Purpose: Recalcitrant infections often require microsurgical free tissue transfer to fill soft tissue and osseous defects. Despite the transfer of vascularized tissue, there is still a 30-40% failure rate due to infection. Antimicrobial peptides are a ubiquitous group of natural molecules with minimal pathogenic resistance. This study examines the utility of using free flaps in a therapeutic as well as reconstructive capacity to deliver the human antimicrobial peptide LL37 ex-vivo.
Methods: Fasciocutaneous flaps were raised in adult fischer rats and transfected ex-vivo with adenovirus-LacZ reporter gene (4.6x109PFU) or adenovirus-LL37 (4.4x109 PFU) +/- vascular endothelial growth factor (VEGF) to improve transduction efficiency. Flaps and periflap tissue were analyzed at days 3,7,14 and 24 for transgene expression by ELISA and/or immunohistochemistry. Distal organs were also analyzed as a measure of toxicity. A reproducible catheter infection model with bioluminescent Staphylococcus Aureus (~5x108 cfu/ml) was also established to determine the biological efficacy of ex-vivo transduction of these flaps with the therapeutic adenovirus-LL37. Non-invasive bioluminescent photon emissions were recorded and final bacterial counts were analyzed at day 7.
Results: Maximal â-galactosidase expression from the adenovirus-LacZ reporter gene was demonstrated immunohistochemically and by ELISA (62ng/ml) at day 7. LL37 expression at day 7 was demonstrated by ELISA (70ng/ml). The addition of VEGF resulted in a 50% increase in transduction efficiency (104ng/ml) and a 30% increase in peri-flap transgene expression. The spleen and liver showed minimal protein expression. Flaps transduced with adeno-LL37 demonstrated superior bacterial clearance compared to controls (~4x105 cfu/ml vs ~3x107 cfu/ml).
Conclusions: We have demonstrated successful transduction of the reporter gene, LacZ as well as the therapeutic gene product, LL37 using ex-vivo gene delivery methods. The gene product was expressed maximally at day 7 and was concentrated in the flap and peri-flap tissues, where recalcitrant infection commonly occurs. Transduction efficiency was improved with the addition of VEGF and biological efficacy was demonstrated in a rat infection model. We believe that the ex-vivo gene transduction of microvascular free flaps with therapeutic genes is feasible, efficacious and safe and has enormous potential in the field of reconstructive plastic surgery.