This work was supported in part by a grant from NIH grant R01 GM087609 (to H.P.L.), a Gift from Ingrid Lai and Bill Shu in honor of Anthony Shu (to H.P.L.), NIH grant U01 HL099776 (to M.T.L.), the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation (to M.T.L. and H.P.L.). G.G.W. was supported by the Stanford School of Medicine, the Stanford Medical Scientist Training Program, and NIGMS training grant GM07365. M.S.H. was supported by CIRM Clinical Fellow Training Grant TG2-01159. W.X.H. was supported by funding from the Sarnoff Cardiovascular Foundation.

NOTE: All procedures described in this paper are performed according to guidelines established by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).
1. Timed Pregnancies &#8211; Superovulation Technique (Figure 1)
NOTE: Precisely timing the gestational age of mouse embryos for fetal surgery at E16.5 and E18.5 is of critical importance. In this section we detail our protocol for timing mouse pregnancies using pregnant mares serum (PMS) and human chorionic gonadotropin (HCG) injections to induce superovulation.
<ol>
	<li>Inject female mice (&#60;5 per cage) intraperitoneally (IP) with 3.0-5.0 international units (IU) of PMS in a volume of 100 &#956;l PBS between 1:00 and 3:00 PM for day 1.</li>
	<li>Between 12:00 and 2:00 PM of day 3 (forty-five to 47 hr after PMS injection), inject female mice IP with 3.0-5.0 IU of HCG in a volume of 100 &#956;l PBS. 
	NOTE: The HCG injection induces ovulation approximately 12 hr post-injection.</li>
	<li>Immediately following HCG injections, mate females with males aged 8-16 weeks. 
	NOTE: We typically place two females into a cage of individual males.</li>
	<li>Separate females from males on the morning of day 4 (6:00 &#8211; 10:00 AM) and record as fetal age E0.5. 
	NOTE: Vaginal plugs can be checked at this time; however, the observation of a vaginal plug does not guarantee pregnancy and females can become pregnant when no plug is observed. Given that pregnancy is typically observable by visual inspection and/or palpation at gestational ages E16.5 and E18.5, checking for vaginal plugs on the morning of day 4 is not strictly necessary. In our experience, and depending on the strain, approximately 30-50% of super-ovulated females become pregnant using the technique described here.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52297/52297fig1highres.jpg" /> 
Figure 1. Schematic for Superovulation Technique.&#160;Schematic showing procedure for superovulation timed pregnancies in mice. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Murine Fetal Surgery (Dorsal Wounding) on E16.5 and E18.5 Embryos (Figure 2)
<ol>
	<li>Before the procedure, clean all surfaces of the operating room and equipment with 70% isopropyl alcohol. In addition, sterilize all surgical supplies and instruments that will be used in the procedure by autoclaving them. Some institutions may allow subsequent use of hot bead sterilization.&#160;Per operation, use sterile packs that include gauze and surgical instruments.</li>
	<li>Induce anesthesia in pregnant mothers (fetal age E16.5 or E18.5) under 2.5% isoflurane/oxygen mixture at 2 L per min followed by maintenance anesthesia at 1 L per min.</li>
	<li>To confirm proper anesthetization, ensure the deep pedal reflexes of the mouse are suppressed and place the mouse in the prone position.</li>
	<li>Apply a vet ophthalmic ointment such as Puralube to prevent eye irritation or dryness during the procedure.</li>
	<li>Prepare abdomen by administering a light application of depilatory cream for no longer than 30 sec (Figure 2A).</li>
	<li>Prepare abdomen for aseptic surgery with povidone-iodine and alcohol (Figure 2B and 2C).</li>
	<li>Perform midline laparotomy under microscope using microsurgical scissors (Figure 2D and 2E).</li>
	<li>Gently expose uterus and fetus selected for surgery (Figure 2F).</li>
	<li>Irrigate surgical field with warm (38 &#176;C) phosphate-buffered saline (PBS) using a blunt-tip needle 
	NOTE: One can be made by carefully bending the tip of a large-bore needle. (Figure 2G and 2H).</li>
	<li>Position the fetus in a manner that allows full access to dorsum.</li>
	<li>Pass a purse string stitch using 7-0 nylon suture through the uterus overlying the site of intended dorsal wounding (Figure 2I). Position purse string over a region of dorsum to the left or right of spinal cord, and in a region of uterine wall devoid of large blood vessels.</li>
	<li>Make a 3 mm incision through uterine wall and amniotic sac in the center of the purse string (Figure 2J).</li>
	<li>Irrigate incision site with warm (38 &#176;C) PBS.</li>
	<li>Using microsurgical scissors, cut a single full-thickness excisional wound, approximately 1 mm in length, in the dorsum of the fetus.</li>
	<li>Gently blot incision site dry with cotton-tip applicator.</li>
	<li>Inject 3 &#956;l volume India ink subcutaneously into the wound site to mark location of wound (Figure 2K).</li>
	<li>Irrigate with warm (38 &#176;C) PBS to ensure ink has been retained within wound site.</li>
	<li>Have surgical assistant inject warm (38 &#176;C) PBS through blunt-tip 10 G&#160;syringe into the amniotic sac as the purse string is closed ( Figure 2L). Retract syringe as purse string closure nears completion (Figure 2M).</li>
	<li>Gently return uterus into the abdominal cavity (Figure 2N).</li>
	<li>Evert skin and peritoneum.</li>
	<li>Have surgical assistant irrigate abdominal cavity with warm (38 &#176;C) PBS.</li>
	<li>Close abdomen quickly by stapling skin and peritoneum closed (Figure 2O).&#160;The standard closure is performed in two layers; peritoneum and abdominal muscle in one layer, subcutaneous tissue and skin in the second layer. For immediate sacrifice and harvesting of the fetus, our demonstration shows closure in one layer.</li>
	<li>Place the animal under observation in a warm incubator set at 37 &#176;C for 30 min&#160;or until the animal regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>Do not return the animal to the company of other animals until it has fully recovered from the procedure.</li>
	<li>Upon awaking from anesthesia and during the subsequent 48 hr, administer subcutaneous injection of buprenorphine (0.05 mg/kg) every 12 hr for analgesia as needed based on pain assessment. Administer carprofen (5 mg/kg) via subcutaneous injection for additional post-operative pain relief as needed.</li>
	<li>Return animals to the cage and provide them with food and water ad libitum.</li>
	<li>Monitor closely for manifestations of pain.</li>
	<li>48 hr post-surgery, sacrifice pregnant mother with an overdose of isoflurane and harvest wounded fetus. In order to do this, adjust isoflurane concentration to 5% or higher and maintain exposure for 1 min after cessation of breathing. Confirm euthanasia with cervical dislocation. Harvest an unwounded embryo for age-matched control.&#160;Late embryos should have a separate method of euthanasia consistent with IACUC recommendations, such as decapitation, cervical dislocation, or chemical injection.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 
Figure 2. Schematic for Murine Fetal Surgery.&#160;General steps for dorsal wounding in E16.5 and E18.5 mouse embryos. (A) Depilation of mouse abdomen. (B and C) Preparation of mouse abdomen. (D) Microscope used for surgical procedure. (E) Midline laparotomy. (F) Exposure of uterus. (G) Creation of blunt-tip needle. (H) Irrigation of uterus with warm saline. (I) Creation of purse string suture. (J) Incision through uterine wall and 1 mm full thickness excisional wound generation. (K) Subcutaneous injection of India ink. (L and M) Closure of purse string suture. (N and O) Closure of abdomen. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Larson, B. J., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scarless+fetal+wound+healing:+a+basic+science+review.">Scarless fetal wound healing: a basic science review.</a> Plastic and reconstructive surgery. 126, 1172-1180 (2010).</li><li>Wilgus, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+healing+in+fetal+skin:+a+review+of+the+literature.">Regenerative healing in fetal skin: a review of the literature.</a> Ostomy/wound management. 53, 16-31 (2007).</li><li>Wulff, B. 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=Mast+cells+contribute+to+scar+formation+during+fetal+wound+healing.">Mast cells contribute to scar formation during fetal wound healing.</a> The Journal of investigative dermatology. 132, 458-465 (2012).</li><li>Lorenz, H. P., Adzick, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scarless+skin+wound+repair+in+the+fetus.">Scarless skin wound repair in the fetus.</a> The Western journal of medicine. 159, 350-355 (1993).</li><li>Longaker, M. T., 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=Wound+healing+in+the+fetus.+Possible+role+for+inflammatory+macrophages+and+transforming+growth+factor-beta+isoforms.">Wound healing in the fetus. Possible role for inflammatory macrophages and transforming growth factor-beta isoforms.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2, 104-112 (1994).</li><li>Longaker, M. T., 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=Studies+in+fetal+wound+healing.+IV.+Hyaluronic+acid-stimulating+activity+distinguishes+fetal+wound+fluid+from+adult+wound+fluid.">Studies in fetal wound healing. IV. Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid.</a> Annals of surgery. 210, 667-672 (1989).</li><li>Colombo, J. A., Napp, M., Depaoli, J. R., Puissant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+influences+of+human+and+rat+amniotic+fluid+on+neural+tube-derived+rat+fetal+cells.">Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells.</a> International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience. 11, 347-355 (1993).</li><li>Colwell, A. S., Longaker, M. T., Peter Lorenz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+differentially+regulated+genes+in+fetal+wounds+during+regenerative+repair.">Identification of differentially regulated genes in fetal wounds during regenerative repair.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16, 450-459 (2008).</li><li>Hu, M. S., 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=Gene+expression+in+fetal+murine+keratinocytes+and+fibroblasts.">Gene expression in fetal murine keratinocytes and fibroblasts.</a> The Journal of surgical research. , (2014).</li><li>Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+mouse+excisional+wound+model+of+scarless+healing.">An in vivo mouse excisional wound model of scarless healing.</a> Plastic and reconstructive surgery. 117, 2292-2296 (2006).</li><li>Wilgus, T. 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=The+impact+of+cyclooxygenase-2+mediated+inflammation+on+scarless+fetal+wound+healing.">The impact of cyclooxygenase-2 mediated inflammation on scarless fetal wound healing.</a> The American journal of pathology. 165, 753-761 (2004).</li><li>Iocono, J. A., Ehrlich, H. P., Keefer, K. A., Krummel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+induces+scarless+repair+in+mouse+limb+organ+culture.">Hyaluronan induces scarless repair in mouse limb organ culture.</a> Journal of pediatric surgery. 33, 564-567 (1998).</li><li>Chopra, V., Blewett, C. J., Krummel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+fetal+to+adult+repair+occurring+in+mouse+forelimbs+maintained+in+organ+culture.">Transition from fetal to adult repair occurring in mouse forelimbs maintained in organ culture.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 5, 47-51 (1997).</li><li>Adzick, N. S., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+study+of+fetal+tissue+repair.">Animal models for the study of fetal tissue repair.</a> The Journal of surgical research. 5, 47-51 (1991).</li><li>Block, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+the+new-born+opossum+(Didelphis+virginianam).">Wound healing in the new-born opossum (Didelphis virginianam).</a> Nature. 187, 340-341 (1960).</li><li>Longaker, M. T., Dodson, T. B., Kaban, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+for+fetal+cleft+lip+repair.">A rabbit model for fetal cleft lip repair.</a> Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons. 48, 714-719 (1990).</li><li>Longaker, M. T., 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+model+for+fetal+cleft+lip+repair+in+lambs.">A model for fetal cleft lip repair in lambs.</a> Plastic and reconstructive surgery. 90, 750-756 (1992).</li></ol>

The authors have nothing to disclose.

The surgical protocol presented here describes an excisional model of fetal murine scarless healing first published in 2006 by our laboratory10. In addition to other established models of excisional wounding11, incisional models of fetal murine scarless healing exist as well12,13. Investigations of fetal scarless wound healing in the monkey, lamb, rabbit, opossum, and rat have been reported14-17. However, mice represent an ideal model for exploring fetal scarless wound healing ...

Fetal skin wounds heal rapidly and scarlessly until late in gestation1. Fetal scarless wound repair is characterized by regeneration of normal tissue architecture and function. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice2,3. In comparison to adult, fetal wound repair is characterized by rapid epithelialization, connective tissue deposition, and fibroblast migration.
Many studies have offered possible explanations for the phenomenon of scarless wound healing during early fetal development. Inflammation is a fundamental component of adult wound repair; however, fetal wounds are characterized by a lack of acute inflammation4. Whether this is a consequence of the functional immaturity of the immune system during fetal stages remains unclear. A recent study suggested that differences in the abundance, maturity, and function of mast cells in E15 vs. E18 fetal skin may be responsible for the transition from a scarless phenotype, at least in the mouse3. Other studies posit that differences in the properties and abundance of fetal and adult wound macrophages are responsible for the reformation of normal extracellular matrix (ECM) during fetal wound repair5.
Differences in environmental factors during fetal and adult development may also affect wound repair. Longaker and colleagues showed that wound fluid from the fetus possesses high levels of hyaluronic acid-stimulating activity compared to none in adult wound fluid6. Consequently, higher levels of hyaluronic acid, a glycosaminoglycan that promotes a microenvironment conducive to cell motility and proliferation, in the fetal wound environment may be responsible for the scarless phenotype seen during early fetal development. Other lines of evidence point to the fact that the fetal wound environment is relatively hypoxemic and submerged in sterile amniotic fluid rich in growth factors7. However, no definitive answer has been provided for a critical event or factor during embryogenesis that triggers the transition from scarless regeneration to fibrotic repair.
Understanding the mechanisms responsible for scarless healing in the fetus necessitates a precise and reproducible model. Here we detail a reproducible model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos. Additionally, minor variations of this model can be utilized to perform a number of further studies, such as gene expression analysis of fetal wounds and skin8,9. Given that precisely timed pregnancies are critical for successful recapitulation of this fetal scarless wound healing model, we also detail our protocol for superovulation timed pregnancies.

<table><tbody><tr><td>7-O MONOSOF Suture</td><td>eSuture</td><td>SN-1647G</td><td></td></tr><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Autoclip 9 mm</td><td>Texas Scientific Instruments</td><td>205060</td><td></td></tr><tr><td>Insulin Syringe</td><td>Thermo Fisher Scientific</td><td>22-272-382</td><td></td></tr><tr><td>Black Pigment</td><td>AIMS</td><td>242</td><td></td></tr><tr><td>BD Safety-Lok 3 ml Syringe</td><td>BD Biosciences</td><td>309596</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>OPMI-MD Surgical Microscope</td><td>Carl Zeiss Surgical Inc</td><td></td><td></td></tr><tr><td>Pregnant Mares Serum (PMS)</td><td>Millipore</td><td>367222</td><td></td></tr><tr><td>Human Chorionic Gonadotropin (HCG)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr></tbody></table>

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.

For histologic analysis, cutaneous wounds in the dorsal skin of E16.5 and E18.5 mouse embryos should be harvested 48 hr post-wounding, fixed in 4% PFA, and paraffin-embedded. In fluorescent transgenic models, cryopreservation with OCT may be appropriate. There are several stains that may be used to visualize cellular and connective tissue architecture. Hematoxylin and eosin is a two-color stain that stains nuclei blue and eosinophilic structures (i.e., cytoplasm and extracellular collagen) various shades of red,...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

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 ...

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12.2K Views.  Stanford University School of Medicine. 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.

Video: A Mouse Fetal Skin Model of Scarless Wound Repair

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

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

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

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

Bioengineering

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 ...

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 ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

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

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

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

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

Immunology and Infection

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

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, ...

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 ...

Biology

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

A Mouse Fetal Skin Model of Scarless Wound Repair

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