الفهرس 
The persistence of epithelial remnants and the viability of palatal connective tissue Graft using single incision method versus de- epithelialization method in the New Zealand rabbits.
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Abstract
The aim of our work was to compare the de-epithelialization graft methods with the
standard single incision technique for harvesting connective tissue grafts. The viability potential
of the graft was examined histologically & immunohistochemically and histomorphometrically.
Necrotic cells were also detected using flow cytometry.
Material and methods:
Twenty adult male Newzealand rabbits were used in this study with an age range from
7 to 8 months and weight range from 2500 to 3000 g. This research was conducted in the Animal
Research center faculty of medicine, Cairo University, Egypt according to the recommendations
and approval of the ethics committee (IACUC) ( approval no.CU-III-F-71-18) on animal
experimentation of the Faculty of Medicine, Cairo University. The animals were divided into two
groups:
group I: 10 rabbits in which the single-incision was done with lancet 15c to perform a
pouch for harvesting the connective tissue graft. The dimensions of the donor area were measured
using a millimeter periodontal probe, 3 mm in width and 10 mm in length.
group II: 10 rabbits in which de-epithelialization was done using round stone, after
marking the extension of the graft as mentioned above.
At each experimental endpoint, the single incision group animals, the reflected flap was
sutured back with interrupted suture using 5/0 absorbable suture material. Regarding, the deepithelialized
group a hemostatic gauze was placed on the palatal wound, immersed with vitamin
K to stop any bleeding, and finger pressure was applied until complete hemostasis was attained.
Then, analgesia was given via the drinking water (100 mg metamizole per kg body weight each
24 h) Mcleod et al.(2009); Martín-Piedra et al.(2017).
Experimental procedure
Single incision prodeure
First, all animals were deeply anesthetized by intramuscular injection of 50mg/kg of
ketamine HCL and xylazine 2%. Then, a #15C scalpel blade was used to make a single horizontal
incision in the existing edentulous area between the 1st molar and the incisors 2 mm from the
gingival margin, The angle of the blade was 90° to the bone. Then, an undermining preparation
toward the median was started within the first incision. The underlying CTG was separated from
the surrounding connective tissue by making incisions to the bone on the mesial, distal, and medial
sides of the graft. The graft was then removed by separating it from the bony surface with a
periosteal elevator.
De-epithelialization procedure
A large flat stone under copious irrigation was utilized to de-epithelialized the graft
with careful handling to ensure that the epithelium was removed uniformly. The thickness of the
graft was standardized in agreement with the visualization of a homogenous and bleeding surface,
indicating total removal of the epithelium. When the connective tissue appeared in regions, the
graft was incised with the aid of a No. 11 blade and removed from the donor area.
Laboratory methods and procedures:
The grafts were fixed in 10% neutral buffered formalin for 24 hours, washed, processed,
embedded in paraffin, and then sectioned.
4 microns thick sections were stained with:
1- Hematoxylin and eosin stain for histological examination to find out the remnants of
epithelium.
2- Immunohistochemical localization of cytokeratin10 in the connective tissue graft.
Flow cytometry analysis for Necrotic cell assay
The cells were trypsinized, washed, and resuspended in PBS then each cell was stained for
20 minutes in the dark with monoclonal antibodies conjugated with phycoerythrin (PE) and 7-
amino-actinomycin D (7 AAD-Sigma Aldrich) was added for 20 minutes in all tubes to ensure
gating on viable cells