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Lab Anim Res 2016: 32(4), 181-186
https://doi.org/10.5625/lar.2016.32.4.181
ISSN 1738-6055 (Print)
ISSN 2233-7660 (Online)
Evaluation of stability and biocompatibility of PHEMA-PMMA
keratoprosthesis by penetrating keratoplasty in rabbits
Yawon Hwang, Gonhyung Kim*
Department of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea
Artificial corneas have been developed as an alternative to natural donor tissue to replace damaged or
diseased corneas. This study was conducted to evaluate the stability and biocompatibility of PHEMA-
PMMA [poly (2-hydroxyl methacrylate)-poly (methyl methacrylate)] keratoprostheses in rabbits following
penetrating keratoplasty. Sixteen male New Zealand White rabbits aged 16 weeks were divided into three
groups. Group I and group II contained six rabbits each, while the control group had four rabbits.
Experimental surgery was conducted under general anesthesia. The cornea was penetrated using an 8
mm diameter biopsy punch. In group I (core 5 mm & skirt 3 mm) and group II (core 6 mm & skirt 2
mm), the keratoprosthesis was placed into the recipient full thickness bed and sutured into position with
double-layer continuous. In the control group, corneal transplantation using normal allogenic corneal
tissue was performed with the same suture method. After four and eight weeks, keratoprosthesis devices
were evaluated by histopathological analysis of gross lesions. Post-operative complications were observed,
such as extrusion and infection in experimental groups. Most corneas were maintained in the defect site
by double-layer continuous suture materials for 4 weeks and kept good light transmission. However, most
artificial cornea were extruded before 8 weeks. Overall, combined PHEMA and PMMA appears to have
sufficient advantages for production of artificial corneas because of its optical transparency, flexibility and
other mechanical features. However, the stability and biocompatibility were not sufficient to enable
application in humans and animals at the present time using penetrating keratoplasty. Further studies are
essential to improve the stability and biocompatibility with or without other types of keratoplasty.
Keywords: Keratoprosthesis, cornea, PHEMA-PMMA, rabbit
Received 16 May 2016; Revised version received 23 September 2016; Accepted 14 November 2016
More than 10 million people suffer from corneal origin
blindness throughout the world, making it the second
most common eye affliction next to cataracts [2,3,4,22].
Corneal blindness is caused by various diseases such as
ulceration, trauma, vitamin A deficiency, keratitis, dry
eye syndrome, and chemical burns [3]. In most forms of
corneal blindness, the successful alternative for visual
regain is corneal transplant using donor tissue [10].
However, there are several risk factors, and corneal
transplants are not always possible due to limitations in
the storage and supply of corneal tissue [15]. In veterinary
medicine, the loss of corneal transparency may occur in
response to various diseases such as corneal pigmentation,
chronic superficial keratitis, severe stromal edema or
fibrosis, keratoconjunctivitis sicca, and ulceration. Long-
term medical or surgical therapies can be applied for
non-treated cases despite initial treatment [7]. In such
cases, the only alternative treatment has been the surgical
application of an artificial cornea (keratoprosthesis) to
restore vision [9]. The first implantation of an artificial
cornea in humans was attempted in 1859 [2]. Kerato-
prostheses have been made of glass, plastics, quartz or
soft rubbers. Recently, carboform, hydrogels and synthetic
polymers have been introduced as materials for kerato-
*Corresponding author: Gonhyung Kim, Department of Veterinary Surgery, Veterinary Medical Center, College of Veterinary Medicine,
Chungbuk National University, Cheongju, Chungdaero 1, Seowon-gu, Cheongju, Chungbuk 28644, Korea
Tel: +82-43-261-3171; Fax: +82-43-261-3224; E-mail: ghkim@cbu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
182 Yawon Hwang, Gonhyung Kim
Lab Anim Res | December, 2016 | Vol. 32, No. 4
prostheses [11,15]. The poly (methyl methacrylate)
(PMMA) based keratoprosthesis was the first polymer to
be used for artificial corneal transplantation [2,4,15].
Since then, keratoprostheses have been improved
significantly, although the complication and failure rates
remain high. The biocompatibility between artificial
cornea and surrounding cornea tissue has also been
found to be poor [4]. The most common complications
associated with keratoprostheses are implant extrusion,
infection, aqueous leakage, corneal melting, epithelial
down growth, glaucoma and reduced visual acuity [1,8,
19]. To reduce the risk of complications, it is important
to improve biocompatibility and cell adhesion [5].
Newly developed keratoprostheses based on poly (2-
hydroxyethyl methacrylate) (PHEMA)-PMMA have
been evaluated to minimize the risk of complications.
PMMA-based materials have continued to be the most
commonly used compounds for keratoprostheses since
1941. PHEMA is a non-toxic polymer of the toxic
monomer HEMA, which was the first hydrogel used in
a biomedical device [15,17]. PHEMA, which has been
utilized as a soft contact lens material since the late
1950s, has a high water content, mechanical strength and
biocompatibility [13]. PHEMA-PMMA based artificial
corneal had more strength the tensile intensity of kerato-
prostheses. PHEMA-PMMA keratoprostheses have a
core and skirt design, in which the core allows trans-
mission of light and the skirt allows integration with
surrounding host tissue.
This study was conducted to evaluate the stability and
biocompatibility of newly developed PHEMA-PMMA
keratoprostheses with two different skirt sizes based on
the penetrating method in rabbits for veterinary and
human medicine.
Materials and Methods
Animals
Sixteen male New Zealand White rabbits aged 16
weeks and weighing 2.6 to 3.1 kg were used in this
animal experiment. Rabbits were housed in a single cage
at the laboratory animal research center of Chungbuk
National University and maintained on a 12/12 hour
light/dark cycle, standard laboratory diet and water. The
experimental protocol was approved by the institutional
animal care and use committee of the laboratory animal
research center of Chungbuk National University.
PHEMA-PMMA keratoprosthesis
The PHEMA-PMMA based artificial cornea (Figure
1) consists of two parts, a central “core” optical component
and a peripheral tissue-integral “skirt”. The thickness of
the artificial cornea is 0.45 mm and the diameter of the
core is about 5.5 mm.
Experimental design
Sixteen rabbits were divided into three groups: group
I; PHEMA-PMMA based artificial cornea with a core
diameter of about 5 mm and a 3 mm skirt implanted in
6 rabbits: group II; PHEMA-PMMA based artificial
cornea with a core diameter of about 6 mm and a 2 mm
skirt was implanted in 6 rabbits: control group; normal
allogenic corneal transplantation was performed in 4
rabbits.
All surgical procedures were performed using an
operating microscope (Zeiss OPMI pico, Carl Zeiss,
Goettingen, Germany). The rabbits were anesthetized
using tiletamine-zolazepam (Zoletil 50, Virbac Laboratories,
Carros, France) at a dose of 10 to 20 mg/kg and xylazine
hydrochloride (Rompun, Bayer Korea, Seoul, Korea) at
a dose of 5 to 10 mg/kg intra-muscular (IM) injection.
Proparacaine hydrochloride (Alcaine 0.5%, Alcon
Laboratories, Rijksweg, Puurs, Belgium) was used for
topical anesthesia. After dilation of the pupil using 1%
tropicamide (Ocutropic, Samil Pharma, Seoul, Korea),
full-thickness corneal trephination was performed with
8 mm diameter biopsy punch and the corneal button was
removed using 360 degree dissection with scissors
(Figure 2).
Figure 1. Gross photographs of PHEMA-PMMA artificial
cornea (front view). Artificial cornea with a diameter of core is 5
mm, with 0.45 mm thick using in group I.
PHEMA-PMMA keratoprosthesis 183
Lab Anim Res | December, 2016 | Vol. 32, No. 4
In the experimental groups, after complete excision of
the cornea the PHEMA-PMMA based device was put
into a corneal bed and artificial corneas were fixed 360
degrees to the surrounding cornea with 9-0 polyglactin
(Vicryl, Ethicon, New Jersey, USA) sutures. Double-
layer continuous sutures were placed between the
remaining cornea and the skirt. In the control group, after
the corneal pocket was created the normal allogenic
cornea from the experimental group was put into a
corneal bed. Double-layer continuous sutures with 9-0
polyglactin (Vicryl) were placed between the allogenic
device and the corneal bed.
Followingtransplantation,blepharorrhaphywasperformed
to protect the surgical area. Antibiotic (Cefazolin;
Chongkundang, Seoul, Korea; 30 mg/kg), and analgesic
(Fluximine; Bomac Laboratories, Auckland, New Zealand;
1.1 mg/kg) were injected IM into each rabbit once a day
for 2 weeks. Postoperatively, topical dexamethasone
with polymyxin B sulfate and neomycin sulfate (Forus;
Samil, Seoul, Korea) and ofloxacin (Ocuflox; Samil,
Seoul, Korea) ophthalmic drops were administered once
a day for 2 weeks.
Gross lesion observation and scoring
Gross lesion evaluation and scoring was performed at
4 and 8 weeks after surgery. Gross lesions were assigned
a score of 1 to 5 depending on extrusion of the device
and infection of the surgical area, where; 1 was extrusion
and infection; 2 partial extrusion and infection; 3 partial
extrusion and minimal infection; 4 no extrusion and
minimal infection; and 5 no complications. Data of
complete extrusion artificial cornea were removed from
gross observation, therefore gross lesion scorings of each
experimental group were evaluated at 4 weeks. For
scoring at 8 weeks, four samples were used for observation
and obtained from both group I and II, because of 5
artificial corneas were extruded completely.
Histological examination
Rabbits were sacrificed 4 weeks and 8 weeks post-
implantation. The animals were anesthetized by IM
injection of 20mg/kg tiletamine-zolazepam, then sacrificed.
After sacrifice, the device-inserted eyes were enucleated
and fixed in 10% buffered formaldehyde. Specimens
were then cut along the sagittal plane and embedded in
paraffin. Sections were cut into 4 µm and hematoxylin
and eosin was used for staining.
Statistical analysis
The gross lesion scores were expressed as the means
±standard error and analyzed using SPSS version 20
(SPSS Inc., Chicago, IL, USA). Groups were compared
by ANOVA followed by Tukey’s test. P values <0.05
were considered to indicate significance.
Figure 2. Creation of the recipient corneal pocket and keratoprosthesis. A. The eye is proptosed by means of gentle pressure while
an 8 mm biopsy punch is used to create corneal pocket. B. Corneal button remove using 360 degree dissection with scissors. C.
The devices are placed into the recipient corneal pocket. D. Double-layer continuous suture is performed using 9-0 polyglactin.
184 Yawon Hwang, Gonhyung Kim
Lab Anim Res | December, 2016 | Vol. 32, No. 4
Results
Post-operative complications
Post-operative complications including death, extrusion
of the device, infection and melting were observed in the
experimental groups. There were no complications
observed in the control group.
Gross lesion and scoring
In the experimental groups, most corneas were maintained
in the defect site by suture materials for 4 weeks and kept
good light transmission. However, all artificial corneas
were partially extruded before 8 weeks. In the control
group, allogenic corneas were maintained well for 8
weeks and vascularization was identified on the trans-
planted corneas (Figure 3).
The gross lesion score of each group and score of each
experimental period are presented in Table 1. In group I
(n=5), group II (n=4) at 4 weeks, the gross lesion score
was 3.00±1.36 and 3.33±1.33, respectively. No significant
differences were observed in the gross lesion scores
Figure 3. Gross photographs of the group I, II and control group at 8 weeks after implantation. A: Group I, partial extrusion of device
is observed. B: Group II, partial extrusion of device is observed. C: Control group, There are no extrusion of implanted allogenic
cornea and no stromal melting. A vascularization to the cornea was observed.
Table 1. Gross lesion scoring of each group and each
experimental period
Group
Score
Mean±SE
4 weeks of group I (n=5) 3.00±1.36
4 weeks of group II (n=4) 3.33±1.30
8 weeks of group I and II (n=4) *2.33±1.30*
8 weeks of Control (n=4) 3.83±0.58
Group I: 4~5 mm diameter of the core, Group II: 5~6 mm diameter
of the core. Data is mean±standard error of gross lesion score.
*P<0.05 when compared to those of other groups.
Figure 4. A: Light microscope photographs of an abnormal cornea that has post-operative infection (black arrow head).
Hematoxylin and eosin staining (H&E), ×400. B: Light microscope photographs of the experimental group that has partial extrusion
of the device. A separation between a skirt and corneal bed is found (black arrow head). H&E, ×100. C: Light microscope
photographs of the experimental group that has complete extrusion of the device after 10 days post-operation. Regeneration of the
stroma is shown (black arrow), but continuity of the epithelium is observed lost (black arrow head). H&E, ×100. D: Light microscope
photographs of the control group. It shows a corneal structure as normal cornea which includes epithelium, stroma, and proliferated
endothelium (black arrow). H&E, ×40.
PHEMA-PMMA keratoprosthesis 185
Lab Anim Res | December, 2016 | Vol. 32, No. 4
between groups. The gross lesion score in control group
(n=4) was 3.83±0.94, while score was 2.33±1.30 in
experimental group at 8 weeks post-operation (n=4). The
gross lesion score in experimental group at 8 weeks was
significantly lower than those of experimental groups at
4 weeks and control group at 8 weeks (P<0.05).
Histological findings
In the experimental groups, complications such as
inflammation of the surgical area, separation of the skirt
and biological cornea due to artificial corneal extrusion,
and partial regeneration of the corneal tissue were
observed. In the control group, the epithelium, stroma
and proliferation of the endothelium were found (Figure
4).
Discussion
Loss of vision can be the final result of chronic corneal
disease and ulceration in both humans and animals.
However, these problems cannot always be prevented or
treated by general medical and surgical therapies [23]. In
such cases, allogenic corneal transplantation (keratoplasty)
may be an option to regain vision. However, trans-
plantation has disadvantages including a high-risk of
surgical failure, shortage of donor corneas, difficulties in
storage, and potential for disease transmission [14].
Moreover, a previous study found that only 20% of
grafts remained clear for 5 years [21]. Because of these
problems, researchers have designed and encouraged the
development of artificial corneas (keratoprosthesis) to
replace the keratoplasty. The ideal keratoprosthesis
would utilize an epithelized material that could be
implanted as in keratoplasty [6]. While various kerato-
prosthesis materials and techniques have been developed,
many complications such as extrusion, melting and
infection have been reported. To reduce the risk of
complications, keratoprosthesis should be stable and
biocompatible.
PMMA has been the primary material used for
keratoprosthesis since the early 1950s. This material has
transparency, high mechanical strength, and is easy to
process [21]. PHEMA is widely used in biomaterials,
including soft contact lenses, because of its flexibility
and high water content. Moreover, this material makes
the composite core-and-skirt keratoprosthesis possible
[6].
The single interrupted suture anddouble-layer continuous
suture methods are commonly used in artificial cornea
transplant or keratoplasty [12,16,20]. Extrusion of the
device was observed frequently in the single interrupted
suture method than in the double-layer continuous suture
in our pre-experimental study (data not shown). Based
on these results, the double-layer continuous suture
method should be used for all procedures. However,
single continuous suturing has become the preferred
method because of its greater safety compared with the
interrupted suturing. According to previous reports,
multiple interrupted sutures can disrupt the even distri-
bution of corneal tension [20].
Overall, three of the animals (3/16, 18%) died after
implantation. Additionally, post-operative inflammation
was observed in one rabbit from group I (1/16, 6%).
Extrusions of artificial cornea were found in most of the
animals in the experimental group. Most corneas were
well maintained in the recipient site by suture materials
for 4 weeks; however, all artificial corneas were extruded
before 8 weeks. There was no extrusion of allogenic
cornea in the control group. Overall, the results of this
study indicate that the biocompatibility of the PHEMA-
PMMA keratoprosthesis is not sufficient to enable
combination with the host tissue during penetrating
keratoplasty. A gross lesion score was applied according
to the degree of inflammation and extrusion of the
device, which are the most important considerations in
post keratoplasty evaluation. When compared with the
gross lesion scores of each group, there were no significant
differences between the experimental groups. However,
the gross lesion scores were significantly lower at 8
weeks after artificial cornea implantation than control.
These findings were likely because of the extrusion of
the most devices at 8 weeks post-operation. Unexpected
cornea healing was also found in three rabbits in the
experimental groups. It is assumed for cornea to be
healed followed by the inner layer of device before the
extrusion of them for 8 weeks.
To evaluate histological changes, we used hematoxylin
andeosinstain.Post-operativeinfection,completeextrusion
of the artificial cornea and partial regeneration of the
corneal tissue after device extrusion were observed upon
histological evaluation. No cell adhesions from the host
tissue to the skirt of the artificial cornea were observed.
These findings indicate that the devices could not be
attached to the recipient host tissue. There was no
difference in skirt size of the devices because separation
of the core and skirt was not confirmed in this study.
186 Yawon Hwang, Gonhyung Kim
Lab Anim Res | December, 2016 | Vol. 32, No. 4
Combined PHEMA and PMMA appears to have
sufficient advantages for production of artificial corneas
because of its optical transparency, flexibility and other
mechanical features. However, the stability and bio-
compatibility were not sufficient to enable application in
humans and animals at the present time using penetrating
keratoplasty. Further studies are essential to improve the
stability and biocompatibility with or without other types
of keratoplasty; however, PHEMA-PMMA based kerato-
prostheses will likely be used commonly in the future.
Conflict of interests The authors declare that there is
no financial conflict of interests to publish these results.
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update. Curr Opin Ophthalmol 2001; 12(4): 282-287.
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Development of a newly designed double-fixed Seoul-type
keratoprosthesis. Arch Ophthalmol 2000; 118(12): 1673-1678.
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Pouliquen YJ. A new fluorocarbon for keratoprosthesis. Cornea
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Evaluation of stability_and_biocompatibility_of_ph

  • 1. 181 Lab Anim Res 2016: 32(4), 181-186 https://doi.org/10.5625/lar.2016.32.4.181 ISSN 1738-6055 (Print) ISSN 2233-7660 (Online) Evaluation of stability and biocompatibility of PHEMA-PMMA keratoprosthesis by penetrating keratoplasty in rabbits Yawon Hwang, Gonhyung Kim* Department of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea Artificial corneas have been developed as an alternative to natural donor tissue to replace damaged or diseased corneas. This study was conducted to evaluate the stability and biocompatibility of PHEMA- PMMA [poly (2-hydroxyl methacrylate)-poly (methyl methacrylate)] keratoprostheses in rabbits following penetrating keratoplasty. Sixteen male New Zealand White rabbits aged 16 weeks were divided into three groups. Group I and group II contained six rabbits each, while the control group had four rabbits. Experimental surgery was conducted under general anesthesia. The cornea was penetrated using an 8 mm diameter biopsy punch. In group I (core 5 mm & skirt 3 mm) and group II (core 6 mm & skirt 2 mm), the keratoprosthesis was placed into the recipient full thickness bed and sutured into position with double-layer continuous. In the control group, corneal transplantation using normal allogenic corneal tissue was performed with the same suture method. After four and eight weeks, keratoprosthesis devices were evaluated by histopathological analysis of gross lesions. Post-operative complications were observed, such as extrusion and infection in experimental groups. Most corneas were maintained in the defect site by double-layer continuous suture materials for 4 weeks and kept good light transmission. However, most artificial cornea were extruded before 8 weeks. Overall, combined PHEMA and PMMA appears to have sufficient advantages for production of artificial corneas because of its optical transparency, flexibility and other mechanical features. However, the stability and biocompatibility were not sufficient to enable application in humans and animals at the present time using penetrating keratoplasty. Further studies are essential to improve the stability and biocompatibility with or without other types of keratoplasty. Keywords: Keratoprosthesis, cornea, PHEMA-PMMA, rabbit Received 16 May 2016; Revised version received 23 September 2016; Accepted 14 November 2016 More than 10 million people suffer from corneal origin blindness throughout the world, making it the second most common eye affliction next to cataracts [2,3,4,22]. Corneal blindness is caused by various diseases such as ulceration, trauma, vitamin A deficiency, keratitis, dry eye syndrome, and chemical burns [3]. In most forms of corneal blindness, the successful alternative for visual regain is corneal transplant using donor tissue [10]. However, there are several risk factors, and corneal transplants are not always possible due to limitations in the storage and supply of corneal tissue [15]. In veterinary medicine, the loss of corneal transparency may occur in response to various diseases such as corneal pigmentation, chronic superficial keratitis, severe stromal edema or fibrosis, keratoconjunctivitis sicca, and ulceration. Long- term medical or surgical therapies can be applied for non-treated cases despite initial treatment [7]. In such cases, the only alternative treatment has been the surgical application of an artificial cornea (keratoprosthesis) to restore vision [9]. The first implantation of an artificial cornea in humans was attempted in 1859 [2]. Kerato- prostheses have been made of glass, plastics, quartz or soft rubbers. Recently, carboform, hydrogels and synthetic polymers have been introduced as materials for kerato- *Corresponding author: Gonhyung Kim, Department of Veterinary Surgery, Veterinary Medical Center, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungdaero 1, Seowon-gu, Cheongju, Chungbuk 28644, Korea Tel: +82-43-261-3171; Fax: +82-43-261-3224; E-mail: ghkim@cbu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • 2. 182 Yawon Hwang, Gonhyung Kim Lab Anim Res | December, 2016 | Vol. 32, No. 4 prostheses [11,15]. The poly (methyl methacrylate) (PMMA) based keratoprosthesis was the first polymer to be used for artificial corneal transplantation [2,4,15]. Since then, keratoprostheses have been improved significantly, although the complication and failure rates remain high. The biocompatibility between artificial cornea and surrounding cornea tissue has also been found to be poor [4]. The most common complications associated with keratoprostheses are implant extrusion, infection, aqueous leakage, corneal melting, epithelial down growth, glaucoma and reduced visual acuity [1,8, 19]. To reduce the risk of complications, it is important to improve biocompatibility and cell adhesion [5]. Newly developed keratoprostheses based on poly (2- hydroxyethyl methacrylate) (PHEMA)-PMMA have been evaluated to minimize the risk of complications. PMMA-based materials have continued to be the most commonly used compounds for keratoprostheses since 1941. PHEMA is a non-toxic polymer of the toxic monomer HEMA, which was the first hydrogel used in a biomedical device [15,17]. PHEMA, which has been utilized as a soft contact lens material since the late 1950s, has a high water content, mechanical strength and biocompatibility [13]. PHEMA-PMMA based artificial corneal had more strength the tensile intensity of kerato- prostheses. PHEMA-PMMA keratoprostheses have a core and skirt design, in which the core allows trans- mission of light and the skirt allows integration with surrounding host tissue. This study was conducted to evaluate the stability and biocompatibility of newly developed PHEMA-PMMA keratoprostheses with two different skirt sizes based on the penetrating method in rabbits for veterinary and human medicine. Materials and Methods Animals Sixteen male New Zealand White rabbits aged 16 weeks and weighing 2.6 to 3.1 kg were used in this animal experiment. Rabbits were housed in a single cage at the laboratory animal research center of Chungbuk National University and maintained on a 12/12 hour light/dark cycle, standard laboratory diet and water. The experimental protocol was approved by the institutional animal care and use committee of the laboratory animal research center of Chungbuk National University. PHEMA-PMMA keratoprosthesis The PHEMA-PMMA based artificial cornea (Figure 1) consists of two parts, a central “core” optical component and a peripheral tissue-integral “skirt”. The thickness of the artificial cornea is 0.45 mm and the diameter of the core is about 5.5 mm. Experimental design Sixteen rabbits were divided into three groups: group I; PHEMA-PMMA based artificial cornea with a core diameter of about 5 mm and a 3 mm skirt implanted in 6 rabbits: group II; PHEMA-PMMA based artificial cornea with a core diameter of about 6 mm and a 2 mm skirt was implanted in 6 rabbits: control group; normal allogenic corneal transplantation was performed in 4 rabbits. All surgical procedures were performed using an operating microscope (Zeiss OPMI pico, Carl Zeiss, Goettingen, Germany). The rabbits were anesthetized using tiletamine-zolazepam (Zoletil 50, Virbac Laboratories, Carros, France) at a dose of 10 to 20 mg/kg and xylazine hydrochloride (Rompun, Bayer Korea, Seoul, Korea) at a dose of 5 to 10 mg/kg intra-muscular (IM) injection. Proparacaine hydrochloride (Alcaine 0.5%, Alcon Laboratories, Rijksweg, Puurs, Belgium) was used for topical anesthesia. After dilation of the pupil using 1% tropicamide (Ocutropic, Samil Pharma, Seoul, Korea), full-thickness corneal trephination was performed with 8 mm diameter biopsy punch and the corneal button was removed using 360 degree dissection with scissors (Figure 2). Figure 1. Gross photographs of PHEMA-PMMA artificial cornea (front view). Artificial cornea with a diameter of core is 5 mm, with 0.45 mm thick using in group I.
  • 3. PHEMA-PMMA keratoprosthesis 183 Lab Anim Res | December, 2016 | Vol. 32, No. 4 In the experimental groups, after complete excision of the cornea the PHEMA-PMMA based device was put into a corneal bed and artificial corneas were fixed 360 degrees to the surrounding cornea with 9-0 polyglactin (Vicryl, Ethicon, New Jersey, USA) sutures. Double- layer continuous sutures were placed between the remaining cornea and the skirt. In the control group, after the corneal pocket was created the normal allogenic cornea from the experimental group was put into a corneal bed. Double-layer continuous sutures with 9-0 polyglactin (Vicryl) were placed between the allogenic device and the corneal bed. Followingtransplantation,blepharorrhaphywasperformed to protect the surgical area. Antibiotic (Cefazolin; Chongkundang, Seoul, Korea; 30 mg/kg), and analgesic (Fluximine; Bomac Laboratories, Auckland, New Zealand; 1.1 mg/kg) were injected IM into each rabbit once a day for 2 weeks. Postoperatively, topical dexamethasone with polymyxin B sulfate and neomycin sulfate (Forus; Samil, Seoul, Korea) and ofloxacin (Ocuflox; Samil, Seoul, Korea) ophthalmic drops were administered once a day for 2 weeks. Gross lesion observation and scoring Gross lesion evaluation and scoring was performed at 4 and 8 weeks after surgery. Gross lesions were assigned a score of 1 to 5 depending on extrusion of the device and infection of the surgical area, where; 1 was extrusion and infection; 2 partial extrusion and infection; 3 partial extrusion and minimal infection; 4 no extrusion and minimal infection; and 5 no complications. Data of complete extrusion artificial cornea were removed from gross observation, therefore gross lesion scorings of each experimental group were evaluated at 4 weeks. For scoring at 8 weeks, four samples were used for observation and obtained from both group I and II, because of 5 artificial corneas were extruded completely. Histological examination Rabbits were sacrificed 4 weeks and 8 weeks post- implantation. The animals were anesthetized by IM injection of 20mg/kg tiletamine-zolazepam, then sacrificed. After sacrifice, the device-inserted eyes were enucleated and fixed in 10% buffered formaldehyde. Specimens were then cut along the sagittal plane and embedded in paraffin. Sections were cut into 4 µm and hematoxylin and eosin was used for staining. Statistical analysis The gross lesion scores were expressed as the means ±standard error and analyzed using SPSS version 20 (SPSS Inc., Chicago, IL, USA). Groups were compared by ANOVA followed by Tukey’s test. P values <0.05 were considered to indicate significance. Figure 2. Creation of the recipient corneal pocket and keratoprosthesis. A. The eye is proptosed by means of gentle pressure while an 8 mm biopsy punch is used to create corneal pocket. B. Corneal button remove using 360 degree dissection with scissors. C. The devices are placed into the recipient corneal pocket. D. Double-layer continuous suture is performed using 9-0 polyglactin.
  • 4. 184 Yawon Hwang, Gonhyung Kim Lab Anim Res | December, 2016 | Vol. 32, No. 4 Results Post-operative complications Post-operative complications including death, extrusion of the device, infection and melting were observed in the experimental groups. There were no complications observed in the control group. Gross lesion and scoring In the experimental groups, most corneas were maintained in the defect site by suture materials for 4 weeks and kept good light transmission. However, all artificial corneas were partially extruded before 8 weeks. In the control group, allogenic corneas were maintained well for 8 weeks and vascularization was identified on the trans- planted corneas (Figure 3). The gross lesion score of each group and score of each experimental period are presented in Table 1. In group I (n=5), group II (n=4) at 4 weeks, the gross lesion score was 3.00±1.36 and 3.33±1.33, respectively. No significant differences were observed in the gross lesion scores Figure 3. Gross photographs of the group I, II and control group at 8 weeks after implantation. A: Group I, partial extrusion of device is observed. B: Group II, partial extrusion of device is observed. C: Control group, There are no extrusion of implanted allogenic cornea and no stromal melting. A vascularization to the cornea was observed. Table 1. Gross lesion scoring of each group and each experimental period Group Score Mean±SE 4 weeks of group I (n=5) 3.00±1.36 4 weeks of group II (n=4) 3.33±1.30 8 weeks of group I and II (n=4) *2.33±1.30* 8 weeks of Control (n=4) 3.83±0.58 Group I: 4~5 mm diameter of the core, Group II: 5~6 mm diameter of the core. Data is mean±standard error of gross lesion score. *P<0.05 when compared to those of other groups. Figure 4. A: Light microscope photographs of an abnormal cornea that has post-operative infection (black arrow head). Hematoxylin and eosin staining (H&E), ×400. B: Light microscope photographs of the experimental group that has partial extrusion of the device. A separation between a skirt and corneal bed is found (black arrow head). H&E, ×100. C: Light microscope photographs of the experimental group that has complete extrusion of the device after 10 days post-operation. Regeneration of the stroma is shown (black arrow), but continuity of the epithelium is observed lost (black arrow head). H&E, ×100. D: Light microscope photographs of the control group. It shows a corneal structure as normal cornea which includes epithelium, stroma, and proliferated endothelium (black arrow). H&E, ×40.
  • 5. PHEMA-PMMA keratoprosthesis 185 Lab Anim Res | December, 2016 | Vol. 32, No. 4 between groups. The gross lesion score in control group (n=4) was 3.83±0.94, while score was 2.33±1.30 in experimental group at 8 weeks post-operation (n=4). The gross lesion score in experimental group at 8 weeks was significantly lower than those of experimental groups at 4 weeks and control group at 8 weeks (P<0.05). Histological findings In the experimental groups, complications such as inflammation of the surgical area, separation of the skirt and biological cornea due to artificial corneal extrusion, and partial regeneration of the corneal tissue were observed. In the control group, the epithelium, stroma and proliferation of the endothelium were found (Figure 4). Discussion Loss of vision can be the final result of chronic corneal disease and ulceration in both humans and animals. However, these problems cannot always be prevented or treated by general medical and surgical therapies [23]. In such cases, allogenic corneal transplantation (keratoplasty) may be an option to regain vision. However, trans- plantation has disadvantages including a high-risk of surgical failure, shortage of donor corneas, difficulties in storage, and potential for disease transmission [14]. Moreover, a previous study found that only 20% of grafts remained clear for 5 years [21]. Because of these problems, researchers have designed and encouraged the development of artificial corneas (keratoprosthesis) to replace the keratoplasty. The ideal keratoprosthesis would utilize an epithelized material that could be implanted as in keratoplasty [6]. While various kerato- prosthesis materials and techniques have been developed, many complications such as extrusion, melting and infection have been reported. To reduce the risk of complications, keratoprosthesis should be stable and biocompatible. PMMA has been the primary material used for keratoprosthesis since the early 1950s. This material has transparency, high mechanical strength, and is easy to process [21]. PHEMA is widely used in biomaterials, including soft contact lenses, because of its flexibility and high water content. Moreover, this material makes the composite core-and-skirt keratoprosthesis possible [6]. The single interrupted suture anddouble-layer continuous suture methods are commonly used in artificial cornea transplant or keratoplasty [12,16,20]. Extrusion of the device was observed frequently in the single interrupted suture method than in the double-layer continuous suture in our pre-experimental study (data not shown). Based on these results, the double-layer continuous suture method should be used for all procedures. However, single continuous suturing has become the preferred method because of its greater safety compared with the interrupted suturing. According to previous reports, multiple interrupted sutures can disrupt the even distri- bution of corneal tension [20]. Overall, three of the animals (3/16, 18%) died after implantation. Additionally, post-operative inflammation was observed in one rabbit from group I (1/16, 6%). Extrusions of artificial cornea were found in most of the animals in the experimental group. Most corneas were well maintained in the recipient site by suture materials for 4 weeks; however, all artificial corneas were extruded before 8 weeks. There was no extrusion of allogenic cornea in the control group. Overall, the results of this study indicate that the biocompatibility of the PHEMA- PMMA keratoprosthesis is not sufficient to enable combination with the host tissue during penetrating keratoplasty. A gross lesion score was applied according to the degree of inflammation and extrusion of the device, which are the most important considerations in post keratoplasty evaluation. When compared with the gross lesion scores of each group, there were no significant differences between the experimental groups. However, the gross lesion scores were significantly lower at 8 weeks after artificial cornea implantation than control. These findings were likely because of the extrusion of the most devices at 8 weeks post-operation. Unexpected cornea healing was also found in three rabbits in the experimental groups. It is assumed for cornea to be healed followed by the inner layer of device before the extrusion of them for 8 weeks. To evaluate histological changes, we used hematoxylin andeosinstain.Post-operativeinfection,completeextrusion of the artificial cornea and partial regeneration of the corneal tissue after device extrusion were observed upon histological evaluation. No cell adhesions from the host tissue to the skirt of the artificial cornea were observed. These findings indicate that the devices could not be attached to the recipient host tissue. There was no difference in skirt size of the devices because separation of the core and skirt was not confirmed in this study.
  • 6. 186 Yawon Hwang, Gonhyung Kim Lab Anim Res | December, 2016 | Vol. 32, No. 4 Combined PHEMA and PMMA appears to have sufficient advantages for production of artificial corneas because of its optical transparency, flexibility and other mechanical features. However, the stability and bio- compatibility were not sufficient to enable application in humans and animals at the present time using penetrating keratoplasty. Further studies are essential to improve the stability and biocompatibility with or without other types of keratoplasty; however, PHEMA-PMMA based kerato- prostheses will likely be used commonly in the future. Conflict of interests The authors declare that there is no financial conflict of interests to publish these results. References 1. Bleckmann H, Holak S. Preliminary results after implantation of four AlphaCor artificial corneas. Graefes Arch Clin Exp Ophthalmol 2006; 244(4): 502-506. 2. Chirila TV. An overview of the development of artificial corneas with porous skirts and the use of PHEMA for such an application. Biomaterials 2001; 22(24): 3311-3317. 3. Cuperus PL, Jongebloed WL, van Andel P, Worst JG. Glass-metal keratoprosthesis: light and electron microscopical evaluation of experimental surgery on rabbit eyes. Doc Ophthalmol 1989; 71(1): 29-47. 4. Guo P, Chen JQ, Huang LN, Wang Z, Wang ZC, Nie DY. Implantation of modified poly 2-hydroxyethy methacrylate- Polymethyl methacrylate Keratoprosthesis in rabbit and monkey corneas. Int J Ophthalmol 2009; 2(4): 310-315. 5. Hicks CR, Chirila TV, Clayton AB, Fitton JH, Vijayasekaran S, Dalton PD, Lou X, Platten S, Ziegelaar B, Hong Y, Crawford GJ, Constable IJ. Clinical results of implantation of the Chirila keratoprosthesis in rabbits. Br J Ophthalmol 1998; 82(1): 18-25. 6. Hicks CR, Fitton JH, Chirila TV, Crawford GJ, Constable IJ. Keratoprostheses: advancing toward a true artificial cornea. Surv Ophthalmol 1997; 42(2): 175-189. 7. Isard PF, Dulaurent T, Regnier A. Keratoprosthesis with retrocorneal fixation: preliminary results in dogs with corneal blindness. Vet Ophthalmol 2010; 13(5): 279-288. 8. Jiraskova N, Werner L, Mamalis N, Rozsival P. Histologic evaluation of AlphaCor keratoprosthesis explanted following various complications. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2014; 158(1): 50-55. 9. Khan B, Dudenhoefer EJ, Dohlman CH. Keratoprosthesis: an update. Curr Opin Ophthalmol 2001; 12(4): 282-287. 10. Lee JH, Wee WR, Chung ES, Kim HY, Park SH, Kim YH. Development of a newly designed double-fixed Seoul-type keratoprosthesis. Arch Ophthalmol 2000; 118(12): 1673-1678. 11. Legeais JM, Rossi C, Renard G, Salvoldelli M, D’Hermies F, Pouliquen YJ. A new fluorocarbon for keratoprosthesis. Cornea 1992; 11(6): 538-545. 12. Liu Y, Gan L, Carlsson DJ, Fagerholm P, Lagali N, Watsky MA, Munger R, Hodge WG, Priest D, Griffith M. A simple, cross- linked collagen tissue substitute for corneal implantation. Invest Ophthalmol Vis Sci 2006; 47(5): 1869-1875. 13. Lou X, Dalton PD, Chirila TV. Hydrophilic sponges based on 2- hydroxyethyl methacrylate: part VII: modulation of sponge characteristics by changes in reactivity and hydrophilicity of crosslinking agents. J Mater Sci Mater Med 2000; 11(5): 319-325. 14. Moffatt SL, Cartwright VA, Stumpf TH. Centennial review of corneal transplantation. Clin Exp Ophthalmol 2005; 33(6): 642- 657. 15. Myung D, Duhamel PE, Cochran JR, Noolandi J, Ta CN, Frank CW. Development of hydrogel-based keratoprostheses: a materials perspective. Biotechnol Prog 2008; 24(3): 735-741. 16. Nardi M, Giudice V, Marabotti A, Alfieri E, Rizzo S. Temporary graft for closed-system cataract surgery during corneal triple procedures. J Cataract Refract Surg 2001; 27(8): 1172-1175. 17. Peppas NA, Huang Y, Torres-Lugo M, Ward JH, Zhang J. Physicochemical foundations and structural design of hydrogels in medicine and biology. Annu Rev Biomed Eng 2000; 2: 9-29. 18. Pereira FQ, Bercht BS, Soares MG, da Mota MG, Pigatto JA. Comparison of a rebound and an applanation tonometer for measuring intraocular pressure in normal rabbits. Vet Ophthalmol 2011; 14(5): 321-326. 19. Sandeman SR, Lloyd AW, Tighe BJ, Franklin V, Li J, Lydon F, Liu CS, Mann DJ, James SE, Martin R. A model for the preliminary biological screening of potential keratoprosthetic biomaterials. Biomaterials 2003; 24(26): 4729-4739. 20. Vajpayee RB, Sharma V, Sharma N, Panda A, Taylor HR. Evaluation of techniques of single continuous suturing in penetrating keratoplasty. Br J Ophthalmol 2001; 85(2): 134-138. 21. Wang L, Jeong KJ, Chiang HH, Zurakowski D, Behlau I, Chodosh J, Dohlman CH, Langer R, Kohane DS. Hydroxyapatite for keratoprosthesis biointegration. Invest Ophthalmol Vis Sci 2011; 52(10): 7392-7399. 22. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective. Bull World Health Organ 2001; 79(3): 214- 221. 23. Wilkie DA, Whittaker C. Surgery of the cornea. Vet Clin North Am Small Anim Pract 1997; 27(5): 1067-1107.