Xenotransplantation Using Lyophilized Acellular Porcine Cornea with Cells Grown in vivo and Stimulated with Substance-P - Biomaterials Science and Engineering - page 388

2. Materials and methods
The animals in this study were treated according to the ARVO Statement for the Use of
Animals in Ophthalmology and Vision Research.
2.1 Preparation of lyophilized acellular porcine cornea
Adult porcine corneas were obtained from a slaughterhouse within 2 hours death, then
transported in a 4 o C moist chamber to the laboratory. Using sterile techniques, the
epithelium of each pig cornea was removed and a 4.0 mm sized stromal button with a
thickness of 300 μm was created from the central pig cornea using a microkeratome
(Automated Corneal Shaper ® ; Chiron Vision, Claremont, CA, USA). The corneal button was
treated in a mixed solution consisting of 40 μ/ml Dnase and 40 μ/ml Rnase (Sigma-Aldrich,
St. Louis, MO, USA) for 30 minutes, followed by distilled water for 2 hours, three freeze-
thaw cycles (-196C liquid nitrogen for 30 minutes, followed by rapid thawing at 37C for 30
minutes), and centrifuged (15000 ×g, 7 minutes) to removed all of the cellular components.
Then, the corneal button was treated in 100 % glycerol (Sigma-Aldrich) at 4 o C for 3 days,
stored at -80 o C for 48 hours, then lyophilized using a lyophilizer (SFDSM06; Samwon
Freezing Engineering Co., Busan, Korea) at -80 o C for 48 hrs. Finally, the corneal button was
irradiated with γ-rays (25 kGy) for sterilization.
2.2 Surgical procedure for in vivo recellularization
Forty-eight New Zealand white rabbits of either sex, weighing 2-3 kg were used for this
study. Rabbits were divided into 3 groups and anesthetized with an intramuscular injection
of mixture of Tiletamine and Zolazepam (Zolatil ® , 12.5mg/Kg; Virbac Lab, Carros Cedex,
France) and xylazine (Rompun ® , 12.5mg/kg, Bayer Korea, Ansan, Korea).
In 16 rabbits, the superior limbal conjunctivae of the left eyes were incised using Westcotts
scissors after topical anesthesia, and lyophilized APCs were inserted under the superior
conjunctivae. The conjunctivae were closed with 8-0 vicryl. One-half of the implants were
treated with substance-P (50 nmol/kg) for 1 hour before grafting. As controls, collagen
sheets (CSs) and 8 sheets of bovine amniotic membranes (AMs) were transplanted (4.0 mm
diameter) under the superior limbal conjunctivae in 16 rabbits using the same procedure.
One-half of the CSs and bovine AMs were also treated with substance-P for comparison. All
rabbits received topical levofloxacin (Cravit ® , Santen, Osaka, Japan) three times daily until
the end of the study.
2.3 Assessment of lyophilized APC with in vivo recellularization
The implants were harvested, and transparency was assessed 3day, and 1, 2, and 3 weeks
after grafting in 2 rabbits per each time.
2.3.1 Optical property
The harvested lyophilized APC was placed on a numeric panel. A 0-4+ scoring system
was devised to describe the transparency semi-quantitatively according to the visibility of
a figure through the implants. Scoring was as follows: 0, clear figure image compared
with the next numeral; 1+, minimally blurred figure; 2+, half of the blurred figure
compared with the next numeral; 3+, intense opacity with the blurred image; and 4+,
complete opacification.