5.
The electrochemical basis of corneal hydration,
swelling, and transparency
Peter M. Pinsky, Xi Cheng
Department of Mechanical Engineering, Stanford University, Stanford, California, USA
1. Abstract
Fluid pressure, water content, and charge concentra-
tion are intimately coupled in the corneal stroma. The
fluid pressure is composed of a hydrostatic pressure
component associated with the intraocular pressure
(IOP) and an osmotic pressure component associated
with the excess concentration of ions in the stroma
compared to the aqueous humor. While the value of
the IOP is fixed, in the living cornea, ion pumps located
in the endothelium actively modulate the osmotic
pressure. The osmotic pressure in turn causes water
exchange between the stroma and aqueous humor.
The resulting hydration of the stroma will have a direct
effect on the collagen fibril lattice, and therefore, on
the transparency of the tissue. The entire system is
driven by the energy of various charges, as well as the
metabolic energy used in ion transport. In this chapter,
we attempt to provide a comprehensive model for
corneal hydration, swelling and transparency. Many
parts of this system have been both experimentally
investigated and modeled in previous groundbreaking
studies. In this work, we describe a macroscopic model
for in-vivo corneal hydration based on a novel energy
approach that can be used for predicting stromal
hydration under various conditions, for example, under
variations in metabolic state, and for improving the
description of corneal biomechanics.
Correspondence: Peter M. Pinsky, Department of Mechanical Engineering, Stanford University, Stanford CA 94305-4040, USA.
E-mail: pinsky@stanford.edu
Biomechanics of the Eye, pp. 63-80
Edited by: C.J. Roberts, W.J. Dupps and J.C. Downs
© 2018 Kugler Publications, Amsterdam, The Netherlands
2. Introduction
The fluid pressure within the corneal stroma is
composed of hydrostatic and osmotic components.
The hydrostatic pressure results from the IOP of the
anterior chamber, whereas the osmotic pressure
results from the interplay of fixed charges and mobile
ions. In fact, from an electrochemical perspective, the
corneal stroma is a highly-hydrated polyelectrolyte gel
consisting of an interacting mixture of fluid, solid, and
ionic phases. Stromal water saturates the collagen and
proteoglycan solid phase, solvates the ionic phase, and
accounts for about 78% of the cornea by weight.1 A small
portion of the water is cellular or bound to the stromal
collagen,2,3 but most of the water is free to flow within
the stroma in response to gradients in fluid pressure.
The solid phase is comprised of a flexible collagen
network, organized as fibrils within lamellae, and
associated proteoglycans (PGs). Stromal PGs — which
in adult stroma include decorin, lumican, keratocan,
and mimecan — have sulfated linear sidechains of
negatively charged disaccharide units called glycos-
aminoglycans (GAGs) that are covalently bound at one
end to the PG core protein.4,5 Common stromal GAGs
include keratan sulfate (KS), dermatan sulfate (DS) and
chondroitin sulfate (CS). At normal pH, the stromal
fixed charge is almost entirely due to GAG ionization.6
The ionic phase includes dissolved salts, primarily Na+
and Cl−, and metabolites such as ​​C​ 3​​​H​ 5​​​O​ ‒3​   ​​ (lactate ion)
and HCO−3 (bicarbonate ion).
Stromal mobile ions interact electrostatically with
the GAG fixed charges and form cloudlike distributions,
64
giving rise to osmotic pressure. The aqueous humor,
filling the anterior chamber, acts as an ionic bath for
the stroma and provides a reservoir of water that is
available for exchange with the stroma. The anterior
stroma is sealed by the tight boundaries of epithelial
cellular layer, making it nearly impermeable to water
(although permeable to O2 and CO2).7 Water transport
across the permeable endothelial layer, which partitions
the stroma and aqueous humor, is driven by the
osmotic pressure difference between the two phases.
When ionic concentration in the stroma exceeds that
in the aqueous humor, a positive osmotic pressure
difference is created and water will tend to flow from
the aqueous chamber into the stroma, and vice-versa
when the osmotic pressure difference is negative. In the
metabolically functioning (in-vivo) cornea, ion pumps,
located in the endothelial layer, actively transport ions
from the stroma into the aqueous humor. This lowers
stromal osmotic pressure and modulates the exchange
of water with the aqueous humor, and is referred to as
the pump-leak mechanism of stromal hydration.8-10
The regulatory system and details of the molecular
mechanisms responsible for active ion transport,
which requires Na+, K+, ATPase, and carbonic anhydrase
activity to transport ​​HCO​ ‒3​   ​​, Cl− and possibly Na+, are
not yet fully understood.10 It may be noted that the
pump-leak mechanism is not unique to the cornea. A
number of tissues employ epithelial and/or endothelial
layers as barriers to separate phases, and to actively
mediate the exchange of solutes and solvent between
those phases. Other examples include the lining of
blood vessels, the kidney, organs of the gastrointestinal
tract, and the choroid plexus in the brain.
Charge can produce powerful forces, either directly
by electrostatics or indirectly by osmotic effects, and the
cornea exploits charge in a variety of remarkable ways.
One of those, we believe, is the way in which charge is
used for transparency. The transparency of the cornea
requires individual collagen fibrils to be maintained in a
quasi-regular lattice with short-range order. The origin
and nature of forces that must necessarily act on fibrils
to maintain the stability of the lattice have long been
the subject of speculation. In this chapter, we apply the
proposed thermodynamic theory of stromal osmotic
pressure to describe how restoring forces can arise from
GAG-based osmotic and electrostatic considerations in
a manner that maintains the lattice even when strong
P.M. Pinsky and X. Cheng
random variations in GAG distributions are considered.
A number of models for corneal swelling have
previously been presented. Notable is the non-equi-
librium model of Klyce and Russell,11 based on the
phenomenological membrane transport theory of
Kedem-Katchalsky,12 and the steady-state model of
Bryant.13 based on the triphasic theory of Lai et al.14
These works and their extensions, for example, Ruberti
et al., Liu et al., and Leung et al.,15,16,17 have modeled the
stroma as one-dimensional coupled flow of solvent
(water) and ionic solutes across the corneal thickness.
In this chapter, a different modeling approach, based
on characterizing the free energy of the stromal poly-
electrolyte gel, will be introduced. The approach is
intrinsically three-dimensional, provides an explicit
expression for the stromal osmotic pressure, accounts
for the nanoscale spatial distribution of GAG-based
fixed charge, and is well-suited to implementation in
finite element codes. It is shown that steady state active
endothelial ion transport reduces stromal ionic con-
centrations, which then occur in a modified Boltzmann
distribution. This leads to a modification of the osmotic
pressure and stromal hydration, manifesting the
macroscopic effects of the pump-leak mechanism.
In Section 3, the stromal fixed charge distribution
is modeled. In Section 3, the stromal free energies
are identified and characterized. In Section 4, the
ex-vivo cornea (no metabolic activity) is analyzed and
the model is applied to stromal swelling pressure
and an investigation of the stability of the collagen
fibril lattice underlying transparency. In Section 5,
the theory is extended to the in-vivo cornea, including
active endothelial ion transport and the pump-leak
mechanism of corneal hydration. In Section 6, the
model is used to investigate the biomechanics of Fuch’s
dystrophy, in which endothelial cells have compromised
ion-pumping capacity, and to examine the effects of
laser-assisted in situ keratomileusis (LASIK) on fluid
pressure in the stroma.
3. GAG-based charge in the stroma
3.1. Corneal hydration measures
Before characterizing the nature of charge distribu-
tions in the stroma, it is first necessary to establish a
suitable definition for the level of tissue hydration. A
The electrochemical basis of corneal hydration, swelling, and transparency 65

(a)
Epithelium

Stroma

IOP = 15 mmHg

Endothelium

(b) (c)
Fig.1. (a) The cornea is a polyelectrolyte gel loaded by the IOP. Stromal lamellae follow the corneal curvature except in the anterior region,
where lamellae exhibit inclination and insert into Bowman’s layer under the epithelium. (b) An illustration of the organization of the
corneal stroma showing several lamellae and a keratocyte cell. Collagen fibrils within lamellae form a lattice with short range order. (c)
Cross-section of a unit cell representing the hexagonal collagen fibril lattice. The GAG-based coating region around fibrils and the inter-
stitial GAG chains are illustrated. (d) The unit cell is a triangular prism (shown in cross-section); the coating and interstitial regions are
indicated.

common definition, denoted Hw, is water weight per
unit dry (collagen) weight. However, this definition is
not convenient for our purpose since we will employ
notions from continuum mechanics. An alternative
measure is the tissue volume dilation, J, which is defined
simply as the swollen volume of the tissue divided by
the normo-hydrated volume. Because we will employ
the dilation J throughout this work to signify hydration
level, it useful to relate the two definitions.
By assuming that keratocytes have the mass density
of water, the hydration Hw of a sample of stromal tissue
can be related to its volume dilation, J, by:
(J ‒ ΦrΦcol)ρW
H W(J) =  __ ΦΦ ρ     (1)
r col col
normal hydration to be set at Hw = 3.26 and J = 1. Using
these conditions in Equation (1), we infer that φr = 0.75.
Therefore, Equation (1) may be simplified to:
H W(J) = 3.94J ‒ 0.74 (2)
The linearity is fully consistent with measurements.20,21
A validation of this relationship is provided by the fact
that the inferred value of φr is in close agreement with
the value of 0.77 reported by Goodfellow.19
In the following subsections, we aim to characterize
the magnitude and nanoscale distribution of the GAG
ionization charge in human stroma. This is an essential
step in describing the stromal polyelectrolyte gel.

where ρw = 1 g/cm3 and ρcol = 1.36 g/cm3 are the mass
density of the water and dry collagen fibrils,18 respec-
tively, and φcol = 0.249 is the collagen fibril volume
fraction at normal hydration.3 The volume fraction of
collagen molecules within the fibrils (which excludes
intrafibrillar bound water) is denoted φr.6,19 We take
3.2. Evidence for a GAG-based fibril coating
X-ray scattering studies under varying tissue hydration2
suggest that some portion of stromal PGs form a
charge-rich and water-binding PG-coating surrounding
each collagen fibril. The radius of the coating has
been measured2 to be rc = 18.25 nm, and this radius is
66
Fig. 2. Disaccharide repeat units of KS, DS, CS4, and CS6.
insensitive to hydration over a wide range. The existence
of such a surface ultrastructure on the collagen fibrils
is corroborated by image studies from Miyagawa et
al.22 and Muller et al.23 In addition, a theoretical study
on transparency by Twersky24 proposed that collagen
fibrils must be centered in a transparent coating and
the coated fibrils occupy approximately 60% of the
matrix volume, giving rc = 21.56 nm. Recent 3-D electron
microscopy reconstructions of corneal collagen and
GAG chains4 also suggests that some GAG chains are in
close association with the fibrils, while the remaining
GAG chains have a random orientation in the interfibril-
lar fluid.
A theoretical study by Cheng and Pinsky,25 based
on comparing predicted stromal swelling pressure
to measurements, indicated that approximately 35%
of stromal fixed charge was in the non-swelling fibril
coating region, and that 65% occupied the interstitial
region between coatings (Fig. 1). The study concluded
that this charge arrangement was capable of producing
osmotic restoring forces that maintain the stability of
the fibril lattice, and that the coating region provided an
effective interfibrillar electrostatic repulsion to prevent
fibril clustering.
P.M. Pinsky and X. Cheng
3.3. Model for GAG charge concentration
The molecular structure of GAG chains is shown in
Figure 2. Each sulfate group carries one negative charge
per group. The KS disaccharides are sulphated at the 6−
carbon position of both the Gal and GlcNAc residues.
The CS disaccharides are sulfated at either the 4− or
6−carbon position of the GalNAc residue, and are
designated CS4 or CS6, respectively (Fig. 2). However,
a detailed fluorophore-assisted carbohydrate electro-
phoresis (FACE) analysis25,26 shows that, on average,
only 72.6% of all available GAG monosaccharides
are actually sulfated and contributing charge. If this
ionization fraction is denoted fion , the stromal average
charge concentration in mM is given by:
 e​Ng​  ​​​L​ c​​​f​ ion​​
​​Cstroma
​  ​​ = _____
​  Fb ​​ (3)
where e is the unit (negative) charge, Ng is the average
number density (per unit volume) of GAG chains with
average contour length Lc, F is the Faraday constant,
and b is the length of a monosaccharide unit. The value
of Cstroma has been measured indirectly,6,27 and the value
used in this study, Cstroma = 38.6 mM, has been taken
from Cheng and Pinsky.25
The corneal stroma is composed of parallel arrays
of collagen fibrils aligned with the lamella direction
and arranged with pseudo-hexagonal packing.4 Based
The electrochemical basis of corneal hydration, swelling, and transparency 67

on the above noted evidence, it is assumed that there 4. Basic polyelectrolyte theory for the
exists a GAG-dense coating around each collagen fibril stroma
surface. The simplest 3-D representative unit cell within
a stromal lamella is that of an equilateral triangular 4.1. Gibb’s free energy
prism with vertices at the center of three fibrils (Fig. 1c). From the point of view of electrochemistry, the stroma
The unit cell is divided into three regions: fibril, fibril is a polyelectrolyte (polymeric electrolyte) gel. It is
coating, and interstitial regions (Fig. 1d). Assuming that in contact, via the endothelium, with the aqueous
collagen fibrils are non-swelling and that keratocyte humor, that therefore acts as an ionic bath for the
cells, which reside between adjacent lamellae, dilate stromal electrolyte. In the living cornea, the pump-leak
with tissue dilation,3,25 the normalized fixed charge mechanism, alluded to in Section 2, has a strong
densities in the fibril coating and interstitial regions influence on the tissue osmotic pressure. In this section,
may be shown to be given by:25 however, we develop basic theory for isolated stroma
without metabolic activity, that is, for in-vitro tissue.
( k f c )
λ 1 ‒ λ ​C​ stroma​​
​​C​ coat(​​​ J)​= ​ __     ​ + ​_
​ J​Φ​ ' ​ ​ ‒ Φ   Φ    ​ ​​ _____
2​C​   ​​  ​​ (4) Extension to the in-vivo case is undertaken in Section 6.
0
The stroma consists of a mixture of solid, fluid, and
( J​Φ​ 'k ​ ​ ‒ Φf ) 2​C​ 0​​
λ ​C​ stroma​​
​​C​ inter(​​​ J)​= ​ __ ​      ​ ​ ​  _____    ​​ (5) ionic phases, immersed in an ionic bath (aqueous
humor) at constant electrostatic potential, φbath,
In these expressions, J is the tissue macroscopic dilation, hydrostatic pressure, Pbath , and ionic concentration, C0+
λ = 0.65 is the interstitial charge partition parameter, φk, = C0− = C0. Since no fixed charge exists in the bath, it is
φf, and φc denote the volume fractions for keratocytes, reasonable to take φbath = 0. We now wish to describe
collagen fibrils, and coatings, respectively, and­ the deformation of the unit cell, introduced in Section
​​Φ​ ‘k ​​  = 1 ‒ Φk ​. The normalization concentration is 2C0, 2, as the tissue undergoes a change in hydration. Let
where C0 is the ionic concentration of the aqueous the reference (normo-hydrated) and current (swollen)
humor. For convenience, we combine the above charge configurations of the unit cell be denoted Ω0 and Ω,
concentrations into a single expression as follows: respectively, with volumes v0 and v, respectively.
Using standard results from continuum mechanics,

{
​​C​ cell​​ = ​ ​  |
Ccoat in the coating region
 
 Cinter in the interstitial region
​  ​ ​​ (6) the motion is denoted ϕ(X): Ω0 → Ω, the deformation
gradient is F = ∂ϕ(X)/∂X, and the displacement field is
u(X) = ϕ(X) −√X. The Cauchy-Green deformation tensor
The expressions for coating and interstitial charge con- is defined by C = FT F and volume dilation J = detC (which
centrations take into account the fact that GAG fixed is related to stromal hydration through (Eq. 2)).
charge cannot occupy the fibril or keratocyte volumes. It is next assumed that the Gibbs free energy of the
Thus, when the unit cell volume decreases, it does so unit cell electrolyte can be additively decomposed28 as
only by reduction in interstitial fluid, which causes a follows:
rapid increase in the interstitial fixed charge concentra-
tion and osmotic pressure. This volume-exclusion effect ​W(​ Φ, C)​= Welectrolyte​(Φ, J)​+ Wbath(​ J)​+ Welastic(​ C)​​
built into the charge model in Equations (4) and (5) is ​​∫Ω   ​​​​  [Шelectrolyte​(Φ, J)​ + Шbath(​ J)​ + Шelastic(​ C)​]​dΩ​ (9)
0

crucial for explaining the strongly non-linear variation

of osmotic pressure with dilation in the cornea. where Шelectrolyte, Шbath , and Шelastic denote the free energy
For future reference, it is noted that the volumes of density (per unit reference volume) of the electrolyte,
the coating and interstitial regions of the unit cell are bath solution, and the elastic fibers of the gel, respec-
given by: tively, and φ is the electrostatic potential.
A suitable form for Шelectrolyte is given by the mean-field
​​v​ coat​​ = Φcv0,​ (7) approximation,29 and is expressed as:
​​v​ inter(​​​ J)​= (​ J​Φ​ k ​ ​ ‒ Φf ‒ Φc)​v0​ (8)
'

​Шelectrolyte​(Φ, J)​= J​[zcellFCcell(​ J)​Φ ‒ 2RTC0

where υ0 is the volume of the normo-hydrated unit cell.
68 P.M. Pinsky and X. Cheng

​(cosh​(_ 2 ​​  ▽Φ ​]​​,

​ RT   ​ Φ)​‒ 1)​‒ ​ _
F εw
| |2 (10) liter), satisfy the Boltzmann distribution given by:

​​C​ i​​​(Φ)​ = C0exp​(‒zi_
​ RT   ​Φ)​, i = +,‒​
F
where zcell = −1 is the GAG-based fixed charge valence (16)
value, and R, T, F, and εw are the gas constant,
temperature, Faraday constant, and dielectric permit- and in which the binary ion valence numbers are z+ = 1
tivity of the electrolyte solvent, respectively. The first and z− = −1. Condition (Eq. (13)) implies31 that:
term on the right-hand side measures the free energy of
the fixed charge, the second term measures the excess ​div σelastic ‒ grad (​ Posmotic + Pbath)​ = 0​ (17)
mean concentration of the mobile ions at any point in
the electrolyte compared to the bath and, after multi- holds over Ω. In this expression, σelastic is the effective
plication by RT, may be interpreted as the osmotic work Cauchy stress, Posmotic is the osmotic pressure defined
of introducing excess ions into the neighborhood of by:
the fixed charges, Ccell, and the last term measures the ∂Шelectrolyte
dielectric free energy. ​​P​ osmotic​​ = ‒​  ___________
∂J     ​
,​ (18)
The bath free energy density, which measures the
potential of the bath hydrostatic pressure, Pbath, to do and Pbath is the bath (aqueous humor) hydrostatic
work associated with configuration volume change, by: pressure defined by:

∂Шbath
​Шbath(​ J)​= ‒Pbath(​ J ‒ 1)​.​ (11) ​Pbath = ‒​ _
∂J   ​​
. (19)

For reasons of brevity, the elastic free energy, Шelastic, Finally, from Equation (17), we may identify the stromal
appearing in Equation (9), which corresponds to the fluid pressure Pfluid as:
elasticity of the collagen fibrils and their 3-D organiza-
tion, is not discussed in this chapter, but full details may ​Pfluid = Posmotic + Pbath​ (20)
be found elsewhere.30
This is a useful formulation because it provides an
4.2. Thermodynamic equilibrium explicit constitutive equation for the osmotic pressure
In thermodynamic equilibrium, electrostatic (Eq. 18). However, the Poisson-Boltzmann equation
equilibrium requires: (Eq. 15) must be solved over the unit cell for the elec-
trostatic potential φ that will be rapidly varying at the
∂W​(Φ, C)​
____
​​  ∂Φ   ​ ​​|​​​ C = constant​​ = 0​ (12) nanoscale. To address this challenge and arrive at a
tractable theory, we modify the above approach by
and mechanical equilibrium requires: employing the concept of energy-based homogeniza-
tion.
∂W​(Φ, C)​
____
​​  ∂C   ​​​| ​​​ Φ = constant​​ = 0​ (13)
4.3. Volume-averaged free energy
where the Gibb’s free energy, W, is given by Equation (9). The idea is to employ a homogenization of the
Condition (Eq. (12)) implies31 that: electrolyte free energy density based on its volume
average over the unit cell. This is given by:
 ​εw   ( ​ RT   ​ Φ)​)​​
​​ ‒Ccell(​ J)​ + 2C0sinh​(_
F F
​▽2Φ = _ (14)
1
​​​ ¯ electrolyte​​​(Φ, J)​ = _
Ш ​​ ​ υ  ​​∫  Ω   ​​Ш
0
​  electrolyte​(Φ, J)​dΩ​ (21)
0
holds over Ω. This result can also be displayed in the
form of the Poisson-Boltzmann equation: where υ0 is the volume of the normo-hydrated
(reference) unit cell Ω0. Now, the homogenized osmotic
( )
 
F
​‒▽2Φ = _
 ​εw ​ ​  ‒Ccell​(J)​ + ​  ∑ 
 ​​z​  iCi​(Φ)​ ​,​ (15) pressure is defined by:
i = +,‒
∂​​¯
    
Ш​​ ​​
​​​ ¯​​  osmotic​​ = ‒​ ______
electrolyte
wherein the mobile ion concentrations, Ci (moles per P  ∂J ​.​ (22)
  
The electrochemical basis of corneal hydration, swelling, and transparency 69

As noted above, the electrostatic potential φ must be
solved from the Poisson-Boltzmann Equation (15).
However, a good approximation for φ is given by the
so-called Donnan potential, φe. The Donnan potential,
φe, is defined to be that potential which satisfies the
Poisson-Boltzmann Equation (15) when the fixed-
charge concentration is uniform (constant). Since Ccell is
piecewise constant over the coating region and inter-
stitial region, the Donnan potential will likewise be
piecewise constant over the unit cell and is given by:
˜ cell(​​​ J)​= ‒ ​ _
​​​Φ​​
RT
F   ​ ​sinh​​  ​Ccell​ (23)
‒1
with:
˜
{Φ​​
˜
​​  coat​​ in the coating region
Φ​​
˜
​​​  cell​​ = ​ ​ 
Φ​​   ​ ​ ​​ (24)
​​  inter​​ in the interstitial region
The accuracy of this approximation has been confirmed
by comparing the Donnan and true electrostat-
ic potentials for the unit cell.30 The electrolyte free
energy density is now found by replacing φ with ˜Φ​​
​​​  cell​​​ in
Equation (10), resulting in:
​˜ cell‒2RTC0​(cosh​(_
​​ ​ electrolyte​​​(J)​=J​[‒FCcellΦ​
Ш ​ RT   Φ​​
(25)
Notice that Шelectrolyte now depends only on J since Ccell
˜ cell​​​(J)​​. This result may be introduced into
(J) and ​​​Φ​​
Equation (21) to arrive at the homogenized electrolyte
free energy density:
1
​​​   electrolyte​​​(J)​ = _
¯
Ш​​ ​ v0  [​​ Шcoatvcoat + Шinter vinter]​,​ (26)
where vcoat and vinter are the reference volumes of the
coating and interstitial regions and given by Equations
(7) and (8), respectively. The homogenized osmotic
pressure may then be determined from Equation (22)
which yields:
the GAGs in the coating region. It is important to note
that the model has no free parameters; the osmotic
pressure is expressed only in terms of experimental-
ly determined quantities, such as collagen fibril and
keratocyte volume fractions as well as stromal average
charge concentration. In the next section, we explore
the application of the presented theory to in-vitro
swelling and the self-organization of the fibril lattice.
5. The stroma in-vitro
5.1. Swelling behavior
To obtain a validation of the model, the above osmotic
theory was incorporated into a finite element model of
|
the cornea, including collagen fibrils aligned according
to X-ray scattering measurements.30 The model was
used to compare predicted osmotic pressure (given by
(Eq. 27) with experimental measurements of swelling
pressure.20,32 Stromal samples, in the form of 7 mm
diameter disks, were immersed in an ionic bath of
physiological concentration C0 = 150 mM, and loaded
by a porous piston across their thickness (Fig. 3a).
The piston load was systematically varied, and the
equilibrium sample thickness recorded against piston
˜
​​​  cell​​)​‒ 1)​]​​
F
load. The experiments were performed in confined32
and unconfined20 experimental conditions. A finite
element model was created to simulate the experi-
mental setup; the model parameters are summarized
in Table 1. The bath hydrostatic pressure Pbath is zero in
Table 1. Parameters of the electrolyte model
Parameter Value
φf, volume fraction of the collagen fibrils (%) 24.9a
φk, volume fraction of the keratocytes (%) 11.2a
φc, volume fraction of the PG-dense coating (%) 13.3a
Cstroma, average charge density of the corneal stroma 38.6b
(mM)
λ, the charge partition parameter 0.65c
Q, dimensionless parameter by the ionic pumping 0.965d ,1.0e
effect

[
_ Φ
¯
​​​    osmotic ​  TC0​ ​(​√ ​Cinter
​ 2  ​ ​+ 1  ​ ‒ 1)​ + ​ ___
c
P​​  
  ​​ = 2​Φ​ 'k  ​R      ​ Cinter
J​Φ​ 'k ​ ​ ‒ Φ
P0, the hydrostatic pressure in bath solution (mmHg) 15.0d, 0e
f
C0, bath concentration (mM) 150

]
 ​(sinh‒1Ccoat ‒ sinh‒1 Cinter)​ ​​ (27) T, temperature (K) 298
a
Values derived Calibrated value
3,25 b 25

The first term in this expression describes a Donnan-like c

Value estimated25
osmotic pressure from the GAGs in the interstitial region d
Values for the in-vivo corneas30
and the second term describes the contribution from
e
Values for in vitro corneas
70 P.M. Pinsky and X. Cheng

Fig. 3. (a) Illustration of confined and unconfined swelling pressure experiments.20,32 In both experiments, a porous piston loads the
cornea in the transverse direction. (b) Comparison of predicted and experimentally measured20,32 values of swelling pressure Ps vs corneal
thickness t.

5
this case (and the fluid pressure in the stromal sample
is purely osmotic pressure).
The stroma sample was meshed with 2,500, 27-node 4
hexahedral elements. In order to model confined and
unconfined experimental conditions, edge boundary 3
conditions were taken as zero radial displacement or
t m /t 0

free, respectively (Fig. 3a). Sample thickness vs osmotic

2
pressure is shown in Figure 3b. The predictions show
good agreement to measurements for the full range
of sample hydration (Hw = 0.05-5.2). At the lowest 1
hydration, the osmotic pressure exceeds 2,000 mmHg.
Swelling pressure predictions for the confined and 0
unconfined cases are nearly identical. This is expected 10
-3
10
-1
10
1
10
3
10
5

because lateral expansion of the sample is restricted by C0 (mM)

the collagen fibrils, and consequently, stromal volume
change in these tests is due to changes in sample
thickness. 6 This study provides confirmation of the
proposed model for the in-vitro case.
The free swelling of the described cylindrical sample
of in-vitro stroma is modeled to investigate the effect of
the bath ionic concentration. Results for edge-confined
free swelling of the stroma sample are shown in Figure 4,
which depicts the ratio of swollen thickness to original
thickness (swelling ratio) vs bath ionic concentration C0.
For dilute bath solutions, the model predicts that the
stroma sample will swell to approximately four times its
original thickness, which may be compared to experi-
mental measurements in deionized water33,34 in which
Fig. 4. Predicted free swelling of stroma in an ionic bath ionic vs
bath concentration C0; the swelling ratio is swollen thickness tm
divided by original thickness t0.
human corneas were observed to swell to approximate-
ly three times their original thickness. For concentrated
bath solutions, the abundance of ions results in ionic
shielding of the fixed charges, reduction in osmotic
pressure ,and minimal swelling. Both limiting states are
captured by the swelling predictions.
The electrochemical basis of corneal hydration, swelling, and transparency
5.2. Self-organization of the collagen lattice and
transparency
The stroma, which comprises about 90% of the corneal
thickness, is organized into lamellae (i.e., fibers) which
contain collagen types I and V in the form of 25-30 nm
diameter fibrils. The collagen fibrils within a lamella
appear in parallel arrays following the direction of the
lamella. In a lamella cross-section, the fibrils assume a
quasi-regular packing arrangement with center-to-cen-
ter interfibrillar distance of 40−60 nm; this arrangement
is referred to as the fibril lattice. The short-range order
of this lattice produces optical interference effects that
render the tissue transparent.35-38
GAGs have an important role in the maintenance
of the fibril lattice. Keratan sulfate, the predominant
stromal GAG component, has been shown to be
involved in modulating the fibril lattice by the knockout
of Chst5 in the mouse.39 Scott40 proposed that two or
more GAG chains, originating at different core proteins
on neighboring fibrils, may form an antiparallel
duplexed association which appears as a bridge-like
structure spanning the interfibrillar distance in electron
microscopy after staining, and that such structures may
be capable of controlling the interfibrillar spacing. The
open question of how negatively charged and mutually
repulsive GAG chains might form durable antiparallel
associations has been discussed by Knupp et al.41 The
idea of an entropic elastic interconnected fibril-GAG
network was employed in the theoretical study on
lattice forces and spatial order by Farrell and Hart,42 who
proposed a model based on a six-fold symmetric elastic
network. A six-fold symmetric structural model was
also proposed by Muller et al.,23 in which GAG bridges
were proposed to link next-nearest-neighboring fibrils.
However, recent 3-D electron tomography imaging4,41
suggests that GAG chains do not appear to exhibit any
approximation to a regular six-fold symmetric bridge
network or any kind of regularity in spatial organi-
zation. Cheng and Pinsky25 modeled the GAGs as a
random network of worm-like chains, and concluded
that the entropic elasticity of these polymers falls far
short of what is needed to maintain the lattice. All of
this suggests that fibrillar bridges cannot mediate the
lattice through a mechanical mechanism.4
Maurice1,38 speculated that repulsive forces must
exist between adjacent fibrils to keep them separated,
and that the origin of these forces was related to the
71
Fig. 5. The GAG-based osmotic restoring force mechanism acts
on fibrils to stabilize the lattice. (a) Shows a set of fibrils in a
regular lattice arrangement with their associated GAG chains
forming fibril coatings and interstitial projections. The distribu-
tion of mobile ions is also illustrated. (b) The osmotic pressure in
each subcell around the undisturbed fibril. Because the osmotic
pressure is uniform, the net restoring force is zero (red dot). (c)
Shows a single fibril perturbed from its lattice position. The GAG
chains attached to the fibril move with the fibril and the GAG fixed
charge concentration increases in advance of the fibril motion and
reduces behind it. (d) The resulting osmotic pressure in each of the
subcells, which is higher where GAG fixed charge concentration
has increased and lower where it has reduced. The forces exerted
by each of the subcells on the fibril act to restore the fibril towards
its regular lattice position (red arrow). This mechanism operates
regardless of the direction of the perturbation.
swelling force within the extracellular matrix. Here
we propose a mechanism for collagen fibril self-orga-
nization based on osmotic pressure gradients that is
robust with respect to the random organization of GAG
chains.25 Consider an individual fibril in the lattice that
is perturbed into to a new position in the lattice while all
other fibrils maintain their positions. A restoring force
must exist if the lattice is stable. The linear GAG chains
are associated with each fibril and attach to that fibril
at one end only via the PG core protein. It is reasonable
that the attached GAG chains will displace with their
supporting fibril. Because the longer chains reach into
the interfibrillar fluid, a local change in position of GAG
charge concentration will be created, such that the con-
centration will increase in advance of the displacement
and reduce behind it (Fig. 5). The shorter fibril coating
GAGs will not play any role in the restoring force until
the coatings become close, at which point electrostatic
repulsion will add an additional component.
72 P.M. Pinsky and X. Cheng

(a) (b)
-3 r = 18.25 nm -3 r c = 21.56 nm
×10 c ×10
6 2.5
λ = 0.3 λ = 0.3
λ = 0.65 λ = 0.65
5 λ = 1.0 λ = 1.0
2

4
(N/m)

1.5

f fibril (N/m)
3
fibril

1
2
f

1 0.5

0 0
0 5 10 15 20 0 5 10 15
r (nm) r (nm)
Fig. 6. Electrostatic restoring force magnitude on a horizontally displaced fibril in the ideal hexagonal cell for two coating radius values:
(a) rc = 18.25 nm; (b) 21.56 nm.

The external work required to move the fibril from its where the k−subcell osmotic pressure is given by:
lattice position r0 to perturbed position r1, while fixing ∂​Ш​ electrolyte
k
  ​​ 
the position of other fibrils in the lattice, is given by: ​​P​ osmotic
k
  ​​ = ‒​  __
∂JK  ​​ (31)
  

​​W​ ext​​ = ​∫r  r​   ​​ffibril(​ r)​

0
1
· dr.​ (28)
In thermodynamic equilibrium, the force ffibril is given
by:
∂Шelectrolyte
​​f​ fibril​​ = ‒​  ___________
∂r     ​​ (29)
which measures the energy cost of moving the fibril
through the electrolyte. In order to estimate this force,
the fibril lattice is assumed to be perfectly hexagonal.
The region between the central (perturbed) fibril and
its six neighbors is divided into six subcells with initial
volume Vk. The central fibril is then displaced from r0
to a new position r, causing a change ∆Vk in the volume
of each subcell (Fig. 5). The GAG fixed charges within
subcells are assumed to be conserved during fibril
displacement, which is consistent with the GAG-fibril
attachment. As the fibril moves into its perturbed
position, the charge concentration in each of the six
lattice subcells can be estimated as a function of r and
the force acting on the fibril is then given by the chain
rule as:
6 ∂​Ш​ electrolyte
k
  ​​  6
​​f​ fibril​​ = ‒​ ∑ ​​ ​ ​  __
 ​ = ​ ∑   ​​​  ​P​ osmotic
∂r     ​
k = 1 k = 1
This result indicates that the local osmotic pressure
variation between the subcells serves as the source of
an interfibrillar restoring force. It may be easily shown
that the vector ∂Jk/∂r is directed toward the center of
the lattice for all subcells.25 Therefore, Equation (30)
describes a set of centrally oriented restoring forces.
When a fibril is perturbed away from its natural lattice
position, the osmotic pressure gradient that results
always produces a restoring force oriented towards
the natural lattice position. When the fibril occupies
that natural lattice position, the osmotic pressure is
uniform, and the restoring force vanishes. A graphical
interpretation of the origin of the lattice restoring force
is provided in Figure 5d.
Since the stromal free energy Welectrolyte has been
modeled, it is possible to develop estimates for the
fibril restoring force ffibril . Consider an ideal hexagonal
cell with fibrils at each vertex (Fig. 5a). The stroma
is assumed to be normally hydrated and GAG fixed-
charge concentrations are uniform and equal in each
subcell. In this case, the fibril restoring force magnitude
may be computed from Equation (30); the result is
depicted in Figure 6 for two assumed fibril coating
∂Jk
k
​ ​ _
∂r   ​​ (30) radius values of 18.25 nm and 21.56 nm. The force-fibril
perturbation relation exhibits two quasi-linear regimes
The electrochemical basis of corneal hydration, swelling, and transparency
2.5 Prediction
Freund et al (1995)
2 6.1. Active ion transport and hydration control
1.5
g(r)
1 the posterior surface of the cornea. It is permeable to
0.5
0
0 50 100 150
r (nm)
Fig. 7. Comparison of the predicted RDF, denoted g(r), from the
fibril dynamics model with the measured RDF from an electron
micrograph of human cornea.43
with a transition where the coatings begin to interact.
For small perturbations, the osmotic-based restoring
force is dominant, but as the perturbation increases
to the point where the PG-coatings begin to overlap,
there is a marked increase in repulsion force between
the fibrils. This provides a mechanism preventing fibril
aggregation.
The lattice restoring force and its role in lattice
self-organization was further studied by using a
transient dynamic model.25 The model included
randomness in the lattice so that the fibrils are never
arranged in perfect hexagonal symmetry. The goal
was to investigate whether the proposed osmotic
restoring forces maintained the lattice in a dynamic
and random environment. The results were interpreted
by computing the predicted radial distribution function
(RDF) of the fibrils, which is a measure of the order of
a lattice.43 RDF results from the dynamic simulation
were shown to closely match measurements based
on electron micrographs,43 and indicated short-range
order in the lattice with correlations between any two
fibrils becoming nearly random after a distance of 200
nm (Fig. 7). The average interfibrillar distance, which
corresponds to the location of the first peak in the RDF,
was maintained at 53 nm in all random cases studied.
These results support the proposed osmotic theory for
lattice self-organization.
73
6. The stroma in vivo
The corneal endothelium is a 4 µm thin cellular
monolayer with a very high density of mitochondria,
indicating high metabolic activity, and is located on
water, metabolic species, including glucose and lactate
ion, and other salt ions. The corneal endothelium
is responsible for maintaining the hydration of the
cornea through a pump-leak mechanism.8 The pump
function is provided by active ion transport across
200
the endothelium9 that regulates the osmotic pressure
of the stroma. The leak function refers to the passive
exchange of water across the endothelium through
water channels in response to the stromal osmotic
pressure.
In this section, we introduce a macroscopic model
for the pump-leak mechanism and use the model
to quantitatively illustrate how stromal hydration is
modulated. The model is
formulated in three steps. First, steady-state (but
non-equilibrium) conditions are used to establish
the jump in electrochemical potential across the
endothelial layer as a function of the steady active ion
fluxes. Then, thermodynamic equilibrium within the
stroma is used to establish a modified Boltzmann dis-
tribution that governs ionic concentrations throughout
the stroma. Finally, a modified free energy provides the
stromal osmotic pressure, describing its dependence
on the endothelial active ion transport.
To assess the electrochemical potential jump across
the endothelial layer, it is treated as an ideal membrane
in which the action of the ion pumps is represented
by an independently specified active steady anion
flux Ja− and cation flux Ja+, transporting ions out of
the stroma and into the aqueous humor.13 Because
the endothelium is thin compared to the stroma, it is
assumed that transport across the endothelium is 1-D.
A coordinate z is introduced with z = z0 at the endotheli-
um-aqueous humor interface and z = z* at the endothe-
lium-stroma interface. At steady-state, net ionic fluxes
resulting from both active and passive transport will
vanish so that:
dμ
​​J​ i​​ = ‒L i_
​  dz i ​ ‒ jai = 0, i= +,‒​ (32)
74 P.M. Pinsky and X. Cheng

must hold across the endothelial layer thickness z Є
[z0 ,z*]. In Equation (32), Li is the membrane permeabili-
ty for ionic species i, µi is the electrochemical potential,
and Jai > 0 is the steady active ionic flux across the
endothelium. Integrating Equation (32) across the
endothelium results in the jump condition for the elec-
trochemical potential:
Jai
​​μ​ *i​ ​ ‒ μ0i = ‒h​_
 L  ​​ (33)
 
i
Qi = 1. Reduced stromal ionic concentrations produce
reduced osmotic (swelling) pressure, and vice versa.
Simply stated, active ionic pumping directly reduces the
swelling tendency of the stroma. In this macroscopic
description, the pump effect is embedded in the
membrane active transport factor Qi.
Based on Equation (38), a modified Poisson-Boltz-
mann equation (see Eq. 16) for the electrostatic
potential in a stromal unit cell is introduced as:

(  ​​​z​  ​ i​​​Q​ i​​​C​ 0​​exp​(‒​z​ i_
​​​ RT  )
)
  FΦ
F
where h = z* − z0 is the thickness of the endothelial layer, ​‒▽2Φ = __
​ ​ε​ w   ​​ ​ ​ ‒​C​ cell​​ + ​  ∑  ​ ​ ​,​ (39)
i = +,‒
and ​​μ​ *i​ ​​ and µ0i are the ionic electrochemical potentials
at z = z* and z = z0 , respectively. Since no active transport The Donnan potential over the coating and interstitial
is taking place within the stroma, thermodynamic regions are solved from Equation (39) by setting the
equilibrium requires µi = ​​μ​ *i​ ​​ where µi is the electrochem- right-hand side to zero, and are given as:
ical potential at any point in the stroma. Noting that: _______
˜ cell​​​(J)​ = ​ _
F   ​ln​(‒​ Q‒   ​ + ​ √ ​Q​ 2​ ​ ​Q​  ​​ )
RT ​Ccell
_ ​  ​​ ​Ccell
​ 2  ​​  ​Q​  ​​
​​​Φ​​  ​ ___  ​ + ​__
  + ​  ​ ​ , ​ (40)
_RT _ FΦ ‒ ‒
μ
​​ ​ i​​ = ​μ​ ref
i​  ​ + ​ M  ​  ln​C​ i​​ + ​z​ i​​​ M  ​,  i = +,‒​ (34)
i i
where the normalized fixed charge concentration Ccell
where M+ and M− are the atomic weights for anions and is given by Equations (4-6). The electrolyte free energy
cations, respectively, results in: density is then:

​˜,   J)​ = J​ ‒F​C​ cellΦ​​

​​​​˜ cell​​ ‒ RT​C​ 0​​​ ​  ∑ 
[ (j = +,‒
RT ​C​ i​​ FΦ  
​​μ​ i​​ ‒ ​μ​ 0i​​= ​ ___ __ ___
​M​   ​​​  ln​ ​ C​   ​​ ​  + ​z​ i​​​  ​M​   ​​​  . ​ (35) ​​Ш​ cell​​​(Φ​  ​​​​ Q​ j​​exp
i 0 i

F˜
Substituting Equation (35) in Equation (33), and (
​ ‒​z​ j_
​​​ RT   Φ​​
)].​ (41)
​​​  cell​​)​ ‒ 2 ​ ​
rearranging gives:
Introducing the Donnan potentials (Eq. 40) into
​​C​ i​​ = ​C​ 0​​exp​(‒​_ ​  ​L​   ​​ ​)
  ​ exp​(‒​z​ i​​​ _
RT )
h ​M​ i​​​J​ ai​​ FΦ
 RT   ​____ ​  ​​. (36) Equation (41), and the result into Equation (26) provides
the homogenized unit cell free energy ¯
i
 
 ​​Ш​​electrolyte . Now,
Observing that the first expression in parenthesis on employing Equation (22) yields the modified unit cell
the right-hand side of Equation (36) is dimensionless, osmotic pressure:
we define the membrane active transport factor Qi:
​  T​C​ 0​​​[​(​√ ​C​ inter
_ ​Φ​  ​​
¯
​​​    osmotic​​ = 2​Φ​ 'k  ​R   ​ ​ + Q ​  ‒ 1)​ + ​_____
c
P​​ 2
 J​Φ​ ' ​ ​ ‒ ​ Φ​   ​​ ​ ​C​ f2​​
​​Q​ i​​ = exp ( ​L​  ​​ ​ )​ · i = +, ‒​
h ​Mi​ ​​​  J​ai  ​​

​​˜   ​​C​ coat​​ ‒ ​​sinh​​​
˜   ​ ​C​ inter​​)​ ​,​
 RT   ​​ ____
​ ‒​_
k f
(37)
(​ sinh​​​ ]
i ‒1 ‒1
(42)
Finally, stromal ionic concentration (Eq 36) may be
written as: in which a modified inverse hyperbolic sine function
was introduced:
​​C​ i​​ = ​Q​ i​​​C​ o​​exp ( RT ​ )​
˜
FΦ
​ ‒​z​ i​​​ _ ,​ (38) _
   ​​(x)​= ln​(x + ​√ ​x​​ 2​ + Q )​  ​,​
‒1
​​​
sinh​​​ (43)
which generalizes the Boltzmann distribution (Eq.
16). Observe that 0 ≤ Qi ≤ 1 and the value of Qi reduces and where Q = Q+Q− is the membrane active transport
with increasing active flux Jai. When no endothelial parameter that describes the combined effect of both
ionic pumping occurs, Jai = 0 and Qi = 1. Equation (38) the cation and anion active transport.
expresses the idea that when active ionic pumping Equation (42) shows that the osmotic pressure is
occurs, Qi < 1 and stromal ionic concentrations are dictated by the combined membrane active transport
reduced compared to when there is no pumping and parameter Q. Estimation of the combined membrane
The electrochemical basis of corneal hydration, swelling, and transparency 75

(a)
700 700
680 680
660 660
640 640
CCT (µ m)

CCT (µ m)
620 620
600 600
580 580
560 560
540 540
520 520
1 1.5 2 2.5 3 3.5 4 4.5 0.2 0.4 0.6 0.8 1
R RJ
L
Fig. 8. The predicted swollen CCT in Fuch’s dystrophy when (a) the endothelial ionic permeability is increased, such that 1 ≤ RL ≤ 4.5 and
with normal active ionic flux RJ = 1, and (b) the active ionic flux rate is reduced, such that 0.2 ≤ RJ ≤ 1 and with normal anionic permeability
RL = 1.

active transport parameter Q is challenging. Based
on imbibition pressure measurements in the rabbit
cornea, a value of Qphysio = 0.965 has been established.30.
Stroma in vitro has no active ionic transport and Q =
1. In this case, the above model reduces to the in-vitro
model described in Section 4.
In the subsections below, we apply the in-vivo osmotic
theory to examine the effects of reduced endothelial
ion pumping (Fuch’s dystrophy) and the fluid pressure
effects of LASIK.
6.2. Effects of reduced endothelial active ion trans-
port
Fuch’s dystrophy is usually characterized by morpho-
logical changes in endothelial cells or by an accelerated
loss of endothelial cells.10,44 In this situation, the cornea
will swell due to increasing endothelial ionic permea-
bility, decreasing active ion flux, or both mechanisms
simultaneously. This situation can be modeled by
modifying the membrane active transport parameter
using Equation (37), such that:
lnQ patho = (__
     )lnQ physio
R J
R (44)
L
where Qpatho is the pathological transport parameter,
Qphysio = 0.965, RJ is the pathological/physiological
fraction of ionic flux, and RL is the pathological/physio-
logical fraction of ionic permeability.31
A finite element model embedding the above in-vivo
osmotic theory and realistic collagen organization was
created,30 and used to analyze a cornea with central
corneal thickness (CCT) of 520 µm, and anterior and
posterior radii of 7.87 and 6.7 mm, respectively. Figure
8a shows the predicted swollen CCT for 1 ≤ RJ ≤ 4.5 and
RJ = 1, and Fig. 8b shows the predicted swollen CCT for
0.2 ≤ RJ ≤ 1 and RL = 1. In both cases, the model predicts
corneal swelling, with predicted maximum swollen
CCT of approximately 690 µm. This lies in the range of
clinical observations for Fuch’s dystrophy.45
The model also predicts that swelling is concen-
trated in the posterior region, a prediction that agrees
with clinical observations45 that the anterior surface
of the cornea is nearly normal among patients with
Fuch’s dystrophy, whereas the posterior surface shows
significant change. Figure 9 shows deformations due to
swelling when RJ = 0.22. The results suggest the stability
of the anterior surface with respect to swelling resulting
from the presence of inclined lamellae.
6.3. Osmotic effects in LASIK
Laser in-situ keratomileusis (LASIK) is commonly used
to correct refractive errors, including myopia and
hyperopia. An anterior circular hinged flap of about
150 µm thickness is first created using a femtosecond
laser. The flap is then folded back to expose the stromal
bed, which is reshaped by excimer laser ablation.
The procedure is completed by returning the flap
to conform with the ablated profile. Although laser
76 P.M. Pinsky and X. Cheng

Fig. 9. Fringe plot of vertical displacements of a cornea with simulated Fuch’s dystrophy. Ion pumping is reduced to 22% of normal (RJ =
0.22). Most swelling occurs in the posterior stroma.

ablation creates a new central corneal curvature with

the goal of achieving emmetropia, the biomechanical
response of the residual (significantly thinned) stromal
tissue is an important issue.46 In a study,31 normal active
endothelium ionic pumping was assumed (Q = Qphysio)
and the osmotic model employed to investigate the
stromal fluid pressure and swelling resulting from a
typical myopic correction.
The ablation depth profile was determined using
Munnerlyn’s theory47 for an intended correction of 5
diopters. The CCT was 520 µm, the flap thickness was
150 µm, and the maximum ablation depth was 87 µm,
resulting in a residual central stromal thickness of 283
µm. The flap is returned after ablation, but it is not
mechanically integrated with the stroma.46
The finite element model consisted of 7,008 27-node
hexahedral elements. The solution was obtained in two
steps:
1. a pre-surgical step in which the normal cornea
Fig. 10. Fringe plots of (a) the fluid pressure and (b) volume dilation
was loaded by an IOP of 15 mmHg and the J of a post-surgical cornea after LASIK designed for an intended
collagen prestress obtained; and correction of 5 diopters.
2. a post-surgical step in which the flap is separated
and ablated tissue removed. = 0.965.30 In this case, the normal pre-surgical fluid
Predicted curvature change, post-surgical corneal pressure is approximately Pfluid = IOP throughout the
radius, and spherical equivalent agreed well with stroma.
clinical measurements by Ortiz et al.48 on 85 eyes with Recalling that the stromal fluid pressure is given by
myopia or myopic astigmatism.30 Pfluid = Pbath +Posmotic (see Eq. 20), where Pbath = IOP, the
As noted in Section 6.1, the active transport post-surgical fluid pressure is determined from Pfluid = IOP
parameter Qphysio was calibrated based on direct exper- +Posmotic , and where Posmotic could be positive or negative.
imental measurement of imbibition pressure in in-vivo The predicted fluid pressure Pfluid for the LASIK model is
rabbit corneas.49 Those measurements imply that in the shown in Figure 10a. It was observed that the osmotic
normal in-vivo (rabbit) cornea, the osmotic pressure is pressure becomes negative and the stromal fluid
approximately zero and the fluid pressure is approx- pressure reduces to approximately half of its normal
imately IOP.30 This normal physiological condition is value in the ablation region. The change in stromal fluid
modeled with the active ion transport parameter Qphysio pressure resulting from the surgical procedure may be
The electrochemical basis of corneal hydration, swelling, and transparency 77

Fig. 11. At the anterior surface, inclined lamellae insert into Bowman’s layer and act as anchors, resisting the stromal fluid pressure. In
the posterior stroma, the stromal and anterior chamber fluid pressures are balanced, and the collagen does not need to be inclined. This
arrangement stabilizes the refractive surface.

inferred from Figure 10a. Since the pre-surgical stromal
fluid pressure is Pfluid = IOP, the change in stromal fluid
pressure can be observed by simply subtracting IOP
= 15 mmHg from the pressure scale in Figure 10a. For
example, at the center of the ablation zone, the pre-sur-
gical fluid pressure is 15 mmHg and the post-surgical
fluid pressure is 4.1 mmHg, which represents a change
of −10.9 mmHg.
The predicted volume dilation J for the LASIK model
is shown in Figure 10b. Modest swelling is indicated in
Figure 10b, and is concentrated in the anterior region of
the residual stroma, particularly where the ablation is
deepest and lamella inclination is lost. However, if IOP
increases, then the swelling increases significantly.30
7. Concluding remarks
This chapter has presented an energy-based theory for
stromal swelling and fluid pressure, and an application
of the theory to demonstrate that GAG-based osmotic
forces are capable of stabilizing the collagen lattice,
which is necessary for the transparency of the cornea.
The model, which has been validated against stromal
swelling experiments, has no ‘free parameters’, and the
physical constants which do appear have been found
through independent measurements.
The precise quantification of the fluid and osmotic
pressures for the in-vivo human cornea remains
an open question, as direct measurements are not
currently feasible. Likewise, measurement of the active
transport parameter Qphysio, (see Eq. 37), which depends
on the endothelial ionic permeability and active ionic
flux, cannot be directly measured. We have employed
experimental measurements of imbibition pressure in
rabbit to assign a value to Qphysio,30 but this is not entirely
satisfactory. Our investigations suggest that stromal
fluid pressure is close to IOP and osmotic pressure is
maintained close to zero by the endothelial ion pumps.
But a final accounting requires more experimental data;
the general theory presented should, however, remain
relevant.
The role of stromal collagen in swelling must also be
considered for a full understanding of how the cornea
responds to swelling forces. Although this important
topic has not been included in the scope of this chapter,
we remark about one interesting aspect. The anterior
stroma terminates at Bowman’s layer, which supports
the epithelium. In and below Bowman’s, the stromal
78
fluid is pressurized, whereas above Bowman’s the fluid
pressure is zero (atmospheric pressure can be cancelled
out). How is the pressure difference equilibrated? This
must be through lamella inclination, as illustrated in
Figure 11. In the posterior stroma, the anterior chamber
IOP and stromal fluid pressure must balance, and
no lamella inclination is needed (or exists). It may be
concluded that anterior lamella inclination stabilizes
the refractive surface of the cornea. A detailed study of
this problem has been conducted by Cheng et al.30
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P.M. Pinsky and X. Cheng
Another aspect of the model not discussed is the
convenience with which it may be implemented in finite
element codes. The finite element method is an ‘energy’
method. The proposed model is also an energy method
and the electrolyte free energy can be readily exploited
in a finite element code. This is a significant advantage
compared to all previous models, which are based on
balance principles for fluxes of solvents and solutes. A
detailed discussion of the finite element implementa-
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