Scleral structure and biomechanics

Highlights

• Reviews the state of the art in structural, biomechanical and in silico techniques for characterising the sclera.

• Presents a comprehensive view of physiological biomechanical loads on the sclera , including fluid pressures and eye movements.

• Discusses recent findings on scleral changes in ageing, glaucoma and myopia.

• Challenges current thinking about the relationship between scleral structure and biomechanical function.

• Summarises recent therapeutic breakthroughs and predicts future areas of progress.

Abstract

As the eye's main load-bearing connective tissue, the sclera is centrally important to vision. In addition to cooperatively maintaining refractive status with the cornea, the sclera must also provide stable mechanical support to vulnerable internal ocular structures such as the retina and optic nerve head. Moreover, it must achieve this under complex, dynamic loading conditions imposed by eye movements and fluid pressures. Recent years have seen significant advances in our knowledge of scleral biomechanics, its modulation with ageing and disease, and their relationship to the hierarchical structure of the collagen-rich scleral extracellular matrix (ECM) and its resident cells. This review focuses on notable recent structural and biomechanical studies, setting their findings in the context of the wider scleral literature. It reviews recent progress in the development of scattering and bioimaging methods to resolve scleral ECM structure at multiple scales. In vivo and ex vivo experimental methods to characterise scleral biomechanics are explored, along with computational techniques that combine structural and biomechanical data to simulate ocular behaviour and extract tissue material properties. Studies into alterations of scleral structure and biomechanics in myopia and glaucoma are presented, and their results reconciled with associated findings on changes in the ageing eye. Finally, new developments in scleral surgery and emerging minimally invasive therapies are highlighted that could offer new hope in the fight against escalating scleral-related vision disorder worldwide.

Introduction

Forming around 85% of the outer tunic of the human eyeball, the sclera is a remarkably resilient and structurally complex connective tissue that performs multiple functions critical to vision. Derived from the Greek word “skleros” (meaning “hard”), the sclera's primary role is to provide a firm and stable substrate for the retina and to protect the other mechanically vulnerable internal structures of the eye, while its opacity prevents off-axial light transmission that could otherwise degrade the retinal image. Scleral and corneal geometry are cooperatively regulated to accurately focus light onto the retina. Although under normal conditions the sclera can be considered metabolically quiescent, it is far from inert in a biomechanical sense. Indeed, it is required to maintain optical stability under highly dynamic loading conditions imposed externally and internally by, amongst other factors, eye movements and a continually fluctuating intraocular pressure (IOP). The sclera's ability to resist deformations that might otherwise impair vision through distortion of the retina or the lens-iris diaphragm relies on biomechanical characteristics imparted by regional specialisations of its connective tissue organisation. In recent years, widening collaboration between clinicians, scientists and engineers has led to significant advances in our understanding of dynamic scleral behaviour. Naturally it follows that we are beginning to perceive with more clarity the central role that the sclera plays in conditions that deteriorate vision. This article aims to summarise and reconcile the findings of these studies as it reviews the state of the art in scleral structure and biomechanics research, considers the implications for ocular ageing and disease, and explores some promising therapeutic avenues in search of novel scleral treatments.

At its anterior boundary the sclera merges with the corneal perimeter at the limbus and extends backward to form an approximate sphere of vertical diameter ~24 mm. The axial length of the emmetropic adult human eye is 24–25 mm. At the back of the eye, the scleral connective tissue fuses with the dural sheath of the optic nerve, whose entry pierces the sclera about 3 mm nasally and 1 mm downward of the posterior pole. Scleral thickness varies with anatomical position, decreasing from 1-1.3  mm at the posterior pole to ~0.5  mm at the equator, before increasing again to ~0.8 mm in the perilimbal sclera. (Fig. 1A).

Tenon's capsule (fascia bulbi) is a compact layer of collagen bundles and elastin fibres that lie parallel to the scleral surface. At its anterior origin, Tenon's capsule is anchored firmly to the underlying episclera and the overlying conjunctiva, before becoming more loosely bound to the episclera further back as the capsule coalesces with the perimysium of the recti muscles (Fig. 2). Biomechanically, the Tenon's capsule fulfils an important function in acting as a pulley system to transfer forces from the ocular muscles to the sclera during eye movements (Roth et al., 2002). Accordingly, the collagen bundles and elastin fibres of the anterior Tenon's run broadly perpendicular to the limbus, consistent with the directions of recti muscle force transduction (Park et al., 2016).

Lying directly beneath Tenon's capsule, the episclera is a thin but dense layer of connective tissue consisting mainly of collagen bundles sparsely populated with elastic fibres, melanocytes and macrophages (Watson and Young, 2004). The collagen bundles of the episclera run largely circumferentially and render the episclera more difficult to distinguish as a distinct layer by their gradual merging into the connective tissue of the underlying stroma.

As the major scleral tissue layer, the stroma (substantia propria) dominates the sclera's biomechanical performance. Stromal material properties can be summarised as non-linear viscoelastic, and stem from its collagen-rich extracellular matrix (ECM) composition and organisation. Bundles of parallel-aligned individual collagen fibrils of diameter 25–230 nm, interspersed in places with elastic microfibrils and fibres, form 0.5–6 μm thick lamellae that lie roughly in the plane of the eyeball surface (Fig. 1B). Scleral lamellae overall demonstrate far more branching and interweaving than those of the neighbouring corneal stroma, and the extent of this varies with both tissue depth and anatomical location (Komai and Ushiki, 1991). Superficially, the scleral collagen fibril bundles merge with tendon fibres at the extraocular muscle insertion sites, while in the deepest stromal layers adjacent to the uvea (the lamina fusca) they taper and branch to intermingle with the underlying choroidal connective tissue, co-localising with increased numbers of elastic fibres (Marshall, 1995). Unlike the eyes of humans and other primates, the scleral stroma of many non-eutherian vertebrates comprises an inner cartilage layer in addition to an outer fibrous layer (Walls, 1942). Further, the anterior sclera of many birds, reptiles and teleost fish contains a ring of bony plates ('ossicles') (Franz-Odendaal, 2008) that are thought to provide leverage for the ciliary muscles in facilitating corneal accommodation (Glasser et al., 1994), and into which meridional fibril bundles of the anterior sclera insert (Boote et al., 2008). In the human sclera, regional specialisations of the stromal architecture, as described below, are of particular biomechanical influence.

On approaching the limbus, the collagen bundles of the deep scleral stroma form a circumcorneal ring-like structure at the scleral spur. Together with the circumferential limbal pseudo-annulus of collagen residing in the posterior one-third of the corneal stroma (Kamma-Lorger et al., 2010; Newton and Meek, 1998), the spur probably helps to maintain the corneal contour in an area of heightened tissue stress imposed by the differing radii of curvature of the cornea and sclera (Boote et al., 2009). Indeed, the use of partial thickness limbal incisions is an established clinical procedure for inducing controlled flattening of the cornea as a means of correcting mild corneal astigmatism. However, not all of the scleral collagen appears to end its frontward course at the limbus, and there is evidence that a significant number of perilimbal scleral collagen bundles continue on into the corneal periphery (Boote et al., 2011), some of which probably originate in the deep sclera (Winkler et al., 2013). At its anterior aspect, the collagen fibrils of the scleral spur taper and become continuous with the connective tissue beams of the corneoscleral trabecular meshwork (Watson and Young, 2004). Here, the innermost layers of the spur, the so-called 'scleral roll', form a bordering substrate for the Schlemm's canal (SC), from whose posterior end an extension of the spur in the direction of the anterior chamber provides an anchor point for attachment of the meridional fibres of the ciliary muscle to facilitate opening of the trabecular beams during aqueous drainage (Hamanaka, 1989). The corneoscleral trabecular beams are further notable for containing significant amounts of elastin (Marshall, 1995; Umihira et al., 1994). These elastic fibres form a tensionally-integrated network that is conserved across species, and appears to be important in tethering the trabecular meshwork to neighbouring tissues - possibly as a design to prevent collapse of outflow pores during IOP fluctuation (Bachmann et al., 2006; Overby et al., 2014; Rohen et al., 1981; Tektas et al., 2010). The trabecular elastin is probably continuous with the elastic fibre networks observed in the deep limbus (Kamma-Lorger et al., 2010) and the pre-Descemet's stroma of the corneal periphery (Lewis et al., 2016), and the idea of a continuous system of elastic fibres covering the majority of the eye tunic and biomechanically linking the posterior pole to the peripheral cornea is an intriguing possibility that warrants further research.

On approaching the optic nerve, superficial layers of the stromal connective tissue merge with the dural sheath of the nerve while the remaining deeper scleral fibres become continuous with the lamina cribrosa (LC) - the highly fenestrated stack of interconnected plates that support the exiting retinal ganglion cell (RGC) nerve axons and central retinal artery (Anderson, 1969). The LC and peripapillary sclera (PPS - the 1–2 mm wide region of sclera bordering the nerve canal opening) collectively form the connective tissue of the optic nerve head (ONH) (Fig. 1D) - a region of key biomechanical interest in glaucoma (Downs, 2015). Here the scleral collagen fibrils are more uniform in diameter, show greater spatial order and associate with increased numbers of elastin fibres compared to other regions of the posterior segment (Quigley et al., 1991). A key biomechanical feature of the PPS is the circumferential pseudo-annulus of collagen that surrounds the LC (Gogola et al., 2018b; Pijanka et al., 2012; Winkler et al., 2010) that is probably necessary to limit canal expansion under IOP-loading (Girard et al., 2009a; Grytz et al., 2011).

The composition of the sclera follows that of other connective tissues in being primarily a scaffold of fibrous collagen in a hydrated interfibrillar matrix of proteoglycans and glycoproteins (Table 1). Notwithstanding notable increases in the lamina fusca, perilimbal sclera and PPS, the overall content of elastin fibres in the sclera is small at around 2% of the dry weight (Watson and Young, 2004). Understandably from a metabolic perspective, the quiescent sclera displays low cellularity with transient increases shown in response to pathology or physical insult.

In the sclera, type I collagen is by far the major contributor at around 95%, with types III, V and VI making up the remaining 5% (Keeley et al., 1984; Thale and Tillmann, 1993). Scleral collagen structure is hierarchical (Fig. 3). Tropocollagen molecules of length ~300 nm are composed of three polypeptide alpha-helix chains of repeating Gly-X-Y amino acid sequences (Bailey et al., 1998). Five such molecules assemble to form ~4 nm diameter collagen microfibrils, in which adjacent molecules are axially staggered by 67 nm (the D-period – see Figs. 1C and 3) (Piez and Miller, 1974). Parallel arrays of microfibrils assemble into fibrils, such that individual microfibrils are slightly inclined (about 5°) to the fibril axis (Yamamoto et al., 2000). Collagen fibrils, in turn, assemble into irregular bundles that ultimately form the scleral lamellae. Scleral collagen fibrils are heterotypic: studies of macular sclera indicated interstitial collagen fibrils of co-polymerised types I/III, with type V residing at the fibril surface and type VI forming inter-bundle filament structures (Marshall et al., 1993). The presence of types V and VI at and between fibril surfaces suggests likely roles in fibril assembly and diameter regulation, as envisaged in other tissues (Izu et al., 2011; Linsenmayer et al., 1993; Wenstrup et al., 2004). In contrast to some other connective tissues, such as tendon, collagen in the sclera does not assemble into discrete structures of a regular size beyond the fibril level. However, the general term “fibre” is used widely in the biomechanics literature to refer to the suprafibrillar collagen arrangement of the sclera and will also be used in this review. It should also be noted that some techniques that utilise visible light to examine suprafibrillar scleral microstructure (see s2.1.2 and s2.1.4) will contain both collagen and elastin components in their “fibre” signal.

Proteoglycans (PGs) inhabit the collagen interfibrillar space and help to mediate fibril size and organisation. PGs consist of a protein core with one or more attached glycosaminoglycan (GAG) sidechains of repeating disaccharide units of either chondroitin sulfate, dermatan sulfate, keratan sulfate or heparin sulfate. The main sulfated PGs present in sclera are aggrecan, decorin and biglycan (Rada et al., 1997, 2000). Sulfate residues on the GAG chains impart negative charge that binds water and creates an incompressible “gel” that is ideal for mediating load transfer between the embedded scleral collagen fibrils. Evidence for the importance of PGs in maintaining scleral structure and biomechanics includes findings from research in knock-out mouse models (Austin et al., 2002; Chakravarti et al., 2003) and from enzyme digestion studies in pig (Murienne et al., 2015; Zhuola et al., 2018) and human (Murienne et al., 2016) sclera. The role of aggrecan in the sclera is not well understood. Aggrecan is a large proteoglycan normally found in cartilage. Due to its many attached glycosaminoglycan sidechains, aggrecan provides osmotic properties that produce a swelling pressure. In cartilage, this swelling pressure plays a critical role in withstanding compression forces, but the importance of swelling pressure in the sclera is unclear.

The elastic fibre network is a proportionately small but functionally important part of the scleral ECM, in particular in the deep tissues of the perilimbal sclera/trabecular meshwork (Marshall, 1995) and throughout the ONH (Quigley et al., 1991) where concentrations are notably increased. Mature scleral elastic fibres consist of an amorphous elastin core sheathed by an aligned scaffold of fibrillin-rich microfibrils. Outside of the trabecular meshwork (see s1.1.4), the significance of scleral elastic fibres to the structural integrity, shape and viscoelastic behaviour of the eyeball is not well understood, but is coming under increasing scrutiny motivated by identified links between systemic microfibril disorders and ocular pathologies such as myopia and glaucoma (Kuchtey and Kuchtey, 2014; Robinson and Booms, 2001).

With the exception of the lamina fusca, most regions of the sclera are sparsely populated by cells until challenged by pathology, injury or infection. The resident cell of the scleral stroma is the fibrocyte, which undergoes transformation into the active fibroblast upon insult. Fibroblasts are responsible for synthesis of all scleral ECM components. They respond to mechanical stimuli from their surrounding ECM and there is growing interest in understanding the extent of the role that fibroblasts might play in dynamically regulating scleral biomechanics via matrix remodelling and contractile responses (Harper and Summers, 2015; McBrien et al., 2009) (see s3.3.2). In addition to mechanical stimuli, fibroblasts control scleral remodelling and alter tissue-level biomechanics in response to a signaling cascade from retina to sclera that is ultimately stimulated by vision. This vision-guided response plays a critical role during eye development, determining the final size of the eye (see s4.3).

The ECM structure and biomechanical properties of the sclera depend on the tissue water content. As mentioned, stromal swelling pressure in the sclera is largely determined by the distribution and sulfation level of constituent PGs (s1.2.2). However, there is also believed to be a significant contribution from free chloride binding ligands, which may help to explain the differential swelling of the corneoscleral tunic in response to changes in its ionic environment (Hodson, 1997; Huang and Meek, 1999). In vitro swelling studies (Huang and Meek, 1999) determined the isoelectric point of the sclera (where the tissue swells least) to be at pH 4, with higher tissue hydrations achievable when the ionic strength of the bathing medium was lower. The sclera loses water as its ages (Brown et al., 1994; Rada et al., 2000), an effect which is suspected to be driven by associated changes in PG composition (see s4.1).

Tissue hydration state is also important in determining the permeability of the sclera to exogenous compounds. While molecular size/shape, surface charge and lipophilicity are the dominant predictors of scleral permeability for a given substance (Ambati et al., 2000; Lee et al., 2004; Olsen et al., 1995), diffusion rates in rabbit sclera have been shown to be elevated with increasing hydration across a range of solutes of varying molecular weight (Boubriak et al., 2000). Enhancing scleral permeability is an active area of research in the field of ocular drug delivery, particularly in the management of posterior segment disease where transscleral delivery is especially challenging due to the limited accessibility and increased thickness of the sclera at the back of the eye (Cabrera et al., 2019; Moisseiev and Loewenstein, 2017; Yamada and Olsen, 2016). Moreover, scleral permeability also has important therapeutic implications in the formulation of exogenous chemical agents for scleral crosslinking treatment (see s5.2).