The Roles of Vitreous Biomechanics in Ocular Disease, Biomolecule Transport, and Pharmacokinetics

Abstract Purpose The biomechanical properties of the vitreous humor and replication of these properties to develop substitutes for the vitreous humor have rapidly become topics of interest over the last two decades. In particular, the behavior of the vitreous humor as a viscoelastic tissue has been investigated to identify its role in a variety of processes related to biotransport, aging, and age-related pathologies of the vitreoretinal interface. Methods A thorough search and review of peer-reviewed publications discussing the biomechanical properties of the vitreous humor in both human and animal specimens was conducted. Findings on the effects of biomechanics on vitreoretinal pathologies and vitreous biotransport were analyzed and discussed. Results The pig and rabbit vitreous have been found to be most mechanically similar to the human vitreous. Age-related liquefaction of the vitreous creates two mechanically unique phases, with an overall effect of softening the vitreous. However, the techniques used to acquire this mechanical data are limited by the in vitro testing methods used, and the vitreous humor has been hypothesized to behave differently in vivo due in part to its swelling properties. The impact of liquefaction and subsequent detachment of the vitreous humor from the posterior retinal surface is implicated in a variety of tractional pathologies of the retina and macula. Liquefaction also causes significant changes in the biotransport properties of the eye, allowing for significantly faster movement of molecules compared to the healthy vitreous. Recent developments in computational and ex vivo models of the vitreous humor have helped with understanding its behavior and developing materials capable of replacing it. Conclusions A better understanding of the biomechanical properties of the vitreous humor and how these relate to its structure will potentially aid in improving clinical metrics for vitreous liquefaction, design of biomimetic vitreous substitutes, and predicting pharmacokinetics for intravitreal drug delivery.

Swindle-Reilly observed areas of stiffened vitreous in older human samples. This data supports the hypothesis that the vitreous phase-separates with age, creating areas of stiffened vitreous gel comprised mostly of collagen and loosened vitreous liquid comprised mostly of hyaluronic acid. 6 Similarly, Silva et al. found that the solid phase of rabbit vitreous was stiffer 4 hours post-dissection compared to the gel phase immediately after dissection, potentially due to the expulsion of aqueous hyaluronic acid from the vitreous gel. 7 Filas et al. found that vitreous digested with hyaluronidase was more elastic-like compared to vitreous digested with collagenase, likely due to the collapse of the collagen network beyond a critical density. 8 This further demonstrated the elastic contribution of collagen to the vitreous humor. Huang et al. quantified the mechanical properties of bovine vitreous humor digested with collagenase and hyaluronidase. 9 Their results, similar to those from , showed a higher stiffness in hyaluronidase-treated vitreous compared to collagenase-treated samples, which they attribute to the loss of tension as hyaluronan is less capable of maintaining the Donnan swelling needed to support the vitreous. 9 These results suggested that the vitreous humor is under tension in its native condition, mostly due to the swelling of hyaluronic acid. The structural integrity of the vitreous results from this tensile state. Upon removal from the eye, the internal tension is broken, resulting in a rapidly degrading vitreous. However, this internal tension hypothesis has not been investigated in vivo, mostly due to limitations in non-invasive measurement techniques. 10 Excised vitreous humor does not experience many natural aspects of ocular physiology that could contribute to biomechanics, such as intraocular pressure, ocular pulse, and vitreoretinal connections. Additionally, it is unknown whether cells within or surrounding the vitreous contribute to this internal tension. These are all potential factors that could affect this internal tension, which appears to be one of the primary influencers of the biomechanical properties of the vitreous humor. Since the biomechanical stiffness of collagen-reinforced tissues increases exponentially with strain, this residual tension could play a key role in determining the in vivo stiffness of the vitreous. Figure 1 gives a lumped parameter model of the role of vitreous biomechanics in the eye. Collagen fibers are represented by an elastic spring, while hyaluronic acid is schematically represented by a dashpot as it contributes primarily to the viscous behavior of the vitreous. Growth and osmotic swelling of the vitreous tamponades the retina and choroid against the sclera; these tissues are otherwise only held in place by surface tension. Liquefaction and/or posterior vitreous detachment (PVD) could diminish the swelling pressure and therefore increase the likelihood of retinal detachment. The internal tension theory suggests that growth and swelling would elongate the collagen spring, thereby increasing the in vivo stiffness of the vitreous. Finally, swelling of the vitreous may be essential to growth of the developing eye: the resulting stresses would be transmitted to the corneoscleral shell. 11

Biomechanical properties of the vitreous humor
The vitreous humor is clearly a highly complex and dynamic tissue, with a variety of interactions between molecular, cellular, and environmental components driving its behavior and role in maintaining the structure of the eye. The clearest example of these intersections is in the biomechanical performance of the vitreous humor. The vitreous humor can be viewed as a protein-polysaccharide interpenetrating polymer network hydrogel, with collagen poly(amino acid) fibrils providing structure and determining the gel's mechanical behavior while hyaluronic acid chains absorb water and swell to maintain spacing between the fibers. 12 Due to this structural complexity, the vitreous can be expected to display viscoelastic behavior as it is exposed to dynamic stresses and strains through regular eye motion and external mechanical insults. Understanding how the vitreous humor responds to mechanical stress can offer valuable insights into the health of the vitreous humor and its ability to support the structure of the eye. These findings can in turn be translated to explore how changes in the vitreous humor due to age and disease can induce pathologies in other tissues of the eye, including the lens and retina and how changes in vitreous biomechanical performance can play a key role in the development of ocular pathologies.

Human vitreous biomechanics
The mechanical properties of the human vitreous humor have been explored through a variety of methods, ranging from standard oscillatory rheometry to novel biomaterial probes. 6,13 However, this wide range of explorations has led to a wide range of measured values for key viscoelastic parameters, some differing by several orders of magnitude depending on the method employed. 6 A recent publication has explored these efforts in great depth, 10 so these efforts will be summarized concisely here. Since 1975, 13 studies have evaluated the mechanical properties of the human vitreous humor, only three of which have done so in situ with the tissue still in the eye, rather than using extracted, in vitro samples of the vitreous humor. 10 Tram et al. (2021) evaluated these studies to identify reports that fully captured storage, loss, and elastic modulus values of the human vitreous humor, finding an average storage modulus (G 0 ) of 5.09 ± 1.86 Pa, loss modulus (G") of 1.81 ± 0.98 Pa, and elastic modulus (E) of 1.17 ± 0.56 Pa for n ¼ 3 papers, using shear rheometry in the 0.1-100Hz frequency range on patients aged 33-92. 6,10,14,15 Selected reports of the human vitreous humor's viscoelastic properties were evaluated to obtain values of storage and loss modulus, which are summarized in Table 1. 14,15 All of these reports were based on shear rheometry, and are in close agreement, with G 0 slightly higher than G 00 . G 0 and G 00 were in the ranges of 0.5-15 Pa and 0.1-7.5 Pa, respectively.
It is important to note that only a small number of published studies reported storage and loss moduli, with most reporting only the dynamic modulus of the vitreous. This may be a result of the variety of testing techniques used to evaluate vitreous properties. Shear rheometry provides useful data for the characterization of viscoelastic behavior but is highly destructive when conducting amplitude sweeps or frequency sweeps beyond the linear viscoelastic region and requires the vitreous to be removed from its native environment. 10 Non-shear rheometry techniques such as viscometry, ultrasound, and MRI-based rheometry give moduli that do not agree with data gathered through physical rheometry of the vitreous humor, with no clear directionality to the differences in properties. 10 Because of these challenges in characterization depending on method, it has been historically difficult to obtain consistent estimates of the vitreous humor's viscoelastic properties. Modern improvements in shear rheometry in addition to new minimally invasive approaches to testing the vitreous humor have begun to enable more consistent and complete characterization of the vitreous.

Animal vitreous biomechanics
Perhaps the greatest challenge in research exploring vitreous biomechanics is the difficulty of obtaining human vitreous humor, particularly from younger eyes, for mechanical evaluation. As a result, there is a much more significant body of research investigating the properties of animal vitreous humor. Previous investigations by our lab (Tram 2021) found that since 1975, 29 studies analyzed the properties of cow, pig, sheep, rabbit, and goat vitreous humors. 10 [16][17][18][19] These studies used a wide variety of techniques to quantify the properties of the vitreous, ranging from direct measurement techniques such as creep rheometry and viscometry to noninvasive techniques such as MRI and ultrasound-based in vivo measurements, obtaining values generally similar to those of the human vitreous. Pig and rabbit samples appear to be the best mechanical analogues, with storage and loss moduli similar to the average mechanical properties of human vitreous. 10 As with studies of the human vitreous, shear rheometry has been the most commonly used method for the evaluation of animal vitreous viscoelasticity in the last decade, with studies in rabbit, pig, cow, and sheep eyes making use of this technique. 7,10,20,21 A summary of animal and human studies on the biomechanics of the vitreous is shown in Table 2. A full list of the studies is available in Supplementary Table 1. The animal studies include 17 run on pig eyes, 15 on cows, 4 on rabbits, 2 on sheep, and 1 on goats. 10 The effect of aging on the vitreous As with any biological tissue, the vitreous humor is subject to the biological, chemical, and mechanical forces involved in aging. The effects of age in the vitreous manifest in a variety of ways unique to its structure, although these effects can be explained by more conventional aspects of tissue aging. As the vitreous ages, its collagen fibrils begin to degenerate and lose their protective layer of type IX collagen, responsible for ensuring proper fibril spacing. 3,22 This results in exposure of the underlying type II collagen layer, causing the collagen fibrils to aggregate into larger clusters, which leads in turn to a reorganization of the interpenetrating network of hydrating hyaluronic acid chains. 23,24 As a result, the regions previously filled by a network of collagen fibrils gradually grow smaller, with lost collagen volume replaced by wells of fluid, aided by concurrent depolymerization of hyaluronic acid chains and release of water into Table 1. Selected recent findings for viscoelastic moduli of human vitreous humor in vitro.

Modulus
Shafaie et al. 13 Schulz et al. 14  these wells. 9,24 This process of gradual separation of the vitreous into a collagen gel and liquid phase is known as vitreous liquefaction, which can develop in over 80% of the population over the age of 60. 3,25,26 While this process may begin in early childhood, it is much more significant after age 40, when liquefaction of the vitreous steadily increases until over 50% of the vitreous has liquefied by the age of 90. 25 This is likely to be related to the 11 year half-life of type IX collagen, which means that nearly 95% of type IX collagen has degraded by age 45, assuming no new type IX collagen synthesis after birth. 3 Interestingly, the onset of significant liquefaction lines up with the age at which presbyopia, or loss of ocular accommodative power, begins to become a significant issue. 27 Starting in small pockets, the progressively increased liquefaction of the vitreous can eventually reach the point where the vitreous begins to detach from the retinal surface, a condition known as posterior vitreous detachment (PVD) that can occur in up to 25% of the global population. 3,24,28 This detachment initiates when the liquefaction process reaches the vitreoretinal interface in the posterior region of the eye and may be accompanied by severe complications that risk blindness or require surgical removal and replacement of damaged tissue. 24 While there has been little research into factors that might accelerate the process of vitreous liquefaction or trigger PVD, some hypothesized stressors include the effects of light, radiotherapy, oxidative damage to the vitreous, ocular trauma, or enzymatic activity. 17,29,30 Biomechanically, the process of vitreous phase separation and degeneration of the vitreous humor's collagen network has a notable impact on its performance as a viscoelastic damper in the eye. Silva et al. (2017) used New Zealand rabbit eyes to evaluate the properties of the liquid and gel phases of an eye undergoing liquefaction, finding that the liquid phase exhibited significantly lower viscoelastic moduli than the gel phase and strong shear thinning. 7 Their analysis of the obtained mechanical data indicates that the liquid and gel phases are likely to be mostly aqueous hyaluronic acid and collagen, respectively, offering insight into how these two phases might interact in the aging eye. Tram et al.
(2018) directly compared human eyes of patients older or younger than 65 to evaluate the impact of age-related changes on the mechanics of the vitreous. Their work found significant increases in the viscosity and dynamic modulus of the solid, collagenous vitreous, while the liquid vitreous showed decreased viscosity and dynamic modulus. 6 Taken together, these findings suggest that the phase separation that occurs during aging of the vitreous simultaneously stiffens remaining gelatinous vitreous humor, while introducing a new, significantly softer liquid phase in the eye. In a comprehensive study with a large number of samples, Schulz et al. (2019) evaluated the viscoelastic behavior of this twophase vitreous humor and found that the viscoelasticity of the whole vitreous significantly decreases with increased age, likely due to both the degraded collagen fiber network and the disruption caused by subsequent liquefaction. 15 Work by Levin and Cohen (2021) to model the impact this phase separation has on the eye through mathematical modeling confirmed the results of Schulz, indicating that much of the reduction in viscoelasticity was due to the emergence of the liquid phase in the aging vitreous. 24 Their models also showed that as the degree of PVD increases, the vitreous significantly softens, with its stiffness dropping to a fraction of its original value by the time of complete detachment. 24 This drop in stiffness with increasing liquefaction and PVD has implications on theories of ocular swelling, as the disconnection of the vitreous from one of its two key attachment points causes it to lose its internal tension. This leads to loss of Donnan swelling and subsequent collapse of the normal collagen network, furthering the progression of PVD according to the theory outlined by Huang et al. 9 In patients with myopia, where the eye is elongated compared to its normal physiology, higher rates of PVD have been observed, likely due to the increased tension experienced by the myopic eye's collagen network from ocular lengthening. 31

Limitations of vitreous mechanical evaluation techniques
While there has been recent significant progress in evaluating the mechanical properties of the vitreous humor, with advances in technique enabling key discoveries in the behavior of the vitreous, there are still limitations in our current ability to evaluate the biomechanics of the vitreous. While animal vitreous humor is commonly used to model the properties of the vitreous when human eyes are unavailable, only certain species, namely pigs and rabbits, appear to have moduli roughly similar to those of humans. 10 Other common models such as cows and sheep, while more readily available, have significant discrepancies in one or more measures of viscoelastic behavior, and are thus somewhat limited in their ability to accurately provide models of the human eye's biomechanical response, both before and after the effects of aging. 10,14 Perhaps more importantly, a significant portion of vitreous biomechanics research is conducted using vitreous samples that have been extracted from the eye for in vitro evaluation. 10 This environment is significantly different from the vitreous in vivo, which is tightly connected with its surrounding tissues and anchors its structural collagen fibers to the inner lamina of the eye. 3,25 The in vivo vitreous humor gel is also kept highly hydrated, at equilibrium swelling, which likely plays a role in its mechanical performance through ensuring adequate collagen fiber spacing. 5,32,33 The destruction of these connections and their effects on vitreous collagen fiber tension is significant and severe, with experimental evaluation of bovine and porcine vitreous revealing that within minutes of extraction from the eye, vitreous samples displayed significant decreases in mechanical performance as measured through shear rheometry, as well as evidence of significant dehydration. 5 This effect can be visualized in Figure 2, which shows how excised vitreous humor from a porcine eye deforms after two hours. Immediately apparent is the non-native shape of the excised vitreous humor after removal, as well as progressive changes to the structure of the vitreous over 120 minutes and clear evidence of fluid loss from the vitreous. These changes are very similar to those found by Nickerson et al., and indicate significant disruption of the vitreous humor's structure and biomechanical behavior following removal from the eye. 12 As much of the work characterizing vitreous biomechanics has been done in vitro, these rapid and significant changes to its properties immediately after excision may indicate wide-scale underestimation of the mechanical properties of the vitreous.
Further, most of the human tissue samples evaluated are extracted from older eyes, and are therefore more likely to be highly liquefied or degenerated. An accurate, noninvasive measuring technique is warranted to effectively evaluate the biomechanics of young and healthy vitreous humor. As an increasing number of vitreous substitutes are being developed, it may be useful to consider this risk of underestimation when designing experiments to evaluate their ability to mimic the properties of the native vitreous. 34

Connecting the vitreous to the retina
The vitreous humor's connections to the retinal tissue of the eye are evidently important in maintaining its performance as a viscoelastic material. Understanding this connection in healthy eyes is thus critical to our ability to fully evaluate the behavior of the vitreous. Vitreoretinal connections in the eye are mediated by the joining of collagen fibrils with the internal limiting lamina (ILL) of the retina, which is secreted by M€ uller cells. 35 The ILL is closely associated with the posterior vitreous cortex, a dense layer of collagen fibrils that forms the outer layer of the vitreous humor. 26 These layers of connective proteins mediate the vitreoretinal connection through two principal methods, which are largely dependent on the organization of collagen fibers relative to the ILL. In the equatorial region of the vitreous, collagen fibrils run parallel to the vitreous and are much finer, so vitreoretinal adhesion is likely to emerge from the efforts of a number of interfacial proteins and molecules which collectively form a molecular "glue" that adheres the vitreous to the ILL. 33,35,36 At the vitreous base in the posterior of the eye, where the more coarse and densely packed collagen fibrils penetrate the vitreous cortex and run perpendicular to the ILL, vitreoretinal adhesion emerges from the insertion of collagen fibers through the ILL to anchor on M€ uller cells, leading to the development of much stronger adhesions. 26,33,36 This posterior anchoring of collagen fibers may be assisted by the formation of complexes with collagen types VI, VII, and XVIII, which have been found to be expressed in the posterior vitreous humor and are known to play a role in anchoring extracellular matrix components. 37 These region-by-region differences in the mechanisms of vitreoretinal adhesion have a significant impact on the mechanical strength that these adhesions provide, which in turn can have significant downstream impacts on how biomechanical insults to the vitreous affect the surrounding retinal tissue. Initial evidence for this difference in adhesive strength was largely qualitative, with work by Gandorfer et al. (2001) finding that treatment of porcine eyes with plasmin led to differential removal of collagen fibrils and detachment of the vitreous cortex depending on the region of the eye examined, with the equator having the most rapid detachment and the vitreous base having no detachment at all. 38 This provided some indication as to the relative density of collagen fibers in these regions and implied the possibility of different adhesion strengths based on these different densities. Pioneering work by Sebag (1991) found evidence for differences in adhesive force based on age and position in the eye based on observations of dissection of the vitreoretinal interface in human eyes; however, this work still relied on qualitative evaluation of separation behavior and examination of histology at the dissected region. 39 Creveling et al. (2018) extended these findings to provide quantitative assessments of vitreoretinal adhesive forces through peeling tests on strips of retinal tissue in the equator and posterior pole of the eye, using a specialized rotational device to collect data through a mechanical testing array. 36 They found quantitative evidence for previously observed qualitative differences in adhesion strength, with overall maximum peeling force at the equator of the eye measured at 7.16 ± 4.08 mN compared to 4.08 ± 2.03 mN at the posterior pole of the eye. 36 They also found significant differences in adhesion force for patients under 60 versus over 60, with a drop in maximum equatorial peeling force from 8.76 ± 4.22 mN down to 4.50 ± 2.00 mN and maximum posterior pole peeling force from 4.08 ± 2.03 mN down to 2.95 ± 1.25 mN. 36

Disruption of the vitreoretinal interface and resulting pathologies
Recently, there has been increasing clinical evidence that vitreoretinal tractional forces can cause macular and retinal tissue damage in patients, with multiple case studies and clinical reports citing vitreoretinal traction as a pathological cause of ocular tissue tears and holes. [40][41][42] In some cases, PVD has been implicated as a causative etiology for these tears, showing the importance of understanding how disruption of the vitreoretinal interface and its impact on force transmission to the retina can potentially lead to ocular pathologies with vision-disrupting consequences. 40 As previously discussed, PVD develops when the process of vitreous liquefaction reaches the posterior of the eye and, in combination with age-related weakening of vitreoretinal adhesions, leads to complete separation of the vitreous cortex from the surface of the retina. 24,28,43 The causes of vitreous liquefaction have been previously discussed. The agerelated weakening of vitreoretinal adhesions is poorly understood, with few well-developed theories on how this process occurs. Studies have found associations between PVD and factors such as gender, age, myopia, diabetic retinopathy, whether a patient has had cataract surgery, and various collagen disorders, but have not to date developed a theory of etiology for PVD. 26,31,33,35,44 PVD can be also artificially induced through surgical vitrectomy, intravitreal gas injections, or injection of proteolytic enzymes such as plasmin and trypsin to cleave vitreoretinal protein linkages, and can occur as a side effect of intravitreal injections, but these cases are generally outside of the accompanying context of vitreous liquefaction and aging and are thus beyond the scope of this review. 38,45,46 PVD can be evaluated through a variety of methods, such as slitlamp biomicroscopy, optical coherence tomography, and ultrasound, that can image the vitreoretinal interface to confirm whether the vitreous remains attached. 35 The disruption of ocular physiology that results from PVD is a significant change in the environment of the eye. This has significant downstream effects that are known or believed to be causative of several other ocular pathologies. Vitreous floaters and flashes are common but relatively benign symptoms of PVD in patients, with a review of research by Gishti et al. (2019) finding significant association between these symptoms and the development of retinal tears secondary to PVD. 47 Vitreous floaters are a visual phenomenon in which opacities in the vitreous humor produce shadows, usually grey lines with dark nodules, that interfere with vision and move with head and eye movements. 33 Most floaters are associated with late-stage PVD, possibly due to accompanying detachment of the inner limiting membrane of the retina or folding of the vitreous cortex as it detaches. 26 Flashes are sudden perceived bursts of light which result from the vitreous pulling on the retina as it begins to detach. 48 While these symptoms cause relatively minor disruptions to patient vision, there are more severe complications, such as retinal tears, macular holes, and other disorders caused by vitreoretinal traction in partial PVD, which can have severe sight-disrupting consequences. 35,47,49 Gishti et al. (2019) highlighted the risk of retinal tears secondary to PVD, as well as concurrent risks of retinal hemorrhage due to small vessel damage in PVD. 47 Another severe complication is the development of macular holes, which according to some hypotheses develop as a result of or are worsened by abnormal traction exerted on the macula during PVD, resulting in a hole that generally requires surgical intervention to repair. 49,50 These surgical interventions can include vitrectomy, internal limiting membrane peeling, and the use of gas or enzymes to separate the vitreous from the retina. 50 Further, proliferative vitreoretinopathy (PVR) is one of the most common complications after retinal detachment, and the contribution of factors released in the vitreous have been recently explored. 51,52 Another significant pathology that emerges from PVD is vitreomacular traction syndrome, in which partial PVD in the macular region causes distortion of macular tissue near remaining vitreomacular adhesions through forces exerted by the vitreous concentrating at these adhesions. 49,53 Work by Koiziumi et al. (2008) to visualize vitreomacular traction revealed that vitreomacular traction can take either a broad or focal form, which can result in the development of different subsequent pathologies at the vitreoretinal interface. 54 The abundance of traction-related disorders related to PVD is likely to be a result of significantly elevated tractional forces that occur at the remnant points of vitreoretinal adhesion in partial PVD, as well as tractional forces resulting from interactions between the liquid and gel phases of the vitreous. 43 The combination of these tractions subjects the remaining areas of vitreoretinal adhesion to elevated mechanical stresses which, based on computational mechanical modeling of eyes undergoing PVD, may predispose them to mechanical failure. 43 This effect can be visualized in Figure  3A, which shows the reduction in vitreoretinal adhesion area with partial PVD. In addition, the vitreoretinal detachment seen in PVD has been linked to several other ocular pathologies, with recent research indicating that detachment of vitreal collagen and subsequent reduction of vitreoretinal traction leads to reduced retinal neovascularization, reduced risk of macular edema, and increased risk of wet AMD. 35 The Impact of biomechanics on biotransport in the vitreous

Native vitreous convection and diffusion
In the native vitreous humor, the movement of biomolecules is influenced by a variety of factors including diffusion through the pores of the collagen matrix, fluid movement due to saccadic eye movements, and transport through circulation of fluids in the vitreous humor. 56,57 Diffusion through the vitreous humor is governed by properties of the vitreous such as pore size, collagen and protein fiber density, the net negative charge of the vitreous humor, and other chemical properties encouraging molecular interactions between diffusing solutes and the vitreous structure. 14,58,59 Interactions with the properties of the vitreous can play a critical role in determining how rapidly molecules are able to diffuse from their source to target tissues, which has significant implications for issues such as intravitreal drug delivery. Saccadic eye movements can also influence the movement of molecules through the eye through induction of flow in the vitreous fluid, leading to advective flow that can significantly impact the distribution of biomacromolecules in the eye. 57 While these effects are difficult to directly visualize in vivo, a number of efforts have been made at mathematical and in vitro modeling of this behavior. Notable findings from these include Repetto et al. (2010), who found that saccadic eye movements induce circulatory flow cells in the vitreous, which help to increase molecule and drug dispersal. 57 Work by Bonfiglio et al. (2013) and Stocchino et al. (2010) found that in a model vitreous humor, saccadic movement induced much more significant drug transport than diffusion and significantly increased molecular transport speed by analyzing the movement of 5 lm glass spheres. 55,60 Notably, Bonfiglio et al. (2013) found that in the vitreous, particles move in a helical pathway as portrayed in Figure 4. 55 Disruption of the vitreous during PVD disrupts this pathway as seen in Figure 3C, leading to reduced intraocular transport speed and efficiency. An analysis by Balachandran and Barocas (2011) similarly found that saccadic movement-induced flow in the eye could significantly increase particle mobility in the vitreous. 61 Other research found that vitreous flow due to saccadic movement also exerted shear stress on the wall of the eye, which could contribute to eventual tissue damage. 62,63 Finally, convective flow in the eye normally occurs to transport molecules from the anterior to posterior of the eye based on analysis of circulation in the vitreous by Smith et al. (2020). 64 By some estimates, this flow may account for up to 30% of intravitreal drug transport in the human eye, mainly affecting larger molecules. This effect is magnified in the case of patients with high IOP, such as those with glaucoma or retinal detachment. 32,65-67

Impact of aging and liquefaction
Aging and subsequent liquefaction of the eye is a dramatic change in the vitreous humor, with significant implications for a variety of ocular behaviors. This includes biotransport through the vitreous, which can potentially be impacted by the changes to pore size resulting from collagen aggregation, disruptions to circulation as the liquid and gel phases of the vitreous develop, and changes to fluid flow dynamics, particularly at the interface of the two phases of the liquefied vitreous. In the case of diffusion, experiments inducing liquefaction using enzymatic digestion of the vitreous have found that the digestion of the vitreous and development of liquid regions generally increases diffusion coefficients for molecules, enabling more rapid movement of molecules. 14,23,68 Research investigating the impact of liquefaction on saccadic movement-induced flow has found that liquefied vitreous displays significantly higher flow, especially in the vertical direction, possibly explaining both increased drug distribution after intravitreal injection and how liquefied vitreous humors undergoing PVD may exert increased forces on the retina. 69 Studies run on liquefied vitreous humor after induced liquefaction have found that convective processes are much stronger and encourage more rapid movement of particles and molecules. 9,70 One particularly important substance whose transport is impacted by liquefaction and aging is oxygen. In the healthy vitreous, oxygen is supplied to the vitreous by the choroidal vasculature at the posterior of the eye, with a gradient of oxygen through the vitreous from high concentrations in the posterior to very low, nearly hypoxic concentrations near the posterior lens capsule due in part to consumption of the oxygen by ascorbate in the vitreous. 71,72 Age-related liquefaction and PVD causes disruption to this gradient, with significant flattening of the gradient leading to much higher oxygen concentrations in regions near the lens. This increases the risk of oxidative damage and subsequent disorders in the lens tissue. 73 Computational modeling work by Filas et al. (2013) to investigate the reasons for this flattened oxygen gradient has found that key contributors to this change are reductions in ascorbate-mediated oxygen consumption and increased oxygen diffusivity through liquefied vitreous compared to intact vitreous. 73 Notably, the case of natural vitreous liquefaction is similar to that of vitreous humor removal and the addition of a vitreous substitute such as silicone oil. In both cases, vitreous ascorbate is lost and the replacement fluid has a higher oxygen diffusivity than the intact vitreous, leading to a flattened oxygen gradient and increased oxygenation of the fluids near the lens. 10

Modeling the vitreous humor
One recurring theme in these biomechanical and biotransport analyses of the vitreous humor is the increasing use of computational models to explore behavior that is difficult to directly observe or measure. The vitreous is often excluded from whole eye models or treated as a liquid. 74,75 The properties of the vitreous humor may play a key role in ensuring the accuracy of these models, making it increasingly vital that we develop precise and accurate techniques for the evaluation of vitreous properties. For example, many of the models of vitreous biotransport during saccadic eye movement rely on accurate measurements of the viscosity and diffusivity of the vitreous humor, which are known to vary depending on factors such as liquefaction state and solute size. 55,62,66 Similarly, computational evaluation of vitreoretinal interactions requires a variety of detailed properties to provide effective digital replication of observed phenomena. Di Michele et al.'s (2020) work on a model of PVD incorporated experimental data on the nonuniformity of vitreoretinal adhesion as well as the shear modulus of the vitreous and rupture force of the retina. 43 Work by Suh et al. (2021) to determine vitreoretinal traction forces after head trauma similarly made use of the shear and bulk modulus of the vitreous obtained by Liu et al. (2013) in their computational model. 76,77 Levin and Cohen's (2021) model evaluating the impact of aging and liquefaction on whole-vitreous biomechanics required the viscoelastic data of both the liquid and gelatinous phase to build a complete and effective model that could be used to test theoretical treatments for age-related mechanical changes. 24 While computational modeling has great utility in developing models capable of predicting physiologic behavior, there is a significant need for experimental validation of these models, whether in vivo or in ex vivo and in vitro models. The vitreous humor is no exception, and to this end a variety of vitreous phantoms and mimics have been developed to evaluate the biomechanics and biotransport properties of the vitreous humor. As early as 2005, work by Repetto et al. (2005) developed a model of the eye, using a spherical Perspex cavity containing glycerol solution to simulate the vitreous humor's viscosity and evaluate the impact of saccadic eye movements on shear stress at the vitreoretinal interface. 63 However, glycerol's fluid nature makes it inadequate to reproduce the elasticity of the native vitreous humor. More recent efforts into developing models of the vitreous have included work by Awwad et al. (2015) developing the PK-eye, a molded plastic device with a spherical central cavity filled with an agar/hyaluronic acid gel and equipped with ports to allow fluid flow-through, simulating aqueous flow through the eye to allow ex vivo estimation of drug clearance from the eye. 78 Loch et al. (2014) have developed the VM and EyeMoS system, a glass sphere filled with viscoelastic synthetic polyacrylamide gel and connected to servos to simulate eye movements, to allow for simulations of post-intravitreal injection drug distribution in pharmacokinetics research. 79 Henein et al. (2019) used a 3D printed model of the vitreous cavity to hold a variety of vitreous substitutes and investigate the hydrodynamics of intravitreal injections using dye injections. 80 Januschowski et al. (2019) used ex vivo pig eyes to test the biocompatibility of a hyaluronic acid-based vitreous substitute through electroretinogram activity testing and PCR for cell mortality markers. 81 Bonfiglio et al. (2013Bonfiglio et al. ( , 2015 have developed a more sophisticated version of Repetto's spherical Perspex model, revising the design to incorporate an indentation representing the lens' intrusion on the vitreous humor and using components found in the native vitreous (hyaluronic acid) and a mechanical analog of the collagen found in the vitreous (agar) to better mimic its viscoelastic behavior. 55,82 This system allows for better exploration of intraocular pharmacokinetics after injection and enables research into the stresses experienced by the vitreous in vivo, potentially allowing for investigation of how these stresses translate into vitreous and vitreoretinal interface disorders. 82 Evaluation of several of the benefits and challenges of several of these vitreous models is shown in Table 3. Several recent studies have made use of noninvasive imaging techniques to study the pharmacokinetics of drugs in the vitreous humor. Luaces-Rodr ıguez et al. (2020) used PET imaging to evaluate how radiolabeled bevacizumab and aflibercept moved through the vitreous and were cleared from a rat eye model, while Fernandez-Ferreiro et al. (2017) used PET imaging of radiolabeled choline, sodium fluoride, and fluorodeoxyglucose to evaluate pharmacokinetics in the vitreous humor. 83,84 Other techniques previously used for biomechanical analysis, such as MRI and ultrasound imaging, may hold potential in evaluating intravitreal biomolecule movement.
In addition to these models and studies designed to explore the pharmacokinetics of intravitreal injection, several efforts have been made to develop models that allow for characterization of the vitreous' mechanical properties and response to injuries. Pokki et al. (2015) used ex vivo pig and human eyes to validate a viscoelasticity measurement system using round magnetic microparticles and externally applied magnetic fields to exert force on the vitreous humor, with camera observation used to evaluate the vitreous humor's deformation response. 13 Evans et al. (2018) recently made use of mice as a model to evaluate how the vitreoretinal interface responds to blast-induced traumatic brain injury (TBI), finding evidence of vitreous hemorrhage and detachment from the retina as well as retinal tissue damage. 85 While the mouse eye is smaller, has a proportionally larger lens, and lacks a fovea compared to human eyes, its anatomical similarities with the human eye make mice a useful model for human retinal disease and injury. 85 Liu et al. (2021) used rabbit eyes in similar evaluations of vitreous response to blast forces, finding slight vitreous hemorrhage at 5000 kPa, but no evidence of other vitreoretinal injury or detachment. 86 Watson et al. (2015) used both computational models and shock tube systems to determine that in blast injuries, the viscoelastic properties of the vitreous played a key role during traumatic loading, as did the strength of adhesion to the retina. 87 Rangarajan  Karimi et al. (2018) have recently developed a computational model of the vitreous and eye that they used to evaluate glass shard collisions with the eye, finding that the vitreous received the lowest stress during collision events and that increasing the speed of the shard decreased the stress experienced. 89 These efforts represent important strides in developing improved computational, biological, and ex vivo models of the human vitreous humor; however, the diversity of properties in the vitreous humor that must be mimicked in any model, and the lack of biomechanical data for young vitreous humor, means there is still significant research effort needed to develop an ideal model for vitreous humor research. 34 Conclusions and future perspectives Clearly, the biomechanical properties of the vitreous humor are dynamic, dependent on a variety of processes, and have the potential to influence ocular health throughout the eye. The degeneration and liquefaction of the vitreous with age is the most common, and perhaps the most significant, event to alter these properties. The effects of this process and subsequent PVD have been directly implicated in a number of significant retinal pathologies with vision-disrupting consequences, as well as having significant impacts on biomolecule transport and ocular pharmacokinetics. Efforts to explore the impact of aging on the vitreous are highly limited, however, as the vitreous humor is a challenging tissue to study in its native environment. Removal from the native environment leads to significant disruptions in mechanical performance, and is likely to also significantly alter its biotransport properties due to loss of interactions with the other tissues and circulatory systems of the eye, making ex vivo and in vitro studies of the vitreous of limited value as simulations of in vivo processes. These limitations leave human vitreous biomechanics and biotransport underexplored, with a limited set of tools in non-native environments or mathematical modeling used to evaluate the vitreous humor in manners that lack key processes and interactions relevant to clinical applications. Previous work demonstrates that porcine and rabbit vitreous samples may be useful in early studies and development, while innovations such as the PK-Eye are promising ex vivo models of the eye that can help fill the modeling gap, particularly with the increase in intravitreal injections and ocular delivery systems. More comprehensive models are needed to fully capture the complexity of the vitreous humor. The development of more biomimetic ex vivo models of the vitreous, up to and including vitreous or ocular organoid systems, can enable microbiologic evaluation of vitreal behavior and stimulus response without needing to evaluate a full eye, allowing research and vitreous substitute development to be conducted at a much more rapid pace.
Improved techniques for visualizing the vitreous and its response to external stimuli in vivo using advances in ocular imaging can allow for accurate measurements of the properties of the vitreous in its native environment, overcoming a long-standing challenge in vitreous humor research. Work by Pokki et al. (2015) serves as a promising example of this type of technique, which could be expanded to enable in vivo characterization of labeled macromolecules or other structures. 13 The development of improved computational models of the eye that fully integrate the vitreous humor can enable more accurate simulation of the complexities of the vitreous, allowing for more effective predictions of its behavior in response to external stimuli. A better understanding of the mechanical properties of the young vitreous humor is needed to inform injury models, in particular, due to the young age and military setting in which most ocular injuries occur. Taking advantage of this capacity with modern computational resources could be an invaluable technique for screening vitreous substitutes for biomechanical and biotransport compatibility among other potential applications.
With significant research effort being spent pursuing the development of a variety of vitreous substitutes, there is a pressing need for a more comprehensive understanding of the biomechanics and biotransport properties of the vitreous humor. 10,34,81 The advances in observation and modeling techniques outlined above can help researchers develop this comprehensive understanding and lead to significant health benefits for future patients by enabling better tracking of ocular disease, prediction of the occurrence of complications such as retinal detachment, and enabling early treatment to address impending severe pathologies. These improvements in our understanding of vitreous behavior and our ability to identify and treat complications arising from its disruption can ensure quality vision for aging patients, free from interference caused by vitreous liquefaction and subsequent PVD. Other relatively unexplored areas include mechanobiology factors related to the vitreous, PVD, and PVR or the influence of other ocular conditions (e.g. myopia) on the vitreous and subsequent PVD. There are opportunities in several key areas for innovation and technological advances that can help improve our understanding of the vitreous humor and its implication in disease pathology and treatments.