Our studies in wound fluids from diabetic subjects have shown that increased MMP-9 activities can predict delayed wound healing but the mechanism of this change and the contribution of infection to this process is not certain. Therefore in this study we used a sterile wound implant model to examine the effect of diabetes MMP-9 expression and activity. The ability of insulin treatment to prevent these changes and the role of the MMP inhibitor TIMP-1 were also investigated.
Diabetes was induced in male Sprague-Dawley rats (D:n=24, STZ:65mg/kg), some rats (n=12) were treated with Insulin (D+Ins, 10IU/day) to maintain blood glucose levels. After 6 weeks, four 1cm2 sponges were implanted subcutaneously and 3 and 12 days later the animals were sacrificed, the sponges were removed and the wound fluids (WF) and cells separated for measurement of MMP and TIMP expression and activities by zymography, RT-qPCR and ELISA. Similarly treated non-diabetic (C:n=12) rats acted as control. TLR2 and TLR4 mRNA levels were determined as markers of infection.
For all animals, MMP-9 activity and TIMP-1 expression decreased with time. Compared with control, diabetes increased the %WF activeMMP-9 (D:41% vs C: 27% at d3, D:39% vs C: 24% at d12 both P<0.02) and activeMMP-9/TIMP-1 ratio at d3 and d12 (both P< 0.02), whilst TIMP-1 levels were unchanged. Insulin treatment prevented the increased %WF activeMMP-9 and activeMMP-9/TIMP-1 ratio at d3 but was less effective at d12 when %activeMMP-9 was decreased (D:39% vs D+Ins: 32%, P<0.01) and TIMP-1 protein was increased (D:463±44 vs D+Ins:638±99 P<0.005). TLR2 and TLR4 were not different between the groups.
This study in a sterile wound healing model suggests that the diabetes induced increase in MMP-9 activity is not caused by bacterial presence or decreased TIMP-1. Further studies to elucidate the mechanism of dysregulation of the MMP-9 activities and its role in poor wound healing in diabetic wounds are required.
Supported by ARH and Rotary District 9685/9680 phD scholarship and NH&MRC.