Fetal Skin Wounds Research Articles

Collagen-Heparin-FGF2-VEGF Scaffolds Induce a Regenerative Gene Expression Profile in a Fetal Sheep Wound Model.

The developmental abnormality spina bifida is hallmarked by missing tissues (e.g. skin) and exposure of the spinal cord to the amniotic fluid, which can negatively impact neurological development. Surgical closure of the skin in utero limits neurological damage, but in large defects this results in scarring and contractures. Stimulating skin regeneration in utero would greatly benefit treatment outcome. Previously, we demonstrated that a porous type I collagen (COL) scaffold, functionalized with heparin (HEP), fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) (COL-HEP/GF) improved pre- and postnatal skin regeneration in a fetal sheep full thickness wound model. In this study we uncover the early events associated with enhanced skin regeneration. We investigated the gene expression profiles of healing fetal skin wounds two weeks after implantation of the COL(-HEP/GF) scaffolds. Using laser dissection and microarrays, differentially expressed genes (DEG) were identified in the epidermis and dermis between untreated wounds, COL-treated wounds and wounds treated with COL-HEP/GF. Biological processes were identified using gene enrichment analysis and DEG were clustered using protein-protein-interaction networks. COL-HEP/GF influences various interesting biological processes involved in wound healing. Although the changes were modest, using protein-protein-interaction networks we identified a variety of clustered genes that indicate COL-HEP/GF induces a tight but subtle control over cell signaling and extracellular matrix organization. These data offer a novel perspective on the key processes involved in (fetal) wound healing, where a targeted and early interference during wound healing can result in long-term enhanced effects on skin regeneration.

Role of Wnt Signaling in Mouse Fetal Skin Wound Healing.

Wnt proteins secrete glycoproteins that are involved in various cellular processes to maintain homeostasis during development and adulthood. However, the expression and role of Wnt in wound healing have not been fully documented. Our previous studies have shown that, in an early-stage mouse fetus, no scarring occurred after cutaneous wounding, and complete regeneration was achieved. In this study, the expression and localization of Wnt proteins in a mouse fetal-wound-healing model and their associations with scar formation were analyzed. Wnt-related molecules were detected by in-situ hybridization, immunostaining, and real-time polymerase chain reaction. The results showed altered expression of Wnt-related molecules during the wound-healing process. Moreover, scar formation was suppressed by Wnt inhibitors, suggesting that Wnt signaling may be involved in wound healing and scar formation. Thus, regulation of Wnt signaling may be a possible mechanism to control scar formation.

Identification of differentially expressed proteins involved in fetal scarless wound healing using a rat model of cleft lip.

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge-shaped cleft lip-like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription-quantitative (RT-q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper-left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper-left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro-inflammatory factors, including TNF-α, were also different between two groups. Label-free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT-qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

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.

Scarless wound healing: Transitioning from fetal research to regenerative healing.

Since the discovery of scarless fetal skin wound healing, research in the field has expanded significantly with the hopes of advancing the finding to adult human patients. There are several differences between fetal and adult skin that have been exploited to facilitate scarless healing in adults including growth factors, cytokines, and extracellular matrix substitutes. However, no one therapy, pathway, or cell subtype is sufficient to support scarless wound healing in adult skin. More recently, products that contain or mimic fetal and adult uninjured dermis were introduced to the wound healing market with promising clinical outcomes. Through our review of the major experimental targets of fetal wound healing, we hope to encourage research in areas that may have a significant clinical impact. Additionally, we will investigate therapies currently in clinical use and evaluate whether they represent a legitimate advance in regenerative medicine or a vulnerary agent. WIREs Dev Biol 2018, 7:e309. doi: 10.1002/wdev.309 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Plant Development > Cell Growth and Differentiation Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells.

MicroRNA-149 contributes to scarless wound healing by attenuating inflammatory response.

A fibrotic or pathological scar is an undesired consequence of skin wound healing and may trigger a series of problems. An attenuated inflammatory response is a significant characteristic of fetal skin wound healing, which can contribute to the scarless healing of fetal skin. According to deep sequencing data, microRNA‑149 (miR‑149) expression was increased in mid-gestational compared with that in late‑gestational fetal skin keratinocytes. It was demonstrated that overexpression of miR‑149 in HaCaT cells can downregulate the expression of pro‑inflammatory cytokines interleukin (IL)‑1α, IL‑1β, and IL‑6 at basal levels and in inflammatory conditions. Furthermore, miR‑149 was revealed to indirectly accelerate transforming growth factor‑β3 and collagen type III expression in fibroblasts, which are essential cells in extracellular matrix remodeling. In a rat skin wound model, miR‑149 improved the quality of the arrangement of collagen bundles and reduced inflammatory cell infiltration during skin wound healing. These results indicate that miR‑149 may be a potential regulator in improving the quality of skin wound healing.

Fibromodulin Is Essential for Fetal-Type Scarless Cutaneous Wound Healing

In contrast to adult and late-gestation fetal skin wounds, which heal with scar, early-gestation fetal skin wounds display a remarkable capacity to heal scarlessly. Although the underlying mechanism of this transition from fetal-type scarless healing to adult-type healing with scar has been actively investigated for decades, in utero restoration of scarless healing in late-gestation fetal wounds has not been reported. In this study, using loss- and gain-of-function rodent fetal wound models, we identified that fibromodulin (Fm) is essential for fetal-type scarless wound healing. In particular, we found that loss of Fm can eliminate the ability of early-gestation fetal rodents to heal without scar. Meanwhile, administration of fibromodulin protein (FM) alone was capable of restoring scarless healing in late-gestation rat fetal wounds, which naturally heal with scar, as characterized by dermal appendage restoration and organized collagen architectures that were virtually indistinguishable from those in age-matched unwounded skin. High Fm levels correlated with decreased transforming growth factor (TGF)-β1 expression and scarless repair, while low Fm levels correlated with increased TGF-β1 expression and scar formation. This study represents the first successful in utero attempt to induce scarless repairin late-gestation fetal wounds by using a single protein, Fm, and highlights the crucial role that the FM-TGF-β1 nexus plays in fetal-type scarless skin repair.

Dynamic Expression of Novel MiRNA Candidates and MiRNA-34 Family Members in Early- to Mid-Gestational Fetal Keratinocytes Contributes to Scarless Wound Healing by Targeting the TGF-β Pathway.

BackgroundEarly- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. Keratinocytes (KCs) have been found to exert important effects on the regulation of fibroblasts. There may be significant differences of gestational fetal KCs at different ages. The advantages in early- to mid-gestational fetal KCs could lead to fetal scarless wound healing.MethodsKCs from six human fetal skin samples were divided into two groups: a mid-gestation group (less than 28 weeks of gestational age) and a late-gestation group (more than 28 weeks of gestational age). RNA extracted from KCs was used to prepare a library of small RNAs for next-generation sequencing (NGS). To uncover potential novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data. Other bioinformatical analyses were used to further validate the novel miRNAs. The expression levels of the miRNAs were further confirmed by real-time quantitative RT-PCR.ResultsA total of 61.59 million reads were mapped to 1,170 known human miRNAs in miRBase. Among a total of 202 potential novel miRNAs uncovered, 106 candidates have a higher probability of being novel human miRNAs. A total of 110 miRNAs, including 22 novel miRNA candidates, were significantly differently expressed between mid- and late-gestational fetal KCs. Thirty-three differentially expressed miRNAs and miR-34 family members are correlated with the transforming growth factor-β (TGF-β) pathway.ConclusionsTaken together, our results provide compelling evidence supporting the existence of 106 novel miRNAs and the dynamic expression of miRNAs that extensively targets the TGF-β pathway at different gestational ages in fetal KCs. MiRNAs showing altered expression at different gestational ages in fetal KCs may contribute to scarless wound healing in early- to mid-gestational fetal KCs, and thus may be new targets for potential scar prevention and reduction therapies.

Gene Expression in Fetal Skin Development and Wounds

Gene Expression in Fetal Skin Development and Wounds

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.

Fetal Wound Healing Biomarkers

Fetal skin has the intrinsic capacity for wound healing, which is not correlated with the intrauterine environment. This intrinsic ability requires biochemical signals, which start at the cellular level and lead to secretion of transforming factors and expression of receptors, and specific markers that promote wound healing without scar formation. The mechanisms and molecular pathways of wound healing still need to be elucidated to achieve a complete understanding of this remodeling system. The aim of this paper is to discuss the main biomarkers involved in fetal skin wound healing as well as their respective mechanisms of action.

Scarless fetal skin wound healing update

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.

Increased expression of Ero1L-alpha in healing fetal wounds

BackgroundAdult mammalian tissues heal injury to the skin with formation of scar; this process quickly seals an injured area, however, excessive scar formation can become a source of persistent pathology, interfering with multiple vital functions. In contrast, mammalian fetal tissue can heal without scar formation. We previously sought to model scarless healing in a rabbit fetal skin wound and identified gene products differentially expressed during fetal wound healing through PCR suppression subtractive hybridization (PCR SSH). One of these transcripts, previously identified simply as clone 11, showed putative increased expression in wounded fetal skin. This study establishes its identity as Ero1L-alpha and confirms its elevated expression in healing fetal wounds.FindingsAfter obtaining further sequence by 5' rapid amplification of cloned ends (RACE) we find that clone 11 is Ero1L-alpha. We determined that clone 11, a differentially expressed transcript in fetal wound healing, comprises the 3' untranslated region (UTR) of an approximately 4 kb transcript in rabbit tissues that corresponds to Ero1L-alpha. We showed that Ero1L-alpha is expressed predominantly as two transcripts in rabbit skin, namely a 1.6 kb transcript and the 4.0 kb transcript recovered in our PCR SSH screen via its 3' UTR sequence. However, a third transcript of 2.9 kb was also detected in Northern blots and was subsequently cloned and confirmed by 3' RACE. Knockdown of the clone 11 sequence in rabbit adult fibroblasts via siRNA resulted in significantly decreased Ero1L-alpha message expression. Increased expression of clone 11 (Ero1L-alpha) in a variety of cell types during the wound healing response was also confirmed by in situ hybridization.ConclusionsEro1L-alpha is one of the previously unknown clones identified in a PCR SSH screen for genes differentially expressed in fetal wounded tissue. In situ hybridization confirms that Ero1L-alpha shows increased expression in multiple cell types after wounding of the fetal integument.

Delayed Wound Closure in Fibromodulin-Deficient Mice Is Associated with Increased TGF-β3 Signaling

Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-β (TGF-β) modulation. Using an adult fmod-null (fmod(-/-)) mouse model, this study further elucidates the interplay between FMOD and TGF-β expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod(-/-) and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod(-/-) mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod(-/-) wounds exhibited early elevation (within 24 hours post-wounding) of type I and type II TGF-β receptors as well as unexpectedly high fibroblast expression of TGF-β3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-β3, fmod(-/-) fibroblasts were significantly less motile than WT fibroblasts. fmod(-/-) fibroblasts were also more susceptible to migration inhibition by TGF-β3, leading to profound delays in dermal cell migration. Increased scarring in fmod(-/-) mice indicates that TGF-β3's antimotility effects predominate over its antifibrotic effects when high TGF-β3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-β bioactivity.

Scarless fetal wound healing: a basic science review.

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.

Cloning and expression of rabbit CCT subunits eta and beta in healing cutaneous wounds

We have previously identified the CCT subunit eta as specifically reduced in healing fetal skin wounds by differential display, and observed that this reduction is not seen with any other CCT subunit. We now report the cloning and characterization of the cDNAs for rabbit CCT-eta and its closest evolutionary homolog, CCT-beta. Quantitative examination of CCT-eta and –beta message expression in healing fetal and adult wounds at 12 h post-injury confirms that CCT-eta mRNA is decreased in fetal wound tissues, but actually elevated in adult wound tissues. CCT-beta mRNA, in contrast, remains unchanged in both fetal and adult wound tissues. CCT-eta mRNA remains persistently elevated in healing adult wounds for 28 days following injury, whereas CCT-beta mRNA remains invariant throughout. CCT-eta protein is similarly increased, whereas CCT-beta protein remains unchanged. -smooth muscle actin (-SMA), a recognized substrate of CCT known to be important in integumentary wound healing, was also measured over the course of wound healing, and both mRNA and protein levels were elevated throughout the 28 days.

Upregulation of Neurodevelopmental Genes During Scarless Healing

Scarless fetal skin wound healing is a paradigm for ideal skin repair and is dependent on peripheral nerve function.To further explore neurogenic mechanisms influence on the scarless skin repair, fetal rats were wounded on gestational days 16 (E16; n = 24) and 18 (E18; n = 8) and wounds were harvested at 1 and 3 days after injury. Unwounded skin at identical gestational age was used for control comparison. The scarless E16 and scarring E18 wounds underwent macroarray gene expression analysis (1172 genes).During the scarless healing period, 53 (4.5%) genes had a statistically significant upregulation post-injury with at least a 2- to 3-fold change 1 day after wounding and 14 (1.2%) genes 3 days after wounding (P < 0.05). Many neurodevelopmental genes were increased during scarless repair on post-injury days 1 and 3. Neuropeptide Y Receptor type I, cJun related Transcription Factor (junD), Synaptophysin, SNAP 25, Neuronal calcium sensor 1 (NCS1), neural visine-like calcium binding protein 1 (NVP1), nerve growth factor-induced gene A (NGFI-A/EGR1), VGF8A protein, p27kip1, and members of the GABA and serotonin family each had 2- to 3-fold expression increases (P < 0.05).We speculate that fetal skin cells express neurotrophins during skin development that regulate peripheral neuron formation. During injury these factors promote the survival and regeneration of peripheral neurons; this interaction of neuropeptides, neuropeptide receptors, and neurotrophins may modulate the fetal scarless repair mechanisms in response to injury. Identification of these neurodevelopmental candidate genes provides insight for new investigation into mechanisms regulating scarless healing.

Expression of Receptor for Activated C Kinase 1 in Healing Skin and Mucosal Wounds

Postnatal (adult) mammalian wound healing results in the formation of scar, whereas fetal mammals are able to effect wound repair without scar. We have investigated the expression pattern of the receptor of activated C kinase 1 (RACK1), a pleiotropic G-protein-like molecule, in healing skin and mucosal wounds in a rabbit model after obtaining a full-length clone of the rabbit RACK1 cDNA. In both adult skin and mucosal wounds, RACK1 mRNA expression is decreased relative to unwounded controls. In contrast, in fetal skin wounds RACK1 expression is unaltered from fetal control. Fibroblasts derived from adult skin tissue express more RACK1 message than fetal skin fibroblasts. These observations suggest that RACK1 may play an important role in distinguishing scarless fetal wound healing from its scirrhous counterpart in adults.

Stromal Collagenase in Melanoma: A Vascular Connection

In this issue, Zigrino et al. report on the role of host-derived mouse collagenase-3 (matrix metalloproteinase (MMP)-13) in melanoma growth and metastasis using a mouse model that lacks MMP-13. The authors demonstrate that vascularization of cutaneous melanomas in these mice is impaired compared with that of controls. This study emphasizes the importance of stromal murine MMP-13, a functional homologue of human MMP-1, in tumor progression.

Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing

Integumentary wound healing in early fetal life is regenerative and proceeds without scar formation. Expressomic analysis of this phenomenon by differential display has previously determined that the eta subunit of the cytosolic chaperonin containing T-complex polypeptide (CCT) is downregulated in the healing fetal wound milieu. We now report that no other CCT subunit shares this distinct pattern of gene regulation as determined by limiting dilution reverse transcriptase polymerase chain reaction (RT-PCR); all seven of the remaining CCT subunits demonstrate no change in messenger RNA (mRNA) expression in healing fetal wounds compared to unwounded control tissue. The alpha subunit, however, did evidence reduced message levels in healing adult wound tissue. We herein report on the cloning and sequence of the complementary DNA (cDNA) for rabbit CCT-alpha and confirm its wound specific decrease in adult tissues through quantitative real-time RT-PCR assay. We also confirm that quantitative evaluation of CCT-alpha and CCT-zeta mRNA expression shows no change in healing fetal wounds.