Interleukin-33 encourages scar formation in murine fetal skin wounds.

The magnitude of the inflammatory response after skin injury is important for determining whether wounds in developing fetal skin will heal scarlessly (minimal inflammation) or with prominent scars (robust inflammation). One class of inflammatory mediators gaining attention for their role in wound inflammation is alarmins. In the current study, the alarmin interleukin-33 (IL-33) was examined in a mouse model of fetal wound healing. IL-33 expression was elevated in scar-forming embryonic day 18 wounds compared to scarless embryonic day 15 wounds. Furthermore, injection of IL-33 into embryonic day 15 wounds caused scarring when wounds were analyzed at 7 days postwounding. The introduction of IL-33 into embryonic day 15 wounds did not induce statistically significant changes in the number of neutrophils, mast cells, or macrophages in vivo. However, IL-33 treatment enhanced collagen expression in cultured fibroblasts derived from adult and fetal murine skin, suggesting that IL-33 may directly stimulate fibroblasts. In vitro studies suggested that the stimulation of collagen production by IL-33 in fibroblasts was partially dependent on NF-κB activation. Overall, the data suggest an association between IL-33 and scar formation in fetal wounds.

Modulation of the inflammatory response by increasing fetal wound size or interleukin-10 overexpression determines wound phenotype and scar formation.

Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro-inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin-10 (IL-10). To test this hypothesis, 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep. A subset of 8-mm wounds were injected with a lentiviral vector containing the IL-10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real-time polymerase chain reaction. In contrast to the scarless 2-mm wounds, 8-mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral-mediated overexpression of the IL-10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL-10 in the transition from fibroplasia to fetal regenerative healing.

The alarmin HMGB-1 influences healing outcomes in fetal skin wounds.

In mice, cutaneous wounds generated early in development (embryonic day 15, E15) heal scarlessly, while wounds generated late in gestation (embryonic day 18, E18) heal with scar formation. Even though both types of wounds are generated in the same sterile uterine environment, scarless fetal wounds heal without inflammation, but a strong inflammatory response is observed in scar-forming fetal wounds. We hypothesized that altered release of alarmins, endogenous molecules that trigger inflammation in response to damage, may be responsible for the age-related changes in inflammation and healing outcomes in fetal skin. The purpose of this study was to determine whether the alarmin high-mobility group box-1 (HMGB-1) is involved in fetal wound repair. Immunohistochemical analysis showed that in unwounded skin, E18 keratinocytes expressed higher levels of HMGB-1 compared with E15 keratinocytes. After injury, HMGB-1 was released to a greater extent from keratinocytes at the margin of scar-forming E18 wounds, compared with scarless E15 wounds. Furthermore, instead of healing scarlessly, E15 wounds healed with scars when treated with HMGB-1. HMGB-1-injected wounds also had more fibroblasts, blood vessels, and macrophages compared with control wounds. Together, these data suggest that extracellular HMGB-1 levels influence the quality of healing in cutaneous wounds.

Novel differences in the expression of inflammation-associated genes between mid- and late-gestational dermal fibroblasts.

While cutaneous wounds of late-gestational fetuses and on through adulthood result in scar formation, wounds incurred early in gestation have been shown to heal scarlessly. Unique properties of fetal fibroblasts are believed to mediate this scarless healing process. In this study, microarray analysis was used to identify differences in the gene expression profiles of cultured fibroblasts from embryonic day 15 (E15; midgestation) and embryonic day 18 (E18; late-gestation) skin. Sixty-two genes were differentially expressed and 12 of those genes are associated with inflammation, a process that correlates with scar formation in fetal wounds. One of the differentially expressed inflammatory genes was cyclooxygenase-1 (COX-1). COX-1 was more highly expressed in E18 fibroblasts than in E15 fibroblasts, and these differences were confirmed at the gene and protein level. Differences in COX-1 protein expression were also observed in fetal skin by immunohistochemical and immunofluorescence staining. The baseline differences in gene expression found in mid- and late-gestational fetal fibroblasts suggest that developmental alterations in fibroblasts could be involved in the transition from scarless to fibrotic fetal wound healing. Furthermore, baseline differences in the expression of inflammatory genes by fibroblasts in E15 and E18 skin may contribute to inflammation and scar formation late in gestation.

Discovery and development of avotermin (recombinant human transforming growth factor beta 3): a new class of prophylactic therapeutic for the improvement of scarring.

Scarring in the skin following surgery or trauma may be associated with adverse aesthetic, functional, growth and psychological effects, such that both physicians and patients regard it as important to minimize the appearance of scars. The prophylactic improvement of cutaneous scar appearance represents a significant opportunity to improve the well-being of patients. Human recombinant transforming growth factor beta 3 (avotermin) is the first in a new class of therapeutic agents to address this medical need. Herein we describe scar-free healing in early embryonic development, including the identification of the cellular and molecular mechanisms underpinning the scarring process. This understanding has led to the discovery of novel therapeutics such as transforming growth factor beta 3, which can be administered to improve scar appearance in human subjects through pharmacological action. We discuss the pioneering development of transforming growth factor beta 3 in this new therapeutic area showing how it has been possible to translate preclinical concepts into clinical application, namely the improvement of scar appearance following surgery.

Identification of differentially regulated genes in fetal wounds during regenerative repair.

During mammalian skin development, wounds heal with regeneration rather than scar. Genomic microarray analysis of fetal (scarless) and postnatal (scarring) cutaneous wounds was performed to identify genes with differential expression and possible proregenerative function. Differentially expressed genes between the scarless and scarring wound transcriptomes were identified with significance analysis of microarrays. At early time points, the fraction of genes with increased expression was greater in the fetal wounds. Conversely, as time after injury increased, the fraction of genes with increased expression in postnatal wounds increased from 0% at 1 hour to 67% at 24 hours. The fetal 1- and 12-hour wound transcriptomes identified genes important in DNA transcription and repair, cell cycle regulation, protein homeostasis, and intracellular signaling. The predominant expression patterns of these genes from 1 to 24 hours predominantly revealed rapid up-regulation, followed by declining expression at 24 hours. Fewer genes with differential expression between the fetal scarless and postnatal scarring wound transcriptomes were identified at 24 hours, most of which had greater expression in the postnatal wound. Our data suggest that multiple gene products may be necessary for the coordination of skin regeneration during wound repair in the fetus.

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats.

OBJECTIVE: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. METHODS: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term = 21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5,705 probes representing 5,705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. RESULTS: Among 5,705 rat genes, there were 53 genes (0.93 percent) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 (FGF2) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. CONCLUSIONS: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.

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.

Scarless fetal wounds are associated with an increased matrix metalloproteinase-to-tissue-derived inhibitor of metalloproteinase ratio.

In contrast to adult cutaneous wounds, early fetal wounds heal scarlessly. Fetal rat skin transitions from scarless repair to healing, with scar formation between days 16.5 (E16) and 18.5 (E18) of gestation. Term gestation is 21.5 days. The composition of the extracellular matrix in fetal skin and wounds differs from that of the adult. Matrix metalloproteinases (MMPs) and their tissue-derived inhibitors (TIMPs) determine the architecture of the extracellular matrix. The authors hypothesized that differential expression of MMPs and TIMPs occurs during the ontogenetic transition to scar-forming repair in fetal skin and wounds. Full-thickness, excisional wounds (2 mm) were created on the dorsum of E16 (n = 42 fetuses) and E19 fetal rats (n = 42 fetuses). Wounds were harvested at 24, 48, and 72 hours. Nonwounded skin from littermates was also harvested as controls. Six E16 and E19 wounds were fixed 72 hours after injury, stained with hematoxylin and eosin, and examined by light microscopy. RNA was isolated from the remaining wounds and skin, and a reduced-cycle, primer-specific, reverse-transcriptase polymerase chain reaction was performed to semiquantitatively determine relative gene expression of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 and of TIMP-1, TIMP-2, and TIMP-3. Significance was determined by unpaired two-tailed t test (p < 0.05) and analysis of variance. In both E16 and E19 wounds, reepithelialization was complete by 72 hours. E16 wounds healed scarlessly, whereas E19 wounds healed with scar. During late gestation, skin expression of MMP-1 and MMP-14 (membrane type-1 MMP) doubled, whereas MMP-2 expression increased nearly 50-fold. Levels of MMP-7 and MMP-9 were unchanged in developing skin. As for the TIMPs, skin expression of TIMP-2 increased more than four-fold, whereas TIMP-1 and TIMP-3 expression was unchanged. In both scarless and scarring wounds, up-regulation of MMP-1 and MMP-9 occurred. However, the maximal increase in MMP-1 and MMP-9 expression occurred much more rapidly and was much greater in the scarless E16 wounds (28-fold versus 23-fold for MMP-1 and 18-fold versus nine-fold for MMP-9). Unchanged in scarless wounds, MMP-2 levels decreased more than three-fold in scarring wounds. MMP-14 (membrane type-1 MMP) expression increased three-fold in scarless wounds but was unchanged in scarring wounds. In contrast, TIMP-1 and TIMP-3 expression in E19 scarring wounds increased six-fold and four-fold, respectively. MMP-7 and TIMP-2 expression did not change in response to injury. E16 scarless wounds have greater MMP relative to TIMP expression than E19 scarring wounds. This favors extracellular matrix turnover, facilitates migration of fetal cells, and promotes scarless repair.

Wound size and gestational age modulate scar formation in fetal wound repair.

The early-gestation fetus heals incisional skin wounds rapidly and scarlessly. The morphology with which the fetus heals excisional skin wounds remains unclear. To characterize excisional fetal wound repair, and to determine whether there is a developmentally regulated wound-size threshold beyond which fetal skin heals with scar, the authors created excisional wounds in fetal lambs of varying gestational age. Time-mated pregnant ewes carrying 22 fetuses at 60 to 90 days' gestation (term, 145 days) underwent laparotomy and hysterotomy. An incisional wound and four circular, punch biopsy wounds of 2, 4, 6, and 10 mm in diameter were placed on the back of each fetal lamb and marked with India ink. The wounds were harvested at 14 days' postwounding and examined grossly and microscopically after serial sectioning and histological staining. Morphological features of all wounds were graded. By 14 days' postwounding all fetal wounds had healed completely. for lambs at each gestational age, increasing wound size was strongly associated with an increase in the frequency of scar. Also, as gestational age increased from 60 to 90 days' gestation the frequency of scarless repair decreased. By understanding the cellular and molecular processes that mediate scar formation with increasing wound size and advancing gestational age, the authors hope to gain further insight into the mechanisms of scarless fetal wound repair.

Differential collagen I gene expression in fetal fibroblasts.

PURPOSE: Fetal wound healing is characterized by the regeneration of normal dermis and the absence of scar. Transforming growth factor beta-1 (TGF-beta1) is a ubiquitous cytokine with potent fibrogenic effects in both postnatal and fetal wounds. Supplementing fetal wounds with TGF-beta1 results in increased fibrosis consisting primarily of collagen I. We hypothesized that the lack of scar formation in fetal wounds may be caused by differential collagen I gene (COL1A1) expression. The authors examined basal collagen Ia gene expression in human fetal, newborn, and adult dermal fibroblasts after stimulation with exogenous TGF-beta1. METHODS: Subconfluent human dermal fibroblasts from fetal, newborn, and adult cell lines were incubated for 24 hours, then stimulated by incubation for 4 hours with 1 ng/mL of human recombinant TGF-beta1, or with media alone for basal collagen gene expression, and then placed in guanidium isothyocyanate buffer. To quantitate COL1A1 gene expression, total cellular RNA was extracted and subjected to northern and slot blot hybridization analysis with Dig-labeled COL1A1 probes. The membrane was exposed to x-ray film for 15 minutes and developed. RESULTS: Scant COL1A1 gene transcript was detected in control fetal fibroblasts. Brief stimulation with of TGF-beta1 upregulated the COL1A1 gene transcription in fetal fibroblasts. Gene expression for COL1A1 in both postnatal cell lines appeared similar in treated and untreated cells. Housekeeping control (GAPDH) confirmed no difference in total amount of RNA at the start or end of the experiment. CONCLUSION: COL1A1 gene expression is notably absent in unstimulated fetal fibroblasts, but is upregulated by TGF-beta1. In contrast, postnatal fibroblasts demonstrate significant constitutive COL1A1 gene expression at baseline and unchanged after TGF-beta1 stimulation. This differential regulation may contribute to the ability of fetal wounds to regenerate without scar and explain the effect of exogenous TGF-beta1 to increase fibroplasia in fetal dermal incisional wounds.

Myofibroblast persistence in fetal sheep wounds is associated with scar formation.

The scarless repair capabilities of the fetus are influenced by the size of the wound and the gestational age of the fetus. Whereas small wounds heal scarlessly, large wounds in the same fetus heal with scar. Myofibroblasts are specialized fibroblasts that express alpha-smooth muscle actin (alpha-SMA), a contractile cytoskeletal protein. The authors hypothesized that small fetal wounds that heal scarlessly will have a relative absence of myofibroblasts, whereas large wounds that heal with scar will have abundant myofibroblasts. In this study, an incisional wound and four punch biopsy wounds of 2, 4, 6, and 10 mm diameter were placed on the backs of 60- to 90-day-gestation fetal sheep (term, 145 days). Fourteen days after wounding, the healed fetal wounds were harvested, the repair morphology was determined (scarless, transitional repair, or scar), and the expression of alpha-SMA was analyzed by immunohistochemistry. In the second part of this study the authors analyzed the temporal expression of alpha-SMA in fetal wounds at 1, 2, 3, and 7 days after wounding in 70-day-gestation fetal sheep. In the 14-day wounds, the authors found that alpha-SMA was not expressed in any incisional or 2-mm wound that healed scarlessly, but it was expressed in all wounds that healed with scar. Overall, alpha-SMA expression strongly correlated with increasing wound size (P < .005). Myofibroblasts were seen as early as 24 hours after wounding, and at 3 and 7 days all wounds showed strong expression of alpha-SMA. These results demonstrate that although myofibroblasts are induced early in fetal wound repair, by 14 days there is a notable lack of myofibroblasts in wounds that heal scarlessly and an abundance of myofibroblasts in those wounds that scar. By determining the factors that regulate the disappearance of the myofibroblast in scarless fetal wounds, the authors hope to gain new insights into the mechanisms of scarless fetal repair.

The in vivo response of foetal tendons to sutures.

The in vivo response of foetal flexor digitorum profundus tendons to tendon sutures was studied macroscopically and microscopically in foetal lambs. No tendon adhesions were noted at any of the examination intervals. 4 days after injury, a mild inflammatory reaction was noted around the suture. The tendon examined at the 4-week interval showed evidence of migration of epitenon cells from the outer surface of the tendon into the suture track. The tendon examined at the 6-week interval showed normal tendon fibres surrounding the suture site. Differences between foetal skin and foetal tendon healing are discussed along with the possible role of amniotic fluid in modulating the healing process in the foetus.

Effects of prostaglandins and indomethacin on the cellular inflammatory response following surgical trauma in fetal rabbits.

This study has examined the effects of prostaglandins E2 and F2a (PGE2 and PGF2a) and indomethacin on cellular inflammation in fetal rabbits. Fetuses heal differently from adults: incisions heal with no scar or inflammation; excisional wounds exposed to amniotic fluid (AF) do not heal or become inflamed, and have high tissue levels of PGE2 and PGF2a; excisional wounds protected from AF do heal and become inflamed, and have low tissue PG levels. The authors inserted slow-release pellets [control, PGE2 (10 micrograms), PGF2a (10 micrograms), indomethacin (10 micrograms)] into subcutaneous pockets in fetal rabbits on day 25 of gestation (one per fetus). Pellets were also placed in subcutaneous pockets in the does. Fetuses and doe tissues were recovered 72 h after surgery. Control pellets in fetuses had a slight inflammatory response, with some cells present. Fetal PGE2 pellets had a layer 5-10 cells thick surrounding the pellet, and fetal PGF2a pellets had a 10-15-cell layer. Fetal indomethacin pellets had no response, with no inflammatory cells present. All pellets placed in does elicited a slight cellular inflammatory response, equal to that seen with control pellet in fetuses. These results show that PGE2 and PGF2a are potent in-vivo promoters of cellular inflammation in fetal rabbits, but not in adult rabbits. Indomethacin suppresses the foreign-body response in fetal rabbits, but not in adult rabbits.

[Fetal wound healing: current status and new perspectives]. / Fetale Wundheilung. Aktueller Stand und neue Perspektiven.

The characteristic of fetal wound healing is scarless wound repair in early gestation. During the last two decades, intensive research efforts have focused on unraveling the molecular regulations underlying the phenomenon of scarless wound healing. Better understanding of synthesis and degradation will enable us to develop important therapeutic options for the prevention and reduction of scarring. The aim of this article is to present an overview, discuss the most important research works of the last two decades on the field of fetal wound healing, and report current therapeutic developments for the modulation of adult wound repair. Recent experimental results using these new therapeutic approaches are very promising and present great possibilities and chances for future surgery.

[A newborn with an unusual lip]. / Een pasgeborene met een bijzondere lip.

A newborn was seen on the suspicion of neonatal schisis. From the upper lip to the right nostril a tiny line was visible, without any other anomalies. The mother had a cheilognathopalatoschisis. The patient had a congenital healed cleft lip, a rare presentation with unknown origin.

Initiation of limb regeneration: the critical steps for regenerative capacity.

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.

Fetal wound healing using a genetically modified murine model: the contribution of P-selectin.

PURPOSE: During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wound repair. METHODS: Linear excisional wounds were created on the dorsa of E15.5 and E17.5 gestation fetuses in wild-type and P-selectin (-/-) mice (term = 19 days). Wounds were harvested at various time-points after wounding and analyzed using histology and immunohistochemistry. RESULTS: The E15.5 wounds in both wild-type and P-selectin (-/-) fetuses healed scarlessly and with minimal inflammation, whereas E17.5 wounds healed with fibrosis and inflammation. However, the scars of the P-selectin (-/-) wounds appeared slightly different than wild-type. There were significantly more inflammatory cells in E17.5 wild-type wounds 6 hours after injury (P < .001), but the difference was no longer significant by 24 hours. Finally, reepithelialization was slower in the E15.5 knockout wounds compared to their wild-type counterparts. CONCLUSIONS: Absence of P-selectin delays inflammatory cell recruitment and reepithelialization of fetal wounds; however, scar formation still occurs in late gestation wounds. The contribution of specific molecules to fetal wound healing can be elucidated using murine knockout or transgenic models.