Quantification of DNA in urinary porcine bladder matrix using the ACTB gene

Abstract

Extracellular matrix (ECM) is a rich network of proteins and proteoglycans that has proved to be very useful in tissue regeneration. Porcine ECM has been proposed as a biological scaffold, and urinary bladder matrix (UBM) has demonstrated superior biological properties; however, its use in human treatment requires ensuring that it is DNA free. Several protocols have been used for decellularization and to demonstrate the absence of DNA, but until now, a porcine housekeeping gene for quantifying DNA by real-time quantitative PCR (qPCR) has been limiting. The aim of this study was to propose a protocol to quantify the DNA content of decellularized UBM by qPCR for the beta-actin gene (ACTB). A total of 20 porcine bladders were used, and each bladder was divided into three pieces: one as a control and the others decellularized with either SDS or Triton X-100 detergent. The presence of DNA was assessed by histology, spectrophotometry, conventional PCR, and qPCR for the ACTB. Histological analysis demonstrated the absence of nuclei using both protocols. Spectrophotometrical evaluation resulted in DNA concentrations of 1561.4 ± 357.1 and 1211.9 ± 635.2 ng of DNA/mg dry weight after the SDS and Triton X-100 protocols, respectively. DNA was not detected in any protocol by conventional PCR. In contrast, using qPCR, we found 3.9 ± 2.8 ng of DNA/mg dry weight in the Triton X-100 protocol. Therefore, the use of qPCR is a reliable method to quantify residual DNA content after decellularization procedures.

Evaluation of Decellularized Matrix and ß-Tricalcium Phosphate as Biomaterials for Bone Neoformation: In vivo Study

“…To avoid this, it is possible to treat the ECM by decellularization (SilvaBenítez et al, 2014;Silva-Benítez et al, 2015), which renders it possible to remove the majority of the antigenic epitopes. The presence of the Gal epitope on the surface of the vascular endothelium is the first cause for rejection of xenogeneic transplants (Badylak & Gilber).…”

“…The models were divided into two experimental groups: lyophilized UBM; beta-TriCalcium Phosphate (β-TCP) (Sigma-Aldrich, St Louis, MO, USA), and a Negative Control (NC) group. Lyophilized UBM was developed as previously mentioned in the literature (Silva-Benítez et al, 2014;Silva-Benítez et al, 2015). Surgical procedure.…”

“…Tissue Engineering (TE) is a multidisciplinary field that applies principles of biology and engineering to the development of tissue substitutes to recover, maintain, or increase the function of diseased, damaged, or lost tissues (Silva-Benítez et al, 2015). The field of TE is focused on the need to provide functional equivalents of native tissues (Burdick & Vunjak-Novakovic, 2009) and to avoid the limitations related with the standard treatments by creating an in vivo local environment, enabling cells to promote their proliferation and differentiation through the utilization of biomaterials (Tabata, 2009).…”

“…There are characteristics that make the ECM suitable for tissue regeneration as follows: it possesses structural and functional proteins that constitute part of the native mammalian ECM; it acts as a controlled-release vehicle of growth factors; it is a rich source of many types of collagen; it participate in the retention of growth factors from the surrounding tissues; it exerts an influence on the modulation of the immune host response; it guides the microenvironmental signals, and it has suitable biomechanical characteristics (Badylak, 2002;Badylak, 2007;Badylak et al, 2009;Liu et al, 2012;Benders et al, 2013). However, when the ECM is used as a biomaterial, there is the possibility of immune rejection (Badylak & Gilbert, 2008, Badylak et al) and a potential inflammatory response from the host Silva-Benítez et al, 2015) related with the antigenic epitopes of the native cellules of the source tissue (Badylak & Gilbert; Badylak et al); nonetheless, the decellularization process of the ECM Silva-Benítez et al, 2015) could be employed to avoid this limitation (Badylak & Gilber;Silva-Benítez et al, 2015).…”

“…The porcine submucosa Urinary Bladder Matrix (UBM) is one of the most commonly ECM used as a biomaterial (Benders et al;Silva-Benítez et al, 2015). It has demonstrated excellent biological properties compared with the ECM obtained from other tissues (Silva-Benítez et al, 2015) due to its composition and structure, in that the UBM mainly comprises collagen (90 %), principally types I, III, IV, V, VI, and VII (Badylak et al), forming fibers with interconnections between them that allow for cell invasion and angiogenesis (Benders et al), which permit the arrival of nutrients, cells, molecules, and growth factors (Zhou et al, 2010).…”