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

15.2The scale of the problem of age-related macular degeneration

AMD is one of the leading causes of blindness in the Western world. In the United States, its prevalence is 0.05% before the age of 50 years and that rises to 11.8% after 80 years of age (Friedman et al., 2004). In Australia, the most common cause of blindness (presenting visual acuity of less than 6/60) was AMD (48%), and the predicted number of Australians who will have low vision or blindness will almost double over the years 2000 to 2024 (Taylor et al., 2005). This estimation was mirrored in the American study with the prevalence of AMD expected to double in the coming decades because of the projected increase in ageing populations (Friedman et al., 2004). The direct health cost for macular degeneration was estimated to be A$19.4 million in 2004 and this did not include the special allocated funding for photodynamic therapy, the best available treatment at the time, which was estimated to be between A$30 and 40 million per annum (Taylor et al., 2006). This estimate will undoubtedly be significantly higher as a number of governments worldwide have approved funding for one of the pharmacological treatments for AMD – intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) ranibizumab (Lucentis). There is clearly an urgent and important need for further research on the management of macular degeneration.

15.3Retinal pigment epitheliun–Bruch’s membrane complex and the effect of ageing

15.3.1 Normal retinal pigment epitheliun

Retinal pigment epithelium (RPE) is a monolayer of pigmented cells derived from the neuroectodermal layer of the optic cup and constitutes the outermost layer of retina. RPE is known to secrete various factors promoting retinal photoreceptor survival and differentiation. The RPE cell exhibits an apical– basal polarity characteristic for a transporting epithelium. The subretinal space which is the extracellular space at the apical RPE surface is filled with inter-photoreceptor matrix. RPE projects specialized microvilli into the subretinal space serving to maintain the retinal adhesion. The infolded basal RPE surface rests on Bruch’s membrane and forms the outer part of the blood–retinal barrier by the tight junctions between RPE cells. The barrier isolates the subretinal space from the porous choriocapillaris, allowing selective transport of nutrients and metabolic end products between the subretinal space and choriocapillaris (Strauss, 2005). There is a geographical difference in the differentiated RPE cells. In the macula, for example, RPE cells are more tightly packed together (14 μm ∞ 12 μm), and contain higher amounts

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of melanin. Higher degradation enzyme activities found in this region allow for the maintenance of greater numbers of macular photoreceptors compared with the peripheral RPE cells, which are larger in size (60 μm) and variable in height (Harman et al., 1997; Panda-Jonas et al., 1996; Strauss, 2005).

RPE helps to absorb light energy focused by the lens on to the retina. The heat generated is dissipated by choriocapillaris perfusion. RPE melanins in the melanosome and lipofuscin which are initially beneficial but later accumulate to toxic levels, help absorb the damaging blue light. RPE plays a critical role in the visual cycle where all-trans-retinol from photoreceptors is isomerized to 11-cis-retinal within the RPE cells and re-delivered back to photoreceptors to bind with opsin and initiates the photo-transduction cascade. RPE is also responsible for photoreceptor outer segment renewal in a circadian-controlled fashion. The shed outer segment is phagocytosed by

RPE via specific binding of apical RPE membrane receptors, such as CD36,

MerTK (receptor tyrosine kinase c-mer) and integrin receptors responsible for regulating the internalization process (Harman et al., 1997; Panda-Jonas et al., 1996; Strauss, 2005).

Growth factor, trophic and paracrine secretory functions of RPE are essential for retinal homeostasis and normal function. The health of RPE is therefore critically important in maintaining the normal retinal function and subretinal homeostasis. Any disturbance to the physiological levels of growth factors may lead to an altered homeostasis of the retinal micro-environment, and such changes in RPE secretory activity are found to be associated with retinal proliferative diseases (Binder et al., 2007).

15.3.2 Ageing retinal pigment epithelium

RPE cells undergo significant age-related changes with observed increase in β-galactosidase staining, telomere loss, mitochondrial deoxyribonucleic acid (DNA) damage, nuclear DNA damage, protein crosslinking and lipid hydroperoxidation, many of which are non-reversible in such post-mitotic cells (de Jong, 2006; Handa, 2007; Zarbin, 2004). RPE in early AMD was observed to contain more melano-lipofuscin and melano-lysosomes than pure melanin and the number of lipofuscin granules increased. Melanosomes contained within the RPE are exposed to a variety of environmental and metabolic insults. There are suggestions that aged human melanosomes are highly phototoxic and can result in RPE dysfunction, while young melanosomes appear to confer photoprotection (Rozanowski et al., 2008). Age-related changes in melanosomes, possibly the result of oxidative damage, include disorientation within the RPE, decline in number after the age of 40 years, increase in melanosome complexes with lysosomes and/or lipofuscin, loss of melanin resulting in fading of eye colour with age, and increases in shorter wavelength blue spectrum absorption.

Preferential accumulation of lipofuscin in ageing RPE within the macula is a heterogeneous mixture of non-degradable lipid peroxidation products. These products originate from conjugates formed by visual cycle retinoid in photoreceptor cells that accumulate in RPE cells due to the inability of the RPE cells to convert all all-trans-retinol into 11-cis-retinal. RPE lipofuscin is a potent generator of reactive oxygen species. It is hypothesized that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis (Ng et al., 2008).

Lipofuscin autofluoresces a yellowish orange colour due to its composition being a heterogeneous mixture of cytotoxic fluorophore N-retinylidene-N- retinylethanolamine (A2E) and its photo-isomers A2E epoxides. There are suggestions of a possible link between A2E’s role in interfering with normal lipid metabolism and a resultant delay in lipid degradation and accumulation, leading to increased RPE sensitivity to blue light. The above degenerative RPE changes ultimately lead to the formation of basal deposits, drusen, RPE cell apoptosis, followed by secondary damage to choriocapillaris and neurosensory retina, and resulting in (de Jong, 2006; Handa, 2007; Zarbin, 2004).

15.3.3 Bruch’s membrane and ageing changes

The anatomy of human Bruch’s membrane displays a penta-laminar structure (1–4 μm thick) composed of a 50 nm, thin acellular RPE basal lamina, an inner collagen layer (ICL), an elastin layer (EL), an outer collagen layer (OCL), and the choriocapillaris basal lamina (Strauss, 2005).

Most dysfunction within AMD starts in the ICL, with drusen-like material accumulating either side of the RPE basal lamina and invasion of the ICL tissue plane by CNVM in advanced AMD. Drusen is the clinical hallmark of AMD. The punctate hard drusen of size <63 μm in diameter is not associated with AMD. However, large (>63 μm) drusen size and, to a lesser extent, the number of indistinct soft drusen are found to correlate positively with progression to advanced AMD. The EL underneath is also found to become pathologically fragmented. Apart from age-related collagen crosslinking change in the OCL, extracellular deposits (drusen, lipid deposits) appear to spare this layer and accumulate mainly within the ICL.

It is thought that, once the EL and ICL are filled with debris, lipoprotein deposits continue to accumulate near the RPE. This possibly explains why no further accumulation was found in the OCL (Huang et al., 2008). Interestingly, CNVM only penetrates through, but does not invade the OCL. Perhaps this is due to the age-related decrease in endostatin levels in these structures, which may be permissive for CNVM formation (Bhutto et al., 2004). In addition, unlike the RPE basal lamina, there is no evidence of deposit formation on either side of the choriocapillaris basal lamina as a function of advancing age.

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15.3.4Relationship between the retinal pigment epithelium and Bruch’s membrane

The intricate relationship between the photoreceptor, RPE, and Bruch’s membrane is fundamental to normal retinal function. Age-related alterations in the molecular composition and ultrastructures of human Bruch’s membrane make it an unfavourable substratum for the attachment and survival of grafted RPE cells (Tezel et al., 1999; Tezel and Del Priore, 1999). Attempts have been made, by way of in vitro re-engineering, to ‘clean’ the Bruch’s membrane (especially the inner collagen layer) with a non-ionic detergent and refurbish it with extracellular matrix (ECM) proteins (laminin, vitronectin, fibronectin) (Tezel et al., 2004). This may be why these attempts to repopulate the RPE defects with native or transplant RPE cells alone have not met with great success. The presence of disease within the host’s Bruch’s membrane, iatrogenic removal of the inner layers of Bruch’s membrane, and immune rejection of the transplant have all been blamed for limiting visual recovery after RPE cell transplantation studies (Del Priore et al., 2006).

15.4Summary of the aetiology and management of age-related macular degeneration

The aetiology of AMD is multi-factorial – including physiological ageing, genetic, inflammatory, and environmental factors – but the end result is a complex series of events that lead to a significant change in the RPE–Bruch’s membrane–choroidal complex, also known as Ruysch’s complex (de Jong, 2006). This is accompanied by loss of RPE and photoreceptors, and eventually fibrovascular membrane and scar formation. Different potential therapies have been tried including: dietary/vitamin supplement; laser treatment with or without photosensitizing dye; submacular membranectomy with or without RPE transplantation or translocation; macular translocation surgery; radiotherapy; gene therapy; and pharmacological treatment (e.g. angiostatic steroid and anti-VEGF therapy). Despite these treatments, many patients lose their vision from chorioretinal fibrovascular scaring following the formation of CNVM, therefore alternative treatments are still being explored (Binder et al., 2007).

Ninety per cent of severe visual loss from AMD is due to the ‘wet’ type of macular degeneration – i.e. CNVM formation (Smith et al., 2001). Unfortunately, simple excision of the CNVM in AMD results in RPE defects because the original RPE is removed along with the neovascular complex; this is because the CNVM found in AMD is situated beneath the ageing native RPE layer (Grossniklaus et al., 1994). The loss of RPE following this surgery leads to progressive loss of the underlying choriocapillaris and the overlying photoreceptors (Del Priore et al., 2006). The end result is similar

to a subtype of dry macular degeneration – geographic atrophy, with equally devastating effects.

15.5Retinal pigment epithelium transplantation from animals to human

In order to address the problem of RPE loss, studies on RPE transplantation in animal models of retinal degeneration were carried out. It has been proven in principle that RPE transplantation can lead to photoreceptor rescue and functional improvement in the Royal College of Surgeons (RCS) rat (Lund et al., 2001), which has retinal degeneration with a mutation in its Mertk gene (D’Cruz et al., 2000). Other cell types have been tried, including stem cells (Schraermeyer et al., 2001) and Schwann cells (Lawrence et al., 2000), but RPE cell transplantation remains the benchmark as most of the other cell types have so far failed to achieve the same degree of rescue and the ethical issues, associated with obtaining these cells, remain a problem. Armed with the success in the laboratory, Peyman and coworkers performed one of the first RPE transplantations in humans, whereby submacular scar excisions were followed by translocation of an autologous RPE pedicle flap or transplantation of an allogenic RPE–Bruch’s membrane explant in two patients (Peyman et al., 1991). This was followed by trials of transplantation of foetal human RPE patches following subretinal membrane removal. These patients were able to fixate over the area of the RPE graft initially but cystoid macula oedema ensued and eventually the grafts were encapsulated by fibrotic scars; these might be a result of immune rejection as none of the patients were immunologically suppressed. Because of the rejection, autologous iris pigment epithelial (IPE) cells have been used to replace the lost or damaged RPE cells in the macular area (Thumann et al., 2000). However, transplantation of suspensions of autologous IPE cells has also not resulted in a prolonged improvement of vision in AMD patients. One of the reasons for this failure was probably because the transplanted IPE cells did not fully differentiate into cells that had the morphological and physiological properties of RPE cells in situ. Binder and coworkers transplanted suspensions of autologous RPE cells into eyes with wet type AMD after removal of CNVM membranes (Binder et al., 2002). They reported that these eyes had significantly better reading acuity than controls with CNVM removal only. However, obtaining sufficient numbers of RPE cells was sometimes difficult, and in some patients the aspirated RPE cells were not transplanted because of insufficient numbers or haemorrhage. In most of these studies, suspensions of isolated cells were injected into the subretinal space, one problem with IPE/RPE cells in the suspension is that photoreceptor cells survived well when a monolayer of pigment epithelial cells was transplanted, but the photoreceptors did not survive when the

therefore might be useful as temporary substrata for subretinal transplantation, few of the materials have been tested in vivo in animal models, and none of the materials has managed to encompass all the qualities for an ideal substratum. In order to mimic the native RPE layer on Bruch’s membrane and to maximize the chance of normal function and survival, the ideal RPE layer for transplantation will need to have a number of properties: (a) it should be a monolayer of RPE cells with good adhesion to a substratum;

(b) it should have the correct orientation/polarity, normal morphology, and expression of differentiated RPE cell features; (c) the substratum should be thin, suitably porous to allow the transport of both nutrients and waste from the underlying tissues to the transplanted RPE monolayer, strong and yet manipulable for ease of introduction to the subretinal space; (d) it should display biostability and biocompatibility, and be immunologically inert so that it does not cause inflammation and rejection. The material may or may not be biodegradable as long as no adverse effects are observed.

15.6.2Biomaterials as substrata for retinal pigment epithelium cell culture and transplantation

The importance for the growth of RPE cells of substrata made of naturally derived and/or synthetic biomaterials was revealed indirectly some time ago. For instance, the effect of the ECM on the proliferation of RPE cells was investigated by using a synthetic polymer substratum (tissue culture polystyrene) coated with collagen, Matrigel™ (a commercially available synthetic basement membrane derived from a mouse sarcoma tumour cell line), poly(d-lysine), or undefined matrices deposited by either RPE cells or retinal glial cells (Williams and Burke, 1990). A crucial study demonstrating that the RPE cells cannot survive and undergo apoptosis when separated from their natural ECM if they do not have the opportunity to reattach to a substratum was also based on the use of tissue culture polystyrene used on its own or coated with ECM components, and of untreated polystyrene used on its own or coated with agents preventing cell adhesion (Tezel and Del Priore, 1997). However, a relatively small range of biomaterials have been proposed and investigated as substrata for transplantable RPE constructs.

In this section we will discuss both the processed biopolymers and the synthetic polymers that have been investigated to date as potential substrata for the growth of RPE cells. Collagen type I was used as a substratum for human foetal RPE cells to create sheet-like constructs, which were then transplanted in vivo into the subretinal space of non-pigmented rabbit eyes (Bhatt et al., 1994). Two different collagen substrata were used, uncrosslinked and crosslinked (by ultraviolet (UV) irradiation). The crosslinked collagen transplants were unsuccessful as a result of the detachment of the RPE cells, which was explained by increased stiffness of the substratum. In the eyes

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containing the uncrosslinked collagen transplants, a layer of pigmented RPE was visible and the retina remained normal until the end of experiments (for 6 weeks). In spite of such promising results, there has been no record of using collagen substrata again until very recently. In this study (Thumann et al., 2006), porcine primary RPE and IPE cells were grown to confluence on a 10 μm thick collagen type I membrane available commercially as ResoFoil® (from RESORBA Wundversorgung GmbH & Co. KG, Nuremberg, Germany). Both RPE and IPE cells readily attached to, and proliferated and formed monolayers on, the collagen substratum, and acquired differentiated properties. These constructs were then transplanted into the subretinal space of enucleated porcine eyes, and further assays showed that the cells maintained viability following this manipulation. In a more recent study (Lu et al., 2007), the attention was focused towards fabricating collagen layers with the same thickness and properties as the natural Bruch’s membrane. Membranes were produced from a collagen available commercially as Vitrogen 100® (Angiotech BioMaterials Corp., Palo Alto, CA, USA) to a thickness around 2.4 μm and having physical properties similar to Bruch’s membrane. Cells from an immortalized human RPE cell line (ARPE-19) were successfully grown on these membranes; they showed normal morphology and intercellular tight junctions, and were able to phagocytize in vitro the photoreceptor outer segments. In spite of such promising results, there is no reported use of collagen substrata in human patients.

Gelatin was the substratum of choice in other studies, and it has to be accoladed as the only material used so far in human clinical trials. In one approach (Ho et al., 1996), ECM was prepared from a layer of RPE cells, coated with a layer of gelatin, and then cooled to 4 °C when the gelatin solidified. Patches of ECM–gelatin were transferred to another tissue culture dish and RPE cells were seeded on to them. In another approach (Ho et al.,

1997), RPE were first cultured to confluence on tissue culture dishes, then covered with liquid gelatin and cooled to 4 °C. The RPE–gelatin blocks were easily cut and transferred to another tissue culture dish. Upon incubation at 37 °C, the gelatin melted and encased the cells, so providing a vehicle for the transplantation of the RPE constructs. Based on these developments, RPE cellular constructs encased in gelatin were transplanted into a human elderly patient affected by AMD (Del Priore et al., 2001). The cells were harvested from a human donor. While the retina remained attached, the patient’s vision did not improve over the follow-up period. As this patient died from unrelated causes about 4 months after operation, a complete histopathological examination of the eye was possible, revealing the presence at the transplant site of clusters of pigmented cells that failed to form a uniform layer. Recently, the transplantation of allogeneic RPE cell sheets encased in gelatin was reported in 12 patients affected by exudative AMD (Tezel et al., 2007). The patients were followed for 1 year. Rejection of

implants was prevented by administration of immunosuppressants, but other postoperative complications were observed, and there was no improvement in visual function.

The best-known and most accessible synthetic biodegradable polymers, the poly(a-hydroxyesters), represented mainly by polylactides and polyglycolides, have attracted much interest as potential substratum materials, to the extent that a review has been possible on this particular topic (Lu et al., 2001b). Biodegradability is an attractive feature of the potential substrata, as it assures that the foreign material will be resorbed and will dissipate in time.

Significant work has been carried out by Mikos’ group (Giordano et al., 1997; Lu et al., 1998; Lu et al., 2001a; Lu et al., 2001b; Thomson et al., 1996). They have developed substrata from poly(l-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA), with a thickness of at least 10 μm, and have demonstrated that foetal or adult RPE cells were able to attach to the polymer surface and to proliferate. At confluence, the cells expressed ZO-1 protein confirming the existence of normal tight junctions between cells. However, the cells appear more elongated prior to reaching confluence.

Based on their own previous investigations regarding the effect of surface micropatterning on RPE cell growth on to glass (Lu et al., 1999), the group demonstrated in a subsequent study that a topography can be created on the PLGA surface that promotes the characteristic cuboidal morphology of the RPE cells (Lu et al., 2001a). This was achieved by using a microcontact printing technique enabling the creation of defined arrays of PLGA (which promotes cell adhesion but not an ideal cell morphology) separated by regions of a block copolymer, poly(dl-lactic acid)/poly(ethylene glycol) (PLA/PEG). The latter is a polymer substratum that inhibits cell adhesion. The resulting pattern promoted a cuboidal morphology of the cultured RPE cells. Other investigators have also focused their attention on these polymers. Porcine and human RPE cell cultures were established from post-mortem sources and grown on PLA or PLGA films, 10–30 μm in thickness, with the aim of evaluating them as substrata for RPE cells (Hadlock et al., 1999). The cells proliferated readily on the films and retained their phenotype and functional characteristics. In another study, commercial poly(dl-lactide-co-glycolide) was used to prepare films of 35–50 μm thickness (Rezai et al., 1999). Sheets of human foetal RPE cells dissected from foetal eyes were attached to these films and incubated in growth media. Within days, the attached cells generated spheroids, which were then dissociated and further characterized. This study was actually designed to investigate the formation of spheroids and to evaluate their long-term behaviour in vitro. The particular use of biodegradable polymers does not really appear to be any more justified than the use of any polymer able to promote cell adhesion. More recently, natural biodegradable polymers have been proposed as an alternative to the synthetic polymers. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), a copolymer produced by

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certain microorganisms, was assessed as a substratum for the RPE cell line D407 (Tezcaner et al., 2003). In order to enhance cell adhesion, the polymer surface was treated in an oxygen plasma reactor. Cell counts showed that this treatment led to an increased number of attached cells. The authors’ suggestion that this is a result of enhanced hydrophilicity of the polymer surface is, however, at odds with the well-known interrelation between cell adhesion and substratum hydrophilicity.

Although rather extensively assessed as substrata for RPE cells, PLA and PLGA were assessed in vivo as substratum materials only in experiments involving different cells − the retinal progenitor cells (RPCs) (Lavik et al., 2005). Murine RPCs were cultured on porous PLA or PLGA scaffolds. Seeded scaffolds were then either co-cultured with degenerating mouse retinal explants or inserted into the subretinal space of rats. The results suggested that the scaffold may assist in the differentiation to photoreceptor phenotype. However, the same group subsequently used ultra-thin layers of laminin-coated poly(methyl methacrylate) (PMMA) as a substratum for RPCs with similar results (Tao et al., 2007). Apparently, the investigators were not concerned by the non-biodegradability of PMMA.

Thermoresponsive polymers constitute another class of polymers, which have been evaluated over the last decade as substrata for the RPE cells. These polymers display a so-called ‘lower critical solution temperature (LCST)’. In principle, at temperatures above the LCST, the polymer is hydrophobic and consequently supports the attachment of cells, which can be grown to confluent sheets. Water is partially displaced from the macromolecular coil, the hydrogen bonds involving water are weakened, and the hydrophobic interactions between polymer segments become dominant, resulting in the polymer chains having a compact conformation that prevents the penetration of water. Below the LCST, the polymer surface turns hydrophilic, as the hydrogen bonding between the hydrophilic segments and water molecules becomes dominant and leads to chains with an extended conformation. As soon as the surface turns hydrophilic, the cells detach (because of a lower tendency of cells to attach to hydrated surfaces) and they can be harvested as single uninterrupted sheets. This technique, coined ‘cell sheet engineering’, has so far been applied in ocular surface reconstruction, myocardial tissue engineering, and other therapies (Yang et al., 2006). It has the advantage that it allows separation of cells without using enzymes.

The polymers based on N-isopropylacrylamide (NIPAAm) include some of the most studied synthetic thermoresponsive materials. The routine cell incubation temperature (37°C) is well above their LCST, which assures normal growth of cells on these surfaces when they are in a hydrophobic state. A copolymer of NIPAAm and aminostyrene, modified with cinnamoyl functions, was studied as a substratum for the growth and fabrication of RPE cell sheets (von Recum et al., 1998a; von Recum et al., 1998b; von Recum