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Biomedical subjects

I J Constable

Publications and source records attributed to I J Constable.

At least 73 records · Page 4Linked to original sources
PubMed1997

Histologic evaluation during healing of hydrogel core-and-skirt keratoprostheses in the rabbit eye.

PURPOSE: We developed two models that are modifications of our original poly(2-hydroxyethyl methacrylate) (PHEMA) core-and-skirt keratoprosthesis. In these keratoprostheses, the mechanical strength of the skirt has been considerably increased with divinyl glycol (DVG) as a cross-linking agent during polymerization. In one (KPro I), methyl methacrylate (MMA) was added as comonomer to increase cell adhesion, and in the other (KPro II), HEMA was polymerized with DVG without comonomer. The aim of this study was to evaluate the process of healing and biocolonization and to ascertain whether KPro I demonstrates better ingrowth than the mechanically stronger KPro II, after implantation in rabbit eyes. METHODS: Ten rabbits were used for each model and studied at five predetermined end points up to 26 weeks. The device was implanted as a full-thickness keratoprosthesis covered with a conjunctival flap. RESULTS: Neither prosthesis demonstrated extrusion or retroprosthetic membrane formation. There was no significant difference between the two types of prosthesis with respect to tissue ingrowth and surrounding tissue melting. Histologically, inflammation was not severe, but calcification was seen in most specimens. Evidence of biodegradation of the prosthesis also was seen. CONCLUSION: In our original keratoprosthesis, fibrovascular invasion had occurred into the prosthetic skirt, but wound dehiscence and low mechanical strength resulted in an unfavorable outcome. In this series, the mechanical properties were improved, and KPro II was stronger than KPro I. Therefore KPro II would be the preferred polymer combination for surgical manipulation. However, biodegradation and calcification require further investigation into the degree and significance of these adverse reactions.

Animals↗
PubMed1997

Effect of crosslinked poly(1-vinyl-2-pyrrolidinone) gels on cell growth in static cell cultures.

Poly(1-vinyl-2-pyrrolidinone) (PVP) and copolymers of 1-vinyl-2-pyrrolidinone are insoluble in water when crosslinked but they can absorb very large amounts of water to become syringe-injectable hydrogels. Such gels have been investigated recently as potential substitutes for the vitreous humour in the eye. In this study, during the cytotoxic evaluation by sulforhodamine B colorimetric assay of variously crosslinked PVP gels, it was found that many of them showed protective/growth promoting effects on 3T3 mouse fibroblasts in static cultures, a phenomenon encountered previously only with aqueous solutions of a limited number of natural or synthetic polymers. Particularly, the gels crosslinked with diethylene glycol dimethacrylate (DEGDMA) induced a significant enhancement of cell proliferation, especially in serum-free cultures. No correlation between this effect and the essential gel properties (chemical composition, viscoelasticity and equilibrium water content) could be established. The study demonstrated that crosslinked PVP hydrogels showed a serum-like growth promoting effect on an anchorage-dependent cell line, which may be due to physical protection, inability of the insoluble gels to penetrate cell membranes, and their ability to mimic the extracellular matrix.

3T3 Cells↗
PubMed1996

Crosslinked poly(1-vinyl-2-pyrrolidinone) as a vitreous substitute.

A hydrogel with a high water content was produced and tested as a possible vitreous substitute. The polymer (poly[1-vinyl-2-pyrrolidinone]) (PVP) was synthesized by free radical bulk polymerization of 1-vinyl-2-pyrrolidinone (VP) in the presence of 0.25% divinyl glycol (DVG) as a crosslinking agent. The fully hydrated polymer, containing about 98% water, was clear, transparent, autoclavable, and easily injected through a small-gauge needle with minimum fragmentation, and without changes in its optical properties. Dynamic mechanical analysis of the hydrogel indicated a covalently crosslinked elastic network both before and after injection. The resilience of hydrogel decreased after being subjected to shear stress during the injection process. A cytotoxicity bioassay of the hydrogel in vitro, using cultured mouse (Balb/ c-3T3) fibroblasts, showed cytostatic but not cytocidal effects. The hydrogel was injected into the vitreous cavity of rabbits and followed up to 4 weeks. The gel was clinically well tolerated, however opacities in the vitreous body were observed following the insertion of the gel. Histopathological examination revealed no adverse reactions to the retina, but the presence of loose polymer particles indicated the possibility of the biodegradation of the polymer. These results suggest the potential use of crosslinked PVP hydrogels as vitreous substitutes, provided that their biodegradation is not significant, a matter that should be further investigated.

3T3 Cells↗
PubMed1996

Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.

The purpose of the present study was to investigate the accumulation of rod outer segment (ROS)-derived debris in cultured human retinal pigment epithelial cells (RPE). The RPE cell layer is responsible for the phagocytosis and digestion of photoreceptor outer segments. Due to the immense volume of photoreceptor-derived material processed by the RPE cells, even minor changes in the efficiency of ROS processing may cause the accumulation of lipofuscin and photoreceptor derived debris. In this work, 17 RPE cultures were established from the globes of eye bank donors whose ages ranged from 18 to 79 years. Third passage cultures were challenged with bovine ROS and the accumulation of an autofluorescent debris was quantified using a flow cytometer. It was demonstrated that ROS challenge greatly increased the rate of autofluorescent debris accumulation. The accumulation of autofluorescent debris varied significantly from culture to culture. This variation was independent of the phagocytosing capacity of individual cultures and was not age dependent. To further investigate the factors which may be responsible for these differences, the presence of cathepsin D, an aspartic protease responsible for 80% of proteolysis of rhodopsin, was analysed by Western blot. Although the 34 kDa active form of cathepsin D was found in all cultures, in 41% of the cultures higher-molecular-weight forms of cathepsin D were additionally present, thus providing a multimer form of cathepsin D in these cultures. The rate of autofluorescent debris accumulation in cultures possessing a multimer form of cathepsin D was significantly greater (mean 42.3, S.D. +/- 19.8) than those in cultures having a singlet active form (mean 18.8, S.D. +/- 5.5) at 34 kDa (Student's t-test, DF = 15, t = 6.834, P < 0.001). The former cultures included one from a donor with age related macular degeneration, the latter cultures included one from a donor with diabetic retinopathy. This study demonstrates that the rate of autofluorescent debris accumulation in cultured RPE cells is not age dependent, but is an intrinsic property of the donor RPE cells that is possibly related to the presence of a multimer form of the lysosomal enzyme cathepsin D.

Animals↗
PubMed1996

Production of neocollagen by cells invading hydrogel sponges implanted in the rabbit cornea.

BACKGROUND: Poly(2-hydroxyethyl methacrylate) sponges are artificial tissue-equivalent matrices with potential value as materials for the peripheral zone of artificial corneas. A keratoprosthetic device was developed incorporating a poly(HEMA) spongy skirt which allowed cellular invasion. The present in vivo study investigated the biosynthetic activity of stromal fibroblasts growing within a poly(HEMA) sponge implanted into the rabbit cornea. METHODS: A porous poly(HEMA) hydrogel was synthesized by polymerization in a large excess of water. Specimens with a pore size larger than 10 microns were impregnated with collagen type I and then implanted into the limbal region of cornea in four rabbits. The animals were followed clinically for 28 days, when they were anaesthetized and new sponge specimens were implanted in their second eye. After 2 h, both eyes were enucleated. The 28-day and 2-h explants were subjected to autoradiographic analysis following labelling with tritiated proline and to an immunostaining technique using antibodies to collagen types I-VI. RESULTS: The autoradiographic analysis showed that the fibroblasts within the 28-day explants continued to be synthetically active and deposited proteins. Using the immunostaining technique, the deposition was most clearly demonstrated by the localization of collagen type III in the tissue invading the sponge. Both techniques failed to indicate any cellular activity in the short-time implants. CONCLUSIONS: The presence of collagen type III is consistent with a normal healing response of the stromal fibroblasts and indicates that poly(-HEMA) sponges are able to function as tissue-equivalent matrices.

Animals↗
PubMed1996

Keratoprosthesis: preliminary results of an artificial corneal button as a full-thickness implant in the rabbit model.

PURPOSE: To develop a prototype artificial cornea and evaluate it in the rabbit model. METHODS: Hydrogel core-and-skirt keratoprostheses were made and were inserted as full-thickness implants covered with conjunctival flaps in the right eyes of eight rabbits. RESULTS: Peroperative complications related to inadequate mechanical strength led to failure in the early postoperative period in three animals, one was euthanased for an unrelated reason and the remaining four have been successful for up to 16 weeks' follow-up. CONCLUSIONS: Full-thickness implantation of an artificial cornea, analogous to penetrating keratoplasty, has been achieved in the rabbit model. Histological findings confirm that integration of the prosthesis with host tissue occurs. The main complications encountered in this preliminary series were related to inadequate strength of the sponge skirt of this prototype device. Work in our laboratories is now concentrated upon improving the mechanical qualities of the hydrogel skirt and on the enhancement of biointegration.

Animals↗
PubMed1996

Poly(1-vinyl-2-pyrrolidinone) hydrogels as vitreous substitutes: histopathological evaluation in the animal eye.

A homopolymer of 1-vinyl-2 pyrrolidinone and its copolymer with 2-hydroxyethyl methacrylate, both cross-linked with divinyl glycol, were produced as possible substitutes for the vitreous body of the eye. The hydrated polymers behaved like viscoelastic gels, displaying excellent physical and optical properties. The sterile gels (0.7-1.5 ml) were injected into the vitreous cavity of rabbits, which previously underwent gas-mediated vitrectomy. Clinically, the eyes were quiet, with the exception of transient opacities in the vitreous. After 4 weeks, the operated eyes were enucleated and subjected to histopathological analysis using light and transmission electron microscopy. The common feature in all sections was the invasion of inflammatory cells. Vacuoles containing granular material, assumed to be polymer, were seen in the intercellular spaces of the neural retina, in the retinal pigment epithelium cells, and in macrophages. These findings indicated the fragmentation and phagocytosis of synthetic gels. It appeared that the biodegradation of the internalized polymers did not proceed further, however, the fate of polymers and their usefulness as vitreous substitutes should be investigated through long-term experiments.

Animals↗
PubMed1996

Polymers of 1-vinyl-2-pyrrolidinone as potential vitreous substitutes: physical selection.

More than 300 polymers of 1-vinyl-2-pyrrolidinone (VP) were synthesized, subjected to hydration, and characterized with the aim to select the most suitable materials as potential artificial substitutes for the vitreous body of the eye. The materials include cross-linked homopolymers, uncross-linked copolymers of VP with 2-hydroxyethyl methacrylate (HEMA), and cross-linked copolymers VP/HEMA. Five different cross-linking agents, both hydrophobic and hydrophilic, were used in this study. The resulting hydrogels, with equilibrium water contents ranging between 66.5 and 99.1%, were first subjected to a selection based on their physical behavior during manipulation, after which only the transparent, viscoelastic gels were further considered. Subsequent injectability and visual acuity tests, as well as the evaluation of light transmission characteristics, reduced further the number of potential candidates for vitreous substitution to only thirteen hydrogels. An eliminatory strategy based on physical properties of the potential vitreous substitutes is essential in order to avoid unnecessary sacrifice of experimental animals for in vivo assessment.

Biocompatible Materials↗
PubMed1996

A simple flow cytometric technique to quantify rod outer segment phagocytosis in cultured retinal pigment epithelial cells.

PURPOSE: The primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A secondary aim was to compare the kinetics of ROS phagocytosis in an immortal human RPE cell line with untransformed human RPE cells. METHODS: Flow cytometry was performed on RPE cells that had been challenged with fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence. RESULTS: Non-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. The kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures. FITC-ROS phagocytosis proceeded at a fairly linear rate for the first 12 h in the 3 human cell cultures studied, but was rapid for the first 3 h before slowing in the D407 cells. Within all cell populations, there was a heterogeneity of phagocytic activity which varied with time. CONCLUSIONS: This automated technique for measuring phagocytosis is rapid, simple, highly accurate, avoids radiation hazards, and permits study of heterogeneity within cell populations. The biochemistry, physiology and pathophysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately quantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor intensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large standard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cytometry, on the other hand, circumvents nearly all of these problems and may prove to be the optimal phagocytosis assay.

Adult↗
PubMed1996

Dynamics of gene transfer to retinal pigment epithelium.

PURPOSE: To examine the nature and dynamics of gene transfer to human retinal pigment epithelium (RPE) using an adenoviral vector and adjuvants that may enhance the uptake of recombinant adenoviruses. METHODS: Human RPE cultures (HRPE7) were transfected in vitro with varying concentrations (4, 20, 40, 120, and 200 pfu/microliter) and for varying periods (1, 2, 4, 16, 24, 48, and 72 hours) with a replication-deficient adenovirus (Ad.RSV. beta gal) containing the bacterial beta-galactosidase transgene (beta gal). The expression of beta gal was monitored by counting after X gal staining. The transgene expression profiles were compared to those of human F2000 fibroblasts under the same conditions. The adjuvant effect of sodium hyaluronate (HA) on the expression of beta gal was tested in F2000 and early and late passage human RPE cells for differing concentrations of HA, viral titers, and incubation times. Immunofluorescent cytochemistry was carried on HRPE7 and F2000 cells for the HA receptors, homing receptor CD44 (CD44), intercellular adhesion molecule 1 (ICAM-1), and the receptor for hyaluronan mediated motility (RHAMM). RESULTS: The number of HRPE7 and F2000 cells expressing the adenoviral transgene increased consistently with increasing incubation time and viral titer. There was a higher uptake of Ad.RSV. beta gal in HRPE7 cells compared to the F2000 fibroblasts under the same conditions. There was an increase of 28.1% and 41.4% in the number of RPE7 cells expressing adenoviral transgene and 16.2% and 15.8% F2000 fibroblast cells expressing the adenoviral transgene in the presence of 0.001% and 0.005% HA, respectively. Significant adjuvant effects on transgene expression also were shown in HRPE51 cells. It appears that the effects of increasing viral titer, length of incubation, and the presence of HA on transgene expression are at least additive. The appearance of CD44 and ICAM receptors on RPE7 and F2000 cells and RHAMM receptors on F2000 cells was similar. The RHAMM receptors in HRPE7 cells, however, were shown preferentially over the nucleus. CONCLUSIONS: On the basis of these results, the authors propose that adenovirus transgene expression increases with increasing incubation time and viral titer in cell culture. The rate of increase of expression differs between human RPE cells and the F2000 fibroblast cells, which may offer a targeting opportunity. The authors propose that the use of HA can offer both an adjuvant effect and a targeting advantage in terms of transferring adenoviral transgenes to human RPE in culture.

Adenoviruses, Human↗
PubMed1995

Laser-induced chorioretinal venous anastomosis for treatment of nonischemic central retinal vein occlusion.

OBJECTIVES: To determine whether a chorioretinal venous anastomosis could be created in humans and to evaluate the influence this has on patients with nonischemic central retinal vein occlusions in whom progressive visual loss developed. DESIGN: Retrospective study. PATIENTS: A total of 24 patients with nonischemic central retinal vein occlusions and progressive visual loss. INTERVENTION: An attempt was made to create a chorioretinal venous anastomosis using laser photocoagulation to enable obstructed venous blood to enter the choroid, thus bypassing the site of occlusion. MAIN OUTCOME MEASURES: Visual acuity, funduscopic appearance, and rapid sequence fluorescein angiograms. RESULTS: A successful chorioretinal venous anastomosis was created in eight cases (33%), with improvement in visual acuity and resolution of the funduscopic appearance of venous occlusion in all eight cases. Of the 16 patients (67%) in whom an anastomosis was not successfully created, the ischemic form of central retinal vein occlusion developed in five (31%), and eight (50%) were left with various degrees of macular damage and reduced visual acuity. CONCLUSIONS: Peripheral chorioretinal venous anastomoses can be created in a nonischemic central retinal vein occlusion and appear to be well tolerated. This technique may have some value in the treatment of patients with this condition; however, to address this fully, a properly constructed randomized prospective clinical trial will need to be performed.

Adult↗
PubMed1995

Initial clinical experience with tissue plasminogen activator (tPA) assisted removal of submacular haemorrhage.

Tissue plasminogen activator (tPA) (250 micrograms/ml) was used to facilitate removal of submacular thrombus in 15 patients. Following a three-port vitrectomy and subretinal tPA injection (0.1 ml) via a 30 gauge needle, blood was evacuated after enzymatic dissolution for 20 minutes. Two injections were required in some cases. Nine women and six men were treated (mean age 75.5 +/- 8.6 years). Duration of symptoms ranged from 2 days to 8 weeks. One case was due to a retinal macroaneurysm, the others to age-related macular degeneration. Vision improved in 13 patients and remained the same or deteriorated in 2 (mean follow-up 11 +/- 4.9 months). Well-defined subretinal neovascular membranes were identified in 2 patients and occult neovascularisation suspected in 2 others. A cataract developed in 1 case and retinal detachments in 2 others; all were treated successfully. The poor visual prognosis associated with submacular haemorrhage may be obviated by the use of the technique we describe.

Aged↗
PubMed1995

Lipofuscin of the retinal pigment epithelium: a review.

Accumulation of lipofuscin is one of the most characteristic features of ageing observed in retinal pigment epithelial (RPE) cells. The lipofuscin found in RPE cells differs from that of other body tissues due to the fact that it is mainly derived from the chemically modified residues of incompletely digested photoreceptor outer segments. It is a heterogeneous material composed of a mixture of lipids, proteins, and different fluorescent compounds, the main fluorophore of which has recently been identified as a derivative of vitamin A. Research interest has variously focussed on the roles of age, light damage, free radicals, antioxidants, visual pigments, retinal locus, lysosomal enzymes, and pigmentation on lipofuscin formation, as well as the effects of lipofuscin on RPE cell function and causation of retinal disease. This article reviews the recent advances in knowledge of the composition, origin, and possible deleterious effects of RPE cell lipofuscin.

Aging↗
PubMed1995

Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

BACKGROUND: To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. METHODS AND RESULTS: Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. CONCLUSIONS: Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

Cells, Cultured↗