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127 
Figure 7.7 
Effect of vitamin C irrigation saline on (A) Anchor Print Ratio (APR), (B) Target Print Ratio (TPR), (C) Swing 
Duration Ratio (SWR) and (D) Limb Idleness Index (LII) on ACLR rats 
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128 
7.2  Effect of GHK-Cu on ACLR 
7.2.1  Animal model 
Among 72 operated rats, 3 rats died during anesthesia and were replaced. Surgical 
accidents  were  recorded,  including  fixation  failure  (1)  and  sub-optimal  tunnel 
placement (4). Two rats (from 0.3 mg/ml and 3 mg/ml GHK-Cu groups respectively) 
were  found  dead  after  the  third  session  of  intra-articular  injection.  Post-surgical 
complications were also recorded as re-sutures of wound  due to  bites (4), patella 
dislocation  (1)  and  infection  (1).  Rats  with  surgical  accidents  or  post-surgical 
complications were excluded for statistical analyses of gait data and mechanical test 
data. The minimum sample sizes are 7 per group for mechanical/gait analysis and 3 
per group for histological scoring. 
7.2.2  Anterior-posterior (A-P) knee laxity test and graft pull-out test 
The A-P knee displacement of the operated knees were significantly higher than 
the contralateral sides (repeated measure ANOVA, p <0.001), and the side-to-side 
differences were significantly reduced from 6 to 12 weeks post operation (p <0.001). 
At 6 weeks post operation, rats treated with 0.3 or 3 mg/ml (p=0.012, 0.049, Scheffe 
post-hoc test) GHK-Cu resulted in a significantly smaller side-to-side difference in 
AP-knee laxity as compared to saline group (ANOVA, p=0.009), but no difference in 
AP laxity was detected at 12 weeks post operation (p=0.531) (Figure 7.8). There was 
no significant difference in failure load of the graft complex between GHK-Cu groups 
and saline group at 6 and 12 weeks post operation (p=0.301, 0.834) (Figure 7.9), 
however, the stiffness of the graft complex was significantly higher in 0.3 mg/ml 
GHK-Cu as compared to saline group at 6 weeks post operation (p=0.007) (Figure 
7.10). All grafts failed at mid-substance during the pull-out test. 
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129 
Figure 7.8 
Effect of GHK-Cu on anterior-posterior (A-P) knee laxity 
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130 
Figure 7.9 
Effect  of  GHK-Cu  on  failure  load  of  femur-graft-tibia 
complex 
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131 
7.2.3 
Histological analysis 
The results of histological scoring were shown in Table 7.3. At 6 weeks week 
post operation, 0.3 mg/ml GHK-Cu group exhibited less graft deterioration and better 
graft  incorporation  as  compared  to  saline  control,  but  the  differences  were  not 
statistically significant at 12 weeks week post operation. Higher dose of GHK-Cu (3 
mg/ml)  exhibited  no  significant  difference  in  graft  degeneration  and  graft 
incorporation as compared to saline control at both 6 and 12 weeks post operation. 
Figure 7.10 
Effect of GHK-Cu on stiffness of femur-graft-tibia complex 
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132 
Bone  tunnel  healing  score  with  respect  to  tunnel  size  exhibited  no  significant 
difference across experimental groups at 6 and 12 weeks post operation.   
Table 7.3 
Results of histological scoring of the study of the effects of 
GHK-Cu 
Time post operation 
Saline 
0.3 mg/ml GHK-Cu  3 mg/ml GHK-Cu 
Graft degeneration 
6 weeks 
6.5 
12 weeks 
Graft incorporation 
6 weeks 
12 weeks 
3.5 
Bone tunnel healing 
6 weeks 
5.5 
12 weeks 
At 6 weeks post operation, a complete bony layer surrounding the graft was noticed in 
GHK treated groups as compared to the leaky tunnel interface (femoral and tibial 
tunnels) in saline control at 6 weeks post operation (Figure 7.11, 7.13). At 12 weeks 
post  operation,  graft  inside  bone  tunnel  was  surrounded  by  bony  shell  and  no 
significant difference was noticed in all experimental groups. Graft incorporation was 
better in femoral tunnel (Figure 7.11) as compared to tibial tunnels (Figure 7.13) in 
all groups, yet graft integrity was better inside tibial tunnel as compared to femoral 
tunnel. Graft degeneration  as shown by decreased collagen birefringence  was  less 
severe in the 0.3 mg/ml GHK-Cu group as compared to saline group, but significantly 
increased cell recruitment to graft mid-substance in 3 mg/ml GHK-Cu group also 
rendered poor graft integrity (Figure 7.12).   
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133 
Figure 7.11 
Histological examination of femoral tunnel under bright field and polarized illumination for evaluation of the 
effect of GHK-Cu on ACLR rat knees. (Optical magnification: 100X) 
6 weeks post operation 
12 weeks post operation 
Saline
0.3 mg/ml GHK
-
Cu
3 mg/ml GHK
-
Cu
134 
Figure 7.12 
Histological examination of intra-articular graft mid-substance under bright field and polarized illumination 
for evaluation of the effect of GHK-Cu on ACLR rat knees. (Optical magnification: 100X) 
6 weeks post operation 
12 weeks post operation 
Saline
0.3 mg/ml GHK
-
Cu
3 mg/ml GHK
-
Cu
135 
Figure 7.13 
Histological examination of tibial tunnel under bright field and polarized illumination for evaluation of the 
effect of GHK-Cu on ACLR rat knees. (Optical magnification: 100X) 
0.3 mg/ml GHK
-
Cu
3 mg/ml GHK
-
Cu
6 weeks post operation 
12 weeks post operation 
Saline
136 
Figure 7.14 
Effect  of GHK-Cu on (A) Anchor  Print  Ratio (APR), (B)Target  Print  Ratio (TPR), (C) 
Swing Duration Ratio (SWR) and (D) Limb Idleness Index (LII) on ACLR rats 
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