View
2
Download
0
Category
Preview:
Citation preview
J. Microbiol. Biotechnol. (2010), 20(4), 803–808doi: 10.4014/jmb.0911.11021First published online 30 January 2010
Effects of Ultra High Molecular Weight Poly-γ-glutamic Acid from Bacillussubtilis (chungkookjang) on Corneal Wound Healing
Bae, Sun-Ryang1, Chung Park
2, Jae-Chul Choi
2, Haryoung Poo
3, Chul-Joong Kim
4, and Moon-Hee Sung
2,5*
1College of Medicine, The Catholic University of Korea, Seoul 150-101, Korea2BioLeaders Corporation, Daejeon 305-500, Korea3Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea4College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea5Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Korea
Received: November 19, 2009 / Accepted: January 9, 2010
Poly-γ-glutamic acid (γ-PGA) is a natural edible polypeptide
in which glutamate is polymerized via γ-amide linkages.
First, we assessed the eye irritancy potential of γ-PGA in
rabbits. Additionally, we studied the effects of γ-PGA on
corneal wound healing, due to the anti-inflammatory
properties and water retaining abilities of γ-PGA. In this
study, the effects of γ-PGA on corneal wound healing after
an alkali burn were evaluated. Thirty eyes wounded by
alkali burning in 30 white rabbits were divided into three
groups: group A was treated with 0.1% 5,000 kDa γ-PGA
for 2 days; group B was treated with 0.1% hyaluronic
acid; and group C was not treated, as a control. The area
of corneal epithelial defect was examined at 12, 24, 30, 36,
42, and 48 h after corneal alkali wounding to determine
initial wound healing. We found that γ-PGA promoted
corneal wound healing, compared with controls, and showed
similar effects to hyaluronic acid. These results indicate
that γ-PGA stimulates corneal wound healing by an
anti-inflammatory effect and enhancing cell migration
and cell proliferation. γ-PGA is a promising biomaterial
that may be a substitute for hyaluronic acid in corneal
wound healing treatment.
Keywords: Poly-γ-glutamic acid (γ-PGA), corneal wound
healing, hyaluronic acid
Poly-γ-glutamic acid (γ-PGA) is an edible and biodegradable
polymer of the amino acid glutamate (Fig. 1). The
molecular mass of γ-PGA ranges from 10 to 10,000 kDa,
depending on γ-PGA producer [2]. γ-PGA is synthesized
primarily by microorganisms [1, 20, 23], including Bacillus
subtilis, the microorganism involved in the fermentation of
chungkookjang, a traditional food in Korea [16]. γ-PGA is
a biodegradable, biocompatible, and water-soluble material,
and can therefore be used as a thickener, calcium absorption
stimulating agent, humectant, and drug delivery matrix
owing to its unique properties [4]. We have succeeded in
the industrial production of γ-PGA with high molecular
weight and in developing new functional applications in
foods and cosmetics. High molecular weight γ-PGA not
only has excellent moisture retaining abilities, but also
relieves allergies and inflammation by inhibiting hyaluronidase,
a penetrating factor that dissolves the cell walls by
unlinking the hyaluronic acid part of the cell wall or
membrane [30]. Recently, we reported that the oral
administration of high molecular weight γ-PGA showed
TLR4- and dendritic-cell-dependent antitumor effects [18,
19].
*Corresponding authorPhone: +82-2-910-4808; Fax: +82-2-910-8550;E-mail: smoonhee@kookmin.ac.kr
Fig. 1. Structures of poly-γ-glutamic acid (γ-PGA): Na salt form(left) and acid form (right).
804 Bae et al.
Cells such as keratocytes and polymorphonuclear leukocytes
(PMNs) are primarily involved in the scrape injury healing
process after corneal alkali burns [24, 25]. Keratocytes
and PMNs produce matrix metalloproteinases, such as
stromelysin, gelatinase, and collagenase, and different
proteases [14], because these enzymes dissolve collagen
fibers.
As such, protease inhibition is important in scrape injury;
various agents have been examined for the treatment of
scrape injury healing in the cornea [7]. Epithelial growth
factor (EGF), matrix metalloproteinase inhibitors, and
hyaluronic acid have been investigated as therapeutics and
their effects have been reported [3, 8, 28].
Hyaluronic acid has been studied as a therapeutic for
corneal alkali burns and corneal surface disease [12, 27,
32]. Hyaluronic acid is a disaccharide polymer that absorbs
water, and is present in the extracellular matrix where it
plays a role in cell movement, and is thus involved in
tissue expansion [31]. It has a different mechanism of
action from fibronectin or EGF, which heal scrape injury
by promoting epithelial movement [22]. Hyaluronic acid
protects the corneal epithelium and endothelium [26, 29],
stimulates epithelial movement and proliferation [15, 21],
and promotes hemidesmosome formation [9]. Endogenous
hyaluronic acid is produced by corneal epithelial cells,
keratocytes, corneal endothelial cells, iris pigment epithelial
cells, and lens epithelial cells [10, 11, 33].
There are various reports regarding agents with efficacy
after administration into eyes with induced corneal wounds,
of which the best known example is hyaluronic acid.
Several recent studies have indicated that hyaluronic acid
is involved in cell protection, cell movement, growth control,
cell division, and tissue morphogenesis. In the present
study, we investigated the effects of γ-PGA in a rabbit
corneal wound model.
MATERIALS AND METHODS
Preparation of Ultra High Molecular Weight Poly-γ-Glutamic
Acid
A 1% culture solution of Bacillus subtilis (chungkookjang) (KCTC
0697BP) was inoculated into 3 l of preparative Basic Medium of
γ-PGA [GS basic medium with 5% L-glutamic acid:glucose 5%,
(NH4)2SO4 1%, KH2PO4 0.27%, Na2HPO4·12H2O 0.42%, NaCl 0.05%,
MgSO4·7H2O 0.3%, vitamin solution 1 ml/l, pH 6.8], and cultured
with stirring at 150 rpm, an aeration rate of 1 vvm, at 37oC for 72 h.
After culturing, a filter press was used to eliminate fungal contaminants,
yielding a sample solution containing γ-PGA.
After adding 2 N sulfuric acid solution to the above sample
solution containing γ-PGA, the sample was left to stand at 10oC for
12 h to sediment the γ-PGA. After cleaning with a sufficient amount
of reverse osmosis (RO) water, γ-PGA was obtained using a
Nutsche filter. The γ-PGA had a molecular mass of 1-15,000 kDa,
and separate experiments were carried out on subfractions with a
range of molecular weights.
Molecular Mass Determination of Ultra High Molecular Weight
Poly-γ-Glutamic Acid
The molecular mass of γ-PGA was determined by gel permeation
chromatography (GPC). Briefly, PGA solution was diluted with
0.1 M NaNO3, and injected into the GPC equipped with a VicoGel
GMPWXL column (7.8 mm×30 cm; Viscotech, Houston, TX, U.S.A.),
which had been equilibrated with 0.1 M NaNO3, at 40oC and a flow
rate of 0.8 ml/min. γ-PGA was detected with a Viscotek LR25 Laser
Refractometer. Polyacrylamide was used as a standard material for
molecular mass determination. Additionally, viscosity was determined
using a viscometer (Brookfield DV+I Viscometer; Brookfield
Engineering, Middleboro, MA, U.S.A.) at 25oC.
We determined the molecular mass using a chemical modification
method, as previously described [30]. Amino groups modified with
FDNB were used for determination of the number of moles and
estimation of the average molecular size of γ-PGA, because every
molecule of γ-PGA had a single free amino group. Both the total
mole number of glutamate monomer and the number of FDNB-
modified glutamate contained in the hydrolysates of FDNB-modified
γ-PGA were assayed. The average number of glutamate units of γ-
PGA could be determined by dividing the glutamate number by the
FDNB-modified glutamate number.
Atomic force microscopy (AFM; XE-100; PSIA Inc., Santa
Clara, CA, U.S.A.) was used to analyze the ultra high molecular
weight γ-PGA, as described previously [30]. Static light scattering
(SLS) was also used for characterization of macromolecules in
solution. SLS makes use of the time-averaged intensity of scattered
light, from which the weight-average molecular weight and second
virial coefficient can be determined.
Eye Irritation Study in Rabbits
Eye irritation tests were performed to assess the irritancy potential
of the test material in the eyes of New Zealand White rabbits. The
method was designed to meet the OECD Guidelines for the Testing
of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24
April 2002) and the Guide to Quasi-Drug and Cosmetic Regulations
in Japan 2006 (February 2006). The ocular route was chosen
because it is a potential route of exposure in humans and has been
the route of choice, based on the method of Draize [13, 17]. Three
animals received 0.1 ml of γ-PGA solution into the conjunctival sac
of the right eye after gently pulling the lower eyelid away from the
eyeball, with the left eye serving as the untreated control. The upper
and lower lids were then gently held together for about 1 s, to
prevent loss of the test substance, and were then released.
The animals were observed for clinical signs once a day and
body weights were measured on day 1 (the day of application) and
day 5 during the observation period, using a balance (HW-60KGL;
A&D, Seoul, Korea). The treated eyes (right eyes) were examined
1, 24, 48, 72, and 96 h after administration of test substance. At
each observation time point, lesions of the cornea, iris, and conjunctivae
were scored separately using a numerical system, based on the
method of Draize [13]. The degree of eye irritation was evaluated
according to the method of Kay and Calandra [17], and the extent
was classified.
Establishment of the Rabbit Corneal Wound Model
An alkali burn caused by placing filter paper (5.5 mm in diameter,
wet with 1 N NaOH) on the cornea is an experimental model for
obstructing voluntary transparency recovery without complications,
EFFECTS OF γ-PGA ON CORNEAL WOUND HEALING 805
such as ulcer, perforation, or neovascularization, and allowing
observation of the alkali burn healing process, similar to that which
occurs clinically. Thus, it is possible to analyze healing of the
epithelium, stroma, and endothelium quantitatively after cornea
alkali burn using this model [5, 6].
General anesthesia was induced in 30 female New Zealand White
Rabbits, 2.5 kg in body mass, by intravenous administration of
pentobarbital, and the ocular surface was anesthetized using 0.5%
proparacaine. After inducing a cornea alkali burn in one eye by
touching the center of the cornea with a circular filter paper (5.5 mm
in diameter, wet with 1 N NaOH) for 60 s, balanced salt solution was
used for irrigation (BSS; Alcon, Fort Worth, TX, U.S.A.) for 120 s.
Treatment of Corneal Wounded Rabbit’s Eyes with γ-PGA
The eyes of 10 of the 20 rabbits in the healing group were treated
with 0.1% hyaluronic acid solution with a molecular mass of
1,200 kDa, whereas the remaining 10 were treated with 0.1% γ-
PGA solution with a molecular mass of 5,000 kDa four times a day.
A further 10 rabbits were treated with phosphate-buffered saline
(PBS) four times a day as a control group.
Assessment of Corneal Wound Healing by γ-PGA Application
The injured corneal epithelium area was stained with 2% fluorescein
solution immediately, 12, 24, 30, 36, 42, and 48 h after inducing
alkali burn injury, and photographs were taken using a Nikon D80
digital camera with a Micro-Nikkor 105 mm 1:4 lens (Nikon, Tokyo,
Japan). In this study, the healing group was compared with the
control group by measuring the area of the epithelial defect using
AutoCAD 2007 (Autodesk, Inc., San Rafael, CA, U.S.A.) and by
determining the healing rate of the epithelium by linear regression
analysis.
Seven days after alkali injury, seven rabbits in each group were
sacrificed to extract the cornea. Six corneas were fixed in 4%
paraformaldehyde, dehydrated in alcohol, and embedded in paraffin.
These specimens were cut into tissue sections and stained with
hematoxylin and eosin. Photographs of the corneal samples were
taken at 400× magnification with an optical microscope to measure
the cell numbers in the cornea. Cell numbers were determined in an
area of 1 mm2 in the central region of the scrape injury and in an
area of the same size around the scraped wound in the total corneal
stroma.
The rest of the cornea was fixed in 4% paraformaldehyde-2.5%
glutaraldehyde in phosphate buffer at 4oC for electron microscopy.
After embedding the tissue in epoxy resin, thermal polymerization
was performed at 60oC for 3 days. The embedded tissue was cut
into semi-thin sections 1 µm thick and stained with 1% toluidine
blue. The lesions were confirmed under an optical microscope, and
the samples were cut into very thin sections on an ultramicrotome
(Leica Microsystems, Wetzlar, Germany). The tissue was double-
stained with uranyl acetate and lead citrate, and then observed by
transmission electron microscopy (JEM-1010; JEOL, Tokyo, Japan).
Statistical Methods
Student’s t-test was used for comparison of the corneal wound
healing rate between the control, hyaluronic acid, and γ-PGA
groups, and the numbers of polymorphonuclear leukocytes (PMNs)
infiltrating the cornea were compared among the groups using
Wilcoxon’s rank sum test. All statistical analyses were performed
using SPSS 12.0. for Windows (SPSS Inc., Chicago, IL, U.S.A.),
and P values <0.05 were deemed to indicate statistical significance.
RESULTS AND DISCUSSION
Molecular Mass Determination of Ultra High Molecular
Weight γ-PGA
The molecular masses of the poly-γ-glutamic acid samples
were 500, 2,000, and 5,000 kDa, as determined by gel
permeation chromatography (GPC) with polyacrylamide
(10-9,000 kDa) as a standard. The viscosity of samples
increased with increasing molecular mass. Atomic force
microscopy (AFM) was also used to determine the
molecular mass. It is possible to calculate the molecular
mass of samples using this method, because it is directly
proportional to the molecular volume. It was confirmed
that the AFM measurement increased with increasing
molecular mass, and that it is possible to calculate the
molecular mass of high molecular weight γ-PGA using this
method on the basis of the number of glutamate monomers
generated by hydrolysis, and quantified with 1-fluoro-2,4-
Table 1. Molecular mass determination of poly-γ-glutamic acid.
GPC [kDa]a Chemical modification [kDa]b Light scattering [kDa]cAFM [nm3](GPC, kDa)d
Viscosity [cP]e
600 - -9.80×10
2
(470)18
2,300 1,600 1,1003.90×104
(1,900)76
5,000 - 2,9001.10×10
5
(6,000)228
- No data.a Determined using GPC (column: Viscotek GMPWXL).b
Determined based on the number of glutamate monomers generated by hydrolysis and with the free amino group quantified using 1-fluoro-2,4-
dinitrobenzene (FDNB).c Determined from the time-averaged intensity of scattered light.d Determined by atomic force microscopy.e Determined by viscometry.
806 Bae et al.
dinitrobenzene (FDNB). The absolute molecular mass of
high molecular weight γ-PGA was successfully determined
by static light scattering (SLS). As shown in Table 1, we
succeeded in determining the molecular mass of three
different types of γ-PGA with various methods, which
were then applied in animal experiments to confirm the
effects of high molecular weight γ-PGA (>5000 kDa).
Eye Irritation Test in Rabbits
This study was performed to evaluate the possible irritation
potential when a single dose of the test substance, γ-PGA
solution, was administered into the conjunctival sac in
three 16-week-old male NZW rabbits. Briefly, 0.1 ml of
γ-PGA solution was administered into the conjunctival sac
of the right eye after gently pulling the lower eyelid away
from the eyeball, with the left eye serving as an untreated
control. The cornea, iris, and conjunctiva were scanned 1,
24, 48, 72, and 96 h after administration. The eye irritation
related to the test substance was calculated according to
Table 2. Irritation scores of poly-γ-glutamic acid in an eyeirritation study.
Test group
No. of animals
Mean total score (MTS)MMTS
1 ha
24 h 48 h 72 h 96 h
γ-PGA 3 0 0 0 0 0 0
MMTS: Maximum mean total score.a Hour after application.
Fig. 2. Effects of ultra high molecular weight poly-γ-glutamic acid on corneal wound healing: (top) clinical signs during the observationperiod; (bottom) wound recovery (%).
EFFECTS OF γ-PGA ON CORNEAL WOUND HEALING 807
the method of Draize [13], and the degree of eye irritation
was classified according to the method of Kay and
Calandra [17]. No eye irritation of the cornea, iris, or
conjunctivae was observed in any of the animals at 1, 24,
48, 72, or 96 h after administration. The mean total score
(MTS) was recorded as 0 and evaluated as “non-irritating.”
There was no abnormal clinical sign, death, or body weight
change attributable to the test substance in any animal
during the observation period. Based on these results, it
was concluded that γ-PGA solution did not induce eye
irritation in rabbits under the conditions used in this study
(Table 2).
Effects of γ-PGA on Corneal Wound Healing in a
Rabbit Model
The early wound defect area was 23.83±0.79 mm2 in the
PBS group, 23.73±1.03 mm2 in the hyaluronic acid group,
and 23.77±0.67 mm2 in the γ-PGA group; there was no
significant difference between the three groups (P=0.957).
The wound healing rate was 0.466±0.059 mm2/h in the
PBS group, 0.490±0.055 mm2/h in the hyaluronic acid
group, and 0.531±0.076 mm2/h in the γ-PGA group. There
was no significant difference between the PBS and hyaluronic
acid groups (P=0.361) or between the hyaluronic acid
group and γ-PGA group (P=0.189). However, the healing
rate of the γ-PGA group was significantly faster than that
of the PBS group (P=0.048; Fig. 1 and 2).
By electron microscopy, we could not observe the
basement membrane after 7 days because there was severe
basement membrane injury in all three groups, so observation
of hemidesmosomes was impossible. During the initial
7 days, the wounds healed without infection or ulcer
formation.
As hyaluronic acid is degraded by hyaluronidase in the
living body, inhibition of hyaluronidase activation, which
leads to increased activation of hyaluronic acid, promotes
corneal wound healing. γ-PGA is known to indirectly
reduce inflammation by inhibiting hyaluronidase activation,
thus hindering hyaluronic acid degradation, although γ-
PGA is not made in the living body, unlike hyaluronic acid
[18]. Additionally, there was no ocular irritation associated
with its administration into the eyes, and in combination
with water, it had superior moisturizing capacity versus
hyaluronic acid as well as a moisturizer activation function
[30].
The results of the present study indicate that corneal
wound healing was significantly faster in the γ-PGA group
than in the control group. However, there was no significant
difference in healing rate between the hyaluronic acid and
control groups, which was similar to the results of previous
studies. It is possible that corneal wound healing can be
promoted directly, rather than indirectly, by inhibition of
hyaluronidase. Thus, γ-PGA is a possible new substitute
for hyaluronic acid for use in corneal wound healing.
Acknowledgments
This work was supported by the Seoul R&BD Program
(10580) and the Research Program 2008 of Kookmin
University in Korea.
REFERENCES
1. Aono, R. 1987. Characterization of structural component of cell
walls of alkalophilic strain of Bacillus sp. C-125. Preparation of
poly (gamma-L-glutamate) from cell wall component. Biochem.
J. 245: 467-472.
2. Ashiuchi, M. and H. Misono. 2002. Poly-γ-glutamic acid, pp.
123-174. In S. R. Fahnestock and A. Steinbüchel (eds.).
Biopolymers Wiley-VCH, Weinheim.
3. Brazzell, R. K., M. E. Stern, J. V. Aquavello, and R. W.
Beuerman. 1991. Human recombinant epidermal growth factor
in experimental corneal wound healing. Invest. Ophthalmol. Vis.
Sci. 32: 336-340.
4. Buescher, J. M. and A. Margaritis. 2007. Microbial biosynthesis
of polyglutamic acid biopolymer and applications in the
biopharmaceutical, biomedical and food industries. Crit. Rev.
Biotechnol. 2: 1-19.
5. Chung, J. H., P. Fagerholm, and B. Lindstrom. 1987. The
behaviour of corneal epithelium following a standardized alkali
wound. Acta Ophthalmol. 65: 529-537.
6. Chung, J. H. and P. Fagerholm. 1987. Endothelial healing in
rabbit corneal alkali wounds. Acta Ophthalmol. 65: 648-656.
7. Chung, J. H. 1988. Experimental corneal alkali wound healing.
Acta Ophthalmol. 66: 1-35.
8. Chung, J. H., P. Fagerholm, and B. Lindstrom. 1989. Hyaluronate
in healing of corneal alkali wound in the rabbit. Exp. Eye Res.
48: 569-576.
9. Chung, J. H., W. K. Kim, J. S. Lee, Y. S. Pae, and H. J. Kim.
1998. Effect of topical Na-hyaluronan on hemidesmosome
formation in n-heptanol-induced corneal injury. Ophthalmic Res.
30: 96-100.
10. Conrad, G., C. Hamilton, and E. Haynes. 1977. Differences in
glycosaminoglycans synthesized by fibroblast-like cells from
chick cornea, heart and skin. J. Biol. Chem. 252: 6861-6870.
11. Dahl, I. 1981. Biosynthesis of proteoglycans and hyaluronate
in rabbit corneal fibroblast cultures: Variation with age of the
cell line and effect of fetal calf serum. Exp. Eye Res. 32: 419-
433.
12. DeLuise, V. P. and W. S. Peterson. 1984. The use of topical
Healon tears in the management of refractory dry-eye
syndrome. Ann. Ophthalmol. 16: 823-824.
13. Draize, J. H. 1959. Appraisal of the safety of chemicals in
foods, drugs and cosmetics, pp. 46-59. The Association of Food
and Drug Officials of the United States, Topeka, Kansas. 46-59.
14. Hibbs, M. S., K. A. Hasty, J. M. Seyer, A. H. Kang, and C. L.
Mainardi. 1985. Biochemical and immunological characterization
of the secreted forms of human neutrophil gelatinase. J. Biol.
Chem. 260: 2493-2500.
15. Inoue, M. and C. Katakami. 1993. The effect of hyaluronic acid
on corneal epithelial cell proliferation. Invest. Ophthalmol. Vis.
Sci. 34: 22313-22315.
808 Bae et al.
16. Kang, S. E., J. H. Rhee, C. Park, M. H. Sung, and I. H. Lee.
2005. Distribution of poly γ-glutamate (γ-PGA) producers in
Korean fermented foods, cheongkukjang, doenjang, and
kochujang. Food Sci. Biotechnol. 14: 704-708.
17. Kay, J. H. and J. C. Calandra, 1962. Interpretation of eye
irritation tests. J. Soc. Cosmet. Chem. 13: 281-289.
18. Kim, T. W., T. Y. Lee, H. C. Bae, J. H. Hahm, Y. H. Kim,
C. Park, et al. 2007. Oral administration of high molecular
weight poly-gamma-glutamate induces NK cell-mediated anti-
tumor immunity. J. Immunol. 179: 775-780.
19. Lee, T. Y., Y. H. Kim, S. W. Yoon, J. C. Choi, J. M. Yang, C. J.
Kim, J. T. Schiller, M. H. Sung, and H. Poo. 2009. Oral
administration of poly-gamma-glutamate induces TLR4- and
dendritic cell-dependent antitumor effect. Cancer Immunol.
Immunother. 58: 1781-1794.
20. Makino, S., I. Uchida, N. Terakado, C. Sasakawa, and M.
Yoshikawa. 1989. Molecular characterization and protein
analysis of the cap region, which is essential for encapsulation
in Bacillus anthracis. J. Bacteriol. 171: 722-730.
21. Nakamura, M., N. Sato, T. I. Chikami, Y. Hasegawa, and T.
Nishida. 1997. Hyaluronan facilitates corneal epithelial wound
healing in diabetic rats. Exp. Eye Res. 64: 1043-1050.
22. Nishida, T. and M. Nakamura. 1991. Hyaluronate stimulates
corneal epithelial migration. Exp. Eye Res. 53: 753-758.
23. Pérez-Camero, G., F. Congregado, J. J. Bou, and S. Muñoz-
Guerra. 1999. Biosynthesis and ultrasonic degradation of
bacterial poly(gamma-glutamic acid). Biotechnol. Bioeng. 63:
110-115.
24. Pfister, R. R., J. L. Haddox, R. W. Dodson, and L. E. Harkins.
1987. Alkali-burned collagen produces a locomotor and
metabolic stimulant to neutrophils. Invest. Ophthalmol. Vis. Sci.
28: 295-304.
25. Pfister, R. R., J. L. Haddox, K. W. Lam, and K. M. Lank. 1988.
Preliminary characterization of a polymorphonuclear leukocyte
stimulation isolated from alkali-treated collagen. Invest. Ophthalmol.
Vis. Sci. 29: 955-962.
26. Reim, M. and V. Lenz. 1984. Behandlung von schweren
Veratzungen mit hochpolymerer Hyaluronsaure (Healon). Fortschr.
Ophthalmol. 8: 323-325.
27. Saric, D. and M. Reim. 1984. Behandlung von Veratzungen des
vorderen Augenabschnitts mit hoch polymerem Na-Hyaluronat
(Healon). Fortschr. Ophthalmol. 8: 588-591.
28. Schultz, G. S., S. Strelow, G. A. Stern, N. Chegini, M. B. Grant,
R. E. Galardy, et al. 1992. Treatment of alkali-injured rabbit
corneas with a synthetic inhibitor of matrix metallo-proteinase.
Invest. Ophthalmol. Vis. Sci. 33: 3325-3331.
29. Stuart, J. C. and J. G. Linn. 1985. Dilute sodium hyaluronate
(Healon) in the treatment of ocular surface disorders. Ann.
Ophthalmol. 17: 190-192.
30. Sung, M. H., C. Park, C. J. Kim, H. Poo, K. Soda, and M.
Ashiuchi. 2005. Natural and Edible biopolymer Poly-g-glutamic
Acid: Synthesis, Production and its Applications. The Chemical
Record. 5: 352-366.
31. Toole, B. P. 1976. Morphogenetic role of glycosaminoglycans
(acid mucopolysaccharides) in brain and other tissue, pp. 275-
329. In S. H. Baronders (ed.). Neural Recognition. Plenum
Press, New York.
32. Wysenbeek, Y. S., N. Loya, I. Ben Sira, I. Ophir, and Y. Ben
Shaul. 1988. The effect of sodium hyaluronate on the corneal
epithelium: An ultrastructural study. Invest. Ophthalmol. Vis.
Sci. 29: 194-199.
33. Yue, B., J. Baum, and J. Silbert. 1978. Synthesis of
glycosaminoglycans by cultures of normal human corneal
endothelial and stromal cells. Invest. Ophthalmol. Vis. Sci. 17:
523-527.
Recommended