Contribution of Subcutaneous Connective Tissues to the Epithelialization and Cyst Formation by the Skin Transplanted Subcutaneously

Skins and hollow organs have been shown to form epithelialized cysts when transplanted into subcutaneous tissue of a recipient animal, expanding their surface areas. This system seems to offer a good potential for regenerating organs. We investigated the functional and structural contribution of epithelia and connective tissue compartments in this regeneration system with two experimental systems.Key Words: skin, epithelialization, transplantation, GFP, epidermis, cystDispase-separated epidermis often forms epithelialized cysts when combined with dermal connective tissue whereas dispase-separated epidermis alone does not form cysts or epithelialize, indicating the functional importance of the dermal connective tissue in the regeneration process.When GFP rats were used as donors for the skin, the donor-derived tissue was composed of whole epidermis and parts of the connective tissue cells and blood vessels under the newly epithelialized portion of the cyst wall. Small capillaries of granulation tissues were shown to be of recipient origin, but some large vessels were of donor origin. These results showed the significant functional and structural contribution of dermal connective tissue in the regeneration of the skin in subdermal transplant.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Wound healing of human skin transplanted onto the nude mouse. II. An immunohistological and ultrastructural study of the epidermal basement membrane zone reconstruction and connective tissue reorganization

The reconstruction of human epidermis during healing of human skin wounded after grafting onto the nude mouse was described in a previous paper (M. Démarchez, P. Sengel, and M. Pruniéras, 1986, Dev. Biol. 113, 90-96). The regeneration of the epidermal basement membrane zone (BMZ) and the reorganization of the connective tissue are the subjects of the present study. They were investigated by two complementary methods: electron microscopy to analyze the BMZ reorganization, and indirect immunofluorescence with species-specific and cross-reacting antibodies directed against laminin, bullous pemphigoid antigen, mouse or human collagens of types I or IV, human elastic fibers, fibronectin, fibrin, actin, and human vimentin, to examine the species origin and distribution of BMZ and connective tissue components during the regeneration process. It is reported that grafted human skin preserves its own immunological markers not only in the epidermis but also in the BMZ and dermis as well, and that, after injury, its regeneration proceeds according to the following sequence of overlapping events: production of a mouse granulation tissue; reepidermization by human cells; reconstruction of a BMZ with human characteristics; formation of a human neodermis. It is concluded that human skin grafted onto the nude mouse is able to regenerate its three structural compartments, namely, the epidermis, BMZ, and dermis. Interestingly, it appeared, also, that the connective tissue regeneration would be a two-step mechanism including the sequential formation of two tissues of distinct sources, namely, a granulation tissue and a neodermis.     

Skin structure in the snout of the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Many fossil lungfish have a system of mineralised tubules in the dermis of the snout, branching extensively and radiating towards the epidermis. The tubules anastomose in the superficial layer of the dermis, forming a plexus consisting of two layers of vessels, with branches that expand into pore canals and flask organs, flanked by cosmine nodules where these are present. Traces of this system are found in the Australian lungfish, Neoceratodus forsteri, consisting of branching tubules in the dermis, a double plexus below the epidermis and dermal papillae entering the epidermis without reaching the surface. In N. forsteri, the tubules, the plexus and the dermal papillae consist of thick, unmineralised connective tissue, enclosing fine blood vessels packed with lymphocytes. Tissues in the epidermis and the dermis of N. forsteri are not associated with deposits of calcium, which is below detectable limits in the skin of the snout at all stages of the life cycle. Canals of the sensory line system, with mechanoreceptors, are separate from the tubules, the plexus and the dermal papillae, as are the electroreceptors in the epidermis. The system of tubules, plexus, dermal papillae and lymphatic capillaries may function to protect the tissues of the snout from infection.     

UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: subsequent connective tissue repair and implications in photoaging

The skin reconstructed in vitro has been previously shown to be a useful model to investigate the effects of UVB exposure (Bernerd and Asselineau, 1997). The present study describes the response to UVA irradiation. Major alterations were observed within the dermal compartment. Apoptosis of fibroblasts located in the superficial area of the dermal equivalent was observed as soon as 6 h after irradiation, leading to their disappearance after 48 h. This effect was obtained without major alterations of epidermal keratinocytes suggesting a differential cell type sensitivity to UVA radiations. In addition, collagenase I was secreted by dermal fibroblasts. The UVA dermal effects could be observed even after removal of the epidermis during the post irradiation period, demonstrating that they were independent of the keratinocyte response. The analysis of the tissue regeneration during the following 2 weeks revealed a connective tissue repair via fibroblasts proliferation, migration and active synthesis of extracellular matrix proteins such as fibronectin and procollagen I. This cellular recolonization of the superficial part of the dermal equivalent was due to activation of surviving fibroblasts located deeply in the dermal equivalent. The direct damage in the dermis and the subsequent connective tissue repair may contribute to the formation of UVA-induced dermal alterations.     

Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability

Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three‐dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer and the epidermis and putatively provide active papillary retraction. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

The role of grafted skin in the regeneration of X-irradiated axolotl limbs

The primary aim of these experiments was to follow the cells descended from limb skin through the process of limb regeneration to determine what range of differentiations these cells may assume. Triploid hindlimb or forelimb skin was grafted to the denuded thighs of diploid host axolotls that had previously received 3000 R of X irradiation across both hindlimbs and the intervening pelvic area. The host limbs were then amputated through their grafts and permitted to regenerate. Cartilage, perichondrium, joint connective tissue, general connective tissue, dermis, and epidermis were present in all the regenerated limbs, but only 10% of the regenerates contained muscle. Tabulation of nucleolar numbers showed that the majority of cells in each regenerated tissue originated from the grafted skin. A strong correlation was demonstrated between the forelimb or hindlimb origin of the skin grafts and the number of digits regenerated.     

Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.     

Dermal fibroblasts contribute to multiple tissues in the accessory limb model

The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non‐GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation‐induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage.     

Some regularities of the regeneration of the musculature of internal organs]

The paper summarizes the data of the author and his co-workers on regulation of the musculature of vessels, digestive tract, uterus, ureters and urinary bladder. The smooth muscle cells have different degrees of differentiation. The muscular tissue of the vessels is most differentiated. The uterus musculature is very plastical. After injury mature myocytes are the first to undergo destruction. The intermedial substance is more stable. Myoblasts, young elements of the fibroblast row and the subendothelial layer cells are the origin of muscular regeneration. Figures of mitosis and amitosis are noted. Mature myocytes and intercellular substance are formed in the process of differentiation of the regeneration. The content of RNP in the regeneration cells is high, but in the process of differentiation of its elements it becomes lower. The DNP level has inconsiderable fluctuations. In early experiments PAS-positive substances are revealed in greater degree than in later ones. The content of acid mucopolysaccharides decreases in the process of fibrillogenesis. In all internal organs under study the muscular tissue regenerated. The degree of differentiation, severity of the lesion and functional peculiarities of the organ determine the completeness of the tissue reparation. The musculature of the intestinal tract and vessels regenerates more completely. Mighty layers of connective tissue with de novo formed blood vessels are disposed among the bundles of the repaired muscular tissue of the uterus and urine bladder wall. Simultaneously a part of regeneration cells are destroyed. These are two sides of a single process of development.     

The influence of subepithelial connective tissues on epithelial proliferation in the adult mouse

Summary Subepithelial connective tissue is capable of modulating the pattern of histodifferentiation of stratified epithelia from adult animals, but it is not known whether the supporting connective tissue also influences epithelial proliferative activity. Epithelial and connective tissues of murine skin and oral mucosa, differing in their morphology and proliferative activity, were separated and heterotypically recombined prior to grafting to histocompatible hosts. After 3 or 8 weeks in situ, mitotic activity was determined following the administration of vinblastine sulfate. Although the mitotic activity in each of the epithelia could be modulated by some connective tissues, there was no distinct pattern of behavior. In combination with connective tissues from tongue or palate, the ear epidermis acquired a significantly increased mitotic activity. In contrast, when oral epithelia with high mitotic activity were recombined with dermal connective tissue, there was usually a significant reduction in proliferative activity. As there was no apparent association between mitotic activity and the induced changes in either organization or histodifferentiation, it is suggested that subepithelial connective tissue is capable of directly influencing the mitotic activity in the overlying epithelium.     

Mechanisms of lymphatic regeneration after tissue transfer

Introduction

Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.

Methods

Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.

Results

Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.

Conclusions

Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.     

Electron microscopy of wound healing in the skin of Gasterosteus aculeatus

The process of healing of a small surgical incision in the skin of Gasterosteus aculeatus has been studied by electron microscopy. The wounds were made in the mid-ventral line where no muscle intervenes between the skin and the peritoneum. The epidermis moved in through the incision and spread outwards beneath the dermis; the migrating epithelial cells were shown to be phagocytic. The wound was closed when cells at the surface of the epidermis met across the gap, forming a plug of epidermal tissue which was then invaded by dermal tissue from either side. Formation of new basement membrane apparently depended on the interaction of epidermal and dermal components. Leucocyte types were identified by electron microscopy; acid phosphatase tests were positive in macrophages and in neutrophil granulocytes, negative in lymphocytes and in eosinophil granulocytes. These four types of leucocyte are present in normal skin; after wounding, more migrated into the epidermis from the blood. The number of neutrophils in the epidermis reached a peak 24 h after wounding and declined during the second day. The number of macrophages rose to a peak by the third day and returned to normal by day 8. The numbers of eosinophil granulocytes and of lymphocytes showed little change. Neutrophil granulocytes were shown to be phagocytic, although not to the same extent as the macrophages.     

The origin of pituitary cysts in the rostral pars distalis of the nine-spined stickleback,Pungitius pungitius L.

Summary Pituitary cysts in the nine-spined stickleback, Pungitius pungitius L., develop next to blood vessels passing through the prolactin zone of the rostral pars distalis to the connective tissue capsule at its periphery. Cysts were most frequent when pituitaries were large compared with body lengths. However, the incidence of cysts could not be closely related to body length alone. As the rostral pars distalis is more highly vascularised in fish with large pituitaries, and as there was no evidence of accumulating secretion around these blood vessels, it is suggested that cysts develop when vascular demands (or supplies) become excessive. Associated with the greater vascularity of the rostral pars distalis in large pituitaries was a remarkable development of non-granulated cells. Indeed cysts may originate as enlarged intercellular spaces between such cells, as the latter often surround blood vessels. The non-granulated cells are also important in enlarging cyst cavities (by phagocytosing prolactin-cell debris) and perhaps for maintaining their structural integrity. It is suggested that the macrophages within cyst cavities are derived from the non-granulated cells.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.     

Nipple connective tissue and its development: insights from the K14-PTHrP mouse

Parathyroid hormone-related protein (PTHrP) regulates a wide variety of developmental processes. Keratin 14 (K14) promoter-mediated overexpression of PTHrP in the epidermis during development converts the entire murine ventral skin to hairless nipple-like skin. In this report, we characterize the morphology and processes that influence the development of nipple connective tissue. The connective tissue of the nipple displayed increased levels of proteoglycans, and collagen bundles with atypical morphology, as well as increased numbers of mast cells, capillaries, nerve fibers and dermal melanocytes. The unique characteristics of nipple connective tissue were not present until mice reach 3-4 weeks of age. The adult male K14-PTHrP mouse has a less dramatic ventral skin phenotype, and does not manifest a nipple-like dermis. Ovariectomy or orchiedectomy prior to sexual maturity had no impact on the ventral skin of the male or female K14-PTHrP mice, but exposure to androgens in utero repressed many of the nipple-like characteristics in the ventral skin of the female K14-PTHrP mice.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Positional information in the forelimb of the axolotl: experiments with double-half tissues

It has been demonstrated recently that upper forelimbs of axolotls comprised of symmetrically arranged soft tissues do not regenerate (P. W. Tank, 1978,J. Exp. Zool.204, 325–336). These double-half forelimb stumps contained skin, muscle, and loose connective tissues in symmetrical arrangement. The present study explores the roles of muscle, skin, and epidermis in the regeneration of double-half forelimbs by grafting them separately to create forelimb stumps bearing symmetrical arrangements of these individual tissues. Forelimb stumps bearing symmetrically arranged flexor and extensor muscles and normally arranged skin underwent complete regeneration (96%). Forelimbs comprised of double-half skin overlying normally arranged muscles and deep tissues formed hypomorphic structures and nonregenerates (56%) with some single and multiple regenerates. Limbs with double-half deep tissues and complete epidermis either regenerated distally incomplete patterns (47%), single patterns (33%), or multiple patterns (20%). Those forelimbs comprised of double-half skin and no muscle regenerated incomplete patterns in the majority of cases (56%) but single and multiple limbs also were formed. Based on these results it can be concluded that no single type of tissue is solely responsible for the regenerative failure experienced by double-half forelimbs in the earlier study. The complete failure of forelimb regeneration occurs only when all types of soft tissues tested (skin, muscle, and deep connective tissues) are present in symmetrical arrangement.     

Structural transformations of the vascular system of the connective tissue layer of the skin

Electron microscopic radioautography was performed in skin biopsies obtained from six surgical patients without skin lesions. It has been discovered that besides typical vessels--arterioles and venules--there are vessels with damaged structure in connective tissue of skin basis. On serial electron microscopic sections vascular wall thinning and gradual disconnection of wall-forming cells can be visualized. The authors suggest that structural changes in connective tissue vessels of skin basis are the physiological phenomenon. As a result of such changes pericytes may separate from the vascular wall and initiate new populations of fibroblasts.     

The 'Abtropfung phenomenon' revisited: Dermal nevus cells from congenital nevi cannot activate matrix metalloproteinase 2 (MMP-2)

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro-matrix metalloproteinase (MMP)-2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP-2 was not in its active form, we used Western blot to study the expression of members of the MMP-2 activation pathway (membrane type 1-MMP and tissue inhibitor of metalloproteinase-2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol-12-myristate-13-acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP-2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.