Teflon Rings Research Articles

Bone Formation on Murine Cranial Bone by Injectable Cross-Linked Hyaluronic Acid Containing Nano-Hydroxyapatite and Bone Morphogenetic Protein.

New injection-type bone-forming materials are desired in dental implantology. In this study, we added nano-hydroxyapatite (nHAp) and bone morphogenetic protein (BMP) to cross-linkable thiol-modified hyaluronic acid (tHyA) and evaluated its usefulness as an osteoinductive injectable material using an animal model. The sol (ux-tHyA) was changed to a gel (x-tHyA) by mixing with a cross-linker. We prepared two sol-gel (SG) material series, that is, x-tHyA + BMP with and without nHAp (SG I) and x-tHyA + nHAp with and without BMP (SG II). SG I materials in the sol stage were injected into the cranial subcutaneous connective tissues of mice, followed by in vivo gelation, while SG II materials gelled in Teflon rings were surgically placed directly on the cranial bones of rats. The animals were sacrificed 8 weeks after implantation, followed by X-ray analysis and histological examination. The results revealed that bone formation occurred at a high rate (>70%), mainly as ectopic bone in the SG I tests in mouse cranial connective tissues, and largely as bone augmentation in rat cranial bones in the SG II experiments when x-tHyA contained both nHAp and BMP. The prepared x-tHyA + nHAp + BMP SG material can be used as an injection-type osteoinductive bone-forming material. Sub-periosteum injection was expected.

Chemo-mechanical properties of the new bioceramic cement PBS CIMMO® compared to MTA REPAIR HP Angelus® cement

Objective: To evaluate the bond strength (BS), pH and solubility (Sol) of a new PBS HP® bioceramic cement compared to MTA REPAIR HP® cement. Material and methods: Twenty human premolars were selected for BS analysis. Roots were cut into thirds (cervical, medium and apical) and filled with one of the cements (n=10): PC (PBS HP) or MC (MTA REPAIR HP). Half of these samples were subjected to 2,000 thermal cycles. After 48 hours, the BS test was performed in a universal testing machine (0.5mm/min). Cylindrical specimens (1.0mm in diameter and 10mm in height) were made with PC or MC and immersed in distilled water for pH analysis. After 0.3 and 24 hours, the pH was measured. Solubility was evaluated through the mass loss of the samples after 24 hours. Teflon rings of 20mm in diameter and 1.5mm in height were filled with PC or MC and kept at 37ºC in 100% humidity. Descriptive and exploratory analyzes of BS, pH and Sol data were performed (p<0.05). Results: There was no significant difference in BS between the two types of cement. Thermo cycling did not affect the BS (p>0.05). MC cement showed significantly higher pH values than the PC at the initial and 3 hours (p<0.05). There was no difference between them (p>0.05) after 24 hours. There was no significant difference regarding solubility (p>0.05). Conclusion: Bioceramic cements have similar properties, as they did not differ in relation to dentin bond strength, pH after 24 hours and solubility.

Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis.

Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.

Keratin Biomembranes as a Model for Studying Onychomycosis.

Difficulties in obtaining human nails that are large enough for examining the penetration of drug formulations led us to produce keratin films regenerated from human hair. We assume that these films can simulate human nail plates in drug penetration and permeation tests and can serve as a biological model for studying onychomycosis. The films were formed from keratin extracted from human hair using dithiothreitol, urea and thiourea. The obtained keratin extract was dispensed into Teflon rings and dried at 40 °C and then cured at 110 °C. The structure, surface morphology, chemical characterization and thermal stability of the films were characterized and were compared to those of human nail, hair and bovine hoof samples using SDS-electrophoresis, scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The structure of the obtained films was found to be closer to human nails than to hair or bovine hooves. The keratin films were infected with Trichophyton rubrum and were proven to be appropriate for serving as a model for studying onychomycosis.

In Vitro Evaluation of ProRoot MTA, Biodentine, and MM-MTA on Human Alveolar Bone Marrow Stem Cells in Terms of Biocompatibility and Mineralization

IntroductionStem cell technology has been a great hope for the regeneration of cells of pulp-dentin complex and dental structures together with surrounding bone and periodontium. The main challenge in the regeneration process is a successful combination of stem cells and efficient inductors such as inductive biomaterials. In this regard, today, manufacturers propose novel tooth filling materials. The current study was aimed to compare the effect of ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK), Biodentine (Septodont, Saint Maur des Fossés, France), and MM-MTA (Micro-Mega, Besançon Cedex, France) on the cell viability, hard tissue deposition capacity, and osteogenic differentiation of human bone marrow stem cells (hBMSCs) derived from mandibular bone. MethodsDental materials were packed into Teflon rings (Grover Corp, Milwaukee, WI) and placed on Transwell inserts (Corning, Corning, NY) to determine the toxicity of tooth filling materials by the 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium assay on days 1, 3, 7, and 14; 20% dimethyl sulfoxide (DMSO) was used as a positive control for the toxicity assay. hBMSCs were characterized by their surface markers with mesenchymal stem cell antibodies. Teflon rings were cocultured with hBMSCs followed by the induction of osteogenic differentiation. The osteogenic differentiation of hBMSCs and hard tissue formation of the materials were evaluated by analyzing the messenger RNA expression levels of osteonectin, Runt-related transcription factor 2, and collagen type 1A by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase activity, and visualization of calcium deposits by alizarin red staining. ResultsMTA, Biodentine, and MM-MTA did not exhibit a cytotoxic effect on hBMSCs after 14 days in culture. Even though all the materials significantly stimulate (P < .05) osteogenic differentiation of hBMSCs compared with the negative control, ProRoot MTA showed greater osteoinductivity than Biodentine or MM-MTA according to the messenger RNA expression, alkaline phosphatase, immunocytochemistry, and alizarin red staining data. ConclusionsAll of the dental materials used in this study show the osteogenic differentiation potential of hBMSCs. Therefore, newly introduced MM-MTA can also be used as a material of choice in routine dental treatment.

Human tooth germ stem cell response to calcium-silicate based endodontic cements

The aim of this study was to compare the cytotoxic effects of endodontic cements on human tooth germ stem cells (hTGSCs). MTA Fillapex, a mineral trioxide aggregate (MTA)-based, salicylate resin containing root canal sealer, was compared with iRoot SP, a bioceramic sealer, and AH Plus Jet, an epoxy resin-based root canal sealer. To evaluate cytotoxicity, all materials were packed into Teflon rings (4 mmµ3 mm) and co-cultured with hTGSCs with the aid of 24-well Transwell permeable supports, which had a pore size of 0.4 µm. Coverslips were coated with MTA Fillapex, iRoot SP and AH Plus Jet and each coverslip was placed onto the bottom of one well of a six-well plate for scanning electron microscopy (SEM) analysis. Before the cytotoxicity and SEM analysis, all samples were stored at 37ºC and at 95% humidity and 5% CO2 for 24 hours to set. The cellular viability was analyzed using MTS test (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium). The cytotoxic effects and SEM visualization of the tested materials were analyzed at 24-hour, 72-hour, one-week and two-week periods. On the 1st day, only MTA Fillapex caused cytotoxicity compared to negative control (NC) group (p<0.008). No significant difference was observed between the other tested materials at this period (p>0.05). After 14 days of incubation with the test materials, MTA Fillapex exhibited significantly higher cytotoxicity compared with iRoot SP, AH Plus Jet and the NC group (P<0.008). In the SEM analysis, the highest levels of cell attachment were observed for iRoot SP and the control group. After 24 hours, MTA Fillapex reduced the number of cells attached to the surface. Within the limitations of this study, sealers exerted different cytotoxic effects on hTGSCs. Although all materials have exerted cellular toxicity, iRoot SP and AH Plus Jet may promote better attachment to hTGSCs.

In vitro comparison of induction capacity and biomineralization ability of mineral trioxide aggregate and a bioceramic root canal sealer

To compare the effect of mineral trioxide aggregate (MTA) and iRoot SP, a bioceramic root canal sealer, on the cell viability, hard tissue deposition capacity and odontogenic differentiation of human tooth germ stem cells (hTGSCs). The dental materials MTA, iRoot SP and Dycal were packed into Teflon rings and placed on transwell inserts for toxicity evaluations by the MTS assay on days 3 and 7. Dycal was used as a positive control for the cell viability assay. Teflon rings were cocultured with hTGSCs, followed by the induction of odontogenic differentiation. The odontogenic differentiation of hTGSCs and biomineralization ability of the materials were evaluated by analysing the mRNA expression levels of dentine sialophosphoprotein (DSPP) and collagen type 1A (COL1A) by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase (ALP) activity and visualization of calcium deposits by von Kossa staining. MTA and iRoot SP exhibited no cytotoxicity, but Dycal caused cytotoxicity (P < 0.05) of almost all of the cells after 7 days. MTA significantly stimulated (P < 0.05) the odontogenic differentiation of hTGSCs compared with iRoot SP. MTA and iRoot SP increased (P < 0.05) the mRNA levels of COL1A and DSPP mRNA compared with noninduced hTGSCs, which served as a negative control (NC). iRoot SP, however, significantly decreased (P < 0.05) COL1A and DSPP mRNA expression levels compared with the PC. MTA and iRoot SP induced hTGSC differentiation into odontoblast-like cells, but MTA might provide more inductive potential and hard tissue deposition compared with iRoot SP.

Human tooth germ stem cell response to calcium-silicate based endodontic cements

ObjectiveThe aim of this study was to compare the cytotoxic effects of endodontic cements on human tooth germ stem cells (hTGSCs). MTA Fillapex, a mineral trioxide aggregate (MTA)-based, salicylate resin containing root canal sealer, was compared with iRoot SP, a bioceramic sealer, and AH Plus Jet, an epoxy resin-based root canal sealer. Material and MethodsTo evaluate cytotoxicity, all materials were packed into Teflon rings (4 mmµ3 mm) and co-cultured with hTGSCs with the aid of 24-well Transwell permeable supports, which had a pore size of 0.4 µm. Coverslips were coated with MTA Fillapex, iRoot SP and AH Plus Jet and each coverslip was placed onto the bottom of one well of a six-well plate for scanning electron microscopy (SEM) analysis. Before the cytotoxicity and SEM analysis, all samples were stored at 37ºC and at 95% humidity and 5% CO2 for 24 hours to set. The cellular viability was analyzed using MTS test (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium). The cytotoxic effects and SEM visualization of the tested materials were analyzed at 24-hour, 72-hour, one-week and two-week periods. ResultsOn the 1st day, only MTA Fillapex caused cytotoxicity compared to negative control (NC) group (p<0.008). No significant difference was observed between the other tested materials at this period (p>0.05). After 14 days of incubation with the test materials, MTA Fillapex exhibited significantly higher cytotoxicity compared with iRoot SP, AH Plus Jet and the NC group (P<0.008). In the SEM analysis, the highest levels of cell attachment were observed for iRoot SP and the control group. After 24 hours, MTA Fillapex reduced the number of cells attached to the surface.ConclusionsWithin the limitations of this study, sealers exerted different cytotoxic effects on hTGSCs. Although all materials have exerted cellular toxicity, iRoot SP and AH Plus Jet may promote better attachment to hTGSCs.

Comparison of antibacterial activities of root-end filling materials by an agar diffusion assay and Alamar blue assay

Background/purposeThe purpose of this in vitro study was to evaluate antibacterial activities of the leachable components of root-end filling materials (calcium silicate cement). Materials and methodsRoot-end filling materials were mixed according to the manufacturers' directions and then placed into 6-mm-diameter Teflon rings with mixed materials; they were allowed to set for 24 hours and then tested. Antibacterial activities of the root-end filling materials were evaluated against Streptococcus sanguinis, Streptococcus mutans and Escherichia coli. The antibacterial activity of the root-end filling materials tested was determined by measuring the diameter of each zone of inhibition (on an agar diffusion test). An Alamar blue assay was used to detect bacterial growth. Statistical analysis was conducted using one-way analysis of variance. ResultsZones of inhibition were observed in the zinc oxide–eugenol cement (IRM) group treating S. sanguinis and E. coli agar plates in the agar diffusion test. Cultures of S. sanguinis and E. coli showed the lowest absorbances with the IRM group at different times of observation (1 hour, 3 hours, 6 hours and 12 hours) (P<0.05). The growth of S. mutans showed no significant difference between controls and any tested materials (P>0.05). ConclusionWe concluded that both the agar diffusion test and Alamar blue assay gave comparable findings of assessing the antimicrobial activity present in root-end filling materials. No antimicrobial activity was detected for mineral trioxide aggregate, calcium silicate cement, or amalgam after coming into contact with S. mutans, S. sanguinis and E. coli. IRM showed high antimicrobial activity against both S. sanguinis and E. coli.

Investigation of Tumor–Peritoneal Interactions in the Pathogenesis of Peritoneal Metastases using a Novel Ex Vivo Peritoneal Model 1

Peritoneal metastasis occurs in up to 30% of patients with gastric cancer. The aim of this experimental study is to develop and validate a novel ex vivo model of the human peritoneum to better identify factors involved in the development of peritoneal metastasis in order to improve its management and prognosis. Peritoneal discs harvested from hernia sacs obtained at inguinal hernia surgery were suspended in media using Teflon rings. Viability of the tissue was investigated using MTS assay, light and scanning electron microscopy (LM and SEM) over 72 h. To assess validity of the model, phenotypic changes in tumor cells were investigated. Changes in matrix metalloproteinases (MMP)-2 and -9 activities in HGC and AGS gastric adenocarcinoma cells after co-culture were investigated using zymography. Modulation of tumor cell adhesion to peritoneum after exposure to heparin was assessed using a fluorometric adhesion assay. Analysis was performed using Kruskal-Wallis for multiple comparisons and Mann-Witney U for comparisons between each group. MTS assay showed reduced viability after 72 h (P = 0.047, compared with 24 h). Mesothelial cell loss at 48 h was demonstrated by LM and SEM, confirming peritoneal viability for at least 24 h after tissue harvesting. Zymography confirmed increased MMP2 and -9 activities in tumor cells and peritoneal tissue during co-culture compared with controls, and heparin significantly reduced tumor cell adherence (P = 0.04), as observed in published in vivo models. A validated complete model of peritoneum was developed that has shown potential to determine realistic mechanisms of peritoneal metastasis.

Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses

AbstractCompartmented neuronal cultures allow experimenters to establish separate fluid environments for neuronal axons and the soma from which they emanate. Physical isolation of cell bodies and axons is achieved by culturing neurons in tri‐chambered Teflon rings. Dissociated ganglia are plated in one end compartment of the trichamber, and axonal growth is guided underneath watertight silicone grease barriers into a separate compartment. Since the axons and cell bodies are located in different compartments, they can be infected and assayed separately. We describe the assembly and use of compartmented neuronal cultures for in vitro study of directional infection of neurons by alpha herpesviruses. Selective application of viral inoculum to only one compartment ensures that the remainder of the neuron is not contaminated by input inoculum. This allows for quantification of viral spread, and unambiguous interpretation of immunofluorescence and electron microscopy images.Curr. Protoc. Cell Biol. 43:26.4.1‐26.4.23. © 2009 by John Wiley &amp; Sons, Inc.

The Influence of Calcium Chloride on the Setting Time, Solubility, Disintegration, and pH of Mineral Trioxide Aggregate and White Portland Cement with a Radiopacifier

This study evaluated the influence of addition of 10% calcium chloride (CaCl 2) on the setting time, solubility, disintegration, and pH of white MTA (WMTA) and white Portland cement (WPC). A test of the setting time was performed following the #57 ADA specifications and a test of the final setting time according to the ASTM. For the solubility tests disintegration and pH, Teflon rings were filled with the cements and weighed after setting. After 24 h in a desiccator, they were once again weighed. Thereafter, they were immersed in MiliQ water for 24 and 72 h and 7, 14, and 28 days, with maintenance in the desiccator and weighing between periods. The pH of water in which the rings were immersed was measured immediately after contact with them and in the other periods. The addition of CaCl 2 provided a significant reduction (50%) in the initial setting time of cements. The final setting time of WMTA was reduced in 35.5% and the final setting time of WPC in 68.5%. The WMTA with CaCl 2 absorbed water and gained weight with time, except for in the 24-h period. The addition of CaCl 2 to the WPC reduced its solubility. The addition of CaCl 2 increased the pH of WMTA in the immediate period and at 24 and 72 h and for WPC in the immediate period and at 24 h. The addition of CaCl 2 to WMTA and WPC reduced the setting times and solubility of both and increased the pH of cements in the initial periods.

A novel model of wound healing in the SCID mouse using a cultured human skin substitute

Studies of skin graft behaviour in rodent excisional wound models are limited by the dominance of wound contracture and graft sloughing as primary healing responses. To slow skin contraction, polytetrafluoroethylene (Teflon) rings were inserted into dorso-lateral full-thickness wounds in SCID mice. Cultured skin substitutes (OrCel), composed of cultured human keratinocytes and fibroblasts in a bovine collagen sponge, were implanted within the rings. Examination and histology of grafts 14 days later showed graft take in four of six recipients, with 90% epithelialization and wound contraction of 31-47%. Immunohistochemical studies, using human-specific antisera to distinguish graft from host tissues, showed that regenerated tissue was predominantly human. Staining with anticytokeratin, revealed a multilayered, stratified neoepidermis. HBG were identified in keratinocytes in all epidermal layers. Langerhans cells were absent. Antihuman vimentin, used as a fibroblast marker, confirmed that cells of the neodermis were primarily of human origin. Neoepidermal keratinocytes, primarily in the basal and suprabasal layers, were also stained. Results suggest that the poly(tetrafluoroethylene) ring inhibited graft sloughing and provided a more favourable environment for the skin substitute to regenerate a substantially normal human skin.

Design of a new cartridge for selective solid phase extraction using molecularly imprinted polymers: Selective extraction of theophylline from human serum samples

This paper describes design of a new cartridge for selective solid phase extraction (SPE) using molecularly imprinted polymers (MIPs). The apparatus which is termed solvent extraction-MISPE (SE-MISPE) cartridge, consisted of a modified conventional micro test tube and has been developed to perform simultaneous forward-extraction of analyte from aqueous sample solution to an organic phase and back-extraction to MIP solid phase. In order to evaluate the performance of the proposed method, extraction of theophylline (THP) from human serum sample was investigated. An appropriate amount of THP-imprinted polymer was placed in the bottom of the micro tube and an organic solvent pipetted onto it and left to swell the polymer completely. A polyethylene frit to secure MIP particles was positioned by two Teflon rings such that it was fixed below the level of the organic layer. Then, aqueous sample solution containing THP was layered over the organic phase and the lid was closed. After completion of extraction, the organic and aqueous phases were removed and the adsorbed analyte was desorbed using a polar organic solvent. In order to reach the highest recovery, the experimental parameters such as the type of organic solvent, pH and ionic strength of aqueous phase, organic to aqueous volume ratio, time of extraction, type and amount of desorbent solvent were optimized. Under the experimental conditions, a plot of HPLC peak areas vs. initial concentrations of THP in the concentration interval of 0.5–30 μg ml −1 showed a good linearity ( r = 0.9974). The limit of detection (LOD) and limit of quantification (LOQ) based on three and ten times of the noise of HPLC profile were 0.09 and 0.3 μg ml −1, respectively. The relative standard deviation (RSD) of the proposed method for the extraction and determination of 5 μg THP from 200 μl standard sample solution for 3 replicate measurements was 3.5%. The results showed that by means of the proposed cartridge, THP could significantly separate from the other structurally related compounds such as theobromine (THB) and caffeine (CAF). The added THP could be quantitatively recovered (79–83%) from the serum samples by the proposed procedure, being thus a guarantee of the accuracy of the SE-MISPE procedure. In addition, the loss of capability of the SE-MISPE cartridge was not considerably observed after 10 times loading and elution cycles.

Timing of Fetal Meconium Absorption by Amnionic Macrophages

This study sought to determine the time interval from fetal defecation to membrane staining and subsequent meconium uptake by amnionic macrophages. Pieces of external membranes were fastened to Teflon rings to create a well. The amnionic surface was exposed to meconium and amniotic fluid for 1 to 48 hours and analyzed microscopically for the presence of free meconium and meconium-laden amnionic macrophages. In each of the experiments, prior to the 12-hour time point, few meconium-laden macrophages were present in the membrane layers. A significant rise in the number of meconium-laden macrophages and a concomitant increase in staining intensity was noted in the membranes at 24 and 48 hours. Contrary to previous reports, our in vitro model of meconium incorporation into placental membranes demonstrated that significant numbers of meconium-laden macrophages were only observed after 24 and 48 hours.

Electron capture dissociation in a linear radiofrequency‐free magnetic cell

Electron capture dissociation in a linear radiofrequency‐free magnetic cell

Reconstruction of a rabbit corneal epithelium on a lyophilized amniotic membrane using tilting air-liquid interface culture followed by tilting submerged culture

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane (CAM) and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane (AM). The lyophilized amniotic membrane (LAM) has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings (Ahn's supporter) were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, followed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

An in vitro system to study trans-neuronal spread of pseudorabies virus infection

The neuronal spread of infection of alpha herpesviruses is controlled by unknown mechanisms. In the natural host, primary infection always leads to invasion of the peripheral nervous system, but rarely results in extensive invasion of the central nervous system. After reactivation of latent infection in the peripheral nervous system, virions are produced and shed from epithelial surfaces, but rarely invade the central nervous system. We have been studying two aspects of the general problem. First, using GFP and mRFP fusion proteins, we have used video confocal microscopy to assess mechanisms that influence spread of pseudorabies (PRV) virion components within axons. Second, and the subject of this report, is the development of a new in vitro cell culture system that enables the study of trans-neuronal spread of infection from neurons to non-neuronal cells similar to what happens after reactivation and spread to epithelial surfaces. We have developed a tissue culture system involving tri-chamber Teflon rings that enables facile analysis of trans-neuronal spread. The system duplicates all the known in vivo correlates of trans-neuronal spread and provides the opportunity to do both quantitative and qualitative assessment of spread of PRV infection from infected neurons to non-neuronal cells.

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.