Occlusion following laser resurfacing promotes reepithelialization and wound healing

One of the critical parameters that has not been examined carefully following laser skin resurfacing is the effect of eschar on the wound healing process. Because occlusive dressings minimize the occurrence of eschar, the present study was undertaken to evaluate the effect of occlusion following laser resurfacing. It is clear that CO2 lasers promote epidermal cell loss and variable amounts of dermal injury. To characterize the wound repair process after laser treatment, biopsy specimens were obtained 2 to 4 days after treatment. Specimens from 15 patients were examined; the preauricular biopsy specimens were paired such that one specimen was from skin that had been occluded and the other specimen (from the same patient) was from skin treated without occlusion. Skin specimens were examined by indirect immunofluorescence using antibodies to specific epidermal and dermal antigens. The results indicate that the keratinocytes that repopulate the epidermis migrate from the hair follicles and express keratin 17, an intermediate filament protein expressed in keratinocytes during the early stages of wound healing. The migration of keratin 17-expressing cells begins 48 hours following laser resurfacing in skin treated with occlusion, whereas cell migration from the follicles of skin treated without occlusion is delayed. In summary, occlusion promotes enhanced cell migration and diminished eschar formation, resulting in more rapid healing.     

Cicatrization of the skin of the 7-day-old chick embryo cultured in vitro

A morphological study of in vitro wound healing has been performed by light, transmission and scanning electron microscopy in dorsal thoraco-lumbar skin of 7-day chick embryos. A circular wound, 750 microns in diameter, was punched out of dorsal skin, removing epidermis and the underlying dense dermis. Wound closure was completed within 96 to 120 hours. Feather bud development was not observed at the wound site. The epidermis began to migrate some 24 h after the wounding; the migration of peridermal cells preceded that of basal epidermal cells by some 12 hours. Mechanisms of the epidermal migration were similar to those observed in situ during wound healing of the integument in 5-day chick embryos (THEVENET, 1981), Superficial epithelization of bare dermis occurred as soon as 12 h after the injury. Cytoplasm of dermal cells exhibited many microtubules and a dilated rough endoplasmic reticulum. During the first 48 h, the epidermal cells established direct contacts and zones of close parallel apposition with epithelized dermal cell processes. The basement membrane lamina densa was maintained at the edges of the wound without retraction or ruffling. It was reconstituted concomitantly with the epidermal migration within 72 h. Cytoplasm of migratory epidermal and epithelized dermal cells exhibited many cytoskeleton structures.     

Healing of skin wounds in the African catfish Clarias gariepinus

The African catfish Clarias gariepinus was used as a model for wound healing and tissue regeneration in a scale-less fish. A temporal framework of histological and cell proliferation markers was established after wound induction in the dorsolateral cranial region, by removing the epidermal and dermal layers, including stratum adiposum (SA). Wound closure and epidermis formation was initiated within 3 h post-procedure (hpp) with migration and concomitant proliferation of epidermal cells from the wound borders. The wound was covered by this primary epidermal front 12 hpp and fusion of the opposing epidermal fronts occurred within 24 hpp. Attachment of the newly formed epidermal layer to the underlying dermis was observed 48 hpp concomitant with a second wave of cell proliferation at the wound edge. Normal epidermal thickness within the wound was achieved 72 hpp. Formation of a basement membrane occurred by 120 hpp with concomitant emergence of the SA from the wound borders. Wound healing in C. gariepinus skin involved closure of the wound and re-epithelization through cell migration with a single wave of early cell proliferation not documented in other species. Furthermore, covering of the wound by epithelium as well as the reappearance of the basement membrane and SA occurred sooner than in other fish species.     

A "traffic control" role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) beta3 levels, which are undetectable in plasma and high in serum, and by TGFbeta receptor (TbetaR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFbeta3 from serum converts serum to a plasmalike reagent. The addition of TGFbeta3 to plasma converts it to a serumlike reagent. Down-regulation of TbetaRII in dermal cells or up-regulation of TbetaRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma-->serum-->plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Cell polarity during wound healing in an insect epidermis

The insect integument displays uniform posterior orientation of cuticular denticles or bristles formed by the epidermal cells. We want to understand how cell polarities become uniformly oriented in the plane of the epidermal sheet. Here we test whether directed cell migration disturbs the orientation of denticles. Burning a circular area of epidermal cells beneath the cuticle causes cells to migrate into the resulting wound and the cuticle pattern observed after the subsequent moult depends on the time interval between burning and ecdysis. After a short wound-healing period cuticular protrusions tend to point away from the wound. With increasing would healing periods they tend to point more and more towards the wound centre. These results suggest that the migrating cells tend to orient cuticular protrusions in the direction of cell movement while continued cell movement will bend nascent cuticular protrusions outwards. Cell shape may also determine denticle orientation. I propose that the asymmetric localization of cell components known to determine the orientation of cell migration may also determine denticle orientation in insect epidermal cells.     

Role of the cytoskeleton during injury-induced cell migration in corneal endothelium

The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming reestablished. When injured corneas are placed in media containing 5 x 10(-7) M cytochalasin B, endothelial cell migration occurs; but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10(-8) M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 micrograms/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.     

Junctional communication is induced in migrating capillary endothelial cells

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.     

Inability of newt epidermal cells to migrate over concanavalin A-coated substrates

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.     

Wound healing ability of Xenopus laevis embryos. II. Morphological analysis of wound marginal epidermis

We previously showed that bisectional wounds made in Xenopus laevis embryos at the primary eye vesicle stage were rapidly closed. In this study, microscopic analyses, including scanning electron microscopy, on the morphology of the epidermis were conducted during wound closure in the half embryos. Bright fluorescence of Texas red-phalloidin showing actin filaments started to be visualized at the cut edge 10 min after wounding. It increased with time, forming a distinguished, though discontinuous, bundle along the wound margin. The wound closure was completely inhibited by 20 microm cytochalasin B, and almost completely by 50 mm 2,3-butanedione 2-monoxime, an inhibitor to myosin ATPase activity. Scanning electron microscopy revealed that the outer epidermal cells became extensively elongated in the radial direction, and the contour of the closing wound edge did not become smoother but remained ragged. Thus, a representative embryonic type of wound closure may be driven in Xenopus embryos by a complex mechanism, involving not only the actin 'purse-string' but also an inward movement of individual cells. Anyhow, the wound closure is a movement of the epidermal sheet maintaining cell-cell contact, and not involving locomotion of single cells separated from the wound edge.     

Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB- treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.     

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé , following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.     

Location of a fibronectin domain involved in newt epidermal cell migration

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.     

The stimulation of DNA synthesis by cytochalasin B in proliferative epidermal and dermal cells

Cytochalasin B influences a variety of cellular events that are associated with the contractile microfilament system and the formation of binucleate cells. Along with the formation of binucleate cells, cytochalasin B also causes an acceleration of cells from G1 to S in the cell cycle. By pulsing the cytochalasin B for 30 minutes and allowing for a previously established lag time (17.5 hours) a stimulation of thymidine incorporation into DNA of proliferative epidermal and dermal cells was found in both control and stripped epidermis. Autoradiographic analysis confirmed that the stimulation was due to an increased number of basal cells accelerated from G1 to S phase. A minimal number of binucleate basal cells, 1 in 300, was observed, which suggests that the stimulated synthesis is independent of binucleate cell formation. The amount of stimulation is maximum with cytochalasin B concentration pulse between 5gamma and 30gamma/ml. The results suggest a possible link in coupling cell membrane and surface events with subsequent increased cell nuclei synthetic activity.     

Organization of the migrating chick epiblast edge: attachment sites, cytoskeleton, and early developmental changes

The radial expansion of the chick extraembryonic epiblast on the inner side of the vitelline membrane in yolk sac formation provides a useful system for study of adhesion and migration of an epithelial cell sheet. A band of specialized cells at the epiblast edge adheres by its dorsal side to the overlying vitelline membrane. The attached edge was examined by scanning electron microscopy. The attachment region (av 0.06 mm wide) extends from the advancing edge to a transitional ridge. The ridge appears to be an area of adhesion and de-adhesion. The attached surface is smooth with small surface projections and filopodia. These become more numerous and prominent with cold treatment. Epiblast cells display a filopodial/lamellipodial mode of migration in vivo and in vitro. The distribution of 4- to 7-nm microfilaments in edge cells is examined using transmission electron microscopy of whole cells. Decoration with heavy meromyosin shows that these components of the cytoskeleton contain actin. Treatment of intact blastoderms and dissociated edge cells with cytochalasin B and cold suggests that microfilaments rather than microtubules are primarily responsible for edge cell morphology. Early blastoderm cells which have not initiated migration respond to cytochalasin B, cold, and colcemid in the same way as migrating edge cells. This suggests that the differentiative change that produces the rapidly migrating edge cells does not involve a shift in the relative contribution of microtubules and microfilaments to the cytoskeleton.     

Tissue abscission and wound healing in the operculum of Pomatoceros lamarckii Quatrefages (Polychaeta: Serpulidae)

Following opercular amputation in Pomatoceros lamarckii Quatrefages, wound healing is initiated from a predetermined point on the peduncle. The events of abscission, cell migration and cuticle deposition during wound healing have been studied by light and electron microscopy. Abscission occurs at a predetermined point on the peduncle indicated by specialized epidermal cells, the easy break-point cells (EBP). Following detachment of tissues distal to the EBP cells, the resultant wound is plugged by a knot of coelomocytes which provide a substratum over which epidermal cells migrate to seal and restore the epidermis. During their migration, the epidermal cells undergo differentiation and deposit a new cuticle. Cuticle formation is initiated by the deposition of a finely filamentous matrix. The fine filaments subsequently coalesce to form thicker fibrils which become aggregated into layers of orthogonally-arranged fibril bundles. The mechanisms involved in abscission, cell migration and cuticle deposition during wound healing of the opercular filament are discussed.     

Effects of cytochalasin B and enucleation on EGF receptor down regulation

The loss of epidermal growth factor (EGF) binding activity on cultured murine 3T3 cells exposed to EGF (EGF receptor down regulation) was determined in colchicine treated cells, cytochalasin B treated cells, and untreated cells. Neither colchicine nor cytochalasin B altered the affinity of the receptor for EGF, but colchicine decreased maximal EGF binding activity by 20%. The maximal extent of EGF receptor down regulation was similar in colchicine treated cells and cytochalasin B treated cells, but the rate of receptor down regulation was higher in cytochalasin B treated cells. Cytoplasts produced by subjecting cytochalasin B treated cells adhering to the substratum to centrifugal force responded to EGF with nearly normal down regulation kinetics. The results suggest that the cytoskeleton is not obligatorily involved in EGF-induced EGF receptor down regulation.     

Cell migration and differentiation during wound healing and regeneration in Microstomum lineare (Turbellaria)

Using transmission electron microscopy and serial sections with light-microscopic autoradiography, I have investigated the ultrastructure of wound healing, the distribution of cells preparing for proliferation, and the fates of cells labelled with exogenous tritiated thymidine ([³H]T) in Microstomum lineare undergoing wound healing and regeneration. Immediately after decapitation the open wound was reduced to a minimum by strong contraction of circular muscle fibers. The wound epidermis was cellular, consisting of thin parts of epidermal cells from the epidermis around the wound. These epidermal cells maintained close adhesive contact with one another through zonulae adherentes and septate junctions. No proliferating cells were found in the old epidermis. The only cells taking up [³H]T were mesenchymal and gastrodermal neoblasts which proliferated and migrated towards the surface. The final epidermis was formed by conjunction of the wound epidermis and newly differentiated epidermal cells. Regeneration in Microstomum, in contrast to that of planarians, occurs mainly by morphallaxis, without the formation of a regeneration blastema, but also through continuous cell proliferation, migration, and differentiation.     

Re‐epithelialization of large wound in paedomorphic and metamorphic axolotls

Axolotls (Ambystoma mexicanum ) may heal their skin wounds scar‐free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re‐epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re‐epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re‐epithelialisation was faster in paedomorphs than in metamorphs. Within these re‐epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU‐labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU‐labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re‐epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re‐epithelialisation period. J. Morphol. 278:228–235, 2017. © 2016 Wiley Periodicals,Inc.