The need for vascular conduits has always been a challenge in the field of vascular surgery, and research has been focused on creation of an alternative to prosthetic grafts. Most of the recent research on tissue engineered vascular grafts is based on electrospun nanofibers using polymers such as poly ε-caprolactone, but there are still many barriers that need to be overcome such as integrity issues, among others. Another alternative for vascular conduits are decellularized vessels obtained from animals. Through a decellularization process, all cells from an animal blood vessel can be removed, leaving an intact extracellular matrix, therefore maintaining the structural properties of a blood vessel, while immunologic responses associated with xenotransplantation can be avoided. Previous reports on decellularized vascular conduits have mainly relied on host cell recullularization after implantation but this can lead to early thrombosis if appropriate endothelialization is not achieved. In this study we used decellularized porcine aorta and devised a method of in vitro recellularization using hMSCs, in combination with SDF-1 and PDGF-BB in an attempt to improve cell reattachment efficiency and potency.

Porcine aorta were harvested from 6-month old pigs and a decellurization protocol consisting of 1% Tripton-X, 1% sodium dodecyl sulfate (SDS) and 1M dimethyl sulfoxide (DMSO) was used for removal of cells. By using DMSO as a penetration enhancer, the effect of SDS and Tripton-X was enhaced, thereby achieving full decellularization in only 3 hours while maintaining the integrity of the extracellular matrix. DNA and ECM protein quantification, as well as histologic analysis of the tissues were performed in order to demonstrate effective decellularization. For the in vitro recellularization procedure, human bone marrow mesenchymal stem cells (hMSC) were cultured and divided into 4 groups: control group, SFD-1 (100ng/ml) group, PDGF-BB (50ng/ml) group, and SDF-1 + PDGF-BB group. Cell proliferation viability was first checked for the 4 groups, and hMSC (1x107 cells) were then seeded onto decellularized tissue surfaces after fibronectin coating under static conditions with either SDF-1, PDGF-BB or a combination of both for 1 week using the respective growth media.

Using our modified DMSO-based decellularization protocol, DNA quantification showed a decrease in DNA content from 20,701.7ng to 113.3ng DNA/10mg dry weight in the decellularized aortic tissue compared to normal tissue (Fig. 1A), while sulfated glycosaminoglycan (sGAG) quantification representing the integrity of the ECM was similar between normal and decellularized aortic tissue (Fig. 1B). Additionally, histologic analysis demonstrated loss of nuclear material on H&E and DAPI stain, and preserved ECM on collagen stain as well as SEM (Fig. 1C and 1D). After cell culture of hMSC with SFD-1 (100ng/ml), PDGF-BB (50ng/ml), and combination of SDF-1 + PDGF-BB, cell proliferation assay demonstrated that the combination group showed the highest cell proliferation at 3 hours, while the control group (native hMSC) showed the lowest proliferation (Fig. 2A). After cells were seeded onto decellularized aortic tissue with the respective growth factors, cell attachment confirmed by live-dead imaging at 2 weeks demonstrated that the combination group had the highest cell reattachment rate compared to the control group (Fig. 2B). This was confirmed by quantification using an image analyzing software (40.5 ± 2.62 vs 22.0 ± 2.54, P<0.01 for combination group vs control, respectively) (Fig 2C).

Our modified protocol for decellularization demonstrated that aortic tissue were effectively decellularized while maintaining structural integrity and in shorter time (3 hours) compared to previously reported protocols. In vitro recellularization of decellularized aortic tissue using hMSCs was significantly improved by a combination of SDF-1 and PDGF-BB. We suggest that the SDF-1/CXCR4 pathway and the increased expression of VCAM-1 by PDGF-BB are likely to play a role in the increased adhesion and migration of MSCs onto the decellularized aortic tissue. Although further in vitro and in vivo studies are required, this preliminary study demonstrates a potential use of decellularized animal blood vessels as an alternative for vascular conduits.