Fistula morphology
After five weeks, no visible fistula orifice was observed in any groups. The external appearance of the anus was mildly deformed in 2 rabbits in the control group, but all animals in SIS and SIS-PRF groups had a normal anal appearance. No discharge was seen at the site of the fistula or anus. No extrusion was seen during these five weeks in the SIS-PRF group.
Scanning electron microscopy
SIS contained relatively straight fibers in a smooth background of tissue on the abluminal side and. It was denser and less fibrous on the stratum compactum side compared to the abluminal side. Abluminal side of the SIS-PRF scaffold showed the same as the abluminal side of the SIS one. But, on the stratum compactum side of the SIS-PRF scaffold, fine and even accumulations of cells (platelets and leukocytes) was seen (Figure3.A).
Biomechanical tests
Tensile strengths showed no significant difference between two groups. Elongation-at-break also was similar between SIS and SIS-PRF groups (Table 1). Load-elongation curve of SIS as a sample can be seen in Figure 4.
Table 1. Biomechanical tests
Groups
|
SIS (n=6)
|
SIS-PRF (n=6)
|
P-value
|
Tensile strength (N)
|
4.49 ± 0.17
|
4.51 ± 0.21
|
0.89
|
Elongation-at-break (mm)
|
1.64 ± 0.10
|
1.63 ± 0.13
|
0.94
|
Histopathological analysis
Although macroscopic investigation showed only two open fistula tracts in the control group, microscopically, there were 4 and 1 remaining fistula tract in the control and SIS groups, respectively. In the control group, moderate and severe inflammation was seen in the site of the fistula tract (figure 5. C). Inflammation was reduced in the SIS group, but the SIS-PRF group was significantly less inflamed than the SIS group (p-value = 0.01). Two rabbits in the control group had severe inflammation with concurrent macroscopic open fistula tract while animals in other groups did not show severity in histological analysis. All the animals in the SIS-PRF group showed mild inflammation. In the SIS group, mild inflammation was seen in 2 animals, and others showed moderate inflammation. The only microscopic open fistula tract in the SIS group had moderate inflammation. There was also a significant decrease in the number of microscopically open fistula tracts in the SIS-PRF group compared to the control group.
A mild foreign body reaction was seen in one of the rabbits in the SIS group, but it was not significantly comparable to other groups. Granulation tissue was observed in one of the animals in the control group, which was one of the rabbits with a macroscopic open fistula tract.
We also compared groups according to neovascularization, but there was no difference between groups.
Connective tissue formation was comparably higher in the SIS-PRF group than SIS (p-value =0.55) and control (p-value=0.22) groups. A brief summary of the histological analysis is shown in Table 2.
Table 2. Histological analysis
Groups
|
control
|
P1-value
|
SIS
|
P2-value
|
PRF-coated SIS
|
P3-value
|
Fistula tract (open or closed)1
|
0.6 (0-1)
|
0.09
|
0.16 (0-1)
|
0.34
|
0 (0-1)
|
0.01
|
Neovascularization2
|
1 (1-3)
|
NA
|
1 (1-3)
|
NA
|
1 (1-3)
|
NA
|
Inflammation severity2
|
2 (1-3)
|
0.49
|
1.6 (1-3)
|
0.01
|
1 (1-3)
|
<0.001
|
Foreign body reaction1
|
0 (0-1)
|
0.34
|
0.16 (0-1)
|
0.34
|
0 (0-1)
|
NA
|
Granulation tissue formation1
|
0.16 (0-1)
|
0.34
|
0 (0-1)
|
NA
|
0 (0-1)
|
0.34
|
Connective tissue formation2
|
1.3 (1-3)
|
0.42
|
1.6 (1-3)
|
0.55
|
1.8 (1-3)
|
0.22
|
P1-values are results of the comparison of control and SIS groups. P2-values are results of the comparison of SIS and PRF-coated SIS groups and P3-values are results of comparison of control and PRF-coated SIS groups
Chi-square test was used for statistical analysis
1 Values are shown as -=0 and +=1
2 Values are shown as 1=mild, 2=moderate and 3=severe