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Dehydration and vitrification of corneal gel

Masahiko Annaka,*ab Toyoaki Matsuura,c Shinji Maruokac and Nahoko Ogatac

Received 19th February 2012, Accepted 26th March 2012

DOI: 10.1039/c2sm25370d

Temporal changes in the weight and elastic properties during the dehydration of pig corneal gel were

investigated. The moisture from the corneal gel evaporated in two stages. At the crossover point

between these two stages, the elastic modulus of the corneal gel showed an anomaly: the loss tangent

(tan d) showed an anomalous peak with the jump of the dynamic elastic modulus, E0. Two fractions of

water exist in the corneal gel: one strongly bound to the relaxation centers, and the other almost freely

diffusible as liquid bulk water. A quantitative analysis shows that the bound fraction of the corneal

water under physiological hydration makes up about 3% of the total water. The abrupt increase in non-

freezing water content, and the distinct change in the bound water fraction h of the cornea were also

observed near the crossover point of weight reduction. From these observations, water exerts

a plasticizing effect on the corneal gel, thereby reducing the elastic stiffness below that of the dry cornea.

The absolute value of the elastic stiffness of the corneal gel is smaller than that for non-crystalline

polymeric glasses by about a few orders of magnitude. It is plausible to explain that the monolayer

coverage of bound water on the lattice of the cornea prevents direct contact of the polymer chains of the

corneal gel.

1 Introduction

In order to adapt the physical properties of living materials to

their biological function, nature has developed gel networks with

outstanding physical behavior. One example is the cornea, which

is the transparent section of the eye’s outer tunic.1 Its most

voluminous component, corneal stroma, is composed of

numerous sheets or layers of highly organized type I collagen

fibrils, i.e. lamella. Within each layer, a hydrated proteoglycan

(PG) and glycosaminoglycan (GAG) matrix that fills the inter-

fibrilar space surrounds the collagen fibrils. Outside this complex

gel matrix are cells scattered throughout. Within each lamellar

layer, however, the collagen fibrils are unidirectionally aligned in

a regular anisotropic order.2 All layers are stacked on each other

in parallel with the lateral surface of the cornea; the collagen

fibers also lie parallel with respect to the corneal surface.

Together with the lens, the cornea serves to form the optical

image on the retina. Of the cornea–lens combination, the cornea

provides about 65% of the refractive power of the eye. The

cornea can have this high refractive power even though it is much

thinner than the lens because the cornea’s anterior surface is in

contact with the air to give a refractive index ratio of 1.377 to

aDepartment of Chemistry, Kyushu University, Fukuoka 812-8581, Japan.E-mail: annaka@chem.kyushu-univ.jp; Fax: +81-92-642-2607; Tel: +81-92-642-2594bInternational Research Center for Molecular Systems, Kyushu University,Fukuoka 819-0395, JapancDepartment of Ophthalmology, Nara Medical University, Nara 634-8522,Japan

This journal is ª The Royal Society of Chemistry 2012

1.000. The cornea can maintain its lucidity and moisture content,

which requires, however, continuous maintenance by the

metabolism. Metabolic defects or diseases lead to turbidity due

to a change in local structure. To protect this highly specialized

tissue from drying out and from contaminants in the air, the

cornea is covered with a continually renewed water film, the tear

film, which also provides an excellent, smooth optical surface.

One index of corneal function or dysfunction is the degrees of

corneal swelling and hydration, and the concomitant loss of

transparency. The transparency of the cornea is related to the

assumption of a constant refraction in the whole tissue. This is

the result of the two-dimensional regular lattice of collagen and

GAG, where water is stored in the intermediate spacing.3 The

refractive index of the cornea is adjusted by the amount of water

and the hydrophilic properties of the GAG are responsible for

this.4 The water also plays an important role due to its influence

on mechanical and rheological properties. Dimensional stability

of the cornea is of primary importance in maintaining clear

vision. The mechanical properties of the tissue that underlie this

stability are determined by the structure of the stroma, which is

made up of several hundred superimposed lamellae. It is well

established that water exists in polymer networks in two different

physical states: free water and bound water. Biological functions

depend on how the water molecules associate with the biopoly-

mers, and moreover the swelling characteristics of corneal

stroma are dominated by the nature of the biopolymers, which

make up the corneal stroma and the states of water.

In this study, therefore, we focused on the change in the state

and dynamics of water, and mechanical properties of the

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cornea induced by dehydration. Of the many techniques that

can be used to study water–biopolymer interactions, nuclear

magnetic resonance (NMR) spectroscopy is unique in that,

depending on the choice of pulse sequence, it can probe the

dynamic states of both the water and the biopolymer on

a variety of time scales. Different states of water in hydrogel

matrices can be specified by measuring the water proton spin–

lattice (T1) and spin–spin (T2) relaxation times. Differential

scanning calorimetory was also used to get a better insight into

the states of water in the cornea, and the concomitant change

in the elastic properties of pig cornea was investigated by

dynamic mechanical analysis.

2 Experimental procedures

2.1 Sample preparation

Enucleated pig corneas with 22 mm diameter were investigated

up to 12 h post mortem. The epithelical and endotherical cells

were removed by scraping with a surgical blade, and the cornea

was excised from the intact globe. The excised tissue was placed

in distilled and de-ionized water and was allowed to it to swell.

The weight w, diameter d (parallel to the corneal surface) and

thickness h (perpendicular to the corneal surface) of the

sample immediately after preparation, which is at physiological

hydration, were w0 ¼ 1.01 g, d0 ¼ 14 mm, and h0 ¼ 1 mm (n¼ 10,

SD ¼ 1.1). A fully hydrated pig cornea appears to be a cylinder

of wFH ¼ 1.01 g, dFH ¼ 14 mm, and hFH ¼ 12 mm in thickness

(n ¼ 10, SD ¼ 1.2).

2.2 Observation of dehydration process

The temporal changes in weight w, diameter d and thickness h of

the cornea were measured simultaneously by utilizing the electric

balance equipped with a CCD camera. The measurements were

carried out at 25 �C, in 36% humidity and at atmospheric pres-

sure. The time zero is established the instant the fully hydrated

cornea was placed on the electric balance. The change in mass is

also expressed as an index of water content, wwater defined as

the amount of water per unit weight w of the cornea: fwater ¼(w � wdry)/w, where wdry is the dry weight of cornea. The pig

cornea used in this study has fwater ¼ 0.80 at physiological

hydration.

Fig. 1 The photograph of enucleated pig cornea (left) and dehydrated

2.3 Differential scanning calorimetry (DSC)

DSC measurements were performed on a Q1000 differential

scanning calorimeter (TA Instruments). Specimens were cooled

to �80 �C at the rate 5 �C min�1 and then were reheated at the

rate 1 �Cmin�1 to 40 �C, and thermograms were recorded at least

twice to ensure reproducibility in the recorded data.

cornea (right), and two-dimensional SAXS intensity profiles for the

corneal gel: (a) immediately after preparation at physiological hydration

and (b) in the dry state measured at 25 �C. SAXS experiments were

carried out with a two-dimensional SAXS spectrometer (BL45XU)

installed at the Japan Synchrotron Radiation Research Institute (JASRI,

SPring 8,Proposal No.2001A0265-NL-np). An incident X-ray beam from

the synchrotron orbital radiation was monochromatized to 1.49 �A.

The scattered X-ray was detected by a two-dimensional CCD camera

positioned 1 m from the sample; the magnitude of the observed scattering

vector ranged from 0.008 to 0.15 �A�1.

2.4 Nuclear magnetic resonance (NMR)

Longitudinal (T1) and transversal (T2) relaxation times of water

protons were measured on the cornea with various hydration

degree as prepared described above at 25 �C. A JNMMu25 pulse

NMR spectrometer (JEOL) with a magnetic field of 0.59 T

(25 MHz, 1H frequency) equipped with a 10 mm proton sensitive

probe was used. T1 was determined by the inversion-recovery

8158 | Soft Matter, 2012, 8, 8157–8163

sequence (p / p/2 / Acq.). T2 was measured by the Curr–Purcell–

Meiboom–Gill (CPMG) spin-echo sequence (p/2 / (p)n / Acq.).

2.5 Elastic properties

Dynamic mechanical measurements were performed on a Rheo-

vibron DDV-25FP mechanical spectrometer (Orientec Co.,

Japan), and a frequency of 11 Hz was applied to the specimens.

The values of storage modulus (E0), loss modulus (E0 0) and loss

tangent (tan d) were obtained. The temperature range covered

were scanned at the rate of 1 �C min�1. For mechanical

measurements, rectangular specimens 5 mm wide and 10 mm

long were cut out of the pig corneas. Our recent small-angle

X-ray scattering (SAXS) measurements revealed that the

periodicity collagen fiber D of the cornea in the dry state

(D¼ 665� 2�A) is same as that for physiologically hydrated state

(D¼ 668� 3�A) within an experimental error as shown in Fig. 1.5

It is confirmed that a collagen fibril in the dry state maintains the

same fine structures as that in a physiologically hydrated cornea

in each sheet, and the directions of the collagen fibrils between

the sheets are randomly distributed as supported by the obser-

vation of the powder pattern. Therefore we did not take the

direction of collagen fibers into consideration when preparing the

specimens.

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3 Results and discussion

Fig. 2 shows the changes in the weight w, the diameter d, and the

thickness h, of the cornea as a function of time during the

dehydration process. These values are normalized by those for

initial fully swollen state, wFH, dFH, and hFH. Two stages of the

dehydration process could be perceived from the plot, in which

the time dependencies of the weight on a logarithmic scale is

roughly represented by two straight lines: the first stage occurs up

to 21 600 s, and the second one with the gentle slope continues

until the end of the plots. The first stage of the evaporation

continued until the weight was reduced to 17% of the fully

swollen state wFH, whereupon the second stage began, which

continued to 13% of the wFH. The thickness decreased in a similar

way to the weight of the cornea. It is interesting to note, however,

no change is observed in the diameter during the process within

the range of experimental error. The corneal gel collapses along

an axis parallel to the optic axis. The dehydration process is

determined by the temporal change in the competitive balance

between the capillary force of water that acts to shrink the gel

network and the rubber elasticity that acts to swell the gel

network. This phenomenon suggests that there are structures,

which lead to shrinkage along the orbital axis. A corneal stroma

is comprised of numerous sheets or layers of highly organized

type I collagen fibrils, lamella.1 Within each lamellar layer,

however, the collagen fibrils are unidirectionally aligned in

regular anisotropic order. All layers are stacked one upon

another in parallel with the lateral surface of the cornea; the

collagen fibers also lie in parallel with respect to the corneal

surface. Within each layer, a hydrated GAG matrix that fills the

interfibrilar space surrounds the collagen fibrils. Because of the

hydrophobic nature of the collagen fibers, stromal swelling is due

almost entirely to the gel pressure exerted by the stromal PG,

acting as polyelectrolyte gel. The dehydration of the GAG

components of the PG is obliged the change in thickness of the

corneal gel in the process of dehydration.

Fig. 3(a) portrays a typical set of endotherms for corneal gels

at different water contents fwater heated at a rate of 1 K min�1

from�80 to 40 �C. The principle features include: (i) the absenceof peaks at fwater < 0.20; (ii) the dehydration produces a increase

in the amount of non-freezing water; (iii) the appearance of

a structure on the endothermic peaks that has been ascribed to

Fig. 2 The temporal change in the weight w, thickness h and diameter

d of the cornea during the dehydration process of the pig cornea at 25 �C.These values are normalized by those at initial fully swollen state, wFH,

dFH, and hFH.

This journal is ª The Royal Society of Chemistry 2012

a distribution of melting points in the sample, and (iv) the fact

that peak area spans a temperature range of �10 to 10 �C,indicating that DSC is insensitive to events below �10 �C.The bound water (non-freezable water) content has been

estimated as the difference between the total water content,

determined gravimetrically, and the amount of free water

(freezable water) computed from the peak area by using the heat

of fusion for bulk water, DHfusion ¼ 320 J g�1 (Fig. 3(b)). It is

important to note that the corneal gels show an abrupt increase

in non-freezing water content in the vicinity of the water content

of the corneal gel fwater ¼ 0.50, at which the temporal change in

the w/wFH enters the second stage of evaporation (Fig. 2). The

dry cornea has the same periodicity of collagen fibers as that for

the physiologically hydrated cornea indicating that the hydration

and dehydration simply cause swelling and deswelling of the

glycosaminoglycan (GAG), which is located between the regular

two-dimensional lattices of collagen fibers, which produced the

change in thickness.5 Therefore, water evaporated in the first

stage could be freely diffusible or weakly bound to GAG, and the

second stage is most likely the slow evaporation of the water

strongly-bounded to GAG.

The insensitivity of DSC to events below �10 �C and to the

thermal processes associated with the onset of mobility in the

bound water fraction is rationalized by Pouchly and coworkers

as follows.6 At low temperatures, where a significant proportion

of the water is immobile, water-rich samples behave in a similar

fashion to those with initially lower water contents, and, such as

the phase transformation below �10 �C, do not proceed in

accordance with equilibrium predictions. They conclude gener-

ally that the fraction of bound water is determined by a number

Fig. 3 (a) The typical DSC endotherms for the pig corneas with different

water content, fwater. (b) Plots of the nonfreezing water fraction in the

corneal gels as a function of water content, fwater.

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of equilibrium and non-equilibrium factors, the latter being

strongly temperature and concentration dependent. More

generally, at water concentrations within the bound water regime

and/or at temperature below �10 �C, the solidification of water

is hampered by kinetic factors arising from reduced water

diffusivity and constrains imposed by the rigid polymer network.

To evaluate the relative amount of the bound water in the

cornea, proton relaxation time T1 and T2 values for water was

investigated at different levels of hydration. To analyze the data,

following assumptions were made; (i) the cornea contains two

types of water, i.e., free water having Ti (i ¼ 1, 2) of pure liquid

water and bound water which is restricted by an interaction with

the constituent polymer, and (ii) observed values of the relaxa-

tion time Ti in the corneal gel are an averaged value of free water

Tif and bound water, Tib, because sufficiently fast exchange

occurs between two types of water on the NMR time scale.

We have7

1

Ti

¼ h

Tib

þ 1� h

Tif

(1)

Since the relaxation rate for the bonded water molecules near the

surface of the gel network polymer is much larger than in the

bulk free water, the effective relaxation rate of exchangeable

water T�1i is directly proportional to the fraction of bonded water

molecules h

h ¼ Nb

Nb þNf

(2)

here Nb is the number of water molecules bound to the surface

and Nf the number of ‘free’ water molecules.

h ¼ KS

V(3)

where S is the biopolymer surface, K is the thickness of the

bounded water surface layer, and V is the total volume of the

water. The change in Ri(¼T�1i ) with degree of dehydration,

therefore, reflects the change in the active gel–water interface.

Fig. 4 shows the changes inR1 and R2 values for water protons in

the corneal gel as a function of water content fwater. With

increasing the water content fwater, R1 becomes smaller due to

the increase in the amount of bound water. R2 also exhibits its

Fig. 4 The transverse relaxation rate, R1 and the longitudinal relaxation

rate, R2 of the corneal gel as a function of water content, fwater. Dashed

line at fwater ¼ 0.5 corresponds to the crossover point between first- and

second-stage of weight-reduction during the dehydration process.

8160 | Soft Matter, 2012, 8, 8157–8163

smallest value in bulk water. The 1H–1H magnetic coupling of

water molecules in the bulk is effectively averaged out, and

therefore the transverse magnetization decays slowly over time.

By increasing the fraction of polymer in the system, the dynamics

of bound water molecules are slowed down, leading to an

increase in R2.8,9 It is worthy to note that R2 increases more

quickly than R1 with increasing polymer fraction, which implies

the transverse relaxation rate, R2 of water protons is more

sensitive to the polymer fraction than the longitudinal relaxation

rate R1. A clear discontinuity for the change in R2 at water

contents fwater between 0.40 and 0.45 is observed, which implies

dehydration below 45% removes progressively more strongly

adsorbed water from the surface of GAG, and results in an

apparent increase in the transverse relaxation rate of the mobile

component and in its associated correlation time. It is worth

mentioning that the corneal gel at fwater z 0.50 corresponds to

the state at which the change in w/wFH moves to the second stage

of the dehydration process (Fig. 2).

For free water T1f z T2f, whereas this is not the case for the

bound water where T1b z T2b. We, therefore, find from eqn (2):

T1

T2

¼ 1þ �T2f=T2b

�h

1þ �T1f=T1b

�h

(4)

Eqn (2) allows for the determination of the bound fraction h if

T1b and T2b are known. If this is not the case one can use the

following approximate procedure. Since T1b[ T2b, one can, for

small water concentrations, neglect the second term in eqn (2).

Though we clearly deal with a distribution of the correlation

times we can assume the validity of the Bloembergen, Purcell and

Pound formula10 with a single average relaxation time, sc as

a first approximation. Assuming that the relaxation of bound

water is of intramolecular origin, the fraction of bound water h in

corneal gel can be deduced from the following equations derived

by Blinc and coworkers:11,12

R1b ¼ 2

3hC

�Jðu0; scÞ þ 2Jð2u0; scÞ

� ¼ R1 � R1f ¼ DR1 (5)

R2b ¼ 2

3hC

�Jð0; scÞ þ 5

3Jðu0; scÞþ 2

3Jð2u0; scÞ

�¼ R2�R2f ¼ DR2

(6)

where

J ¼ sc1þ u2s2c

(7)

u0 is the proton Larmor frequency, and the dipolar coupling

constant C is 2.5 � 1010 s�1 for water. If we divide eqn (6) by (7),

we can determine sc form the DR1/DR2 ratio and then using

known sc, we obtain h from the measured T1 and T2 values. In

this study the proton relaxation times for the free state are

set equivalent to the bulk water value, T1f z 3000 ms and

T2f z 2000 ms. From these parameters, the fraction of bound

water h was estimated as a function of water content. As shown

in Fig. 5, h increases with polymer fraction. It is noteworthy that

the bound water fraction h of the cornea under physiological

conditions (fwaterz 0.80) is only 3% in terms of the total amount

of water. The observation of the distinct change in the bound

water fraction h of the cornea at a water content fwater below

0.40 suggests that this water content corresponds to monolayer

This journal is ª The Royal Society of Chemistry 2012

Fig. 5 Calculated dependence of the bound water fraction h with water

content, fwater in the corneal gel. Dashed line at fwater ¼ 0.5 corresponds

to the crossover point of the weight-reduction during the dehydration

process.

Fig. 6 (a) Dynamic elastic modulus, E0, dynamic loss modulus, E0 0 andloss tangent, tan d for physiologically hydrated pig corneal gel (w/w0 ¼ 1)

as a function of temperature at the frequency 11 Hz. (b) Temporal change

in weight normalized by that for physiologically hydrated state, w/w0, the

dynamic storage modulus, E0 and loss tangent, tan d during the dehy-

dration process of pig cornea at 25 �C. The values of w/wFH corre-

sponding to w/w0 are given in parentheses on the w/w0-axis for

comparison.

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coverage of the polymer chains of the corneal gel. This is

consistent with the observation that the transverse relaxation

rate R2 of the water protons exhibit a dramatic decrease at water

contents in excess of 0.40, presumably because of the increased

mobility of water associated with the formation of hydration

multilayers. Proton exchange become significant as multilayer

water is introduced.13

Measurements of the relaxation times of water protons

support our assumption that the corneal water exists in two

different conditions: a large fraction of bulk water and a smaller

fraction of motion-restricted water, which is bound to the

relaxation center. There exists a brisk exchange between these

two states. In the physiological hydration state, corneal water is

almost freely diffusible in the collagen–GAG lattice, and

approximately 3% of this water is found in the environment of

the relaxation center, which most likely to be negatively charged

groups of PG. The remaining fraction of water is not attributed

to the fixed lattice of the cornea, but belongs to the tissue as

a whole. These physicochemical properties are considered to

have important clinical implications. The nutritional supply of

the cornea, which depends on diffusion, is understandable on this

background. Also the fast and reversible cloudiness of the

cornea, produced by pressure on the bulbus, can be interpreted.

According to the pressure gradient, the free water is displaced

into the zones of smaller pressure and irregularities develop in the

collagen lattice. These irregularities lead to collagen free lakes,

which are responsible for the growing cloudiness.14,15

Upon dehydration, it is generally observed that gels convert

into a glass-like transparent substance (gel-to-glasslike transi-

tion). Since the first report on the vitrification of a heat-treated

egg white gel,16 we have studied the evolution of the properties of

gels during dehydration. The dry gels look like plastics and show

several features that are commonly observed in the non-crystal-

line polymer, e.g. transition features in the thermal and elastic

properties.17,18 In the process of the gel-to-glasslike transition,

a large mechanical change from soft corneal gel to hard glasslike

state of the cornea is expected, which may directly provide

information on the change in the interaction between the

constituent polymer and water. However the change in the

mechanical properties during the dehydration process have been

rarely reported. The dynamic response of the cornea at low

This journal is ª The Royal Society of Chemistry 2012

frequency is also of great physiological importance.19Mechanical

properties measured under small strains varying sinusoidally at

low frequencies may provide insight into the macromolecular

organization of the cornea and into changes caused by small

strains. If a relationship to the mechanical properties of the

cornea during the in vivo deformations is to be established, then

the time scale used to measure the dynamic optomechanical

properties must be the same as the accommodation time (1 s or

less). In the in vivo cornea one is concerned with vibrations in the

order of 0.1–10 Hz, which may have amplitudes ranging from

0.01 mm to 1 mm. The origin of these vibrations are threefold: (a)

rapid contraction and relaxation of individual muscle fibers

(microvibration), (b) pressure wave transmitted from the heart to

the tissues by the blood vessel system, and (c) vibrations coming

from the environment.20 Therefore dynamic mechanical

measurements were performed at a frequency of 11 Hz.

In Fig. 6(a), the dynamic modulus E0, the dynamic loss

modulus E00 and the loss tangent (tan d) for physiologically

hydrated pig cornea (w/w0 ¼ 1, fwater ¼ 80%) are plotted against

temperature. There is very little temperature dependence at all.

The results show no maxima or minima, and therefore no

absorption due to a particular mechanical relaxation can be

assigned. Temporal weight changes and the dynamic modulus E0

for physiologically hydrated pig cornea are shown in Fig. 6(b).

The values of weight are normalized by the weight for initial

physiologically hydrated state, w0 and plotted on a logarithmic

scale. The first data point in Fig. 6(b) (w/w0 ¼ 1) corresponds

approximately to that at 15000 s in Fig. 2. At the early stage of

dehydration, the dynamic modulus E0 increased gradually with

time, and the value of E0 exhibited an abrupt increase by two

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orders of magnitude at �6000 s close to the time at which

temporal change in the w/w0 enters the second stage of evapo-

ration. The loss tangent, tan d, showed an anomalous peak with

the jump of E0, and it remained constant after �6000 s. Since the

free water between lamella layers is considered to diffuse almost

freely, it buffers the mechanical motion of macromolecular

components. Therefore free water may have a key role to

maintain the characteristic properties of the corneal gel in the

physiologically hydrated state. As the amount of the free water is

decreased, the macromolecular components come into contact

with each other through the bound water, which leads to the

increase in the dynamic modulus E0 of the cornea, and the change

in the rate of weight reduction. Abrupt change in the dynamic

modulus observed at �6000 s is likely to be due to the evapo-

ration of the water which is interacted weakly with PG, although

the detailed mechanism is not clear at present. It is worth

mentioning that dynamic modulus E0, loss tangent tan d and

weight w/w0 become constant simultaneously at approximately

6000 s, which indicates the elastic stiffness closely correlates with

the amount of free water. The evaporation of the free water

exerts much influence on the mechanical properties of the corneal

gel, as well as its weight and dimension. The sharper peak of

elastic loss tangent of the cornea is observed compared with the

heat-treated egg white gel18 or randomly cross-linked synthetic

gels.19 Moreover, the absolute value of the dynamic modulus in

dry state is about 10 times larger than that for the heat-treated

egg white gel.18 It is likely to results from the ordered structure of

cornea.

Fig. 7 Dependence of he dynamic storage modulus, E0 and loss tangent,

tan d on water content, fwater during the dehydration of the pig cornea at

25 �C. Dashed line at fwater ¼ 0.5 corresponds to the crossover point of

the weight-reduction during the dehydration process.

Fig. 8 Schematic representation of dehydration of corneal stroma. (a) The rigi

compose the lamellae of the corneal stroma in the physiological hydrated state. (

of dehydration. (c) Evaporation of the water bounded to PG evaporated for t

water and the subsequent deswelling of PG,which is located between the regular

8162 | Soft Matter, 2012, 8, 8157–8163

The temporal change in elastic properties depends on the

initial weights and sizes of the samples. The mechanical prop-

erties of the cornea is determined by the state of the stroma,

which is deeply related to the amount and the state of water

associated with the constituent biopolymers, therefore water

content fwater dependence of the elastic properties has more

physical meaning. By replacing the dehydration time with water

content fwater by using the relation fwater ¼ (w � wdry)/w, where

wdry is the dry weight of cornea, we obtained the dynamic

modulus E0 and the loss tangent (tan d) as a function of water

content fwater as shown in Fig. 7. E0 decreases sharply in the

range of water content from 0.40 to 0.50, and this is followed by

a small decrement with further increase of water content up to

0.8. It is noted that the range of water content where the sharp

decrease in E0 occurs, corresponds to the crossover point between

first- and second-stage of the weight-reduction during the dehy-

dration process (Fig. 6(b)). The abrupt increase in bound water

content, and the distinct change in the bound water fraction h of

cornea were also observed in this range of water content. From

these observations, water exerts a plasticizing effect on the

corneal gel, thereby reducing the elastic stiffness below that of

the dry cornea. The absolute value of the elastic stiffness of the

corneal gel is smaller than that for non-crystalline polymeric

glasses by about a few orders of magnitude. It is plausible to

explain that the monolayer coverage of bound water on the

lattice of the cornea prevents direct contact of the polymer chains

of the corneal gel.

These characteristics are commonly observed in glasses. On

the process of the dehydration, the evaporation of water brings

each collagen sheet into close proximity by the capillary force of

the remaining water. This leads to an increase in the attractive

interaction between the neighboring collagen sheets, and hinders

their thermal motion. This behavior is considered to be similar to

the freezing of the micro-Brownian motion in amorphous poly-

mers, which is observed in the glass transition with decreasing

temperature.21 Although more intensive studies must be needed,

the gel-to-glass transition probably occurs in the dehydrated

cornea.

4 Clinical implications

Change in the properties of the cornea due to dehydration is

important to the interpretation of its clinical appearance. It has

been reported that reversible cloudiness or haze was caused by

photorefractive keratectomy.22 This is typically observed during

d collagen type I fibrils and smaller strands of hydrated proteoglycans (PG)

b) Evaporation of freewater between the lamellae occurred in the first-step

he second-step of dehydration of the corneal stroma. Evaporation of free

two-dimensional lattices of collagenfibers, caused the change in thickness.

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surgery such as an excimer laser assisted in situ keratomileusis23

or laser thermal keratoplasty.24 The sclera is opaque and appears

white due to the high water content and disordered array of

transverse and oblique collagen fibers that make up its coating.

Unlike the cornea, which has a very orderly array of parallel

bundles that are layered on top of one another, the sclera is

designed for strength and not clarity.1,2 If the water content of the

sclera is reduced below 40%, it begins to appear clear like the

cornea,25–27 which closely resembles the gel-to-glasslike transition

observed in egg white gel.16 The changes in the properties of

ocular tissues induced by dehydration may bring new insights in

the understanding of the physicochemical basis of diseases and

clinical appearance.

5 Conclusions

Temporal changes in weight and elastic properties during the

dehydration of pig corneal gel were investigated. The corneal gel

collapses anisotropically along an axis parallel to the optical axis,

and no change is observed in the diameter during the dehydra-

tion process. During the dehydration process, the moisture from

the corneal gel evaporated in two stages. At the crossover point

between these two stages, the elastic modulus of the corneal gel

showed an anomaly: the loss tangent (tan d) showed an anom-

alous peak with a jump of the dynamic elastic modulus, E0. Twotypes of water exist in the corneal gel: one strongly bound to

relaxation centers of glycosaminoglycan (GAG) of the proteo-

glycan (PG) component, and the other almost freely diffusible as

liquid bulk water. Corneal water is mainly held by the GAG

located between the regular two-dimensional lattice of collagen

fibers, and the dehydration simply causes deswelling of the GAG

(Fig. 8). A quantitative analysis shows that the bound fraction of

the corneal water under physiological hydration makes up about

3% of the total water. Therefore, water evaporated in the first

stage could be freely diffusible or weakly bound to GAG, and the

second stage is most likely the slow evaporation of the water

strongly-bound to GAG. The abrupt increase in non-freezing

water content, and the distinct change in the bound water frac-

tion h of the cornea were also observed near the crossover point

of weight reduction. From these observations, it can be

concluded that water exerts a plasticizing effect on the corneal

gel, thereby reducing the elastic stiffness below that of the dry

cornea. The absolute value of the elastic stiffness of the corneal

gel is smaller than that of non-crystalline polymeric glasses by

a few orders of magnitude. It is plausible to explain this in that

the monolayer coverage of bound water on the lattice of the

cornea prevents direct contact of the polymer chains of the

corneal gel.

This journal is ª The Royal Society of Chemistry 2012

Acknowledgements

The work was partly supported by a Grant-in-Aid for Scientific

Research (B), and a Grant-in-Aid for the Global COE Program,

‘‘Science for Future Molecular Systems’’ from the Ministry of

Education, Culture, Science, Sports and Technology of Japan.

We are grateful to Prof. Kazuhiro Hara, Kyushu University, for

valuable discussions.

References

1 D. M. Maurice, The Cornea and Sclera, in The Eye, ed. H. Davison,Academic Press, New York, 1984, pp. 1–158.

2 I. Fatt, Physiology of the Eye, Butterworth, Boston, 1971, pp. 92–188.3 D. M. Maurice, The Cornea, in Biochemistry of the Eye, ed. C. N.Graymore, Academic Press, New York, 1970; pp. 1–103.

4 B. O. Hadbys, Curr. Eye Res., 1961, 1, 81.5 T. Matsuura, H. Ikeda, N. Idota, R. Motokawa, Y. Hara andM. Annaka, J. Phys. Chem. B, 2009, 113, 16314.

6 J. Pouchly, J. Biros and S. Benes, Makromol. Chem., 1979, 180, 180.7 J. R. Zimmermann and W. E. Brittin, J. Phys. Chem., 1957, 61,1328.

8 D. G. Gadian, Nuclear Magnetic Resonance and Its Applications toLiving Systems; The Alden Press, Oxford, 1984.

9 M. B. Smith, K. K. Shung and T. J. Mosher, in Principles of MedicalImaging, ed. K. K. Shung, M. B. Smith, B. Tsui, Academic Press: SanDiego, 1992, pp. 213–273.

10 N. Bloembergen, B. M. Purcell and R. V. Pound, Phys. Rev., 1948, 73,679.

11 R. Blinc, V. Rutar, I. Zupancic, A. Zidansek, G. Lahajar and J. Slak,Appl. Magn. Reson., 1995, 9, 193.

12 A. Blinc, G. Lahajar, R. Blinc, A. Zidansek and A. Sepe, Magn.Reson. Med., 1990, 14, 105.

13 M.-C. Vackier, B. P. Hills and D. N. Rutledge, J. Magn. Reson., 1999,103, 361.

14 T. Seiler, N. M€uller-Stozenburg and J. Wollensak, Graefe’s Arch.Clin. Exp. Ophthalmol., 1993, 221, 122.

15 B. R. Masters, V. H. Subramanian and B. Cgance, Curr. Eye Res.,1982, 2, 317.

16 E. Takushi, L. Asato and T. Nakada, Nature, 1990, 345, 298.17 A. Nakamura, K. Hara, A. Matsumoto and N. Hiramatsu, Jpn. J.

Appl. Phys., 1998, 37, 4391.18 M. Sugiyama, M. Annaka, K. Hara, M. E. Vigild and G. E. Wignall,

J. Phys. Chem. B, 2003, 107, 6300.19 D. Kaplan and F. A. Bettelheim, Biophys. J., 1972, 12, 1630.20 E. A. Balaz, Aging of Connective and Skeletal Tissue, Thule

International Symposia, Nordiska Bokhandeln, Stockholm, 1969.21 E. Donth, The Glass Transition, Springer, Berlin, 2001.22 P. J. Douherty, K. L. Wellish and R. K. Maloney, Am. J.

Ophthalmol., 1994, 118, 169.23 D. K. Joo and T. G. Kim, J. Cataract Refractive Surg., 1999, 25, 1165.24 M. H. Feltham and F. Stapleton, Clin. Exp. Ophthalmol., 2000, 28,

185.25 K. M. Meek, The Cornea and Sclera, in Collagen: Stracture and

Mechanics, ed. P. Fratzi, Springer, Potsdam, 2008.26 D. S. Schultz, J. C. Lotz, S. M. Lee, M. L. Trinidad and J. M. Stewart,

Invest. Ophthalmol. Visual Sci., 2008, 49, 4232.27 K. Trier, Adv. Organ Biology, 2005, 10, 353.

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