Marco político y los objetivos a alcanzar

The type of cells used in the preparation of TEVGs is an extremely important consideration, since the cell type can directly affect the structure of the graft and how it performs in vivo. Autologous adult vascular cells such as SMCs, ECs, and fibroblasts have been used in many cases. There are drawbacks to the use of these cells, however, extracting them requires blood vessel biopsies which are very invasive, cause donor site morbidity, and may even be impossible due to impaired vessel quality or availability. Additionally, adult cells in general are limited in replicative and regenerative capacities due to their age.59 As a result, the use of adult stem cells in tissue engineering and

regenerative medicine has gained a lot of importance, especially due to their potential to improve scaffold remodeling and integration.81 Stem cells, by definition, are able to self- renew, exist in a undifferentiated state, and are capable of differentiation along multiple lineages.82 There are several types of stem cells which include progenitor cells, bone marrow mononuclear cells (BM-MNCs), mesenchymal stem cells (MSCs), adipose, muscle-derived stem cells and induced pluripotent stem cells (iPSCs).59

Mesenchymal stem cells (MSC), with their multipotent capacity, are the most well characterized source of adult stem cells for application in tissue engineering. These are a heterogeneous population of fibroblast-like cells that are found in most adult organs.81 MSCs are defined by three main criteria which were established by the International Society for Cellular Therapy: adherence to plastic dishes, specific surface antigens (CD73+, CD90+, CD105+, CD45-, CD34-, CD14 or CD11b-, CD79- OR CD19- , HLA-DR), and in vitro ability to give rise to adipocytes, osteoblasts, and

chondrocytes.83 MSCs can be isolated from a variety of sources including bone marrow, umbilical cord blood, and adipose tissue.81

Adipose-derived stem cells are particularly advantages for tissue engineering applications. These cells have been shown to differentiate into both SMCs and ECs and they can be easily harvested in high quantities from adipose tissue aspirate.59 _{They have}

shown the ability to secrete several pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF); and they can

differentiate into contractile SMCs with a combination of TGF-β1 and bone

application in TEVGs include their multipotency, secretory functions and

immunomodulatory properties.83 These cells can maintain their stemness for several passages, and subcutaneous adipose tissue is abundant and easy to harvest. Liposuction is a common surgical procedure that is safe, and a large number of cells can be obtained with minimal risk. Isolation of ASCs can be achieved using a simple procedure in which the adipose tissue is minced and digested in collagenase, followed by centrifugation which results in a cell pellet known as the stromal vascular fraction (SVF). The SVF provides a rich source of adipose derived stem cells (ASCs). Furthermore, repeatable access to subcutaneous adipose tissue offers an obvious benefit over the isolation of MSCs from bone marrow.81 Approximately 1 in 30 cells from the SVF become the ASC population, with an estimated yield of 100,000 to 1 million ASCs per gram of fat. It has also been determined that gender, BMI, renal failure, diabetes, smoking, cardiovascular disease and peripheral artery disease does not significantly affect ASC yield.84

Several studies have demonstrated that cell seeding promotes TEVG patency and longevity, however, the exact mechanism by which the cells promote neo-vessel

formation is not entirely known. There is still no known correlation between the number of cells seeded and results in long term graft function. There is are a variety of seeding techniques that have been developed, but many of them are lengthy and limited in clinical applicability. In order to mass produce small-diameter, off-the-counter vascular grafts, it is necessary to develop a cost-effective, reliable, and efficient seeding technique. The most common method used is static seeding, in which a concentrated cell suspension is injected onto a scaffold. There are many limitations to this technique that result in low

seeding efficiency and minimal cell penetration of scaffold walls, however, it is the simplest and fastest method. After application of the cell suspension, the construct is incubated with media to allow cell attachment. Statically seeded cells are incubated with the scaffold from several hours to several days to maximize seeding efficiency. Other seeding techniques include dynamic, magnetic, vacuum, electrostatic and centrifugal as well as incorporation of biological glues such as fibrin or fibronectin.57 _{Key features for}

any seeding techniques include preserving cell viability, providing a uniform cell distribution, attaining high seeding efficiency and minimizing the seeding time.67

ASCs are increasingly being studied for their low immunogenicity and

immunomodulatory properties including immune stimulation and immunosuppressive effects. ASCs have the ability to reduce inflammation and tissue damage, and also exert effects on both the innate and acquired immune systems with profound implications for monocyte differentiation, macrophage response, and T and B lymphocyte behavior.85

Suppression of chronic inflammation is key for successful TEVG integration. Stem cell immunomodulation is advantageous for tissue engineered construct integration for reducing inflammatory cytokine production, suppressing cytotoxic T-cell proliferation, and stimulating the secretion of anti-inflammatory cytokines such as IL-4 and IL-10. ASCs may also regulate macrophage polarization by reduction of classically activated pro-inflammatory M1 macrophage phenotype in favor of the alternatively activated pro healing M2 macrophage phenotype.86