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

9.2 Spherical lens – lens regenerate is spherical following closure of the anterior capsulotomy. Nucleus has poor clarity with a ‘starshaped’ appearance. The cortex has good structure and clarity.

internal scaffold to maintain capsule tautness and separation of the anterior and posterior capsule is necessary.

9.3.4Controlling lens epithelial proliferation and differentiation

In mammals, the lens vesicle is formed during embryological development by invagination of the lens placode, which is induced from surface ectoderm by signals from the retina primordium. Some ectodermal cells remain inside the lens vesicle after invagination and closure, possibly acting as a scaffold for the initial orientation of lens fibers. These cells eventually cytolyse and disappear. Initially, epithelial cells proliferate along the anterior and posterior capsule, followed by elongation of the posterior epithelial cells, anterior migration and eventual loss of fiber nuclei, and differentiation into lens fibers at the equatorial zone. The lens fibers form the fetal lens nucleus and are gradually compacted as differentiation proceeds from the equator of the maturing lens. In the regenerating lens provided with an intact capsular bag, lens fiber differentiation also begins with formation of a monolayer of epithelium across the anterior and posterior capsule, followed by elongation of the posterior epithelial cells, anterior migration of fiber nuclei and loss of nuclei. Subsequently, these lens fibers form the nucleus, and are gradually compacted as differentiation proceeds from the lens equator (Piatigorsky, 1981; Gwon, 2006). The regenerated lenses have been shown to contain all

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three major crystallins – alpha, beta and gamma – in proportions similar to fetal or normal lenses (Gwon et al., 1989).

However, the rate of lens differentiation during regeneration may vary in the different parts of the capsule bag resulting in abnormal alignment of the earliest regenerating lens fibers (Gwon et al., 1990). In order to synchronize the regenerative process, it is desirable to delay lens differentiation until the entire capsule is covered by a confluent monolayer of lens epithelium.

Ideally, one would aim to replicate the embryological environment by creating a scaffold of amniotic fluid or similar constituents normally present during lens invagination in embryogenesis. An initial attempt by Sikharulidze showed the rate of regeneration and the quality of the regenerated lens material was improved if a fragment of partially cytolyzed fetal tissue was inserted within the capsule at the time of lens evacuation (Sikharulidze, 1956). Subsequently, Stewart demonstrated that the regenerated lens had an optical density (refractive power) similar to that of the normal crystallin lens when fetal tissue had been implanted, i.e. 5–10 diopters in the 6-month-old rabbit (Stewart and Espinasse, 1959; Stewart, 1960). Other investigators had mixed results using cytolized ectodermal tissue (Chanturishvili, 1958; Binder et al., 1962; Petit, 1963; Agarwal, 1964; Angra et al., 1973). A review of their work can be found in a paper by Gwon (Gwon, 2006).

9.4Scaffolds

Tissue engineering techniques generally require the use of a porous bioresorbable scaffold to serve as a three-dimensional template for initial cell attachment and subsequent tissue formation both in vitro and in vivo (Hutmacher et al., 2001). The porosity of the scaffold often serves to allow the flow of nutrients and in some cases can control the integration of cells within its matrix.

The ocular lens is a crystalline, transparent biconvex structure whose sole function is to transmit and refract light on the retina. As an organ, the lens is unique in its derivation from one cell type, in its retention throughout life of all cells that are ever produced, in having no blood or nerve supply and in synthesizing unique proteins (Rafferty, 1985). Since the lens is avascular, its pathology is simpler than most other tissues and primary inflammatory processes do not occur (Duke-Elder, 1969a). Thus, scaffolds for lens engineering have specific requirements. Foremost is the need for transparency to support visual function in the postoperative period. It must be biocompatible with the lens epithelium and capsule. Immunogenic processes are not a major concern as the closed capsule is a barrier to inflammatory processes. The avascularity of the lens may be beneficial in limiting cellular infiltration but may also limit the ability to degrade a bioresorbable scaffold.

Matching the lens differentiation rate with the degradation and resorption rate

of the scaffold is perhaps the biggest challenge to successful regeneration of an optically clear lens. Alternatively, a non-degradable scaffold can offer transparency and refractive ability that can be retained within the surrounding regenerating lens tissue. Both biodegradable and non-degradable scaffolds have been evaluated in the rabbit lens regeneration model.

9.4.1Biodegradable scaffolds

Tissue-engineered biodegradable scaffolds have been developed as a threedimensional template for initial cell attachment and subsequent tissue formation for both in vitro and in vivo applications. As outlined by Hutmacher, the ideal scaffold should have the following characteristics: (a) be porous for cell growth and transport of nutrients and metabolic waste; (b) be biocompatible and bioresorbable with controllable degradation and a resorption rate to match tissue replacement; (c) have suitable surface chemistry for cell attachment, proliferation and differentiation; (d) have mechanical properties to match the tissue site; and (e) have a reproducible process for fabrication. Thus, many of the biodegradable scaffolds that have been utilized contain natural polymers such as collagen and glycosaminoglycans as components (Hutmacher et al., 2001).

Natural biological materials have several advantages and disadvantages over synthetic materials. Because they are identical or similar to substances already found in the body, the likelihood of toxicity or chronic inflammation may be reduced. They generally have inherent biological activity, which can be utilized for cell signaling. They are susceptible to naturally occurring enzymes and are thus biodegradable. They have the disadvantage of being frequently immunogenic and are difficult to manipulate or process. Some of the more commonly studied natural materials for tissue engineering are collagen, elastin, hyaluronic acid (HA), agarose, alginate, chitosan and fibrin gels (Mann, 2003). Of these, HA is the only optically clear material that can maintain its clarity in the lens capsule bag.

Hyaluronic acid

HA is particularly attractive as a scaffold in lens engineering because of its long history of biocompatibility and use in the eye. It is a naturally occurring glycosaminoglycan distributed widely in the extracellular matrix throughout the body and contributes significantly to cell proliferation and migration.

It is soluble and interacts with binding proteins, proteoglycans and growth factors. It actively contributes to the regulation of water balance (Hutmacher et al., 2001). As mentioned, it can retain its clarity in the lens capsule bag. It is found in high concentrations in the developing fetus and is implicated in the fetal ability to heal without scarring (Longaker et al., 1989; Longaker

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et al., 1990; Longaker and Adzick, 1991; Longaker et al., 1991; Mast et al., 1991). HA is naturally broken down by hyaluronidase and is readily available as a viscosurgical tool for ocular surgery.

In a series of unpublished studies, various HA-based products have been evaluated for their ability to enhance the regenerative process in the rabbit lens. Of these, the cohesive HA, Healon“ ophthalmic viscosurgical device (OVD) (Advanced Medical Optics, Santa Ana, CA, USA), has given the most consistent results to date. When Healon“ OVD was given at doses of 0.05–0.1 ml, the capsule bag remained distended during the regenerative process. New lens regrowth filling 5–20% of the capsule bag was first noted at days 19–21 and generally filled the capsule bag by 41–54 days. In some eyes the capsule bag flattened by 1 week indicating early dissolution of HA and resulting in some abnormally shaped lens regenerates. In eyes where the capsule bag remained distended during the regenerative process the lenses were spherical with abnormal early growth resulting in an irregular star-shaped nucleus and fairly good cortical structure and clarity (Fig. 9.2). The abnormal nucleus has been partially attributed to variations in the maturation process in different parts of the capsule bag in the early stages of regeneration.

Cross-linked hyaluronic acid

In the search for a longer-lasting scaffold to maintain the tautness of the capsule and control the proliferative process in the lens capsule, a cross-linked HA gel was evaluated. The HA gel products tested were Restylane® and Perlane® (Q-Med Scandinavia, Inc., Princeton, NJ, USA). They were chosen because of their ability to degrade slowly over a 6- to 12-month period when injected intradermally for soft tissue augmentation (Duranti et al., 1998). However, when injected into the capsule bag at the time of lens removal, we saw no evidence of degradation of either cross-linked HA gel over a 12-month period. Instead the cross-linked HA gel became compacted in a spherical ball-like nucleus in the center of the regenerating tissue. However, the regenerative process was synchronized for 360° around the cross-linked HA. Early lens regrowth was inversely related to the amount of cross-linked HA injected and could be seen at 8–12 days with a dose of 0.025–0.05 ml and at 3 weeks with a dose of 0.1 ml. As time progressed, the regenerated lenses were spherical and the regenerated lens cortical structure was clear with normal lens fiber alignment around the spherical compacted residual

HA material (Fig. 9.3) (Gwon and Gruber, 2005).

The ability of the regenerating lens to dissolve a cohesive or dispersive HA and not a cross-linked HA has yet to be explained. It may be that, once lens fiber differentiation has occurred, hyaluronidase is unavailable to breakdown exogenous cross-linked HA used in these studies. HA and its metabolism by hyaluronidase has been studied in various tissues. In these

9.3 Synchronized lens regeneration – regenerated lens is spherical with clear normal cortex surrounding spherical nucleus of compacted cross-linked HA (7 months postoperative).

studies, undifferentiated, rapidly proliferating cells have high HA levels. It is thought that hyaluronidase may mediate the process by which cells lose their HA matrix and slow proliferation before differentiation can commence (Stern, 2004).

Hyaluronidase

While the cross-linked HA failed to dissolve over time, it provided a means to understand the residence time desired for a biodegradable scaffold. By providing hyaluronidase at various times during the postoperative period following implantation of a cross-linked HA, we were able to study the regenerative process with a biodegradable scaffold. When hyaluronidase was given intravitreally at 0, 3, 5, 7, 9 and 10 days postoperatively, limited lens regeneration was observed as early as 2–4 weeks post-operation. Full lens regrowth was seen by days 42–67 in the 0, 3, 5 and 10 day groups and by day 109 in the 7 and 9 day groups. Regenerated lens clarity was greatest in the 0, 3 and 5 day groups compared with the 7, 9 and 10 day groups (Gwon and Gruber, 2008). While a clear lens regenerate has not been established in the rabbit model, these initial studies suggest that a gradual dissolution of the HA scaffold over a 2- to 4-week time period improves the clarity and structure of the lens regenerate.

In an alternative method of addressing small opacities and residual scaffold material, an attempt was made to treat one regenerated lens with focal photocoagulation. Focal photocoagulation provided limited removal

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of retained HA and possibly some lens tissue (Gwon, 2005; Gwon and Gruber, 2005; Gwon, 2007; Gwon and Gruber, 2007a; Gwon and Gruber, 2007b).

In summary, lens regeneration following endocapsular lens/cataract extraction in the rabbit may be enhanced by providing a suitable internal and external scaffold. The external scaffold, i.e. lens capsule, can be sealed by placement of a collagen/silicone patch or fibrin adhesive after lens/cataract extraction through a small, approximately 1–2 mm, capsulotomy. The ideal internal scaffold has yet to be designed. However, the data suggest that HA provides a good biodegradable scaffold that can be modified for optimum retention/dissolution time to enhance the alignment of the earliest regenerating lens epithelium.

9.4.2Non-degradable scaffolds

Synthetic non-degradable scaffolds for tissue engineering may offer some advantages over natural degradable biomaterials. They can typically be reproducibly and reliably manufactured. They can be tailored for specific application and sterilization is often easier (Mann, 2003; Dvir et al., 2005).

In comparison to scaffolds for replacement of other body tissues, the ocular environment has additional, specific requirements for a non-degradable scaffold. In addition to being biocompatible with the lens epithelium and capsule, it must be transparent and have the spectral transmission and refractive properties of the natural lens. By correcting for a patient’s aphakia and refractive error, it should permit a quicker postoperative recovery time and restoration of functional vision. In order to limit any visual distortion related to the interface between the synthetic polymer and the naturally regenerating lens tissue, it should ideally have the same refractive index as the natural lens.

Foldable lens

The concept of providing an internal scaffold for the lens epithelium by implantation of a semi-permeable synthetic foldable lens was suggested by Gwon and co-workers in 1998. In that study, an Acuvue® (etafilcon A, 58% H2O, Vistakon, Jacksonville, FL, USA) contact lens was modified for intralenticular implantation in the capsule bag in the rabbit. While lens epithelial proliferation and differentiation occurred around the synthetic polymer, the regenerated lens anterior and peripheral to the implant was clear with excellent structure, while the posterior regenerated lens structure was poor, except in one eye. In that one eye, lens differentiation was seen anterior and posterior to the intralenticular implant forming two lenses within

the capsule bag. Lens structure and clarity were excellent permitting a good view of retinal structures (Gwon et al., 1998). It is unknown why the posterior regenerating lens tissue was opaque. It may be that the intralenticular lens blocked transport of nutrients from the anterior lens epithelium necessary for the posterior differentiating fibers and/or blocked the metabolic waste products from being released.

Injectable lens

The difficulty of implanting a foldable lens through a 1–2 mm capsulorrhexis has led many investigators to pursue an injectable polymer for lens refilling.

The concept of injecting a synthetic polymer to replace the natural or cataractous crystalline lens was first suggested in 1964 by Kessler (Kessler,

1964; Kessler, 1966; Kessler, 1975). Since that time numerous investigators have worked on developing a suitable polymer that would have the flexibility of the natural lens and be capable of restoring accommodation in the presbyopic and/or cataractous eye. These polymers are generally transparent and liquid for easy injection into the capsule bag through a small, 1–2 mm capsulotomy. Once in the capsule bag, they polymerize to create a lens that conforms to the shape of the bag. Investigators have used: silicone polymers of low modulus, such as polydimethylsiloxane; or hydrogels – including poly(1-hydroxy-1,3-propandiol), polyether (such as poly(ethylene glycol) (PEG)), polyalcohol, poly(vinyl pyrrolidone) (PVP) and poly (acryl amide) hydrogels (Agarwal et al., 1967a,b; Parel et al., 1981, 1986; Haefliger et al., 1987; Deacon, 1988; Hettlich et al., 1994; Nishi et al., 1998b; deGroot et al., 2001; Han et al., 2003; Koopmans et al., 2003; Wong et al., 2007; Aliyar et al., 2005; Kwon et al., 2005; Yoo et al., 2007). Recent reviews by Norrby and Yoo discuss the current state of injectable polymers for lens refilling (Norrby, 2005; Yoo et al., 2007).

To date, advances with lens refilling have had moderate success with limited accommodation shown in primate models. Primary failure has been linked to the development of scarring and folds in the lens capsule, epithelial cell proliferation and secondary (PCO). While most investigators have regarded the development of PCO as a hindrance to the development of an injectable accommodating lens (Agarwal et al., 1967a,b; Parel et al., 1981, 1986; Hettlich et al., 1994; Haefliger et al., 1987; Nishi et al., 1998b; deGroot et al., 2001; Han et al., 2003; Koopmans et al., 2003; Aliyar et al., 2005; Kwon et al., 2005; Wong et al., 2007), it is also possible that the residual lens epithelium may be encouraged to regenerate and differentiate around a synthetic polymer and develop a tissue-engineered lens.

In a study by Gwon, coating the capsule bag with HA prior to injection of a silicone polymer in rabbits resulted in less capsule fibrosis and a clear regenerative tissue surrounding the silicone polymer anteriorly and peripherally

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and slightly opaque tissue posterior to the synthetic scaffold. Lens regeneration was observed as early as 15 days postoperatively. Thus, if the clarity of the posterior regenerating tissue could be improved, an injectable synthetic polymer has the potential to offer restoration of good functional vision in the adult (Gwon, 2007; Gwon and Gruber, 2007a,b; A. Gwon, L. Gruber, S. Norrby, T. Terwee, H. Weber and S.A. Koopmans, unpublished observations, 2008). It is well known that silicone intraocular lenses are associated with more capsular fibrosis than acrylic polymers. Whether the HA provided a barrier between the silicone polymer and the capsule or had a molecular role in inhibiting capsular fibrosis are yet to be discerned.

9.5Potential human application

The first evidence of the regenerative ability of the natural lens in humans was reported by Sommering in 1828 (Duke-Elder, 1969b) when he observed the development of a ring-like transparent mass in the lens capsule following extracapsular lens extraction in humans. The so-called ‘Elschnig pearls/ Sommering ring’ formation and pco continue to be common complications of modern extracapsular cataract surgery, yet may provide the necessary initial cells for engineering a replacement lens.

Further evidence of the regenerative ability of the human lens is provided in a report by Marcus Gunn. He observed the growth of new lens fibers in an adult after spontaneous absorption of a traumatic cataract sustained in early life (Gunn, 1888). Subsequently, Randolph reported that Becker had noted a transparent new formation in an 80-year-old female patient at the site of the crystalline lens, which had been removed 11 years previously (Randolph, 1900).

9.6Conclusions

Tissue engineering has the potential to restore all the functional properties of the lens, including accommodation, as the regenerating process is initiated with reformation of the fetal lens and recreation of a young flexible lens. With minimal modification of current surgical techniques it is possible to restore the lens capsule and the residual lens epithelial cells left in the capsule bag can be directed to grow with the well-defined spatial order of the natural lens (Fig. 9.4).

While the insertion of a biodegradable HA-based scaffold has improved the structure of the regenerating lens, the alignment of the earliest regenerating lens fibers and the optimum balance between regenerating fibers and degradation of the scaffold remain a challenge. A further understanding of the molecular role of HA and the mechanical properties affecting the regenerative process is the subject of ongoing research.

9.4 Spherical regenerated lens with good structure and clarity after dissolution of HA scaffold (2 months postoperative).

Because of time requirements for age-related cytogenesis rates, it is anticipated that the use of a biodegradable scaffold for the treatment of cataract/refractive errors would be more applicable to the pediatric population and might entail the use of serial spectacles and/or contact lenses during the regenerative process. It may also be possible to accelerate the regenerative process in adults by molecular or cellular means.

Alternatively, the natural regenerating lens tissue may be directed to grow in the concentric pattern of the normal lens around a suitably flexible and biocompatible synthetic polymeric scaffold with refractive properties that would be suitable for treatment of cataract and refractive errors and provide true accommodation and correction of presbyopia in the adult.

9.7Future trends

The current trend in cataract surgery is toward minimally invasive techniques that will restore accommodation in both the cataract and presbyopic patient. Tissue engineering of the lens can be accomplished through the design of new materials to control the regenerative process or through molecular stimulation of the spontaneously regenerating lens. In the former instance, work with synthetic refractive polymers shows great promise. The latter approach provides a basic understanding of the genes and molecular mechanisms involved which could potentially lead to a non-engineered approach to regenerating the lens in vivo.

The ultimate goal of these approaches will be to restore all the natural properties and functions of the lens, including transparency, focusing power, spectral transmission and accommodative ability.

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9.8Acknowledgements

The author wishes to thank the following: Brad Gray and Lawrence Gruber for their invaluable assistance in manuscript and laboratory support; Carolyn Bates, Shelley Buchen, John Lally and Jim Deacon for their advice and support; Jane Rady and the late Irving H. Leopold for their support and encouragement; and numerous colleagues and friends who have contributed their research efforts and expertise in the area.

9.9References

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