Collagen Fibril Orientation in the Human Corneal Stromaand Its Implication in Keratoconus

Albert Daxer*^ and Peter Fratzl-fX

Purpose. The kind and the degree of preferred collagen fibril orientation in normal humancorneal stroma were investigated as important qualities of the cornea with respect to itsmechanical properties and, hence, to refractive surgery. To determine whether this informa-tion is relevant to corneal disease, the authors investigated collagen fibril orientation in severalcorneas with keratoconus.Methods. By means of low-angle x-ray scattering, 17 normal human corneas and four corneasof eyes with keratoconus were investigated.Results. Collagen fibrils in the normal human corneal stroma showed two preferred orienta-tions orthogonal to each other. These were the horizontal and the vertical directions. Theauthors defined a degree of orientation y, determined to be y = 0.49 ± 0.10 (mean ± SD).This means that the excess of the preferentially oriented fibrils in relation to the total numberof fibrils was approximately 49%. It follows from this value that approximately two thirds ofthe fibrils (66%) were within in a 45° sector (±22.5°) around the horizontal and verticalmeridians, whereas approximately one third (34%) is oriented in the oblique sectors inbetween. No statistically significant variation of y within a central 7 mm zone could bedetected in normal corneas. The orthogonal arrangement of the collagen fibrils was, however,profoundly altered in keratoconus, in which nonorthogonal orientations were found insidethe apical scar.

Conclusions. The normal human corneal stroma shows a considerable degree of structuralanisotropy. It is characterized by two preferred collagen fibril orientations orthogonal to eachother. Alteration of the regular orthogonal arrangement of the fibrils in keratoconus may berelated to the biomechanical instability of die tissue. Invest Ophthalmol Vis Sci. 1997; 38:121-129.

J. he human corneal stroma is composed of approxi-mately 200 successively stacked lamellae of type I colla-gen fibrils. Within each lamella, the collagen fibrilsrun parallel to each other and show a regular interfi-brillar spacing.1"4 Orientation of the fibrils is constantwithin each lamella but varies throughout successivelayers in a way that may depend on species, as well asother conditions.5"8 Although the regular arrange-ment of the fibrils within each lamella is consideredto be responsible for the transparency of the tis-

From the * Universitfitsklinik fur Augenheilkunde, Innsbruck, the flnstilut fiirMaterialphysik der Universita't, and the %Ludwig Boltzmann-Institut furOsteologie, Vienna, Austria.Presented in part at the Xhh ICER 1994, New Delhi, India, and at the 1995 and1996 meetings of the Association for Research in Vision and Ophthalmology, FortLauderdale, Florida.Received for publication September 14, 1995; revised July 22, 1996; acceptedAugust 27, 1996.Proprietary interest category: N.Reprint requests: Albert Daxer, Universitdtsklinik fur Augenheilkunde, Anichstrasse35, A-6020 Innsbruck, Austria.

sue, ' ' the orientation of the successive fibril layersthroughout the entire cornea is an important factordetermining the mechanical properties of the cor-nea. 11,12

Although many local details of the corneal struc-ture have been obtained by electron microscopicmethods,cg"5 information about typical structuralcharacteristics of the entire cornea, such as preferredor random orientation of the layers, is obtained morereadily by scattering techniques.48'13 In particular,Meek et al8 reported x-ray scattering patterns fromnormal human corneas, indicating preferred orienta-tion of collagen fibrils along the horizontal and verti-cal meridians. Nevertheless, the collagen fibril orienta-tion in the human corneal stroma has never beencharacterized quantitatively. Such quantitative infor-mation is essential for an understanding of the biome-chanical properties of the cornea and, hence, for theoptimization of refractive surgery.14"16

Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1Copyright © Association for Research in Vision and Ophthalmology 121

122 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1

In this study, we used x-ray scattering to determinequantitatively the distribution of collagen fibril orien-tation in 17 normal human corneas at five positions(central, nasal, inferior, temporal, superior) within acentral 7 mm zone. For comparison, we investigatedfour corneas of eyes affected by keratoconus, bothinside and outside the lesion. In these eyes with kerato-conus, considerable changes were found in the pre-ferred angles between the collagen fibrils.

MATERIALS AND METHODS

Specimens and PreparationSeventeen human corneas with no apparent sign ofcorneal disease were removed within 5 hours of death,and four corneas of patients with keratoconus wereobtained after penetrating keratoplasty. Corneas weremarked at the anatomic 12 o'clock position with onestitch of a 10-0 nylon suture. All corneas were storedat — 70°C until measurement on the low-angle x-rayapparatus was performed. In agreement with otherstudies,4'17 preliminary experiments have shown thatthe freezing procedure had no detectable influenceon the scattering patterns of the corneas. The 17 nor-mal corneas were fully transparent, and no lesionscould be detected on slit lamp examination immedi-ately before and after explantation. Each of the fourkeratoconus corneas had typical scars. Donors (15women and two men) ranged in age from 38 to 89years (median, 71 years). The state of hydration wasestimated during the x-ray scattering experiment us-ing the position of the main maximum of the scatter-ing pattern: The interfibrillar distance, d, was approxi-mately the same in all samples (d = 59.1 ± 2.2 nm;mean ± SD), indicating a hydration only slightlysmaller than normal.4 The tenets of the Declarationof Helsinki were followed, and approval of the respon-sible institutional human experimentation committeewas granted. Keratoconic corneas were taken after in-formed consent was obtained.

Apparatus and MeasurementAs described elsewhere,4 we used a 12 kW x-ray genera-tor and a pinhole x-ray camera with a fixed distanceof 100 cm from the sample to the detector. The detec-tor was a two-dimensional position-sensitive propor-tional counter (Siemens AG, Karlsruhe, Germany).The x-ray beam (diameter, 0.5 mm) had a wavelengthof X = 1.54 A and was directed parallel to the opticalaxis at various corneal positions. In the normal humancorneas, the positions were the center, nasal, inferior,temporal, and superior margins of a central 7 mmzone. Measurements were performed without furtherpreparation in a native state of the corneas at roomtemperature. For measurements, the hydrated cor-neas were fitted carefully into an air-tight container

made of welded plastic and were mounted into a vac-uum chamber of the x-ray scattering apparatus. Thisprocedure leaves the corneal buttons at approximatelythe correct curvature. An optical window in the wallof the vacuum chamber permitted visual observationof the tissue during the experiment. Typically, x-rayspectra were collected for 30 minutes and were cor-rected for absorption and parasitic pinhole scattering.The apparatus was calibrated by measuring the De-bye-Scherrer rings of Ag-Behenate.18

Analysis of X-Ray Scattering PatternsTwo-dimensional, small-angle, x-ray scattering pat-terns were collected as schematically shown in Figure1. Examples for such two-dimensional patterns areshown in Figure 2. These two-dimensional spectrawere described as functions J (#,</>) of the scatteringangle 9 and the rotation angle 4> (for definition of theangles, see Fig. 1). In a first step, these spectra wereused to obtain the curve 1(0) by summing all valuesof](9,<f>) inside a ring between 9 and 9 + d9 (with cf)varying from 0° to 360°). A typical result is shown inFigures 3A and 3B. The maximum in the region be-tween 9 = 0.35° and </> = 0.60° (shaded area, Fig. 3A)corresponds to the first subsidiary maximum visible ingreen in Figure 2 (red arrow), which, according toWorthington and Inouye,19 was caused by the firstmaximum of the equatorial scattering from collagenand contains a contribution from the sharp meridio-nal collagen reflections (that is, the sharp third-orderreflection located at 0.41°). For cylindrical fibrils, theequatorial scattering is approximately the Bessel func-tion (2 J](ka)/ka)2 multiplied by the interferencefunction of the cylinder centers L(k), with k = 4TT/X.sin(0/2). Although the peak should, in principle, becaused by collagen, the background scattering belowwould arise from matrix proteins other than collageninside the corneal stroma. Indeed, because the maxi-mum corresponds to the oscillation of the Bessel func-tion, the scattering from collagen fibrils should be-come zero on both sides of the maximum in 1(0). Thecontribution from the interference function resultingfrom fibril packing L(k) has been shown to be approx-imately constant and proportional to the number offibrils in the k-range beyond the first zero of the Besselfunction.19 Consequently, the value of 1(0) at thesetwo mimima of the scattering intensity should becaused by the background from matrix proteins otherthan collagen. We have interpolated between thesetwo points to obtain a reasonable estimate for thebackground, as shown in Figure 4. The fitted back-ground function is shown as a solid line in Figures 3Band 4. It corresponds to a power law behavior withthe related intensity proportional to 9K, where K =-1.02 ± 0.072 (mean ± SD) (ra = 67). Figure 5 showsthe integrated scattering intensity 1(9) subtracted bythe background function. It can be seen that the mini-

Collagen Fibril Orientation in the Cornea 123

x-ray source

e

cornea

FIGURE l. Schematic drawing of the experimental setup. Thedetector is shown from two views. Side view demonstrates thefunctional connection to the other experimental elements,such as x-ray source and sample, as well as to the scatteringangle 6. Front view (as seen from the x-ray source), showsthe scattering angle 6 and the rotation angle <f>.

Two-dimensional positionsensitive x-ray detector

0 scattering angle

c|> rotation angle (orientation)

mum at 9 = 0.35° is entirely determined by the fibriltransform, which is fitted (solid curve in Fig. 5) to thedata by (2 Ji(ka)/ka)2 (see also above). The meridio-nal scattering of the collagen fibrils caused by the axialpacking of their molecules, indicated by the arrowslabeled 3 and 5 in Figure 5, contributes as sharp peakswithin the subsidiary maxima.

In a second step, a curve 1(0) was obtained bysumming up all the values of J(0,0) at a given 0, for9 between 0.35° and 0.60°, corresponding to the re-gion of scattering angles of the first subsidiary maxi-mum. Generally, the scattering intensity must fulfillthe condition 1(0) = 1(0 + 180°). Because 1(0) ismeasured in fact for O s ^ s 360°, we averaged 1(0)and 1(0 + 180°) to improve the counting statisticsand, therefore, the accuracy of the measurement. Atypical result is shown in Figures 3C and 3D. The oscil-lation of the curve in these figures corresponds to thefact that the height of the first subsidiary maximumdepends considerably on the rotation angle 0. Thisoscillatory part is seen clearly above a constant contri-bution, which is shown shaded in Figure 3D. 1(0) canbe fitted by a constant (dashed line in Figure 3D)added to a sinus function (solid line in Figs. 3C and3D). For each normal cornea, the height of the fourmaxima in Figures 3C and 3D was equal within theaccuracy (counting statistics) of our x-ray measure-ments. This was not the case, however, for keratoconiccorneas. Hence, the quantitative evaluation proceduredescribed here was only applied to normal corneas.

In summary, the shaded areas in Figure 3 havethe following significance:

Areas I] and I3 are the total amount of scatteringbelow the subsidiary maximum between 9 = 0.35°and 9 = 0.60°, including the signal from all colla-gen (preferentially oriented or not) as well asfrom other matrix proteins.Area I2 is the scattering caused by noncollagenousproteins only, and area I4 is the scattering causedby randomly oriented collagen as well as by noncol-lagenous proteins. It should be noted that I3-I4(oscillations above the constant contribution inFig. 3D) represents the excess of fibrils oriented inthe preferred directions.

RESULTS

Structural Anisotropy in Normal CorneasX-ray scattering spectra were obtained from 17 normalcorneas at five locations each. An example is shownin Figure 2A, in which maxima in four orthogonaldirections are visible. The four inner peaks (brown)are caused by interfibrillar spacing, whereas the fourouter peaks (green) are related to oscillations of thefibril transform with a contribution from the third-order peak of the axial packing period of the collagenfibrils.4'819 The orthogonal fourfold symmetry of thecollagen-related peaks in the scatteiing patterns ob-

124 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1

FIGURE 2. Two-dimensional x-ray scattering patterns of a nor-mal cornea (top) and a keratoconic cornea in the apical scar(bottom). Inner reflections (brown) represent the scatteringintensity caused by interference between the collagen fibrils,and their position (i.e., scattering angle measured as dis-tance from the center) is related to the interfibrillar dis-tance. The outer reflections (green, indicated by the redarrow) represent the first subsidiary maximum. It resultsfrom the scattering of both, the geometry of the individualcollagen fibrils (fibril transform) (i.e. equatorial scattering),and the third-order reflection of the axial collagen period(i.e., meridional scattering). The presentation of the inten-sity distribution is based on a logaridimic scale. The normalcornea shows orthogonal fourfold symmetry (top); this sym-metry is disturbed in the keratoconus (bottom).

tained from normal human corneas indicates pre-ferred orientation of the collagen fibrils in two orthog-onal directions. Moreover, within the counting statis-tics, all spectra for normal corneas were symmetricwith respect to horizontal and vertical directions, im-

plying that an equal amount of collagen fibrils wereoriented vertically and horizontally. This remains trueeven we take into account that meridional (from theaxial collagen period) and equatorial (from the fibriltransform) scattering of the collagen fibrils contributeto the peaks of the first subsidiary maximum. Thisis because the main direction of the collagen fibrilorientation found in the normal cornea are orthogo-nal; hence, the meridional and equatorial contribu-tions superimpose at the same four positions. The fourorthogonal peaks in Figure 2 are located on top ofan intensity ring that resulted from collagen fibrilsoriented in any direction. Although the preferred col-lagen fibril orientation in the vertical and horizontaldirections is evident from the two-dimensional scatter-ing pattern of normal human cornea (Fig. 2A), fur-ther analysis is required to obtain quantitative infor-mation regarding the amount of preferentially ori-ented collagen fibrils.

To quantify the degree of collagen fibril orienta-tion (that is, the relation between the amount of pref-erentially oriented fibrils and the total amount of col-lagen fibrils), the two-dimensional scattering patterns(Fig. 2A) were integrated, as described above, yieldingcurves such as those in Figure 3 for each set of data.The ratio denned as a = (I] — I2)/Ii [Ii and I2 indicat-ing the areas of the shaded regions in Figs. 3A and 3B,respectively] represents the contribution of collagen(oriented as well as disordered) to the total scatteringbetween 9 = 0.35° to 0.60°. The total scattering I,includes scattering from oriented and disordered col-lagen, as well as from other matrix proteins. A secondratio that may be defined is /? = (I3_i4)/I3, whichdescribes the amount of scattering from oriented colla-gen fibrils alone, relative to the total scattering be-tween 9 = 0.35° to 0.60° (Is and I4 indicating theshaded areas in Fig. 3C and 3D, respectively). Al-though ft can be thought as a lower bound for thedegree of orientation of collagen fibrils, the "true"degree of orientation is defined as y = P/cx. = (1 —I4/I3)/(l — I2/Ii). The ratio y measures the amountof oriented collagen, relative to the total amount ofcollagen present in the corneal stroma. It is zero if allcollagen is randomly oriented within the plane of thecornea and y = 1 if all collagen is oriented superiorto inferior and nasal to temporal.

Figure 6 and Table 1 show the degree of orienta-tion y for each cornea at each investigated position.y varies widely and ranges from y = 0.29 to y = 0.75.In particular, we determined the following values forthe degree of orientation y at the different topo-graphic positions:

y (central) = 0.47 ± 0.09 (mean ± SD, n = 15);y (nasal) = 0.52 ± 0.11 (n = 14);y (inferior) = 0.51 ± 0.11 (n = 13);y (temporal) = 0.50 ± 0.08 (n = 11);

Collagen Fibril Orientation in the Cornea 125

100

FIGURE 3. (A,B) Sphericallyintegrated intensity I (9).The maximum at 0.41° corre-sponds to the first subsidiarymaximum. The shaded areaIi represents the total inte-grated intensity. The shadedarea I2 represents the inte-grated intensity correspond-ing to the matrix elementsother than collagen. (C,D)Integrated intensity of the ^first subsidiary maximum ob- 3tained by integration from 6 3= 0.35° to 9 = 0.60°. As in i! ^in A, the shaded area I3 rep- —resents the total integratedintensity. The shaded area LI4 represents the integratedintensity caused by randomlyoriented collagen fibrils, to-gether with noncollagenousmatrix elements.

0.2 0.4 0.6 0.8 1.0scattering angle 6 (degree)

0.2 0.4 0.6 0.8 1.0scattering angle 9 (degree)

200

150 -

100

200

3CO

0 90 180 270 360rotation angle <j> (degree)

270 360rotation angle <\> (degree)

y (superior) = 0.45 ± 0.11 (n = 14);y (overall) = 0.49 ± 0.10 (TO = 67).The intraindividual variation of the degree of ori-

entation y (that is, the values of y at different topo-graphic positions within a central 7 mm zone of thesame cornea) was not statistically significant.

Figure 7 shows y averaged for each cornea overthe investigated positions. The bars in Figure 7 indi-cate the standard deviations of the measurements. Al-though few corneas show considerable variation of ybetween the different positions, it is apparent fromFigure 7 and Table 1 that a low or high degree ofcollagen fibril orientation is primarily a property ofthe entire cornea and does not much depend on thelocation in the cornea.

The typical average value of y « 0.5 indicates thatdiere is an excess of fibrils of approximately 50% or-dered vertically and horizontally over a background offibrils, pointing with equal probability in any direction.Considering the sinusoidal variation of the intensity(Fig. 3D) and, therefore, of the number of fibrils, theprobability that a fibril points in the direction <£ willbe approximately p($) = [1 + y cos(4 $)]/2TT, if $is expressed in radian (0 < (f> < 2TT). Consequently,

the total proportion of fibrils pointing away from dievertical and the horizontal by more than n/8 radians

(= 22.5°) will be 4 / ^ j | p ( $ ) d $ = 1/2 - y/ix. With7T/8

die experimentally determined value for y = 0.49, diismeans that in normal human corneas, only approxi-mately one third (34%) of the collagen fibrils pointaway from die vertical or the horizontal by more dian22.5°. In odier words, approximately two thirds (66%)of the fibrils are oriented in a 45° sector around thevertical and horizontal meridians, whereas only onethird (34%) are oriented in the oblique sectors in be-tween.

Keratoconus

In contrast to normal corneas, the situation in corneasobtained from keratoconic eyes appears more com-plex. Figure 2B shows an example of a two-dimen-sional x-ray scattering pattern from the scarred regionin keratoconus.

The orthogonality of the collagen-related peaks,which is a characteristic quality of normal human cor-neas (see Figs. 2A and 3) has changed to an irregularscattering pattern in the center of the scarred region

126 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1

100

3

c2

10

"common tangent construction"o

contact points

0.10 1.00scattering angle 6 (degree)

FIGURE 4. (circles) 1(6) in a double logarithmic plot, (solidline) This shows how a background under the peak at 0.41°was interpolated. It is defined by the two contact points onboth sides of the maximum. The slopes of 1(0) and of thebackground are the same at these two contact points, andafter background subtraction, they correspond to minimaof 1(9). In this double logarithmic plot, the line representsa power law behavior of the background. Typically, the back-ground as determined also describes reasonably well thebehavior of 1(6) at higher scattering angles 9.

(Fig. 2B). In particular, the four distinct maxima ofthe inner reflections (interference peaks) (brown inFig. 2A) appear confluent at their periphery becauseof the relative rotation in Figure 2B. In the apicalscar, the interference peaks (brown in Fig. 2B) showpreferred angles of approximately 120°/60°, and theyappear unequally intense. In keratoconus, the orienta-tional behavior of the collagen fibrils is best recog-nized qualitatively by the interference peaks (mainmaximum (brown) in Fig. 2B), whereas it cannot beobtained easily from the first subsidiary maximum(green in Fig. 2B). Because of the deviation from ar-rangement in two orthogonal directions, the equato-rial contribution (caused by the fibril transform) andthe meridional contribution (caused by the axial colla-gen period) had to be considered separately. This iswhy a quantitative analysis of the degree of orientationwas not attempted for keratoconus.

DISCUSSIONAll investigated normal human corneas showed colla-gen-related intensity profiles characterized by orthog-onal fourfold symmetry (Figs. 2A and 3), indicatingthe preferred orientation of the collagen fibrils alongthe horizontal (nasal-temporal) and the vertical (in-

ferior-superior) directions. This behavior was foundnot only in the center of the cornea but also at everyother investigated position (nasal, inferior, temporal,superior) at the margin of a central 7 mm zone. Inthis context, it is noteworthy that the two preferredorientations correspond to the directions of the inser-tion of the four musculi recti oculi, present in mostanimals.20 The preferentially orientated fibrils in thecornea appear as biomechanical elements of a com-plex optokinetic system that is able to take up themechanical forces of the musculi recti along the cor-neal trajectories. Small-angle light-scattering experi-ments performed on rabbit and bovine corneas alsoshowed such preferred orientation,13 whereas x-rayscattering experiments in different species only re-vealed a preferred collagen fibril orientation in thehuman cornea.8

To decide whether the preferred orientation ofthe collagen fibrils may have some impact for biome-chanical consideration, in particular for the develop-ment of models of the corneal tissue for refractivesurgery, it is a prerequisite to determine quantitativelythe amount of fibrils that contribute to the anisotropy.Indeed, current models for the simulation of refrac-tive surgery procedures are based on complex assump-tions of the corneal ultrastructureeg'14"16 because de-tailed information about the orientational arrange-

scattering angle 6 (degree)FIGURE 5. 1(6) after subtraction of the background (circles).The first subsidiary maximum between 0.35° and 0.6° isclearly visible. The solid curve corresponds to the fibril trans-form and is proportional to (Ji(ka)/ka)2 (JA is the Besselfunction of the first kind). The arrows labeled 3 and 5 corre-spond to the position of the sharp third- and fifth-orderpeaks of die axial scattering of the collagen fibrils (meridio-nal reflections). Some contribution within the subsidiarymaxima caused by this axial fibril scattering is visible.

Collagen Fibril Orientation in the Cornea 127

0.0

•2JoQ.

O>

O>Q.

FIGURE 6. Degree of preferred orientation y for each normalcornea at each of the investigated positions within a central7 mm zone. The bars show the mean value of each y-distri-bution.

ment of the structural elements in the corneal stromais not available.

Our results show a considerable degree of pre-ferred orientation of 7(mean) = 0.49, which meansthat the excess of collagen fibrils that represent thepreferred orientated portion is 49%. Another valuethat characterizes the degree of structural anisotropyin the human cornea is the relation between the num-ber of collagen fibrils oriented in the 45° sectors

around the horizontal and vertical meridians to thenumber of collagen fibrils oriented in the obliquesectors between them, which was calculated for 7 =0.49 to be approximately 2:1. In other words, approxi-mately two thirds (66%) of the fibrils are within thehorizontal and vertical sectors, whereas only one third(34%) is in the oblique sectors between. The relatedformula for the probability to find collagen fibrils ori-ented in a particular direction (see Results) directlypermits the implication of the structural data into bio-mechanical models for the simulation of refractivesurgery procedures.

To avoid fractional forces on the specimen causedby mounting, and in accordance with earlier experi-ments,4'81721 our measurements were conducted with-out the application of transcorneal pressure. It hasbeen reported, however, that under conditions inwhich corneal tension is released, lamellar shape maybecome wavy.22 The amplitudes of the waves wereshown to be directed perpendicular to the cornealsurface, and the periodicity was approximately 10/zm.22 Because of this geometry, the effect of wavinesson the scattering patterns, which were collected withthe x-ray beam perpendicular to the corneal surface,were small. One would expect some smearing of theintensity profile l((f>) and, therefore, a little underesti-mation of 7.

For unknown reasons, two of the 17 investigatednormal corneas (corneas 6 and 9) had a relatively loworientational anisotropy (Fig. 7). Although there wasno detectable lesion, the presence of a corneal orsystemic disease that may have altered the structureof the corneal stroma cannot be excluded. Alterationof the normal development of the collagen fibril ori-entation pattern can be induced by the application of

TABLE 1. Degree of Preferred Collagen Fibril Orientation

CorneaCoding

123456789

1011121314151617

Central

0.530.520.480.410.460.310.520.47—

0.550.480.390.540.460.64—

0.33

Nasal

0.560.630.750.450.570.320.530.42—

0.45—

0.460.590.540.58—

0.40

7

Inferior

0.610.66—

0.45—

0.570.59—

0.540.510.490.580.300.610.410.37

Temporal

0.510.570.480.450.36——

0.58—

0.620.550.49—

0.42—

0.43—

Superior

0.450.640.44—

0.630.35—

0.400.290.48—

0.440.320.440.630.410.43

128 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1

1.0

0.9

0.8cosb

O)

0.7

0.60.5

0.4

0.3

0.2

0.1

0.010

cornea15

FIGURE 7. Degree of orientation y averaged over the investi-gated positions for each of the 17 normal corneas. Barsindicate the standard deviation computed for each cornea.The horizontal line at y — 0.49 is a guide for the eye andindicates the mean value for the degree of orientation.

specific drugs, as shown in domestic fowls.6'7 Despitesuch experiments, almost no information is availableconcerning the possible connection between the ori-entational behavior of collagen fibrils and human cor-neal disease.

With this in mind, we also investigated four cor-neas affected by keratoconus by comparing the orien-tation of the collagen fibrils inside and outside thescarred region. The striking result was that the orienta-tions of the fibrils inside the lesion were altered dra-matically in each of the four specimens, whereas out-side the diseased region, the orientational arrange-ment of the fibrils corresponded to that obtained innormal corneas (i.e., orthogonal arrangement). Inparticular, the angles between the main fibril direc-tions changed to typically 60° and 120° in the apicalscar of the keratoconus, and the intensity correspond-ing to one of the two fibril directions was reducedconsiderably (see the inner reflections coded in brown[main maximum] in Fig. 2B), indicating an unequalnumber of collagen fibrils along the main orienta-tions. In other words, the regular orthogonal arrange-ment of the collagen fibrils, as observed in the normalcorneas and in keratoconus outside the lesion, is de-stroyed within the apical scar of the keratoconus.

Keratoconus is a noninflammatory corneal diseasecharacterized by progressive thinning of the centralcornea and accompanied by increased corneal curva-ture and apical scarring.23'24 Increased proteolytic ac-tivity is probably one of the major events in keratoco-

nus.25 Proteolytic degradation of collagen fibrils, oreven entire lamellae, may be a possible explanationfor the abnormal collagen fibril layer arrangement inthe apical scar. Some contribution may come fromreactive fibrosis in the focal scars within the depth ofthe stroma.23 It has been suggested that the adhesive-ness of the corneal lamellae may be less than normalin keratoconus.23 Consequently, one may speculatethat the increased degradation process of the extracel-lular matrix preferentially starts from the lamellar bor-ders. The fact that the orientational behavior of thecollagen fibril layers in keratoconus is dramaticallyaltered, whereas changes in other structural parame-ters of the fibrils are much more subtle,21 also seemsto indicate that ultrastructural disease primarily occursin interlayer bonding rather than inside the layers.

Key Words

collagen, corneal biomechanics, corneal stroma, keratoco-nus, refractive surgery

Acknowledgments

The authors thank Professors Mikuz and Gottinger for theirsupport in obtaining the corneas and Klaus Misof for helpfuldiscussions.

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Collagen Fibril Orientation in the Cornea 129

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