Epithelial Sheet Formation Research Articles

Establishment of perpendicular protrusion of type I collagen on TiO2 nanotube surface as a priming site of peri-implant connective fibers

Natural teeth are supported by connective tissue collagen fibers that insert perpendicularly in the tooth cementum. Perpendicular insertion plays an important role in the maintenance of the junction between the oral epithelium and the periodontal connective tissue. Most titanium dental implant surfaces have no micro or macro structure to support perpendicularly oriented collagen attachment. Without this tight biologic seal to resist bacterial invasion and epithelial downgrowth, progressive bone loss in peri-implantitis is seen around dental implants. The purpose of this study was to establish the perpendicularly oriented collagen attachment to titanium oxide nanotube (TNT), and to assess its binding stability. TNT was prepared on the titanium-surface by anodization. Scanning electron microscopy (SEM) showed a regularly aligned TNT with an average 67 nm-diameter when anodized at 30 V for 3 h. Subsequently, collagen type I (CoI) was electrophoretically fused to anodic TNT in native polyacrylamide gel system where negatively charged CoI-C term was perpendicularly navigated to TNT. SEM and atomic force microscopy (AFM) were used to analyze CoI on the TiO2 and TNT surface. Several tens of nanometers of CoI protrusion were recorded by AFM. These protrusions may be long enough to be priming sites for cell-secreted CoI. CoI laid parallel to the titanium surface when fused by a chemical linker. Binding resistance of CoI against drastic ultrasonication was measured by Fourier-transform infrared spectroscopy attenuated total reflection (FTIR-ATR). The electrophoretically fused CoI in the titanium nanotube (TNT–CoIEPF) showed the significantly greatest binding resistance than the other groups (P < 0.01, a 1-way ANOVA and Tukey HSD post hoc test). Furthermore, TNT–CoIEPF surface rejected epithelial cell stretching and epithelial sheet formation. Chemically linked horizontal CoI on titanium oxide (TiO2) facilitated epithelial cell stretching and sheet formation.

Substrate deformations induce directed keratinocyte migration.

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.

Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4–activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP–modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP–modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)–expressing Escherichia coli was cultured onto PAR4-AP–modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP–modified titanium.

Development of a biomaterials-based functional epithelial graft for tracheal regeneration

Event Abstract Back to Event Development of a biomaterials-based functional epithelial graft for tracheal regeneration Ratna Varma1, 2, Golnaz Karoubi2 and Thomas K. Waddell1, 2 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 Toronto General Hospital, Latner Thoracic Surgery Research Laboratories, Canada Tracheal injury, stenosis, and malignancy demand surgical management or tracheal reconstruction [1],[2], however, the latter is an unmet clinical need as tracheal grafts and transplants fail due to the lack of a functioning epithelium and immune rejection. Tracheal immune rejection is largely associated with the epithelium, therefore we believe that the best method to optimize the use of donor tracheae is to decellularize only the epithelium and maintain the remaining tracheal structure, to replace it with a recipient-derived epithelium. We aim to develop a donor-recipient hybrid tracheal model by de-epithelializing the donor trachea and engrafting a pre-developed biomaterial-based recipient epithelial graft. The absence of a functioning, ciliated, pseudostratified epithelium is a primary concern in tracheal regeneration. Clinically applied biomaterials demonstrate limited success in allowing proper airway epithelium growth and function [3]-[5]. Promising biomaterials have not been studied systematically. Therefore, we propose to perform the first comparative study to determine the optimal biomaterial for this context. Hyaluronan-Poly-ethylene glycol (HA-PEG), Chitosan-Collagen, Collagen Vitrigel Membrane, Gelatin, Silk Fibroin, and Fibrin-PEG diacrylate, were screened to identify substrates that promote airway epithelium (cell lines: BEAS-2B, A549, CALU-3, and primary human tracheal epithelial cells: HTECs) attachment, viability and function while possessing mechanical strength. Cell viability and proliferation on the biomaterials are assessed through Live/Dead staining and Ki-67 immunocytochemistry, respectively, while cell attachment is assessed via focal adhesion kinase and vinculin immunocytochemistry. Tight junction (zonula occludins-1) formation and differentiation of HTECs into ciliated (acetylated α-tubulin) and secretory cells (mucin 5AC) in air-liquid-interface culture is examined via immunocytochemistry. Preliminary results of phase contrast microscopy revealed no attachment of BEAS-2Bs and HTECs on HA-PEG hydrogels (HA-PEG, HA-PEG with functionalized IKVAV peptide, and HA-PEG with functionalized RGD peptide) by day 7. There was limited attachment of A549s and BEAS-2Bs and no attachment of CALU-3s on Chitosan-Collagen by day 14, and successful attachment with epithelial sheet formation with all cell types on Silk Fibroin by day 14 of culture. Irregular tight junction formation on HTECs on Silk Fibroin is detected as early as day 14 of air-liquid-interface culture and organizes by day 45. While Silk Fibroin is a promising biomaterial with exceptional mechanical strength [6], it demonstrates delayed maturation of HTECs and no ciliated cell differentiation by day 45 as opposed to day 21 on the Collagen I control. Silk Fibroin can be coated with other matrix proteins to promote enhanced epithelial cell attachment and differentiation. A 72-hour extracellular matrix screen of thirty individual or matrix protein combinations demonstrates BEAS-2B and HTEC attachment to Fibronectin (50 µg/ml) + Laminin (100 µg/ml) and RGD Peptide (50 µg/ml) + Laminin (50 µg/ml), and little attachment to Collagen I (200 µg/ml or 400 µg/ml) which is often used as a standard control. With further investigation, an optimal biomaterial will be selected to develop an airway epithelium graft for human tracheal regeneration. This study will have significant clinical implications for viable tracheal transplants in future and can serve as a stepping stone for regenerating other organs comprising an epithelium. Dr. Molly Shoichet; Alexander Baker

Substrate Stiffness Affects Human Keratinocyte Colony Formation.

Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned along the periphery of an evolving cell colony.

Par3 controls epithelial spindle orientation by aPKC-mediated phosphorylation of apical Pins.

Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens. We now show that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. The Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation.

Serum response factor is essential for the proper development of skin epithelium

Mammalian epidermis is a stratified epithelium that serves as a barrier protecting the organism from mechanical stress and dehydration. Previous studies have demonstrated the importance of the actin cytoskeleton in the establishment of a functional skin epithelium. Despite what is known about the actin cytoskeleton in epithelial sheet formation, the molecules important for controlling the actin cytoskeleton during epidermal development have not been determined. Serum response factor (SRF) is a transcription factor that is considered to be an important regulator of the actin cytoskeleton. To examine the role of SRF in the developing mouse epidermis, we have employed gene targeting to ablate Srf in keratinocytes. Conditional inactivation of Srf during the embryonic timepoint leads to a defect in the organization of the epidermis. Immunohistochemical analyses demonstrated a marked loss of the filamentous actin cytoskeleton and E-cadherin localization in epidermis, as well as an aberration in the localization of tight junction proteins. Moreover, impairment of the "inside-out" epidermal barrier was shown. Srf conditional knockout keratinocytes are unable to establish proper intercellular connections or form an epithelial sheet as shown by histological examination and induced keratinocyte differentiation experiments. Our results demonstrate that Srf is essential for the actin-mediated sealing of epithelial cell-cell contacts and the development of functional stratified skin epithelium in vivo.

Modulation of the Tumor Suppressor Protein α-Catenin by Ischemic Microenvironment

Dysregulation or mislocalization of cell adhesion molecules and their regulators, such as E-cadherin, beta-catenin, and alpha-catenin, usually correlates with loss of polarity, dedifferentiation, invasive tumor growth, and metastasis. A subpopulation of alpha-catenin-negative cells within the DLD-1 colorectal carcinoma cell line causes it to display a heterogeneous morphological makeup, thus providing an excellent model system in which to investigate the role of alpha-catenin in tumorigenesis. We re-established expression of alpha-catenin protein in an alpha-catenin-deficient subpopulation of the DLD-1 cell line and used it to demonstrate that loss of alpha-catenin resulted in increased in vitro tumorigenic characteristics (increased soft agarose colony formation, clonogenic survival after suspension, and survival in suspension). When the cells were used to form tumor xenografts, those lacking alpha-catenin showed faster growth rates because of increased cellular cycling but not increased tumor microvascular recruitment. alpha-Catenin-expressing cells were preferentially located in well perfused areas of xenografts when tumors were formed from mixed alpha-catenin-positive and -negative cells. We therefore evaluated the role of the ischemic tumor microenvironment on alpha-catenin expression and demonstrated that cells lose expression of alpha-catenin after prolonged exposure in vitro to hypoglycemic conditions. Our findings illustrate that the tumor microenvironment is a potent modulator of tumor suppressor expression, which has implications for localized nutrient deficiency and ischemia-induced cancer progression.

Desmoplakin Is Essential for Epidermal Sheet Formation

Vasioukhin et al. (2001) provided a dramatic demonstration that the desmosomal cytolinker protein desmoplakin (Dp) is essential for maintaining the integrity of epithelial cell sheets. Furthermore, they show a requirement for desmosomes in efficient maturation of adherens junctions (AJs). Dp belongs to the plakin family and contains a carboxy-terminal tail incorporating the intermediate filament (IF) binding site and a desmosome association site in the amino-terminal domain. Early embryonic lethality at E6.5 had previously demonstrated the crucial importance of Dp (Gallicano et al., 1998). Lethality was due to the breakdown in IF association with desmosomes and the failure of desmosomes to assemble and stabilize. Rescue of this lethal phenotype was achieved by fusion of knockout (KO) and wild-type embryos. This restored Dp+/+ extraembryonic tissue postponing lethality to E10 with defects in the heart, neuroepithelium and skin. The microvasculature does not contain desmosomes but was also affected because of disruptions to endothelial junctions comprising Dp, plakoglobin and VE-cadherin (Gallicano et al., 2001). The breakthrough with Dp came from the generation of mice with conditional KO of Dp in the epidermis by Vasioukhin and co-workers. The skin of newborns was very fragile and peeled off with mild mechanical stress, leading to death within a few hours. The number and size of desmosomes corresponded to wild type, but they differed ultrastructurally, lacking an inner dense plaque and attachment to the keratin cytoskeleton, which thus became detached from the cell periphery. This led to disruptions of the epidermis through loss of cell–cell adhesion. Studies on cultured KO keratinocytes unveiled additional differences, as these were incapable of sheet formation owing to inefficient clustering of desmosomal and AJ components at cell–cell borders. Rescuing with fulllength Dp restored desmosome–IF connections, but expression of only the head domain did not, supporting the need for the IF-binding site and desmosome–IF connections in epidermal sheet formation. The discovery of a patient who was compound heterozygous for Dp mutations causing severe skin fragility reinforces this Dp function (Jonkman et al., 2005). The mutations led to a truncation of Dp at the carboxyl terminus rather than complete absence of Dp. This gave rise to a loss of desmosome–IF connections and consequent severe fragility of the skin and mucous membranes, with a substantial loss in body fluids. Early postnatal death ensued. Vasioukhin et al. (2001) also revealed the interdependence of adherens junctions (AJs) and desmosomes. AJs precede and are required for desmosome formation but the ablation of Dp caused a reduction in the number of AJs. This has led to a hypothesis that desmosomes act as molecular clamps holding immature AJs in place for a second phase of maturation during epithelial sheet formation. The integrity of the epidermis is essential for survival, and Dp plays a key role in this vital function.

Monitoring of monolayer and multilayer growth for epithelial sheet formation

The profiles of monolayer and multilayer growth of epithelial cells were kinetically analyzed based on the rates of cell proliferation and glucose consumption. The cells were classified into two populations in proliferative and non-proliferative states, and in the monolayer growth most of the cells were regarded as being proliferative. In the multilayer growth, the proliferative cells decreased with elapsed time, causing an increase in the non-proliferative cells. The kinetic analysis indicated that the proliferative cells consumed glucose for cell mass production (anabolism) and metabolic maintenance (catabolism), while the non-proliferative cells utilized glucose exclusively for the latter. Employing the kinetic equations with the determined parameters, the population balance of proliferative and non-proliferative cells could be estimated throughout the monolayer and the multilayer growth periods. The bioreactor system which permits to determine glucose concentration automatically was constructed, and the monitoring in the culture of epithelial cells was performed in a non-invasive manner by considering glucose consumption of the two populations in the epithelial sheets. It was found that the kinetic analysis was valid for monitoring the growth profiles of cells in the bioreactor, giving fair agreement between calculated and experimental values.

Type II platelet‐activating factor‐acetylhydrolase is essential for epithelial morphogenesis in C. elegans

Type II platelet-activating factor-acetylhydrolase (PAF-AH (II)) is an N-myristoylated enzyme that contains a lipase/esterase catalytic motif and selectively hydrolyzes the sn-2 acetyl ester of PAF and other short-chain acyl groups attached to phosphoglycerides. However, the physiological role of this enzyme remains to be elucidated. PAF-AH (II) is conserved in a variety of species ranging from a simple multicellular organism, Caenorhabditis elegans, to mammals. C. elegans possesses two homologous PAF-AH (II) genes named paf-1 and paf-2. In this study, we generated these two loss-of-function mutants to elucidate the in vivo PAF-AH (II) function. Surprisingly, mutants of paf-2, a major isoform of C. elegans PAF-AH (II)s, exhibits gross defects in epithelial sheet formation, resulting in unsuccessful subsequent morphogenesis with complete penetrance. Moreover, paf-2 RNAi worms show a variable abnormal morphology (Vab) including ectopic protrusions and a lumpy shape at the late embryonic and early larval stages due to epithelial organization defects. Consistent with these phenotypes, PAF-AH (II) is predominantly expressed in epithelial cells of C. elegans. This study demonstrates for the first time that PAF-AH (II) is essential for epithelial morphogenesis.

Dysfunction of keratinocyte adhesion.

Dysfunction of keratinocyte adhesion.

New experimental models for the in vitro reconstitution of human bladder mucosa

This study describes two experimental models for the in vitro reconstitution of the human bladder mucosa (neo-bladder): human urothelial stabilized cell lines were cultured on three-dimensional matrices, collagen or platelet-fibrin gels, containing murine fibroblast 3T3-J2 cells. Low-density seeding (2x10(4) cells/ml) of both normal (TCA-48) and neoplastic cell lines (TCA-47) on collagen matrix gave rise to isolated papillar colonies, while high-density seeding (3.75x10(6) cells/ml) led to the formation of wide pluristratified epithelial sheets, resembling the normal transitional epithelium. In contrast, high-density seeding (5x10(5) cells/ml) on platelet-fibrin matrix did not allow the formation of epithelial sheets: only isolated voluminous colonies of normal TCA-48 cells, and sparse and small colonies of neoplastic TCA-47 could be observed. Growth assays and cytotoxicity reduction tests showed that the growth inhibitory effect of platelet-fibrin gel on urothelial cells was probably due to the aspecific activation of the complement contained in the plasmatic fraction, whose precipitation forms fibrin-glue. Collectively, these findings allow us to draw the following conclusions: i) neobladders obtained by culturing urothelial cells on collagen matrix reproduce normal bladder mucosa and could be utilized in pharmacological studies; and ii) platelet-fibrin gels, that specifically inhibit neoplastic urothelial cell growth, could be used as scaffolds in surgical bladder reconstitution.

Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans.

Type II platelet-activating factor-acetylhydrolase [PAF-AH (II)] is an N-myristoylated enzyme that contains a lipase/esterase catalytic motif and selectively hydrolyzes the sn-2 acetyl ester of PAF and other short-chain acyl groups attached to phosphoglycerides. However, the physiological role of this enzyme remains to be elucidated. PAF-AH (II) is conserved in a variety of species ranging from a simple multicellular organism, Caenorhabditis elegans, to mammals. C. elegans possesses two homologous PAF-AH (II) genes, named paf-1 and paf-2. In this study, we generated these two loss-of-function mutants to elucidate the in vivo PAF-AH (II) function. Surprisingly, mutants of paf-2, a major isoform of C. elegans PAF-AH (II)s, exhibits gross defects in epithelial sheet formation, resulting in unsuccessful subsequent morphogenesis with complete penetrance. Moreover, paf-2 RNA interference worms show a variable abnormal morphology, including ectopic protrusions and a lumpy shape at the late embryonic and early larval stages due to epithelial organization defects. Consistent with these phenotypes, PAF-AH (II) is predominantly expressed in epithelial cells of C. elegans. This study demonstrates that PAF-AH (II) is essential for epithelial morphogenesis.

Mammalian formin-1 participates in adherens junctions and polymerization of linear actin cables.

During epithelial sheet formation, linear actin cables assemble at nascent adherens junctions. This process requires alpha-catenin and actin polymerization, although the underlying mechanism is poorly understood. Here, we show that formin-1 interacts with alpha-catenin, localizes to adherens junctions and nucleates unbranched actin filaments. Furthermore, disruption of the alpha-catenin-formin-1 interaction blocks assembly of radial actin cables and perturbs intercellular adhesion. A fusion protein of the beta-catenin-binding domain of alpha-catenin with the actin polymerization domains of formin-1 rescues formation of adherens junctions and associated actin cables in alpha-catenin-null keratinocytes. These findings provide new insight into how alpha-catenin orchestrates actin dynamics during intercellular junction formation.

Desmoplakin is essential in epidermal sheet formation.

We have generated an epidermis-specific desmoplakin (DP) mouse knockout, and show that epidermal integrity requires DP; mechanical stresses to DP-null skin cause intercellular separations. The number of epidermal desmosomes in DP-null skin is similar to wild type (WT), but they lack keratin filaments, which compromise their function. DP-null keratinocytes have few desmosomes in vitro, and are unable to undergo actin reorganization and membrane sealing during epithelial sheet formation. Adherens junctions are also reduced. In vitro, DP transgene expression rescues these defects. DP is therefore required for assembly of functional desmosomes, maintaining cytoskeletal architecture and reinforcing membrane attachments essential for stable intercellular adhesion.

Hydra regeneration from recombined ectodermal and endodermal tissue. I. Epibolic ectodermal spreading is driven by cell intercalation.

Cell-cell interaction and cell rearrangement were examined in the process of epithelial sheet formation during regeneration from hydra cell aggregates. The ectodermal and endodermal epithelial cell layers of Hydra magnipapillata were separated by procaine treatment. Each of the separated layers was then dissociated into single cells and reaggregated to produce ectodermal or endodermal cell aggregates. When the two aggregate types were recombined, a firm adhesion was quickly established between them. This was followed by a vigorous spreading of the ectodermal epithelial cells as a thin layer over the endoderm in a manner similar to the 'epiboly' in some developing embryos. Cell movement in this spreading process was examined using fluorescent dyestaining. It revealed that cells initially located in the inside of the aggregate migrated to intercalate themselves among the cells originally present in the contact surface. This radial cell intercalation took place continuously in the contact surface of both the ectodermal and endodermal aggregates, and produced a rapid growth of the contact surface, eventually leading to complete envelopment of the entire endoderm by the ectoderm. The resulting structure was a small sphere having a two-layered epithelial organization as in normal hydra. This sphere regenerated into a complete hydra a few days later. A tryptic extract of hydra membrane fraction specifically inhibited the ectodermal spreading over the endoderm, but not the initial adhesion or the later regeneration processes. These observations suggest that radial cell intercalation at the contact surface plays a crucial role in producing ectodermal spreading and establishing epithelial sheet organization in the recombined aggregates. The intercalation is presumably activated by a signal exchange through the contact surface. The inhibitory effect of the membrane extract suggests that it contains a factor that is involved in some way in this signaling mechanism.

The influence of scattering on temporo-regional proliferation in the cultured Madin-Darby canine kidney (MDCK) cells.

An epithelial Madin-Darby canine kidney (MDCK) cell line was grown in two kinds of standard media and a medium conditioned by MRC-5 embryonic lung cells, and temporo-regional differences in cell proliferation were examined by immunohistochemical detection of incorporated bromodeoxyuridine (BrdU). MRC-5-conditioned medium is known to contain scatter factor, which induces scattering of MDCK cells. In a proliferation assay, the BrdU labeling index (percentage ratio of BrdU-positive to total nuclei) was highest in day 2 cultures in the standard media, but on day 3 in the conditioned medium. MDCK cells in the standard media formed multiple epithelial sheets by day 2. In the conditioned medium, however, epithelial sheet formation was retarded and observed only after day 3. Once epithelial sheets had formed, cells close to the edge of the sheet were more proliferative than those distant from the edge in both standard and conditioned media. These findings suggest that proliferation of MDCK cells is suppressed during the scattering induced by the conditioned medium, and that their DNA synthesis becomes most active when cell scattering has ceased and epithelial sheets have begun to form.

Cultured epithelium for the treatment of skin defects

A review of the clinical applications of cultured keratinocyte sheets in skin and epithelial defects is reported. The improvement in keratinocyte cell culture techniques and formation of epithelial sheets has allowed the use of these cultured sheets for the grafting of skin defects. Application of cultured epidermal grafts in extensive burn lesions seems to be the most obvious use. However, acceleration of wound healing is also important in other types of wounds to decrease morbidity, hospitalization time and cost of treatment. Both autogeneic and allogeneic cultured keratinocytes have been used to cover several types of wounds. They provide a temporary barrier controlling evaporation and preventing wound infection. Clinical studies with allografts indicate that keratinocytes produce wound healing factor(s) resulting in rapid heating of chronic ulcers and in reduction of hypertrophic scar formation. The secretion of such growth factor(s) survives deep freezing. Therefore cryopreservation seems to be worth...

Ameloblastoma and keratinizing ameloblastoma in dogs.

Ameloblastomas reviewed in this report were locally invasive neoplasms arising from the epithelial structures of the dental lamina, and were characterized histologically by features which are unique to dental lamina epithelium. These include the formation of epithelial sheets in which the cells nearest the stroma form a palisading row aligned perpendicularly to the basement membrane and the cells toward the center separate from each other except at desmosomal attachments. This is similar to the appearance of th stellate reticulum of the early enamel organ. Other features include epithelial cords which branch and interconnect, and the intimate association of epithelial structures and collagenous matrix. In our dogs, other important features were the deposition of inclusions similar in appearance to enamel matrix between the cells of the epithelium and various degrees of keratinization. All ameloblastomas studied were locally invasive tumors which occurred at various sites on the gingiva. The average age of the dogs was 8.7 years and the age range was three to 13 years. Radiographically, all of the tumors studied resulted in periodontal osteolysis. Six dogs were treated with radiation therapy, but details of the radiotherapy of two dogs could not be located. Of the other four dogs, one is alive 48 months after radiotherapy with no evidence of tumor regrowth. Regrowth of the oral tumor was apparent in the other three dogs six, 21, and 34 months after completion of the radiotherapy. Three dogs were treated by radical mandibulectomy; all are alive with no evidence of tumor recurrence at two, 20, and 28 months postoperatively. Two dogs had local dissection (curettage) of tumors and the tumors recurred at 12 and 15 months after surgery. One dog was euthanatized after diagnosis of the oral tumor because of a progressive neuropathy.