We would like to thank Mr Paolo Matteuci and Mr George Smith for providing patient tissue. We are also grateful to Miss Amber Rose Stafford for assisting with tissue collection and the Daisy Appeal for providing laboratory facilities.

Human skin was obtained from patients undergoing reconstructive surgery at Castle Hill Hospital and Hull Royal Infirmary (Hull, UK) under full informed, written patient consent, institutional guidelines, and ethical approval (LRECs: 17/SC/0220 and 19/NE/0150). Non-diabetic skin was collected from patients undergoing routine surgery (mean age = 68). Diabetic skin was selected from donors who had established type II diabetes and a history of ulceration (mean age = 81). Samples from surgery were transported in holding media and processed immediately upon arrival at the laboratory. All experimental steps using unfixed human tissue were performed at Biosafety Level-2 (BSL-2) in a class II laminar flow biosafety cabinet.
1. Preparation of skin culture media and staining reagents
NOTE: All reagent and consumable details are provided in the Table of Materials. Ensure all reagents and equipment used for the processing and culture of human tissue are sterile. Sterilize instruments prior to the use and decontaminate with disinfectant following contact with the tissue. Decontaminate waste products in 1% disinfectant before disposal.
<ol>
	<li>Holding media: Supplement high glucose Dulbecco&#39;s Modified Eagle Medium (DMEM) with 2 mM L-glutamine and 4% (v/v) antibiotic-antimycotic solution.</li>
	<li>Hank&#39;s balanced salt solution (HBSS) with antibiotics: Add 4% (v/v) antibiotic-antimycotic solution to HBSS. Store at 4 &#176;C until use.</li>
	<li>Dulbecco&#39;s phosphate buffered saline (DPBS): Prepare DPBS by dissolving 9.6 g of DPBS powder per liter of distilled water (dH2O). Autoclave to sterilize and store at 4 &#176;C until use.</li>
	<li>Human skin growth media: Supplement high glucose DMEM with 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic solution and 10% (v/v) fetal bovine serum. Store at 4 &#176;C until use.</li>
	<li>Skin fixative: To 450 mL of dH2O, add 40 mL of formaldehyde solution, 10 mL of glacial acetic acid, 4.5 g of sodium chloride and 0.25 g of alkyltrimethylammonium bromide. Store at room temperature (RT) and use within a few days. 
	CAUTION: Fixative is hazardous (irritant and flammable). Handle with care and dispose of via an appropriate route.</li>
	<li>Phosphate buffered saline (PBS): Prepare PBS for whole-mount staining by adding 6 g of sodium chloride to 100 mL of phosphate buffer solution and 900 mL of dH2O.</li>
	<li>Staining wash buffer: Dissolve 0.5% (v/v) Triton X-100 in PBS.</li>
	<li>Blocking buffer: Add 0.2% (w/v) sodium azide and 2% (v/v) animal serum to staining wash buffer. Store at 4 &#176;C for up to two weeks. 
	NOTE: Block in the serum of the secondary antibody host species. Sodium azide will prevent bacterial growth during incubation.</li>
	<li>DAPI working solution: Prepare a 5 mg/mL stock of 4&#8242;,6-diamidino-2-phenylindole (DAPI) in dimethyl sulfoxide. Dilute the stock 1:1,000 in staining wash buffer to give a 5 &#181;g/mL DAPI working solution.</li>
	<li>Peroxidase block: Add 0.3% (v/v) hydrogen peroxide to staining wash buffer. Store at 4 &#176;C until use. Keep in the dark to prevent decomposition.</li>
	<li>ABC-HRP Kit:
	<ol>
		<li>HRP-conjugated secondary antibody: 1 drop of biotinylated rabbit anti-goat IgG in 5 mL of staining buffer. Store at 4 &#176;C for up to two weeks. 
		&#8203;NOTE: The kit/secondary used will depend on the host species of the primary antibody.</li>
		<li>Avidin-biotin complex (ABC) reagent: 2 drops of reagent A and 2 drops of reagent B in 5 mL of staining wash buffer. ABC reagent should be prepared at least 30 min prior to use. Store at 4 &#176;C for up to two weeks.</li>
	</ol>
	</li>
	<li>Peroxidase substrate: 3 drops of reagent 1, 2 drops of reagent 2, 2 drops of reagent 3 and 2 drops of hydrogen peroxide in 5 mL of dH2O. Peroxidase substrate should be freshly prepared immediately before use and cannot be stored.</li>
</ol>
2. Preparation of skin for wounding
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
<ol>
	<li>Collect the skin in holding media and transport to the BSL-2 cabinet.</li>
	<li>Place the skin dermis side down within a 90 mm sterile Petri dish and remove adipose tissue with sterile scissors.</li>
	<li>Place the skin in a 50 mL tube containing 25 mL of HBSS with antibiotics for 10 min at RT. Shake intermittently to remove any residual blood and adipose tissue.</li>
	<li>Repeat step 2.3 using a new 50 mL tube.</li>
	<li>Place the skin in a fresh 50 mL tube containing 25 mL of HBSS, this time without antibiotics for 10 min at RT. Shake as in step 2.3.</li>
	<li>Perform a final skin rinse by placing skin in a new tube with 25 mL of DPBS. The skin is now ready to wound.</li>
</ol>
3. Creating ex vivo human skin wounds
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
<ol>
	<li>Prepare the skin culture dishes prior to wounding. In a 60 mm Petri dish, stack two sterile absorbent pads and add 4 mL of human skin media via the side of the dish. Place a sterile nylon filter membrane onto the absorbent pad stack. 
	NOTE: Skin media can be altered depending on the required treatment conditions. Up to three wound explants may be cultured on each stack.</li>
	<li>Dry the dermal side of the skin on sterile gauze in a 90 mm Petri dish to remove residual DPBS. 
	NOTE: This prevents the skin from sliding around when wounding.</li>
	<li>Place the skin dermis side down on a clean 90 mm Petri dish lid and dab the epidermis dry with fresh sterile gauze. 
	NOTE: It is easier to wound the skin in a Petri dish lid than the base. Subsequent work should be carried out quickly to prevent the skin drying out.</li>
	<li>Holding the skin taut, press a 2 mm biopsy punch against the skin and twist gently. Do not punch entirely through the skin. 
	NOTE: Partial thickness wounds are designed to punch through the epidermis and partially into the dermis. There may be donor-to-donor and site-to-site variability in the force required to create the partial thickness wound.</li>
	<li>Use curved toothed tissue forceps to pick up each side of the 2 mm wound and hook curved iris scissors under the 2 mm wound to cut it out uniformly.</li>
	<li>Biopsy around the central 2 mm wound using a 6 mm biopsy punch to create a 6 mm explant with a partial thickness 2 mm wound in the center. 
	NOTE: A 6 mm biopsy punch may be used to score the skin to mark out where each 2 mm wound should be. Take care not to pierce through the tissue entirely. Create wound explants in a honeycomb pattern to reduce wastage.</li>
	<li>Place wound explants epidermis side up on the nylon filter membrane stack (prepared in step 3.1). 
	&#8203;NOTE: When handling wound explants, be careful not to damage the central wound. Use small forceps and pick up each explant at opposite sides.</li>
	<li>Incubate wounds at 32-37 &#176;C and 5% CO2 in a humidified atmosphere (90-95%) for 1-7 days. Replace the media every 2-3 days.</li>
</ol>
4. Whole-mount staining of ex vivo wounds
NOTE: This section describes immunofluorescence and immunoperoxidase staining methods. Mix all reagents well before use.
<ol>
	<li>Fluorescent staining method
	<ol>
		<li>Collect wound explants in 1.5 mL microcentrifuge tubes containing 500 &#181;L of skin fixative and incubate at 4 &#176;C overnight. 
		NOTE: The fixative used in this protocol works well for the described antibodies. Optimization will be required for other antibodies. Tissue fixation longer than 24 h may lead to over-fixation.</li>
		<li>The following day remove the fixative and replace with 1 mL of staining wash buffer. Biopsies can be stored in staining wash buffer at 4 &#176;C up to 2 weeks prior to staining. 
		&#8203;NOTE: For all wash buffer steps, use a serological pipette or pipette tip, taking care not to damage the wound.</li>
		<li>Aspirate the staining wash buffer and perform one more rinse with 1 mL of staining wash buffer.</li>
		<li>Calculate the amount of blocking buffer required for steps 4.1.5-4.1.6 (number of samples x 300 &#181;L = amount of blocking buffer in &#181;L). Make extra buffer if required.</li>
		<li>Add 150 &#181;L of blocking buffer to each sample and incubate for 1 h at RT. For all staining steps, ensure each sample is sufficiently covered and that there are no bubbles covering the biopsy wound surface. 
		&#8203;NOTE: This step onwards can be performed in 1.5 mL microcentrifuge tubes or in a 48 well plate. If using a 48 well plate, incubate the wounds face down in each well.</li>
		<li>Dilute the primary antibody in the remaining blocking buffer. 
		&#8203;NOTE: Anti-mouse keratin 14 (K14) diluted 1:1,000 in blocking buffer works well. Optimize this step for use with other antibodies or multiple probes.</li>
		<li>Aspirate the blocking buffer and add 150 &#181;L primary antibody per well/microcentrifuge tube. Incubate wound explants in primary antibody at 4 &#176;C overnight.</li>
		<li>The next day, aspirate the primary antibody and rinse in staining wash buffer containing 0.2% sodium azide for 1 h at RT (500 &#181;L per sample).</li>
		<li>Perform three more rinsing steps using staining wash buffer (30 min per wash, 500 &#181;L per sample).</li>
		<li>Dilute the fluorescently conjugated secondary antibody in staining wash buffer (e.g., goat anti-mouse 488 at 1:400 dilution).</li>
		<li>Calculate the required amount of secondary antibody (number of samples x 150 &#181;L = amount in &#181;L).</li>
		<li>Add 150 &#181;L of secondary antibody to each well/microcentrifuge tube. Incubate for 1 h at RT. Perform incubation steps 4.1.10 - 4.1.16 in the dark as the secondary antibody is light sensitive. 
		&#8203;NOTE: This step can be performed at 4 &#176;C overnight if required. Optimize the concentration of secondary antibody required for adequate signal and limited background staining.</li>
		<li>Remove the secondary antibody and perform 3 x 30 min rinses with staining wash buffer (500 &#181;L per sample).</li>
		<li>Discard the leftover wash buffer and calculate the amount of DAPI working solution required (as per step 4.1.11).</li>
		<li>Counterstain each explant with 150 &#181;L of DAPI working solution for 10 min at RT. 
		&#8203;NOTE: DAPI will stain cell nuclei blue. Hoechst dye can be used as an alternative to DAPI.</li>
		<li>Perform two final 30 min washes with staining wash buffer (500 &#181;L per sample). Biopsies can be stored in staining wash buffer at 4 &#176;C in the dark up to two weeks prior to imaging.</li>
	</ol>
	</li>
	<li>Brightfield staining method.
	<ol>
		<li>Perform steps 4.1.1 - 4.1.3.</li>
		<li>Quench endogenous peroxidase activity with peroxidase block at 4 &#176;C overnight. 
		NOTE: This step is important when using an HRP-conjugated antibody to reduce non-specific background staining from the tissue. Highly vascularized tissue will contain more endogenous peroxidase activity.</li>
		<li>Discard the peroxidase block and rinse twice for 30 min in staining wash buffer.</li>
		<li>Perform steps 4.1.4 - 4.1.8. 
		&#8203;NOTE: Washes after step 4.1.7 are particularly important to remove sodium azide from the samples. If sodium azide is not adequately removed, it will inactivate the HRP and interfere with staining detection.</li>
		<li>Add 150 &#181;L HRP-conjugated secondary antibody to each well/microcentrifuge tube and incubate overnight at 4 &#176;C or 1 h at RT.</li>
		<li>Remove the secondary antibody and perform 3 x 30 min washes in staining wash buffer.</li>
		<li>Add 150 &#181;L ABC reagent to each well/microcentrifuge tube and incubate overnight at 4 &#176;C or 1 hour at RT.</li>
		<li>Aspirate the ABC reagent and perform 3 x 30 min washes in staining wash buffer.</li>
		<li>Add 150 &#181;L peroxidase substrate to one explant and determine the time required to detect a noticeable color change. 
		&#8203;NOTE: Choose a sample where strong staining is expected. In this case, a red ring to show the migrating epidermis (K14). 3,3&#39;-diaminobenzidine-4, or any other appropriate chromogenic substrate, may be used as a replacement for this peroxidase substrate.</li>
		<li>Once a color change is observed, remove the peroxidase substrate, and replace with 1 mL of dH2O.</li>
		<li>Repeat the peroxidase substrate detection for the other explants, incubating for the time determined in step 4.2.11.</li>
		<li>Rinse all explants with 1 mL of dH2O to remove residual peroxidase substrate. Although explants may be stored up to one week at 4 &#176;C prior to imaging, it is better to image them as soon as possible to prevent leaching of the peroxidase substrate into the dH2O over time.</li>
	</ol>
	</li>
</ol>
5. Imaging and quantification
<ol>
	<li>Fluorescent imaging 
	NOTE: Fluorescent imaging is performed using a confocal laser-scanning microscope. However, an inverted fluorescent microscope may be sufficient for acquiring 2D images to quantify wound closure rates. When selecting secondary antibodies, ensure that the chosen fluorochromes are compatible with the excitation and emission spectra of the microscopy equipment available.
	<ol>
		<li>Use a confocal laser-scanning microscope equipped with a 2.5x, 10x and 20x objective, x-y-z motorized stage, digital camera, and acquisition software. Switch on the transmitted light detector (TPMT) to allow easy visualization of each biopsy and to enable measurement of total wound closure. Alternatively, measure each wound via brightfield microscopy following fluorescence imaging.</li>
		<li>Place a 60 mm Petri dish base on the imaging platform and add a thin layer (around 1 mL) of DPBS. 
		&#8203;NOTE: If too much DPBS is used, the biopsy will move around during imaging. Alternatively use a 48 well plate if a plate holder is available.</li>
		<li>Use small tissue forceps to transfer wound explants from wells/microcentrifuge tubes to the Petri dish containing DPBS. Place the biopsy wound side down in the Petri dish.</li>
		<li>Use the eyepiece and fluorescent lamp to locate and focus on the wound. If bubbles are trapped under the sample in the field of view, pick up the wound with tissue forceps and reposition.</li>
		<li>Set up the imaging software, ensuring equal pinhole size between channels for optimum confocality. For this, check the value of one airy unit for each channel and select the largest value. Select scan speed, image quality and averaging. 
		&#8203;NOTE: The fluorochromes of the conjugated secondary antibodies and the chosen counterstain (e.g., DAPI) will dictate the channels required.</li>
		<li>Switch on the live acquisition software and adjust the laser power and gain of each channel to the levels required to visualize staining. Reduce background noise by increasing the digital offset.</li>
		<li>Position the wound in the center of the imaging plane. 
		&#8203;NOTE: If the wound does not fill the entire image due to using a smaller objective or creating a larger wound, take a panel of images and stitch them together (manually or with a tiling function in the relevant imaging software).</li>
		<li>Acquire images of the wound biopsies. Use the same imaging settings between explants. 
		NOTE: Higher power images will allow assessment of tissue structures and cellular marker expression and location.</li>
		<li>Collect serial Z stacks through the wound, especially where the tissue is not completely flat against the Petri dish. Use analysis software to collapse the Z stack into a single maximum intensity projection image.</li>
	</ol>
	</li>
	<li>Brightfield imaging 
	NOTE: Brightfield imaging of immunoperoxidase stained biopsies can be performed in multiple ways.
	<ol>
		<li>Inverted microscope imaging: Prepare wound explants for imaging by placing them in a Petri dish/well as described in steps 5.1.2-5.1.3. Acquire digital images under brightfield illumination on an inverted microscope equipped with a digital camera. Stitch together multiple images if required.</li>
		<li>Wireless digital microscope imaging: Use a wireless digital microscope connected to a phone or laptop to obtain high quality images in a cost-effective manner. Place explants wound side up onto some tissue and remove any residual dH2O (or staining wash buffer) from sample storage. Position the wound explant in the center of the microscope field of view. Acquire images using the connected camera.</li>
	</ol>
	</li>
	<li>Quantification 
	&#8203;NOTE: Percentage wound closure can be quantified in any software that allows freehand shapes to be drawn and measured. ImageJ can be used to perform quantification as follows:
	<ol>
		<li>Open the image to be quantified in ImageJ software.</li>
		<li>Use the freehand shape tool to draw around the outside of the re-epithelialized wound where it meets the normal skin. Press M (or Analyze | Measure) to acquire an &#39;outer&#39; area measurement. 
		NOTE: The re-epithelialized wound tissue texture differs from normal skin. The images do not need to be scaled prior to this type of analysis.</li>
		<li>Use the freehand shape tool to draw around the open wound area. This is where the open wound meets the inside edge of the re-epithelializing tissue. Press M (or Analyze | Measure) to acquire an &#39;inner&#39; area measurement.</li>
		<li>Use the following equation to deduce percentage wound re-epithelialization/closure: 
		% Closure = (Outer Wound Area - Inner Wound Area) / (Outer Wound Area) x 100 
		NOTE: Percentage area coverage of antibodies can be deduced in the same way (e.g., K14) or as a percentage of the total wound area. Percentage intensity can also provide semi-quantitative information about tissue level expression of markers of interest, while high power imaging presents expression data at the cellular level.</li>
	</ol>
	</li>
</ol>

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(110), e52564 (2016).</li><li>Pupovac, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+immunocompetent+3D+skin+models.">Toward immunocompetent 3D skin models.</a> Advanced Healthcare Materials. 7 (12), 1701405 (2018).</li><li>Ata&#231;, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+hair+on-a-chip:+in+vitro+skin+models+versus+ex+vivo+tissue+maintenance+with+dynamic+perfusion.">Skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.</a> Lab on a Chip. 13 (18), 3555-3561 (2013).</li><li>Kim, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscale,+full-thickness,+human+skin+on+a+chip+assay+simulating+neutrophil+responses+to+skin+infection+and+antibiotic+treatments.">A microscale, full-thickness, human skin on a chip assay simulating neutrophil responses to skin infection and antibiotic treatments.</a> Lab on a Chip. 19 (18), 3094-3103 (2019).</li><li>Jardet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+human+Th17-driven+ex+vivo+skin+inflammation+model.">Development and characterization of a human Th17-driven ex vivo skin inflammation model.</a> Experimental Dermatology. 29 (10), 993-1003 (2020).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metformin+attenuates+diabetes-induced+tau+hyperphosphorylation+in+vitro+and+in+vivo+by+enhancing+autophagic+clearance.">Metformin attenuates diabetes-induced tau hyperphosphorylation in vitro and in vivo by enhancing autophagic clearance.</a> Experimental Neurology. 311, 44-56 (2019).</li><li>Demirovic, D., Rattan, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+induces+stress+response+and+hormetically+modulates+wound+healing+ability+of+human+skin+fibroblasts+undergoing+ageing+in+vitro.">Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.</a> Biogerontology. 12 (5), 437-444 (2011).</li><li>Wilkinson, H. N., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+senescence:+A+functional+link+between+diabetes+and+ageing.">Wound senescence: A functional link between diabetes and ageing.</a> Experimental Dermatology. 30 (1), 68-73 (2020).</li><li>Fisher, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fragmentation+promotes+oxidative+stress+and+elevates+matrix+metalloproteinase-1+in+fibroblasts+in+aged+human+skin.">Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.</a> The American Journal of Pathology. 174 (1), 101-114 (2009).</li><li>Quan, T., Little, E., Quan, H., Voorhees, J. J., Fisher, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+matrix+metalloproteinases+and+collagen+fragmentation+in+photodamaged+human+skin:+impact+of+altered+extracellular+matrix+microenvironment+on+dermal+fibroblast+function.">Elevated matrix metalloproteinases and collagen fragmentation in photodamaged human skin: impact of altered extracellular matrix microenvironment on dermal fibroblast function.</a> Journal of Investigative Dermatology. 133 (5), 1362 (2013).</li></ol>

The authors declare no conflicts of interest.

In this experimental protocol, we describe an optimized method for evaluating wound closure in human ex vivo skin using whole-mount tissue staining. This is an important resource to allow critical evaluation of potential wound treatments, and to provide better understanding of the human wound repair response. We have published healing assessment in ex vivo skin wounds previously12,13, yet in these reports the whole-mount staining approach was not used to measure wound closure. Whole-mount staining is far easier and requires less technical experience than standard histology, which involves paraffin or OCT embedding and sectioning of samples. The whole-mount procedure also reduces experimental variability, allowing quantification of the entire wound and not just a single transverse section at a defined position within the tissue (see Figure 4B for comparative illustration). We fully support the importance of quantifying healing of the entire non-symmetrical wound structure, as clearly outlined by Rhea and Dunnwald for murine acute wounds14. These authors showed the importance of serially sectioning in vivo excisional wounds for reproducible and precise measurements of wound morphology. Serial sectioning could equally be applied to human ex vivo wounds; however, for accurate quantification of wound closure and re-epithelialization, high throughput whole-mount staining should be the preferred method. We note that this whole-mount staining protocol should also be compatible with subsequent processing (wax or OCT) for traditional histological analysis.
Whole-mount staining is not without disadvantages. While it affords higher reproducibility in wound healing experiments, it does require the use of more tissue for analysis than standard histological techniques. This may be an issue where tissue access is limited, particularly where multiple antibodies need to be assessed. An alternative approach would be to employ an incisional wounding method where wound width is relatively uniform and variability is reduced (as shown in mouse and human wounds15,16). However, excisional wounds remain more applicable to most pathological wound types17.
In this study, 2 mm partial thickness wounds were created within the center of 6 mm skin explants. This method may be optimized for alternative excisional wound and explant sizes at different skin depths18. In addition, the force required to generate wounds will vary between donors, where aged skin will require less force to biopsy. We would also avoid using skin displaying prominent stretch marks or other structural alterations. We have validated a range of antibodies to consider different aspects of the ex vivo healing response. This protocol may also be used with other skin-relevant antibodies, where antibody concentrations and incubation times will need to be optimized. Nevertheless, we believe our protocol is most suited to absolute quantification of total wound closure, followed by spatial assessment of specific proteins of interest. While whole-mount provides reduced resolution of immunolocalization versus standard histological analysis of tissue sections, it provides additional 3D information that is missing from standard 2D histology.
One caveat of assessing healing in ex vivo skin versus in vivo models is that it lacks a systemic response. An important aspect of wound repair is inflammation and subsequent tissue granulation, which is caused by an influx of inflammatory cells and endothelial cells from the vasculature19. Despite this limitation, ex vivo skin still provides a better recapitulation of clinical healing than cell-based wound assays. In vitro experiments in general involve single cell type monolayers or co-cultures grown on tissue culture plastic, whereas ex vivo skin provides a native environment to explore cell behavior. More recently, a number of skin equivalent systems have emerged, where skin is grown in a laboratory setting from artificial matrix and isolated skin cells20,21. Although these models mimic human skin better than most in vitro approaches, they still do not fully simulate the native tissue environment and are generally too fragile to injure reproducibly. Additionally, we (and others) have demonstrated that ex vivo human skin tissue retains resident immune cells, which will no doubt contribute to repair22,23. Future work should now focus on extending the viability and immunocompetency of the ex vivo model for late-stage healing assessment24. One option is further advancement of promising organ-on-a-chip technologies capable of prolonging tissue viability and maintaining native skin architecture for up to two weeks in culture25. Ex vivo models have also begun to consider the importance of the skin inflammatory response by successfully incorporating immune cells, such as neutrophils, into the host tissue26 or injecting host tissue with antibodies to elicit an immune reaction27. We expect that these findings will pave the way for development of more refined and translatable methods in the future.
A major benefit of using ex vivo skin to measure wound closure is the ability to compare healing rates in healthy (e.g., non-diabetic) versus pathological (e.g., diabetic or aged) tissue. Here we showed that re-epithelialization and barrier formation are indeed impaired in diabetic versus healthy ex vivo&#160;wounds. Indeed, this provides a route for pre-clinical assessment of pathological repair, where ageing and diabetes are major risk factors for developing chronic wounds1. While in vitro pathological models exist, such as cells isolated from aged and diabetic tissue, or cells cultured in high glucose to mimic hyperglycemia28,29, these cells can quickly lose their phenotype once removed from the in vivo microenvironment. An important component of the extrinsic pathological healing environment is the dermal matrix, which is altered in both ageing and diabetes30. Indeed, this perturbed matrix affects the behavior of resident and na&#239;ve fibroblasts31,32. Thus, the importance of studying cells in their host tissue environment cannot be underestimated.
In summary, our protocol provides an important platform to quantify human wound re-epithelialization, explore regulatory factors and to test the validity and efficacy of potential therapeutics12,13. While pre-clinical testing does still require in vivo approaches, a combined strategy using ex vivo human tissue and in vivo murine wounding should refine the pre-clinical pathway, reducing animal use while increasing cross-species translatability.

Chronic, non-healing wounds, which are highly prevalent in the elderly and diabetic, are a majorly unappreciated area of clinical unmet need. These wounds present a major physical and psychological burden to patients and cost healthcare providers billions each year to treat1. Despite improved understanding of wound biology and advancements in technology, up to 40% of chronic wounds still fail to heal following best standard care2. Thus, 14-26% of patients with diabetic foot ulcers subsequently require amputation3, while 5-year post-amputation mortality rate stands at approximately 70%4. As a result, there is an urgent requirement to develop efficacious new therapies to improve patient quality of life while reducing the substantial healthcare burden imposed by poor healing wounds. Poorly predictive pre-clinical models remain a significant hurdle to the development of effective new therapies.
Wound repair is a dynamic and multifaceted process involving a diverse range of cell types, countless levels of communication and a tissue environment that is temporally remodeled. Skin healing is underpinned by four major reparative stages: hemostasis, inflammation, proliferation, and matrix remodeling. These stages ultimately act to prevent blood loss and infection, close the wound surface (a process termed re-epithelialization) and return the skin to an uninjured state5. Chronic wounds are associated with diverse etiology and widespread perturbation to healing processes6, further complicating the identification of therapeutic targets. Nevertheless, a broad range of models have been developed to both elucidate the molecular and cellular drivers of wound pathology and test new therapeutic approaches7.
The most used wound repair model is acute wounding in the mouse. Mice are highly tractable for mechanistic studies and provide validated models of ageing and diabetes8. Despite the general similarities shown amongst mouse and human healing, between-species differences in skin structure and healing dynamics remain. This means most murine wound research does not easily translate to the clinic9. Consequently, there has been a push towards human in vitro and ex vivo systems with high applicability and translatability10,11.
Here we provide an in-depth protocol for performing partial thickness excisional wounds in ex vivo human skin. We&#160;also&#160;outline our whole-mount staining approach as a highly reproducible method of evaluating ex vivo human skin healing. We show the trajectory of epidermal repair (re-epithelialization) and subsequent barrier formation, evaluating the rate of wound closure in healthy versus diabetic human skin. Finally, we demonstrate how whole-mount staining can be adapted for use with a range of antibodies to assess various aspects of the healing response.

<table><tbody><tr><td>50 mL Falcon Tubes</td><td>Falcon</td><td>352070</td><td>&lt;em&gt;For skin washing&lt;/em&gt;</td></tr><tr><td>1.5&amp;nbsp;ml TubeOne Microcentrifuge Tubes, Natural (Sterile)</td><td>Starlab</td><td>S1615-5510</td><td>&lt;em&gt;For whole-mount staining&lt;/em&gt;</td></tr><tr><td>48-Well CytoOne Plate, TC-Treated</td><td>Starlab</td><td>CC7682-7548</td><td>&lt;em&gt;For whole-mount staining&lt;/em&gt;</td></tr><tr><td>Acetic Acid Glacial</td><td>Fisher Chemical</td><td>A/0400/PB15</td><td>&lt;em&gt;Part of fixative&lt;/em&gt;</td></tr><tr><td>Alkyltrimethylammonium Bromide</td><td>Sigma-Aldrich</td><td>M7635</td><td>&lt;em&gt;Part of fixative&lt;/em&gt;</td></tr><tr><td>Anti-Alpha Smooth Muscle Actin Antibody [1A4]</td><td>Abcam</td><td>ab7817</td><td>&lt;em&gt;Stains blood vessels&lt;/em&gt;</td></tr><tr><td>Anti-Collagen I Antibody</td><td>Abcam</td><td>ab34710</td><td>&lt;em&gt;Stains collagen&lt;/em&gt;</td></tr><tr><td>Anti-Cytokeratin 14 Antibody [LL002]</td><td>Abcam</td><td>ab7800</td><td>&lt;em&gt;Stains epidermis&lt;/em&gt;</td></tr><tr><td>CD1A Antibody (CTB6)</td><td>Santa Cruz Biotechnology</td><td>sc-5265</td><td>&lt;em&gt;Stains Langerhans cells&lt;/em&gt;</td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole, dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>62247</td><td>&lt;em&gt;Counterstain for cell nuclei&lt;/em&gt;</td></tr><tr><td>Falcon 60mm Petri dishes</td><td>Falcon</td><td>353004</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Fibronectin Antibody (EP5)</td><td>Santa Cruz Biotechnology</td><td>sc-8422</td><td>&lt;em&gt;Stains fibronectin&lt;/em&gt;</td></tr><tr><td>Formaldehyde, Extra Pure, Solution 37-41%, SLR</td><td>Fisher Chemical</td><td>F/1501/PB17</td><td>&lt;em&gt;Part of fixative&lt;/em&gt;</td></tr><tr><td>Gauze Swabs</td><td>Medisave</td><td>CS1650</td><td>&lt;em&gt;To clean skin&lt;/em&gt;</td></tr><tr><td>Gibco&amp;trade; Antibiotic-Antimycotic Solution</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Gibco DMEM, high glucose, no glutamine</td><td>Thermo Fisher Scientific</td><td>11960044</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Gibco Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10500064</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Gibco HBSS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>14170088</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Gibco L-Glutamine (200 mM)</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich</td><td>H1009-100ML</td><td>&lt;em&gt;For immunoperoxidase staining&lt;/em&gt;</td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health</td><td>N/A</td><td>&lt;em&gt;For image analysis&lt;/em&gt;</td></tr><tr><td>Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Mouse, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A11001</td><td>&lt;em&gt;Secondary antibody used depends on required fluorochromes and primary antibody&lt;/em&gt;</td></tr><tr><td>Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>A11012</td><td>&lt;em&gt;Secondary antibody used depends on required fluorochromes and primary antibody&lt;/em&gt;</td></tr><tr><td>Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Thermo Fisher Scientific</td><td>L3224</td><td>&lt;em&gt;For viability assessment of tissue&lt;/em&gt;</td></tr><tr><td>Iris Forceps, 10 cm, Curved, 1x2 teeth</td><td>World Precision Instruments</td><td>15917</td><td>&lt;em&gt;To create wounds&lt;/em&gt;</td></tr><tr><td>Iris Scissors, 11 cm, Curved, SuperCut, Tungsten Carbide</td><td>World Precision Instruments</td><td>501264</td><td>&lt;em&gt;To create wounds&lt;/em&gt;</td></tr><tr><td>Iris Scissors, 11 cm, Straight, SuperCut, Tungsten Carbide</td><td>World Precision Instruments</td><td>501263</td><td>&lt;em&gt;To remove adipose tissue&lt;/em&gt;</td></tr><tr><td>Keratin 1 Polyclonal Antibody, Purified</td><td>Biolegend</td><td>905201</td><td>&lt;em&gt;Stains epidermis&lt;/em&gt;</td></tr><tr><td>Keratin 14 Polyclonal Antibody, Purified</td><td>Biolegend</td><td>905301</td><td>&lt;em&gt;Stains epidermis&lt;/em&gt;</td></tr><tr><td>LSM 710 Confocal Laser Scanning Microscope</td><td>Carl Zeiss</td><td>Discontinued</td><td>&lt;em&gt;For fluorescent imaging&lt;/em&gt;</td></tr><tr><td>Merck Millipore Absorbent pads</td><td>Merck Millipore</td><td>AP10045S0</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Merck Millipore Nylon Hydrophilic Membrane Filters</td><td>Merck Millipore</td><td>HNWP04700</td><td>&lt;em&gt;Human ex vivo culture&lt;/em&gt;</td></tr><tr><td>Normal Goat Serum Solution</td><td>Vector Laboratories</td><td>S-1000-20</td><td>&lt;em&gt;Animal serum used depends on secondary antibody&lt;/em&gt;</td></tr><tr><td>Phosphate Buffer Solution</td><td>Sigma-Aldrich</td><td>P3619</td><td>&lt;em&gt;For wash buffer&lt;/em&gt;</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>&lt;em&gt;For blocking buffer&lt;/em&gt;</td></tr><tr><td>Sodium Chloride</td><td>Fisher Bioreagents</td><td>BP358-212</td><td>&lt;em&gt;Part of fixative&lt;/em&gt;</td></tr><tr><td>Sterilisation Pouches</td><td>Medisave</td><td>SH3710</td><td>&lt;em&gt;To sterilise instruments&lt;/em&gt;</td></tr><tr><td>Stiefel 2mm biopsy punches</td><td>Medisave</td><td>BI0500</td><td>&lt;em&gt;For partial thickness wound&lt;/em&gt;</td></tr><tr><td>Stiefel 6mm biopsy punches</td><td>Medisave</td><td>BI2000</td><td>&lt;em&gt;For outer explant&lt;/em&gt;</td></tr><tr><td>Thermo Scientific Sterilin Standard 90mm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>101VR20</td><td>&lt;em&gt;To prepare skin&lt;/em&gt;</td></tr><tr><td>Triton X-100</td><td>Fisher Chemical</td><td>T/3751/08</td><td>&lt;em&gt;For wash buffer&lt;/em&gt;</td></tr><tr><td>VECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG)</td><td>Vector Laboratories</td><td>PK-6101</td><td>&lt;em&gt;For immunoperoxidase staining; HRP kit used depends on primary antibody&lt;/em&gt;</td></tr><tr><td>Vector NovaRED Substrate Kit, Peroxidase (HRP)</td><td>Vector Laboratories</td><td>SK-4800</td><td>&lt;em&gt;For immunoperoxidase staining&lt;/em&gt;</td></tr><tr><td>Wireless Digital Microscope</td><td>Jiusion</td><td>N/A</td><td>&lt;em&gt;For brightfield imaging&lt;/em&gt;</td></tr></tbody></table>

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.

In this report, we present a novel ex vivo skin wounding and whole-mount staining approach to assess factors that influence the human skin repair response. Figure 1A shows a schematic of the procedural pipeline, which can be performed in 3-10 days, depending on wound incubation times. The partial thickness wounds are cultured on membrane stacks at the air : membrane interface and can be collected for whole-mount staining, embedded in paraffin or OCT medium for general histology, or frozen in liquid nitrogen for biochemical analysis (Figure 1B). We generally create 2 mm partial thickness wounds within the center of 6 mm explants. However, the size of the wound and surrounding explant may be altered depending on requirements. The whole-mount procedure has been successfully adapted for both immunoperoxidase and immunofluorescence staining methods (Figure 1C).
Immunofluorescence allows for the probing of tissue with multiple antibodies. For this, we advise using primary antibodies raised in different species, and species-matched fluorescently conjugated secondary antibodies to limit cross-species reactivity. Antibody concentrations and incubation times will need to be optimized. If background staining is observed, reduce antibody concentrations, increase wash steps, and add blocking buffer to the secondary antibody. Fresh tissue viability can be directly assessed with commercial viability dyes (see Table of Materials). We also show that tissue may be fixed post viability staining and successfully imaged when it is practically suitable (Figure 1D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62326/62326fig01.jpg" /> 
Figure 1: The human ex vivo wounding and whole-mount staining approach. (A) Pipeline depicting the procedural workflow from collecting skin and performing ex vivo wounding, to staining tissue and analyzing data. (B) Diagram demonstrating the human ex vivo skin wound culture system with analyses routinely performed on the tissue. (C) Whole-mount staining can be employed using both immunoperoxidase and immunofluorescence techniques. K14 = keratin 14. (D) Live tissue may be stained with commercial viability dyes and imaged successfully post fixation. Bar = 100 &#181;m. This staining was performed in non-diabetic skin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62326/62326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The most widely applicable use for whole-mount staining of wounds is to determine wound closure rate in a more reproducible manner than can be provided via histological sectioning. Percentage closure was quantified as percentage re-epithelialization of the wound surface, as demonstrated in Figure 2A. Percentage area coverage of specific markers can be measured from the total wound area or as a percentage of the re-epithelialized wound. We characterized healing in healthy (non-diabetic) versus diabetic skin across a time course of seven days, collecting wounds at each day post-wounding (representative images, Figure 2B). Healthy skin wounds closed over time as expected, with full closure observed in most samples by day 4-5. On the contrary, diabetic skin wounds failed to close fully within the seven-day analysis period (Figure 2C). A significant delay in wound closure was observed between healthy and diabetic skin wounds when comparing healing rates at each time-point post-injury (P &#60; 0.001 to day 6, P &#60; 0.05 at day 6 and P &#60; 0.05 to P &#60; 0.001 at day 7).
Following assessment of overall wound closure rates; we measured the percentage of the entire wound area (outer area in Figure 2A) where K14 positive cells could be visualized (green staining in Figure 2B). Interestingly, we observed that in healthy ex vivo skin wounds, K14 staining peaked at day 2 and then rapidly declined (significance at each time-point versus the day 2 peak, Figure 2D). This is likely reflecting re-formation of the early epidermal barrier, excluding K14 antibody penetration through differentiated epidermal layers (see Figure 2E schematic). During the re-epithelialization process, basal layer (K14+ve) keratinocytes migrate inwards over the open wound such that the epidermis closer to the outer wound edge forms earlier than the epidermis closer to the inner wound edge (migrating front). While the front edge of the newly formed epidermis continues to migrate to close the remaining open wound, the outer edge epidermis begins to differentiate to reform the other epidermal layers. In early healing, we would thus expect to see most of the re-epithelialized area consists of basal (K14+ve) cells, while in later repair K14 staining is lost as the epidermis differentiates from the outside inwards (see whole-mount images in Figure&#160;2E). Therefore, the decline in K14 staining shown in Figure 2D (downward arrows) correlates with increased epidermal differentiation. Interestingly, visible K14 staining peaked earlier in healthy (day 2) versus diabetic (day 4) wounds, further demonstrating that re-epithelialization and subsequent epidermal differentiation are delayed in diabetic skin wounds.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62326/62326fig02.jpg" /> 
Figure 2: Whole-mount staining reveals perturbed healing rates in diabetic versus healthy skin. (A) The method used to quantify wound closure from outer and inner wound measurements. Brightfield images show keratin 14 (K14) staining in red. Bar = 300 &#181;m. (B) Representative images of healing over time (day post-wounding) in healthy and diabetic skin. Bar = 500 &#181;m. K14 = green. DAPI = blue nuclei. (C) Quantification of wound closure rates (percentage re-epithelialization) showing that ex vivo wounds from healthy skin close significantly faster than ex vivo&#160;wounds from diabetic skin. H = healthy. Db = diabetic. (D) Percentage K14 staining peaks earlier in healthy versus diabetic skin and then declines in line with increased epidermal differentiation (down arrows). (E) K14 (basal epidermal cell) staining is lost as the epidermis differentiates. D = differentiated. ND = not differentiated. White dotted lines depict inner and outer wound edges. White arrows = direction of migration. n = 6 wounds per donor, per time point. Mean +/- SEM. * = P &#60; 0.05, ** = P &#60; 0.01 and *** = P &#60; 0.001. Healthy and diabetic compared at each healing time point in C (P value for least significant comparison). Temporal change in K14 staining compared to peak for each donor in D. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62326/62326fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We next used whole-mount staining to explore tissue expression and localization of other wound-relevant markers in non-diabetic skin (Figure&#160;3). All antibodies used and their working concentrations are provided in the Table of Materials. Blood vessels in the open wound stained positively with alpha smooth muscle actin (a-SMA) antibody, used in combination with K14 to delineate the epidermal edges in lower power images (Figure 3A). The dermal matrix was stained with antibodies against collagen type I (COL 1) and fibronectin (Fn). Here collagen was observed as abundant thick fibers while fibronectin fibers were sparse, wavy, and thin (Figure 3A). Our whole-mount staining approach is also able to provide cell level resolution of staining, as demonstrated for K14-positive keratinocytes (Figure 3B).
Finally, we show that human ex vivo wounds possess resident immune cells, with Langerhans cells detected around newly formed epidermis at day 3 post-wounding (Figure 3C). Indeed, these results suggest that whole-mount staining may be used to investigate key features of the healing response including inflammation, proliferation, and the extracellular matrix (Figure 4A). Taken together, our data reveal that the combined ex vivo skin wounding and whole-mount staining procedure is a valid method to assess various aspects of healthy and diabetic (pathological) human skin repair.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62326/62326fig03.jpg" /> 
Figure 3: Optimization of the whole-mount staining approach for use with other antibodies. (A) Blood vessels were stained with alpha smooth muscle actin (&#945;-SMA, green) and keratin 14 (K14, red), while matrix fibers were stained with collagen I (COL 1, red) and fibronectin (Fn, green). (B) The whole-mount procedure provides up to cell level resolution of localization (K14, green; K1, red). (C) CD1a+ve Langerhans cells (green) observed in newly formed epidermis. DAPI = blue nuclei. Bar = 100 &#181;m. White dotted lines show inner and outer wound edges and separate wound from epidermis. This staining was performed in non-diabetic skin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62326/62326fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62326/62326fig04.jpg" /> 
Figure 4: Validity of the whole-mount staining procedure for assessing wound healing. (A) Illustration depicting how the whole-mount staining technique can evaluate wound-relevant processes. Antibodies used = red text. K14 = keratin 14. COL 1 = collagen 1. Fn = fibronectin. (B) The whole-mount staining procedure (blue arrows) introduces less variability to wound closure measurements than standard histological analysis (red arrows). S1 = section 1. WE = wound edge. Bar = 300 &#181;m. This staining was performed in non-diabetic skin.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62326/62326fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.8K Views.  University of Hull. 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.

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

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

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

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 ...

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

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 ...

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 ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

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

Summary

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Chapters in this video

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

Murine Model of Wound Healing

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

A Mouse Fetal Skin Model of Scarless Wound Repair

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

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

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft