The influence of differing connective tissue substrates on the maintenance of adult stratified squamous epithelia

Summary The interaction between adult stratified squamous epithelium and its supporting connective tissue possibly involves both permissive and directive influences. To examine the effect of vitality and specificity of connective tissue on the maintenance of epithelial structure and histo-differentiation, specimens of skin and oral mucosa from various regions of adult mice were separated using either EDTA or trypsin. Prior to transplantation, the epithelium was recombined with either inverted homologous connective tissue or with connective tissue that had been killed either by heating or repeated freeze-thawing. Epithelial sheets were also transplanted onto the graft bed alone or in combination with striated muscle or tendon.Normal patterns of cytodifferentiation were maintained when the epithelium was recombined with inverted or frozen-thawed subepithelial connective tissue but there was a loss of spatial organization on the frozen-thawed connective tissue. In contrast, heat-killed or trypsin-treated frozen-thawed subepithelial connective tissue and non-dermal connective tissue failed to maintain a viable epithelium. These observations suggest that subepithelial connective tissues (dermis, lamina propria) but not deep connective tissues facilitate epithelial proliferation and histodifferentiation.Supported by NIH/NIDR RO1 DEO5190     

Stress and matrix‐responsive cytoskeletal remodeling in fibroblasts

In areolar “loose” connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross‐sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large “sheet‐like” morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch‐induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues. J. Cell. Physiol. 228: 50–57, 2013. © 2012 Wiley Periodicals, Inc.     

EDTA separation and recombination of epithelium and connective tissue of human oral mucosa. Studies of tissue transplants in nude mice

A possible epithelial-mesenchymal interaction in determining epithelial histologic features of human oral mucosa was examined. The study comprised 74 biopsies of normal buccal mucosa and 54 biopsies of normal palatal mucosa. Epithelium was separated from connective tissue by the use of 1 mM ethylenediamine tetraacetate dihydrate. Self-recombined and cross-recombined epithelial and connective tissues and connective tissue sheets alone were transplanted to subcutaneous sites of nude mice. Histologic examination of cross-recombined palatal epithelium/buccal connective tissue transplants showed a change in keratinization pattern but no major change in number of epithelial cell layers as the result of connective tissue influence. Transplanted sheets of connective tissue after growth for 14 days showed that complete separation of biopsies from buccal mucosa had been obtained. However, palatal mucosa had been incompletely separated as evidenced by re-epithelialization of most of the connective tissue transplants. The consequences of the incomplete palatal epithelium-connective tissue separation are discussed.     

Study of antibodies to heterologous connective tissue, isolated from sera of patients with rheumatism]

Antibodies against connective tissue elements of various bovine organs were isolated from the sera of rheumatic fever patients with the aid of immunosorbents (bovine connective tissue extract and erythrocyte stroma). The antibody preparations obtained were not identical and contained antibodies against different antigens of bovine connective tissue. The antibody preparations failed to react with human connective tissue components.     

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.     

结缔组织铺片脂肪组织油红染色在组织学实验教学中的应用

目的在传统结缔组织铺片基础上开展脂肪组织油红染色方法在医学本科生组织学实验教学中的应用。方法学生先进行疏松结缔组织铺片,并施行脂肪组织油红o-甲苯胺兰-伊红三重染色,然后镜下观察。结果油红o染色把结缔组织中的脂肪细胞内脂滴保存下来并染上红色。脂肪组织中央的细胞脂滴均匀红染,充满胞浆,周边的脂肪细胞胞浆中油红染色很少,细胞呈空泡状,显示出脂肪细胞亚群存在。甲苯胺兰染色使得疏松结缔组织中肥大细胞染成紫红色,胞核染色浅,细胞数量多、成群分布。伊红可使得结缔组织内除脂肪细胞、肥大细胞意外的其他细胞的胞浆和胶原纤维染成淡红色。结论传统的组织学平铺片技术基础上引入脂肪油红o-甲苯胺兰-伊红三重染色,可增强学生动手能力,并能很好地了解输送结缔组织中细胞的不同表型和分布,丰富组织学内容,把教学、科研连接一起,达到提高实验教学质量的目的。     

Connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse

Summary Epithelial-mesenchymal interactions play an important role during embryogenesis but it is uncertain whether such interactions influence the maintenance of epithelial structure in the adult. To examine this problem, separated epithelial and connective tissue components of skin and mucosae from various regions of adult mice were homoor heterotypically recombined and transplanted to histocompatible hosts. The patterns of tissue architecture and keratinization of the resultant epithelia were examined for changes indicative of mesenchymal influences on the epithelial phenotype. Each type of epithelium, in some recombinations, fully conserved its normal pattern of phenotypic expression indicating that subepithelial connective tissue from all regions is permissive and that regionally-specific connective tissue influences are not necessary for conservation of epithelial specificity. In other recombinations, however, the epithelium acquired features of tissue architecture or keratinization typical of the epithelium normally associated with the connective tissue component, indicating directive influ ences from the connective tissue. The patterns of epithelial response observed suggest that there may be separate connective tissue influences on epithelial architecture and cytodifferentiation and that there is a regionally-related variation in the competence of epithelia to respond to these influences.Supported by NIH NIDR 2 R01 DE05190     

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.     

Viscoelastic properties of bovine orbital connective tissue and fat: constitutive models

Reported mechanical properties of orbital connective tissue and fat have been too sparse to model strain–stress relationships underlying biomechanical interactions in strabismus. We performed rheological tests to develop a multi-mode upper convected Maxwell (UCM) model of these tissues under shear loading. From 20 fresh bovine orbits, 30 samples of connective tissue were taken from rectus pulley regions and 30 samples of fatty tissues from the posterior orbit. Additional samples were defatted to determine connective tissue weight proportion, which was verified histologically. Mechanical testing in shear employed a triborheometer to perform: strain sweeps at 0.5–2.0 Hz; shear stress relaxation with 1% strain; viscometry at 0.01−0.5 s⁻¹ strain rate; and shear oscillation at 1% strain. Average connective tissue weight proportion was 98% for predominantly connective tissue and 76% for fatty tissue. Connective tissue specimens reached a long-term relaxation modulus of 668 Pa after 1,500 s, while corresponding values for fatty tissue specimens were 290 Pa and 1,100 s. Shear stress magnitude for connective tissue exceeded that of fatty tissue by five-fold. Based on these data, we developed a multi-mode UCM model with variable viscosities and time constants, and a damped hyperelastic response that accurately described measured properties of both connective and fatty tissues. Model parameters differed significantly between the two tissues. Viscoelastic properties of predominantly connective orbital tissues under shear loading differ markedly from properties of orbital fat, but both are accurately reflected using UCM models. These viscoelastic models will facilitate realistic global modeling of EOM behavior in binocular alignment and strabismus.     

Effect of Aeromonas proteases on the binding of Aeromonas hydrophila strains to connective tissue proteins

125I-labelled connective tissue protein binding to cells of Aeromonas hydrophila, A. caviae, and A. sobria strains isolated from diseased fish, was correlated with the Aeromonas protease degradation of 125I-labelled collagen types I and IV, fibronectin and laminin, immobilized on tissue culture microtitre plates. An inverse relation between 125I-labelled connective tissue protein binding to cells of Aeromonas strains and proteolytic degradation of immobilized connective tissue proteins by Aeromonas proteases was established. Inhibition of the Aeromonas proteolytic activity by protease inhibitors enhances the 125I-labelled connective tissue protein binding to cells of Aeromonas hydrophila strains. Culture conditions were found to influence both expression of proteolytic activity and binding properties.     

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.

Dispase-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.     

Inductive action of epithelium on differentiation of intestinal connective tissue of Xenopus laevis tadpoles during metamorphosis in vitro

The action of the epithelium on differentiation of connective tissue cells of Xenopus small intestine during metamorphosis was investigated by using culture and morphological techniques. Connective tissue fragments isolated from the small intestine at stage 57 were cultivated in the presence or absence of homologous epithelium. In the presence of the epithelium, metamorphic changes in the connective tissue were fully induced by hormones including thyroid hormone (T₃), as during spontaneous metamorphosis, whereas they were partially induced in the absence of the epithelium. Macrophage-like cells showing non-specific esterase activity in the connective tissue were much fewer in the absence of the epithelium than in the presence of it, and aggregates of fibroblasts possessing well-developed rough endoplasmic reticulum developed only in the presence of the epithelium. Just before the aggregation of the fibroblasts, the connective tissue close to the epithelium became intensely stained with concanavalin A (ConA) and wheat germ agglutinin (WGA). The present results indicate that the epithelium plays important roles in the differentiation of intestinal connective tissue cells, which in turn affect the epithelial transformation from larval to adult form during anuran metamorphosis. Thus, the tissue interaction between the epithelium and the connective tissue in the anuran small intestine is truly bidirectional.     

Conception of a Bioelectromagnetic Signal System via the Collagen Fibril Network; Biochemical Conclusions and Underlying Coherent Mechanism. II. Energetic Aspects,Acid and Neutral Proteases,and the Phenomenon of Coherence

This paper presents a bioelectrical conception of connective tissue regulation in bone, cartilage, and tendon, as well as other mechanically stressed connective tissues, based on the biological hypothesis of a biosensor and nerve-like signal conducting function of the native collagen fibril in the extracellular matrix. The various levels of existing conceptions of bioelectrical connective tissue regulation as well as some questions of classical connective tissue research (e.g., neutral and acid protease activity) are discussed from this electrophysiological point of view. Part I presented the topic in the form of classical biophysics and physicochemistry. This paper, Part II, makes use of the concept for a discussion of the “living state” of the extracellular matrix, biochemical aspects of acid and neutral protease activity, and nanoelectronic, relativistic, and coherent aspects of connective tissue regulation.     

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.     

Glucocorticoid action on connective tissue: from molecular mechanisms to clinical practice

Glucocorticosteroids are highly effective in treating various acute and chronic diseases, but their long-term use is often accompanied by side effects, such as osteoporosis of skeleton and bones and atrophy of the skin. Clinically, many of these side effects involve changes in connective tissue. Glucocorticoid effects on connective tissue metabolism are, however, sometimes beneficial for instance, in the treatment of keloids or autoimmune connective tissue diseases. Recent advances in the biochemical technology have provided tools to examine the molecular mechanisms by which glucocorticoids affect connective tissue. These studies have shown distinct alterations in the extracellular matrix as a result of glucocorticoid treatment. This knowledge is useful for the further development of glucocorticosteroids with desirable action spectrum and with minimal side effects.     

The connective tissue index of <Emphasis Type="Italic">Helix aspersa</Emphasis> as a metal biomarker

The digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island (Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantify the relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, through light microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cu and Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetric density of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein is suggested as a biomarker of Cu exposure in terrestrial mollusks.     

Uptake of colloidal thorium dioxide by the mouse connective tissue mast cell.

The ability of the mouse mast cell to phagocytize colloidal thorium dioxide, Thorotrast, was investigated employing the mouse connective tissue air pouch. The connective tissue mast cell of mouse was found to have limited capability to ingest the particulate Thorotrast in comparison to the rat peritoneal mast cell which ingested this material readily. Fibroblasts and macrophages in the connective tissue removed injected Thorotrast very rapidly in contrast to mast cells that demonstrated limited phagocytic capabilities. The tissue mast cell of the mouse, therefore, should not be considered a part of the reticuloendothelial system.     

Stretching of the back improves gait, mechanical sensitivity and connective tissue inflammation in a rodent model

The role played by nonspecialized connective tissues in chronic non-specific low back pain is not well understood. In a recent ultrasound study, human subjects with chronic low back pain had altered connective tissue structure compared to human subjects without low back pain, suggesting the presence of inflammation and/or fibrosis in the low back pain subjects. Mechanical input in the form of static tissue stretch has been shown in vitro and in vivo to have anti-inflammatory and anti-fibrotic effects. To better understand the pathophysiology of lumbar nonspecialized connective tissue as well as potential mechanisms underlying therapeutic effects of tissue stretch, we developed a carrageenan-induced inflammation model in the low back of a rodent. Induction of inflammation in the lumbar connective tissues resulted in altered gait, increased mechanical sensitivity of the tissues of the low back, and local macrophage infiltration. Mechanical input was then applied to this model as in vivo tissue stretch for 10 minutes twice a day for 12 days. In vivo tissue stretch mitigated the inflammation-induced changes leading to restored stride length and intrastep distance, decreased mechanical sensitivity of the back and reduced macrophage expression in the nonspecialized connective tissues of the low back. This study highlights the need for further investigation into the contribution of connective tissue to low back pain and the need for a better understanding of how interventions involving mechanical stretch could provide maximal therapeutic benefit. This tissue stretch research is relevant to body-based treatments such as yoga or massage, and to some stretch techniques used with physical therapy.     

A rapid connective tissue stain for glycol methacrylate embedded tissue

No reliable connective tissue stains for GMA sections were available until recently. However, the use of toluidine blue in combination with basic fuchsin appeared to be a rapid and reliable connective tissue stain for glycol methacrylate (GMA) embedded tissue.     

Intracellular and extracellular activity of cathepsin D in the liver in cirrhosis and its involution

The distribution of cathepsin D in liver with CCl4 induced cirrhosis and its involution in rats was investigated by ultrastructural cytochemistry. Besides intracellular, it was revealed the extracellular activity of cathepsin D. The reaction product was on collagen fibers near the hepatocytes and connective tissue cells as well as on the hepatocytes microvilli and on the outside part of cellular membrane of connective tissue cells (macrophage, fibroblast, Ito cells). Hence the source of extracellular cathepsin D in liver are the parenchymatous as well as nonparenchymal cell elements. The results testify that under the cirrhosis and its involution, the cathepsin D takes part in intracellular proteolysis and is secreted by hepatocytes and connective tissue cells in the intracellular space; it also takes part in extracellular catabolism of connective tissue.