The authors would like to thank Kevin Carlson of the Brown University Flow Cytometry and Sorting Facility for consultation and assistance with flow cytometry experiments. Images in Figure 1B and C were created with BioRender. Kayla Lee and Gregory Serpa are thanked for their photographic assistance. This work was supported by grants from the following: Defense Advanced Research Projects Agency (DARPA) YFAA15 D15AP00100, Dean&#8217;s Areas of Emerging New Science Award (Brown University), National Heart Lung and Blood Institute (NHLBI) 1R01HL126887-01A1, National Institute of Environmental Science (NIES) T32-ES7272 (Training in Environmental Pathology), and the Brown University Research Seed Award.

All animal studies described here were approved by the Brown University Institutional Animal Care and Use Committee and carried out in accordance with the Guide for the Care and Use of Animals of the National Institutes of Health.&#160; NOTE: in the video, the surgical drape has been omitted for demonstration purposes.&#160;
1. Subcutaneous implantation of PVA sponges
<ol>
	<li>Use scissors to cut sheets of PVA sponge into 8 mm x 8 mm x 4 mm pieces. Rehydrate the pieces of PVA sponge by submerging them in sterile 1x PBS in a beaker.</li>
	<li>Autoclave the sponges in 1x PBS to sterilize, and cool completely. Store autoclaved sponges in sterile 1x PBS at 4 &#176;C.</li>
	<li>Prior to performing surgical implantation, aliquot the necessary number of sponges into a sterile culture dish under sterile conditions in a laminar flow hood. Store the extra sponges in sterile 1x PBS at 4 &#176;C for future use. Ensure that the sponges swell to 1 cm x 1 cm x 0.5 cm after rehydration. An image of dehydrated PVA sponges is shown in Figure 1A. 
	NOTE: To maintain the sterility of the surgical site, the PVA sponge implantation procedure requires one individual to handle the mouse and prepare the surgical site, and a second person to perform the implantation.</li>
	<li>Anesthetize an 8&#8211;12 week-old male C57BL/6J mouse by intraperitoneal injection of 80 mg/kg ketamine.&#160;Administer 40 mg/kg of xylazine intraperitoneally for analgesia.&#160; Confirm the depth of anesthesia by the loss of a pedal reflex. Prepare the surgical site by removing the hair along the dorsum with clippers. Using sterile gauze, apply povidone-iodine solution to the shave area 2x, followed by 70% ethanol.</li>
	<li>Place the mouse on sterile surgical drapes. Place a heated pad below the surgical drapes to maintain the mouse&#8217;s core temperature.</li>
	<li>Wear sterile surgical gloves for performing the surgery. Pull the dorsal skin away from the underlying tissue with forceps, and using sterile surgical scissors, make a 2 cm incision along the dorsal midline approximately 2 cm anterior to the base of the tail. While holding the incision open with sterile forceps, use sterile curved, blunt-tipped surgical scissors to form a subcutaneous pocket along the dorsum in one of the positions indicated in Figure 1B.</li>
	<li>Continue to use forceps to lift the skin away from the underlying tissue. Using sterile surgical scissors, gently squeeze one PVA sponge in the culture dish to remove excess PBS. Pick up the PVA sponge by one corner using sterile surgical scissors and, leading with the corner held by the scissors, place the sponge into the subcutaneous pocket formed in step 1.4.</li>
	<li>Repeat this process 5x, placing a total of six sponges into subcutaneous pockets as shown in Figure 1B.</li>
	<li>Pinch the incised dorsal skin together with sterile forceps and close the incision with two stainless steel wound clips.</li>
	<li>Use a new set of sterile surgical instruments for each mouse. Alternatively, use a bead sterilizer to sterilize instruments between mice.</li>
</ol>
2. Isolation of fluids from PVA sponges
<ol>
	<li>To assess the acute wound cytokine milieu, isolate sponges between 1&#8211;14 days postimplantation.</li>
	<li>Prepare a fluid collection tube by nesting the barrel of a 5 mL syringe into a 16 mL culture tube.</li>
	<li>Euthanize a mouse with implanted PVA sponges by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Remove the surgical staples and open the incision with toothed forceps. Use scissors to extend the incision along the dorsal midline.</li>
	<li>Using forceps, extract one sponge from its subcutaneous pocket and transfer it to the prepared syringe barrel, being careful to minimize pressure to the sponge. Disassociate any connective tissue that remains adhered to the surface of the sponge. Use scissors to dissect the sponge from the surrounding tissue if necessary. Repeat the sponge removal process with the remaining sponges. Place the tube containing the sponges on ice.</li>
	<li>To isolate the wound fluid, centrifuge the culture tube containing the 5 mL syringe with the sponges at 700 x g for 10 min.</li>
	<li>After the centrifugation, discard the syringe and the sponges. The wound fluid will have collected in the 16 mL culture tube. A representative image of fluid collected from a day 7 wound is shown in Figure 1D. Transfer the wound fluid to a clean tube for long-term storage at -80 &#176;C.</li>
</ol>
3. Isolation of cells from PVA sponges
<ol>
	<li>To assess the acute wound healing cellular milieu, collect sponges between 1&#8211;14-days post-sponge implantation. Sponges obtained from a wound 7 days after implantation are shown in Figure 1E.</li>
	<li>Prepare collection medium containing 1x HBSS (+calcium/+magnesium/&#8211;phenol red) supplemented with 1% FBS, 1% HEPES, and 1% penicillin-streptomycin.</li>
	<li>Aliquot 5 mL of HBSS collection medium into a 15 mL conical tube.</li>
	<li>Extract the PVA sponges from euthanized mice as described in 2.2&#8211;2.4. Place the sponges into the conical tube containing 5 mL of HBSS collection medium.</li>
	<li>Transfer the sponges and the HBSS collection medium to an 80 mL blender bag by pouring. Hang the blender bag from the hatch of the paddle blender, positioned so that the paddles strike the sponges and media. Adjust the settings of the paddle blender to run on high for 60 s. Press start.</li>
	<li>Transfer the media from the blender bag back to the 15 mL conical tube with a pipette, leaving the sponges behind in the bag. Squeeze the sponges to thoroughly release the media. Adjust the settings of the paddle blender to run for 30 s on high. Add 5 mL of media to the blender bag and repeat the stomaching process 2x for a total of three sponge washes.</li>
	<li>Centrifuge the conical tube at 250 x g for 5 min. A red pellet of cells and red blood cells will be visible at the bottom of the conical tube after centrifugation.</li>
	<li>Discard the supernatant and perform a red blood cell lysis: add 900 &#956;L of dH2O to the cell pellet and pipette to mix. Neutralize with 100 &#956;L of 10x PBS. Add 4 mL of 1x PBS to the tube to fully neutralize the lysis, then centrifuge at 250 x g for 5 min. 
	NOTE: The lysis step should be brief; leaving the water in contact with the cells for more than 3&#8211;5 s could result in lysis of non-red blood cells.</li>
	<li>After centrifugation, a white cell pellet containing wound cells devoid of red blood cells will be present at the bottom of the conical tube. Discard the supernatant and resuspend the cell pellet in the desired medium for downstream analyses.</li>
</ol>
4. Optional flow cytometry analysis of innate leukocytes isolated from PVA sponge wounds
<ol>
	<li>Resuspend 0.5&#8211;1 x 106 wound cells in 25 &#956;L of a 2x solution of blocking antibody containing 10 &#956;g/mL anti-CD16/CD32 FcR&#947;II/III antibody diluted in 1x PBS + 1% BSA. 
	NOTE: All antibodies should be titrated to determine optimal concentrations for flow cytometry analysis.</li>
	<li>Incubate the cells with blocking antibody for 10 min on ice.</li>
	<li>Make a 2x master mix of antibodies containing 2.5 mg/mL of PerCP-eFluor710-Ly6G, 5 mg/mL of FITC-Ly6C, APC-Fire750-CD45.2, APC-R700-Siglec-F, and 10 mg/mL of eFluor660-F4-80 diluted in 1x PBS + 1% BSA. Directly add 25 &#956;L of the antibody cocktail to the cells incubating in blocking antibody.</li>
	<li>Incubate the cells for 20 min on ice, protected from light.</li>
	<li>Wash the cells 2x with 1x PBS.</li>
	<li>Make a master mix of amine-reactive fixable viability dye-eFluor506 diluted 1:1,000 in 1x PBS. Resuspend the cell pellet in 50 &#956;L of the viability dye solution. 
	NOTE: Serum must be excluded from the fixable viability step, as it will prevent binding of the dye to endogenous proteins. Incubate the cells for 20 min on ice, protected from light.</li>
	<li>Wash the cells 2x with 1x PBS. Resuspend the cell pellet in 50 &#956;L of 1% paraformaldehyde.</li>
	<li>Incubate the cells with 1% paraformaldehyde for 15 min on ice, protected from light.</li>
	<li>Wash the cells 1x with 1x PBS and resuspend the pellet in 1x PBS for flow cytometric analysis.</li>
</ol>
5. Tail skin excision
<ol>
	<li>Anesthetize an 8&#8211;12 week-old male C57BL/6J mouse by intraperitoneal injection of 80 mg/kg of ketamine.&#160;Administer 40 mg/kg of xylazine intraperitoneally for analgesia.&#160;Confirm the depth of anesthesia by the loss of a pedal reflex.</li>
	<li>Prepare the surgical site on the tail of the anesthetized mouse by using sterile gauze to apply povidone-iodine solution 2x, followed by one application of 70% ethanol.</li>
	<li>Define a 10 mm x 3 mm section on the dorsal surface of the tail, 10 mm from the base of the tail (Figure 1C) using a permanent marker to trace from a premade template.</li>
	<li>Place the anesthetized mouse on sterile surgical drapes.</li>
	<li>Wear sterile surgical gloves to perform the surgical procedures. With a sterile scalpel blade, make a full thickness incision along the right, bottom, and left edges of the wound area.</li>
	<li>Using sterile forceps, peel the excised skin away from the tail, and use sterile surgical scissors to cut away the top edge of the wound area.</li>
	<li>Apply pressure with sterile gauze to stop bleeding.</li>
	<li>Apply a spray barrier film to the wound bed.</li>
	<li>Photograph the wounds from a fixed distance at regular time intervals. Analyze photographs by planimetric analysis to determine wound area measurements. Alternatively, use calipers to obtain wound length and width measurements.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIP-1alpha+as+a+critical+macrophage+chemoattractant+in+murine+wound+repair.">MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.</a> Journal of Clinical Investigation. 101 (8), 1693-1698 (1998).</li><li>Wetzler, C., K&#228;mpfer, H., Stallmeyer, B., Pfeilschifter, J., Frank, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+Sustained+Induction+of+Chemokines+during+Impaired+Wound+Healing+in+the+Genetically+Diabetic+Mouse:+Prolonged+Persistence+of+Neutrophils+and+Macrophages+during+the+Late+Phase+of+Repair.">Large and Sustained Induction of Chemokines during Impaired Wound Healing in the Genetically Diabetic Mouse: Prolonged Persistence of Neutrophils and Macrophages during the Late Phase of Repair.</a> Journal of Investigative Dermatology. 115 (2), 245-253 (2000).</li><li>Kim, D. J., Mustoe, T., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing+in+aging+small+mammals:+a+systematic+review.">Cutaneous wound healing in aging small mammals: a systematic review.</a> Wound Repair and Regeneration. 23 (3), 318-339 (2015).</li></ol>

The authors have nothing to disclose.

This article describes two tractable murine wound models that allow for the assessment of the acute wound healing response. The first method involves the surgical implantation of PVA sponges in the dorsal subcutaneous space. This approach offers a distinct advantage over biopsy-based wound models for studying the cellular wound healing response due to the large number of cells and quantity of wound fluids obtained from the isolated sponges. For the successful execution of this procedure, maintaining a sterile surgical fi...

There are many murine model systems available to examine wound healing processes, each possessing specific advantages and limitations1,2. The following methods present two murine wound models, each of which addresses a particular aspect of the wound healing response, and which can be used to identify the cause and effect of perturbations in the response to injury. The process of wound healing occurs in distinct phases. The first phase is inflammatory, characterized by the rapid influx of platelets, neutrophils, and monocytes/macrophages, as well as the production of proinflammatory cytokines and chemokines. Following resolution of inflammation, the environment transitions to a more reparative state with the induction of profibrotic and proangiogenic cytokines and growth factors. Granulation tissue is deposited and neovessels form with the migration of myofibroblasts, fibroblasts, epithelial cells, and endothelial cells. In the final stages, the provisional extracellular matrix is remodeled, and scar formation and wound closure proceeds2,3,4,5,6,7,8.
No single murine model provides a system to study all stages of wound healing2. Here, two surgical wound models are described: one elucidates acute cellular and cytokine wound healing responses, and the other allows for the assessment of wound closure as well as histological analyses. These two methods may be employed in a complementary fashion to assess the effects of a perturbation or comorbidity on different aspects of the wound healing response. The dorsal subcutaneous implantation of polyvinyl alcohol (PVA) sponges is a system that has been used in rodent models for decades to elucidate numerous aspects of cellular and granulation tissue responses9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. This approach allows for the retrieval of cytokine-rich wound fluids and cellular infiltrates. In this model, 1 cm x 1 cm x 0.5 cm pieces of PVA sponge are placed into subcutaneous pockets through a 2 cm incision made at the posterior dorsal midline. The incision is closed with surgical clips, and the sponges can be retrieved at later time points for cell and fluid isolation. The cellular and cytokine milieu of isolated sponges reflects the normal stages of acute wound healing up to about 14 days postimplantation. At later time points the model is more advantageous for studying granulation tissue formation and the foreign body response1. With this system, it is possible to isolate &#62;106 cells, which offers a distinct advantage for phenotypic and functional assays and RNA isolation, over isolating cells from other biopsy-based methods1,22,23,25,26.
The rate of wound closure is determined using the tail skin excision model. In this model, as initially described by Falanga et al. and reported by others27,28,29,30, a 1 cm x 0.3 cm full thickness section of tail skin is removed near the base of the tail. The wound area is easily visualized and can be measured over time. Alternatively, tail tissue can be isolated for histological analysis. This approach can be used as an alternative to or in conjunction with the well-established dorsal punch biopsy method. The primary distinctions between these two models are the rate of wound closure, the presence or absence of fur, and the skin structure2,31,32. Tail skin wounds offer a longer timeframe in which to assess wound closure, as it takes approximately 21 days for full closure to occur. This is opposed to unsplinted dorsal punch biopsies, which heal much faster (~7&#8211;10 days), primarily by contraction due to the action of the panniculus carnosus. Splinted dorsal punch biopsies heal more slowly and diminish the effects of contractile healing, but rely on the presence of a foreign body to restrict contractile-based mechanisms1,2,27,30,31,33.
The described wound models are informative for understanding normal wound healing processes in the absence of perturbation. While the healing of rodent skin differs in very significant ways from human skin, including loose structure, reliance on contractile healing, and other anatomical differences, the murine system offers certain advantages for mechanistic and screening studies. Foremost among these is the availability of inbred strains and genetic mutants, genetic tractability, and lower cost. Mechanistic insight gained from murine studies can be translated to complex animal models that more closely mimic human skin healing, such as the porcine system2,31.
In addition to examining wound healing responses in the steady state, these models can be combined with comorbid conditions to understand the basis of wound healing defects at the cellular, cytokine, and gross tissue level. It is in this particular setting that the two models can be used in concert to assess the effects of a particular comorbid condition, such as postoperative pneumonia, on both the acute cellular wound healing response and the rate of wound closure30.

<table><tbody><tr><td>10x Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>BP3991</td><td></td></tr><tr><td>15 mL centrifuge tubes, Olympus</td><td>Genesee</td><td>28-103</td><td></td></tr><tr><td>1x HBSS (+Calcium, +Magnesium, &amp;ndash;Phenol Red)</td><td>ThermoFisher Scientific</td><td>14025076</td><td></td></tr><tr><td>5ml Syringe</td><td>BD</td><td>309646</td><td></td></tr><tr><td>Anti-mouse CD45.2-APC Fire750</td><td>BioLegend</td><td>109852</td><td>Clone 104</td></tr><tr><td>Anti-mouse F4/80-eFluor660</td><td>ThermoFisher Scientific</td><td>50-4801-82</td><td>Clone BM8</td></tr><tr><td>Anti-mouse Ly6C-FITC</td><td>BD Biosciences</td><td>553104</td><td>Clone AL-21</td></tr><tr><td>Anti-mouse Ly6G-PerCP-eFluor710</td><td>ThermoFisher Scientific</td><td>46-9668-82</td><td>Clone 1A8-Ly6g</td></tr><tr><td>Anti-mouse Siglec-F-APC-R700</td><td>BD Biosciences</td><td>565183</td><td>Clone E50-2440</td></tr><tr><td>Autoclip Stainless Steel Wound Clip Applier</td><td>Braintree Scientific</td><td>NC9021392</td><td></td></tr><tr><td>Autoclip Stainless Steel Wound Clips, 9mm</td><td>Braintree Scientific</td><td>NC9334081</td><td></td></tr><tr><td>Blender Bag, 80mL</td><td>Fisher Scientific</td><td>14258201</td><td></td></tr><tr><td>Culture Tube, 16mL, 17x100</td><td>Genesee Scientific</td><td>21-130</td><td></td></tr><tr><td>Fetal Bovine Serum - Standard</td><td>ThermoFisher Scientific</td><td>10437028</td><td></td></tr><tr><td>Fixable Viability Dye eFluor506</td><td>ThermoFisher Scientific</td><td>65-0866-14</td><td></td></tr><tr><td>Hepes Solution, 1M</td><td>Genesee Scientific</td><td>25-534</td><td></td></tr><tr><td>ImageJ Software</td><td>NIH</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin (5000 U/mL)</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Polyvinyl alcohol sponge - large pore size</td><td>Ivalon/PVA Unlimited</td><td></td><td>www.sponge-pva.com</td></tr><tr><td>Povidone-iodine solution, 10%</td><td>Fisher Scientific</td><td>3955-16</td><td></td></tr><tr><td>Spray barrier film, Cavilon</td><td>3M</td><td>3346E</td><td></td></tr><tr><td>Stomacher 80 Biomaster, 110V</td><td>Seward</td><td>0080/000/AJ</td><td></td></tr></tbody></table>

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.

Systemic inflammatory response following PVA sponge implantation 
The PVA sponge implantation surgery generated a systemic inflammatory response, as demonstrated by the induction of IL-6 in the plasma 1 day after wounding (Figure 2A). Other proinflammatory cytokines including TNF-&#945; and IL-1&#946;, as well as an array of chemokines including CCL2 and CXCL1 were induced systemically in the first 7 days post-PVA sponge implantation, and have been described elsewhere<s...

Watch this Scientific Journal Video about Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models. at JoVE.com

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

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 ...

assessment-acute-wound-healing-using-dorsal-subcutaneous-polyvinyl

Research

JoVE Journal

Immunology and Infection

8.3K Views.  Brown University. Deze modellen kunnen worden geïmplementeerd om verschillende aspecten van de wondgenezingsrespons te beoordelen, waaronder cellulaire en cytokinetiek en wondsluiting. De PVA spons model maakt het herstel van miljoenen wond leukocyten voor fenotypische en functionele analyses, terwijl de staart huid excisie model maakt de gemakkelijke visualisatie van langzaam genezende wonden. De PVA spons en excisional staart wond modellen kunnen worden gebruikt in combinatie met comorbide voorwaarden, zoal...

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

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing ...

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

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. ...

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 ...

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, ...

Developmental Biology

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 ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

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 ...

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

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

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