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Biosensors and Bioelectronics 18 (2003) 381 Á/387 www.elsevier.com/locate/bios A study of piezoelectric and mechanical anisotropies of the human cornea A. Champa Jayasuriya a,*, Snehasish Ghosh a, Jerry I. Scheinbeim a, Virginia Lubkin b, Greg Bennett b, Phillip Kramer b a Department of Chemical and Biochemical Engineering, Polymer Electroprocessing Laboratory, College of Engineering, Rutgers-The State University of New Jersey, 98 Brett Road, Piscataway, NJ 08854-8058, USA b Aborn Laboratory, New York Eye and Ear Inﬁrmary, 310 East 14th Street, New York, NY 10003, USA Received 19 July 2001; accepted 23 July 2002 Abstract The piezoelectric and dynamic mechanical properties of human cornea have been investigated as a function of drying time. As expected, the piezoelectric coefficient, d31, and the Young’s modulus, Y , were found to be extremely sensitive to water content. d31 decreased with dehydration of the corneal tissue and Y increased with dehydration. While these results are significant, the discovery of the unprecedented mechanical and electromechanical anisotropy exhibited by the cornea are the major findings of this study and indicate that the collagen fibrils comprising the cornea are highly oriented. The piezoelectric responses of corneas observed in this study are: diagonally cut samples starting at an average piezoelectric coefficient value of 2250 pC/N, followed by the vertically cut samples, with an average starting value of about 600 pC/N and finally the horizontally cut samples with an average starting value of about 200 pC/N. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cornea; Collagen; Piezoelectricity; Young’s modulus 1. Introduction procedures as well as the pathogenesis of certain corneal disease states. A better understanding of the biomechanical proper- Both corneal and scleral tissue share many simila- ties of the human cornea is certainly warranted based on rities. Both are primarily collagenous hydrogels consist- the recent explosion in the numbers of refractive surgical ing of collagen fibrils; however, the scleral tissue is procedures performed. Various procedures such as opaque while the cornea is transparent to visible light radial keratotomy, photorefractive keratectomy, laser (Maurice, 1957). It was suggested that both the size and in situ keratomileusis, and astigmatic keratotomy, rely some ordered distance between fibrils allowed for the on the ‘average’ corneal structure to develop ‘normo- forward transmission or scattering of light. Others grams’ which guide surgical planning. Disease states, suggest that corneal transparency is a function of the such as keratoconus, result from surgically induced or refractive indices of the collagen fibrils and interfibril congenital mechanisms, which cause unwanted corneal matricies (Smith, 1969). It is also believed that collagen steeping and irregular astigmatism. Understanding cor- fibrils in scleral tissue are assumed to be of random size neal elasticity and the corneal response to forces which and orientation and, therefore, cannot transmit light deform it (intraocular pressure, variable atmospheric (Komai and Ushiki, 1991). While it is easy to accept the pressure, extraocular pressure from lid disease, etc.), reasoning behind the inability of scleral tissue to may offer insight to more predictable refractive surgical transmit light, it is less obvious that a full understanding of cornea transparency exists. To contribute to the understanding of corneal tissue and differences between corneal and scleral tissue, we began an investigation into * Corresponding author. Tel.: '/1-732-445-3660 the piezoelectric and mechanical properties of the 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 1 4 4 - 6 382 A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 cornea in hopes of observing any significant differences from them. Five human corneas were sectioned for between the behavior of cornea and sclera. We also this purpose. hoped to provide additional insights into the general 2) For the second set, the roughly circular corneas structure/properties relationships of the cornea itself to were marked as the hour hand on the face of the allow for further understanding of its transparent clock. The positions for 12 and 3 o’clock were optical properties and its mechanical stiffness. determined by the location of the eye muscles and Piezoelectricity in organic polymers (Fukada, 1974) marked with sutures at 12 and 3 o’clock. Rectan- and in certain biological tissues (Shamos and Lavine, gular strips were then sectioned and labeled by 1967), such as collagen (Fukada and Yasuda, 1964; appointing 12 Á/6 o’clock sections as vertical; 3Á/9 Fukada et al., 1976; Christiansen and Silver, 1992; Pins o’clock sections as horizontal and diagonals as and Silver, 1995) and bone (Fukada and Yasuda, 1957; either 01:30 Á/07:30 or 10:30 Á/05:30 (see Fig. 1). Netto and Zimmerman, 1975) has been extensively investigated. Piezoelectric phenomena have been studied extensively in bone. Mechanical energy can induce electrical potentials of significant magnitude to theore- tically exert a wide range of clinical effects. These include potentially, control of cell mutation, enzyme activation or suppression, and orientation of intra-and- extra cellular macromolecules (Andrew, 1968). Although some work has been done in determining mechanical properties of collagen in general, no studies have been done on the mechanical or electromechanical properties of the cornea. However, a significant body of literature does exist on both structural and microstruc- tural studies of the cornea. Wide-angle X-ray diffraction studies have been performed on collagen fibrils from connective tissues to determine the orientation distribu- tion function and preferred orientation of collagen (Aspden and Hukins, 1979) as well as to measure the angular distribution within the tissue (Aspden and Hukins, 1981). It was reported anisotropies in values of the piezo- electric d-coefficient of human scleral collagen (Ghosh et al., 1998) which depend on both the specific region of the eye from which the sample is taken and the direction the mechanical force is applied. In the current study we present the discovery of the unprecedented piezoelectric response exhibited by human cornea compared to sclera. In addition, the data show that the piezoelectric response differs significantly depending on the orienta- tion of the corneal samples tested. 2. Materials and methods 2.1. Corneal samples Human corneal samples were sectioned from eyes obtained from the Eye-Bank for Sight Restoration, Inc., New York. The results presented here are from the following two sets of samples. 1) In the first set studied, corneal samples were sectioned into rectangular strips without any knowl- edge of the orientation of the strips to find out the Fig. 1. Human cornea showing three different sections: vertical, magnitude of the piezoelectric response obtained horizontal, and diagonal. A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 383 Fourteen corneas were used for this part of the study, seven were sectioned diagonally, four hor- izontally and three vertically. 2.2. Measurement of the piezoelectric-coefﬁcient and Young’s modulus The piezoelectric d-coefficient of a material is defined as the incremental change in electric polarization that occurs as a result of an incremental change in mechan- ical stress, s . Stress is defined as the force per unit area applied to the material. For a linear elastic material, this stress produces an elastic strain o , given by the relation s0/Eo , where E , the elastic modulus, is also called the Young’s modulus. Silver print conductive electrodes (GC Electronics, Fig. 2. Piezoelectric d -coefﬁcient versus time for ﬁrst set of ﬁve human Rockford, IL) were painted on opposite surfaces of the corneas. corneal strip and allowed to air dry. To compensate for any dehydration that might occur in the corneal strip and falls somewhat asymptotically to roughly the same during the painting and drying of the electrodes, these value after about 40 min. Interestingly enough, there strips were rehydrated in saline for a 30-min period of was a surprisingly large amount of scatter from sample time. The rehydrated samples were then placed between to sample. Cornea No. 3 had the highest starting value the grips of the measuring device in the sample chamber. of d :/1600 pC/N (picoCoulombs per Newton); cornea Care was taken not to let the samples dry out No. 2 around 1200 pC/N; cornea No. 1 about 700 pC/N; significantly because experience indicates that unlike corneas No. 4 and 5 being the two lowest with a starting scleral collagen (Pratzl and Daxer, 1993), corneal value of about 300 pC/N. All of the samples decrease to collagen does not rehydrate if allowed to dry out a similar d -coefficient value of about 50 pC/N after 40 significantly. min. An interesting feature of the drying curve is that The system used to measure the piezoelectric coeffi- from an initial high value, the curves fall steeply at first cient and Young’s modulus was a Rheolograph Solid† , and then level off to a plateau before finally making a which was manufactured by Toyo Seiki Seisaku-Sho second asymptotic decline to the final (dry) value of 50 Ltd., Japan. The piezoelectric response of the cornea, pC/N. Whether this represents the suggested two-stage both its magnitude and anisotropy are easily measured drying process of the cornea remains to be determined in a reproducible manner as this instrument was (Roveri et al., 1979). Not only is there enormous scatter designed to measure much smaller piezoelectric activity. from sample to sample in piezoelectric d-coefficient A dynamic stress, s0/5 N peak to peak, was applied to apparent from Fig. 2, but the drying curve for cornea each strip at a frequency of 104 Hz (default value) and the piezoelectric d -coefficient as well the elastic modulus No. 3 is much steeper compared to that of the other were continuously monitored as the sample dried out corneas. Also, even at the end of 40 min, the samples with time. still show a response that is higher than the piezoelectric response obtained from synthetic piezoelectric polymers (Winsor et al., 1996; Furukawa, 1989; Mei et al., 1993). 3. Results and discussion Further, measurements of the elastic modulus showed a similarly wide scatter of values. The scattered values in 3.1. Human cornea piezoelectric response and Young’s modulus led to the suspicion that the corneas may, in fact, not be direc- Since this study began with the assumption that tionally isotropic. human cornea was mechanically isotropic in nature, This prompted a second investigation of the human we proceeded to measure the piezoelectric d -coefficient corneas. In the second set of corneas studied, each of the and Young’s modulus without paying any particular corneas was directionally specified (see Section 2) in Fig. attention to the direction in which corneal samples were 1. Fig. 3a represents the piezoelectric d-coefficient as a cut from the cornea. function of drying time for four human corneas cut Fig. 2 shows the piezoelectric d -coefficient as a horizontally. Even for similar horizontal sections there function of drying time obtained from five human is a fair amount of scatter from cornea to cornea. corneas. The qualitative behavior is similar for all Cornea No. 8 starts at the lowest value of :/100 pC/N; samples studied. The response starts at a high value cornea No. 7 starts at :/200 pC/N, while corneas No. 6 384 A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 Fig. 3. (a) Piezoelectric d -coefﬁcient versus time for horizontally cut corneal sections. (b) Piezoelectric d -coefﬁcient versus time for vertically cut corneal sections. (c) Piezoelectric d -coefﬁcient versus time for diagonally cut corneal samples. (d) Piezoelectric d -coefﬁcient versus time for horizontal, vertical and diagonal corneal sections averaged over seven human corneas. and 9 start at :/275 pC/N. The difference between large amount of scatter in the initial values of the d- corneas No. 6 and 8 is almost 300%. The drying coefficients. Corneas No. 14, 15 and 16 all start at values behavior is also different. Corneas No. 7, 8 and 9 all around 1400 pC/N. Corneas 17 and 18 starts at around dry out to the same final piezoelectric d -coefficient value 2000 pC/N while corneas No. 13 and 19 have initial of around 30 pC/N. Cornea No. 6 follows a very values between 3500 and 4000 pC/N. All of the different drying pattern and is still decreasing with a diagonally cut corneas show similar drying behavior value of :/125 pC/N at the end of the drying time. and asymptote to around 100 pC/N except for cornea Fig. 3b shows the drying behavior for the three No. 19 which is still decreasing with a value of 1000 pC/ corneal samples sectioned vertically. Their initial piezo- N at the end of the measurement time. electric d -coefficient starting values are similar and lie Fig. 3d shows the averaged piezoelectric d-coefficient between 600 and 800 pC/N. Although all three samples as a function of drying time for the differently oriented decrease over time to around 40 pC/N, their drying sections together. The difference in their respective pattern is very different as is evident from Fig. 3b. response is evident: the diagonally cut samples starting Cornea No. 12 appears to show clear evidence of a two at an average piezoelectric coefficient value of 2250 pC/ stage drying process. Fig. 3c represents the piezoelectric N, followed by the vertically cut samples, with an behavior with drying time for the seven corneal samples average starting value of about 600 pC/N and finally sectioned diagonally. All of the starting values of the horizontally cut samples with an average starting piezoelectric d -coefficient obtained from these diagonal value of about 200 pC/N. Not only are the d-coefficient sections are astonishingly high. Once again there is a values of the diagonal sections considerable, but they A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 385 are an order of magnitude greater than the response time for the horizontally cut samples. Corneas No. 6 from the horizontally cut sections and more than three and 9 which exhibit the highest initial piezoelectric d- times that of the vertically cut sections. These represent coefficient values (see Fig. 3a) have the lowest initial significant electromechanical anisotropies in the cornea. value of Young’s modulus: about 2 MPa. Cornea No. 7 In addition, if one considers that the highest piezo- has a 4 MPa Young’s modulus as the starting value and electric response known for synthetic polymers e.g. cornea No. 8 has the highest initial value of Young’s P(VDF-TrFE) copolymers (Furukawa, 1989) at room modulus at 5 MPa. The nature of the drying curves is, temperature and nylon (Mei et al., 1993) at higher however, very different. Corneas No. 6 and 9 show a temperatures are on the order of 30Á/50 pC/N, one can linear slowly increasing trend. However, the curve for truly appreciate the magnitude of this response. cornea No. 8 increases almost linearly from 5 to 6 MPa The nature of the drying curves of the diagonally cut for about 35 min and then starts rising sharply to 8 sections is also different from both the vertically cut and MPa. Cornea No. 7 shows a sharper climb in general horizontally cut sections. The diagonally cut samples from 4 to 7 MPa for 30 min and an even steeper climb to dry out to a final average d-coefficient value of :/275 almost 12 MPa by 40 min. pC while the other two sections dry out an average value Fig. 4b shows the drying behavior of the Young’s of :/100 pC/N, although at sufficiently long drying modulus for the vertically cut samples. All three samples times, all three sets of values may converge. Fig. 4a start at an initial value of :/1.3 MPa. Corneas No. 10 presents the Young’s modulus as a function of drying and 11 show a very similar linear increase to about 2.5 Fig. 4. (a) Young’s modulus versus time for horizontally cut corneal sections. (b) Young’s modulus versus time for vertically cut corneal sections. (c) Young’s modulus versus time for diagonally cut corneal sections. (d) Young’s modulus versus time for horizontal, vertical and diagonally cut corneal sections averaged over seven human corneas. 386 A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 MPa for the first 30 min and continue to gradually when subjected to a mechanical stress, we anticipated increase. However, cornea No. 12, which had the lowest that for any particular corneal section, the lowest initial piezoelectric d -coefficient (see Fig. 3b), shows a piezoelectric response would be generated by the section much steeper initial rate of increase and then increases that exhibits the lowest response to mechanical stress, to a final value of :/7 MPa after 40 min. i.e. the one with the highest Young’s modulus. In other Fig. 4c represents Young’s modulus as a function of words, the direction in which the cornea is stiffest will drying time for the diagonally cut corneal samples. generate the lowest piezoelectric response. There is a large amount of scatter in the initial and final Also human corneas exhibit significant mechanical values of Young’s modulus for all the samples. Cornea anisotropy with a starting values of Young’s modulus No. 13 has the lowest initial value starting at 0.05 MPa for horizontally cut samples with 3 MPa, a vertically cut while cornea No. 14 has the highest initial value of 0.75 samples with 1 MPa and a diagonally cut samples with MPa. The final values are between 0.6 MPa for cornea 0.3 MPa. This difference in the value of Young’s No. 17 and 1.5 MPa for cornea No. 15. The nature of Modulus suggests a higher degree of fiber orientation the drying curves is qualitatively similar for most along specific directions of the cornea. One may samples. Only cornea No. 13 shows a very rapid and speculate that the highest modulus direction has the steep increase in Young’s modulus with time. greatest number of collagen fibrils oriented in that Fig. 4d shows the averaged Young’s modulus with direction while the lowest modulus direction has the drying time for the three differently oriented sections. least number of collagen fibrils oriented in that direc- The response shown in Fig. 4d is the reverse of the tion. averaged response of the piezoelectric d -coefficients shown in Fig. 3d. The average Young’s modulus of the horizontally cut samples, with the smallest piezo- References electric response, has the highest initial value of :/3 MPa and increases to around 6 MPa. These values are Andrew, C.B.L., 1968. Biologic signiﬁcance of piezoelectricity. Calc. more than three times the values obtained from the Tiss. Res. 1, 252 Á/272. vertically cut sections, which initially exhibit a value of 1 Aspden, R.M., Hukins, D.W.L., 1979. 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Effect of water on piezo- an average starting value of about 600 pC/N and finally electricity in bone and collagen. Biophys. J. 15, 573 Á/576. Pins, G.D., Silver, F.H., 1995. A self-assembled collagen scaffold the horizontally cut samples with an average starting suitable for use in soft and hard tissue replacement. Mater. Sci. value of about 200 pC/N. Since the piezoelectric Eng. C3, 101 Á/107. response of a polymer is usually generated by the Pratzl, P., Daxer, A., 1993. Structural transformation of collagen reorientation of the electric dipoles within the polymer ﬁbrils in corneal stroma during drying. Biophys. J. 64, 1210 Á/1214. A.C. Jayasuriya et al. / Biosensors and Bioelectronics 18 (2003) 381 Á/387 387 Roveri, N., Ripamonti, A., Bigi, A., Volpin, D., Gino, M.G., 1979. X- Smith, J.W., 1969. The transparency of the corneal stroma. Vis. Res. 9, ray diffraction study of bovine lens capsule collagen. Biochemica et 393 Á/396. Biophysica Acta 576, 404 Á/408. 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