Preferential interactions of calcium ions in poly(2-hydroxyethyl methacrylate) hydrogels.

An investigation of the preferential interaction of calcium ions with oxygen atoms in poly(2-hydroxyethyl methacrylate) (PHEMA)-based hydrogels has been carried out. The formation of polymer-Ca complexes was achieved by exposing powdered or fully hydrated samples with 5 mM, 0.1-0.5 M, or saturated CaCl2 solutions for certain periods of time. The characteristics of the polymer-Ca complexes were deduced from the effect of the solute on the equilibrium water content, and from NMR, atomic absorption and infrared spectroscopies. The absence of significant changes in the NMR chemical shift and infrared vibrational wavenumbers for the various functional groups confirmed that polymer complexation with Ca2+ ions involves only weak interactions, possibly electrostatic or ion-dipole interactions. Among the three types of oxygen atoms in PHEMA, hydroxyl oxygen atoms seem to be the most sensitive to the presence of Ca2+ ions. Complexation at the ester oxygen atoms was also evidenced by a new band in the infrared spectra at 1,550 cm(-1). On the other hand, there were no indications that the hydrophobic domains in the backbone and the methyl groups at the side chain of PHEMA interact significantly with Ca2+ ions.

In-vitro study of the spontaneous calcification of PHEMA-based hydrogels in simulated body fluid.

In-vitro calcification of poly(2-hydroxyethyl methacrylate) (PHEMA)-based hydrogels in simulated body fluid (SBF) under a steady/batch system without agitation or stirring the solutions has been investigated. It was noted that the formation of calcium phosphate (CaP) deposits primarily proceeded through spontaneous precipitation. The CaP deposits were found both on the surface and inside the hydrogels. It appears that the effect of chemical structure or reducing the relative number of oxygen atoms in the copolymers on the degree of calcification was only important at the early stage of calcification. The morphology of the CaP deposits was observed to be spherical aggregates with a thickness of the CaP layer less than 0.5 microm. Additionally, the CaP deposits were found to be poorly crystalline or to have nano-size crystals, or to exist mostly as an amorphous phase. Characterization of the CaP phases in the deposits revealed that the deposits were comprised mainly of whitlockite [Ca(9)MgH(PO(4))7] type apatite and DCPD (CaHPO4.2H2O) as the precursors of hydroxyapatite [Ca(10)(PO(4))6(OH)2]. The presence of carbonate in the deposits was also detected during the calcification of PHEMA based hydrogels in SBF solution.

Corneal replacement using a synthetic hydrogel cornea, AlphaCor: device, preliminary outcomes and complications.

PURPOSE: Clinical assessment of outcome of corneal replacement with a synthetic cornea, AlphaCor, in patients considered at too high risk for conventional penetrating keratoplasty with donor tissue to be successful, but excluding indications such as end-stage dry eye that might be suited to traditional prosthokeratoplasty. METHODS: All patients in the multicentre clinical trial were managed according to an approved protocol, with Ethics Committee approval in each centre. Preoperative visual acuity ranged from perception of light (PL) to 6/60 (20/200). Implantation was by means of an intralamellar technique, with a conjunctival flap in most cases. Tissues anterior to the optic were removed as a secondary procedure. RESULTS: Up to 30 November 2001, 40 AlphaCor devices had been implanted in 38 patients, of mean age 60 years. Follow-up ranged from 0.5 months to 3 years. There had been one extrusion (2.5%) and four cases (10%) where a device had been removed due to melt-related complications. All five of these cases received a donor corneal graft after the device was removed, with these grafts remaining anatomically satisfactory and epithelialised to date. Corneal melts in AlphaCor recipients were found to be strongly associated with a history of ocular herpes simplex infection. Two further devices (5%) were removed owing to reduced optic clarity after presumed drug-related deposition, and have been successfully replaced with second devices. Mean preoperative best-corrected visual acuity was hand movements. Visual acuities after surgery ranged from PL to 6/6(-2) (20/20(-2)). CONCLUSIONS: Early results suggest that the AlphaCor, previously known as the Chirila keratoprosthesis (Chirila KPro), has a low incidence of the complications traditionally associated with keratoprostheses and can be effective in restoring vision in patients considered untreatable by conventional corneal transplantation. Importantly, the device can be replaced with a donor graft in the event of development of a significant complication. A history of ocular herpes simplex is a contraindication to AlphaCor implantation. Ongoing monitoring of clinical outcomes in all patients will allow the indications for AlphaCor, as opposed to donor grafts, to be determined.

An overview of the development of artificial corneas with porous skirts and the use of PHEMA for such an application.

An overview of the efforts to develop functional polymeric artificial corneas (keratoprostheses) by incorporating a porous skirt is presented. The development of such a device by the author's group using poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels, as a combination of their homogeneous and heterogeneous states, and the rationale of this choice are also discussed. The latest results of the clinical trials with the PHEMA keratoprosthesis in human patients indicate a lower risk of the complications traditionally associated with the implantation of artificial corneas.

Synthetic hydrogels as carriers in antisense therapy: preliminary evaluation of an oligodeoxynucleotide covalent conjugate with a copolymer of 1-vinyl-2-pyrrolidinone and 2-hydroxyethyl methacrylate.

A major challenge of the antisense therapeutic strategies is the development of improved systems for the delivery of antisense oligodeoxynucleotides (AS ODNs) in order to enhance the cellular uptake, to assure a better efficiency in reaching the target tissue, and to provide sustained delivery over longer periods of time. Because the current methods for delivery (liposomes and cationic polymers) present some disadvantages, the attention was directed toward the use of neutral polymers as carriers for the AS ODNs. Based on our previous work on synthetic hydrogels for vitreous substitution, we developed a poly[1-vinyl-2-pyrrolidinone-co-(2-hydroxyethyl methacrylate)] hydrogel as a potential carrier for AS ODNs. We have previously demonstrated that such hydrogels are not cytotoxic, and they may have growth-promoting effects on cultured fibroblasts. This copolymer also has the advantage of being injectable. In this study, a specific AS ODN was synthesized and then covalently bound to the copolymer via carbodiimide coupling method. The resulting conjugate was subjected to in vitro release experiments over 46 days in the presence of bovine vitreous humor. Compared with the control (no enzyme present), a significant amount of covalently bound ODN was released from the ODN-hydrogel conjugate, suggesting the possibility of using such systems for the sustained delivery of AS ODNs.

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: a 3-month study.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.

Hydrophilic sponges based on 2-hydroxyethyl methacrylate: part VII: modulation of sponge characteristics by changes in reactivity and hydrophilicity of crosslinking agents.

Despite previous unsuccessful attempts to use hydrated poly(2-hydroxyethyl methacrylate) sponges as implantable biomaterials, recently these materials became important as peripheral components in an artificial cornea of the core-and-skirt design. The low mechanical strength of sponges prompted this study on possible improvement of tensile properties by the use of a variety of crosslinking agents. Three vinylic (dimethacrylates) and two allylic compounds were used at different concentrations (0.1 to 2% (mol)) as crosslinking agents in the production of sponges. Their influence on the mechanical properties, porous morphology and swelling behavior of resulting sponges was evaluated. The onset of phase separation during polymerization was also measured by visible spectrophotometry. The results suggested an inherent heterogeneity of sponges, i.e. pores of non-uniform size and structural inhomogeneities. While the effects of changes in the nature and concentration of crosslinking agents on the equilibrium water content of sponges were ambiguous, some of the mechanical properties, such as toughness and elasticity, were improved by crosslinking with allylic agents. Scanning electron microscopic examination suggested that the mechanical effect is related to the variation of size of the polymer particles constituting the sponge structure, which was proved to be dependent upon the onset of phase separation during polymerization.

Swelling behavior and mechanical properties of chemically cross-linked gelatin gels for biomedical use.

Cross-linked gelatin gels are used as biomaterials in living tissues, either as bioadhesives or as devices for sustained drug release. As these applications involve surgical insertion of gels, the effect of cross-linking on mechanical properties is relevant. The effect of cross-linking on the gel water uptake is also relevant for the kinetics of drug release. Equally important is the influence of initial amounts of gelatin in the gelling solutions. We investigated the influence of cross-linking (with glutaraldehyde) and of gelatin content upon the equilibrium water content and tensile properties of resulting gels. A range of gels was prepared with gelatin contents of 10 to 40% wt, and cross-linked with 2.5 to 50% wt glutaraldehyde. The increase in gelatin content and degree of cross-linking reduced the water uptake of gels from about 60-65% wt to about 50% wt. The tensile characteristics were differentially affected. While the increase of cross-linking induced a decrease of toughness and elasticity and an increase of stiffness, it did not affect the ultimate strength of gels. On the contrary, the increase of gelatin content induced a definite increase of the ultimate strength but did not significantly affect the other properties.

Development of a poly(2-hydroxyethyl methacrylate) orbital implant allowing direct muscle attachment and tissue ingrowth.

PURPOSE: To develop a poly(2-hydroxyethyl methacrylate) orbital implant that allows tissue ingrowth and direct muscle attachment to minimize the risk of extrusion and to enhance cosmesis. METHODS: Assessment of clinical outcomes and histologic findings after implantation of 18 prototype prostheses into rabbits. The implants were not wrapped with other tissues or materials. RESULTS: One case of infection was observed but there were no extrusions, with up to 21 months follow-up. Biocolonization was confirmed histologically. Good movement was observed when a cosmetic shell was fitted. CONCLUSIONS: The prototype prosthesis appears promising, with particular advantages being the direct attachment of extraocular muscles, good cosmesis and movement, and a low complication rate in this pilot study.

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.

Synthesis, physical characterization, and biological performance of sequential homointerpenetrating polymer network sponges based on poly(2-hydroxyethyl methacrylate).

A limitation in the use of hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) sponges as implantable devices is their inherently poor mechanical strength. This precludes proper surgical manipulation, especially in the eye where the size of the implant is usually small. In this study a new method was developed to produce mechanically stronger PHEMA sponges. Sequential homointerpenetrating polymer network (homo-IPN) sponges were made by using HEMA as the precursor for generating both the first network and the successive interpenetrated networks. Following the formation of network I, the sponge was squeezed to remove the interstitial water, soaked in the second monomer (also HEMA), and squeezed again to remove the excess monomer from the pores before being subjected to the second polymerization leading to the formation of network II. Two two-component IPN sponges (K2 and K4) with increasing HEMA content in the network II and a three-component IPN sponge (K3) were produced, and their properties were compared to those of a homopolymer PHEMA sponge (control). Apart from elongation, the tensile properties were all significantly enhanced in the IPN sponges; the water content was the same as in the control sponge, except for sponge K4, which was lower. Light microscopy revealed similar pore morphologies of the control and IPN sponges K2 and K3, and the majority of the pores were around 25 microm. Sponge K4 displayed smaller pores of around 10 microm. Cellular invasion into the sponges was examined in vitro (incubation with 3T3 fibroblasts) and in vivo (implantation in rabbit corneas). Although the in vitro assay detected a change in the cell behavior in the early stage of invasion, which was probably due to the formation of IPNs, such changes were not reflected in the longer term in vivo experiment. There was a proper integration of sponges K2 and K3 with the corneal stroma, but much less cellular invasion and no neovascularization in sponge K4. We concluded that IPN formation is a valid method to enhance the strength of PHEMA sponges, provided that the content of HEMA in the successive networks is not too high.

The origins of the artificial cornea: Pellier de Quengsy and his contribution to the modern concept of keratoprosthesis.

There has been little recognition of the French ophthalmologist Guillaume Pellier de Quengsy and his contribution to the problem of artificial cornea (keratoprosthesis). This fact that he was the first to propose such a device was seldom acknowledged, and usually as a secondary reference. Based on the examination of original texts (1789), this study demonstrates that Pellier not only proposed an essentially correct keratoprosthesis, but also suggested a porous prosthetic skirt, a revolutionary concept which is currently fundamental to artificial cornea research.

Correlation of histological findings with gadolinium enhanced MRI scans during healing of a PHEMA orbital implant in rabbits.

BACKGROUND/AIMS: To investigate a poly(2-hydroxyethyl methacrylate) (PHEMA) orbital implant with a spongy anterior hemisphere and a smooth gel posterior hemisphere, by histology correlated with magnetic resonance images. METHODS: Following enucleation, eight rabbits received PHEMA implants to which the muscles were directly sutured, and underwent gadolinium enhanced magnetic resonance imaging (MRI) from 3 to 52 weeks. After the rabbits were killed, the implants were removed, cut in a plane corresponding to the scan, and processed for light and electron microscopy. RESULTS: All eight rabbits retained their implant to the end of the study period without complications. The scans demonstrated muscle attachment to the anterior half of the implant, and enhancement was seen on injection of gadolinium chelate. Histology confirmed muscle attachment, and cellular and vascular ingrowth. Over time, a transformation from reactive inflammatory to relatively non-vascular scar tissue was seen within the implant. Calcium deposits in one implant were detected by imaging and histology. CONCLUSION: The implants are readily visualised on MRI. Muscle attachment and fibrovascular ingrowth into the anterior hemisphere are seen, while encapsulation of the posterior hemisphere is minimal. Histological findings confirm the progress of the healing response, with initial inflammation and marked vascularisation, developing later into quiescent scar tissue predominantly of fibroblasts.

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.

Implantation of collagen IV/poly(2-hydroxyethyl methacrylate) hydrogels containing Schwann cells into the lesioned rat optic tract.

Poly (2-hydroxyethylmethacrylate) (PolyHEMA) hydrogels, when combined with extracellular matrix molecules and infiltrated with cultured Schwann cells, have the capability to induce CNS axonal regrowth after injury. We have further investigated these PolyHEMA hydrogels and their potential to bridge CNS injury sites. Collagen IV-impregnated hydrogels containing Schwann cells were implanted into the lesioned optic tract in 14 rats. On examination 2-4 months later, there was good adherence between the implants and CNS tissue, and large numbers of viable Schwann cells (S100+, GFAP+, Laminin+, and LNGFR+) were seen within the hydrogel matrices. Immunohistochemical analysis showed that the collagen IV-impregnated PolyHEMA hydrogels preferentially supported the transplanted Schwann cells and not host glial cells such as astrocytes (GFAP+) or oligodendroglia (CAII+). Macrophages (ED1+) were also seen within the sponge structure. Eighty-three percent of the implanted hydrogels contained RT97+ axons within their trabecular networks. Regrowing axons were associated with the transplanted Schwann cells and not with the small number of infiltrating astrocytes. RT97+ axons were traced up to 510 microm from the nearest host neuropil. These axons were sometimes myelinated by the transplanted Schwann cells and expressed the peripheral myelin marker Po+. WGA/HRP-labeled retinal axons were seen within transplanted hydrogel sponges, with 40% of the cases growing for distances up to 350-450 microm within the polymer network. The data indicate that impregnating PolyHEMA sponges with collagen IV can modify the host glial reaction and support the survival of transplanted Schwann cells. This study thus provides new information on how biomaterials could be used to modify and bridge CNS injury sites.

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.

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.

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.

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.