order (RS)-CPP Studies have demonstrated that unseeded

Studies have demonstrated that unseeded Dacron or Gore-Tex grafts are incorporated in the host blood vessels through a process which is generally referred to as graft healing resulting in endothelialization of the synthetic graft (Pasquinelli et al., 1990). Endothelialization can occur from blood vessel stems at either anastomosis. However, in humans, endothelial cell (EC) ingrowth is very limited, that is, a few millimeters in years (Guidoin et al., 1993). Another mechanism of endothelial healing is, EC coming from circulating order (RS)-CPP (Shi et al., 1994; Onuki et al., 1997; Graham et al., 1980; Clowes et al., 1986; Poole-Warren et al., 1996). We and others have shown that circulating ECs, endothelial progenitor cells including the myeloid/monocytic lineages contribute to re-endothelialization (Nowak et al., 2004; Asahara et al., 1997; Bailey et al., 2006). Furthermore, progenitor cells for smooth muscle cells (SMCs) are also present in circulating blood (Simper et al., 2002). In contrast to experimental animals, the flow surface of synthetic vascular grafts remains unhealed in humans, particularly in the small caliber conduits. Marked differences in graft healing exist between animals and man; and the usual mechanisms of graft endothelialization are partially ineffective in man (Pasquinelli et al., 1990). It has been shown that EC seeding of synthetic grafts prior to implantation has improved the patency of such grafts when compared to unseeded grafts (Wang et al., 1993; Deutsch et al., 2009). Strategies for repopulation of vascular conduits have usually used in vitro expanded cells derived from bone-marrow or peripheral blood. Recently, we successfully transplanted a tissue engineered allogeneic vein with in vitro expanded autologous bone-marrow derived stem cells. The tissue-engineered graft showed good patency in vivo, thus proving a promising and safe clinical approach (Olausson et al., 2012). However, the disadvantage with this approach is that the collection, isolation and large scale expansion of stem cells from tissues or bone-marrow in a limited time with low variability is highly challenging. All these observations led us to hypothesize that the use of peripheral whole blood (known to contain circulating ECs/endothelial progenitor cells (EPCs)) would result in efficient recellularization (RC) and endothelialization of acellular veins, without the need to isolate and expand subpopulations of angiogenic progenitor cells from bone-marrow or whole blood. Furthermore, an in vitro complete and preformed endothelial lining at the time of implantation would help to increase the patency of these engineered vein grafts. Thus, we aimed to develop an easy procedure that would increase the clinical use of the technique and make it globally accessible. We report the clinical transplantation of tissue-engineered allogeneic veins using autologous peripheral whole blood (APWB), in two pediatric patients with portal vein thrombosis.
Materials and Methods
Results
Discussion Recellularization using blood is advantageous over the use of in vitro expanded autologous stem cells as one may reduce the risk of spontaneous mutations that may be associated with expanded cells. One clinical study (Hibino et al., 2010) reports successful outcomes in their thoracic venous conduit using a bio-absorbable graft incubated for 2h with an inoculum of bone marrow prior to implantation without the need for extended ex vivo culture. However, aspiration of bone-marrow is an invasive technique, needing anesthesia and specialized personnel to perform the procedure and may also be associated with risk for infection. The short incubation time with isolated cells will not permit the formation of an EC layer, which is a crucial factor for clinical patency, and function of the blood vessel. Our approach promises a simplified convenient technique. We speculate that the ECM proteins and growth factors retained in the tissues after DC may enhance the attachment and growth of the recipient cells in the tissue (Li et al., 2004). At present, the definition of EPC remains controversial and is not yet consistent (Yoder and Ingram, 2009). Most commonly, marker combinations for identifying the putative circulating EPC comprise certain hematopoietic lineage markers, such as, CD133, CD34, VEGFR-2, Tie-2, and UEA-1 lectin (Yoder and Ingram, 2009; Peichev et al., 2000) and circulating myeloid cells expressing CD14 (Hristov et al., 2007; Venneri et al., 2007) — corresponding to a heterogeneous cell population possessing an overlapping phenotype with endothelial cells and hematopoietic progenitors. PBMC are also capable of developing into fibrocytes — potential vascular progenitors (Bucala et al., 1994). Fibrocytes express the common leukocyte antigen CD45 and are variably reported to express CD14 and CD34 (Medbury et al., 2008). These cells are also known to express collagen I and the alpha-smooth muscle actin. In the present study, phenotypic characterization of the cells from PWB repopulating the veins demonstrated that the majority of the cells expressed VEGFR-2, while a small fraction was also double positive for the monocyte marker CD14 (VEGFR-2+/CD14+), indicating that the major cell types are derived from the monocytic/macrophage lineage, confirming results from several published reports (Nowak et al., 2004; Asahara et al., 1997; Bailey et al., 2006; Simper et al., 2002). Interestingly, cells that migrated into the media were found to be CD45+ which also stained positive for the marker smooth muscle cell alpha actin, indicating that this cell population may include precursors of smooth muscle cells. Thus, regeneration of blood vessels can attract a variety of endothelial and smooth muscle cell precursors that may differentially express the VEGFR-2, CD45 and CD14 receptors. Perfusion with endothelial cell medium, increased, although not significantly, the numbers of VEGFR-2+ cells, indicating proliferation of these cells. We also observed the formation of a complete EC monolayer. Thus, perfusion with PWB is the most important step for endothelialization, since perfusion with PWB will permit binding of many endothelial precursor cells, while perfusion with EC medium (which contains VEGF and other growth factors) may help in proliferation and formation of an EC monolayer.