Transitional Cell Layer Research Articles

The application of tissue-engineered preputial matrix and fibrin sealant for urethral reconstruction in rabbit model

To introduce the role of fibrin sealant and preputial acellular matrix (PAM) as a new source of inert collagen matrix for urethral reconstruction. A ventral urethral segmental defect was created in 24 male rabbits divided into four groups. In group 1 (G1), urethrotomy was closed in layers. In group 2 (G2), closure was followed by applying fibrin sealant. In groups 3 (G3) and 4 (G4), urethroplasty was performed with a patch graft of PAM, and in G4, fibrin sealant was also applied. Serial urethrography was performed before and after the operation. Then, the animals were euthanized, and their urethra was excised 1, 3, and 9 months postoperatively for further electron microscopic examination, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique, and immunohistochemical (IHC) staining with CD34, CD31, desmin, SMA, and α-actin. In G1 and G2, the fistula repair failed in all the time points. In G3 and G4, serial urethrography confirmed the maintenance of a wide urethral caliber without signs of strictures or extravasations. Satisfactory vascularity was observed in G3 and G4 during the whole study, which was more significant in G4 after 9 months of follow-up. The presence of a complete transitional cell layer was confirmed over the graft in G3 and G4 in all time points. IHC staining confirmed the effectiveness of fistula repair in G3 and G4, 3 months postoperatively. This rabbit model showed that PAM combined with fibrin sealant may herald a reliable option for repairing segmental urethral defects.

P3.449* The promise of estriol cream for prevention of vaginal infection in premenopausal women

BackgroundVaginal epithelium thins during the luteal phase of the menstrual cycle, increasing susceptibility to vaginal infection. Exogenous oestrogen thickens vaginal epithelium in ovariectomized monkeys and protects against SIV transmission. While...

P2.192 Vaginal Epithelial Thickness and Serum Hormone Levels by Body Mass Index at the Luteal and Follicular Phases of the Menstrual Cycle

BackgroundObesity is increasing in the United States and is associated with reproductive disorders. Little is known regarding the precise mechanisms by which obesity impacts reproductive health, but many studies have...

Penile urethra replacement with autologous cell-seeded tubularized collagen matrices

Acellular collagen matrices have been used as an onlay material for urethral reconstruction. However, cell-seeded matrices have been recommended for tubularized urethral repairs. In this study we investigated whether long segmental penile urethral replacement using autologous cell-seeded tubularized collagen-based matrix is feasible. Autologous bladder epithelial and smooth muscle cells from nine male rabbits were grown and seeded onto preconfigured tubular matrices constructed from decellularized bladder matrices obtained from lamina propria. The entire anterior penile urethra was resected in 15 rabbits. Urethroplasties were performed with tubularized matrices seeded with cells in nine animals, and with matrices without cells in six. Serial urethrograms were performed at 1, 3 and 6 months. Retrieved urethral tissues were analysed using histo- and immunohistochemistry, western blot analyses and organ bath studies. The urethrograms showed that animals implanted with cell-seeded matrices maintained a wide urethral calibre without strictures. In contrast, the urethras with unseeded scaffolds collapsed and developed strictures. Histologically, a transitional cell layer surrounded by muscle was observed in the cell-seeded constructs. The epithelial and smooth muscle phenotypes were confirmed with AE1/AE3 and α-actin antibodies. Organ bath studies of the neourethras confirmed both physiological contractility and the presence of neurotransmitters. Tubularized collagen matrices seeded with autologous cells can be used successfully for long segmental penile urethra replacement, while implantation of tubularized collagen matrices without cells leads to poor tissue development and stricture formation. The cell-seeded collagen matrices are able to form new tissue, which is histologically similar to native urethra.

Long-term study of male rabbit urethral mucosa reconstruction using epidermal cell

To investigate the transformation of characteristics of epidermal cells from foreskin which were used to reconstruct male rabbit anterior urethra in combination with acellular collagen matrices. In three rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and seeded (inoculated) onto a tubular acellular collagen matrix, acquired from allogeneic rabbit bladder submucosa. A urethral mucosal defect was created, and urethral reconstruction was performed with the tubular acellular collagen matrix seeded with epidermal cells. On gross examination at 12 months following the procedure, the mucosa of the urethral grafts appeared lubricous and smooth. Urethrography showed that a wide urethral caliber had been maintained without any sign of strictures. Histological examination showed a transitional cell layer in the graft without evidence of a margin between the graft and the host tissue at 12 months postoperatively. Epidermal cells seeded onto acellular collagen matrices can be successfully used to reconstruct urethras that have defects and are transformed to transitional epithelial cells.

Ultraviolet B‐induced alterations of the skin barrier and epidermal calcium gradient

Ultraviolet irradiation induces a variety of cutaneous changes, including epidermal permeability barrier disruption. In the present study, we assessed the effects of ultraviolet B (UVB) irradiation in epidermal barrier function and calcium distribution in murine epidermis. Adult hairless mice were exposed to a single dose of UVB (0.15 J/cm(2)). Barrier function was evaluated by transepidermal water loss (TEWL), lanthanum perfusion. The morphological alterations were examined by histology, immunohistochemistry and electron microscopy using ruthenium tetroxide (RuO(4)) postfixation. For evaluation of the effect on epidermal calcium distribution, the ion-capture cytochemistry was employed. UVB irradiation caused a significant increase in TEWL, which peaked at day 4. In parallel, the increased number of sunburn cells and the changes in epidermal hyperplasia and proliferation were observed. Electron microscopic observation demonstrated that the water-soluble lanthanum tracer was present in the extracellular stratum corneum domains, and the increased intercellular permeability was correlated with defective organization of the extracellular lipid lamellar bilayers of the stratum corneum. Moreover, UVB irradiation also caused an appearance of calcium precipitates in the stratum corneum and transitional cell layers as well as the increased cytosolic calcium in the lower epidermis, reflecting the alterations of the epidermal calcium gradient. These results suggest that the changes of the epidermal calcium distribution pattern may correlate with the perturbation of the epidermal barrier induced by UVB irradiation.

Urethral replacement using epidermal cell‐seeded tubular acellular bladder collagen matrix

To investigate the feasibility of replacing urinary epithelium cells with foreskin epidermal cells to reconstruct engineered anterior urethra with an acellular collagen matrix. Acellular collagen matrices were generated from allogeneic rabbit bladder submucosa. In nine rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and labelled with 5-bromo2'-deoxy-uridine (BrdU) before seeding onto a tubular acellular collagen matrix (1.5x1 cm). In male rabbits, a urethral mucosal defect was created, and urethroplasty performed with a tubular acellular collagen matrix seeded with epidermal cells (nine rabbits) or with a matrix with no cell seeding (nine rabbits; control group). Urethrography was done at 1, 2 and 6 months after grafting. The urethral grafts were harvested and analysed grossly and histologically. In the control group, gross views and urethrography revealed stricture of repaired defects at the different sample times. In the experimental group, a wide urethral calibre was maintained with no sign of strictures. Histology in the control group showed a single layer of epithelium cells with disorganized muscle fibre bundles in the submucosa layer at 1 month after grafting, and a transitional cell layer surrounded by disorganized muscle fibre bundles at 2 and at 6 months. Grafts seeded with epidermal cells formed a single-layer structure by 1 month, and at 2 and 6 months there were several layers of epidermal cells with abundant vessels in the submucosa. There was an evident margin between graft epidermal cells and host epithelium at 6 months. The implanted cells expressed keratin, shown by staining with anti-pancytokeratins. Immunofluorescence for BrdU confirmed the presence of implanted epidermal cells at 1 month after grafting; there were fewer positive cells at the implantation site at 2 months. At 6 months, there were several layers of epidermal cells with no signs of BrdU staining. Urethral reconstruction was better with an acellular collagen matrix seeded with epidermal cells than with the acellular collagen matrix alone. Foreskin epidermal cells seem adequate in replacing urethral epithelium cells for urethral reconstruction.

Urethral replacement using cell seeded tubularized collagen matrices.

Acellular collagen matrices derived from bladder submucosa have been used successfully as an off-the-shelf biomaterial for urethral replacement, experimentally and clinically in an onlay fashion. We investigated whether collagen matrices, either alone or with autologous cells, could be used for tubularized urethral replacement. Acellular collagen matrices were processed and tubularized. Ten rabbits underwent an open bladder biopsy with subsequent cell expansion. Autologous bladder cells were grown and seeded onto the pre-configured tubular matrices. A 1 cm. long urethral segment was excised in 24 male rabbits. Urethroplasty was performed with the tubularized collagen matrices seeded with cells in 12 animals and without cells in 12. Serial urethrography was performed preoperatively and at 1, 2, 3 and 6 months postoperatively. Retrieved urethras were analyzed grossly, histologically, immunocytochemically and with Western blots. Contractility and the presence of neurotransmitter receptors were confirmed with organ bath studies. Serial urethrography confirmed the maintenance of a wide urethral caliber without any signs of strictures in animals implanted with the cell seeded matrices. The urethral segments replaced with the collagen scaffolds without cells demonstrated strictures and graft collapse at all time points. The implanted cell seeded matrices had a normal urethral architecture by 1 month, consisting of a transitional cell layer surrounded by muscle cell fiber bundles with increasing cellular organization with time. Epithelial and smooth muscle phenotypes were confirmed immunocytochemically and with Western blot analyses using pancytokeratins AE1/AE3 and smooth muscle specific alpha-actin antibodies. Formation of a transitional cell layer was confirmed in the matrices implanted without cells but only scant unorganized muscle fiber bundles were present, mostly at the anastomotic sites. Organ bath studies demonstrated the capacity for contractility along with cholinergic and adrenergic specific receptors in the tissue engineered scaffolds compared to controls. These results show that collagen matrices seeded with cells form normal urethral tissue can be used for tubularized replacement, whereas tubularized collagen matrices alone without cells lead to poor tissue formation and strictures. The collagen matrices seeded with cells may offer a useful alternative in the future for patients requiring a tubularized urethral segment replacement.

Acellular collagen matrix as a possible “off the shelf” biomaterial for urethral repair

Objectives. To determine whether a naturally derived collagen-based tissue substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model. Several urethral conditions often require nongenital tissues for reconstruction, such as skin grafts or mucosal grafts from the bladder or buccal regions. However, the use of these tissues for urethroplasty may be associated with additional procedures for graft retrieval, prolonged hospitalization, and morbidity. Methods. A ventral urethral defect was created in 10 male rabbits. The acellular collagen matrix, obtained and processed from porcine bladder submucosa, was trimmed and used to replace the urethral defect in an onlay fashion. Serial urethrography was performed pre- and postoperatively at 0.5, 1, 2, 3, and 6 months. The animals were sacrificed 0.5, 1, 2, 3, and 6 months after surgery. The retrieved implants were analyzed grossly, histologically, and with immunocytochemistry. Results. All animals survived until being sacrificed without any noticeable voiding dysfunction. Serial urethrograms confirmed the maintenance of a wide urethral caliber without any signs of strictures. Gross examination at retrieval showed normally appearing tissue without any evidence of fibrosis. Histologically, the implanted matrices contained host cell infiltration and generous angiogenesis by 2 weeks after surgery. The presence of a confluent transitional cell layer was confirmed by immunocytochemical analyses using pancytokeratin antibodies. Anti-alpha actin antibodies demonstrated the migration of unorganized muscle fiber bundles 2 months after implantation and organized muscle bundles 6 months after implantation. Conclusions. The acellular collagen matrix appears to be a useful material for urethral repair in the rabbit. The matrix can be processed easily, has good characteristics for tissue handling and urethral function, and has the advantage of being an “off the shelf” material.

The Changes of Epidermal Calcium Gradient and Transitional Cells after Prolonged Occlusion Following Tape Stripping in the Murine Epidermis

Disruption of the epidermal permeability barrier causes an immediate loss of the calcium gradient, and barrier recovery is parallel with the restoration of the calcium gradient in the epidermis. Artificial restoration of the barrier function by occlusion with a water vapor-impermeable membrane abrogate the expected increase in lipid synthesis and retard the barrier recovery, as well as block the normalization of the epidermal calcium gradient. To clarify the long-term effects of occlusion after acute barrier perturbation, we studied the calcium distribution and epidermal keratinocytes response after occlusion with a water vapor-impermeable membrane immediately following tape stripping in the murine epidermis. Acute barrier disruption caused an immediate depletion of most calcium ions in the upper epidermis, obliterating the normal calcium gradient. When the skin barrier function was artificially corrected by occlusion, the return of calcium ions to the epidermis was blocked. After 2 h of air exposure or occlusion, the density of epidermal calcium precipitates remained negligible. The transitional cell layers appeared with occlusion, but not or negligibly with air exposure. By 6 h though, calcium precipitates could be seen, the density of the calcium precipitates with occlusion was more sparse than with air exposure. With the air exposure, the thickness of the stratum corneum had normalized and the calcium gradient nearly recovered to normal after 24 h. The longer the occlusion period, the greater was the increase of transitional cells. By 60 h of occlusion, the thickness of the stratum corneum had increased and the transitional cell layers had disappeared, in parallel with the calcium gradient which was almost normalized. These results show that prolonged occlusion of tape-stripped epidermis induced transitional cells and delayed the restoration of the epidermal calcium gradient, the stratum corneum was then restored, transitional cells having disappeared, in parallel with normalization of the epidermal calcium gradient.

Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease.

Polyomavirus (PV) exceptionally causes a morphologically manifest renal allograft infection. Five such cases were encountered in this study, and were followed between 40 and 330 d during persistent PV renal allograft infection. Transplant (Tx) control groups without PV graft infection were analyzed for comparison. Tissue and urine samples were evaluated by light microscopy, immunohistochemistry, electron microscopy, and PCR. The initial diagnosis of PV infection with the BK strain was made in biopsies 9+/-2 mo (mean +/- SD) post-Tx after prior rejection episodes and rescue therapy with tacrolimus. All subsequent biopsies showed persistent PV infection. Intranuclear viral inclusion bodies in epithelial cells along the entire nephron and the transitional cell layer were histologic hallmarks of infection. Affected tubular cells were enlarged and often necrotic. In two patients, small glomerular crescents were found. In 54% of biopsies, infection was associated with pronounced inflammation, which had features of cellular rejection. All patients were excreting PV-infected cells in the urine. PV infection was associated with 40% graft loss (2 of 5) and a serum creatinine of 484+/-326 micromol/L (mean +/- SD; 11 mo post-Tx). Tx control groups showed PV-infected cells in the urine in 5%. Control subjects had fewer rejection episodes (P<0.05) and stable graft function (P = 0.01). It is concluded that a manifest renal allograft infection with PV (BK strain) can persist in heavily immunosuppressed patients with recurrent rejection episodes. PV mainly affects tubular cells and causes necrosis, a major reason for functional deterioration. A biopsy is required for diagnosis. Urine cytology can serve as an adjunct diagnostic tool.

Spatial distribution of nerve processes and beta-adrenoreceptors in the rat atrioventricular node.

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and beta1 and beta2-adrenoreceptors. Serial 12 microm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE-reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE-reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. Beta1-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. Beta2-adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of beta2-ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype-specific pharmacological agents may have distinct effects upon AV nodal conduction.

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats.

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg-1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration.

Reconstruction of the Urinary Bladder Mucosa in Three-Dimensional Collagen Gel Culture: Fibroblast-Extracellular Matrix Interactions on the Differentiation of Transitional Epithelial Cells

The purpose of this study was to reconstruct a urinary bladder mucosa in three-dimensional collagen gel culture conditions that included fibroblasts. Transitional epithelial cells and fibroblasts, isolated respectively from the epithelial and lamina propria layers of porcine urinary bladder, were cultured in monolayer. These fibroblasts were embedded and cultured within a collagen gel matrix to reconstruct a lamina propria. The isolated transitional epithelial cells were then seeded in vitro on this reconstructed lamina propria on which the transitional epithelial cells formed a stratified urothelium composed of basal, intermediate and superficial layers. Urothelial differentiation was observed only on the fibroblast-containing collagen matrix. Differentiation did not occur on a cell-free collagen matrix through use of fibroblast conditioned medium. Thus, urothelial differentiation depended upon a fibroblast-extracellular matrix interaction. The differentiated transitional epithelial cell layer thus produced closely resembled a urothelium in vivo. This culture model may provide a useful system in which to study various diseases of the urinary bladder.

Distribution of sulphydryl groups and disulphide linkages in tissue proteins of quail uropygial glands

1. The distribution of sulphydryl (-SH) groups and disulphide (S-S) linkages in the tissue proteins of quail uropygial glands was histochemically observed by a staining method using N-[7-dimethylamino-4-methyl-3-coumarinyl]maleimide. 2. In the uropygial gland, the -SH groups of the tissue proteins of glandular cells displayed a progressive change to S-S linkages concomitant with the shift from the transitional cell layer to the degenerating cell layer, suggesting that cell destruction was occurring with the products being released as secretions. This phenomenon was apparently more pronounced in the deep compared with the shallow portion of secretory tubules. 3. The above results indicated that a mechanism similar to epidermal differentiation operates in the process of holocrine secretion in the uropygial gland.

Selective Staining of the Superficial Cells of Mouse Urinary Bladder Epithelium by Horseradish Peroxidase (HRP)

A simple method for staining the superficial cells of mouse urinary bladder epithelium with horseradish peroxidase (HRP) is presented. HRP solution was instilled into the bladder lumen in vivo. After fixation with glutaraldehyde, the bladder was treated with Triton X-100 and then reacted with diaminobenzidine (DAB). Intense DAB reactions were detected throughout the whole of the cytoplasm of superficial cells by both light and electron microscopy. No reaction was observed in intermediate or basal epithelial cells. This simple method will be useful for the three-dimensional reconstruction of cell shape and identification of the stratification of transitional epithelial cell layers.

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Prenatal developmental process of human temporomandibular joint

The prenatal development of 20 human TMJs from the fourteenth gestational week to full term was studied and the following observations were made. Radiologic findings: at the sixteenth gestational week, the outline of the TMJ as radioopaque structures appeared. Curvature of the articular eminence of the temporal components was observed at gestational week 33, and convexity of the condylar head of the condylar process was observed at the thirty-first week. Histologic findings: at the fifteenth gestational week, thin intramembranous ossification of the temporal components and endochondral ossification of the condylar process were observed, and secondary cartilage occupied the whole condylar process. The articular disk was distinguishable and was composed of fine collagen fibers. At the seventeenth gestational week, endochondral ossification of the condylar process appeared, the formation of joint cavities was fairly completed, and synovial tissues were easily observed. From gestational week 17 through 21, endochondral ossification of the condylar process developed rapidly and the layer structures, which consisted of a fibrous covering layer, transitional cell layer, hypertrophic layer, and erosive layer, were observed. At the twenty-first gestational week, Meckel's cartilage disappeared and hematopoietic foci appeared in the temporal components and the condylar process. Week 21 seemed to be the greatest turning point of the prenatal development of the human TMJ. Secondary cartilage of the condylar process diminished gradually throughout fetal life, but was vestigial at full term.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of vaginal keratinization in estrogen treated immature BALB/cCRGL mice.

In 10-day-old Balb/cCrgl mice, the subcutaneous injection of 0.1 μg of estradiol in distilled water per animal per day resulted in the conversion, over a 4 day period, of the original 3 cell layered cuboidal epithelium to a stratified, multilayered, fully keratinized epithelium. By light microscopy, there was development of a prominent stratum germinativum and of a mucinified surface on the 1st day, followed by the sequential formation of a stratum spinosum, a stratum granulosum, and a stratum corneum. By electron microscopy, the principal early modifications consisted of a marked increase in ribosomes, desmosomes, and 70 A cytoplasmic filaments, the latter being aggregated into approximately 700 A fibrils. The subsequent establishment of a keratin layer was preceded by the appearance of keratohyaline granules and the disappearance of mitochondria and endoplasmic reticulum in cells immediately above the stratum spinosum and by the development of a transitional cell layer in which there was progressive aggregation of cytoplasmic filaments and disappearance of nuclei, keratohyaline granules, and free ribosomes. In the upper stratum granulosum, transitional cell layer, and stratum corneum there were distinctive modifications in desmosomal structure (composite and modified desmosomes). The morphological and physiological significance of these observations is discussed.

Ultrastructural changes induced by ultraviolet light in human epidermis: Granular and transitional cell layers

Ultrastructural changes induced by ultraviolet light in human epidermis: Granular and transitional cell layers