric forehead reconstruction: a 13-year experience.</a> Plast Reconstr Surg. 124, 1559-1570 (2009).</li><li>Neumann, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expansion+of+an+area+of+skin+by+progressive+distention+of+a+subcutaneous+balloon:+Use+of+the+Method+for+Securing+Skin+for+Subtotal+Reconstruction+of+the+Ear.">The expansion of an area of skin by progressive distention of a subcutaneous balloon: Use of the Method for Securing Skin for Subtotal Reconstruction of the Ear.</a> Plast Reconstr Surg. 19, 124-130 (1957).</li><li>De Filippo, R. E., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+and+growth:+the+molecular+and+physiologic+influences+of+tissue+expansion.">Stretch and growth: the molecular and physiologic influences of tissue expansion.</a> Plast Reconstr Surg. 109, 2450-2462 (2002).</li><li>Buganza Tepole, A., Joseph Ploch, C., Wong, J., Gosain, A. K., Kuhl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+skin:+A+computational+model+for+skin+expansion+in+reconstructive+surgery.">Growing skin: A computational model for skin expansion in reconstructive surgery.</a> J Mech Phys Solids. 59, 2177-2190 (2011).</li><li>LoGiudice, J., Gosain, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+Tissue+Expansion:+Indications+and+Complications.">Pediatric Tissue Expansion: Indications and Complications.</a> J Craniofac Surg. 14, 866-866 (2003).</li><li>Rivera, R., LoGiudice, J., Gosain, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+expansion+in+pediatric+patients.">Tissue expansion in pediatric patients.</a> Clin Plast Surg. 32, 35-44 (2005).</li><li>Marcus, J., Horan, D. B., Robinson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+expansion:+Past,+present,+and+future.">Tissue expansion: Past, present, and future.</a> J Am Acad Dermatol. 23, 813-825 (1990).</li><li>Patel, P. A., Elhadi, H. M., Kitzmiller, W. J., Billmire, D. A., Yakuboff, K. 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F., Huber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different-shaped+tissue+expanders+on+transluminal+pressure,+oxygen+tension,+histopathologic+changes,+and+skin+expansion+in+pigs.">Effects of different-shaped tissue expanders on transluminal pressure, oxygen tension, histopathologic changes, and skin expansion in pigs.</a> Plast Reconstr Surg. 76, 731-736 (1985).</li><li>van Rappard, J. H., Molenaar, J., van Doorn, K., Sonneveld, G. J., Borghouts, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-area+increase+in+tissue+expansion.">Surface-area increase in tissue expansion.</a> Plast Reconstr Surg. 82, 833-839 (1988).</li><li>Pusic, A. L., Cordeiro, P. 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Here we presented a protocol to characterize the deformations induced during a tissue expansion procedure in a porcine model using multi-view stereo (MVS) and isogeometric kinematics (IGA kinematics). During tissue expansion, skin undergoes large deformations going from a smooth and relatively flat surface to a dome-like 3D shape. Skin, like other biological membranes 34, responds to stretch by producing new material, increasing in area that can be then used for reconstructive purposes <sup class=...

Tissue expansion is a common technique in plastic and reconstructive surgery that grows skin in vivo for the correction of large cutaneous defects 1. Neumann, in 1957, was the first surgeon to document this procedure. He implanted a balloon below the skin of a patient and inflated it gradually over a period of several weeks to grow new tissue and resurface an ear 2. Skin, like most biological tissues, adapts to applied forces and deformations in order to reach mechanical homeostasis. When stretched beyond the physiological regime, skin grows 3,4. One of the central advantages of tissue expansion is the production of skin with proper vascularization and the same hair bearing, mechanical properties, color, and texture as the surrounding tissue 5.
After its introduction six decades ago, skin expansion has been widely adopted by plastic and reconstructive surgeons and is presently used to correct burns, large congenital defects, and for breast reconstruction after mastectomy 6,7. Yet, despite its widespread use, skin expansion procedures can lead to complications 8. This is partly due to the lack of sufficient quantitative evidence needed to understand the fundamental mechanobiology of the procedure and to guide the surgeon during preoperative planning 9,10. Key parameters in this technique are the filling rate, filling volume per inflation, the selection of the shape and size of the expander, and the placement of the device 11,12. Current preoperative planning relies largely on the physician&#39;s experience, resulting in a wide variety of arbitrary protocols that often differ greatly 13,14,15.
To address the current knowledge gaps, we present an experimental protocol to quantify expansion-induced deformation in a porcine animal model of tissue expansion. The protocol relies on the use of multi-view stereo (MVS) to reconstruct three-dimensional (3D) geometries out of sequences of two-dimensional (2D) images with unknown camera positions. Employing splines, representation of smooth surfaces leads to the calculation of the corresponding deformation maps by means of an isogeometric (IGA) description. The analysis of the geometry is based on the theoretical framework of continuum mechanics of membranes having an explicit parameterization 16.
Characterizing physiologically relevant deformations of living materials over long periods of time still remains a challenging problem. Common strategies for imaging of biological tissues include stereoscopic digital image correlation, commercial motion capture systems with reflective markers, and biplane video fluoroscopy 17,18,19. However, these techniques require a restrictive experimental setup, are generally expensive, and have been primarily used for ex vivo or acute in vivo settings. Skin has the advantage of being a thin structure. Even though it consists of several layers, the dermis is largely responsible for the mechanical properties of the tissue and thus the surface deformation is of primary importance 20; reasonable kinematical assumptions can be made regarding the out of plane deformation 21,22. Moreover, skin is already exposed to the outside environment, making it possible to use conventional imaging tools to capture its geometry. Here we propose the use of MVS as an affordable and flexible approach to monitor in vivo deformations of skin over several weeks without interfering majorly with a tissue expansion protocol. MVS is a technique that extracts 3D representations of objects or scenes from a collection of 2D images with unknown camera angles 23. Only in the last three years, several commercial codes have appeared (see list of materials for examples). The high accuracy of the model reconstruction with MVS, with errors as low as 2% 24, makes this approach suitable for the kinematic characterization of skin in vivo over long periods of time.
To obtain the corresponding deformation maps of skin during tissue expansion, points between any two geometric configurations are matched. Conventionally, researchers in computational biomechanics have used finite element meshes and inverse analysis to retrieve the deformation map 25,26. The IGA approach employed here uses spline basis functions which offer several advantages for the analysis of thin membranes 27,28. Namely, the availability of high degree polynomials facilitates representations of smooth geometries even with very coarse meshes 29,30. Additionally, it is possible to fit the same underlying parameterization to all the surface patches, which circumvents the need for an inverse problem to account for non-matching discretizations.
The method described here opens new avenues to study skin mechanics in relevant in vivo settings over long periods of time. In addition, we are hopeful that our methodology is an enabling step towards the ultimate goal of developing computational tools for personalized treatment planning in the clinical setting.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yucatan miniature swine</td>
			<td style="width:263px;">Sinclair Bioresources, Windham, ME</td>
			<td style="width:141px;">N/A</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Antibiotics</td>
			<td>Santa Cruz Animal Health, Paso Robles, CA</td>
			<td style="width:141px;">sc-362931Rx</td>
			<td>Ceftiofur, dosage 5mg/kg intramuscular</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chlorhexidine-based surgical soap</td>
			<td style="width:263px;">Cardinal Health, Dublin, OH</td>
			<td>AS-4CHGL(4-32)</td>
			<td>4% chlorhexidine gluconate surgical hand scrub</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tattoo transfer medium&#160;</td>
			<td style="width:263px;">Hildbrandt Tattoo Supply, Point Roberts, WA</td>
			<td>TRANSF</td>
			<td>Stencil thermal tattoo transfer paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine with epinephrine</td>
			<td style="width:263px;">ACE Surgical Supply Co, Brockton, MA</td>
			<td style="width:141px;">001-1423</td>
			<td>Lidocaine Hcl 1% (Xylocaine) - Epinephrine 1:100,000, 20ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td style="width:263px;">ZooPharm, Windsor, CO</td>
			<td style="width:141px;"></td>
			<td>1 mg/ml sustained release, dosage 0.01 mg/kg intramuscular</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital camera</td>
			<td style="width:263px;">Sony</td>
			<td style="width:141px;">Alpha33&#160;</td>
			<td>Standard digital camera with 18-35mm lens, 3.5-5.6 aperture. Used in automatic mode, no flash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tape measure</td>
			<td style="width:263px;">Medline, Mundelein, Illinois</td>
			<td style="width:141px;">NON171330</td>
			<td>Retractable tape measure, cloth, plastic case, 72inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue expanders</td>
			<td style="width:263px;">PMT, Chanhassen, MN</td>
			<td style="width:141px;">03610-06-02</td>
			<td>4cm x 6cm, rectangular, 120cc, 3610 series 2 stage tissue expander with standard port</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ReCap360</td>
			<td style="width:263px;">Autodesk</td>
			<td style="width:141px;">N/A</td>
			<td>MVS Software, Web application: recap360.autodesk.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blender</td>
			<td style="width:263px;">Blender Foundation</td>
			<td style="width:141px;">N/A</td>
			<td>Computer Graphics Software, open source: blender.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SISL</td>
			<td style="width:263px;">SINTEF</td>
			<td style="width:141px;">N/A</td>
			<td>C++ spline libraries, open source: https://www.sintef.no/projectweb/geometry-toolkits/sisl/</td>
		</tr>
	</tbody>
</table>

quantification of strain in a porcine model of skin expansion using multi-view stereo and isogeometric kinematics

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and giant congenital nevi. Despite its widespread use, planning and executing an expansion protocol is challenging due to the difficulty in measuring the deformation imposed at each inflation step and over the length of the procedure. Quantifying the deformation fields is crucial, as the distribution of stretch over time determines the rate and amount of skin grown at the end of the treatment. In this manuscript, we present a method to study tissue expansion in order to gain quantitative knowledge of the deformations induced during an expansion process. This experimental protocol incorporates multi-view stereo and isogeometric kinematic analysis in a porcine model of tissue expansion. Multi-view stereo allows three-dimensional geometric reconstruction from uncalibrated sequences of images. The isogeometric kinematic analysis uses splines to describe the regional deformations between smooth surfaces with few mesh points. Our protocol has the potential to bridge the gap between basic scientific inquiry regarding the mechanics of skin expansion and the clinical setting. Eventually, we expect that the knowledge gained with our methodology will enable treatment planning using computational simulations of skin deformation in a personalized manner.

This methodology has been successfully employed to study the deformation induced by different expander geometries: rectangle, sphere and crescent expanders 31,32. The results corresponding to the sphere and crescent expanders are discussed next. Figure 2 illustrates the three steps of MVS model reconstruction. The starting point is a collection of photographs from a static scene. The animal with the tattooed grids...

Watch this Scientific Journal Video about Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics at JoVE.com

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and ...

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.

quantification-strain-porcine-model-skin-expansion-using-multi-view

Research

JoVE Journal

Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...