Absolute quantification of residual DNA in a new extracellular matrix derived from human reticular dermis (HADM) using real-time TaqMan® MGB-PCR.

Abstract

The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis. The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA. It was compared the incubation of the dermis in NaOH (0.06N) and in the standard culture medium "Dulbecco Modified Eagle Medium" (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA. It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA. Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.