Introduction
Diabetic wounds represent a common problem for the reconstructive surgeon.  Although the etiopathogenesis is thought to relate to impaired blood flow, recent evidence implicates deranged molecular signaling in the development and persistence of these wounds.  Specifically, these wounds are characterized by an increased rate of cell death (apoptosis), potentially mediated through hyperglycemic activation of p53 tumor suppressor.  We targeted this pathway through a novel topical gene silencing delivery system to affect wound healing.
Methods
A novel gel-based siRNA delivery system was developed to locally treat nondelimited cutaneous wounds.  Paired four millimeter diameter wounds were created on the depilitated dorsum of a db/db mouse, stented with a twelve millimeter O-ring, and covered with a transparent sterile occlusive dressing.   p53 siRNA (complexed with liposomal transfection reagent) was incorporated into an agarose matrix, applied onto the wound bed and allowed to gel.  Reapplication was performed on day 5.  Wounds were harvested every third day post-treatment for histologic and Western analysis.  A second group was followed for time-to-closure with photodigital analysis of the wounds.  Lung, liver, and spleen homogenates were examined for relative p53 suppression.
Results
Local silencing of p53 in diabetic wounds yielded greatly improved wound healing, with closure at 18 days (± 1.3d) in treated wounds vs. 28 days (± 1d) in sham treated controls (p<0.05).  Immunohistochemistry and Western blot analysis demonstrated near complete knockdown of p53 in the wound bed at 8 days.  Immunohistochemistry one month after complete healing revealed normal levels of p53, an important finding in light of the potential mutagenicity of prolonged p53 suppression.  There was no systemic suppression of p53.
Conclusion
Topical silencing of p53 markedly improved diabetic wound healing.  Silencing is near-total yet entirely local and transient, suggesting near-term clinical applicability.  Further elucidation of pathway dysregulation may help identify more specific downstream gene targets, improving both our molecular understanding of this clinical challenge and our therapeutic alternatives.