H.P. Lorenz

Fetal wound healing.

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.

Adult Skin Wounds in the Fetal Environment Heal with Scar Formation

ObjectiveThis study investigated the influence of the fetal environment on the healing characteristics of adult skin. Summary Background DataThe 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. MethodsFull-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 allogeneic 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. ResultsThe wounds in the adult skin grafts healed with scar formation. This observation contrasts strongly with the scarless healing of the incisional fetal skin wounds. ConclusionsThis study suggests that scarless fetal skin healing properties are intrinsic to fetal skin and are not primarily the result of the fetal environment.

Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential

Fibroblasts in fibrosis Excess fibrous connective tissue, similar to scarring, forms during the repair of injuries. Fibroblasts are known to be involved, but their role is poorly characterized. Rinkevich et al. identify two lineages of dermal fibroblasts in the dorsal skin of mice (see the Perspective by Sennett and Rendl). A fibrogenic lineage, defined by embryonic expression of Engrailed-1, plays a central role in dermal development, wound healing, radiation-induced fibrosis, and cancer stroma formation. Targeted inhibition of this lineage results in reduced melanoma growth and scar formation, with no effect on the structural integrity of the healed skin, thus indicating therapeutic approaches for treating fibrotic disease. Science, this issue 10.1126/science.aaa2151; see also p. 284 An embryonic fibroblast lineage deposits connective tissue in wounds. [Also see Perspective by Sennett and Rendl] INTRODUCTION Fibroblasts are the predominant cell type that synthesizes and remodels the extracellular matrix in organs during both embryonic and adult life and are central to the fibrotic response across a range of pathologic states. Morphologically, they are most commonly defined as elongated, spindle-shaped cells that readily adhere to and migrate over tissue culture substrates. However, fibroblasts exhibit a variety of shapes and sizes, depending on the physiologic or pathologic state of the host tissue, and represent a heterogeneous population of cells with diverse features that remain largely undefined. In cutaneous tissues, fibroblasts display considerable functional variation during wound repair, depending on developmental time, and between anatomic sites. For example, wounds in the oral cavity remodel with minimal scar formation, whereas scar tissue deposition within cutaneous wounds is substantial. The mechanisms underlying this diversity of regenerative responses in cutaneous tissues have remained largely underexplored. RATIONALE The effective development of treatments for fibrosis depends on a mechanistic understanding of its pathogenesis. The identification and characterization of distinct lineages of fibroblasts, based on functional role, hold potential value for developing therapeutic approaches to fibrosis. We employed a nonselective depletion-based fluorescence-activated cell sorting strategy to isolate fibroblasts from a murine model that labels a particular lineage of cells based on the gene expression of Engrailed-1 (En1) in its embryonic progenitors. Using this reporter mouse, we reveal the presence of at least two functionally distinct embryonic fibroblast lineages in murine dorsal skin and characterize a single lineage that plays a primary role in connective tissue formation. RESULTS Genetic lineage tracing and transplantation assays demonstrate that a single somitic-derived fibroblast lineage that is defined by embryonic expression of En1 is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induces ectopic dermal architectures that mimic their place of origin rather than their site of transplantation. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin leads to diminished connective tissue deposition in wounds and significantly reduces melanoma growth in the dorsal skin of mice. Tensile strength testing reveals that, although scar formation is significantly reduced in wounds treated with diphtheria toxin to ablate the En1 lineage, as compared with control wounds, tensile strength in lineage-ablated wounds is not significantly affected. Using flow cytometry and in silico approaches, we identify CD26/dipeptidyl peptidase-4 (DPP4) as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity in the wound bed of wild-type mice during wound healing results in diminished cutaneous scarring after excisional wounding. CONCLUSION We have identified multiple lineages of fibroblasts in the dorsal skin. Among these, we have characterized a single lineage responsible for the fibrotic response to injury in the dorsal skin of mice and demonstrated that targeted inhibition of this lineage results in reduced scar formation with no effect on the structural integrity of the healed skin. Furthermore, these studies demonstrate that intra- and intersite diversity of dermal architectures are set embryonically and are maintained postnatally by distinct lineages of fibroblasts in different anatomic locations. These results hold promise for the development of therapeutic approaches to fibrotic disease, wound healing, and cancer progression in humans. Schematic showing reduced scarring with targeted ablation/inhibition of En1 fibroblasts. Fibroblasts derived from embryonic precursors expressing En1 are responsible for most connective tissue deposition in skin fibrosis. Targeted ablation/inhibition of this lineage leads to a reduction in fibrosis during wound repair and tumor stroma formation. These findings may lead to the elimination of scarring and other types of fibrotic tissue disease. Green cells, En1-positive fibroblasts; red cells, En1-negative fibroblasts. CREDIT: SILHOUETTES FROM PHYLOPIC.ORG Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

Outcomes of complex abdominal herniorrhaphy: experience with 106 cases.

Purpose:Reconstruction of abdominal wall defects is a challenging problem. Often, the surgeon is presented with a patient having multiple comorbidities, who has already endured numerous unsuccessful operations, leaving skin and fascia that are attenuated and unreliable. Our study investigated preoperative, intraoperative, and postoperative factors and techniques during abdominal wall reconstruction to determine which variables were associated with poor outcomes. Methods:Data were collected on all patients who underwent ventral abdominal hernia repair by 3 senior-level surgeons at our institution during an 8-year period. In all cases, placement of either a synthetic or a biologic mesh was used to provide additional reinforcement of the repair. Results:A total of 106 patients were included. Seventy-nine patients (75%) had preoperative comorbid conditions. Sixty-seven patients developed a postoperative complication (63%). Skin necrosis was the most common complication (n = 21, 19.8%). Other complications included seroma (n = 19, 17.9%), cellulitis (n = 19, 17.9%), abscess (n = 14 13.2%), pulmonary embolus/deep vein thrombosis (n = 3, 2.8%), small bowel obstruction (n = 2, 1.9%), and fistula (n = 8, 7.5%). Factors that significantly contributed to postoperative complications (P < 0.05) included obesity, diabetes, hypertension, fistula at the time of the operation, a history of >2 prior hernia repairs, a history of >3 prior abdominal operations, hospital stay for >14 days, defect size > 300 square cm, and the use of human-derived mesh allograft. Factors that significantly increased the likelihood of a hernia recurrence (P < 0.05) included a history of >2 prior hernia repairs, the use of human-derived allograft, using an overlay-only mesh placement, and the presence of a postoperative complication, particularly infection. Hernia recurrences were significantly reduced (P < 0.05) by using a “sandwich” repair with both a mesh overlay and underlay and by using component separation. Conclusions:A history of multiple abdominal operations is a major predictor of complications and recurrences. If needed, component separation should be used to achieve primary tension-free closure, which helps to reduce the likelihood of hernia recurrences. Our data suggest that mesh reinforcement used concomitantly in a “sandwich” repair with component separation release may lead to reduced recurrence rates and may provide the optimal repair in complex hernia defects.

Primary and secondary orbit surgery: the transconjunctival approach.

The transconjunctival approach to the orbit is underutilized because of concern regarding inadequate exposure and higher postoperative rates of lower eyelid shortening and ectropion. All patients who had a transconjunctival incision performed for orbital surgery over the last 6 years (1990 to 1996) were studied. Patients who had a transconjunctival blepharoplasty were excluded. A total of 35 patients, average age 32 years, had 45 transconjunctival incisions performed. Lateral canthotomy or cantholysis was not done. Operations fell into three categories: fracture plating alone, 10 (22 percent); split-calvarial bone graft placement with or without plating, 26 (58 percent); and orbital decompression, 9 (20 percent). The overall incidence of ectropion was 6.7 percent (3 of 45). One patient (2 percent) had transient ectropion, and two patients (4 percent) had persistent ectropion, which required surgical correction. Ectropion occurred only in those lower eyelids that had a previous transcutaneous incision (3 of 18 = 17 percent). None occurred in those eyelids that had no prior incision or only a previous transconjunctival incision. The transconjunctival approach without a lateral canthotomy provides safe access to the orbit in eyelids that have not had a previous transconjunctival incision.

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.

Gene expression in fetal murine keratinocytes and fibroblasts.

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.

Excess Dermal Tissue Remodeling In Vivo: Does It Settle?

Background: Surgical manipulation of skin may result in undesired puckering of excess tissue, which is generally assumed to settle over time. In this article, the authors address the novel question of how this excess tissue remodels. Methods: Purse-string sutures (6-0 nylon) were placed at the midline dorsum of 22 wild-type BALB/c mice in a circular pattern marked with tattoo ink. Sutures were cinched and tied under tension in the treatment group, creating an excess tissue deformity, whereas control group sutures were tied without tension. After 2 or 4 weeks, sutures were removed. The area of tattooed skin was measured up to 56 days after suture removal. Histologic analysis was performed on samples harvested 14 days after suture removal. Results: The majority of excess tissue deformities flattened within 2 days after suture removal. However, the sutured skin in the treatment group decreased in area by an average of 18 percent from baseline (n = 9), compared to a 1 percent increase in the control group (n = 10) at 14 days after suture removal (p < 0.05). This was similarly observed at 28 days (treatment, −11.7 percent; control, 4.5 percent; n = 5; p = 0.0243). Despite flattening, deformation with purse-string suture correlated with increased collagen content of skin, in addition to increased numbers of myofibroblasts. Change in area did not correlate with duration of suture placement. Conclusions: Excess dermal tissue deformities demonstrate the ability to remodel with gross flattening of the skin, increased collagen deposition, and incomplete reexpansion to baseline area. Further studies will reveal whether our findings in this mouse model translate to humans.

Discussion: Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

www.PRSJournal.com 508 R complex soft-tissue injuries remains a challenge to plastic surgeons. Reduced regenerative capacity is attributed in part to the lack of mesenchymal stem cells and epithelial stem cells in full-thickness wounds. Without this stem cell niche, subsequent progeny, and inability to polarize surrounding cells, regeneration of the tissue layers is not possible. Consequently, Lough et al. isolated leucine-rich, repeat-containing, G-protein–coupled receptor 6 (LGR6+) epithelial stem cells, seeded them onto scaffolds in an in vivo burn wound model, and characterized the subsequent pro-angiogenic response, thereby demonstrating a clinically applicable stem cell–based scaffold that improves healing and hair growth in full-thickness wounds. Prior research has shown that LGR6+ epithelial stem cells in the follicular bulge are capable of differentiating into all cellular lineages of the skin and interfollicular epidermis.1 The authors aimed to exploit these cells by isolating via fluorescenceactivated cell sorting and seeding them onto simple collagen-based scaffolds for use in full-thickness wounds. Lough et al. utilized green fluorescent protein–expressing LGR6+ epithelial stem cells and confocal microscopy to demonstrate the ability to adhere to and proliferate on acellular matrices. Next, they placed LGR6+ epithelial stem cell scaffolds on 3-mm burn wounds on the dorsum of athymic nude mice. After 10 days, burn injuries treated with the scaffolds healed significantly more than untreated wound beds and matrix controls. In addition, wounds treated with the scaffolds produced nascent hair follicles by day 14, corroborating published literature describing the hair-producing capability of LGR6+ epithelial stem cells.2 Finally, the authors combined stromal vascular fraction—rich in myriad cell types such as adipose-derived stromal cells/mesenchymal stem cells, endothelial progenitor cells, preadipocytes, T cells, B cells, mast cells, and macrophages—and LGR6+ epithelial stem cells and showed a synergistic augmentation of pro-angiogenic transcripts with the combination of the two entities. These data are important, but not surprising given the previous reports regarding adipose-derived stromal cells promoting angiogenesis.3 However, what is rather remarkable is the polarization of cells, whereby green fluorescent protein–expressing LGR6+ epithelial stem cells aggregate at the surface of the scaffold while red fluorescent protein– expressing stromal vascular fraction favors an internal subepithelial position. Lough et al. postulate that the epithelial stem cells and adiposederived stromal cells/mesenchymal stem cells in stromal vascular fraction initiate a primitive dermal-epidermal interface within the three-dimensional environment of the matrix. In their article, the authors effectively present a novel methodology for a hybrid graft alternative that utilizes an epithelial stem cell subpopulation capable of producing all elements of functional skin. This promises to circumvent current limitations, such as requiring large donor sites of healthy skin. Moreover, this concept improves on cultured epithelial autografts by enriching for the essential multilineage stem cell population—LGR6+

Abstract 140: identification, characterization, and prospective isolation of a fibroblast lineage contributing to dermal development, cutaneous scarring, radiation fibrosis, and cancer stroma.

PurPose: Fibroblasts represent a heterogeneous population of cells with diverse functional and phenotypic features. Such diversity remains largely undefined due to phenotypic drift in vitro and a lack of unique surface markers for functional subclasses of fibroblasts. Here we track the contributions of two distinct fibroblast lineages, defined by embryonic expression of Engrailed-1 (En1) and Wnt1, to connective tissue formation in the context of dermal development, cutaneous wounding, radiation fibrosis, and cancer stroma.