Nutritional Requirements of the Corneal Epitheliumand Anterior Stroma: Clinical Findings

Deborah F. Sweeney, Ruo Zhong Xie, Daniel J. O'Leary, Antti Vannas, Ross Odell,Klaus Schindhelm, Han Ying Cheng, John G. Steele, and Brien A. Holden

PURPOSE. TO monitor the health of the epithelium and the anterior stroma when porous membranesare implanted into the feline cornea and to determine membrane diffusivity characteristics neededto maintain corneal integrity.

METHODS. Filtration membranes in a range of effective pore sizes of less than 15 nm (groups 1 and2, n = 11), 25 nm (group 3,n = 8), 50 nm (group 4, n = 16), and 100 nm (group 5, n = 15) wereimplanted into an interlamellar corneal pocket of the stroma. The implanted membranes ranged inthickness from 6 nm to 15 nm and were between 8 mm and 12 mm in diameter. Animals weremonitored for clinical signs of intolerance to the implants.

RESULTS. At 1 month, thinning and ulceration had occurred in the epithelium and the anteriorstroma of all animals in groups 1 and 2; epithelial changes, anterior stromal thinning, and ulcerationhad developed in 75% of animals of group 3; 50% of animals showed vascularization and only 7%showed epithelial degeneration in group 4; and local anterior stromal thinning was observed in 7%of animals in group 5, indicating clinical acceptance of the implanted membrane. In the long term(greater than 50 days), 30% and 73% of the group 4 and 5 corneas, respectively, were clinicallyquiet. Analysis of the failure times indicated an inverse relation between failure rate and pore size:less than 15 nm > 25 nm > 50 nm > 100 nm. The difference between the 100-nm and 50-nmmembranes was significant (P = 0.03).

CONCLUSIONS. A corneal implant must have a porosity greater than that provided by 50-nm mem-branes. The 100-nm membranes used in this study establish the porosity needed to satisfy thenutritional requirements of the cornea. (Invest Ophthalmol Vis Sci. 1998;39:284-291)

There is much interest in developing a successful meansof visual correction by altering the refractive power ofthe cornea.' Surgical approaches for correction of re-

fractive error, particularly for high levels of error, are notentirely satisfactory because of postsurgical regression, forma-tion of haze, scarring, or irregular astigmatism.2 Furthermore,the results cannot be reversed if patients experience poor orunstable vision or other complications.

One approach to overcoming these problems involves theplacement of a synthetic corneal onlay onto the basementmembrane of the central cornea with subsequent regrowth ofthe epithelium over the onlay. However, the use of cornealinlays or onlays as a means of refractive error correction mayreduce the normal passage of nutrients and growth factorsthrough the cornea, thus interfering with epithelial health.

The cornea is transparent and avascular, making it aunique tissue in the body. Oxygen, essential for normal cornealmetabolism, is supplied by the atmosphere through the tears.3

The exact sources and types of nutrients that are essential forcorneal health are not precisely known but are thought to be

From the Cooperative Research Centre for Eye Research andTechnology, The University of New South Wales, Sydney, Australia.

Partly supported by the Australian Federal Government throughthe Cooperative Research Centre's Program.

Submitted for publication April 8, 1997; revised July 31, 1997;accepted September 19, 1997.

Proprietary interest category: N.Reprint requests: Deborah F. Sweeney, Cooperative Research Cen-

tre for Eye Research and Technology, The University of New SouthWales, Randwick NSW 2052, Australia.

supplied mainly by the aqueous humor4 and possibly by thelimbal vasculature.

Tears contain potassium, calcium, magnesium, and so-dium chloride, which are all thought to be necessary for main-taining epithelial surface integrity.5 In comparison, tears con-tain virtually no glucose, and the epithelial glucose uptakefrom this source is low.6 Glucose is present in the aqueoushumor in sufficient quantities to supply the whole cornea, andthe limbal vessels alone could supply as much as 20% of thecorneal glucose requirements.7

Various deprivation experiments have attempted to dem-onstrate the importance of the different routes,8'9 namely aque-ous humor, limbal vessels, and tears, for providing the nutri-ents essential for corneal health. Ablation of the limbal bloodvessels does not adversely affect the clinical appearance of thecornea, so this route of supply is not considered essential.

It has been reported that insertion of an impermeablemembrane into the stroma to interfere with nutrition from theaqueous humor invariably leads to the degeneration of boththe stroma and the epithelium anterior to the membrane.1011

Even when the membrane is permeable to glucose, the ante-rior stroma only survives when the membrane is small and isimplanted in the posterior of the stroma.12"14 This allowsnutrients to diffuse around the membrane from the aqueoushumor, with possibly some supply from the limbal vascula-ture.15

The aim of this study was to clinically investigate changesin the cornea, particularly the anterior stroma and the epithe-lium, when thin porous membranes of different effective poresizes and porosities were implanted into feline corneas. The

284Investigative Ophthalmology & Visual Science, February 1998, Vol. 39, No. 2Copyright © Association for Research in Vision and Ophthalmology

IOVS, February 1998, Vol. 39, No. 2

TABLE 1. Membrane Materials and Specifications

Corneal Nutritional Requirements 285

Group

1

2

3

4

5

Membrane

Cuprophan

GambraneNuclepore 15

(NP15)Poretics 25

(PO25)Nuclepore 50

(NP50)Nuclepore 100

(NP100)

Number ofAnimals

3

26

8

16

15

Materials

Regeneratedcellulose

PolycarbonatePolycarbonate

Polycarbonate

Polycarbonate

Polycarbonate

Pore Size(nm)

—15

25

50

100

Pore Density(pore/cm2)

6 x 108

NA

6 x 108

3 X 108

Porosity(ca%)

—0.1

NA

1.2

2.4

Thickness(nm)

15

156

NA

6

6

porous membranes used in this study included membranes thatwould permit effective diffusion of low-molecular-weight mol-ecules, such as glucose, and components with higher molecu-lar weight (including proteins of the size of albumin, Mr

67,000, and larger). This allowed for the critical membranediffusivity characteristics of the nutritional requirements of theepithelium and of the anterior stroma to be determined.

METHODS

Membranes

The details and characteristics of the membranes implanted arelisted in Table 1. The membranes were selected because theywere commercially available, known to be biocompatible withminimal cytotoxicity, and covered a range of pore sizes andporosities sufficient to evaluate the effects of diffusion of mol-ecules of a variety of sizes on anterior corneal health.

All the materials were composed of polycarbonate, exceptthe blood dialysis membrane (Cuprophan; Membrana AG,Wuppertal, Germany), which was composed of regeneratedcellulose. This allowed the effect of the material itself to beignored as a variable in the responses of the different porositymembranes in this series. The pore size is variable for Cu-prophan (Membrana AG) because it is a regenerated cellulosenetwork with a molecular weight range of 300 to 3000. An-other dialysis membrane used in the study, Gambrane (Gam-bro, Lund, Sweden), had a molecular weight cut-off of approx-imately 20,000.

The membranes were cut with a trephine of 8-, 10-, or12-mm diameter to form lenticules. Membranes (Nuclepore;Costar, Cambridge, MA, and Poretics; Medos, Sydney, Austra-lia) were rigorously washed before implantation by sonicatingin the following series of solutions: 5 minutes in distilled water,10 minutes in 0.1% Triton X-100 in distilled water, 5 minutes indistilled water, and 5 minutes in sterile saline. The membraneswere then sonicated in four alternating 5-minute washes ofdilute HC1 (pH 3) and then in distilled water. The washedmembranes were left overnight in dilute HC1 (pH 3), rinsed for5 minutes in sterile saline or Hartmann's solution, and auto-claved in sterile saline. The Cuprophan (Membrana AG) andGambrane (Gambro) membranes were washed and sterilized ina similar series of solutions but were excluded from the alter-nating HC1 washes-distilled water rinses.

Mass TransferThe diffusive permeabilities of native and explanted mem-branes to glucose, inulin, and albumin were measured using atwo-chamber device. The 14-ml chambers were stirred on avertical axis, and the ports between which the membrane wasclamped were heavily chamfered to enhance mixing near diemembrane surface. The exposed membrane area was 0.126cm2. Experiments were conducted at room temperature(23°C ± 1°C).

The permeabilities to glucose and inulin were measuredsimultaneously using [l4C] glucose and [3H] inulin in phos-phate-buffered saline (PBS) containing 0.925 g/1 unlabeled glu-cose and 0.011 g/1 unlabeled inulin. The albumin permeabilitywas measured using [125I]albumin in PBS with 1.0 g/1 unla-beled albumin.

Albumin was labeled using the chloramine T method.16

Free l25I label was removed by gel nitration (using SephacrylS-200 High Resolution, Pharmacia Fine Chemicals, Uppsala,Sweden) before each experiment. The fraction of free activityin the starting material, as determined by trichloroacetic acidprecipitation, was consistently less than 0.6%.

The solution containing labeled solute was placed inchamber 1, and the unlabeled solution was placed in chamber2. Samples (1 ml, with replacement by 1 ml unlabeled solution)were taken from chamber 2 after 5 minutes and at varioustimes over the next 1 (glucose-inulin) to 3 hours (albumin).Smaller samples (0.1 ml, with replacement) were taken fromchamber 1. The activities of l4C and 3H labels were measuredby liquid scintillation counting, and 125I activity was measuredin a y counter. Free 125I label in samples was quantified bytrichloroacetic acid precipitation.

Because of the small membrane surface area, large cham-ber volumes, and short experiment duration, die concentrationin chamber 2 (C2), was much less than the concentration inchamber 1 (C,) and, after correction for sampling, was seen toincrease linearly with time as shown in Figure 1. Therefore, thepermeability (P) was calculated from die slope17 using:

V2P = =CXA dt'

where V2 is die volume of chamber 2, A is the exposed mem-brane area, and Cj is the mean concentration in chamber 1.

To correct for the diffusion resistance of the two fluidboundary layers, experiments were conducted at a range of

286 Sweeney et al. IOVS, February 1998, Vol. 39, No. 2

2 -

.22

1000/rpm

FIGURE 1. Example of a Wilson plot to correct for fluid-phasediffusion resistances. Error bars represent uncertainty causedby counting error ( ± 1 SD).

stirrer speeds (250-1200 rpm), and the true membrane per-meability, Pm, was estimated from a Wilson plot17 as illustratedin Figure 1. The reciprocal of the measured permeability (theresistance) was plotted against the reciprocal of stirrer speed,a straight line was fitted, and Pm was calculated as the recip-rocal of the intercept according to:

1 1 1- = —- + constP Pm rpm

(2)

Expected permeabilities (at 37°C) of die Nuclepore mem-branes were calculated from the nominal pore diameter dp,pore density, membrane thickness Zm (Table 2), and the freediffusion coefficients of the solutes, assuming straight cylindri-cal pores normal to the membrane surface and using an equa-tion given by Bean18:

(3)

where Af is the total pore cross-section area fraction, D° is thefree diffusion coefficient of species i, dt is the molecular diam-eter, the term in brackets is a correction for steric interactionof pore and solute, and the hydrodynamic interaction term,/(di/dp), approaches one as djdp approaches zero. Permeabili-ties measured at 23°C were converted to 37°C using:

P2y = 0.7UPir, (4)

which is derived from the Stokes-Einstein equation19 and theviscosity of water at the two temperatures. The free diffusioncoefficients in water at 37°C were taken to be: glucose, 90 X10~6 cm2/second20; inulin, 2.9 X 10~6 cm2/second2'; albu-min, 0.85 X 10~6 cm2/second.20

Animals

All cats were obtained through the University of New SouthWales Animal Breeding Unit, and the clinical procedures and

TABLE 2. Expected (Theoretical Calculation) and Measured Membranes Permeabilities (with Standard Deviations)

Membrane

CUPROPHAN

NP15

PO25

NP50

NP100

Native orExplanted

Native

Native

Native

Explant

Native

Explant

Native

Explant

DaysImplanted

N/A

N/A

N/A

16-23

N/A

13-68

N/A

28

Parameter

ExpectedMeanSDnExpectedMeanSDnMeanSDnMeanSDnExpectedMeanSDnMeanSDnExpectedMeanSDnMeanSDn

Glucose

3.403.200.3440.110.100.0560.640.2130.660.0931.486.590.3653.320.9142.958.462.6055.260.933

Permeability (cm/s

Inulin

0.120.330.0340.0200.0310.01660.1600.05030.0860.00730.442.390.2950.810.2240.882.550.3751.490.273

Albumin

0.002

0.0080.00330.0050.00130.160.320.0730.150.0840.330.610.3140.280.062

x I0~4)

Inu/Glu

0.370.1020.00440.1820.3250.02360.2510.00730.1330.02630.2980.3630.04850.2440.01940.2980.3100.07450.2840.0143

Alb/Glu

0.0120.00330.0080.00230.1100.0480.00830.0520.04240.1100.0730.03540.0530.0032

Measurements were made at 23°C and adjusted to 37°C using equation 4.

IOVS, February 1998, Vol. 39, No. 2 Corneal Nutritional Requirements 287

TABLE 3« Grading Scale for Clinical Tolerance ofMembranes

Clinical Findings

Edema

DepositsInfiltrates

Neovascularization*

Epithelial or anterior stromalthinning overlying theimplant

Corneal ulcer in the stromaanterior to the implant

Grade

SlightBullae

FocalDiffuse1

3

ClinicalScore

16126126

6

6

*Neovascularization: grade 1, neovasculature only at incision site;grade 2, neovasculature migrated towards the membrane; grade 3;neovasculature reached the membrane.

animal handling were approved by the University's AnimalCare and Ethics Committee. Fifty cats, ranging in age from 1 to6 years, were used in this study.

Clinical Procedures

The nictitating membranes of all eyes were removed. Seven to14 days after this procedure, each animal was assigned ran-domly to a group to receive a lenticule from one of themembrane groups. Group data, including average pore size,thickness of the material implanted, and number of animalsassigned to each, are presented in Table 1.

Baseline measurements of corneal integrity, which in-cluded a slit lamp evaluation, were conducted by one observer.Details of corneal transparency, vessel hyperemia, and pene-tration were assessed using the grading scales in Table 3-

Intrastromal implantation was performed by one of twosurgeons (RZX, AV), and the eye to undergo implantation waschosen at random. All surgery was performed between Febru-ary 1993 and August 1994.

A diamond knife with incision depth control was used tomake a 6-mm incision just inside the superior limbus. A cornealdissector with a sharp edge was used to prepare an interlamel-lar pocket large enough to hold an 8-mm to 12-mm diametermembrane (45 of the 50 membranes were of 8-mm diameter).The aim was to prepare the pocket at one-third depth of totalcorneal thickness. When difficulty was encountered in separat-ing the anterior corneal lamellae, a mid-stromal dissection wasperformed using minimal force. The membrane was insertedinto the pocket with the use of plastic-tipped forceps. Theincision was closed with 8-0 nylon by interrupted sutures.Figure 2 presents the clinical picture of an eye after surgery.The eyes were assessed daily with a slit lamp in the first weekafter implantation and were then examined at least weekly forthe duration of the observation period. The observations andsurgery were all performed without the surgeons' knowledgeof the materials implanted.

Topical steroids (Sofradex; 0.5% framycetin sulfate and0.5% dexamethasone sodium) were used after surgery in someanimals from all groups to control vessel growth associatedwith the limbal incision. Drops were usually administered

three times a day. They were used generally for less than 7 daysbut in five eyes were continued for up to 1 month after surgeryif necessary to control new vessel growth. Their use began theday after surgery.

A clinical tolerance rating was developed for the evalua-tion of the outcome of the implantation (Table 3). A single orcumulative score of six was denned as clinical intolerance. Theanimal was usually killed at this point, and the cornea wasprocessed for histologic examination. At various other timepoints, animals with clinically quiet eyes were killed for eitherhistologic examination or assessment of membrane permeabil-ity.

The times to failure, where failure was denned as a scoreof 6 or greater, were used to construct Kaplan-Meier survivalcurves22 for each membrane type. If an animal was killed forhistologic assessment or for membrane permeability measure-ment or if the membrane developed a split while the corneawas healthy, the case was considered censored. The influenceof membrane type on the failure rate was assessed using dieCox proportional-hazard regression model,22 with membranetype as a categorical variable and simple contrasts with the100-nm membrane as the reference category (SPSS; StatisticalPackage for Social Scientists, Chicago, IL).

The explanted membranes were stored in cacodylatebuffer at pH 7.2. Permeabilities to glucose and inulin weremeasured on the day of explantation. Permeability to albuminwas measured on the second day, and the permeabilities toglucose and inulin were measured again on the third day.

RESULTS

PermeabilityThe permeabilities of the native and explanted membranes areshown in Table 2. These have been adjusted to 37°C as de-scribed above. The permeabilities of the explanted membrjinesare also plotted in Figure 3.

The permeability of the native Nuclepore 15-nm mem-brane was low for all solutes, a consequence of its extremelylow porosity (approximately 0.1%). Explanted 15-nm mem-branes were, as a result, not tested. Explanted Cuprophan

FIGURE 2. Picture of an eye with an intracorneal implantedmembrane 60 days after surgery. The eye was clinically quiet,and the cornea was clear. The nontranslucent membrane onlyreflects light.

288 Sweeney et al.

10"3 F

10"4

5 10-sCD

10"6

IOVS, February 1998, Vol. 39, No. 2

10"7

102103 104

Molecular weight

105

FIGURE 3- The permeabilities of explant membranes (O,100-nm Nuclepore; D, 50-nm Nuclepore; A, 25-nm Poretics)and native membranes (V, 15-nm Nuclepore; O, Cuprophan).

o.o

• NP100A NP50• PO2S• NP15• Cuprophan• (Censored)

25 50 75 100

Time (days)

125 160 175

FIGURE 5. Kaplan-Meier survival curves for eyes with im-planted membranes. Survival was defined as a clinical score

membranes were also not tested, because they could not berecovered with sufficient intact area to allow measurements.

Explanted 50-nm and 100-nm membranes tended to beless permeable than native membranes, but there was littledifference between native and explanted Poretics 25-nm mem-branes. Permeability did not appear to be related to the dura-tion of implantation. When the explanted 50-nm and 100-nmmembranes were retested after 2 or more days, during whichtime they were stored in buffer, the glucose and inulin perme-abilities were generally higher than for the initial measure-ments.

Clinical Results

The clinical results for each group are presented in Figure 4.The corneal tolerance for groups 1 and 2 was shortest; all eyeshad a clinical score of >6 by day 25. For the same time period,clinical tolerance increased with larger pore sizes (25, 50, and100 nm) to 38%, 71%, and 93%, respectively. Only the groups

FIGURE 4. Incidences of intolerant clinical features for groups1 through 5.

with 50-nm and 100-nm pore sizes (groups 4 and 5) maintaineda clinical tolerance for implanted membranes for more than 50days; these outcomes were 31% and 73%, respectively.

The depth of the surgical pocket in the corneal stromawas recorded with the slit lamp after surgery. In 40 eyes, thepocket was in the anterior one third, and in 10 eyes it was inthe mid-stroma. The cornea! tolerance or survival was notinfluenced by this variation of depth of implantation in thedifferent groups. In group 3, the membranes were all in theanterior one third of the stroma, making the depth factornegligible.

Cumulative survival curves of all eyes with implantedmembranes are shown in Figure 5. In general, survival wasbetter with larger pore diameters. The Cox regression coeffi-cient estimates are shown in Table 4 along with the corre-sponding failure rates relative to the 100-nm membranes.There was an inverse relation between the regression coeffi-cient and pore diameter. The coefficients for all membraneswith a pore size of 50 nm or less were significantly differentfrom zero; that is, the failure rates were higher than for the100-nm membranes.

All membranes were flat, resulting in small, peripheralfolds on implantation because of corneal curvature. Depositswere observed in these areas in all groups. A slight to moderateinflammatory reaction with limbal hyperemia and cornealedema was observed after implantation in all groups, whichsubsided within 1 to 4 days.

A common adverse reaction was corneal neovasculariza-tion. Limited vascularization associated with the incision sitewas present in all groups within 2 weeks of surgery. Laterstromal neovascularization progressed to the implant and wasaccompanied by an inflammatory reaction by the end of 1month in 40% and 50% of group 1 and 2 eyes, respectively. Ingroup 3, new vessel growth was not as extensive. In group 4,50% showed vascularization at 1 month. For group 5, vascular-ization regressed in all but one case (71%) between 20 and 40days, indicating clinical acceptance of the 100-nm membranes.

Degenerative changes were observed in the epitheliumand anterior stroma, resulting in anterior stromal thinning in allanimals in groups 1, 2, and 3, and less frequently in groups 4and 5. Epithelial ulceration was often accompanied by the

IOVS, February 1998, Vol. 39, No. 2 Corneal Nutritional Requirements 289

TABLE 4. Cox Proportional-Hazard Regression Parameter Estimates (B) and Their Standard Errors

Membrane

CuprophanNP15PO25NP50NP100

PoreDiameter(nm)

5152550

100

B

5J73.332.251.30

Se(B)

0.780.740.630.60

P

<0.001<0.001<0.001

0.030

RelativeFailure Rate

43.528.09.53.661

GlucosePermeability(cm x 10~4)

3.20.10.26.68.5

The P value pertains to the null hypothesis; B = 0. The failure rate, relative to NP100, is given by exp(B). Also shown are the permeabilitiesto glucose of the native membranes. The pore diameter for Cuprophan is taken to be twice the diameter of the inulin molecule.

above changes. Ulceration appeared early and was most fre-quent in groups 1,2, and 3-

DISCUSSION

Clinical FindingsThe purpose of the clinical scoring used was to evaluate cor-neal health and to be able to kill the animals when clinicalexamination indicated that the corneal integrity had clearlysuffered. The goal was to discontinue the follow-up earlyenough in order that secondary changes, such as neovascular-ization accompanied by inflammation, would not mask theunderlying disease and malfunction of the cornea. However,some eyes were followed up to assess whether improvementin the clinical condition occurred after the score exceeded 6.

Earlier studies have shown that corneal inlays imperme-able to solutes are tolerated only if the diameter of the inlay issmall and is placed in a deep corneal pocket.23 In this study,the corneal pockets were prepared "free hand," which resultedin small variations in the pocket depth. However, the cornealtolerance of the implanted membranes did not seem to beinfluenced by the variation in pocket depth in this series. It isthought that the large pore size of the membrane and itsanterior location negate the effects of lateral diffusion of sol-utes in this model.

The clinical findings after the implantation of differentporous membranes included epithelial abnormalities and irreg-ularity, anterior stromal thinning, and infiltration of inflamma-tory cells. The incidences of corneal epithelial abnormalities,anterior to the implanted membrane, were inversely correlatedwith the pore sizes of the implants. These clinical manifesta-tions appear to reflect a nutritional deficiency in the corneaanterior to the implant.

The observation that eyes containing implants with100-nm pore sizes were clinically quiet for more than 100 daysafter implantation indicates that the surgical model is suitablefor evaluation of implants and that polycarbonate membranesare well tolerated in the anterior cornea. This is further evi-dence that the decreased pore size and density are the mainfactors causing corneal intolerance in groups 1 through 4. Wecan, therefore, postulate that the pore size or diffusivity of themembrane to solutes of a larger molecular weight than glucoseis the main factor causing clinical intolerance.

The limited initial corneal neovascularization observedwas not associated with the pore sizes of the implants and waslikely to have been caused by an inflammatory reaction thatwas, in turn, caused by the surgical procedure and the im-

planted membrane. Therefore, inhibition of corneal inflamma-tion is important for achieving and maintaining a successfulmodel.

Mild corneal edema after implantation was observed in theoperated eyes for 1 to 4 days. This edema response appeared tobe caused mainly by surgical irritation. Severe edema in thecornea anterior to the implanted membrane was observed in alleyes in group 1 within 2 weeks. It was also accompanied bycorneal ulceration in 80% of eyes in this group. This indicatesthat severe edema and inflammation are clinical manifestationsof acute nutritional deficiency in the anterior stroma.

Mass TransferExplanted membranes were tested to determine their in vivopermeabilities. The permeability of the explants was lowerthan that of the native membranes and increased with storagetime. One possible explanation for the reduced permeability isthe infiltration of macromolecular constituents of the extracel-lular matrix into the membrane pores.

The 50-nm and 100-nm membranes were substantiallymore permeable to glucose and inulin than expected. Oneexplanation may be that the membrane porosity was greaterthan the value calculated from the nominal pore size and poredensity. Based on measurements of hydraulic permeability andscanning electron microscopy, Comper and Williams2'* esti-mated porosities 50% to 250% higher than the nominal values.

The permeability of Cuprophan (Membrana AG) to glu-cose after adjustment to 37°C was similar to the value reportedby Klein et al.25 The permeability to inulin was higher than thevalue reported by those authors.

ImplicationsThese studies showed that the membranes with 100-nm poreswere clearly superior to membranes with 50-nm, 25-nm, or15-nm pores and were superior to Cuprophan (MembranaAG)-Gambrane (Gambro) for the support of corneal tissueintegrity anterior to the implant during the observation period.Because the track-etched membranes were otherwise identi-cal, it appears that the superiority was a result of the largerpore diameter, the larger total pore cross-sectional area, orboth, and that the cause of failure was interference with thetransport of nutrients across the cornea.

The movement of nutrients from the aqueous to the epi-thelium and the impact of this movement for a permanentintracorneal implant are not fully understood. Among the fac-tors that determine the extent to which the implant will reducethe flux of a particular nutrient are the permeability of theimplant, the diffusion coefficient in the stroma and the stromal

290 Sweeney et al. IOVS, February 1998, Vol. 39, No. 2

10 10 6 105 10'4 10-3

Implant permeability (cnVs)10-2

FIGURE 6. The predicted effect of an intracorneal implant onglucose flux at the epithelium as a function of implant perme-ability. The y axis is the ratio of the flux (at the epithelium)with implant present to the flux with no implant. (•) Themodel of McCarey and Schmidt.7 The line represents the as-sumption that diffusion through the stroma is limiting.

thickness, the permeability of the endothelium, and the rate ofconsumption of certain nutrients at the epithelium and withinthe stroma as a function of the local concentration.

The most complete published model for nutrient require-ment focused on glucose metabolism is that of McCarey andSchmidt.26 They calculated the glucose concentration as afunction of position within the stroma under the followingassumptions: The glucose concentration anterior to the endo-thelium was 880 jag/ml; the glucose diffusion coefficientwithin the stroma, Ds, was 2.5 X 10~6 cm2/second; the stromalthickness, Ls, was 0.05 cm; the epithelial glucose consumption(per cm2) was proportional to the glucose concentration witha proportionality constant, k, of 1.17 X 10~5 cm/second; andkeratocyte glucose consumption was a nonlinear function ofglucose concentration. The effect of implants with a thickness(Zm) of 0.015 cm but with various fractions of open pore area(or porosity, Af) on glucose porosity was calculated, assumingthat the diffusion coefficient within the pores was the same asin the stroma. The porosity can be converted to a permeabilityusing:

(5)

Their results for four animals are plotted in Figure 6 (closeddots) as the ratio of the glucose concentration (or flux) at theepithelium with implant compared with no implant. Also plot-ted (as the line in Fig. 6) is a simpler and more conservativemeasure of the impact of the implant, the ratio of the perme-ability of stroma plus membrane (/*stm) to the permeability ofthe stroma (P^ alone, calculated from:

* stm

Ls/Ds+\Pm(6)

According to these models, the Nuclepore 50-nm and 100-nmmembranes, with glucose permeabilities (at 37°Q of 3 X 10~4

cm/second to 5 X 10 A cm/s after explantation, should reduceglucose delivery to the epithelium by 10% at most.

The glucose permeability of (native) Cuprophan (Membra-na AG) was not significantly lower, and Figure 3 suggests thatglucose flux should have been reduced only 10% to 15% by theimplant. The Poretics 25-nm membrane and the Nuclepore15-nm membrane would reduce glucose flux by 30% to 50%and greater than 70%, respectively. The failure of the latter twomembranes was consistent with their estimated impact onglucose flux, but it is unlikely that the different failure rates ofthe 100-nm, 50-nm, and Cuprophan membranes were causedby their differential effects on the flux of glucose or other smallmolecules.

It appears, then, that the failure rates in the observationperiod of the study are better interpreted in terms of pore sizethan in terms of small molecule permeability. The permeabilityof Cuprophan is strongly dependent on molecular weight, andit is effectively impermeable to molecules larger than Mr

10,000 or so. It is not surprising that the failure rate of Cu-prophan was high. The implication of the inferior performanceof the 50-nm membranes is that the minimum implant pore sizeconsistent with epithelial health is greater than 50 nm. Thisseems large, but it should be noted that 50-nm pores willinterfere with the diffusion of solutes larger than approxi-mately 10 nm.27

Epikeratophakia with human stromal lenticules and withsynthetic collagen lenticules provides epithelial regrowth overthe onlay.26'29 The synthetic collagen permeability is in thesame range as rabbit corneal stroma.28 Our results are consis-tent with this, because implants that interfered the least withthe stromal permeability for small and large molecular weightmolecules presented the best corneal tolerance.

This study suggests that permeability to solutes with mo-lecular weights larger than glucose is necessary for maintainedepithelial and anterior stromal integrity. For synthetic implants,pore diameter and total pore area (porosity) are importantcriteria for maintaining the long-term nutritional needs of thecornea.

Acknowledgments

The authors thank Denise Lawler for her excellent work, and theythank the staff at the Cooperative Research Center for Eye Researchand Technology for its contribution to this study.

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2. Schallhorn ST, Blanton CH, Kaupp S, et al. Preliminary results ofphotorefractive keratectomy in active-duty United States Navy per-sonnel. Ophthalmology. 1996; 103:5-22.

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