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Background: Osteoarthritis (OA) is a debilitating disease process, affecting mobility and overall health of millions. Current treatment is for symptomatic relief and discovery of approaches to halt or reverse damage is imperative. Deletion of developmental endothelial locus-1 (Del1) has been shown to increase severity of OA in knockout mice. We examined the intracellular pathways involved in the ability of DEL1 to protect chondrocytes from apoptosis and anoikis and hypothesized that it functioned via integrin signaling., Materials and Methods: Primary human chondrocytes were treated with various inducers of apoptosis, including anoikis, in the presence of added DEL1 or bovine serum albumin as control. Various inhibitors of integrin binding were examined for their effect on DEL1 activity. Downstream signaling pathway components were detected by immunoblotting., Results: The addition of DEL1 protected chondrocytes from multiple inducers of apoptosis as measured by cell survival, terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase 3/7 assays (P < 0.05). The effect of DEL1 was blocked by RGD peptides and by antibodies directed to integrin α V β 3 , but not by controls or antibody to integrin α1 (P < 0.05). Treatment with DEL1 promoted ERK and AKT activation when cells were attached, but only AKT activation under conditions of anoikis., Conclusions: DEL1 protected chondrocytes from apoptosis in response to activators of either the intrinsic or extrinsic pathways, and to anoikis. This effect was mediated primarily through integrin α V β 3 . This represents a therapeutic target for therapies to prevent cartilage degeneration in OA., (Published by Elsevier Inc.)

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.)

Recent evidence suggests that mechanical forces can significantly impact the biologic response to injury. Integrated mechanical and chemical signaling networks have been discovered that enable physical cues to regulate disease processes such as pathologic scar formation. Distinct molecular mechanisms control how tensional forces influence wound healing and fibrosis. Conceptual frameworks to understand cutaneous repair have expanded beyond traditional cell-cytokine models to include dynamic interactions driven by mechanical force and the extracellular matrix. Strategies to manipulate these biomechanical signaling networks have tremendous therapeutic potential to reduce scar formation and promote skin regeneration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs)., Methods: KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence., Results: There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence., Conclusion: In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy., (Published by Elsevier Inc.)

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.

Background: Developmental endothelial locus (Del)1 is a secreted extracellular matrix-associated protein that stimulates angiogenesis through integrin binding and is implicated in vasculogenesis. We hypothesized that increased expression of an angiogenic factor would lead to enhanced wound healing., Materials and Methods: Transgenic mice had Del1 cloned behind a keratin 14 promoter (K14-Del1) to drive constitutive expression in basal keratinocytes. Transgenic animals and wild-type litter mates underwent excisional wounding or depilation, and tissues were harvested at various time points. Wound healing and hair regrowth were assessed by photography, histology, and immunohistochemistry. For injection experiments, purified Del1 protein was injected in the flanks of wild-type mice with carrier on the contralateral flank as a control. Del1 expression during hair development was performed using transgenic mice with a LacZ cassette introduced downstream from the native promoter., Results: K14-Del1 animals appeared normal and healed excisional wounds normally but demonstrated an increased rate of hair regrowth after wound healing. Using depilation experiments to specifically address hair follicle growth, we found increased hair regrowth was independent of wounding. This was confirmed by injection of purified Del1 protein. During normal hair anagenesis, Del1 is expressed in the root of the hair follicle., Conclusions: Constitutive expression of Del1 in skin does not affect skin vascularity or improve wound healing. Surprisingly, we found the primary effect of constitutive Del1 expression in the basal keratinocytes was increased hair growth following induction of anagenesis. During normal hair anagenesis, we see expression of Del1 in the root of the hair follicle suggesting it may function there to stimulate hair growth.

Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.

In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.

Background: Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs., Materials and Methods: Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively., Results: Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs., Conclusions: KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

Background: Fibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects., Materials and Methods: Human foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein., Results: Terminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 +/- 03 versus 5.3 +/- 1.7% (P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2-4 h after matrix detachment., Conclusions: FAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.

Purpose: Fibroblast survival within an in vitro collagen matrix is dependent on matrix anchorage to a rigid substratum. The purpose of this study was to determine whether granulation tissue survival in vivo also is dependent on matrix anchorage. We hypothesized that splinting an excisional wound (i.e., anchoring the wound edges) would promote granulation tissue survival and that desplinting a splinted wound would produce granulation tissue apoptosis., Methods: Eighteen Wistar rats (3 months, 350 g) underwent excisional wounding (2 x 2 cm, dorsal skin) with immediate wound splinting (a metal template affixed with sutures) on day 0. On day 6, rats (n = 6 per group) underwent splint removal (desplinted), splint removal with circumferential incision of the wound edge (desplint/release), or no intervention (splinted); sacrifice of all animals was on day 7. Frozen sections of granulation tissue were stained with TUNEL or H and E; data were analyzed with ANOVA and the unpaired t test., Results: The cross-sectional and surface area of the desplinted and desplint/release granulation tissue both decreased compared to the splinted granulation tissue (*P < 0.05). The nuclear density of the desplint/release granulation tissue was 25% less compared to the splinted granulation tissue (*P < 0.05). The desplinted and desplint/release apoptotic rates were twice and >10x greater than the splinted apoptotic rate, respectively (*P < 0.05)., Conclusions: The rate of cell death in a splinted wound (an in vivo equivalent of an anchored FPCM) is minimal to nil, which is consistent with our hypothesis. Desplinting and releasing the wound edge of a previously splinted wound (the in vivo equivalent of a detached FPCM) results in granulation tissue regression and a large increase in apoptosis. Desplinting a wound alone results in changes somewhat intermediate to the splinted and desplint/release conditions. Loss of wound anchorage acutely promotes granulation tissue apoptosis.

Background: In several mammalian animal models, early-gestational-age fetal wounds heal without scar, but wounds of late gestational age heal with scar. This change in wound healing phenotype can be a result of both intrinsic (i.e., cellular characteristics) and extrinsic (i.e., environmental) factors. Our question was: Does amniotic fluid (AF) influence the change from scarless to scar-forming repair in the rat?, Methods: Rat AF was investigated for its modulation of fibroblast-populated collagen lattice (FPCL) contraction and morphological changes of adult fibroblasts. AF was also assayed for transforming growth factor beta (TGF-beta) levels. Adult rat dermal fibroblasts in monolayer and incorporated into FPCLs were incubated with AF additions from gestational age 14, 16, 18, and 21 days at 10% (v/v)., Results: Day 14 AF significantly stimulated FPCL contraction, but AF of 16, 18, and 21 days inhibited FPCL contraction. Fluorescence histology identified microtubules and microfilaments in AF treated adult rat dermal fibroblasts. The staining pattern of microtubules in Day 14 AF-treated fibroblasts showed denser structures at the cell center. Cells incubated with Day 16 or 18 AF showed fine peripheral microtubules. A mink lung epithelial cell bioassay was used to analyze concentrations of TGF-beta in AF. TGF-beta levels were greatly elevated in Day 14 AF, but were relatively low in Day 16, 18 and 21 AF. The inhibitor of FPCL contraction from AF of Days 16, 18, and 21 was not identified., Conclusion: It is proposed that the robust expression of TGF-beta or cytoskeletal changes induced by Day 14 AF contributes to enhanced FPCL contraction., (Copyright 2001 Academic Press.)

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators., (Copyright 2001 Academic Press.)

Background: We have recently proposed that the "distal esophagus" in esophageal atresia with tracheo-esophageal fistula (EA/TEF) is actually embryologically derived from the middle branch of a trifurcation of the embryonic lung bud, which subsequently grows caudally in the foregut to connect with the developing stomach. We hypothesized that differential mRNA expression of the lung-specific patterning transcription factor, thyroid transcription factor 1 (TTF-1), in the developing fistula tract in TEF relative to the bronchi (the other branches of the lung bud trifurcation) might explain the unique nonbranching pattern of growth of the fistula tract., Materials and Methods: EA/TEF was induced in Sprague-Dawley rat embryos via intraperitoneal injection of 2.2 mg/kg adriamycin into pregnant dams on Days 6-9 of gestation. The foregut from embryos developing EA/TEF and from control embryos (no adriamycin) were isolated on Gestational Days 13.5, 15.5, and 17.5 (term = 21 days). Some were processed for whole-mount in situ hybridization for TTF-1, while others were embedded and sectioned for histologic analysis via in situ hybridization for TTF-1., Results: The expression of the respiratory-specific transcription factor TTF-1 is conserved in the epithelium of the developing fistula tract in TEF. The pattern of expression of TTF-1 in the fistula tract mirrors the expression in the large airways of the developing lungs, despite the fact that the fistula tract does not form secondary branches to give rise to a lung., Conclusions: The fistula tract in TEF is a respiratory-derived structure that expresses the lung-specific transcription factor TTF-1 throughout its development in the foregut. Contrary to the patterning role that it normally plays in the developing lung, TTF-1 does not induce branching morphogenesis in the fistula tract. Thus, the nonbranching pattern of growth of the fistula tract may be attributable to local mesenchymal-epithelial interactions that override TTF-1 patterning activity., (Copyright 2000 Academic Press.)

Background: Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membrane-rich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro., Materials and Methods: We microdissected embryonic pancreases at Embryonic Days (E)9.5-11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10. 5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel., Results: Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel., Conclusions: These data validate our in vitro system as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis., (Copyright 2000 Academic Press.)

Background: Physiologically programmed cell death or apoptosis occurs during the natural balance between cellular proliferation and demise., Materials and Methods: We compared the expression of 64 apoptosis-related genes in keloids and normal scars to investigate the potential role of apoptosis in keloid formation. Two sets of mRNA were isolated from keloids excised from four previously untreated patients and four normal scar patients separately. Human cDNA arrayed hybridization was performed to compare the apoptosis-related gene expression between these two groups. In addition, TUNEL assays were performed to evaluate the percentage of apoptotic cells in keloids (center and periphery) versus normal scars., Results: Eight of the sixty-four apoptosis-related genes studied were significantly underexpressed in keloid tissue. The underexpressed genes and their relative expression compared with normal scar were defender against cell death 1 (DAD-1) (34.1% of normal scar); nucleoside diphosphate kinase B (c-myc transcription factor) (24.7%); glutathione S-transferase (17.9%); glutathione S-transferase microsomal (28.1%); glutathione peroxidase (47.2%); tumor necrosis factor receptor 1-associated protein (TRADD) (51.0%); 19-kDa interacting protein 3 (NIP3) (36.0%); and cytoplasmic dynein light chain 1 (HDLC1) (47.7%). Spatial analysis of apoptosis using TUNEL assays revealed apoptosis indices of 0.83 for keloid periphery and 0.63 for keloid center., Conclusions: In this study we demonstrated underexpression of apoptosis-related genes in human keloid tissue and decreased apoptotic activity in fibroblasts derived from keloids versus normal scars. We hypothesized that keloid fibroblasts fail to undergo physiologically programmed cell death and, thus, continue to produce and secrete connective tissue beyond the period expected in normal scar formation, accounting for the progressive and hypertrophic nature of keloids. This mechanism leads to new possibilities for treatment of keloids through induction of apoptosis., (Copyright 1999 Academic Press.)

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound., (Copyright 1999 Academic Press.)

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.

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.