Biomedical subjects

V Trinkaus-Randall

Publications and source records attributed to V Trinkaus-Randall.

At least 37 records · Page 2Linked to original sources

PubMed1998

In vivo comparison of three different porous materials intended for use in a keratoprosthesis.

AIM: The goal was to compare the biological response of the corneal stroma with three porous materials: a melt blown microfibre web of polybutylene:polypropylene (80:20); a polyester spun laced fabric (polyethylene terephthalate), and an expanded polytetrafluoroethylene. Fifty per cent of each of the materials were modified using argon radio frequency. METHODS: Discs (6 mm in diameter) were inserted into interlamellar stromal pockets and followed for a period of 12 weeks. Clinical evaluations were performed weekly. At 6 and 12 weeks, fibroplasia and the distribution of matrix proteins and growth factors (bFGF and TGF-beta) were evaluated immunohistochemically. The characterisation of glycosaminoglycans was determined after selective extraction and digestion. RESULTS: The response to the disc resembled that of a wound with a decrease in keratan sulphate and an increase in dermatan sulphate. Pretreatment of the disc reduced corneal oedema and neovascularisation. Heparan sulphate, not normally detected in the corneal stroma, was detected in the region immediately surrounding the disc and in the discs of some materials. The presence of glycosaminoglycans and collagens in the disc indicated that cells had migrated into the disc and deposited a complex matrix in all three materials. The collagen response was not surface specific. bFGF and TGF-beta were detected in the region between the disc and the stroma in the polybutylene material and became diffuse with time. CONCLUSION: Fibroplasia occurred most rapidly into the polyester discs but was accompanied by a large number of inflammatory cells. While the distribution of collagens was not altered by the material, the expression and distribution of growth factors was material dependent. bFGF was expressed transiently and occurred before that of TGF-beta. It is predicted that the transient expression of growth factors mediates the regulation of matrix proteins.

Animals↗

PubMed1997

Advances in polyvinyl alcohol hydrogel keratoprostheses: protection against ultraviolet light and fabrication by a molding process.

Our goal was to: 1) modify poly(vinyl alcohol) hydrogels intended for use in a synthetic cornea to absorb ultraviolet light and 2) develop molding procedures for the fabrication of the device. Two ultraviolet light-absorbing monomers were incorporated by copolymerization. The resulting hydrogels protected against ultraviolet light, were not toxic to corneal fibroblasts, and the protection did not diminish during prolonged aqueous exposure. A novel keratoprosthesis molding procedure was developed that made use of the phase transition properties of mixtures of poly(vinyl-trifluoroacetate) and acetonitrile. In this procedure the keratoprosthesis was shaped and a fibrous skirt was bonded to it in a single operation. Composite keratoprostheses were designed and prepared for in vivo testing.

Animals↗

PubMed1997

Chondrocyte survival and differentiation in situ are integrin mediated.

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.

Animals↗

PubMed1997

Plasma surface modification of artificial corneas for optimal epithelialization.

We have demonstrated that the optimal surface treatment of a polyvinylalcoholcopolymer hydrogel for epithelial cell migration and proliferation is an argon radio frequency (rf) plasma treatment. The surface chemistry of the material was determined prior to each cellular evaluation, allowing us to compare the biological response with a known surface chemistry. The cellular response was carried out in a consistent manner a minimum of three separate runs. We found that the optimal conditions required culturing the cells under constant rotation. Cells became confluent on argon-plasma-treated surfaces coated under several different reactions pressures, and after 2 weeks they became multilayered. Our experiments demonstrated that cells proliferated and extracellular matrix and adhesion proteins were present only when the surface was treated with an argon rf plasma; acetone- and ammonia-treated surfaces did not yield the desired results. Organ culture experiments further demonstrated the efficacy of the argon-treated surfaces. In these experiments, intact keratoprosthetic devices with modified hydrogel surfaces were implanted into rabbit corneas. The excised corneas containing the devices were cultured, and 3 weeks later, using confocal laser scanning microscopy, confluent epithelium was detected on the modified hydrogel surface. This is the first demonstration that rabbit limbal epithelial cells can migrate onto a synthetic cornea containing a modified hydrogel-treated surface and form a confluent surface of epithelium.

Animals↗

PubMed1997

The expression of integrin subunits alpha 6 and beta 4 by corneal epithelial cells on modified hydrogel surfaces.

Our goal was to quantitate the expression and localization of integrin subunits alpha 6 and beta 4 by corneal epithelial cells on defined synthetic substrates. Previously we demonstrated that the cytoplasmic pH and translocation of the alpha 6 integrin subunit to the cell membrane was modified by ionic interactions. These results suggest that changes in the ionic interactions at the cell-substrate interface not only alter the intracellular milieu but ultimately affect the expression of adhesion proteins. To test this hypothesis, hydroxyethylmethacrylate (hema) hydrogels were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate) or carboxyl moieties (methacrylic acid). Changes in the distribution of mRNA and protein were monitored using confocal laser scanning microscopy. The steady state level of integrin mRNA was evaluated, and the results indicate that while the plating efficiency was identical on all surfaces, the expression and localization of integrin subunits was surface dependent. Alpha 6 and beta 4 proteins were localized along the basal surface of nonpermeabilized cells cultured on laminin, on surfaces with amine moieties, and on those with amine and carboxyl moieties. The level of diffuse cytoplasmic staining increased with the presence of carboxyl moieties. Alpha 6 and beta 4 integrin subunits were negligible when the surfaces contained carboxyl moieties alone. The expression of alpha 6 and beta 4 mRNA was higher on surfaces containing amine moieties than on surfaces containing only carboxyl moieties. These results indicate that the characteristics of the substrate and the resulting cell-matrix interaction alter protein and mRNA expression of integrin subunits.

Animals↗

PubMed1997

Expression of integrin receptors on plasma membranes of primary corneal epithelial cells is matrix specific.

Modulation of cell behavior may occur through cell adhesion receptors that bind domains of extracellular matrix molecules and mediate cell-substrate signal transduction. It was hypothesized that while primary corneal epithelial cells seeded onto laminin and fibronectin express and synthesize integrin receptors, they are not detected on the plasma membrane until the appropriate ligand is present. The integrin subunits (alpha-6, beta-4 and beta-1) present on the plasma membrane after adherence to laminin and fibronectin were compared with changes that occurred in mRNA expression and protein synthesis. Prior to seeding, the percentage of cells expressing integrin receptors and matrix proteins on their plasma membrane was determined. Negligible laminin and fibronectin (0-7%) were present on the plasma membrane while the population of epithelial cells expressing beta-4 and beta-1 on the plasma membrane was low (21-23%). After 3 hr of adherence the cell population expressing integrin subunits was substrate dependent. The percentage of cells adherent to LM expressing beta-4 was four-fold greater than cells adherent to FN. After 24 hr the percentage of cells cultured on fibronectin expressing beta-4 increased significantly indicating ligand deposition. The expression and protein synthesis of alpha-6 and beta-4 was evaluated and an increase in the synthesis of alpha-6 and beta-4 was not detected until 18 hr on LM and 21 hr on FN. The present results demonstrate that expression and transport of integrin receptors to the plasma membrane of primary corneal epithelial cells after adhesion is regulated by the presence of specific ligands.

Animals↗

PubMed1997

Implantation of a synthetic cornea: design, development and biological response.

Our goal was to evaluate 3 different designs of synthetic corneas in vivo. All devices had a transparent hydrogel center molded to a porous peripheral skirt. Over 30 devices were implanted into rabbits and followed for up to 6 months. The devices were preseeded with rabbit stromal fibroblasts, which enhanced the rate of fibroplasia. The anterior surface of the hydrogel was modified using argon rf plasma treatments. Clinical examinations were performed, and histological analyses were conducted on tissue throughout the time course. Our optimal model ranged from 4.5 to 6 mm and had an extended porous skirt increasing the surface area for fibroplasia and ultimate anchorage of the device. Fibroplasia occurred in this model, and collagen was detected by 28 days. The anterior chamber was normal with no detectable leakage of aqueous humor. Glycosaminoglycans were detected and followed the time course outlined previously when porous material itself was inserted into the stroma. We present the first demonstration that rabbit limbal epithelial cells can migrate onto the synthetic cornea in vivo.

Animals↗

PubMed1995

Synthesis of stromal glycosaminoglycans in response to injury.

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14, 42, and 84 days. The tissue and/or discs were removed and labeled with 35S-sulfate for 18 h; GAGs were extracted with 4 M guanidine-HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS-PAGE. The nonbound fractions from the chromatography were assayed for TGF-beta using Western blot analysis and for hyaluronic acid using an 125I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-beta that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-beta. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling.

Animals↗

PubMed1995

Distribution of F-actin, vinculin and integrin subunits (alpha 6 and beta 4) in response to corneal substrata.

Our goal was to determine the early response of corneal epithelial cells to living modified stromal substrates. We examined the distribution of integrin subunits (alpha 6 and beta 4), vinculin and the organization of F-actin in epithelial cells after cell-matrix and cell-cell hypothesized that the distribution of proteins in the cell matrix attachment complex would be altered if the substrate was modified. Integrin subunits, alpha 6 and beta 4, were chosen as they play a role in cell matrix adhesion and adhesion site formation. Corneal epithelial cells were cultured from explants and seeded on three corneal substrates (a stroma containing an intact basal lamina, a cornea lacking a basal lamina and a cornea treated with alkali). After 3 h of incubation, the tissue was fixed and stained with monoclonal antibodies specific for vinculin and for the integrin subunits alpha 6 and beta 4. The organization of F-actin was assessed using rhodamine phalloidin. The localization of the proteins was recorded with confocal laser scanning microscopy. Filamentous actin delineated the lateral cell membranes of corneal epithelial cells. The organization of actin and distribution of vinculin and integrin subunits of epithelial cells cultured on stromal substrates containing a basal lamina mimicked a simple epithelial organization. In contrast, when cells are cultured on the substrate lacking a basal lamina and alkaline treated corneal substrates the distribution of the specific proteins examined was altered. Vinculin and alpha 6 were present along membranes of cells cultured on substrates lacking a basal lamina and were diffuse in cells cultured on the alkaline substrates. These studies demonstrate that changes in the distribution of adhesion and cytoskeletal proteins in response to different surfaces may contribute to the healing dynamics in different wounds.

Actins↗

PubMed1994

Age-related changes of scleral hydration and sulfated glycosaminoglycans.

Human scleras were analyzed to determine if tissue hydration changed in an age-related fashion, and whether this change could be related to decreased levels of glycosaminoglycans. The anterior half of normal human scleras were examined with donor ages ranging from newborn to 99 years (n = 40). Tissue hydration was found to decrease by 1.06% per decade (P = 0.0012). Glycosaminoglycans were extracted from scleras with guanidine hydrochloride, purified by anion exchange chromatography and quantitated using dimethylene blue and selective enzymatic digestion. The glycosaminoglycans of the sclera were primarily dermatan sulfate and chondroitin sulfate with small quantities of hyaluronic acid. While no loss of chondroitin sulfate with age was detected, a significant age-related loss of dermatan sulfate was found (P = 0.0006, n = 14). An examination of the relationship between glycosaminoglycan concentration and scleral hydration showed that increased levels of sulfated glycosaminoglycans were associated with increased levels of scleral hydration (P < 0.02, n = 14). No improvement in the correlation could be achieved by considering either dermatan sulfate or chondroitin sulfate individually. Our results suggest that, as in other connective tissues, aging is associated with a loss of glycosaminoglycans, and their loss is coincident with decreased tissue hydration.

Adolescent↗

PubMed1994

Effect of pretreating porous webs on stromal fibroplasia in vivo.

We have previously demonstrated that keratocytes penetrate and deposit collagen after a porous web is inserted into interlamellar corneal pockets. In these studies our goal was to determine whether pretreatment of the porous discs would enhance wound healing. We have evaluated four methods of pretreating the porous disc prior to its placement in the stroma. The pretreated discs were followed in vivo for a period of 42 days. The criteria we used to determine whether pretreatment affected wound healing were: collagen deposition, extent of fibroplasia, the synthetic rate of keratocytes within the disc, and lack of edema. Our results indicate that preseeding with stromal keratocytes enhanced the overall synthetic rate and specifically enhanced the amount of collage deposited within the web.

Animals↗

PubMed1994

Expression of integrin and organization of F-actin in epithelial cells depends on the underlying surface.

PURPOSE: To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS: Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS: The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS: This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.

Actins↗

PubMed1993

Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging.

PURPOSE: The purpose of this study was to examine changes in the distribution of integrin subunits, alpha 6 and alpha 4, in the normal human cornea with age. METHODS: Thirty normal corneas were examined and divided into three groups; corneas from children younger than 2 years, corneas from adults 29 to 70 years, and corneas from adults older than 70 years. The corneas were frozen and the sections were cut, double-stained with monoclonal antibodies to the integrin subunits, and visualized with Texas Red or fluorescein using confocal laser scanning microscopy. Computer imaging was conducted to determine differences. RESULTS: The alpha 6 subunit was generally localized along the basal and lateral surfaces of basal epithelial cells and projected into Bowman's membrane. The beta 4 subunit was only present along the basal surface. Overall, the major age-related difference was the loss of continuous alpha 6 and beta 4 subunits along the basal surface of basal epithelial cells. When reconstructed images from corneas of individuals older than 70 years were optically sectioned en face, the alpha 6 subunit appeared discontinuous. If the same optical images were viewed from corneas of younger individuals, the staining was continuous. The number and distribution of hemidesmosomes along the basal lamina did not change with age in the corneas examined. CONCLUSIONS: Using computer imaging associated with confocal laser scanning microscopy, we have demonstrated that there is an age-related change in the localization of the alpha 6 and beta 4 subunits.

Adult↗

PubMed1991

Localization of integrin and syndecan in vivo in a corneal epithelial abrasion and keratectomy.

We compared the localization of integrin, a glycoprotein adhesion receptor, and syndecan, a transmembrane proteoglycan receptor, in vivo during the process of corneal epithelial wound healing to determine if the contact between migrating epithelium and substrata influence the expression of these two adhesion proteins. The expression of three integrin subunits of the alpha/beta heterodimer complex, beta 1, alpha 5, and alpha 6, were examined over a 48 hr in response to an abrasion. We also examined their expression in a keratectomy and compared their two conditions. The beta 1 subunit, which is expressed basolaterally in a normal cornea, was localized during epithelial migration over the basal lamina and keratectomy. Negligible levels of the fibronectin binding receptor subunit, alpha 5, were detected in a normal cornea. The levels of staining between a normal cornea, an abrasion and a keratectomy did not differ. Detection of alpha 6, the laminin binding receptor subunit, was expressed most intensely along the basal side of basal cells in a normal cornea. During the course of wound healing, alpha 6 was only detected in the basal cells and by two days the localization resembled that seen in the normal cornea. Epithelial staining for the alpha 6 subunit was also detected during the migration of cells over the keratectomy, more intensely than the other two subunits. Syndecan, which acts as a more permanent adhesion receptor in stratified epithelium, was only detected 48 hr post-abrasion, throughout all layers of epithelium, as was expected. These results indicate a predominance of alpha 6 expression by corneal epithelial cells at all times of in vivo wound healing and the presence of syndecan only upon restratification of the epithelium.

Animals↗

PubMed1991

In vivo fibroplasia of a porous polymer in the cornea.

We have developed a melt-blown fibrous construction of polybutylene/polypropylene in which we previously demonstrated keratocyte ingrowth and collagen synthesis in vitro. In the present studies, we evaluated this material in vivo in interlamellar corneal pockets for periods of up to six months. By day 42, the porous interstices of the disc were heavily populated with keratocytes. Extracellular matrix deposition occurred and there was a 5000-fold increase in total protein and a 1000-fold increase in total collagen over background. The cells within the disc continued to be synthetically active for the six months of our study. Discs remained in corneas for periods of up to one year without any extrusion. This material has great promise as a porous peripheral component of a keratoprosthetic device.

Animals↗

PubMed1991

Quantification of stromal destruction in the inflamed cornea.

An intrastromal injection of endotoxin lipopolysaccharide (LPS) in one eye of New Zealand albino rabbits induced a prominent keratitis characterized clinically and microscopically by edema and infiltration. Polymorphonuclear leukocytes (PMNs) constituted the primary invading leukocytic element. Collagen synthesis was measured by pulsing the corneas with 3H-proline before inducing inflammation. The invasion of the cornea by leukocytes did not alter the conversion of proline to hydroxyproline significantly in the stroma during the 14-day observation period, signifying that there were only negligible changes in the rate of collagen synthesis. However, the percentage of total stromal protein represented by collagen (ie, collagen/total protein) was only 50% of that in comparable corneas receiving an injection of phosphate-buffered saline. Some animals were rendered leukopenic by intravenous nitrogen mustard before intrastromal LPS injection caused a less severe corneal inflammatory response, characterized microscopically by fewer infiltrating leukocytes. Similarly, in nonleukopenic rabbits, topical therapy with 1% prednisolone acetate markedly reduced the corneal inflammatory response which also was characterized by fewer invading leukocytes. In neither instance was there extreme collagen loss, suggesting that the loss of stromal collagen is related to PMN infiltration.

Animals↗

PubMed1990

The synthesis and role of integrin in corneal epithelial cells in culture.

Cell-substratum interaction and recognition is essential for cellular adherence and protein synthesis. To understand the mechanisms underlying diseases related to corneal erosion, it is necessary to examine the role integral proteins play in cellular adherence and spreading. In the current study, we used antibodies to evaluate the accumulation of integrin by corneal epithelial cells in tissue culture. We demonstrated that while the protein was incorporated into the cell layer at 6, 24, 48, and 72 hr, a far greater amount was secreted into the media. The total amount of integrin did not change with time, and the level of mRNA did not increase from 24 to 48 hr. During this time period, fibronectin was not detected. Antibodies to the fibronectin-binding receptor inhibited cellular spreading over a period of 18 hr. These results indicate that rabbit corneal epithelial cells do synthesize integrin and that integrin plays a critical role in cellular spreading.

Animals↗

PubMed1990

In vitro evaluation of fibroplasia in a porous polymer.

We evaluated a polybutylene/polypropylene blend in a blown microfiber configuration in vitro for potential use as the peripheral area of a keratoprosthetic device. Material properties such as ultimate tensile strength and ultimate elongation were measured. Stromal fibroblasts were seeded onto the material in vitro, and cell proliferation between uncoated and Type I collagen coated discs did not differ significantly. Fibroblasts could be seen migrating along the fibers and also traversing the fibers. The synthesis of connective proteins was examined. Laminin, fibronectin, and Type I collagen were detected by day 8. The experiments demonstrate that stromal fibroblasts can adhere onto the fibers, proliferate, and synthesize connective tissue proteins. Experiments are now being conducted to further evaluate the material in vivo.

Animals↗