In this study the synthetic peptide TP508 was tested in a mouse model mimicking high energy-fracture conditions with soft tissue injuries, and showed positive effects on enhancing fracture healing. The time to union in mouse fracture models is about 3 weeks [6, 7], but in the current study, most of the animals in the control group did not achieve fracture union at 5 weeks, suggesting a delayed fracture union. However, the mechanical, radiographic and histological data demonstrated a superior fracture healing in the group receiving an injection of 100 μg TP508 into the fracture gap. This is in agreement with previous studies showing the benefit of TP508 in enhancing healing of various musculoskeletal tissues [8, 9]. The group receiving 10 μg TP508 into the fracture gap did not lead to a significant improvement of the fracture healing, suggesting that the dose of TP508 administration is important. The positive effects of TP508 on tissue repair appear to be dose-dependent. Previous studies had used various doses of TP508, ranging from 0.1 μg in excision wounds in rats  to 300 μg in rabbit distraction osteogenesis studies . In a rat closed femoral fracture study, Wang et al  noted a TP508 dose dependant increase in fracture strength, 1 μg TP508 group increased the fracture strength by 21% and 10 μg TP508 group by 36% relative to the control group. Since most of the studies have used TP508 in a soluble injection form and given at the same time as the injury, and only a small amount of TP508 could retain their bioactivities to the repair phases, therefore a higher dose of TP508 is needed to show the positive effects. Recently, studies have shown that TP508 given in a slow release microsphere form is more effective in enhancing bone repair and consolidation even at a reduced dose . In the present study, we have used two doses of TP508 (100 and 10 μg/ml) in PBS delivery form based on the data from previous studies, and the data showed that the higher dose 100 μg/ml resulted in significant promoting effects of fracture healing. The use of controlled slow release form of TP508 with the same dose in the similar animal model will be the subject for future investigation.
We have also used one group where TP508 (100 μg/ml) was administrated into the crushed muscle and it was hoped that TP508 will help to reduce the adverse effects of the pro-inflammatory cytokines released from the traumatised muscles and enhance fracture healing. In vitro and in vivo studies have shown that TP508 altered the inflammatory response through an increase in the expression of IL-1 and IL-2 , and to recruit endothelial cells and osteoblasts through chemotaxis to the wounded areas [13–15]. Wang et al showed in a rat closed femoral fracture model, a single percutaneous injection of TP508 improved the resultant biomechanical properties of the healing fracture, and TP508 induced gene expression of early growth factors, inflammatory response modifiers and angiogenesis-related factors . The immune genes and growth factors that have been down-regulated by TP508 included several MHC Class II genes, Interferon-γ, IL 1β receptor type 2, IL10 and IL12 . The ability of TP508 to alter the immune response was also found in the dermal tissues, in several wound healing studies it was found that TP508 could suppress inflammatory responses at later stages of healing [1, 10]. These findings are in agreement with those of Ryaby et al and Li et al who in a rat closed diaphyseal fracture mode  and in a rabbit distraction osteogenesis model [11, 12] described a significant reduction in the number of inflammatory cells at the later stages of healing. Although there was no statistical difference between Group II and the control group in fracture callus volume and mechanical properties, there was significant reduction of scar tissue formation in the crushed muscles in group II, suggesting that TP508 may have a positive effect on muscle repair and regeneration, and this may in turn to facilitate soft tissue recovery and angiogenesis following high energy fracture. The use of TP508 to aid soft tissue healing needs future careful investigation.
As angiogenesis is an essential part of fracture repair  and early studies have noted that TP508 may have positive effect on angiogenesis. TP508 was shown to promote both the size and number of blood vessels in the chick chorioallantoic model  and TP508 enhances angiogenesis throug up-regulation of the c-Fos and c-Jun genes and not the VEGF or MMP-2 genes . This agreed with Vartanian et al who used a model of angiogenic sprouting and showed that TP508 did not increase VEGF gene expression . In their assay, TP508 stimulated angiogenic sprouting to an extent similar, to the intact thrombin molecule, but the proteolytically active receptor agonists had no effect on angiogenic sprouting, thus TP508 may promote angiogenesis through its non-proteolytic receptor pathways . In the present study, we have found that there was increased blood vessel formation in the crushed muscles and fracture gap areas and significantly reduced scar formation in the groups receiving TP508 (regardless the dose) comparing to the saline control group, indicating that the enhancement of fracture repair by TP508 is partially associated with the enhanced angiogenesis induced by TP508.
In conclusion, local administration of TP508 (100 μg) into the fracture gap has promoted fracture repair in a mouse model of high-energy fracture. The effect appears to be dose dependent and is associated with reduced inflammation and enhanced angiogenesis in the surrounding soft tissues and in the fracture gap. TP508 may therefore be used to argument high energy fracture healing and more research work is needed to determine the best form and dose of TP508 delivery for its potential clinical applications.