Inflammation can be influenced by thrombus formation. If a thrombus has been formed, the activated platelets release granules containing fibrinogen, coagulation factors that activate and amplify the coagulation cascade, and molecules responsible for the complement cascade activation. As a result, platelets initiate not only thrombogenic events but also inflammatory responses ^[80][81]. A crosstalk between the complement cascade and the platelets enhances the procoagulant responses mediated by inflammation. The activated platelets release P-Selectin, chemokine ligand 5 (CCL5), and chemokine ligand 5 (CXCL4), which are leukocyte chemoattractants. The presence of different GP receptors in platelets favor the interaction with the Mac 1 receptors on the leukocytes, thereby creating platelet–leukocyte aggregates (PLA) on the VG. Figure 6 shows a brief representation of the PLA generation on VGs. The PLA crosstalk increases the inflammatory and thrombogenic response by releasing factors of the coagulation and complement cascades ^[81][82]. Although this specific event represents a gap in the literature on VGs, it is recognized that PLA aggregates are very common in the development of cardiovascular diseases in which the alteration of the endothelium will promote this cascade of events, in which patients have shown an increase in PLA markers such as P-Selectin from the platelets and the P-selectin glycoprotein ligand-1 from the immune cells. For example, Esposito et al. showed that 41% of patients undergoing peripheral vascular surgery with synthetic vascular grafts, although non immediately, presented graft occlusion characterized by an increase in the monocyte–platelet and neutrophil–platelet conjugates ^[18][19].

Figure 6. Proposed scheme for platelet–leukocyte aggregates generated in vascular grafts. Platelet–leukocyte aggregates might be favored if there is a significant damage of the adjacent tissues during the implantation, as well as an inadequate hemodynamic behavior of the VG with a thrombogenic surface. In this case, the first step is protein adsorption. Due to the surgery, the F3 coagulation factor will be released, activating the extrinsic coagulation pathway. On the other hand, kininogen, prekallikrein, and factor 12 (F12) will attach to negatively charged surfaces, activating the intrinsic pathway and, finally, under low shear stress conditions, vWF is unfolded, thereby interacting with the platelet receptors. Active platelets adhere to the surface of the vascular graft and promote coagulation by autoamplification mechanisms. Active platelets also release leukocyte chemoattractants and leukocyte-binding proteins such as the P-selectin. The leukocytes adhered through the P-selectin glycoprotein ligand-1 will release a series of pro-inflammatory cytokines, which enhances the inflammatory response even further (Created with BioRender.com, accessed on 7 November 2021).

3.3.2. Complement Cascade

The complement cascade is a well-known protein-mediated system that interacts through enzymatic mechanisms to attract immune cells to the damaged tissue. Three different pathways activate the complement system. The classical pathway where C1q proteins recognize fragments of damaged cells, the lectin pathway where mannose-binding lectin (MBL) binds to sugars on the damaged cells (or to bacteria if there is an infection), and the last pathway involve small amounts of C3 protein that have been hydrolyzed to bind to other complexes. All the pathways converge in the activation of the C3 convertase, which cleaves C3 to generate C3a and C3b. While C3 is a direct chemoattractant of leukocytes, C3b joins to a different complex to generate C5a and C5b. C5b promotes the lysis of damaged cells or bacteria, but C5a is a leukocyte chemoattractant with potent platelet activator capabilities, through interactions with leukocyte and platelet receptors. Accordingly, the PLA complex is mainly responsible for the activation of the alternative pathway with the platelets to induce C3 hydrolysis, followed by the C5 auto-activation ^[82][83]. Recent reports show that VGs exhibit a low platelet aggregation and a reduction in the amount of C3a compared non-endothelialized controls and PTFE ^[83][84].

Protein adsorption on the VG’s surface regulates the interaction between the cells and the biomaterial surface, thereby largely inhibiting cell adhesion. This adsorption processes occur during the early stages after VG implantation due to their affinity to specific moieties on the surface and their surfactant behavior. Hydrophobic materials such as polyurethanes (PU) and PET easily attract and bind host proteins from the plasma and interstitial fluid. The surfactant power of certain proteins is the result of their large size, the presence of a wide array of side chain function groups, and a partial protein unfolding to obtain stability under physicochemical conditions ^[84][85]. These properties led to noncovalent interactions between the proteins and the biomaterial surface, including electrostatic, van der Waals, and entropic interactions. Additionally, stronger interactions might be induced by the denaturation of proteins on the hydrophobic surfaces, and unique hydrophobic interactions, which generally last for the first weeks and prevent cell adhesion ^[68][85][69,86].

C3a inhibition has also been shown to decrease vein graft atherosclerosis in mice models due to a decreased leukocyte attraction ^[86][87]. Additionally, increased levels of C5b have been found in PET VG compared to polymeric TEVGs ^[12][13]. These findings confirm not only the participation in the activation of the complement cascade in VGs’ failure but also the regulatory role of the endothelial cells (ECs) in thrombogenic and proinflammatory responses and the importance of endothelialization.

3.3.3. Cytokines and Degradation Products

Regarding the specific inflammatory process and cell recruitment after the VG implantation surgery, it has been reported that some of the degradation proteins from the ECM, such as hyaluronic acid, are released and are also known as damage-associated molecular patterns (DAMPs). These molecules are recognized by the toll-like receptors (TLR) or by the triggering receptor expressed on myeloid cells 1 (TREM-1), causing monocyte attachment and activation. Monocyte activation promotes the release of the macrophage colony-stimulating factor (M-CSF) and monocytes chemoattractant protein 1 (MCP1), promoting the leukocyte colonization on the lumen of the VG ^[27][28]. On the other hand, during the first 24 h post implantation, neutrophils from the perivascular tissue have been reported to infiltrate into the graft wall and amplify the signal, thereby leading to the release of MCP1 for the monocyte chemiotaxis ^[87][88]. Such an effect has been consistently observed in VG, where the number of monocytes is correlated with the number of present neutrophils ^[14][15].

In animal models, it has been reported that, on the first days, the predominant cells on the VG walls are neutrophils, but there are also infiltrated monocytes that differentiate towards macrophages ^[88][89][89,90]. It is known that both neutrophils and macrophages are responsible for the biomaterial degradation through the release of matrix metaloproteinases (MMPs) and reactive oxygen species (ROS). MMPs are themselves adhesive molecules to other monocytes. In saphenous vein grafts, in vitro experiments have shown an increase in the MMPs 2 and 9 in a ROS-dependent manner ^[90][91]. ROS are free radicals intended to break chemical bonds apart in the material to finally degrade it. However, ROS levels increase if the material resists degradation. Elevated ROS act as a second messenger interacting with redox-sensitive proteins, including receptors and transcriptional factors. In leukocytes, ROS induce the expression of intercellular adhesion molecules (ICAM-1), promoting the leukocyte-binding and -infiltration. The inflammatory response is further enhanced by the increased concentrations of M-CSF and MCP1 ^[91][92].

The transitional flow and low shear stress at the anastomosis have also been shown to increase the ROS levels. Under such conditions, the expression of proinflamatory proteins such as the tumor necrosis factor-α-mediated factor (TNF-α) is exacerbated by the activation of the JNK (c-Jun N-terminal kinase)/p38 kinase pathway ^[92][93]. TNF-α is well known to act as a signaling molecule recognized by the TNF receptor on the surface of leukocytes to activate proinflammatory and proliferative pathways.

Even though allergenic responses have only been reported in a few VG cases ^[93][94][94,95], activated mast cells have been observed in the perivascular tissue of TEVGs but fail to infiltrate into the graft itself ^[95][96]. Mast cells are known to release granules with histamine, chemokines and adhesive molecules to promote leukocyte infiltration ^[95][96]. In this sense, a local shock-like inflammatory response might be rapidly auto-amplified, thereby causing a disproportionate immune reaction to induce blood vessel contraction. This event, added to the previous thrombus formation, marks the early patency loss of VGs due to an excessive inflammatory response. For example, it has been reported that patients that undergo endovascular aneurysm repair with vascular grafts (PET) have a higher risk (14 to 60% of the total of patients) of developing a dangerous acute-phase systemic inflammatory response ^[96][97].

3.3.4. Bacterial Infection

Although rare, infection is a possible cause of failure upon graft implantation. The most common pathogens during VG implantation are staphylococcal species and E.-coli-abundant patient skin. After implantation, there are two possible infection peaks. The first one can occur during the early postoperative period, in which the infection is the result of contamination in surgery, while the second one occurs later on because of bacteremia seeding on the graft. This second type of infection will cause the instability of the immune response and does not allow for a proper microenvironment for regeneration. This is especially important because infections coming from bacteria in the graft will start showing symptoms and affecting the systemic response from the body when the proliferation of cells within the graft should start to promote vascular regeneration. In consequence, the infection resolution will compete with the regeneration of the vascular wall.

Infections happen in 0.2 to 5% of intracavitary grafts (in the abdomen and thorax) and in 6% of the cases in extracavitary grafts ^[35][36]. When infection does happen, the usual protocol is to withdraw the graft, because mortality rates range from 24 to 75% in the intracavitary cases and in about 17% in the extracavitary ones. The most common site for post-operative graft infection is the groin, where the infection can even result in limb loss ^[97][98]. When a graft is removed, bypasses might be needed. For instance, in the case of arteriovenous grafts, removing the graft will delay the starting point for dialysis, which is critical in patients with chronic kidney disease. The starting point is of special importance because bypass maturation periods oscillate from 33 to 54 days [3].

The probability of infection is related to the type of graft that is being implanted, as it depends on surface characteristics such as hydrophobicity, electric charge, functional groups, micro-pattering, roughness, and porosity of the material ^[98][99]. PTFE grafts are the least susceptible to infection, while grafts from biological origin and PET are respectively 5 and 50 times more prone to infection ^[98][99]. VG endothelialization has proven to reduce bacterial adhesion in animal models ^[99][100], and therefore tissue-engineered VGs represent an alternative to prevent infections.

3.4. CASE C: The Graft Is Successfully Implanted at the Intervention Site and the Inflammatory-Remodeling Processes Begin

It has been reported that if the TEVG successfully overcome the peri-implantation period, hematopoietic stem cells (HSCs) and already differentiated monocytes—macrophages—infiltrate into the material during the first 2 to 10 days. This infiltration occurs as a response of the MCP1 activity from monocytes interacting with DAMPs ^[27][28] and from mast cells in the periphery ^[95][96]. Another reported mechanism is the release of the macrophage inflammatory protein 1-alpha (MIP-1) produced by natural killer cells (NK) that infiltrate the vascular wall after the first days of implantation ^[100][101].

3.4.1. Macrophage’s Polarization

In physiological conditions, in response to local stimuli during the wound healing process, macrophages can modulate their polarization into M1 and M2 phenotypes. While M1 phenotype macrophages are associated to the proinflammatory pathways (inflammation, fibrosis, and material encapsulation), the M2 phenotype macrophages are associated with an anti-inflammatory response that triggers cellular recruitment, differentiation and proliferation, angiogenesis, and the degradation of bioresorbable materials ^[41][101][42,102].

The literature has shown that during the first weeks after implantation, macrophages within the VG have been reported to develop a M1 phenotype due to the proinflammatory cytokines released during previous events such as interleukin 1 (IL-1). Such phenotype is maintained by the TLR and TNF-α signaling. Although this event has not been studied in detail in vascular grafts, it is known that M1 macrophages have different functions. Firstly, M1 appear to be related to an increase in the leukocyte recruitment by the release of MCP1 and other proinflammatory cytokines. Secondly, M1 macrophages exhibit a high phagocytic activity toward material degradation via matrix metalloproteinases (MMPs) and ROS release. Finally, even in the situations of exacerbated inflammatory responses responsible for VG failure, M1 have been shown to release endoglins and semaphorins that are chemoattractant for endothelial cells. In fact, one of the sources of cells for endothelial lining in VG comes from transmural capillary infiltration through porous grafts from a granulated tissue capsule ^[102][103]. Figure 7 shows a proposed scheme of the role of acute inflammation in TEVG’s endothelialization.

Figure 7. Proposed scheme for the acute inflammation role in TEVG’s endothelialization. Monocyte infiltration occurs during the first days after implantation. The inflammatory response due to different cytokines and TNFα induces monocyte differentiation towards M1 type macrophages. M1 macrophages will initiate the biomaterial degradation process through MMP release and by ROS generation and auto-amplification signals. During the first weeks, M1 will release endoglins and semaphorins, both of which are chemoattractants for endothelial progenitors, to begin the endothelialization in the lumen and the angiogenesis in the vascular wall (Created with BioRender.com, accessed on 7 November 2021).

Other types of inflammatory cells have also been shown to be attracted by the perivascular tissue in VGs, including dendritic cells and lymphocytes T and B ^[87][88]. However, they have not been correlated with vascular graft failure, which suggests that an innate immunity response plays a more significant role than humoral immunity in the performance of vascular grafts ^[100][101].

After the first weeks, the cell response to the material and the hemodynamic conditions are the main determinants in chronic inflammation or regeneration. Chronic inflammatory outcomes typically induce intimal hyperplasia and atherosclerosis. However, regenerative pathways promote reendothelialization, which is one of the most important steps towards recovering the function of the replaced artery. We will review these two possible outcomes separately.

3.4.2. Inflammation Resolution

After the first weeks, the massive death of neutrophils and macrophages has been reported to occur in VG, having two main effects. First, the release of their inner contents activates signaling cascades from the other leukocytes, and ECs responds to promote the production of IL-4 and IL-13. Second, the interaction of macrophages with apoptotic cells inhibits the NADPH oxidase and, consequently, leads to a decrease in ROS production by the macrophages. However, this mechanism has been shown to improve the phagosomes’ proteolytic activity. Under these conditions, macrophages are no longer able to secrete molecules to degrade the material, but acquire a phenotype that allows for the phagocytosis of the necrotic tissue. This switching process usually occurs within the first month after implantation ^[103][104].

This process is known as macrophage polarization, where macrophages develop an alternative activated phenotype or M2, which is thought to mediate the remodeling of the new tissue in four different ways ^[67][68]. First, M2 release growth factors such as the transforming growth factor beta (TGFβ) and other molecules, such as Arginase I (Arg I) and MCP-1, which induce the migration and proliferation of SMCs. The interaction between M2 and SMCs through the MCP1 and its receptor (CCR2) maintains the M2 state. Second, M2 release the vascular endothelial growth factor (VEGF), the insulin-like growth factor binding protein (IGFBP-3), and the stromal cell derived factor 1 (SDF1) to induce endothelialization. Mature ECs maintain the M2 state through the NO activity and the release of IL-4. Third, M2 release anti-inflammatory cytokines such as IL-10. Fourth, extracellular matrix components are produced, including collagen type I and IV, tropoelastin, fibronectin, and glycosaminoglycans ^[95][96]. Once the endothelium is formed, the NO and the PGI induce the expression of the functional phenotype of SMCs (contractile). Contractile SMCs maintain the expression of cyclopentenyl cytosine (CPEC), which inhibits SMC proliferation but promotes EC proliferation. Figure 8 shows a proposed scheme of the macrophage polarization effects on the cell phenotype development during the regeneration process of the vascular wall.

Figure 8. Macrophage polarization effects on the cell phenotypes in the regeneration process of the vascular wall. During the first weeks, M1 macrophages stimulates EC attachment and proliferation through the release of endoglins, semaphorins, and the ECM products during the phagocytosis. After the first weeks, inflammation resolution begins with macrophage populations switching towards an M2 phenotype due to the IL-4 and MCSF released by functional ECs as well as an increase in the NO levels produced by the ECs. M2 macrophages also induce vascular wall remodeling by increasing ECs recruitment and maturation through the release of VEGF, IGFBP3, and SDF1. M2 also induce the SMCs’ expression profile towards a contractile phenotype through the release of TGFβ, ArgI, and MCP1, while contractile SMCs help the maintenance of the M2 population with the interaction of the CCR2. The interplay between mature ECs and contractile SMCs contributes to maintaining the vascular wall tone and patency (Created with BioRender.com, accessed on 7 November 2021).

Although many authors have designed TEVGs with less porous outer layers to avoid leakage or to maintain the mechanical properties, it is necessary to understand that the cells that will remodel the TEVG have two sources: the blood and the peripheral tissues. However, there is more recent evidence that the cells in the vascular wall come mainly from the peripheral tissue, and this will only happen if the vascular wall is surrounded by granular tissue ^[67][68]. For that reason, the TEVG microstructure affects cell infiltration and subsequent remodeling, and one of the main features of a TEVG should be the porosity of the conduit. This will determine its capacity to support the microcapillary formation through the vascular wall to allow cell migration from the perivascular tissue towards the graft lumen ^[42][43]. It has been found that VG with pores ranging from 30 to 90 µm are more likely to allow cell infiltration and maintain longer patency rates ^[67][68]. On the contrary, either capillary growth will be impaired or the infiltration of fibrous tissue will be enhanced.

Furthermore, to overcome the inflammatory responses and start a physiological regeneration process of the vascular wall, the microenvironment of the VG should be optimized not only to allow for cell infiltration, but also to induce reendothelialization. This means that the structure of the VG needs to be tailored for cell adhesion and migration through the balance of inflammatory cues that promote progenitor endothelial cells towards a functional endothelium, due to its modulatory roles over macrophages and SMCs.

Reendothelialization, which is the formation of a lining of endothelial cells over the surface of a VG, is a process that is still under study, and this step is critical to provide homeostasis in the vascular wall, given the multiple endothelium function in controlling the vascular wall permeability, compliance, and inflammation. Further details on vascular wall regeneration and endothelial formation can be studied separately. Figure 9 and Figure 10 shows a brief summary of the cell-biomaterial interaction required for the reendothelialization mechanism as a result of immune response activation over TEVGs, as well as the possible outcomes of the interaction of TEVGs with the native microenvironment if the failure mechanisms are activated within the early stages of integration.

Figure 9. General idealized and summarized cell biomaterial interaction for the reendothelialization mechanism as a result of immune response activation over TEVGs. On the desired outcome, during the first stages after the implantation of regenerative vascular grafts, neutrophils and monocytes adhere to the inner and outer surfaces. The chemoattractant responses of these cells induce the infiltration of different kinds of cells, including hematopoietic stem cells. The acute inflammation stages induce the attachment of endothelial precursors. After one month, infiltrated cells on the vascular wall will differentiate towards functional smooth muscle cells and fibroblasts, and the adhered endothelial cells on the lumen will maturate. Therefore, a healthy endothelium will provide the required homeostasis for maintaining vascular graft patency (Created with BioRender.com, accessed on 7 November 2021).

Figure 10. Possible outcomes of the interaction of TEVGs with the native microenvironment if the failure mechanisms are activated within the early stages of integration. (A) Instant thrombus formation causes a significant reduction of the lumen of the graft, (B) while an exacerbated inflammatory state reduces the possibilities to initiate a regenerative pathway. (C) If infection occurs, the pathogens will repopulate the graft instead of the native cells. (D) If after acute inflammation, the microenvironment stabilizes; the graft becomes a suitable substrate for tissue remodeling (created with BioRender.com, accessed on 7 November 2021).