Sarojini Vijayasekaran

Poly(2-hydroxyethyl methacrylate) sponges as implant materials: in vivo and in vitro evaluation of cellular invasion

The pore size and the in vivo behaviour of four poly(2-hydroxyethyl methacrylate) sponges were investigated. The sponges were synthesized by polymerization of monomer in 70, 80 and 90 wt% water, respectively. In one of the formulations, a high amount of initiator was added. The average pore diameter was calculated with Ferrys equation and the results compared to those obtained by examination of samples using environmental scanning electron microscopy. The use of the equation greatly underestimated the size of pores. We also showed that the pores in polymers obtained in 70 wt% water were not interconnected, whilst the pores in polymers obtained in 80 and 90 wt% water, respectively, were larger and interconnected throughout the polymer. When implanted subcutaneously in rabbits, only the latter polymers allowed invasion and proliferation of cells. Penetration and proliferation of cells in these sponges were also assessed by an in vitro method using cultured human fibroblasts. The procedure included the overlaying of a glass plate covered by confluent cultured cells on to the surface of polymer impregnated with collagen. The depth of migration and number of sections needed to be cut to count a fixed number of invading cells were measured after incubation for 2 wk and used as indicators in comparing the ability of various sponges to allow cellular invasion. The assay showed that more cells invaded a hydrogel sponge produced in 80 wt% water than one produced in 90 wt% water. It also showed that the cut polymer surfaces allowed a greater cellular invasion than the moulded ones.

Development and clinical assessment of an artificial cornea

Keratoprosthesis research has been a gradual, rather fragmentary process with advances being made by isolated groups of researchers. This has arisen partly because of poor funding in the area; research groups which have achieved commercial support have often had constraints upon the full disclosure of their findings. Despite these difficulties there has been real progress over the last decade by several independent groups. This article concentrates upon our own development of a hydrogel core-and-skirt keratoprosthesis, the Chirila KPro, in order to illustrate the scientific and clinical problems common to keratoprosthesis research. Pilot data from a clinical trial is presented and the priorities for future research are discussed.

The Chirila Keratoprosthesis: phase I human clinical trial

OBJECTIVE To undertake a preliminary safety and performance evaluation of an artificial cornea, the Chirila Keratoprosthesis, in human patients. DESIGN A prospective, interventional case series. PARTICIPANTS Fourteen consecutive patients with blindness of corneal origin not treatable by repeated standard penetrating keratoplasty. METHODS Keratoprostheses were manufactured and implanted. The patients, all with preoperative visual acuity of light perception to count fingers (CF), were followed clinically in adherence to a protocol. MAIN OUTCOME MEASURES Safety (keratoprosthesis retention, incidence of serious complications) and performance (visual acuity, comfort, appearance). RESULTS Ninety-three percent of keratoprostheses were retained to the date of reporting, up to 2.5 years. One keratoprosthesis (7%) was removed in a manner that restored the patients preoperative condition. All but one patient maintained their preoperative level of visual acuity or improved on it, with most achieving their estimated full potential visual acuity, (range, count fingers - 20/20). CONCLUSIONS This keratoprosthesis is acceptably safe and has demonstrated an ability to restore vision in cases in which alternative management would have had a poor prognosis. More extensive trials are warranted.

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.

Poly(I-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.

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.

Effect of triamcinolone acetonide on vascular endothelial growth factor and occludin levels in branch retinal vein occlusion.

PURPOSE To investigate the molecular mechanism by which triamcinolone acetonide (TA) may reduce edema in a porcine model of branch retinal vein occlusion (BRVO). DESIGN Animal study. METHOD After baseline ophthalmoscopic examination and fundus photography, a BRVO was created photothrombotically in each eye of 6 pigs, using argon green photocoagulation and intravenous Rose Bengal. Following this, the left eye was injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined. Fluorescein angiography, in addition to ophthalmoscopy and fundus photography, was performed. Following sacrifice of the animals, the eyes were enucleated and processed. The distribution of vascular endothelial growth factor (VEGF), occludin, and glial fibrillary acidic protein (GFAP) were localized by immunofluorescence cytochemistry on 10 microm frozen retinal sections of TA-treated and untreated eyes. RESULTS Retinal VEGF levels were significantly lower in the TA-treated eyes as compared with the untreated eye (P = .002). Conversely occludin levels were significantly higher in the treated eye (P = .026). There was also a significant reduction in GFAP immunoreactivity in the Muller cells of the treated eyes (P = .015) with no statistical significance in the astrocytes (P = .065). CONCLUSION Intravitreal TA down regulates VEGF, which may prevent a decrease in occludin and also inhibits an increase in GFAP expression in Muller cells. These events may contribute to a reduction in the blood retinal barrier breakdown that occurs in BRVO and promote resolution of the associated retinal edema.

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.

Synthetic Polymers as Materials for Artificial Vitreous Body: Review and Recent Advances:

In spite of a century of recorded attempts to replace the vitreous body of the eye with a foreign material, there is no permanent substitute currently available, and the success of some materials as temporary substitutes is still very limited. Among the large number of materials proposed or tested as vitreous substitutes, synthetic polymers have been episodically considered during the past four decades. This article will review these attempts, their outcomes, and recent progress in this field. There were only seven synthetic polymers and a few semisynthetic polymers (modified biopolymers) so far proposed or tested as vitreous substitutes. It appears that probably a synthetic hydrogel with very high water content would eventually be the material of choice for a permanent vitreous substitute, but the ideal material has not yet been found.

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.Purpose We compared the in vitro susceptibility of gram-positive bacteria to ciprofloxacin and ofloxacin using human corneal susceptibility levels. Methods The concentrations of ciprofloxacin and ofloxacin that can be attained in 99% of human corneas (Cornea99) after topical administration were calculated statistically from reported data. The minimal inhibitory concentrations (MICs) were determined for 95 corneal isolates of gram-positive bacteria (51 Staphylococcus aureus, 16 Streptococcus pneumoniae, 16 Streptococcus viridans group, and 12 coagulase-negative staphylococci). Susceptibility was interpreted by comparing the MICs with the respective Cornea99 for each antibiotic. Time-kill studies of representative gram-positive bacteria were tested using the Cornea99 and the maximal corneal concentrations reported for ciprofloxacin and ofloxacin. Results The Cornea99 of ciprofloxacin and ofloxacin were calculated to be 3.57 μg/ml (n = 22) and 2.22 μg/ml (n = 20), respectively. The reported mean corneal concentrations of ciprofloxacin (6.90 ± 6.20 μg/ml) and ofloxacin (5.71 ± 6.15 μg/ml) were comparable (p = 0.54). All gram-positive bacteria were equally susceptible to ciprofloxacin and ofloxacin (p = 0.54) based on the Cornea99. The time-kill studies determined that 8–24 h were required for both ciprofloxacin and ofloxacin to reach bactericidal levels. Conclusion Ciprofloxacin and ofloxacin demonstrated comparable penetration into the cornea and provided equivalent in vitro efficacy against representative gram-positive bacteria. Time-kill studies indicated that 8–24 h of continual corneal concentrations of ciprofloxacin and ofloxacin were necessary in this study to reduce susceptible gram-positive bacteria by 99.9%.