Search

Background: Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis--the keratinocyte and fibroblast--during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration., Materials and Methods: Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate<2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways., Results: By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts., Conclusions: Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early-gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment., (Copyright © 2012 Wiley Periodicals, Inc.)

Summary: Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.

Background: Mammalian fetal skin injury heals scarlessly. The intrinsic differences between embryonic and adult fibroblasts that underlie this observation are poorly understood. Several studies have linked Wnt proteins with skin morphogenesis. The authors' study aimed to establish a correlation between beta-catenin-dependent (canonical) Wnt protein, transforming growth factor (TGF)-beta1, and the expression of hyaluronan synthesis enzymes during scarless versus scarring wound healing., Methods: Wnt signaling was quantified after 1.5-mm skin wounds were created in BAT-gal fetal (e16.5) and postnatal (p1) mice. Canonical Wnt signals were localized by X-gal staining and quantified with quantitative real-time polymerase chain reaction. Primary embryonic and postnatal mouse dermal fibroblasts were treated with recombinant Wnt3a or TGF-beta1. Proliferation was assayed by bromodeoxyuridine incorporation. Gene expression of enzymes that regulate hyaluronan production and turnover was examined by quantitative real-time polymerase chain reaction (hyaluronan synthases or HAS1-3, hyaluronadase-2), as well as other target genes for Wnt and TGF-beta (Axin2, TGF-beta1, TGF-beta3, type 1 collagen, proliferating cell nuclear antigen)., Results: Canonical Wnt signaling increased following wounding in postnatal, but not fetal, mice. In vitro, rmWnt3a increased postnatal fibroblast proliferation but not in embryonic cells. Both Wnt3a and TGF-beta1 induced HAS2 and HAS3 gene expression in embryonic fibroblasts, while HAS1 and Hyal2 were induced in postnatal fibroblasts. Finally, rmWnt3a significantly increased type I collagen expression, particularly in postnatal fibroblasts, and influenced expression of TGF-beta isoforms., Conclusions: Increased canonical Wnt signaling occurs during postnatal but not fetal cutaneous wound repair. Fetal and postnatal fibroblasts have a disparate response to rmWnt3a in vitro. rmWnt3a affects postnatal fibroblasts in a similar fashion to rhTGF-beta1, a known profibrotic cytokine.

The developing fetus has the ability to heal wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix (ECM) architecture, strength, and function. In contrast, adult wounds heal with fibrosis and scar. Scar tissue remains weaker than normal skin with an altered ECM composition. Despite extensive investigation, the mechanism of fetal wound healing remains largely unknown. We do know that early in gestation, fetal skin is developing at a rapid pace and the ECM is a loose network facilitating cellular migration. Wounding in this unique environment triggers a complex cascade of tightly controlled events culminating in a scarless wound phenotype of fine reticular collagen and abundant hyaluronic acid. Comparison between postnatal and fetal wound healing has revealed differences in inflammatory response, cellular mediators, cytokines, growth factors, and ECM modulators. Investigation into cell signaling pathways and transcription factors has demonstrated differences in secondary messenger phosphorylation patterns and homeobox gene expression. Further research may reveal novel genes essential to scarless repair that can be manipulated in the adult wound and thus ameliorate scar.

Evidence is provided pointing out certain basic similarities, though not an identity, between the mechanisms of early fetal regeneration and induced organ regeneration in adults. These similarities favor a model of induced organ regeneration in which biologically active scaffolds block wound contraction and scar formation.

The developing fetus has the remarkable ability to heal dermal skin wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix architecture, strength, and function. The biology responsible for scarless wound healing in skin is a paradigm for ideal tissue repair. This regenerative capacity is lost in late gestation when fetal wounds heal with fibrosis and scar. Early in gestation, fetal skin is developing at a rapid pace in a unique environment. Investigation of normal skin embryogenesis and comparison between early scarless and late scarring fetal wounds has revealed distinct differences in inflammatory response, cellular mediators, wound contraction, cytokines, growth factors, and extracellular matrix modulators. The knowledge gained from comparative observational studies has served as a base for experimental interventions in animal models to induce or ameliorate scar. Although much progress has been made over the past decade, the mechanism of fetal wound healing remains largely unknown and attempts to mimic the scarless wound phenotype have not been completely successful. Identification of more key genes involved in skin regeneration may have implications in adult skin wounds and repair in other organ systems.

Vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein that is a potent endothelial cell-specific mitogen with increased expression during adult cutaneous wound healing. VEGF activity is mediated by two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed primarily in vascular endothelial cells. Initiation of profibrotic cytokine expression likely coordinates the transition from scarless healing to scar formation in fetal wounds. Angiogenesis is an important component of the scarring repair process, but the function of VEGF and degree of angiogenesis during scarless repair has not been investigated. We hypothesize that VEGF and its receptors are differentially expressed in scarless compared with scarring fetal wounds because VEGF is implicated in angiogenesis during skin development and adult wound healing. Excisional wounds were created on fetal rats at gestational ages 16.5 days (E16) and 18.5 days (E18) (term = 21.5 days). Wounds were harvested at 24 and 72 hours (n = 12 wounds per time point). Nonwounded fetal skin (E17, E19, and E21) was used as control. Reduced-cycle, specific-primer, reverse-transcriptase polymerase chain reaction was performed to determine the expression of VEGF and its receptors, VEGFR-1 and VEGFR-2. Wounds at 72 hours and fetal skin controls were examined under high-power microscopy for blood vessel counts. Unpaired two-tailed t test was used (p < 0.05 was considered significant). VEGF expression increased 2.4-fold (p < 0.001) during normal skin development from E17 to E19. In scarless wounds (E16), VEGF expression increased 2.8-fold (p < 0.02) at 72 hours. No increased expression occurred in the scarring wounds (E18). VEGFR-1 and VEGFR-2 expression increased over 2-fold during normal skin development from E17 to E21. However, each was down-regulated 30 to 50 percent in scarless (E16) and scarring (E18) wounds. There is a 2-fold increase in mean vessel counts per high-power field in scarless (E16) wounds at 72 hours compared with age-matched control skin (p < 0.02) and a 1.7-fold increase in mean vessel count in scarring fetal wounds (E18) compared with age-matched control skin (p < 0.05). There is no difference in the total number of vessels found in scarless versus scarring wounds or between 19.5-day versus 21.5-day fetal skin. VEGF and its receptors, VEGFR-1 and VEGFR-2, increase expression during skin development and dermal differentiation. VEGF expression quickly elevates during scarless compared with scarring repair, which likely contributes to the more rapid scarless fetal repair rate. Similar numbers of new ves-sels are formed during scarless and scarring fetal repair.

The developing fetus has the ability to heal wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix (ECM) architecture, strength, and function. In contrast, adult wounds heal with fibrosis and scar. Scar tissue remains weaker than normal skin with an altered ECM composition. Despite extensive investigation, the mechanism of fetal wound healing remains largely unknown. We do know that early in gestation, fetal skin is developing at a rapid pace and the ECM is a loose network facilitating cellular migration. Wounding in this unique environment triggers a complex cascade of tightly controlled events culminating in a scarless wound phenotype of fine reticular collagen and abundant hyaluronic acid. Comparison between postnatal and fetal wound healing has revealed differences in inflammatory response, cellular mediators, cytokines, growth factors, and ECM modulators. Investigation into cell signaling pathways and transcription factors has demonstrated differences in tyrosine phosphorylation patterns and homeobox gene expression. Further research may reveal novel genes essential to scarless repair that can be manipulated in the adult wound and thus ameliorate scar.

Surgical intervention is currently performed on highly selected fetuses with anatomical deformities that have a high mortality or severe morbidity when treated postnatally. In the future, in utero surgical intervention for non-life-threatening disease may become possible as fetal surgery becomes safer for the mother and fetus. Fetal cleft repair is an attractive intervention for plastic surgeons because it affords the potential to provide a scarless repair and correct the primary deformity. Furthermore, scarless fetal lip and palate repairs may prevent the ripple effect of postnatal scarring with its resultant secondary dentoalveolar and midface growth deformities. These potential benefits can dramatically reduce the number of postnatal reconstructive procedures in children with facial clefts. The rationale for a prenatal treatment approach to the patient with cleft lip/palate and the experimental evidence to support in utero intervention are discussed in this article.

In utero surgical intervention is an exciting frontier in medicine. Fetal surgeons strive to treat congenital anomalies definitively while organogenesis is still occurring. Many of these anomalies pose such a threat to the viability of the affected fetus that waiting until after the child is born to treat them is frequently not satisfying and too often unsuccessful. We review the embryology of selected systems that have associated aberrancies of development for which fetal surgery is particularly applicable. The surgeon can more effectively launch an assault against congenital anomalies when armed with a solid appreciation of normal development. Recognizing the critical period for the development of a system allows him or her to formulate the optimal time and mode of intervention.

The early-gestation fetus heals dermal wounds rapidly and scarlessly. This phenomenon appears to be intrinsic to fetal skin and independent of the intrauterine environment. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to scarless repair. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults.

Early in gestation, fetal wounds are capable of healing scarlessly. Scarless healing in the fetus is characterized by regeneration of an organized dermis with normal appendages and by a relative lack of inflammation. Although there is a transition period between scarless and scar-forming repair, scarless healing also depends on wound size and the organ involved. The ability to heal scarlessly, furthermore, appears to be intrinsic to fetal skin. Unique characteristics of fetal fibroblasts, inflammatory cells, extra-cellular matrix, cytokine profile, and developmental gene regulation may be responsible for the scarless phenotype of early gestation fetal wounds. With the current knowledge, only minimal success has been achieved with the topical application of neutralizing antibodies, antisense oligonucleotides, and growth factors to improve wound-healing outcomes. Thus, further investigation into the mechanisms underlying scarless repair is crucial in order to devise more effective therapies for scar reduction and the treatment of cirrhosis, scleroderma, and other diseases of excessive fibrosis.

There are a growing number and variety of fetal disorders that may benefit from intervention prior to birth. Despite the diversity, there are common pathophysiologic denominators or patterns that tie together many seemingly disparate disorders. The purpose of this article was not to review disorders presented in other, accompanying articles but, rather, to present pathophysiologic patterns that common influence fetal surgery within a cohesive framework.

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.

Craniosynostosis occurs in approximately 1:2000 live births. It may affect the coronal, sagittal, metopic and lambdoid sutures in isolation or in combination. Although non-syndromic synostoses are more common, over 150 genetic syndromes have been identified. Recent advances in genetic mapping have linked chromosomal mutations with craniosynostotic syndromes. Despite the identification of these genetic mutations, the fundamental biomolecular mechanisms mediating cranial suture biology remain unknown. Today, many laboratories are investigating murine cranial suture biology as a model for human cranial suture development and fusion. Normal murine cranial suture biology is very complex, but evidence suggests that the dura mater provides the biomolecular blueprints (e.g. the soluble growth factors), which guide the fate of the pleuripotent osteogenic fronts. While our knowledge of these dura-derived signals has increased dramatically in the last decade, we have barely begun to understand the fundamental mechanisms that mediate cranial suture fusion or patency. Interestingly, recent advances in both premature human and programmed murine suture fusion have revealed unexpected results, and have generated more questions than answers.

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.

The human fetus is capable of healing cutaneous wounds without scar up to the third trimester of development This process of tissue repair is more akin to newt limb regeneration than classic adult scar forming wound repair. Regeneration of the newt limb is dependent on neural input in its early stages. This study was an attempt to determine whether a similar dependence on neural input exists for mammalian fetal wounds to heal without scar. The left hind limb of six fetal lambs was denervated during the early second trimester of development (day 55; term = 145 days). Two weeks after denervation, the animals were again exposed to create bilateral incisional and 6-mm-diameter excisional wounds on their innervated right and denervated left lower extremities. Five days after creation of these defects, the wounds were examined for alterations in repair. Four fetal lambs survived, and three were suitable for evaluation. There were marked alterations in wound healing seen after denervation. Excisional wounds on the innervated side contracted and decreased their surface area by 14 percent. In contrast, the denervated wounds not only failed to contract, but increased in size by 60 percent. Changes in the incisional wounds were equally distinctive. Innervated incisional wounds healed completely without scar and had a wound breaking strength comparable to that of normal skin (Table I). In contrast, two of the three denervated incisional wounds dehisced and failed to heal, even in the regions where the skin was approximated by suture. The third denervated incisional wound did heal but with a significant amount of scar. Electron microscopy confirmed this finding by clearly demonstrating thickened and irregular collagen deposition in the extracellular matrix of all the denervated incisional specimens. In summary, like the regenerating newt limb, scarless fetal skin wound repair requires neural stimulation for tissue regeneration to occur. Therefore, in the mammal, the primary regulator for this unique type of tissue repair may have a central neural, rather than a local, tissue origin.

Successful open repair of a cleft lip in utero has the advantage of scarless wound healing in the fetus. Unfortunately, no long-term outcome studies have been performed to evaluate the efficacy of these repairs. Moreover, no study to date has compared the long-term results of an in utero cleft lip repair to a similar, control-matched, newborn cleft repair. This study was performed to evaluate the 9-month outcome of in utero cleft lip surgery compared with an identical cleft lip repair performed on infant lambs. In utero epithelialized cleft lips were created through an open hysterotomy in sixteen 65-day-old fetal lambs (term = 140 days) using methods described by Longaker et al. Eight of 16 animals underwent subsequent in utero repair of these clefts at 90 days gestational age. The repair of the remaining eight animals was delayed until 1 week postpartum. At 9 months, the animals were analyzed for changes in lip contour and for the degree of scarring by hematoxylin and eosin and Masson's trichrome collagen staining. Two animals in each group died from preterm labor. Of the animals that survived to term, all repaired lips had some degree of abnormality postoperatively. One of six lips repaired in utero dehisced before delivery. Three of six neonatal repairs dehisced in the first postoperative month. In the remaining animals with intact lip repairs, the vertical lip height on the repaired side was an average of 9 to 12 mm shorter than the normal lip in both the in utero and neonatally repaired animals. Phenotypically, the postnatally repaired animals had more lip distortion and visible notching. Histologically, the in utero repair was scarless and the neonatal repairs had scar throughout the entire vertical height of the lip with an associated loss of hair in this region. Maxillary growth was also evaluated. There was no inhibition of maxillary growth in the animals that underwent in utero cleft lip repair. However, in the neonatal repair group, significant maxillary retrusion was evident. Compared with the cleft side of the maxilla, horizontal growth was decreased by 11 percent (p = 0.01). Compared with the intrauterine repair group, there was a 17-percent decrease in horizontal maxillary width (p = 0.01). Straight-line in utero repair of a cleft lip produces a better long-term result in terms of maxillary growth than a similar repair performed postnatally in the ovine model. There was no diminution in maxillary growth in the animals treated in utero. Histologically, in utero repair of clefts was indeed scarless. However, both lip repairs produced lips that were significantly shorter than their contralateral noncleft sides. This degree of lip shortening would require a secondary lip revision, thereby defeating the purpose of performing an intrauterine repair. Comparisons now need to be made between in utero and neonatal repairs using a Millard-type rotation advancement technique before intrauterine treatment can be considered to be more beneficial than our current treatment modalities.

In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

Thrombospondin 1 (TSP-1), an adhesive glycoprotein, plays an important role in platelet adhesion, inflammation, cell-cell interaction, and angiogenesis. TSP-1 is expressed by endothelial cells, fibroblasts, and macrophages. The unique cysteine-serinevaline-threonine-cysteine-glycine (CSVTCG) binding domain of TSP-1 also plays an important role in cell binding and modulation of cellular processes. The purpose of this study was to evaluate histologically and quantitatively TSP-1 and its CSVTCG receptor in fetal skin wounds over time. Pregnant ewes underwent laparotomy and hysterotomy. At 65 days gestation (term, 145 days), incisional and excisional wounds were created on the fetal back in a similar position on each animal. The uterus and laparotomy were closed. The wounds were harvested on days 1, 3, 7, 21, and 28. Expression of TSP-1 and its CSVTCG receptor was evaluated immunohistochemically and quantitated by computer image analysis in units of absorbance. Immunoglobulin G (negative) controls were performed and subtracted from the TSP-1 sample to eliminate background absorbance readings. Serum (negative) control was used for the CSVTCG receptor. Platelet concentrates were used as the positive control: TSP-1, 63.43; CSVTCG, 58.72. Results are expressed as absorbance+/-SEM. Results of TSP-1 are as follows: day 1, 33.02+/-0.26; day 3, 22.21+/-0.14; day 7, 20.56+/-1.07; day 21, 7.76+/-0.40; and day 28, 5.99+/-0.03. TSP-1 displays an early peak during fetal skin repair, followed by a steep decrease over the viewed time period. Results of CSVTCG receptor are as follows: day 1, 26.19+/-2.43; day 3, 30.20+/-0.64; day 7, 24.56+/-0.80; day 21, 24.70+/-0.40; and day 28, 21.65+/-1.39. Thus, CSVTCG receptor displays a slowed decrease in expression over time during fetal repair. No significant differences were noted between incisional and excisional samples. Temporal and histological differences exist in the localization and expression of TSP-1 and its CSVTCG receptor during fetal wound repair. TSP-1 is upregulated in tissues early. This corresponds with the known role of TSP-1 in cell-cell interaction, including potentiation of growth factor activity. TSP-1 also modulates matrix, allowing scar-free provisional matrix in the earlier stages of repair deposited by platelets. The potentiation of cell-associated protease activity by TSP-1 can support tissue and matrix turnover. This activity of TSP-1 may contribute to the formation of a scarless wound. TSP-1 destabilizes extracellular matrix contacts, and facilitates mitosis and migration. The action of TSP-1 as an adhesive protein allows numerous different cells to adhere to the extracellular membrane. CSVTCG receptor expression decreases during fetal repair as the cells migrate to the epithelial surface, suggesting a significant role of the CSVTCG receptor in keratinocytic maturation, differentiation, and epithelization.

Background/purpose: The transforming growth factor-beta (TGF-beta) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-beta cytokine isoforms (TGF-beta1, beta2, and beta3), as well as that of the type II TGF-beta receptor (TbetaRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ., Methods: Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistrywas performed using polyclonal antibodies to TGF-beta1, beta2, and beta3, and TbetaRII., Results: The patterns of expression of TGF-beta1, beta2, and beta3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TbetaRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TbetaRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells., Conclusions: TGF-betas may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-beta1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TbetaRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

The cleft nasal deformity, a combination of malpositioned cartilage and tissue and postrepair scarring, is a difficult problem to correct. To harness the potential of scarless fetal wound healing, in utero repair of cleft lip and palate deformities has been studied but the fetal cleft nose deformity has not been addressed. The purpose of this study was to manipulate the fetal nasal shape in utero as a first step toward restoration of normal nasal form in cleft nasal deformities. To do this, preformed hypertonic sponges were placed into the right nostril of eight fetal lambs during the second trimester (when scarless cutaneous wound repair is known to occur). Then, the size and shape of fetal nasal structures were analyzed after selected time periods (1, 2, and 6 weeks) with measurements, routine histologic examination, and three-dimensional computed tomographic scans of the experimentally expanded noses compared with the control nonexpanded noses of the birth twins or age-matched specimens. Results showed that experimentally expanded nasal structures had markedly increased in septal length measurement, in nostril area (doubled), and in intranasal volume (more than doubled). Histology showed normal cellular elements without scarring in the tissue sections from the expanded nasal areas. In conclusion, the shape of nasal tissue can be manipulated without scarring in second-trimester fetal lambs after placement of a nasal expansion device. This study is an experimental first step toward restoring normal nasal form by repositioning alar cartilages and soft tissue during fetal cleft repair.

Fetal wounds heal without a scar early in gestation, and may hold the key to scarless repair. Several important concepts central to the fetal wound-healing response have been determined. The fetal fibroblast modulates the wound-healing response through collagen deposition, extracellular matrix deposition, and growth factor secretion. Fetal repair is both gestational-age and wound-size dependent, with a transition from scarless to scarring repair occurring during fetal life. Fetal fibroblasts manifest a decreased ability to induce dermal appendage formation from fetal epithelium at the same time that scarring in the fetus begins, suggesting that epithelial-mesenchymal interactions play an important role in scarless fetal repair. The fetal immune response during wound healing differs from the adult response, with a primarily mononuclear cell infiltrate and decreased activity and presence of polymorphonuclear leukocytes, whereas the cytokine profile of the fetal wound differs markedly from that of the adult wound. Patterning genes (homeobox genes) involved in organogenesis may prove integral to fetal healing, and are emerging as an active area of research. Once the biology of fetal wound healing is fully determined, attempts to manipulate the adult wound undoubtedly will progress rapidly, and scarless repair may become a clinical reality in children and adults.

We performed the first in utero correction of a unilateral right coronal craniosynostosis using 70-day gestation fetal lambs. The craniosynostosis was created in eight fetuses by excising their right coronal sutures, and then placing demineralized bone powder, transforming growth factor-beta, and bone morphogenetic protein-2 into the defect. Twenty-one days later, after suture fusion had occurred, four of the eight sheep were treated with a 4 mm x 12 mm strip craniectomy to open the entire synostosed right coronal suture. The edges of the excision were wrapped with 100-microm-thick Gore-Tex (W. L. Gore & Associates, Flagstaff, Ariz.) sheets to prevent bony refusion. All eight lambs then progressed to term (140 days). The skulls of four normal, unoperated, term lambs were used as controls. At 140 days, all four treated lambs had a widely patent strip craniectomy site without any evidence of bone regeneration. This in utero correction led to a marked improvement in craniofacial morphology of three of four animals when compared with the uncorrected controls with significant (p < 0.01) correction in orbital position, skull length, and shape of the frontal bone. This was in sharp contrast to the uncorrected animals, which had marked orbital elevation, compression of the anteroposterior length of the cranial vault, frontal bone flattening, and shortening of the cranial base. The fourth corrected animal also showed evidence of improvement but had some abnormal calvarial changes secondary to the development of horns, which displaced the calvaria in a downward vector. We conclude that the in utero correction of craniosynostosis is feasible and provides a significant benefit by decreasing the severity of many of the associated deformities seen with this disorder.

Fetal mammals heal skin wounds through the second trimester of development without evidence of scar. We have investigated the role of bone morphogenetic protein 2 (BMP-2), which is a member of the TGF-beta superfamily, in normal skin development and fetal wound healing. We first used RNA in situ hybridization to demonstrate that BMP-2 was expressed at low levels in the developing hair follicles and in the epidermis of normal human fetal skin. We then created an in vivo model to test how exogenous BMP-2 would affect fetal skin development and wound healing. Fifty micrograms of BMP-2 was implanted into the subcutis of five 70-day-old fetal lambs through a full-thickness linear incision. The BMP-2 was placed beneath the right half of the wound, whereas the left half served as an untreated control. In two of the five animals 1 microgram of TGF-beta was placed into the same position in addition to the 50 micrograms of BMP-2. Twenty days later (90 days gestation, term = 140 days) all the fetal wounds were examined for evidence of cellular hyperproliferation and scar formation. BMP-2 induced massive dermal and epidermal growth when compared with controls. This finding was characterized by marked epidermal thickening and keratinization, a dramatic increase in the number of hair follicles, and more than 50 percent thickening of the dermis. The dermal thickening was the result of both increased cellularity and deposition of large irregular collagen bundles. Wounds treated with both BMP-2 and TGF-beta healed also with an adult-like pattern of scar formation. Surprisingly, the wounds with BMP-2 alone healed with an equal pattern of scar, indicating that there was not an additive effect of combining BMP-2 and TGF-beta. We conclude that BMP-2 is a pleomorphic growth factor that induces cellular growth, maturation, and fibroplasia in both the dermis and epidermis. Further analysis of this growth factor in both fetal and adult wound healing may lead to important discoveries regarding the control of scar formation and fibrosis in many adult tissues.

The lack of scarring and fibrosis in healing fetal skin wounds may relate to a prolonged presence of hyaluronan (HA). It has been suggested that fetal wounds may lack hyaluronidase, but the hyaluronidase levels in fetal wounds remain unknown. The size of HA influences its biological action, especially in relation to angiogenesis, which is also reduced in fetal wound healing. The present study determined the levels and size of HA, as well as hyaluronidase levels, in fetal and adult lamb wounds. Wire mesh cylinders, or polyvinyl acetate sponges, were placed subcutaneously in fetal lambs at 75, 100 or 120 days gestation. Wound fluid and wound tissue were harvested 3, 7 or 14 days later. Samples were digested with papain and both HA and hyaluronidase activity were determined in a competitive ELISA assay. Size distribution of HA was estimated using a Sephacryl S1000 column and fractions were collected for HA determination. Adult wound fluid HA remained low (4-5 micrograms/ml) over the 14 days. Fetal fluids were similar on day 3, but increased to 15-25 micrograms/ml by day 7. In 75/100-day wounds, HA remained elevated at 14 days, but in 120-day fluids decreased to levels similar to adult fluid. The HA in all fluids was polydisperse with a main peak at 200 kDa. Hyaluronidase levels were detected in all samples, reaching a peak 7 days post-wounding. In adult wound fluids hyaluronidase was much higher than the fetal wound fluids. These data suggest that lower hyaluronidase levels in fetal wounds may underlie the different pattern of HA deposition seen in fetal wounds.

Human fetal skin heals without scar formation when it is transplanted to a subcutaneous location on an adult athymic mouse and subsequently wounded. In contrast, human fetal skin of identical gestational age heals with scar formation when transplanted to a cutaneous location on the athymic mouse recipient. To determine if mouse (adult) or human (fetal) fibroblasts are healing the graft wounds, we performed indirect immunohistochemistry for mouse and human collagen types I and III. Full-thickness skin grafts (n = 51) from human fetuses at 18 weeks' (n = 4) or 24 weeks' (n = 2) gestational age were placed onto athymic mice in two locations: cutaneously onto a fascial bed and subcutaneously in a pocket under the murine panniculus carnosus. Linear incisions were made in each graft 7 days after transplantation. Grafts were harvested at 7, 14, and 21 days after wounding and stained with hematoxylin and eosin or Mallory's trichrome. Immunohistochemistry for either human collagen type I or type III or for mouse collagen type I was performed. The subcutaneous grafts healed with human collagen types I and III in a scarless pattern. The wound collagen pattern was reticular and unrecognizable from the surrounding dermis. Hair follicles and sebaceous gland patterns were unchanged in the wounded dermis. Conversely, the cutaneous grafts healed with mouse collagen in a scar pattern with disorganized collagen fibers and no appendages. Mouse collagen scar was present along the base of the cutaneous grafts and as a thin capsule around the subcutaneous grafts. We conclude that (1) subcutaneous grafts heal with human fetal collagen and no scar formation, and (2) cutaneous grafts heal with mouse collagen in a scar pattern. Fetal fibroblasts can heal fetal skin wounds without scar despite being perfused by adult serum and inflammatory cells in an adult environment. These data suggest that the fetal fibroblast is the major effector cell for scarless fetal skin repair.

Objective: This study investigated the influence of the fetal environment on the healing characteristics of adult skin., Summary Background Data: The remarkable ability of the fetus to heal without scarring is poorly understood. The unique qualities of fetal wound healing may be caused by the fetal environment, the fetal tissues, or a combination of both. There are numerous differences between the prenatal and postnatal environments that may play a role in the unique fetal response to injury., Methods: Full-thickness adult sheep skin was transplanted onto the backs of 60-day-gestation fetal lambs (term, 145 days of gestation). The adult skin grafts were thus perfused by fetal blood and bathed in amniotic fluid. Previous work has demonstrated that, before midgestation, fetal lambs do not reject allogenic skin grafts. Forty days later (100 days of gestation), incisional wounds were made on both the adult skin graft and the adjacent fetal skin. The wounds were harvested 14 days postwounding and analyzed by both light microscopy and immunohistochemical testing using antibodies to collagen types I, III, and VI., Results: The wounds in the adult skin grafts healed with scar formation. This observation contrasts strongly with the scarless healing of the incisional fetal skin wounds., Conclusions: This study suggests that scarless fetal skin healing properties are intrinsic to fetal skin and are not primarily the result of the fetal environment.

Sequential surgical procedures in cleft lip/palate (CL/P) patients result in scar formation that is believed to be associated with midface growth retardation. By use of a previously developed fetal lamb model, wound healing characteristics were investigated after in utero CL repair. It is hypothesized that scarless healing after fetal CL repair occurs without inflammation and scar formation. CL wounds were created in mid-second-trimester fetal lambs and either repaired in three layers (mucosa, muscle, and skin) or left unrepaired. Fetuses were then harvested at 7, 14, and 21 days postoperatively, and the wound site was examined microscopically. When created at 75 days' gestation (term = 145 days), fetal lamb CL wounds heal rapidly without inflammation and scar formation. By day 21 postoperatively, there was complete regeneration of skin, muscle, and mucosa, as well as epidermal appendages. With this model, it will be possible to compare the effects of scarless fetal CL repair with those of postnatal repair on midface growth.

Wound healing in the fetus occurs by a different process from that in the adult. Instead of healing with scar formation, fetal cutaneous wounds heal by regeneration that results in complete restoration of normal skin architecture. The mechanisms responsible for this remarkable phenomenon involve factors in the fetal environment and properties intrinsic to fetal cells. Hyaluronic acid (HA) is a major component of the fetal extracellular matrix (ECM) and is believed to play an important role in this process. In this study, HA and HA-stimulating activity (HASA) in fetal and adult wound fluid were examined using sensitive, newly developed assays. In an ovine model, higher levels of HA and HASA were observed in fetal as compared with adult wound fluid. This difference was most prominent in wound fluid from fetal lambs at 75 and 100 days gestation (term = 145 days); these samples contained persistently elevated HA and HASA levels for up to 2 weeks after wounding (HA peak levels 145 micrograms/mL and 110 micrograms/mL, respectively). In contrast, wound fluid from 120-day-gestation fetuses had significantly lower levels (P < .001) that were transient and similar to that in the adult (HA peak levels 70 micrograms/mL and 10 micrograms/mL, respectively). These observations confirm an ontogenic transition in wound HA metabolism from a fetal to an adult-like phenotype. Levels of HASA as a function of time after wounding correlated with levels of HA, suggesting a role for HASA in controlling HA deposition during tissue repair. Two patterns of HASA and HA synthesis were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: This study determined how scar formation develops in a non-human primate model of fetal skin repair., Summary Background Data: A transition from healing scarlessly to healing with scar formation characterizes skin repair in rat and sheep fetuses. New knowledge of the regulatory processes occurring in the fetal wound at the initial stages of scar formation may provide insights into the early mechanisms of scar formation., Methods: Full-thickness wounds were made in fetal rhesus monkey lips from 75 through 114 days gestation (n = 6, term = 165 days). Wounds were harvested at 14 days postwounding and processed for histology (hematoxylin & eosin, Masson's trichrome) as well as immunohistochemistry (human type I or type III collagen)., Results: Wounds healed with complete restoration of normal tissue architecture in the 75-day gestation fetus. However in the 85-100 day gestation fetuses, wounds healed with an absence of hair follicles and sebaceous glands, but the dermal collagen pattern remained reticular and similar to that in unwounded dermis. At 107 days, a thin scar was present in the wound, thereby demonstrating a transition to scar formation between 100 and 107 days gestation (early 3rd trimester) in the non-human primate., Conclusions: In the non-human primate fetus, a transition from scarless repair to adult-type repair with scar formation occurs in the early third trimester. These data provide insight into the transition process; the ontogeny of scar formation is characterized initially by wounds healing without the presence of epidermal appendages but with a normal reticular dermal collagen pattern, which we term the "transition wound."

Fetal wounds are abundant in hyaluronic acid (HA), but little is known as to the total HA content of fetal tissues as a function of gestational age. Previous studies demonstrated scarless healing prior to approximately 130 days gestation, after which disorganized collagen deposition became prevalent. Glycosaminoglycans (GAGs) have been shown to play an important role in wound healing, cytodifferentiation, and morphogenesis. In this study, we examined the HA and GAG content of fetal sheep skin of increasing gestational age. We found that the total amount of HA and GAGs declined from a high at 80 days gestation (528 +/- 9 micrograms/gm) to a low at 130 days (174 +/- 11). Analysis of the various GAG species revealed that HA comprised the largest fraction (75-96%). The sulfated GAGs, Heparan Sulfate (HS) and Dermatan Sulfate (DS), were not present in the extracellular matrix until 120 days gestation. Both the trough of HA content and the appearance of sulfated GAGs in the extracellular fraction correspond to the appearance of scarring in fetal sheep wound repair.

In utero repair of several life-threatening malformations in the human fetus is now a clinical reality, yet fetal surgery still poses significant risks to both the mother and the unborn child. Preterm labor is a major problem and is directly related to the large hysterotomy required for fetal exposure. Endoscopic surgical manipulation of the fetus, through small uterine "ports," solves this problem and may eventually permit fetal intervention for non-life-threatening malformations. In this pilot study we demonstrated the feasibility of performing endoscopic surgery on the fetus in situ. A lip incision was created and repaired using endoscopic microsurgical techniques in midgestation fetal lambs. This represents the first application of this technique for in utero fetal intervention.

Fetal wounds heal without inflammation and scar formation. This phenomenon may, in the future, be applicable to human cleft lip and palate repair. However, extensive experimental work must first be done to document the benefits of in utero repair. We developed a large animal model for creation and repair of a complete cleft lip and alveolus using fetal lambs. The cleft lip and alveolus deformity was created in eight 75-day-gestation fetuses (term = 145 days) and either repaired in three layers or left unrepaired. There were four sham-operated fetuses, and all animals were alive at harvest. Repaired, unrepaired, and control fetuses were harvested at 7, 14, 21, and 70 days following surgery. The unrepaired fetuses demonstrated a complete cleft lip and alveolus with an oronasal fistula. The maxilla was asymmetrical, with the greater segment deviated toward the cleft and with decreased anterior maxillary width. In contrast, repaired cleft lip and alveolus animals showed no scar, normal thickness of the lip, and a symmetrical maxilla. Histologic analysis of the repaired wounds showed evidence of tissue regeneration without scar formation. The results of this preliminary study indicate that the fetal lamb cleft lip and alveolus model is technically feasible with an excellent survival rate. Healing occurs without scar formation. In the repaired animals, the maxilla was symmetrical. This model will be used to document facial growth following in utero repair of a cleft lip and alveolus.

The purpose of this report is to call attention to the fetal wound healing process as a blueprint for ideal tissue repair. Wound healing in the fetus is fundamentally different from healing in the adult. Fetal tissue repair occurs rapidly and in the absence of scar formation. Because scarring and fibrosis dominate some diseases in every area of medicine, an understanding of fetal wound healing should help develop therapeutic strategies to avert the devastating consequences of excessive scar formation.

Recent experimental and clinical evidence suggests that the fetus responds to injury in a fashion fundamentally different from that of the adult. Acute inflammation is almost always absent, hyaluronic acid is a prominent component of the wound matrix, and collagen is deposited in a scarless manner. Using a variety of animal models and techniques, numerous investigators have begun to analyze the constituents of the fetal wound healing process in an attempt to understand the control mechanisms that endow the fetus with unique healing abilities. Since scarring and fibrosis dominate some diseases in almost every medical specialty, the ultimate clinical aim is to delineate the biological principles of fetal wound healing and then apply them to modulate adult wound healing problems.

Clinical observations and experimental data suggest that fetal wound healing is very different from adult wound healing. An understanding of the biology of scarless fetal wound healing has tremendous clinical potential for modulating postnatal wound problems. In this study, the fetal lamb model was used to assess excisional fetal skin wound contraction in utero. Full-thickness 9-mm punch biopsy wounds were created on fetal lambs at 100 days' gestation (term, 145 days). Half of the wounds remained exposed to amniotic fluid, whereas the other half were covered by a silastic patch to exclude amniotic fluid. Wounds were harvested 3, 7, or 14 days later and wound areas were calculated. Exposure to amniotic fluid retarded wound contraction significantly at 3 days, but by 14 days all wounds had completely contracted and reepithelialized. Myofibroblasts are an important cellular element of wound contraction. The presence of wound myofibroblasts was documented by both transmission electronmicroscopy and immunocytochemistry with antimuscle actin antibody. It is concluded that fetal lamb wounds contract in utero and exposure to amniotic fluid appears to retard fetal skin wound contraction only during the early healing process.

Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, we tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104, 114, 124, and 141 days' gestation (term gestation for the fetal lamb is 145 days), as well as from 10-day-old neonatal sheep and adult sheep. Labeled S aureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonize bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth.

We have previously described a model for in utero cleft lip repair in rabbits. Cleft lip and alveolus (CL) were created in fetal rabbits at 24 days gestation (term, 31 days). In this study, postnatal maxillary growth was evaluated in three groups of animals: 1) unoperated controls, 2) unrepaired CL, and 2) repaired CL. The animals were killed at 4, 12, and 26 weeks after birth. Direct cephalometry was performed on dry skulls to evaluate premaxillary width, anterior maxillary length and width, and posterior maxillary width. The results of this study indicate that rabbits that undergo an in utero CL procedure, with or without repair, exhibit no significant decrease in maxillary length and width when compared with controls.

Midgestation fetal wound healing is characterized by healing without fibrosis or scar formation. The mechanisms that underlie this remarkable process are mediated in part through a fetal wound extracellular matrix rich in hyaluronic acid. In this study a newly developed assay was used to determine the hyaluronic acid levels in fetal and adult wound fluid. Adult wound fluid had a rapid increase in hyaluronic acid, which peaked at 3 days and decreased to 0 by 7 days. In contrast levels of hyaluronic acid in fetal wound fluid increased rapidly and remained significantly elevated for 3 weeks. This prolonged presence of hyaluronic acid in the matrix of fetal wounds creates a 'permissive' wound environment that promotes fetal fibroblast movement and proliferation and inhibits cytodifferentiation. Such a matrix environment promotes healing by regeneration rather than by scarring. This observation has therapeutic implications. The prolonged application of hyaluronic acid or hyaluronate protein complexes to wounds in children or adults may modulate healing in a manner that makes the wounds more fetal-like.

Fetal wound healing is fundamentally different from wound healing in the adult. Although experimental work in mice, rats, rabbits, monkeys, and sheep has demonstrated that fetal healing occurs without inflammation and scarring, all of these studies have been limited to fetal skin wounds. Whether all fetal tissues heal in a regenerative-like fashion is unknown. Amniotic fluid exposure may play an important role in scarless fetal skin wound healing, but the effect of amniotic fluid on fetal mesothelial wound healing has not been characterized. To investigate these questions we created bilateral linear diaphragmatic wounds in 100-day gestation fetal lambs (term = 145 days). The right thoracotomy was closed to exclude amniotic fluid. In contrast, the left thoracotomy was fashioned into an Eloesser flap which permitted the left diaphragmatic wound to be continually bathed in amniotic fluid. Wounds were harvested after 1, 2, 7, or 14 days and analyzed by light microscopy and immunohistochemistry with antibodies to collagen types I, III, IV, and VI. Whether bathed in or excluded from amniotic fluid, the mesothelial-lined diaphragm healed with scar formation and without evidence of muscle regeneration. Interestingly, diaphragmatic wounds exposed to amniotic fluid were covered by a thick fibrous collagen peel similar to that seen in gastroschisis bowel. These findings indicate that not all fetal tissues share the unique scarless healing properties of fetal skin.