Organ culture preservation for corneal tissue. Technical and quality aspects.

INTRODUCTION: The technical and quality aspects of organ culture as a storage method for human donor corneas are described. MATERIALS AND METHODS: Data electronically stored since 1989 of > 41,000 corneas, processed in the Cornea Bank Amsterdam, are analysed. The technical information of eye banks collected in the Directory of the European Eye Bank Association (EEBA) is used as comparison. European Union (EU) directive for tissue banking and EEBA technical guidelines are references for the quality aspects. RESULTS: Organ culture allows the storage of donor corneas up to 4-5weeks. The storage phase is followed by a generally much shorter phase of 1-7 days, to reverse the corneal swelling occurring in the first phase and to transport the tissue to the clinic. Selection of the corneas based on inspection of the endothelium after storage as well as microbiological testing of the storage solution after a quarantine period are mandatory for this technique. General agreement exists about the outline of the method, but technical variations are applied to suit local circumstances and preferences of corneal surgeons. Agreement exists about a minimum endothelial cell count as selection criterion in case the donor endothelium is meant to be grafted. The use and cutoff points of other selection parameters for the cornea, e.g. the endothelial cell mosaic, are varying. According to EU regulations, a quality management system should be installed. This way each bank is able to issue a standardized product, while the production process is monitored with quality registrations. With the clinical outcome of the graft, the quality of the selection and storage procedures is verified. With the notification of adverse reactions such as primary graft failure and endophthalmitis, minimum risks will be assessed. CONCLUSION: The organ-cultured cornea is a well-documented product concerning microbiological safety and quality of the tissue. However, variations in performance and materials and no definite cut-off points for selection do not make an organ-cultured cornea a generally standardized product. The corneal surgeons have to ascertain themselves of the safety and quality of the followed procedure. It is up to an organization such as the EEBA to formulate tissue-specific additions to the EU regulations such as training opportunities, technical guidelines and criteria based on science.

Students' knowledge of oral hygiene vs its use in practice.

PURPOSE: The purpose of the work is to estimate the knowledge connected with the rules of the oral hygiene and its correlation with everyday habits among the students of Dental studies and Medical Studies at Medical School in Lublin and Polytechnics of Lublin. MATERIAL AND METHODS: A survey was conducted among 483 students: 58 2nd-year and 88 5th-year students of dentistry, 97 2nd-year and 51 5th-year students of medicine and 108 2nd-year and 81 5th-year students from The Polytechnics of Lublin. RESULTS: The study revealed that 50% students of dentistry, 32.43% students of medicine and 26.6% students of polytechnics brush their teeth after every meal; 94.23% students of dentistry, 89.91% students of medicine and 78.8% students of polytechnics know-how often teeth should be brushed. Students had better knowledge of how frequent they should change a toothbrush: 71.8% students of dentistry, 61.49% students of medicine and 54.4% students of polytechnics change their toothbrushes every 3 months, however, 84.61%, 62.16% and 49.42% students respectively have knowledge concerning the frequency of changing a toothbrush. The study also revealed that 13.46% students of dentistry, 10.14% students of medicine and 6.49% students of polytechnics visit dental clinic every 3 months, however, 4.49%, 13.51% and 14.05% students respectively go to see the dentist less than once a year. The reason for making a dental appointment was pain in 7.05% students of dentistry, 16.22% students of medicine and 22.22% students of polytechnics and a check-up in 64.74%, 62.84% and 51.85% students respectively. CONCLUSIONS: Students' knowledge of oral hygiene does not always correlate with practice.

Hygienic habits and the dental condition in 12-year-old children.

PURPOSE: The aim of the study was the description of the dental condition of hygienic routines in 12-year-old children in urban and rural areas of Lublin voivodship. MATERIAL AND METHODS: The study comprised 274 children at the age of 12 (152 girls and 122 boys). 95 girls and 92 boys came from the urban area; 57 girls and 30 boys came from the rural area. RESULTS: On the basis of clinical examination it was concluded that 11.96% of boys and 18.95% of girls from the urban area and 6.67% boys and 8.77% of girls from the rural area brush their teeth after every meal; 60.87% of boys and 68.42% of girls from the urban area and 43.33% of boys and 50.88% of girls from the rural area brush their teeth twice; 22.83% of boys and 11.58% of girls from the urban area and 26.67% of boys and 28.07% of girls from the rural area brush their teeth once daily. DMF count was for boys from the urban area--4.12/girls--3.92 and for boys from the rural area 4.50/girls--4.29. The treatment indicator was for boys from the urban area--0.56/ girls - 0.47 and for boys from the rural area 0.35/girls--0.67. CONCLUSIONS: On the basis of the research conducted in the study, it was concluded that tooth brushing is more frequent with urban area children than in children from rural area. This leads to a conclusion that the action for improvement of the health awareness is a dire need among 12-year-olds both from urban and from rural areas.

Histocompatibility and corneal transplantation.

BACKGROUND: HLA typing and matching have been poorly implemented in corneal transplantation, mainly because of inconclusive or contradictory analytical results. Consequently, we studied the immune response of corneal transplant recipients to HLA histoincompatibilities in a large homogeneous study. METHODS: All corneal transplantations were performed by a single surgeon in a single center between 1976 and 1996. Population genetic and other statistical analyses were performed. Simulation studies assessed the effects of HLA-DR mistypings on analytical results. RESULTS: Mono- and multivariate analyses identified retransplantation, degree of vascularization, HLA-AB and -DR match grades, endothelial cell count, graft size, recipient gender, storage method and panel-reactive antibodies as significantly influencing the survival of corneal transplants. Simulation studies showed that the beneficial effect of HLA-DR matching is abrogated by HLA-DR mistypings. CONCLUSIONS: Corneal transplant recipients have a normal immune response to HLA incompatibilities. Demonstration of that fact requires accurate HLA typings.

The specific architecture of the anterior stroma accounts for maintenance of corneal curvature.

AIM: To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability. METHODS: Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer. RESULTS: After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states. CONCLUSIONS: The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas.

PURPOSE: Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. METHODS: Human corneas were organ-cultured in MEM for 7 (n = 17, 3 pairs), 14 (n = 18, 9 pairs) and 21 days (n = 18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. RESULTS: Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. CONCLUSION: With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21 days after organ-culture.

The application of synthetic hydroxyapatite in children and adolescents in various clinical cases.

The possibility of application of HA-BIOCER synthetic hydroxyapatite in the treatment of enamel hypoplasia in children and adolescents manifested by mineralisation disorders, enamel underdevelopment, enamel deficiency and oversensitivity to mechanical, thermal and chemical stimuli was evaluated. The possibility of applying the same preparation in case of enamel fractures and teeth injuries type I according to Ellis was also examined. It was found that the application of hydroxyapatite stimulates processes of remineralization in decalcified places. It also causes removal of tooth oversensitivity to thermal and mechanical stimuli by closing open dentinal tubules or decrease in their size. HA-BIOCER preparation brings about smoothing and lighting of hypoplastic foci, improves aesthetic appearance and is not toxic to patients.

Preliminary clinical results of posterior lamellar keratoplasty through a sclerocorneal pocket incision.

PURPOSE: To report the preliminary results of a surgical technique for transplantation of posterior corneal tissue through a sclerocorneal pocket incision for corneal endothelial disorders. DESIGN: Retrospective, noncomparative, interventional cases series. PARTICIPANTS AND INTERVENTION: In seven sighted human eyes, a deep stromal pocket was created across the cornea through a 9.0-mm superior scleral incision. A 7.0- or 7.5-mm diameter, posterior lamellar disc was excised and replaced by a 'same size' donor posterior disc, without suture fixation. The scleral incision was sutured. MAIN OUTCOME MEASURES: Intra- and postoperative complications, best spectacle-corrected visual acuity, keratometry, topography, biomicroscopy, pachymetry, and endothelial cell density were evaluated. RESULTS: Six to 12 months after surgery, all transplants were clear and in position. Best spectacle-corrected visual acuity was limited by preexisting maculopathies in two eyes and varied from 20/80 to 20/20. Postoperative astigmatism averaged 1. 54 diopters (D; standard deviation [SD] +/- 0.81 D), pachymetry averaged 0.49 mm (SD +/- 0.09 mm), and postoperative endothelial cell density averaged 2520 cells/mm(2) (SD +/- 340 cells/mm(2)). In one eye, a microperforation occurred during stromal pocket dissection so that the procedure was converted into a penetrating keratoplasty. CONCLUSIONS: Posterior lamellar keratoplasty through a sclerocorneal pocket incision is a feasible surgical approach to manage corneal endothelial disorders.

Beneficial effect of HLA-DR matching on the survival of corneal allografts.

BACKGROUND: Although HLA typing and matching have been used for 3 decades, that practice has been poorly implemented in corneal transplantation, mainly because of inconclusive or contradictory analytical results. Consequently, we studied the immune response of corneal transplant recipients to HLA histoincompatibilities in a large homogeneous study. METHODS: All corneal transplantations performed by a single surgeon between 1976 and 1996 were studied. HLA-AB matching was used for recipient selection. All HLA typings were performed by a single experienced laboratory. Population genetic techniques were used to assess the validity of the HLA typings. Mono- and multivariate analyses were performed to identify the factors which significantly influence the survival of corneal allografts. Simulation studies were carried out to demonstrate the effects of mis-typed donor and recipient HLA-DR typings on analytical results. RESULTS: Retransplantation, degree of vascularization, HLA-AB and DR matching, endothelial cell count, graft size, recipient gender, and storage method were identified as significant factors by our monovariate analyses. A Cox proportional hazards survival analysis model identified degree of vascularization and HLA-AB and DR matching as significant prognostic factors when all immunological rejection episodes were used, P=0.000001. When only irreversible immunological rejection episodes were used, panel reactive antibodies, retransplantation, and number of rejection events were also identified, P=0.000001. Simulation studies showed that the effects of HLA-DR matching are abrogated by poor HLA-DR typings. CONCLUSIONS: Corneal allograft recipients have a normal alloimmune response to histoincompatibilities. Demonstration of that fact requires accurate HLA typings.

Biodegradable three-dimensional networks of poly(dimethylamino ethyl methacrylate). Synthesis, characterization and in vitro studies of structural degradation and cytotoxicity.

In ophthalmology, there is a need for novel degradable biomaterials for e.g. controlled drug release in the vitreous body. These degradable materials should feature both excellent biocompatibility, and well-defined kinetics of degradation. In most cases, poly(D,L-lactic acid), or poly(lactic-co-glycolic acid) are used. These materials, however, suffer from some serious drawbacks, since the degradation kinetics are difficult to control, especially since the so-called 'burst-degradation' occurs. Here, we describe a set of novel polymeric networks which largely consist of poly(dimethylamino ethyl methacrylate) (poly(DMAEMA)); these materials are crosslinked via a dimethacrylate molecule that contains two carbonate groups. This system is susceptible to hydrolytic scission. The degradation products do not exert a catalytic effect on the ongoing degradation reaction (i.e. there is no burst effect). We describe the synthesis of three of these materials, which differ merely with regard to the crosslinker content. These materials were characterized through DMTA, 1H NMR and FT-IR spectroscopy, and scanning electron microscopy. The reaction DMAEMA + 2-hydroxyethyl methacrylate (HEMA) was studied in detail, using 1H NMR spectroscopy, and these experiments revealed that the reaction of DMAEMA and HEMA produces a random (Bernouillian-type) copolymer. From this, we contend that the new materials have more or less uniform distribution of the crosslinks throughout their volume. Structural degradation of the three materials was studied in vitro, at pH 7.4, 9.1 and 12.0. It is found that the materials exhibit smooth hydrolysis, which can be controlled via the crosslink density and the pH, as was expected a priori. It should be noted that degradation of these materials produces non-hydrolysable, but water-soluble, oligo(DMAEMA) and poly(DMAEMA) molecules. We subsequently performed in vitro studies on the biocompatibility of these materials. The MTT cytotoxicity assay revealed that the materials were cytotoxic to chondrosarcoma cells. This is most probably due to local increase of the pH due to the basic character of the pending dimethylamino groups. Cytotoxicity remained virtually unchanged after extended washing with water. This indicates that the cytotoxicity is an intrinsic property of the material and was not caused by remnants of free monomer. Cytotoxicity was also seen in cell cultures (human fibroblasts isolated from donor corneas) which were grown in contact with the materials. It is concluded that the new materials have attractive degradation characteristics, but their cytotoxicity makes them unsuitable for applications in ophthalmology.

Microbial decontamination of human donor eyes with povidone-iodine: penetration, toxicity, and effectiveness.

BACKGROUND/AIMS: Povidone-iodine (PVP -I) is applied for microbial decontamination of human eyes donated for transplantation. Concentrations and immersion times vary greatly. The effectiveness and toxicity of PVP-I were assessed for different decontamination protocols. METHODS: Human donor eyes and corneas were immersed in different concentrations (5-100 mg/ml) of PVP-I for different times (2-30 minutes). The penetration of iodine into the corneal tissue was assessed by x ray microanalysis. Microbial contamination was determined by taking cultures of the limbal areas and storage solutions and by incubation of the corneoscleral buttons in antibiotic-free culture medium. Cytotoxicity of PVP-I for corneal fibroblasts in culture was assessed using the MTT assay. RESULTS: Depending on concentration and immersion time iodine was found to penetrate into the epithelium, Bowman's layer, and stroma in amounts equivalent to 2-40 mg/ml PVP-I. The MTT assay demonstrated that 2.5 mg/ml PVP-I caused total damage to fibroblasts in vitro. Rinsing eyes with tap water and subsequent immersion in PVP-I reduced the rate of contamination from 82 out of 106 to 69 out of 106 and 37 out of 106, respectively. Antibiotics in the storage medium further reduced contamination from about 40% to 3%. Microbial contamination was not reduced by increasing the concentration and immersion times beyond 5 mg/ml PVP-I for 2 minutes. CONCLUSION: Immersion of human donor eyes in 5 mg/ml PVP-I solution for 2 minutes significantly reduces microbial contamination of donor corneas without relevant penetration of iodine into the corneal layers. Higher PVP-I concentrations and longer immersion times do not further reduce contamination, whereas the amount of iodine penetrating the corneal layers is elevated above the level cytotoxic for corneal fibroblasts. In view of this, concentrations above 5 mg/ml of PVP-I and immersion periods over 2 minutes are not recommended for reduction of the contamination rate of donor eyes.

A surgical technique for posterior lamellar keratoplasty.

PURPOSE: To design a surgical technique for transplantation of posterior corneal tissue, while leaving the recipient anterior cornea intact. METHODS: In human cadaver eyes, and in a cat and monkey model, recipient eyes had an 8.0-mm limbal incision made with a diamond blade set to 50% of central pachymetry. A stromal pocket was created across the cornea, and a 6.0-mm diameter posterior lamellar disc was excised. A donor posterior disc was implanted into the recipient opening, and the limbal incision was sutured. The procedure was evaluated with keratometry, biomicroscopy, endothelial (supra)vital staining, and light microscopy. RESULTS: In human cadaver eyes, post-operative astigmatism averaged 1.2 D (SD, +/- 0.6 D). Posterior transplants showed an intact endothelial cell layer with 1.0% (SD, +/- 1.2%) of cell death. In the animals, six (75%) eyes had clear transplants 2 weeks after surgery; one of these eyes later developed an allograft rejection. Two (25%) eyes showed corneal decompensation, because of inverted implantation of the donor disc. Microscopy showed minimal scarring at the donor-to-host interface and a normal wound-healing response at the posterior stromal wound edges. CONCLUSION: In experimental models, posterior lamellar keratoplasty can be performed through a limbal incision and a mid-stromal pocket. The procedure may be a potential alternative in the surgical management of corneal endothelial disorders.

Traumatic injuries to permanent teeth in children treated in Department of Pedodontics, Medical Academy in Lublin.

The traumatic dental injuries in children represent the second, after dental caries, most common reason of attending the dental surgeries. The paper presents the statistical analysis of the patients attending the Department of Pedodontics, Medical Academy in Lublin due to the traumatic dental injuries.

Detection of endotoxin in media from sterile corneal organ cultures.

Lipopolysaccharides (LPS) are elements of the cell wall of gram-negative bacteria. They are also called endotoxins and are known to stimulate various inflammatory reactions by interaction with cytokines and macrophages. Additionally, they may have a direct effect on other cells. As contamination of sterile organ-culture media with bacterial substances may influence the donor-tissue prognosis, we investigated a series of culture media drawn from organ culture for the presence of endotoxin. A total of 341 samples of sterile organ-culture media from 3 different cornea banks using either organ culture or the refrigerated-storage technique were tested for endotoxin. The assay was performed using the Limulus amebocyte-lysate test. A level of endotoxin above the background threshold was found in 99/341 (29%) culture media. The incidence of endotoxin ranged from 14% up to 50%, depending on the cornea bank and culture system used. Endotoxin detected in sterile corneal organ cultures probably derives from nonreplicating bacterial postmortem donor-tissue contamination. The presence of endotoxin-positive cultures varied between the eye banks but was not related to a given storage method. As endotoxin may directly influence graft viability or trigger inflammatory host responses, these findings may have significance for the clinical results of corneal grafting.

Corneal endothelium and growth factors.

Human corneal endothelium is characterized by a low regenerative capacity, mainly because of a low mitotic activity, and therefore complete regeneration of the endothelial layer after injury is precluded. A decrease in endothelial cell density and an abnormal cell mosaic, which may occur after many conditions, can compromise the integrity of the endothelial monolayer, resulting in corneal decompensation with reduced vision and eventual need for penetrating keratoplasty. It would be beneficial to have growth factors that can help to maintain or restore the integrity of this delicate endothelial monolayer by maintaining or increasing the endothelial cell density or to stimulate the regeneration during wound healing. Growth factors represent a group of signalling peptides which influence diverse cellular processes, including cell proliferation, differentiation, migration and survival. One of the areas that has received great interest is its enhancement of wound healing. In this review the effects of three most effective growth factors (EGF, PDGF, FGF) on corneal endothelium, especially on wound healing in human corneal buttons, will be discussed.

Synergistic action of heparin and serum on basic fibroblast growth factor-modulated DNA synthesis and mitochondrial activity of cultured bovine corneal endothelial cells.

Basic fibroblast growth factor (bFGF) is a major mitogen and chemoattractant for many cell types. The synergistic role of fetal bovine serum (FBS) and heparin on the modulation of tissue-cultured bovine corneal endothelial cells by bFGF was studied. Cell modulation was assessed by DNA synthesis ([3H]thymidine incorporation), mitochondrial activity (MTT assay), mitochondrial volume (transmission electron microscope study), and cell shape in mitotically competent cells and cells mitotically inhibited by 5-fluorouracil, mitomycin C, and irradiation. The dose-dependent stimulation of bFGF was found to be different for DNA synthesis and mitochondrial activity, the maximal effective dose being 1 and 10 ng/ml, respectively. Supplementation of bFGF with FBS enhanced both DNA synthesis and mitochondrial activity and caused a shift in the dose response to lower bFGF concentrations for the DAN synthesis, but not for the mitochondrial activity. Supplementation of bFGF with heparin resulted in an additional response for the mitochondrial activity, but not for the DNA synthesis. In addition, bFGF increased mitochondrial volume and induced elongation of the cells. These processes were further enhanced by the addition of heparin to bFGF. These results suggest that serum and heparin have a differential effect on mitogenic and non-mitogenic cells processes modulated by bFGF.

Toxic keratopathy due to the accidental use of chlorhexidine, cetrimide and cialit.

Due to economical reasons some ophthalmologists are using an irrigating solution made by the hospital pharmacy instead of the commercially available solutions. These irrigating solutions come in bottles which are identical to the ones used for other solutions. During the last three years bottles were accidentally mixed up five times. Consequently, bottles containing solutions such as chlorhexidine, cetrimide, chlorhexidine/centrimide and cialit solutions were used during cataract surgery. This resulted in immediate corneal edema which, in its turn resulted in a bullous keratopathy. Four patients underwent a penetrating keratoplasty. In one patient the cornea was covered with a conjunctival flap. Light microscopy of the corneas included epithelial edema, loss of keratocytes, and a disrupted and sometimes absent endothelial cell layer.

Histopathologic effects of mitomycin C after trabeculectomy in human glaucomatous eyes with persistent hypotony.

We evaluated the histopathologic findings in seven patients who underwent surgical revision of the filtration site after trabeculectomy with mitomycin C because of persistent hypotonous maculopathy. Light microscopic examination of subconjunctival tissue and sclera demonstrated hypocellularity of fibroblasts and disruption of the normal architecture. Tissue fragments at the margin of the bleb wall demonstrated scarring and contained multiple fibroblasts. Additionally, we investigated the histopathologic changes in an eye obtained from a patient who died one week after a trabeculectomy with mitomycin C. Transmission electron microscopy showed myelin figures, increased melanolipofuscin granules, vacuolated cytoplasm, and disrupted mitochondria of the ciliary body epithelium underlying the site of mitomycin C application. On the basis of these findings, both overfiltration because of tissue disorganization of the filtering bleb and aqueous hyposecretion because of ciliary body toxicity might be involved in the causes of persistent hypotony after mitomycin C trabeculectomy.