We acknowledge all the co-authors of Jiang et al. 2020 for contributing to the development of SCAD methodology13. We thank Dr. Steffen Dietzel and the Bioimaging core facility at the Ludwig-Maximilans-Universit&#228;t for access to the Multiphoton system. Y.R. was supported by the Else-Kr&#246;ner-Fresenius-Stiftung (2016_A21), the European Research Council Consolidator Grant (ERC-CoG 819933), and the LEO Foundation (LF-OC-21-000835).

The model presented below provides a detailed step-by-step description of the generation of SCAD assay as briefly described in Jiang et al., 202013. SCAD sample preparations were performed after sacrificing the animals as per the international and the Government of Upper Bavaria guidelines. Animals were housed at the animal facility of the Helmholtz Centre Munich. The rooms were maintained with optimal humidity and constant temperature with a 12 h light cycle. Animals were supplied with food and water ad libitum.
1. SCAD tissue preparation
<ol>
	<li>Sacrifice newborn pups onpostnatal day 0 or day 1 (P0 or P1).</li>
	<li>Carefully excise at least 1.5 cm x 1.5 cm full-thickness dorsal back skin until the skeletal muscle layer using a sterile surgical scalpel.</li>
	<li>Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.</li>
	<li>Wash the excised tissue with 50-100 mL of cold DMEM/F-12 media to remove contaminating blood.</li>
	<li>Wash once with Hanks&#39; Balanced Salt Solution (HBSS) to maintain tissue and cell viability.</li>
	<li>Place the skin upside down (superficial fascia on top) in a 10 cm Petri dish containing DMEM/F12 media.</li>
	<li>Using a disposable 2 mm biopsy punch, excise full-thickness round skin pieces, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis to generate SCAD tissues.</li>
	<li>Prepare and fill 200 &#181;L of DMEM/F12 (without phenol red) complete media-DMEM/F12 media supplemented with 10% FBS, 1x GlutaMAX, 1x MEM non-essential amino acids, and 1x Penicillin/streptomycin into individual wells of a 96-well plate.</li>
	<li>Using sterile forceps, carefully transfer and fully submerge individual SCAD tissue upside down (fascia facing up) to the wells of a 96-well plate.</li>
	<li>Transfer the plate to a cell culture incubator maintained under standard conditions (37 &#176;C, 21%(v/v) oxygen, 5% (v/v) C02, and 95% humidity).</li>
</ol>
2. SCAD - Tissue culture
<ol>
	<li>Culture SCADs in an incubator for up to 5 days.</li>
	<li>On Day 2 and Day 4 of culture, replace the media with 200&#956;l&#160;of fresh pre-warmed DMEM/F12 complete media to ensure continued cell and tissue viability conditions. Replace treatment compounds during each media change. 
	NOTE: Ensure to leave 10 &#181;L of media in the well to avoid tissues sticking to the wells.</li>
	<li>Prepare SCADs for the following experiments: Live imaging (section 3), and Tissue Harvesting and 2D/3D immunofluorescence staining (section 4).</li>
</ol>
3. Live imaging of SCADs
<ol>
	<li>Prepare a minimum of 30 mL of 2%-3% (w/v) low melting agarose solution in PBS in a glass bottle by heating it in a microwave until boiling.</li>
	<li>Immediately transfer the bottle and cool the liquid agarose solution in a 40 &#176;C water bath.</li>
	<li>Transfer the SCAD tissue with fascia/Scar facing upward onto the center of a 35 mm dish.</li>
	<li>Embed the SCAD at room temperature (RT) by slowly transferring 40 &#176;C liquid agarose onto the 35 mm dish using a Pasteur pipette. Agarose polymerizes within 2 min.</li>
	<li>Add 2 mL of pre-warmed DMEM/F12 complete media (without phenol red indicator).</li>
	<li>Acquire time-lapse Images of day 0 SCADs up to 48 h using a confocal or a multiphoton microscope equipped with a suitable incubation system&#160;set to 37 &#176;C, 21% (v/v) oxygen, 5% (v/v) C02, and 95% humidity.</li>
</ol>
4. Tissue harvesting and 2D/3D immunofluorescence staining
<ol>
	<li>Wash the tissues at relevant time points by replacing the media with sterile PBS.</li>
	<li>Using sterile forceps, carefully transfer individual SCADs to 1.5 mL microcentrifuge tubes containing 500 &#181;L of 2% paraformaldehyde to fix the tissues overnight at 4 &#176;C.</li>
	<li>Wash the tissues three times with PBS and proceed with 2D/3D immunofluorescence staining.</li>
	<li>3D immunofluorescence staining
	<ol>
		<li>Permeabilize the tissues by incubating in a 1.5 mL microcentrifuge tube containing 500 &#181;L of PBS supplemented with 0.2% gelatin, 0.5% Triton-X100, and 0.01% Thimerosal (PBSGT)&#160;at RT for 24 h.</li>
		<li>Prepare an adequate amount of primary antibodies as per the manufacturer&#180;s instructions and incubate SCAD tissues in 150 &#181;L of PBSGT solution for 24 h at RT. 
		NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control tissues using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).</li>
		<li>Gently wash the SCADs three times with PBS to remove the unbound primary antibody.</li>
		<li>Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT.</li>
		<li>Gently wash the tissue for 30 min in PBS three times to remove excess unbound secondary antibodies.</li>
		<li>Transfer the tissue onto a 35 mm glass-bottom dish for confocal or multiphoton imaging. 
		NOTE: When using water immersion objectives, embed the SCADs in 2 % low melting point agarose to immobilize the tissue to prevent drifting during image acquisition</li>
	</ol>
	</li>
	<li>2D immunofluorescence staining
	<ol>
		<li>Transfer the tissue to a cryo-mold and gently fill with optimal cutting temperature (OCT) compound to completely immerse the tissue.</li>
		<li>Gently adjust the orientation of the tissue, ensuring the absence of air bubbles to obtain a cross-section or a vertical section.</li>
		<li>Place the mold on dry ice for 20-30 min and incubate the block at -80 &#176;C overnight.</li>
		<li>Set the blade temperature to -25 &#176;C and specimen block temperature to -17 &#176;C. Prepare 6 &#181;m cryo-section using a cryostat, transfer the sections onto an adhesion slide, and store the slides in -20 &#176;C freezer.</li>
		<li>Rinse the slides three times in PBS and incubate&#160;the slides in 5% Bovine Serum Albumin (BSA) w/v in PBST (PBS supplemented with 0.05% Tween) for 1 h.</li>
		<li>Add an appropriate amount of primary antibody to 150&#956;l PGST, and incubate overnight at 4 &#176;C 
		NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control sections using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).</li>
		<li>Gently wash the sections three times with PBS to remove the unbound primary antibody.</li>
		<li>Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT for 2 h.</li>
		<li>Gently wash the tissue for 5 min in PBS three times to remove excess unbound secondary antibodies.</li>
		<li>Mount the slides with a mounting medium containing DAPI and dry the slides in the dark at RT.</li>
		<li>Image the sections using a fluorescence microscope.</li>
	</ol>
	</li>
</ol>

<ol><li>Longaker, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+skin+wounds+in+the+fetal+environment+heal+with+scar+formation%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">Adult skin wounds in the fetal environment heal with scar formation.</a> Annals of Surgery. 219 (1), 65-72 (1994).</li>
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<li>Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+healing--Aiming+for+perfect+skin+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Wound healing--Aiming for perfect skin regeneration.</a> Science. 276 (5309), 75-81 (1997).</li>
<li>Correa-Gallegos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patch+repair+of+deep+wounds+by+mobilized+fascia%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Patch repair of deep wounds by mobilized fascia.</a> Nature. 576 (7786), 287-292 (2019).</li>
<li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+wounds+and+its+burden%3A+An+updated+compendium+of+estimates%5BTitle%5D)+AND+%22Advances+in+Wound+Care%22%5BJournal%5D)">Human wounds and its burden: An updated compendium of estimates.</a> Advances in Wound Care. 8 (2), 39-48 (2019).</li>
<li>Rinkevich, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+isolation+of+a+dermal+lineage+with+intrinsic+fibrogenic+potential%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.</a> Science. 348 (6232), 2151(2015).</li>
<li>Leavitt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prrx1+fibroblasts+represent+a+pro-fibrotic+lineage+in+the+mouse+ventral+dermis%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Prrx1 fibroblasts represent a pro-fibrotic lineage in the mouse ventral dermis.</a> Cell Reports. 33 (6), 108356(2020).</li>
<li>Driskell, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+fibroblast+lineages+determine+dermal+architecture+in+skin+development+and+repair%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Distinct fibroblast lineages determine dermal architecture in skin development and repair.</a> Nature. 504 (7479), 277-281 (2013).</li>
<li>Walmsley, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+fibroblast+harvest+reveals+surface+marker+shift+in+vitro%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+C%2C+Methods%22%5BJournal%5D)">Live fibroblast harvest reveals surface marker shift in vitro.</a> Tissue Engineering. Part C, Methods. 21 (3), 314-321 (2015).</li>
<li>Hakkinen, K. M., Harunaga, J. S., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+comparisons+of+the+morphology%2C+migration%2C+cell+adhesions%2C+and+actin+cytoskeleton+of+fibroblasts+in+four+different+three-dimensional+extracellular+matrices%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+A%22%5BJournal%5D)">Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices.</a> Tissue Engineering. Part A. 17 (5-6), 713-724 (2011).</li>
<li>Jiang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Injury+triggers+fascia+fibroblast+collective+cell+migration+to+drive+scar+formation+through+N-cadherin%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Injury triggers fascia fibroblast collective cell migration to drive scar formation through N-cadherin.</a> Nature Communications. 11 (1), 5653(2020).</li>
<li>Wan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connexin43+gap+junction+drives+fascia+mobilization+and+repair+of+deep+skin+wounds%5BTitle%5D)+AND+%22Matrix+Biology%3A+Journal+of+the+International+Society+for+Matrix+Biology%22%5BJournal%5D)">Connexin43 gap junction drives fascia mobilization and repair of deep skin wounds.</a> Matrix Biology: Journal of the International Society for Matrix Biology. 97, 58-71 (2021).</li>
<li>Molbay, M., Kolabas, Z. I., Todorov, M. I., Ohn, T. -L., Ertürk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guidebook+for+DISCO+tissue+clearing%5BTitle%5D)+AND+%22Molecular+Systems+Biology%22%5BJournal%5D)">A guidebook for DISCO tissue clearing.</a> Molecular Systems Biology. 17 (3), 9807(2021).</li>
<li>Ueda, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+clearing+and+its+applications+in+neuroscience%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">Tissue clearing and its applications in neuroscience.</a> Nature Reviews Neuroscience. 21 (2), 61-79 (2020).</li>
<li>Ertürk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nature Protocols. 7 (11), 1983-1995 (2012).</li>
<li>Wilhelm, K. -P., Wilhelm, D., Bielfeldt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+of+wound+healing%3A+an+emphasis+on+clinical+studies%5BTitle%5D)+AND+%22Skin+Research+and+Technology%3A+Official+Journal+of+International+Society+for+Bioengineering+and+the+Skin+%28ISBS%29+%5Band%5D+International+Society+for+Digital+Imaging+of+Skin+%28ISDIS%29+%5Band%5D+International+Society+for+Skin+Imaging+%28ISSI%29%22%5BJournal%5D)">Models of wound healing: an emphasis on clinical studies.</a> Skin Research and Technology: Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). 23 (1), 3-12 (2017).</li>
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All authors declare no competing interests.

Several models have already been developed to understand scar formation after injury. While a lot of advances have been rendered in this regard but, actual mechanisms are still not clear. In contrast to the previous technique, the SCAD model incorporates all cell types and cutaneous layers, thereby maintaining the complexity of native skin18,19. This methodology is capable of generating fundamental datasets that are important in understanding molecular mechanisms...

Wound healing is a process of restoration of breached wounds. Tissue injuries in invertebrates result in partial or complete regeneration. In contrast, mammals respond to deep injury by scarring, a process tailored to quickly seal wounds with dense plugs of matrix fibers that minimize the breached area and at the same time permanently deform the injured site1,2,3. Large skin burns or deep open wounds in mammals result in pathological phenotypes such as hypertrophic or keloid scars4,5. These exuberant scars cause a tremendous burden on clinical and global healthcare systems. In the US alone, scar management costs about $10 billion annually6,7. Therefore, the development of relevant methodologies are required to better understand the fundemental processes and mechanisms involved in scar formation.
In recent years, a wide range of studies in mice has revealed heterogeneous fibroblast populations with distinct functional potencies based on their origins in certain skin locations8,9,10. In back skin, Rinkevich et al., 2015, identified that a specific fibroblast population with an early embryonic expression of Engrailed-1 (En1), termed EPF (Engrailed positive fibroblast) contributes to cutaneous scarring upon wounding. Conversely,&#160; another fibroblast lineage with no history of engrailed expression, Engrailed negative fibroblast (ENF), does not contribute to scar formation8. Fate mapping of these En1 lineages using Cre-driven transgenic mouse lines crossed to fluorescence reporter mouse lines such as R26mTmG (En1Cre x R26mTmG) allows visualization of EPF and ENF populations.
Studying fibroblast migration in vivo over several days is limited by ethical and technical constraints. Furthermore, compound, viral and neutralizing antibody library screens to modulate pathways involved in scarring is technically challenging. Previously used in vitro or ex vivo models lack the ability to visualize fibroblast migration and scar formation in genuine skin microenvironments, uniformity in scar development, as well as tissue complexity that emulates in vivo skin environments11,12. To overcome the above limitations, we developed an ex vivo scarring assay termed SCAD (SCar-like tissue in A Dish)13,14. This simple assay can be performed by excising 2 mm full-thickness skin containing the epidermis, the dermis, and the subcutaneous fascia regions and culturing them in serum-supplemented DMSO media for up to 5 days. Scars generated from SCAD reliably replicate transcriptomic and proteomic hallmarks of in vivo scars. In addition, SCADs generated from relevant transgenic mouse lines (e.g., En1 mice) crossed with fluorescent reporter mouse lines allow the visualization of fibroblast migration dynamics and scar development at an unprecedented resolution. Furthermore, this model can be easily adapted for any high throughput applications (e.g., compound library, antibody library, or viral screening)13,14. In this article, we describe an optimized protocol to generate SCADs and subsequent downstream processing applications to study cellular and matrix dynamics in scar development.

<table><tbody><tr><td>10% Tween 20, Nonionic Detergent</td><td>Biorad Laboratories</td><td>1610781</td><td></td></tr><tr><td>Bovine serum albumin, Cold ethanol fract</td><td>Sigma</td><td>A4503-50G</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red-500 mL</td><td>LIFE Technologies</td><td>11039021</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190169</td><td></td></tr><tr><td>Epredia Cryostar NX70 Cryostat</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Epredia SuperFrost Plus Adhesion slides</td><td>Fisher scientific</td><td>J1800AMNZ</td><td>Adhesion slides</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin-500 mL</td><td>LIFE Technologies</td><td>&amp;nbsp;10500064</td><td></td></tr><tr><td>Fluoromount-G with DAPI</td><td>Life Technologies</td><td>00 4959 52</td><td>Mounting medium with DAPI</td></tr><tr><td>Forceps curved with fine points with guidepinstainless steel(tweezers)125 mm length</td><td>Fisher Scientific</td><td>12381369</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G2500-100G</td><td></td></tr><tr><td>GlutaMAX Supplement-100 mL</td><td>LIFE Technologies</td><td>35050038</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red-500 mL</td><td>LIFE Technologies</td><td>14025092</td><td></td></tr><tr><td>Ibidi Gas incubation system for CO&lt;sub&gt;2&lt;/sub&gt; and O&lt;sub&gt;2&lt;/sub&gt;</td><td>Ibidi</td><td>11922</td><td></td></tr><tr><td>Ibidi Heating system</td><td>Ibidi</td><td>10915</td><td></td></tr><tr><td>Leica SP8 upright microscope - Multiphoton excitation 680&amp;ndash;1300 nm</td><td>Leica</td><td></td><td>Equipped with a 25x water-dipping objective (HC IRAPO L 25x/1.00&amp;thinsp;W) in combination with a tunable laser (Spectra-Physics, InSight DS&amp;thinsp;+&amp;thinsp;Single)</td></tr><tr><td>Non Essential Amino Acids</td><td>LIFE Technologies</td><td>11140035</td><td></td></tr><tr><td>NuSieve GTG Agarose ,25 g</td><td>Biozym /Lonza</td><td>859081</td><td></td></tr><tr><td>OCT Embedding Matrix</td><td>Carlroth</td><td>6478.1</td><td></td></tr><tr><td>Paraformaldehyde, 16% W/V AQ. 10 x10 mL</td><td>VWR International</td><td>43368.9M</td><td></td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Stiefel Biopsy-Punch 2 mm</td><td>Stiefel</td><td>270130</td><td></td></tr><tr><td>Straight Sharp/Sharp Dissecting Scissors 11.4 cm</td><td>Fisher Scientific</td><td>15654444</td><td></td></tr><tr><td>Thimerosal Bioxtra, 97%&amp;ndash;101%</td><td>Sigma-Aldrich</td><td>T8784-1G</td><td></td></tr><tr><td>Zeiss Axioimager M2 upright microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing scar development using scad assay - an ex-situ skin scarring assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or &#34;SCar-like tissue in A Dish&#34; that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids&#160;researchers in understanding fundamental processes and mechanisms of wound repair,&#160;directly exploring the effects of modulators on wound healing outcomes.

Generation of SCADs can be separated into three essential steps: Harvesting&#160;back skin from P0-P1 mice, generating of full-thickness biopsy punches, and subsequent culture of individual scads up to 5 days in 96-well plates. As a readout, this assay can further be applied to analyze the spatial and temporal aspects of scarring. The spatial analysis utilizes 2D and 3D immune-labeling of tissues to study spatial localization of cellular and matrix components within developing scar tissue. Spatiotemporal studies allow vi...

Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. ...

visualizing-scar-development-using-scad-assay-an-ex-situ-skin

Research

JoVE Journal

Biology

2.8K Views.  Helmholtz Zentrum München. This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Video: Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Medicine

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 ...

Bioengineering

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 ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Developmental Biology

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 ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Immunology and Infection

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

To closely mimic the mode of burn injuries requires the interplay between clinical observation and studies in animal models. In this study, a swine model of severe burn injury was established to assess an experimental dressing in physiological and pathophysiological settings.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

Summary

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In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

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Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

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Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing