Biomedical subjects

T V Chirila

Publications and source records attributed to T V Chirila.

At least 19 recordsLinked to original sources

PubMed1998

Biodegradation in vitro and retention in the rabbit eye of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogel as a vitreous substitute.

To elucidate the relatively short retention of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogels in the eye when used as potential vitreous substitutes, a 14C-labeled hydrogel was produced and subjected to both in vitro biodegradation assays and in vivo experiments. The polymer was synthesized by the free-radical copolymerization of 99% 1-vinyl-2-pyrrolidinone with 1% 14C-methyl methacrylate in the presence of ethylene glycol dimethacrylate (0.1%) as crosslinking agent. The in vitro protocol for assessing the biodegradation included the incubation of hydrogel with hydrolases (trypsin or collagenase), followed by examination of changes in its physical characteristics and by monitoring its residual radioactivity, as well as by detection of possible degradation products. Within the maximum duration of experiments (4 weeks), none of the procedures indicated biodegradation of polymer. The hydrogel was also injected into the vitreous humor of rabbits and followed up to 4 weeks. Residual radioactivity measurements of the vitreous contents indicated that 50% of the polymer was removed by the end of this period. Histopathologic examination revealed cell infiltrates of the mononuclear phagocyte system in both vitreous and retinal tissue. A possible phagocyte-mediated mechanism for the dissipation of hydrogel is discussed.

Animals↗

PubMed1998

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea.

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.

Animals↗

PubMed1998

Assessment of anticollagenase treatments after insertion of a keratoprosthetic material in the rabbit cornea.

PURPOSE: This study was performed to evaluate the enzyme production in response to implantation of the hydrogel material used in the experimental Chirila keratoprosthesis (KPro) and to assess the effects of five topical drugs on enzyme production and activity. KPros may be extruded from the cornea as a result of tissue melting, a process that involves excessive enzyme activity. To reduce the possibility of implant loss for the hydrogel Chirila KPro, a number of antiinflammatory drugs that have been used to treat other corneal melting conditions were investigated for their effect on initial collagenase activity after the implantation of KPro material into the rabbit cornea. METHODS: Poly(2-hydroxyethyl methacrylate) sponge pieces were implanted into rabbit corneas. Prednisolone, tetracycline, medroxyprogesterone, acetylcysteine, and sodium citrate were assessed for effects on gelatinolytic activity and stromal collagenase [matrix metalloprotease-1 (MMP-1)] production in vivo and in vitro by using zymography and Western blotting techniques. RESULTS: Whereas all five anticollagenase drugs were effective in reducing gelatinolytic activity in vitro, many were ineffective in vivo. However, medroxyprogesterone caused a reduction of gelatinolytic activity in vivo. The amount of MMP-1, as measured by immunoblotting, also was reduced by medroxyprogesterone treatment when compared with untreated controls. An increase in the apparent molecular weight of MMP-1 in operated corneas appears to be the result of the association of MMP-1 with collagen fragments resulting from the surgical trauma. CONCLUSION: This study indicates that topical medroxyprogesterone may be a useful adjunctive therapy after prosthokeratoplasty.

Acetylcysteine↗

PubMed1998

Clinical results of implantation of the Chirila keratoprosthesis in rabbits.

AIMS/BACKGROUND: An ideal keratoprosthesis (KPro) would closely resemble a donor corneal button in terms of its surgical handling, optics, and capacity to heal with host tissue in order to avoid many of the complications associated with the KPros which are currently in clinical use. This study was carried out to assess the long term clinical outcomes on implantation of the core and skirt poly(2-hydroxyethyl methacrylate) KPro in animals. METHODS: 20 KPros were made and implanted as full thickness corneal replacements into rabbits and followed for up to 21 months to date. RESULTS: 80% of the prostheses have been retained, with a low incidence of complications such as cataract, glaucoma, and retroprosthetic membrane formation which are frequently associated with KPro surgery. CONCLUSIONS: KPros of this type may offer promise in the treatment of patients for whom penetrating keratoplasty with donor material carries a poor prognosis. Refinement of the KPro and further animal trials, including implantation into abnormal corneas, are however mandatory before human implantation could be planned.

Animals↗

PubMed1998

The modulation of cellular responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases macrophage collagenase production in vitro.

We examined the regulation of collagenase production by the monocyte/macrophage THP-1 cell line when these cells were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies. Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response. Copolymer hydrogels containing 2-ethoxyethyl methacrylate (EMA) or methyl methacrylate (MMA) also induced a high response, while PHEMA hydrogels induced a low level response and the phosphorylated hydrogel induced no response. This pattern was altered when the morphology of the hydrogels was changed to that of a sponge. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels. Sponges containing EMA and MMA produced low level response relative to the TCP control. PHEMA and phosphorylated sponges produced little and no response respectively. The dramatically reduced enzyme response to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge.

Biocompatible Materials↗

PubMed1998

Implantation of PHEMA keratoprostheses after alkali burns in rabbit eyes.

PURPOSE: We have previously examined histologically the healing of a PHEMA core-and-skirt keratoprosthesis (the Chirila KPro) as a full-thickness implant in healthy animal corneas. The present study was carried out to determine whether a diseased cornea could also generate biocolonization of the skirt region of a KPro. METHODS: Ten KPros were placed as full-thickness corneal implants under conjunctival flaps in 10 alkali-burned rabbit corneas. Histological findings at intervals from 2 weeks to 6 months postoperatively were compared with earlier findings in 10 rabbits that had received identical KPros without prior alkali injury. RESULTS: Despite severe corneal injury and the reduced keratocyte population present, there were no clinically detected complications in 60%. Histological findings established that, compared with healthy host tissue, skirt biocolonization and KPro-cornea healing after an alkali burn were impaired, with evidence of epithelial downgrowth in 40%. One animal required euthanasia earlier than the planned end point, but no KPro extrusions occurred. CONCLUSION: Biocolonization of a KPro skirt is reduced but not prevented in an alkali-induced corneal inflammation model. Although no extrusions occurred, close follow-up and anticollagenolytic medication would be required to minimize the complication rate.

Animals↗

PubMed1997

Keratoprosthesis results in animals: an update.

BACKGROUND: The report presented is an update on continuing development work on modified PHEMA core-and-shirt KPros in animals. METHODS: Two variations (improved wet-eye, and dry-eye) of a prototype core-and-skirt Chirila KPro are described. The clinical success rate on implantation of these versions of the Chirila KPro was assessed. RESULTS: It was found that a significant improvement in retention rate was shown in the improved model but that the dry-eye model failed early in two of the three implanted. CONCLUSIONS: The significance of the improved strength and the reasons for disappointing results with the early dry-eye KPros are discussed. Ongoing work is briefly outlined.

Animals↗

PubMed1997

In vitro assessment of the biological activity of basic fibroblast growth factor released from various polymers and biomatrices.

The kinetics of controlled release of basic fibroblast growth factor (bFGF) from polymers (sutures, polycarbonate, Hydron, and Elvax), biopolymers (alginate), and biomatrices (lens capsules), and conditions for storage of bFGF (temperature, plastic type, heparin) were evaluated in vitro. Tissue culture proliferation bioassays with 3T3 fibroblasts, showed that only lens capsules with bFGF had a sustained release of bFGF for up to three weeks. The other materials released all of the 'bound' bFGF with two hours or produced an inflammatory response in vivo. Therefore, the lens tissue had the most potential for controlled long-term delivery of bFGF in vivo. These studies emphasise the importance of in vitro analysis of release kinetics of growth factors from a range of materials as a basis for potential in vivo applications.

3T3 Cells↗

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

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

Enhancement of neovascularization in regenerating skeletal muscle by the sustained release of erucamide from a polymer matrix.

The angiogenic agent erucamide (cis-13-docosenamide), incorporated into a polymeric biomaterial (Elvax 40P, a copolymer of ethylene and vinyl acetate), was used to determine whether angiogenesis can be increased in the regenerating skeletal muscle, and whether the enhanced revascularization improves the new muscle formation. The angiogenic nature of this lipid was confirmed in a rat cornea-micropocket assay, prior to insertion of small strips of the polymer containing either 3 micrograms, 300 micrograms erucamide or only polymer as a control into the mid-region of crush-injured tibialis anterior (TA) muscles of forty-five adult male BALB/c mice. All TA muscles were sampled ten days after injury and analyzed morphometrically. Statistical analyses of the mean blood vessel area density in lesions from twelve perfused TA muscles (three from each of the erucamide-treated or control group), revealed a dose-dependent angiogenic effect of erucamide: a dosage of 3 micrograms increased mean blood vessel area density to 5.1% compared to 2.0% in controls, due to numerous large caliber, thin-walled vessels, whereas the mean vessel area density in both the 30-micrograms (3.5%) and 300-micrograms (1.5%) doses were similar to controls. However, at all three doses tested, erucamide did not significantly alter the degree of new muscle formation, connective tissue deposition, or removal of necrotic debris.

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 controversial episode in the history of artificial cornea: the first use of poly(methyl methacrylate).

The introduction of the synthetic plastic poly(methyl methacrylate) as a material for artificial cornea (keratoprosthesis) is usually credited to William Stone, Jr. who allegedly performed the first experiments in 1947 and reported the results in 1953. As this plastic marked a revival in the development of artificial cornea, it is important to establish who was the first to use it and actually deserves the credit. This study demonstrates that at least three other ophthalmic surgeons (Wünsche in Germany, Franceschetti in Switzerland, and Györffy in Hungary) performed trials with keratoprostheses made from poly(methyl methacrylate) and published their results prior to Stone, Jr.

Cornea↗

PubMed1995

Axonal growth within poly (2-hydroxyethyl methacrylate) sponges infiltrated with Schwann cells and implanted into the lesioned rat optic tract.

Porous hydrophilic sponges made from 2-hydroxyethyl methacrylate (HEMA) have a number of possible biomedical applications. We have investigated whether these poly(HEMA) hydrogels, when coated with collagen and infiltrated in vitro with cultured Schwann cells, can be implanted into the lesioned optic tract and act as prosthetic bridges to promote axonal regeneration. Nineteen rats (20-21 days old) were given hydrogel/Schwann cell implants. No obvious toxic effects were seen, either to the transplanted glia or in the adjacent host tissue. Schwann cells survived the implantation technique and were immunopositive for the low affinity nerve growth factor receptor, S100 and laminin. Immunohistochemical studies showed that host non-neuronal cells (astrocytes, oligodendroglia and macrophages) migrated into the implanted hydrogels. Astrocytes were the most frequently observed host cell in the polymer bridges. RT97-positive axons were seen in about two thirds of the implants. The axons were closely associated with transplanted Schwann cells and, in some cases, host glia (astrocytes). Individual axons regrowing within the implanted hydrogels could be traced for up to 900 microns, showing that there was continuity in the network of channels within the polymer scaffold. Axons did not appear to be myelinated by either Schwann cells or by migrated host oligodendroglia. In three rats, anterograde tracing with WGA/HRP failed to demonstrate the presence of retinal axons within the hydrogels. The data indicate that poly(HEMA) hydrogels containing Schwann cells have the potential to provide a stable three-dimensional scaffold which is capable of supporting axonal regeneration in the damaged CNS.

Animals↗