Morphometrical results of Tibialis cranialis muscles in different groups and time periods
Diameter of muscle fibers
The mean diameter of muscle fibers in the second week showed a significant decrease in Tr group compared to the other treatment groups (p<0.05). In the fourth week, Tr group was significantly different from the control and S groups (p<0.05). Also, in the 8th week, all treatment groups had the highest diameter that only MC + MSC group showed a significant increase compare to the control and Tr groups (p<0.05). (Table 1).
The ratio of the number of muscle fibers nuclei to the number of fibrocytes nuclei (mn/fn)
The average of this ratio in the second week showed a significant decrease in Tr group compared to control group (p<0.05), but in the S and cell receiving groups, although there was an increase of the ratio, there was no significant difference with Tr group. In the 4th week, the lowest ratio observed in Tr groups that has a significantly different from other groups (p<0.05). Also, the highest mean ratio was observed in control and MC + MSC groups. In the 8th week, there was a significant decrease in (mn/fn) in Tr group compared to all groups (p<0.05) (Table 1).
The ratio of the number of muscle fibers nuclei to the number of muscle fibers (mn/mf)
The results of this ratio in the second and fourth week showed a significant decrease in the Tr group compared to other groups (p<0.05), but in the fourth week, this decrease was not significant with S group. In the 8th week, control group with the highest ratio had a significant increase compared to the other groups (p<0.05) and the lowest ratio belonged to the Tr group (Table 1).
The ratio of the number of blood vessels to the number of muscle fibers (v/mf)
The v/mf in the second and fourth weeks was the lowest in Tr group compared to the other groups (p<0.05), but it increased in cell receiving and S groups, but not up to the control group and there was a significant difference between these groups with control group (p<0.05). In the eight weeks, the cell and scaffold receiving groups increased the (v/mf) ratio but they were significantly different with control group (p<0.05) (Table 1).
The number of muscle mast cells
In the present study, the mean number of mast cells in the fourth and eighth weeks in the Tr group showed a significant increase compared to other groups (P <0.05) and in the second week, there was no significant difference but only with S group. In all weeks, the MC + MSC group had the lowest average number of mast cells among the other treatment groups (Table 2) (figure 2).
The percentage of sarcoplasmolysed muscle fibers
The results of this study showed that Tr group had the highest mean number of percentage of sarcoplasmolysis in all 3 time periods, which was not significantly different from S group only in the second week (P <0.05). Furthermore, the use of scaffold and cells was able to reduce the rate of sarcoplasmolysis, especially in the MC + MSC group, which had a significant decrease in the eighth week compared to other groups (P <0.05) (Table 2) (figure 2).
The mean percentage of number of necrotic muscle fibers
Results obtained from the percentage of number of necrotic fibers showed that this parameter was significantly increased in Tr group compared to the other groups in 2, 4 and 8 weeks (P <0.05). The lowest percentage of necrotic fibers was observed in MC + MSC group, which was significantly different from to the other groups in the second and fourth weeks (P <0.05) and in the eighth week, there was no significant difference, but only with MC group (Table 2) (figure 2).
The ratio of the Schwann cells number to the fibrocytes number (scn/fn) in the proximal nerve
This study showed that the mean ratio number of scn/fn in the Tr group decreased compared to the control group and had a significant difference (P <0.05) that these conditions were similar in all three time periods. However, the mentioned ratio in other groups was almost similar to the Tr group and had no significant difference with that group (Table 3).
The ratio of Schwann cells number to fibrocytes number (scn/fn) at the site of nerve transected
In the Tr group there was the lowest number of scn/fn in periods of 2 to 8 weeks, while in other groups this ratio had a significant increase compared to the Tr group (P <0.05). In the second week, the groups in which the cell or scaffold was used had no significant difference with each other, but in weeks 4 and 8, the MC + MSC group had a significant difference with the other intervention groups (P <0.05) and only in eighth week there was no significant differences with MC group (Table 3).
The mean percentage of myelinated nerve fibers number at the site of nerve transected
This evaluation showed that there was no significant difference between Tr and intervention groups in the second and fourth weeks, while in the eighth week there was a significant difference (P <0.05) (Table 3).
Evaluation of nerve fascicles formation at the site of nerve transected
The formation of nerve fascicles in 2 and 4 week periods was not observed in all groups except MC + MSC group in the fourth week. Also, in the eighth week, nerve fascicles were formed, but only in cell groups, this formation was complete (Figure 3).
The ratio of Schwann cells to fibrocytes (scn/fn) in the distal nerve
The mean data obtained from the ratio of scn/fn showed a significant decrease in the tr group compared to other treatment groups in all three time periods (P <0.05) except S group in the second week. However the use of scaffold and cell in other groups increased this ratio and especially in the MC + MSC group which was significantly different in the eighth week with most intervention groups (P <0.05) (Table 4).
The number of spheroid nerve fibers in the distal part
The highest mean number of spheroid fibers showed in the Tr group, so that there was a significant difference with other groups (P <0.05). Also, the use of scaffold and cells at the site of transection reduced this mean number in all groups, and the MC + MSC group had the lowest mean number. These results were the same in all three time periods (Table 4) (figure 4).
The mean number of distal nerve mast cells
Evaluation of this parameter showed a significant increase in 2 and 4 weeks in the Tr group, which also in eighth week had a significant difference with other groups (P <0.05) except the MSC group. In Scaffold and cell receiving groups, the mean number of this parameter decreased, so that in the eighth week, MC + MSC group had a significant decrease compared to other treatment groups (P <0.05) (Table 4).
The mean Percentage of myelinated nerve fibers in the distal part
The average percentage of myelinated fibers in the control group was higher than other groups in all time periods and showed a significant difference with them (P <0.05). But there were no significant difference observed between the other groups (Table 4) (figure 5).
The mean number of myelinated nerve fibers per microscopic area unit in the distal part
In this evaluation, increasing or decreasing number of myelinated nerve fibers in a fixed microscopic field indicates changes in their diameter. This mean in the control group, there was a significant decrease with other groups in the second and fourth weeks, which indicates the greater diameter of the fibers and the lower number of them per surface unit (P <0.05). In addition, Tr group had a significant increase compared to the other groups in eighth week (P <0.05) also, scaffold and cell groups decreased the average number by increasing the diameter of the fibers (Table 4) (figure 5).
The mean ratio of perineurium thickness to nerve fascicle thickness (per/fas) of distal nerve section
The mentioned ratio increased in Tr group in 2 and 4 week periods so that it had a significant difference with other groups (P <0.05). Despite that, in the intervention groups, the ratio decreased and showed a significant difference with the control and Tr groups (P <0.05). Also, in the eighth week, no significant differences were observed between any of the groups (Table 4).
Assay of S100 protein in distal part of the nerve
The results of this study on immunohistochemical staining showed that Schwann cell accumulation in the Tr group in the second and fourth weeks was significantly reduced compared to the other groups and in the eighth week, only with the MSC and MC + MSC groups (P <0.05). Cell and scaffold receiving groups were also able to increase Schwann cell accumulation in the distal part, so that there was a significant difference in MC + MSC group at 4 and 8 weeks with all treatment groups and with the control group there was no significant difference (P <0.05). Furthermore, in all intervention groups, this parameter increased over time in all time periods of each group (Fig 6 and 7).
Evaluation of muscle and nerve tissue parameters of each group at different time periods
This study shows the changes of each group in three time periods which is evident in most groups, especially in the eighth week, as muscle and nerve tissue regeneration over time. The results are as follows:
1) The results obtained from the average thickness of muscle fibers confirmed a significant increase in the eighth week compared to other weeks in all groups (p<0.05) except the MSC group, which did not differ significantly from the second week despite the increase in the mean of the eighth week (Table 1).
2) The average of mn\fn showed that in most groups the second and fourth weeks have no significant difference but with the eighth week is significant (p<0.05). However, in the MC group, the eighth week was not significantly different from the second, and in the MC+MSC group, also the eighth week was not significantly different from the fourth (Table 1).
3) The results of mean mn/mf ratio, mean percentage of necrosis muscle fibers, ratio of scn/fn at the site of nerve transected, mean percentage of myelinated nerve fibers at the site of nerve transected, ratio of scn/fn in the distal nerve, number of spheroid nerve fibers and mast cells in the distal part showed that in all groups the second and fourth weeks were not significantly different, but the eighth week was significantly different from the previous weeks (p<0.05) (Table 1, 2, 3, 4).
4) The ratio of vf/mf and the percentage of sarcoplasmolysis muscle fibers, in the eighth week of all groups had a significant difference with other weeks (p<0.05). However, the ratio of vf/mf in Tr and MC groups and the percentage of sarcoplasmolysis muscle fibers in Tr group in the second and fourth weeks were significantly different (p<0.05) (Table 1 and 2).
5) The number of muscle mast cells decreased over time so that in all groups the eighth week had a significant decrease compared to the second and fourth weeks (p<0.05), but in the MC + MSC group there was a significant difference only with the fourth week (p<0.05) (Table 2).
6) The ratio of scn/fn in the proximal nerve and ratio of peri/fas in distal nerve section in most groups showed no significant difference between 2, 4 and 8 weeks (p<0.05). Despite that, the ratio of scn/fn in the eighth week of the MSC group had a significant increase compared to the other weeks and in the MC + MSC group in the eighth week there was a significant difference only with the second week (p<0.05). Also, the ratio of peri/fas in the eighth week only in the MC + MSC group was significantly different from other weeks (p<0.05) (Table 3 and 4).
7) The mean Percentage of myelinated nerve fibers in all groups except S group and the mean number of myelinated nerve fibers per microscopic area unit in the distal part in all groups except Tr group in the eighth week had a significant difference with previous weeks (p<0.05) (Table 4).