Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts

Abstract

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient’s cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

“…Another explanation for the insufficiency of VEGF-A 165 to stimulate the ADSC proliferation is a lack of the VEGFR2 receptor on human ADSCs, resulting in a decreased sensitivity of these cells to VEGF, as suggested in a study by Bassaneze et al (2010) [ 48 ]. Nevertheless, in our recent study, VEGF-A 165 attached to a decellularized pericardium through a fibrin mesh increased its recellularization with ADSCs and subsequent endothelialization in comparison with a pericardium modified only with fibrin [ 13 ].…”

“…Recent preclinical applications of VEGF-A 165 include therapy of spinal cord injury in a rat model [ 7 ], regeneration of sciatic nerve in rats, where VEGF-A 165 was combined with FGF-2 [ 8 ], functionalization of artificial blood vessel replacements for promoting spontaneous endothelialization of these grafts in an ovine model [ 9 ], treatment of placental insufficiency [ 10 ], and healing of cutaneous wounds in mice [ 11 ]. VEGF-A 165 has also been widely used for experimental work in vitro, e.g., creation of vascularized tissue-engineered constructs, intended for bone regeneration [ 12 ] or functionalization of a decellularized pericardial matrix, intended for cardiovascular tissue engineering, in order to promote its recellularization with endothelial and stem cells [ 13 ]. In this context, it is worth mentioning that VEGF-A 165 immobilized on cell cultivation substrates can also act as an extracellular adhesion molecule, binding to integrin adhesion receptors on cells [ 14 , 15 ].…”

Complexation of CXCL12, FGF-2 and VEGF with Heparin Modulates the Protein Release from Alginate Microbeads

“…The presence of Hep in scaffold/delivery systems can prolong GF/chemokine release from days to weeks. This outcome is very important, since a prolonged release of FGF-2 or VEGF supported in vitro ingrowth of adipose tissue-derived stem cells and their differentiation towards smooth muscle cells, as well as attachment of ECs on the pericardium surface [ 70 ], improved in vivo long-term colonization of grafts with ECs and smooth muscle cells with excellent graft patency after 18 months [ 71 ], or stimulated intensive in vivo vascularization of polylactide scaffolds within four weeks [ 72 ]. Furthermore, a long-term CXCL12 release prolonged islet/beta cell graft function without systemic immunosuppression [ 28 , 32 ].…”

Spatiotemporal Extracellular Matrix Modeling for in Situ Cell Niche Studies

“…During the development of the StEMM, the main focus was on the log‐ and lag‐phases of a well‐defined MPC culture, where the cell density and cell marker expression of CD46 in culture of periods between 8 and 96 hours was compared, thus capturing the association of ECM composition with cellular phenotypes and thereby identify regions of interest. Surface manipulation of decellularized ECM using exogenous matrices or specific enzymatic removal of endogenous molecules alters cellular responses during culture 58,59 . Retaining native complex matrices after decellularization is therefore of great importance for such studies, where not only the gross macrostructures need to be preserved, but also basement membrane proteins and bound paracrine factors and cytokines.…”