Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

the disadvantages of the LASEK procedure, which include post-operative discomfort and delayed visual recovery resulting from the 4–7 day epithelial closure time (Taneri et al., 2004).

In epi-LASIK, the epithelial sheet is removed mechanically using an epikeratome with a blade to avoid the use of alcohol. The cleavage plane and the viability of the epithelial sheet after epi-LASIK procedures have been examined by a range of techniques (Tanioka et al., 2007; Chen et al., 2008; Choi et al., 2008a; Choi et al., 2008b). Immunohistochemistry of the epithelial flap has shown that the cleavage plane in epi-LASIK procedures depends on the type of epikeratome used in human eyes (Choi et al., 2008b). A comparison of different commercially available epikeratomes using immunohistochemistry and electron microscopy has shown that each epikeratome tested had successfully cleaved through the basement membrane zone without damage to the stromal surface in the pig and human corneas used (Herrmann et al., 2008). Trypan Blue staining of the epithelial flaps created in the abattoir-sourced pig eyes in this study revealed minimal damage to the cells of the central portion of the flaps (with <12% damaged) but this outcome might be different in the corneas of live subjects where the epithelium is more difficult to cleave. In vivo confocal microscopy used to monitor wound healing in patients following epi-LASIK treatment for myopia showed that cells in most of the epithelial flaps were damaged during the first few days and were rapidly replaced by new cells during the healing process (Chen et al., 2008). The possibility that the presence of the flap of epithelium, whether vital or not, might confer any benefit – such as reduction in post-operative pain, promotion of epithelial healing and/or impact on apoptosis of keratocytes in the underlying stroma – is the subject of current discussion. The role of the epithelial flap in epi-LASIK was the subject of a recent study in 56 human subjects being treated for myopia where the flap was replaced in one eye and removed in the contralateral eye (Kalyvianaki et al., 2008). Both techniques revealed similar epithelial wound-healing responses with equivalent haze scores, and visual and refractive outcomes but lower subjective pain scores were noted at the 2 hours post-operative time point in the eyes without flaps, indicating that retention of the flap may not offer benefit in this respect. This is consistent with earlier studies that compared various epithelial removal techniques for PRK and showed that post-operative pain, sub-epithelial opacity and visual acuity were similar regardless of the epithelial removal procedure (Lee et al., 2005; Sakimoto et al., 2006). It has been suggested that combining these techniques of LASEK and epi-LASIK, using alcohol with an epikeratome, may improve the quality of the epithelial flap and hinge (Camellin and Wyler, 2008).

Butterfly LASEK is another method of epithelial removal for surface ablation; the method was introduced to try and improve the viability of the epithelial flap tissue to facilitate wound healing and post-operative recovery.

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This technique involves making two epithelial pockets created on either side of a central corneal epithelial incision where the epithelium has been eased back using alcohol to expose the stromal surface for laser ablation (Vinciguerra and Camesasca, 2002; Vinciguerra et al., 2003). Interestingly, re-epithelialisation of the stromal surface occurs more rapidly after PRK without the use of alcohol, than with butterfly LASEK where alcohol is used, and PRK also showed lower post-operative pain scores (Ghanem et al., 2008).

LASIK arose in the 1990s and combined photoablation using the excimer laser with a lamellar cut to create a hinged corneal flap aimed at preserving the integrity of the central anterior cornea including an intact epithelium (Pallikaris et al., 1990). The procedure involved use of the microkeratome to cut through the central cornea creating a corneal flap approximately 120–180 microns thick and 8–10 mm in diameter. The flap was lifted to expose the underlying stroma for ablation with the laser to achieve the desired change of shape and the flap was repositioned without sutures. LASIK is currently the most frequently performed refractive surgical procedure; reports indicate that

700 000 procedures are performed annually and over 15 million people are treated using LASIK worldwide, with 6 million of those in the USA (AAO,

2008). The popularity of this laser-based refractive procedure is related to the perceived advantages for the patient – including rapid visual rehabilitation, reduced post-operative discomfort and a reduced wound-healing scenario that is likely to provide a more stable outcome (Ambrósio et al., 2008).

Problems with LASIK are associated with the creation of a hinged flap in the anterior one-third of the cornea and include dry eye, glare, epithelial ingrowth, corneal haze and diffuse lamellar keratitis (Kramer et al., 2005; Sandoval et al., 2005). LASIK-associated dry eye is the most commonly reported problem affecting approximately 50% (Ambrósio et al., 2008) to

95% (Sandoval et al., 2005) of LASIK patients. The fact that not all LASIK patients suffer from dry eye is curious and may be related to factors such as the size and thickness of the corneal flap, the depth of ablation with the laser, underlying sub-clinical conditions and/or the questions asked in surveys. Dry eye symptoms are caused by a combination of transection of corneal nerve axons with the microkeratome changing the function of the lacrimal gland–ocular surface unit and an altered distribution of the tear film due to the changed corneal curvature (Ambrósio et al., 2008). Inflammation caused by LASIK may also contribute to dry eye and would explain the reported efficacy of treatment with topical anti-inflammatory drugs such as cyclosporine A for up to 6 months after surgery (Ambrósio et al., 2008). This timing correlates with studies that showed that the return of corneal sensation, substantially diminished immediately after LASIK, was restored over the 6–12 months that it took the sub-epithelial and sub-basal nerve plexus to regrow. However, some studies report that the total length and

morphology of nerve fibres after LASIK is never completely restored to pre- operative levels (Vesaluoma et al., 2000; Lee et al., 2002; Dawson et al., 2005; Moilanen et al., 2008). Confocal microscopy of post-LASIK corneas out to 12 months has revealed decreased keratocyte numbers on both sides of the lamellar cut (Vesaluoma et al., 2000; Mitooka et al., 2002), increased numbers of activated keratocytes compared with PRK (Mitooka et al., 2002) and undulations in Bowman’s layer (Vesaluoma et al., 2000; Mitooka et al., 2002). Histology of post-LASIK corneal tissue has shown incomplete stromal wound healing and scar formation months to years after procedures (Anderson et al., 2002; Dawson et al., 2005; Schmack et al., 2005). Issues with flap thickness related to the difficulties of cutting of the cornea with a mechanical microkeratome underlie many of the problems observed with

LASIK (Binder, 2006). Additionally, biomechanical issues are of concern with LASIK (and PRK) procedures, as chronic interlamellar and interfibrillar slippage akin to keratoconus may result in biomechanical failure and ectasia (Pallikaris et al., 2001; Dawson et al., 2008).

Some of these issues may be reduced by the development of new instruments such as the femtosecond laser introduced in 2002 (e.g. the IntraLase FS laser from IntraLase Corp, USA), which allows the surgeon greater precision and consistency in cutting predictable and uniform flaps in the cornea (Binder, 2006). The use of lower raster energy levels with femtosecond LASIK procedures reduces keratocyte activation and postLASIK haze (Petroll et al., 2006; Netto et al., 2007). Newer versions of the femtosecond laser that require less energy to make flaps combined with laser software updates may improve flap accuracy and outcomes in the future. Thin-flap-assisted-in-situ-keratomileusis uses the femtosecond laser to make a much thinner customised corneal flap just beneath Bowman’s layer in a procedure named ‘sub-Bowman’s keratomileusis’ (SBK). This is aimed at reducing the flap thickness, thereby leaving more residual stromal tissue which enables safer/greater ablation of stromal tissue in high myopes. SBK also reduces the corneal biomechanical stability effects reported with conventional LASIK procedures and reduces the pain associated with PRK techniques (Azar et al., 2008; Slade, 2008). Some regard this technique as the new generation of corneal surgery. Femtosecond lenticule extraction (FLE or FLEX) is a new approach for myopia that uses the femtosecond laser to create a flap and carve a stromal lenticule, which is manually removed from the cornea to give the desired correction (Sekundo et al., 2008). Overall, there is still a need for alternative or parallel approaches that offer the cosmesis and convenience of the refractive surgeries, but which are more predictable and reversible with no permanent damage to the corneal structure and tissue.

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4.4Additive approaches to correct refractive error: corneal implants

An alternative approach is to correct refractive errors by inserting an implant made from synthetic and/or biological materials into the corneal tissue. Changed corneal power can be achieved by altering the curvature of the anterior corneal surface by using a pre-shaped implant made from an iso-refractive material, or by inserting a lens made from a material with a different refractive index than the corneal tissue, or by a combination of these approaches. Additive technologies have the potential to correct hyperopia, myopia, astigmatism and presbyopia. The implant may be placed within the stromal tissue (keratophakia, intracorneal rings, intracorneal lens, corneal inlay) or immediately beneath the epithelium (epikeratophakia, subepithelial, corneal onlay). Additive technologies offer several advantages over currently used subtractive refractive surgical techniques, paramount of which is the fact that the implant may be removed, making the procedures adjustable and reversible.

4.4.1Early use of materials for corneal implants

The original work on corneal implants was performed to explore new treatments for clinical problems and to correct refractive error. The choice of materials was based on what was available at the time that offered good optical properties and might be tolerated by corneal tissue. Barraquer (1949) first demonstrated that it was possible to alter corneal curvature, with the potential to correct refractive errors, by surgically implanting lenses of biconvex flint glass and Plexiglas into the corneal stroma of rabbits using a freehand lamellar pocket. Both materials were solid and, while tissue posterior to the implant remained clear, tissue forward of the lenses developed necrosis and resulted in extrusion of the implants. Despite the clinical failure, the outcome was instructive as Barraquer recognised that lenses made of impermeable materials blocked a metabolic exchange that occurred from the posterior to anterior cornea, which was essential to the health of corneal tissue forward of the implanted lens. During this time, Krwawicz (1960) implanted impermeable plastic lenses in rabbits, which eventually eroded out, but they demonstrated that corneal curvature could be altered by an intracorneal implant. These outcomes with impermeable materials informed clinicians such as Choyce, Brown and Dohlman of the opportunity to use membranes to reduce the fluid flow from the aqueous humour through the cornea, which would enable treatment of conditions such as endothelial dystrophies, which resulted in stromal and epithelial oedema (Brown and Dohlman, 1965; Choyce, 1968;

Choyce, 1982). Silicone membranes implanted in rabbits to test this idea did result in dehydration of the stromal tissue anterior to the implant (Brown and

Dohlman, 1965). These investigators also noted that the material had good qualities as it was transparent, flexible and able to be sterilised. In order to address the issue of permeability in intracorneal implants for the correction of refractive error, Barraquer implanted a ‘large size interlamellar inclusion with a large central perforation’ to allow for metabolic exchange that was aimed at modifying the corneal curvature to alter its power (Barraquer,

1966). The clinical failure of these implants was attributed to their large size and to the fact that they were a different shape to the cornea and had exerted compression on the corneal tissue. A greater understanding of the causes of failure of impermeable materials implanted in the cornea came later from modelling work presented originally by Maurice (1969) and refined by

McCarey and Schmidt (1990), which considered the movement of glucose through the corneal tissue in the presence of an intracorneal implant made from a non-permeable material. Modelling showed that an impermeable material could only allow glucose into the stromal tissue anterior to the implant via lateral flow if the intracorneal lens was implanted deep in the stroma (McCarey and Schmidt, 1990).

4.4.2Corneal implants made from biological tissue

Barraquer concluded in his article that the best material to be included in the cornea was ‘the corneal parenchyma itself’ (Barraquer, 1966) and we note that this is a permeable material. Barraquer went on to question issues of maintaining corneal transparency if a tissue lenticule was used, the possibility of the lenticule being reabsorbed, the cells of the recipient invading the lenticule, the size of the ‘inclusion’ and the possibility that the cornea might modify its form. These ideas were tested by implanting lenticules of corneal tissue in interlamellar pockets in the stroma of rabbits which were maintained for up to one year (Barraquer, 1966). Clinical data from this trial showed changed curvature of the anterior face of the cornea which was ‘in perfect relation to the curve of the lenticule included in the thickness of the cornea’ (Barraquer, 1966). Histological evaluation showed that donor cells had died and the lenticule was slowly repopulated with cells of the host. Problems with the lathing of the tissue lenticules were noted and it was suggested that lenticules should be of minimal thickness and be implanted as superficially as possible (Barraquer, 1966). Later, lenticules were cut from the patient’s own tissue, which was frozen and lathed to the desired shape, then sutured back into the patient’s cornea, giving that cornea new refractive power in a procedure named ‘keratomileusis’. Some patients were implanted with desiccated positive lenticules cut from human corneal stroma using a superficial interlamellar pocket and were maintained for up to 11 months (Barraquer, 1966). The outcomes of the human trial were similar to the rabbit trial except for the presence of some opacities that formed at the

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lenticule interface and a less marked change in corneal curvature, which was attributed to the ‘lesser elasticity’ of the rabbit tissue due to the absence of Bowman’s layer present in the human cornea. Barraquer named this procedure of implanting tissue lenticules into the cornea ‘keratophakia’ (Barraquer,

1966). Significantly, he suggested that it should be possible to manufacture lenticules ‘with a foreign substance’, possibly with a high refractive index, which would allow for diminished thickness of the intracorneal implant

(Barraquer, 1966).

In the early 1980s, Kaufman and Werblin modified the keratophakia technique to a more superficial procedure named ‘epikeratophakia’, aimed at correcting refractive error associated with aphakia by placing a lenticule of donor stromal tissue lathed to a specified dioptric power on to the debrided surface of the recipient’s cornea to be incorporated into the cornea by the regrowth of the epithelium (Kaufman, 1980; Werblin and Kaufman, 1981; Werblin and Klyce, 1981). This technique offered several advantages over keratophakia including a simple surgical technique, minimal damage to the central optical zone, maintaining central Bowman’s intact and the possibility of reversibility (Kaufman, 1980; McDonald and Dingeldein, 1988). An early study sutured shaped donor lenticules on to debrided primate corneas with an annular keratectomy groove, which were maintained for up to 25 months (Yamaguchi et al., 1984a; Yamaguchi et al., 1984b). Histology showed that the host epithelium covered the donor lenticules with hemidesmosomes formed at the epithelial–lenticule interface. Interestingly, they also found that the host keratocytes repopulated the donor lenticule through surgical breaches of Bowman’s layer in the host. Clinical data from later epikeratophakia studies by McDonald et al. (McDonald et al., 1987; McDonald and Dingeldein,

1988) showed that a predictable optical correction was not achieved because of inaccuracies with stromal tissue cryolathing associated with the effect of freezing and thawing of allograft tissue, remodelling of the implanted lens tissue, graft haze and difficulties in maintaining epithelial cover. Other studies – which tested the potential of epikeratoplasty in the treatment of aphakia, myopia and/or keratoconus – revealed stromal scarring (Lass et al.,

1987) and difficulties in maintaining epithelial growth over the surface of the tissue lenticule (Lass et al., 1987; Rao et al., 1987) that were attributed to the possibility of corneal hypesthesia (diminished nerve response) (Rao et al., 1987). The main reason for clinical failure of epikeratophakia was imperfect re-epithelisation of the implanted tissue lenticule (Young et al., 1994). Failed human epikeratoplasty lenticules were histologically examined and structural abnormalities were identified at all levels of the donor lenticules including irregular epithelium, changes in the basement membrane, focal breaks and undulations in Bowman’s layer, changes in keratocyte population and stromal collagens (Binder and Zavala, 1987). Another histological study on failed epikeratoplasty tissue removed from patients treated for keratoconus

and aphakia revealed corneal scarring and accumulation of electron-dense material associated with the keratocytes around the implanted stromal tissue (Grossniklaus et al., 1989). This outcome was similar to that reported on explanted tissue from epikeratoplasty used in the treatment of keratoconus, which showed problems with epithelial irregularity and sub-epithelial fibrosis as well as folds in Descemet’s membrane and stromal scars (Rodrigues et al., 1992).

Biological materials other than corneal stroma have also been considered as intracorneal implants to correct refractive error. In one study, human placental collagen IV stabilised by aldehyde treatment was implanted in the corneas of dogs for 2 years and showed good clinical biocompatibility with clear corneas and no inflammatory response (Dupont et al., 1989). Histology conducted on implanted corneas showed thinned epithelium forward of the implants, degenerated keratocytes near the implant and foreign material at interfaces. Alternative materials were also considered including collagen types I, III, or IV, collagen–hydrogel copolymers, bioactive synthetics, and coated hydrogels, such as poly(2-hydroxyethyl methacrylate) (PHEMA) (Thompson et al., 1991). It was recognised that a suitable material should offer optical clarity, support of epithelial migration and adhesion, be permeable to glucose and other solutes and not be degraded (Thompson et al., 1991). In addition, it was also suggested that attachment strategies to fix the synthetic implants to the cornea without cutting Bowman’s layer (such as adhesives) might improve the efficacy of the procedure, named ‘synthetic epikeratoplasty’

(Thompson et al., 1991). A study of human placental collagen IV discs, stabilised by aldehyde treatment, implanted in the corneas of rhesus monkeys using the epikeratoplasty technique ensued (Thompson et al., 1993). These implants remained clear and maintained epithelial cover for up to 30 months with some formation of cell–matrix junctions at the epithelial–lenticule interface during that time, but all showed degradation of the lenticule with time that involved neutral protease activity which degraded the collagen. As with the use of tissue lenticules, the stability of the collagen material in the corneal environment was recognised as an issue that needed to be addressed. Problems with the use of tissue lenticules in keratophakia and epikeratophakia procedures focused attention on alternative materials such as those of synthetic origin, and alternative designs and surgical procedures in the intracorneal implant area.

4.4.3Corneal implants made from impermeable synthetic materials

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lenses (Barraquer, 1966). Researchers at the time recognised polysulphone as a candidate material as it had a high refractive index (1.633) permitting the creation of thin lenses to give refractive correction without altering the shape of the corneal surface. In addition, polysulphone had excellent optical properties, could be easily moulded or lathed, absorbed ultraviolet and infrared light, and was already known to be biocompatible for biomedical applications, including intraocular lenses (McCarey et al., 1988). On this basis, lenses made of polysulphone and other materials, such as poly(methyl methacrylate) (PMMA) (refractive index of 1.49), were implanted intrastromally in animals and humans (Choyce, 1982; Kirkness et al., 1985; Lane et al., 1986; Climenhaga et al., 1988; Deg and Binder, 1988; McCarey et al., 1988; Rodrigues et al., 1990). Some implants were tolerated for months to years with few reports of inflammation being a problem (Choyce, 1982; Deg and Binder, 1988;

Rodrigues et al., 1990). The impermeable nature of the materials tested may have overwhelmed any subtlety in response that might have been noted among the different materials tested; these included glass (Barraquer, 1949), polysulphone (Choyce, 1982; Choyce, 1985; Kirkness et al., 1985; Lane et al., 1986; Climenhaga et al., 1988; Deg and Binder, 1988; McCarey et al., 1988; Rodrigues et al., 1990) and PMMA (Choyce, 1982; Rodrigues et al.,

1990). The influence of the type of material used in the tissue response was considered by some authors (Choyce, 1982; Kirkness et al., 1985; Deg and Binder, 1988; Rodrigues et al., 1990), although it was difficult to distinguish any real evidence that one material offered any benefit over another. Typical complications included interface opacities, epithelial thinning and stromal necrosis which resulted in lens extrusion. Concern about the chemical stability of the material and its effect on the corneal tissue during the period of implantation were also noted (Barraquer, 1966; Kirkness et al., 1985; Deg and Binder, 1988). Opacities associated with the implant interface, particularly lipid or crystalline deposits, were a frequently reported occurrence (Choyce, 1982; Choyce, 1985; Lane et al., 1986; Climenhaga et al., 1988; Deg and Binder, 1988; McCarey et al., 1988; Rodrigues et al., 1990). While some consideration was given to the inappropriate stiffness of the material used

(Barraquer, 1966; Deg and Binder, 1988), failures were, in general, attributed to inadequate nutrition of the tissue anterior to the lens owing to the solid nature of the material used (Barraquer, 1949; Choyce, 1982; Choyce, 1985; Kirkness et al., 1985; Lane et al., 1986; Deg and Binder, 1988; McCarey et al., 1988; Rodrigues et al., 1990). Attempts to improve the permeability of polysulphone, such as the addition of large holes (Barraquer, 1966; Choyce,

1982) or small fenestrations (McCarey et al., 1988), offered some benefit in reducing opacification of corneal tissue forward of the implant and in improved retention time of the devices in the cornea, but failed to completely resolve these problems. Implanting the lens deeper in the stromal tissue close to Descemet’s membrane enabled some implants to be retained for longer

but resulted in other problems such as tearing of Descemet’s membrane and protrusion of the inlays into the anterior chamber (Choyce, 1985; McCarey et al., 1988; Rodrigues et al., 1990).

4.4.4Corneal implants made from permeable synthetic materials

The difficulties encountered with intracorneal lenses made from solid materials and the growing significance of material permeability and modulus gave rise to concurrent activity with alternative materials such as hydrogels. Hydrogels are three-dimensional hydrophilic polymer networks capable of swelling in water and retaining large amounts of fluid between the polymer chains, which impart solute permeability to this class of materials. The water content is generally accepted to reflect the permeability of the material and hydrogels can be manufactured with a range of water contents, although those with a great deal of water have a low modulus and can be difficult to handle. The permeability and optical properties of hydrogels were already understood in the 1960s from their use as materials for the manufacture of contact lenses.

Hydrogels could also be made with a refractive index matching that of the cornea (1.376), offering the potential to be shaped to change the corneal curvature and achieve the desired refractive outcome. Dohlman et al. (1967) used a hydrogel material, poly(glycerol methacrylate) (PGMA; refractive index 1.353) with 88% water content, as an intracorneal implant; this was well tolerated by the corneal tissue (no inflammation), but implants were extruded in a response that was attributed to insufficient permeability and mechanical issues due to their flat form. Mester et al. (1972) demonstrated that corneal curvature and the dioptric power of the cornea could be changed using implants made from hydrogels, mostly PHEMA (refractive index 1.44). Following those initial trials, various hydrogel materials with a range of water contents, originally developed as contact lens materials, were tested as intracorneal implants in animal and human trials. Included in these trials were Perfilcon A, Lidofilcon A, Lidofilcon B, Surfilcon A and Etafilcon A

(see Table 4.1 for details).

Generally, each of these hydrogel implants was reported to have been well tolerated by the corneal tissue for extended periods of time (Mester et al., 1972; McCarey and Andrews, 1981; Werblin et al., 1983; Koenig et al., 1984; Samples et al., 1984; Yamaguchi et al., 1984b; Beekhuis et al., 1986; McCarey et al., 1990; McCarey, 1991; Parks and McCarey, 1991; Werblin et al., 1992a; Werblin et al., 1992b; McDonald et al., 1993). Early work with hydrogels concentrated on testing the significance of the water content of the materials on the biological and optical outcomes. Biocompatibility was linked to the water content of the implanted material following work in rabbits where implants of a low water content material (glycerol methacrylate;

GMA) failed quickly because of non-inflammatory ulceration while implants of a high water content hydrogel, Perfilcon A (70%), were tolerated for 12 months (Sendele et al., 1983). Longer-term testing of two high water content materials, Lidofilcon A (70%) and Lidofilcon B (79%), implanted in lamellar pockets in monkey eyes, showed that both materials were equally well tolerated by the cornea for up to 5 years (McDonald et al., 1993). During this period, McCarey and Schmidt (1990) modelled glucose distribution in the cornea in the presence of intracorneal lenses of varying diameter, depth, permeability and thickness. They identified that the glucose permeability of the material was more significant in maintaining corneal health than the diameter or depth of the implanted lens.

Refractive data from many of the studies suggested that the type of hydrogel material was perhaps less significant than the species used to test it and the depth at which the implant was placed in the cornea. Optical data from studies that compared lenses made of hydrogels with different water contents (various hydrogels ranging between 38 and 79% water content) (Binder et al., 1981/82) and also similar water contents (Lidofilcon A and Perfilcon A, both with 70% water content) (Werblin et al., 1983) that were