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 chori...

<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 border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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,...

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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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12.1K visualizações.  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.