Thrombomodulin Promotes Corneal Epithelial Wound Healing

Purpose

To determine the role of thrombomodulin (TM) in corneal epithelial wound healing, and to investigate whether recombinant TM epidermal growth factor-like domain plus serine/threonine-rich domain (rTMD23) has therapeutic potential in corneal epithelial wound healing.

Methods

TM localization and expression in the murine cornea were examined by immunofluorescence staining. TM expression after injury was also studied. The effect of rTMD23 on corneal wound healing was evaluated by in vitro and in vivo assays.

Results

TM was expressed in the cornea in normal adult mice. TM expression increased in the early phase of wound healing and decreased after wound recovery. In the in vitro study, platelet-derived growth factor-BB (PDGF-BB) induced TM expression in murine corneal epithelial cells by mediating E26 transformation-specific sequence-1 (Ets-1) via the mammalian target of rapamycin (mTOR) signaling pathway. The administration of rTMD23 increased the rate of corneal epithelial wound healing.

Conclusions

TM expression in corneal epithelium was modulated during the corneal wound healing process, and may be regulated by PDGF-BB. In addition, rTMD23 has therapeutic potential in corneal injury.     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

Hindlimb unloading depresses corneal epithelial wound healing in mice.

C57BL/6 mice were subjected to hindlimb unloading (HU) for a period of 3 wk to determine the possible effects on epithelial wound healing. A standardized corneal epithelial wound was performed, and parameters of the inflammatory response and reepithelialization were analyzed over an observation period of 96 h. Wound closure was significantly retarded in mice during HU with reepithelialization being delayed by approximately 12 h. Both epithelial migration and cell division were significantly depressed and delayed. The inflammatory response to epithelial wounding was also significantly altered during HU. Neutrophils, as detected by the Gr-1 marker, were initially elevated above normal levels before wounding and during the first few hours afterward, but there was a significant reduction in neutrophil response to wounding at times where neutrophil influx and migration in controls were vigorous. A similar pattern was seen with CD11b+CD11c+ cells (monocyte lineage). Langerhans cells are normally resident within the peripheral corneal epithelium. They respond to injury by initially leaving the epithelial site within 6 h and returning to normal levels by 96 h, 2 days after reepithelialization is complete. During HU, this pattern is distinctly different, with Langerhans cell numbers slowly diminishing, reaching a nadir at 96 h, which is significantly below normal. Evidence for systemic effects of HU is provided by findings that collagen deposition within subcutaneous sponges was significantly reduced during HU. In conclusion, HU, a ground-based model simulating some physiological aspects of spaceflight, impairs wound repair of corneas. Multiple factors, both local and systemic, likely contribute to this delayed wound healing.     

Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closure

The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.     

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.     

Corals use similar immune cells and wound-healing processes as those of higher organisms

Sessile animals, like corals, frequently suffer physical injury from a variety of sources, thus wound-healing mechanisms that restore tissue integrity and prevent infection are vitally important for defence. Despite the ecological importance of reef-building corals, little is known about the cells and processes involved in wound healing in this group or in phylogenetically basal metazoans in general. A histological investigation into wound healing of the scleractinian coral Porites cylindrica at 0 h, 6 h, 24 h and 48 h after injury revealed differences in cellular components between injured and healthy tissues. Cell counts of the obligate endosymbiont, Symbiodinium, and melanin volume fraction analysis revealed rapid declines in both Symbiodinium abundance and tissue cross-sectional area occupied by melanin-containing granular cells after injury. Four phases of wound healing were identified, which are similar to phases described for both vertebrates and invertebrates. The four phases included (i) plug formation via the degranulation of melanin-containing granular cells; (ii) immune cell infiltration (inflammation); (iii) granular tissue formation (proliferation); and (iv) maturation. This study provides detailed documentation of the processes involved in scleractinian wound healing for the first time and further elucidates the roles of previously-described immune cells, such as fibroblasts. These results demonstrate the conservation of wound healing processes from anthozoans to humans.     

兔机械性角膜上皮损伤后伤口愈合的形态学动态变化

目的观察兔机械性角膜上皮损伤后伤口愈合的形态学动态变化。方法选取新西兰大白兔12只,随机分为A、B两组,均建立机械性角膜上皮损伤模型（刮除直径为8mm的角膜中央上皮）,术后给予盐酸林可霉素滴眼液滴眼,3次/日,1滴/次。A组在建模后第0、1、4、7天共4个时间点采用前节裂隙灯照相系统进行眼表照相,并计算损伤面积。B组在建模后第1、4、7天,按随机数字表法取2只兔的实验眼角膜,行透射电镜及病理检查。结果前节裂隙灯照相系统记录了不同观察时间点损伤范围（伤口愈合面积）的动态变化。造模后第1天,角膜上皮全层缺如,损伤边缘处上皮细胞胞膜向损伤区内伸出褶皱的伪足;造模后第4天,上皮自周边向缺损区域爬行生长,覆盖整个角膜,可见2～3层细胞,主要为基底细胞和多角细胞,细胞连接松驰;造模后第7天,再生上皮分化较完全,约5～6层细胞,极向齐,连接较紧密,可见均匀分布的半桥粒结构。结论兔机械性角膜上皮损伤后伤口愈合的动态形态学变化包括上皮细胞向心性移行、增殖,以及随后的分化,连接。     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Expression of vimentin by rabbit corneal epithelial cells during wound repair

Summary Intermediate filaments of epithelial cells generally consist of specific combinations of keratins. However, cultured epithelial cells from certain tissues and some epithelial tumors have been shown also to express vimentin. In the present study, the expression of vimentin by epithelial cells in healing corneal wounds (partial thickness penetrating wounds) and in tissue culture was analyzed. Both immunohistochemical and immunotransblot analyses indicated that although vimentin was not detected in the normal rabbit corneal epithelium in vivo, cultured rabbit corneal epithelial cells co-express keratins and vimentin. At 1 day post-wounding, vimentin was not detectable in the epithelial cells that had covered the denuded stroma. However, at 2 days post-wounding, the epithelium at the base of the epithelial plug immunoreacted with both anti-vimentin and antikeratin monoclonal antibodies. Immunotransblot analyses of the extracts of the epithelial plugs confirmed the presence of vimentin (M_r=58k). The 58k band was not detected in the extract of normal rabbit corneal epithelium. At day/5, vimentin was no longer detectable in the epithelium. This study demonstrated that corneal epithelial cells transiently co-express vimentin and keratins in vivo during wound healing and in tissue culture. The time-course of the transient expression of vimentin suggests that the vimentin expression in the epithelial cells during healing is not linked to cell proliferation or to the centripetal migration of the epithelium during early stages (first 24 h) of healing, but may be linked to cell-matrix interactions or the migration of basal cells in the upward direction at the following stage of healing.     

Distinct roles of JNK-1 and ERK-2 isoforms in permeability barrier repair and wound healing

c-Jun N-terminal kinases (JNKs, also called stress activated protein kinases) and the extra-cellular signal responsive kinases (ERKs) exert different functions in mitogenesis, maturation and differentiation of immune and epithelial cells. We investigated specific functions of individual JNK and ERK isoforms in skin permeability barrier repair and in wound healing. JNK1, but not JNK2 or JNK3, deficient mice revealed a delay in the permeability barrier repair after superficial injury to the skin (tape-stripping) as well as a delay in the healing of full skin thickness wounds. Skin barrier injury induced an increase in epidermal JNK1 enzyme activity in mouse skin in vivo, and JNK1 activity correlated with the degree of differentiation in organotypic keratinocyte cultures. Skin injury activated epidermal ERK2 enzyme activity with biphasic maxima after 30 min and 3h, and the activity was independent from the differentiation state in keratinocyte culture. In summary, superficial and deep wound healing depends on the differential activity of MAP kinases such as JNK1 in epidermal differentiation and ERK2 in proliferation.     

CXC chemokines and their receptors: a case for a significant biological role in cutaneous wound healing

Wound healing requires a complex series of reactions and interactions among cells and their mediators, resulting in an overlapping series of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and scar formation. Cytokines and chemokines promote inflammation, angiogenesis, facilitate the passage of leukocytes from circulation into the tissue, and contribute to the regulation of epithelialization. They integrate inflammatory events and reparative processes that are important for modulating wound healing. Thus both cytokines and chemokines are important targets for therapeutic intervention. The chemokine-mediated regulation of angiogenesis is highly sophisticated, fine tuned, and involves pro-angiogenic chemokines, including CXCL1-3, 5-8 and their receptors, CXCR1 and CXCR2. CXCL1 and CXCR2 are expressed in normal human epidermis and are further induced during the wound healing process of human burn wounds, especially during the inflammatory, epithelialization and angiogenic processes. Human skin explant studies also show CXCR2 is expressed in wounded keratinocytes and Th/1/Th2 cytokine modulation of CXCR2 expression correlates with proliferation of epidermal keratinocytes. Murine excision wound healing, chemical burn wounds and skin organ culture systems are valuable models for examining the role of inflammatory cytokines and chemokines in wound healing.     

The effect of the treatment of skin wounds in rats with hydrolytic enzymes on the synthetic activity of the epidermocytes]

Epidermis cells synthetic activity was studied by a luminescent microscopy method. Different conditions of skin wound healing in intrascapular region were investigated on 84 rats-males. Daily effect of 0.2% trypsin or 0.1% ronidase solution on the healing surface for 20 min has been shown to increase basal layer cells synthetic activity of forming epidermis in comparison with control. However, maximum raise of epidermis cells functional activity after ronidase influence was observed already on the 4th day, while after trypsin action--only on the 7th. Epithelial cells synthetic activity decreases several days before total skin defect epithelialization. In all experiments their activity stabilizes not less than one month after the operation. We have come to a conclusion that the mechanism of healing acceleration due to proteolytic enzymes depends on basal layer cells synthetic activity of forming epithelium.     

Keratinocytes derived from embryonic stem cells induce wound healing in mice

The skin plays an important role in defending the body against the environment. Treatments for burns and skin injuries that use autologous or allogenic skin grafts derived from adult or embryonic stem cells are promising. Embryonic stem cells are candidates for regenerative and reparative medicine. We investigated the utility of keratinocyte-like cells, which are differentiated from mouse embryonic stem cells, for wound healing using a mouse surgical wound model. Mice were allocated to the following groups: experimental, in which dressing and differentiated cells were applied after the surgical wound was created; control, in which only the surgical wound was created; sham, in which only the dressing was applied after the surgical wound was created; and untreated animal controls with healthy skin. Biopsies were taken from each group on days 3, 5 and 7 after cell transfer. Samples were fixed in formalin, then stained with Masson’s trichrome and primary antibodies to interleukin-8 (IL-8), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), collagen-1 and epidermal growth factor (EGF) using the indirect immunoperoxidase technique for light microscopy. Wound healing was faster in the experimental group compared to the sham and control groups. The experimental group exhibited increased expression of IL-8, FGF-2 and MCP-1 during early stages of wound healing (inflammation) and collagen-1 and EGF expression during late stages of wound healing (proliferation and remodeling). Keratinocytes derived from embryonic stem cells improved wound healing and influenced the wound healing stages.     

Finasteride accelerates prostate wound healing after thulium laser resection through DHT and AR signalling

Objectives

Urinary tract infection, urinary frequency, urgency, urodynia and haemorrhage are common post‐operative complications of thulium laser resection of the prostate (TmLRP). Our study mainly focuses on the role of finasteride in prostate wound healing through AR signalling.

Materials and methods

TmLRP beagles were randomly distributed into different treatment groups. Serum and intra‐prostatic testosterone and DHT level were determined. Histological analysis was conducted to study the re‐epithelialization and inflammatory response of the prostatic urethra in each group. We investigated the role of androgen in proliferation and inflammatory response in prostate. In addition, the effects of TNF‐α on prostate epithelium and stromal cells were also investigated.

Results

Testosterone and DHT level increased in testosterone group and DHT decreased in finasteride group. Accelerated wound healing of prostatic urethra was observed in the finasteride group. DHT suppressed proliferation of prostate epithelium and enhanced inflammatory response in prostate. We confirmed that DHT enhanced macrophages TNF‐α secretion through AR signalling. TNF‐α suppressed proliferation of prostate epithelial cells and retarded cell migration. TNF‐α also played a pivotal role in suppressing fibroblasts activation and contraction.

Conclusion

Testosterone treatment repressed re‐epithelialization and wound healing of prostatic urethra. Finasteride treatment may be an effective way to promote prostate re‐epithelialization.

     

PDGF在大鼠断层供皮区创面愈合过程中表达变化的研究

实验研究已经证明Ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ（ＰＤＧＦ）能够促进各种类型的伤口愈合,然而在伤口愈合过程中内源性ＰＤＧＦ表达变化的研究却少有报道,为探讨ＰＤＧＦ对伤口愈合的影响,我们应用原位杂交、斑点杂交技术观察了内源性ＰＤＧＦ在大鼠创面愈合过程中的表达变化,结果发现：在创面愈合过程中,肉芽组织中的成纤维细胞,毛细血管内皮细胞及创缘真皮内的毛囊上皮细胞均能表达ＰＤＧＦ－ＢＢ基因,在伤后６天,组织修复的高峰期,ＰＤＧＦ－ＢＢ基因表达达到最强,伤后１２天,伤口完全上皮化,ＰＤＧＦ的基因表达也恢复正常,说明ＰＤＧＦ的基因表达和伤口愈合时间有密切的关系。提示ＰＤＧＦ在创面愈合过程中可能起着重要的调控作用。     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Suppression of keratin 15 expression by transforming growth factor beta in vitro and by cutaneous injury in vivo

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine which plays an important role in cutaneous wound repair. To gain insight into the mechanisms of action of this growth and differentiation factor in the skin, we searched for genes which are regulated by TGF-beta1 in cultured HaCaT keratinocytes. Using the differential display RT-PCR technology we identified a gene which was strongly downregulated by TGF-beta1. The identified cDNA includes sequences of the keratin 15 (K15) gene which encodes a component of the cytoskeleton of basal cells in stratified epithelia. Surprisingly, our cDNA also included an unknown sequence. Since this cDNA lacks an open reading frame, the corresponding mRNA is likely to be nonfunctional. However, we also demonstrate a strong negative regulation of the expression of the published, functional K15 variant. Expression of K15 was also suppressed by tumor necrosis factor alpha (TNF-alpha) and to a lesser extent by epidermal growth factor (EGF) and keratinocyte growth factor (KGF). By contrast, the major basal type I keratin, K14, was upregulated by TGF-beta1, whereas TNF-alpha, EGF, and KGF had no effect. Consistent with the in vitro data, we found a significant reduction of the K15 mRNA levels after skin injury, whereas K14 expression increased during the wound healing process. Immunostaining revealed the presence of K15 in all basal cells of the epidermis adjacent to the wound, but not in the hyperproliferative epithelium above the granulation tissue. These data demonstrate that K15 is excluded from the activated keratinocytes of the hyperthickened wound epidermis, possibly as a result of increased growth factor expression in injured skin.     

Cellular mechanisms for diminished scarring with aging

The study of an age-dependent spectrum of scar formation is driven by the desire to understand and recapitulate scarless healing. Although focus in the past has been directed toward scarring in the fetus, less exuberant scarring is a common clinical observation in the elderly. Cell turnover is a major contributor to the development of scar tissue and is governed by the proliferative and apoptotic cellular fractions within a healing wound. We hypothesize that the balance between cell proliferation and apoptosis during late stages of excisional wound healing is, at least in part, responsible for age-related variations in scarring potential. Full-thickness 7-mm ulcers (four per ear), exposing bare cartilage, were made on the inner surface of the ear on 12 young and 12 aged New Zealand White rabbits. Analyses were performed at days 15, 21, and 28 postwounding. A previously described Scar Elevation Index was derived from histomorphometric analysis, along with the quantification of epithelial ingrowth and total cellularity. Apoptotic cellular fractions were derived from TdT-mediated dUTP nick end-labeling assay-stained histologic sections; proliferative fractions were derived from proliferating cell nuclear antigen-labeled serial sections. Young rabbits demonstrated significantly greater scar elevation/area. Apoptosis was strongly associated with progress of epithelialization in both groups. Significantly higher proliferative indices were seen in the young and were sustained through day 28, by which time levels had substantially declined in the aged. No differences in apoptotic indices were demonstrated between groups at any time point. The clinical observation of less exuberant scarring with aging is supported by this animal model. Apoptosis follows the progression of epithelialization but does not appear to independently influence scar morphology. A diminished proliferative response during later stages of healing is an important contributing mechanism for the decrease in scar formation seen in the elderly.     

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.     

Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.