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International Symposium on LIGAMENTS AND TENDONS XIII

Friday, 18 October 2013 Italy, Tuscany, Arezzo

INTRA-ARTICULAR INJECTION OF TRIPEPTIDE COPPER COMPLEX GHK-CU (II) IMPROVED GRAFT HEALING IN ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION Sai-Chuen Fu,Yau-Chuk Cheuk, Wai-Hang Cheng, Shu-Hang Yung, Christer G Rolf, Kai-Ming Chan 1 1 Department of Orthopaedics and Traumatology, Faculty of Medicine, Faculty of Medicine, The Chinese University of Hong Kong. Department of Orthopaedic Surgery, Huddinge University Hospital, CLINTEC, Karolinska Institutet, Stockholm, Sweden

INTRODUCTION
Anterior cruciate ligament reconstruction (ACLR) is the standard treatment to restore knee function. However, the biological healing of the graft is often slow and poor healing can lead to ACLR failure and excessive knee laxity. If this process can be further enhanced, it will reduce the time of maturation of the graft, and hence earlier returns to full activities and sports. In the present study, the possibilities of biological augmentation of graft healing in ACLR were investigated. Recent findings show that bioactive small molecules such as matrikines may also take part in tissue remodeling. Matrikines are small peptides liberated by partial proteolysis of extracellular matrix macromolecules, which are able to regulate cell activities. Among different classes of matrikines, Glycyl-Histidyl- Lysine (GHK) tripeptide and its copper (II) chelated form (GHK-Cu) exhibit profound involvements in the tissue remodeling processes. GHK have been extensively used in the cosmetic industry for skin tissue remodeling for years with its high safety profile in humans. GHK-Cu can simultaneously activate the in vivo synthesis of matrix components, and the in vivo production of degradative enzymes and the inhibitors. GHK-Cu can promote bone healing and promote implant attachment to bony tissues. It can also act as chemoattractant for repair cells and induce wound angiogenesis. It is possible that GHK-Cu can promote graft healing in ACLR.

METHODS
The animal experiments in this study were approved by the Animal Experimentation Ethics Committee in authors’ institution (Ref. no.: 11/054/GRF and 460611). The procedures of ACLR were performed on the right knee according to our previous study. Seventy-two male Sprague Dawley rats (12 weeks old, 400-450g) were used. Intra-articular injection (50μl per injection) of saline or GHK-Cu solution (0.3 or 3 mg/ml) was performed weekly from 2nd week to 5th week post operation. At 6 or 12 weeks post- operation, the rats were euthanized to harvest knee specimens for static anterior-posterior (AP) knee laxity test and graft pull-out test (n=8). Histological scoring on H&E stained sections was performed (n=4) with polarization microscopy. A 2-way ANOVA was used to analyze the effect of time and treatment on the outcome measures. Statistical significance was accepted at α=0.05 RESULTS At 6 weeks post operation, rats treated with 0.3 or 3 mg/ml GHK-Cu resulted in a significantly smaller side-to-side difference in AP-knee laxity as compared to saline group, but no difference in AP laxity was detected at 12 weeks post operation (p=0.531) (Figure 1). There was no significant difference in pull-out strength of the graft complex between GHK-Cu groups and saline group at 6 and 12 weeks post operation (p=0.301, 0.834), 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). All grafts failed at mid-substance during the pull-out test. Histological examination showed that graft incorporation and bone healing inside tunnels was significantly better in the GHK-Cu treated groups. 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 2).

DISCUSSION
Our study suggests that GHK-Cu may improve graft healing in ACLR. GHK-Cu may improve tissue remodeling in graft mid- substance and graft tunnel interface, but extensive tissue remodeling may not be beneficial as shown in 3 mg/ml GHK-Cu group. Further studies are necessary to investigate the underlying mechanisms for the observed effects of GHK-Cu. As restoration of A-P knee laxity after ACLR was improved at earlier time point, biological enhancement in graft healing by GHK-Cu may imply earlier return to normal activity after operation. However, as the pull-out strength was not improved, high demand activities may not be warranted safe. There are still plenty of rooms for biological augmentation of graft healing in ACLR.


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