Alloderm as a Topical Gene Therapy Matrix to Improve Graft Vascularity

Background Alloderm acellular dermal matrix is widely used for structural or dermal replacement purposes1-3. Given its highly biocompatible quality, its infection resistance, and its potential to vascularize4, we explored the possibility of Alloderm to function as a siRNA delivery matrix. Specifically, we sought to improve Alloderm vascularization by siRNA mediated inhibition of Prolyl hydroxylase domain-2 (PHD2), a cytoplasmic protein that regulates the HIF-1Ą pathway of angiogenic factor upregulation and neovascularization. Methods A solution of fluorescently labeled siRNA was used to rehydrate thin implantable Alloderm and Immunofluorescence (IFL) and release kinetics were performed. 12mm sections of graftable Alloderm were reconstituted in PHD2 siRNA containing solution (nonsense siRNA = control) and applied to 12mm diameter dorsal wounds of FVB mice. Grafts were sewn in, bolstered, and covered with occlusive dressings. Photographs were taken at Days 0, 7, and 14. Wounds were harvested at days 7 and 14, and analyzed (mRNA, protein, H+E, and immunohistochemistry (IHC). ANOVA determined significance (p<.05). Results IFL revealed rapid, even distribution of siRNA throughout the Alloderm. Release kinetics were first order with 80% release by 6 hours. By day 14, PHD2-containing Alloderm appeared viable, lighter colored, and adherent whereas controls were dusky and non-adherent. RT-PCR demonstrated near complete knockdown of PHD2 (fold change of 0.03) while VEGF and FGF-2 were increased 2.3 and 4.7 fold. On ELISA, VEGF was increased more than fourfold and SDF doubled. HIF-1Ą was increased in treated groups by Western Blot. H+E suggested improved graft incorporation in treated groups. IHC demonstrated increased vascularity measured by Ą-smooth muscle actin, as well as increased new cell proliferation by denser PCNA staining in treated versus controls. Conclusion Alloderm is an effective matrix for local delivery of siRNA. Strategies to improve the Alloderm matrix and/or genetically alter the local tissue environment can be envisioned.

References 1. Maurice SM, Skeete DA. Use of human acellular dermal matrix for abdominal wall reconstructions. J Surg. 2009 Jan;197(1):35-42 2. Breuing, KM and Warren, SM. Immediate bilateral breast reconstruction implants and inferolateral Alloderm slings. Annals of Plastic Surgery. Sept; 55:3, 2005. 3. Jung, SN, Chung, JW, Yim, YM, and Kwon, H. One stage skin grafting of the exposed skull with acellular human dermis (Alloderm). J Craniofacial Surgery. Nov; 19:6, 2008. 4. Wong, AK, Schonmeyer, BH et al. Histologic analysis of angiogenesis and lymphangiogenesis in acellular human dermis. Plast Reconstr Surgery. Apr; 121:4. 2008.