Current Eye V(dimic 1 L nurnhcr 10 1992,
Research
98;-986 ~~
Concentration and molecular weight dependency of rabbit corneal epithelial wound healing on hyaluronan 'Mawtsugu Nakamura, Mitsushi Hikida and Tsutornu Nakano
Central Research Laboratories, Santen Pharmaceutical Co. Ltd., 9-19, Shirnoshinjo, 3-chome, Higashryodogawa-ku, Osaka 533, Japan
ABSTRACT Hyaluronan (hyaluronic acid) is a hi h molecular weight viscoelastic polymer which has een postulated to enhance wound healing. We investigated the dose and molecular weight (9 x 1O4 - 280 x 10 ) dependent effects aluronan on the rate of migration of rabbit corneal elium in organ culturo and on wound closure in vivo debridement with n-heptanol. When corneal blocks were cultured with hyaluronan for 20 hours, distances of epithelial migration significantly increased over exposed stroma in proportion to hyaluronan concentration. However, there was no difference in the stimulatory action of hyaluronan on epithelial migration when corneal blocks were cultured at 1 m g h l of hyaluronan irrespective of chan es in the molecular weight range between 9 x 1O4 an 280 x 104. Glycosaminoglycans other than hyaluronan (chondroitin, chondroitin sulfate, keratan sulfate and heparan sulfate) failed to increase the epithelial migration. When hyaluronan eye drops were instilled after corneal epithelial removal with nheptanol, hyaluronan stimulated wound closure in a dose-dependent manner, but its stimulatory efficacy was not dependent on molecular weight.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
i
i
INTRODUCTION Hyaluronan has been used to protect corneal epithelial cells, especially in dry eye patients (1-6).Its protective effects are thought to be related to its spongelike structure of polysaccharidechains containing entrapped water and non-Newtonianfluid behavior similar to that of tear mucous glycoprotein (1,6-8). Recently, hyaluronan has been shown to stimulate corneal epithelial migration in vitro (9,10), and healing of a corneal epithetial defect in vivo (11). Therefore, it was suggested that hyaluronan acts on corneal epithelial cells as well as other cell types (12,13), and stimulates corneal wound healing by its interaction with specific binding sites on the cell surface. However, it is not known if these effects of hyaluronan are either dose or molecular weight dependent. In this paper, we examined the effects of dose and
molecular weight of hyaluronan on rates of rabbiit corneal epithelial wound healing. This was done by measuring its effects on epithelial migration in an organ culture and on wound closure after corneal epithelial denudation with n-heptanol in vivo. MATERIALS AND METHODS
Hvaluronan Hyaluronans prepared from rooster comb were used and dissolved either in TC-199 (in vifro)(the Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka) or 25 mM phosphate buffer saline (pH 7.4, PBS)(in vivo). These samples were sterilized and free from pyrogen and protein ( 4 0pghnl).
Animal Albino rabbits (Japanese White) weighting 2 to 3 kg were obtained from Kitayama Labes Co. Ltd. (Kyoto, Japan). Animal experimentation was performed in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23).
Cornealeoithellalmiarationinvitca Corneal epithelial migration was measured according to Nishida et at. (14).Briefly, each corneal block (2x 4 mm) was placed in one well of a 24-well tissue culture plate (#3424,Costar, Cambridge, MA, USA.), and incubated for 20 hours at 37'C under humidified 5% CO, and 95% air. To the culture medium (TC-199), different molecular weights or doses of hyaluronan were added (0.1,0.3, 0.5,1 mg/ml, molecular weight: 80 x 104). The various molecular weights included: (9 x lo4, 26 x 104, 68 x lo4, 80 x lo4, 84 x lo4, 206 x lo4 or 280 x lo4). To investigate whether or not the stimulatory effect of hyaluronan is unique among various other glycosaminoglycans, the stimulatory effect of hyaluronan
Receivcd o n Fchiitary 19, 1992, accepted on September 14, 1992
Oxford klnirervty Press
98 1
Current Eye Research was compared with those of 1 mg/ml of chondroitin from whale cartilage, chondrotin sulfate from whale cartilage, keratan sulfate from bovine cornea or heparan sulfate from bovine kidney. At the end of cultivation, specimens were fixed, embedded and stained with hematoxylinsosin. The distances of corneal epithelial migration, extending down the side of a cultured block, were measured with a computer assisted digitizer under a light microscope.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
Cornealedthelialwoundclasurei0The animals were anesthetized by intravenous injection of pentobarbital and by topical application of oxybuprocaine eye drops. The corneal epithelium was denuded with n-heptanol according to Cintron et al.(15). In brief, a filter paper (6 mm in diameter) immersed with n-heptanol was placed on the top of each cornea for 1 minute and then degenerated corneal epithelial cells were washed away with saline. In this model, the initial wound area was 34.9 f 1.5 mm2. Complete coverage of surface occurred in 56.4 i 1.5 hours, and the rate of
Figure 1. Micr raph of corneal epithelium migrating over the cut su ace of the stroma cultured in unsupplemented TC-199 medium (A) and in TC-199
7
982
healing was 0.71 f 0.09 mm2/hourfor the no-treatment group. These data were in a good agreement with Cintron et al.(l5). In the first experiment, the animals were divided into 3 groups (4 eyes/group). Each of the groups topically received either PBS, hyaiuronan solution (1 mg/ml, molecular weight: 80 x lo4), or no-treatment. One drop of either PBS or hyaluronan was administered immediately after corneal denudation and again at 2,4, 6,24,26,28 and 30 hours after denudation. In the second experiment, the animals were divided into 4 groups (6 eyes/group) and they topically received hyaluronan solution (molecular weight: 80 x lo4) at concentrations of 0 (control), 0.1,0.5 or 1 mg/ml at 0, 2, 4 and 6 hours after denudation. In the third experiment, the animals were divided into 4 groups (5 eyes/group) and they topically received various molecular weights (65 x lo4, 111 x lo4 or 280 x lo4) of hyaluronan (1 mg/ml). One drop of a test solution was applied 0 , 2 , 4 and 6 hours after denudation with n-heptanol. The areas of an epithelial defect were measured with a computer assisted
containin 1 mg/ml of hyaluronan (B) at 20 hours in culture. he bar represents 200 pm.
B
Current Eye Research digitizer after staining with 1% fluorescein and taking photographs.
Statlsticalanalvsls Statistical analysis was done by Dunnett test.
RESULTS Morphological changes seen in the migrating corneal epithelial layer after 20 hours’ cultivation are shown in Figure 1 When the corneal block was cultured in an unsupplemented medium, the epithelial layer extended down the cut side of the block,over the stroma, as a monolayer of cells (Figure 1 . A). With the addition of hyaluronan (1 mg/ml), the length of the path of corneal epithelial layer increased (Figure 1 . 6). We could not observe any stromal swelling following the addition of hyaluronan. Figure 2 shows the quantitative effect of hyaluronan on the length of the path of the corneal epithelial layer. The addition of hyaluronan significantly stimulated corneal epithelial migration in a dose-dependent fashion. Provided its concentratiorr was either 0.5 or 1 mg/ml. We also investigated tlie effect of various molecular weights of hyaluronan on corneal epithelial migration in vifro. As shown in Table 1, irrespective of the molecular weights of hyaluronan, all of them after 20 hours of
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
~
h
E
1°00
1
**
**
cultivation were equally effective in stimulating corneal epithelial migration. Of the different glycosaminoglycanstested, only hyaluronan increased the length of the path of the corneal epithelium (Table 2). Chondroitin, chondroitin sulfate and keratan sulfate did not affect the length of the path, but heparan sulfate instead decreased the length of the path of the corneal epithelium. Figure 3 shows the changes in wound area after denudation in vivo of the corneal epithelium. Eighteen hours after denudation, there were no differences among all of the test groups. However, after 24 hours, hyaluronan significantly stimulated corneal epithelial wound closure relative to both the untreated and vehicletreated groups. After 48 hours, the cornea treated with
Table 1 . Molecular weight dependency of hyaluronan on corneal epithelial migration. molecular weight of hyaluronan
epithelial migration
control 9 x lo4 26 x lo4 69 x lo4 80 x lo4 84 x lo4 206 x lo4 280 x lo4
463 f 17 570 f 27** 576k 5** 61 8 f 30** 593f 7** 631 i 3 6 * * 587 f 33** 588 2 21 ’*
(v)
mean f S.E. (n=6) **: pc0.01 vs control Cornea was cultured in the medium containing 1 mg/ml of hyaluronan for 20 hours.
Table 2. Effects of glycosaminoglycans on corneal epithelial migration.
Concentration of hyaluronan (mg/ml) Figure 2. Dose dependent effects of hyaluronan on stimulation of rabbit corneal epithelial migration. Cornea was cultured in medium containing (0 - 1 mg/ml, molecular weight: 80x1041 hyaluronan for 20 hours. Each hatched bar represents tl-e mean 1 S.E. of 14 determinations *‘:p
Research
98;-986 ~~
Concentration and molecular weight dependency of rabbit corneal epithelial wound healing on hyaluronan 'Mawtsugu Nakamura, Mitsushi Hikida and Tsutornu Nakano
Central Research Laboratories, Santen Pharmaceutical Co. Ltd., 9-19, Shirnoshinjo, 3-chome, Higashryodogawa-ku, Osaka 533, Japan
ABSTRACT Hyaluronan (hyaluronic acid) is a hi h molecular weight viscoelastic polymer which has een postulated to enhance wound healing. We investigated the dose and molecular weight (9 x 1O4 - 280 x 10 ) dependent effects aluronan on the rate of migration of rabbit corneal elium in organ culturo and on wound closure in vivo debridement with n-heptanol. When corneal blocks were cultured with hyaluronan for 20 hours, distances of epithelial migration significantly increased over exposed stroma in proportion to hyaluronan concentration. However, there was no difference in the stimulatory action of hyaluronan on epithelial migration when corneal blocks were cultured at 1 m g h l of hyaluronan irrespective of chan es in the molecular weight range between 9 x 1O4 an 280 x 104. Glycosaminoglycans other than hyaluronan (chondroitin, chondroitin sulfate, keratan sulfate and heparan sulfate) failed to increase the epithelial migration. When hyaluronan eye drops were instilled after corneal epithelial removal with nheptanol, hyaluronan stimulated wound closure in a dose-dependent manner, but its stimulatory efficacy was not dependent on molecular weight.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
i
i
INTRODUCTION Hyaluronan has been used to protect corneal epithelial cells, especially in dry eye patients (1-6).Its protective effects are thought to be related to its spongelike structure of polysaccharidechains containing entrapped water and non-Newtonianfluid behavior similar to that of tear mucous glycoprotein (1,6-8). Recently, hyaluronan has been shown to stimulate corneal epithelial migration in vitro (9,10), and healing of a corneal epithetial defect in vivo (11). Therefore, it was suggested that hyaluronan acts on corneal epithelial cells as well as other cell types (12,13), and stimulates corneal wound healing by its interaction with specific binding sites on the cell surface. However, it is not known if these effects of hyaluronan are either dose or molecular weight dependent. In this paper, we examined the effects of dose and
molecular weight of hyaluronan on rates of rabbiit corneal epithelial wound healing. This was done by measuring its effects on epithelial migration in an organ culture and on wound closure after corneal epithelial denudation with n-heptanol in vivo. MATERIALS AND METHODS
Hvaluronan Hyaluronans prepared from rooster comb were used and dissolved either in TC-199 (in vifro)(the Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka) or 25 mM phosphate buffer saline (pH 7.4, PBS)(in vivo). These samples were sterilized and free from pyrogen and protein ( 4 0pghnl).
Animal Albino rabbits (Japanese White) weighting 2 to 3 kg were obtained from Kitayama Labes Co. Ltd. (Kyoto, Japan). Animal experimentation was performed in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23).
Cornealeoithellalmiarationinvitca Corneal epithelial migration was measured according to Nishida et at. (14).Briefly, each corneal block (2x 4 mm) was placed in one well of a 24-well tissue culture plate (#3424,Costar, Cambridge, MA, USA.), and incubated for 20 hours at 37'C under humidified 5% CO, and 95% air. To the culture medium (TC-199), different molecular weights or doses of hyaluronan were added (0.1,0.3, 0.5,1 mg/ml, molecular weight: 80 x 104). The various molecular weights included: (9 x lo4, 26 x 104, 68 x lo4, 80 x lo4, 84 x lo4, 206 x lo4 or 280 x lo4). To investigate whether or not the stimulatory effect of hyaluronan is unique among various other glycosaminoglycans, the stimulatory effect of hyaluronan
Receivcd o n Fchiitary 19, 1992, accepted on September 14, 1992
Oxford klnirervty Press
98 1
Current Eye Research was compared with those of 1 mg/ml of chondroitin from whale cartilage, chondrotin sulfate from whale cartilage, keratan sulfate from bovine cornea or heparan sulfate from bovine kidney. At the end of cultivation, specimens were fixed, embedded and stained with hematoxylinsosin. The distances of corneal epithelial migration, extending down the side of a cultured block, were measured with a computer assisted digitizer under a light microscope.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
Cornealedthelialwoundclasurei0The animals were anesthetized by intravenous injection of pentobarbital and by topical application of oxybuprocaine eye drops. The corneal epithelium was denuded with n-heptanol according to Cintron et al.(15). In brief, a filter paper (6 mm in diameter) immersed with n-heptanol was placed on the top of each cornea for 1 minute and then degenerated corneal epithelial cells were washed away with saline. In this model, the initial wound area was 34.9 f 1.5 mm2. Complete coverage of surface occurred in 56.4 i 1.5 hours, and the rate of
Figure 1. Micr raph of corneal epithelium migrating over the cut su ace of the stroma cultured in unsupplemented TC-199 medium (A) and in TC-199
7
982
healing was 0.71 f 0.09 mm2/hourfor the no-treatment group. These data were in a good agreement with Cintron et al.(l5). In the first experiment, the animals were divided into 3 groups (4 eyes/group). Each of the groups topically received either PBS, hyaiuronan solution (1 mg/ml, molecular weight: 80 x lo4), or no-treatment. One drop of either PBS or hyaluronan was administered immediately after corneal denudation and again at 2,4, 6,24,26,28 and 30 hours after denudation. In the second experiment, the animals were divided into 4 groups (6 eyes/group) and they topically received hyaluronan solution (molecular weight: 80 x lo4) at concentrations of 0 (control), 0.1,0.5 or 1 mg/ml at 0, 2, 4 and 6 hours after denudation. In the third experiment, the animals were divided into 4 groups (5 eyes/group) and they topically received various molecular weights (65 x lo4, 111 x lo4 or 280 x lo4) of hyaluronan (1 mg/ml). One drop of a test solution was applied 0 , 2 , 4 and 6 hours after denudation with n-heptanol. The areas of an epithelial defect were measured with a computer assisted
containin 1 mg/ml of hyaluronan (B) at 20 hours in culture. he bar represents 200 pm.
B
Current Eye Research digitizer after staining with 1% fluorescein and taking photographs.
Statlsticalanalvsls Statistical analysis was done by Dunnett test.
RESULTS Morphological changes seen in the migrating corneal epithelial layer after 20 hours’ cultivation are shown in Figure 1 When the corneal block was cultured in an unsupplemented medium, the epithelial layer extended down the cut side of the block,over the stroma, as a monolayer of cells (Figure 1 . A). With the addition of hyaluronan (1 mg/ml), the length of the path of corneal epithelial layer increased (Figure 1 . 6). We could not observe any stromal swelling following the addition of hyaluronan. Figure 2 shows the quantitative effect of hyaluronan on the length of the path of the corneal epithelial layer. The addition of hyaluronan significantly stimulated corneal epithelial migration in a dose-dependent fashion. Provided its concentratiorr was either 0.5 or 1 mg/ml. We also investigated tlie effect of various molecular weights of hyaluronan on corneal epithelial migration in vifro. As shown in Table 1, irrespective of the molecular weights of hyaluronan, all of them after 20 hours of
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
~
h
E
1°00
1
**
**
cultivation were equally effective in stimulating corneal epithelial migration. Of the different glycosaminoglycanstested, only hyaluronan increased the length of the path of the corneal epithelium (Table 2). Chondroitin, chondroitin sulfate and keratan sulfate did not affect the length of the path, but heparan sulfate instead decreased the length of the path of the corneal epithelium. Figure 3 shows the changes in wound area after denudation in vivo of the corneal epithelium. Eighteen hours after denudation, there were no differences among all of the test groups. However, after 24 hours, hyaluronan significantly stimulated corneal epithelial wound closure relative to both the untreated and vehicletreated groups. After 48 hours, the cornea treated with
Table 1 . Molecular weight dependency of hyaluronan on corneal epithelial migration. molecular weight of hyaluronan
epithelial migration
control 9 x lo4 26 x lo4 69 x lo4 80 x lo4 84 x lo4 206 x lo4 280 x lo4
463 f 17 570 f 27** 576k 5** 61 8 f 30** 593f 7** 631 i 3 6 * * 587 f 33** 588 2 21 ’*
(v)
mean f S.E. (n=6) **: pc0.01 vs control Cornea was cultured in the medium containing 1 mg/ml of hyaluronan for 20 hours.
Table 2. Effects of glycosaminoglycans on corneal epithelial migration.
Concentration of hyaluronan (mg/ml) Figure 2. Dose dependent effects of hyaluronan on stimulation of rabbit corneal epithelial migration. Cornea was cultured in medium containing (0 - 1 mg/ml, molecular weight: 80x1041 hyaluronan for 20 hours. Each hatched bar represents tl-e mean 1 S.E. of 14 determinations *‘:p
