Which of these responses is general inflammatory or immune

INFLAMMATION

Inflammatory responses to biomaterials are characterized by immune cell infiltration and adhesion to the materials and their subsequent production of inflammatory cytokines. In its acute state, inflammation is characterized by swelling as blood pours into the injured site along with the emigration of neutrophils and other leukocytes from the circulation into the implant site [95]. Neutrophils, attracted by the soluble factors (e.g., PDGF and TGF-β) released by platelets and complement, are responsible for engulfing microorganisms, releasing degradative enzymes and reactive oxygen intermediates ROIs, and the clearance of bacteria and foreign materials [95]. Monocytes are recruited following the initial influx of neutrophils, and may differentiate into macrophages. Macrophages are very important cells in repairing injury [13, 63]. These leukocytes will adhere to the protein monolayer through ligandreceptor interactions. Monocytes and then macrophages attempt to phagocytose the foreign biomaterial while releasing a variety of growth factors—including TGF-β, PDGF, and GM-CSF—that promote the migration, proliferation, and the activation of additional macrophages [13, 171].

The inability of the immune system to clear biomaterial implants is hypothesized to result in “frustrated phagocytosis,” which causes chronic inflammation as macrophages may actively persist throughout the lifetime of the biomaterial [13]. The accumulation and activation of macrophages will continue as long as they are unable to phagocytose and rid the body of the biomaterial, and macrophages will increase their production of degradative enzymes and later fuse to form foreign body giant cells (FBGCs) to increase their capacity to engulf the material [14]. The macrophages continue to produce the proinflammatory cytokines TNF-α, IL-1, and IL-6, which up-regulate the expression of adhesion molecules on inflammatory cells and facilitate the recruitment of a large number of cells to the implant site [63, 117, 171]. Infiltrating macrophages and dendritic cells (DCs) are the antigen-presenting cells that are most responsible for generating immunocompetent responses to a particular antigen, but in reactions involving biomaterials the lack of proper antigen and activation signaling may induce these cells to continually produce reactive oxygen molecules and proteases in attempts to destroy and break down the biomaterial [88, 110, 114, 140]. (See Table 31.2.)

TABLE 31.2. The important phases, cells, and chemical mediators in the foreign body response to biomaterials.

3.3 Pathophysiological role of mechano-transcription in gut inflammation

Inflammatory response in the GI tract involves numerous cellular and molecular events that are orchestrated by cytokines, chemokines, and other inflammatory mediators such as prostaglandins, nitric oxide and cell surface adhesion molecules (Papadakis and Targan, 2000; Stadnyk, 2002). These pro-inflammatory mediators can be secreted by inflammatory cells or non-inflammatory cells such as epithelial cells, myofibroblasts, and smooth muscle cells in the gut (Stadnyk, 2002; Powell et al., 1999; Shi and Sarna, 2005). Although gut inflammation and infections are the most visible causes of production of pro-inflammatory mediators, recent studies have shown that mechanical force is a potent pro-inflammatory stimulus in the gut (Wehner et al., 2010; Lin et al., 2014b).

Wehner et al. reported that static mechanical strain significantly induced iNOS, COX-2, and IL-1β gene expression levels in cultured intestinal smooth muscle cells and peritoneal macrophages (Wehner et al., 2010). Mechanical stimulation also amplified LPS-induced iNOS and IL-1 gene expression in intestinal smooth muscle cells, and similarly COX-2 and IL-6 mRNA expression in macrophages. Lin et al. further investigated mechano-sensitive expression of pro-inflammatory mediators in vitro, in vivo, and ex vivo in primary culture of colon smooth muscle cells and found that static stretch significantly induced mRNA expression of iNOS, COX-2, IL-6, and MCP-1 (Lin et al., 2014b). Gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In an in vivo model of partial colon obstruction, they found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, compared to the distal segments or sham control animals. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of pro-inflammatory transcription factor NF-κB p65 leading to increased mRNA expression of more inflammatory mediators in naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators, indicating a critical role of mechanical stress-induced IL-6 in the secondary activation of pro-inflammatory transcription factors. These studies demonstrate that mechanical stress upregulates gene expression of certain cytokines, chemokines, and inflammatory mediators i.e. iNOS, COX-2, IL-6, and MCP-1 in gut smooth muscle cells and other cells in the intestine. The mechanical stress-induced mediators are well recognized in inflammatory response. MCP-1 is a potent chemoattractant recruiting lymphocytes, monocytes and other inflammatory cells into the local site (MacDermott, 1999; Gijsbers et al., 2006). IL-6 is a well-defined pro-inflammatory mediator involved in immune cell activation and differentiation and plays a key role in the development of inflammatory bowel disease (Papadakis and Targan, 2000; Mudter and Neurath, 2007; Pawłowska-Kamieniak et al., 2021). Needless to say, iNOS and the COX-2/prostaglandin E synthase/PGE2 axes are among the best-described pathways implicated in inflammatory regulation (Krause and DuBois, 2000; Mohajer and Ma, 2000; Mancini and Di Battista, 2011).

Recent studies have tested if mechanosensitive expression of certain pro-inflammatory mediators has a role in immune response in Crohn's disease (CD). Osteopontin (OPN) is a secreted glycoprotein with many demonstrated roles in the regulation of immune response on multiple levels (Uede, 2011; Rittling and Singh, 2015). Sato et al. found that active CD patients demonstrated significantly higher plasma OPN levels than normal or UC patients, and the elevated plasma OPN levels in CD patients were significantly correlated with disease activity (Sato et al., 2005). These findings were later confirmed by other groups (Agnholt et al., 2007; Komine-Aizawa et al., 2015). Further, OPN was found to facilitate production of IL-12 from lamina propria mononuclear cells (LPMC) and is closely involved in the Th2 immune response in CD (Sato et al., 2005; Ashkar et al., 2000). However, the cellular source and mechanisms of increased plasma OPN in CD are not clear. We found that expression of OPN was dramatically upregulated in the mechanically distended colon in bowel obstruction (Lin and Shi, 2021). OPN level was also increased in the plasma in rats with bowel obstruction. In the rat model of stenotic Crohn's colitis, OPN expression was found increased not only at the inflammation site, but at the distended pre-inflammation site (Lin and Shi, 2021). Plasma OPN level was significantly increased in stenotic Crohn's colitis rats. However, prevention of inflammation-associated obstruction with liquid diet eliminated OPN expression in the pre-inflammation site and normalized plasma OPN level. These results suggest that OPN expression in Crohn's colitis is largely regulated by mechanical stress, and the plasma OPN levels in colitis are closely related to the extent of bowel distention.

5.3.2.2 TOPSi particles

The inflammatory response of thermally oxidized-PSi (TOPSi) particles has also been investigated using RAW 264.7 macrophage cells. The results obtained highlight the prospect of macrophage activation and consequent inflammatory response, increasing progressively with particle size. In this study, PSi microparticle fractions (size: 1–10, 10–25 μm; specific surface area: 202 m2/g) and nanoparticle fractions (size: 97, 125, and 164 nm; average zeta potential: − 33.67 mV) were prepared (Bimbo et al., 2011). The in vitro biocompatibility of these fractions was subsequently investigated, by observing the behavior of the PSi particles on the Caco-2 and RAW 264.7 murine macrophage cells.

It was reported in this study that the nanoparticles that possessed an average size of 164 nm elicited a lower amount of oxidative stress, because they produced the least amount of ROS, compared to the other particles (Bimbo et al., 2011). Further indicators of potential oxidative stress, such as the production of nitric oxide, and TNF-α were also examined. Higher production of nitric oxide and TNF-α was observed from the microparticles, indicating that oxidative stress may show a progressive increase as the size of the particles increases. In the bid to validate these preliminary observations, the authors proceeded to examine the toxic responses induced by the PSi particles on the Caco-2 cells and the RAW 264.7 macrophages, using fluorescence and luminescence assays (Bimbo et al., 2011). These assays revealed the toxicity of the PSi particles, with the microparticles reported to be more toxic and disruptive of the Caco-2 cell membranes than the nanoparticles and triggering a subsequent decrease in the metabolic activities of the cells.

Host Inflammatory Response

The inflammatory response manifests primarily as acute (minutes-to-days) and chronic (weeks-to-months) responses based on the duration and intensity of inflammatory stimuli and its mitigation in situ. Generally, the acute inflammatory response to biomaterials resolves quickly, usually within a week, depending on the extent of injury at the implant site and the type of biomaterial in the IMD. Chronic inflammation is less uniform histologically, resulting from constant and variable inflammatory stimuli from the implant’s presence, mechanical irritation as implant–tissue micro-motion, or degradation components produced by the implant. The chronic inflammatory response to biomaterials is usually confined to the implant site and can range from weeks to months to years (Anderson, 1988). In fact, the host response can be expected to persist for as long as the biomaterial remains in the individual. Multiple cell types, both resident within and recruited to the tissue around the implant site, as well as diverse molecular mediators, are involved in propagating, sustaining, and resolving the inflammatory response.

The predominant cell type presents in the inflammatory response varies with the age of the injury. Neutrophils (polymorphonuclear leukocytes, PMNs) characterize the acute inflammatory response. In general, neutrophils dominate during the first several days following injury and then are replaced by infiltrating blood-derived monocytes/macrophages as the predominant cell type. Neutrophils are short-lived cells that attack pathogens and foreign materials at the wound site and disintegrate after 24–48 h of wound formation. Neutrophils are often accompanied by host mast cells in acute inflammatory phases. Mast cell activation results in degranulation, with histamine release and fibrinogen adsorption known to mediate acute inflammatory responses to implanted biomaterials (Tang et al., 1998). The extent of release of cytokines interleukin-4 (IL-4) and IL-13 from mast cells in degranulation processes plays a significant role in subsequent development and degree of the FBR (Zdolsek et al., 2007). Biomaterial-mediated inflammatory responses may be modulated by histamine-mediated phagocyte recruitment and phagocyte adhesion to implant surfaces facilitated by adsorbed host fibrinogen, among many other possible host proteins (Anderson and Patel, 2013). Monocytes arriving at the implantation site following earlier PMNs undergo phenotypic changes, differentiating into macrophages. Monocyte infiltration depends on chemotactic cues from tissue injury as well as inflammatory signals secreted by PMNs. That this recruitment depends on the implanted biomaterial characteristics and tissue site is arguable: it appears to be relatively ubiquitous. Chronic inflammation is characterized by the presence of precursor monocytes, macrophages, and lymphocytes adhered to the biomaterial in addition to the proliferation of blood vessels associated with both macrophage and endothelial cell actions, and abundant connective tissue produced by late arriving myofibroblasts.

The progression of events in host inflammation and eventual FBR requires the extravasation and migration of monocytes/macrophages to the implant site. The guided movement of monocytes/macrophages to the implant occurs in response to evolving presence of multiple cytokines, chemokines, and other chemoattractants produced at the implant site upon injury, resulting acute hemostasis, and associated immediate acute inflammatory cell responses. Following blood–material interactions associated with acute wounding (surgery and implant placement naturally always produces wounding, even if minimally invasive as discussed in Chapter 2), platelets in the resulting clot release chemoattractants such as transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), CXCL4 (platelet factor, PF4), leukotriene (LTB4), and IL-1. These agents can direct blood monocytes and tissue-resident macrophages to the wound site (Broughton et al., 2006). Interaction of implant-adsorbed proteins with adhesion receptors present on inflammatory cell populations constitutes the major cellular recognition system for implantable synthetic materials and medical devices (Hu et al., 2001). Adsorbed wound-site proteins such as albumin, fibrinogen, complement, fibronectin, vitronectin, globulins, and many others are implicated in modulating host inflammatory cell interactions and are thus linked to subsequent inflammatory and wound healing responses (Jenney and Anderson, 2000). Understanding protein adsorption in vivo is complicated by the number and different types of proteins present, and that their adsorptive interactions with biomaterials surfaces vary with time, often independent of their relative mass fractions present in biological milieu (i.e., the so-called Vroman effect, Bamford et al., 1992) and Chapter 5. That these proteins likely change their compositional fractions and resulting wound-site reactivities further confounds interpretations of their involvement in the aged FBR response. Most Vroman effects with biomaterials have been studied in the context of blood coagulation. Little is known about the alterations in the Vroman response or protein alterations of the FBR as a function of age.

Recruitment of macrophages to the implant site further propagates chemoattractant signals. Macrophage activation in situ prompts production of PDGF, tumor necrosis factor (TNF-α), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) attracting more macrophages to the wound site (Broughton et al., 2006). Monocyte chemotactic protein (CCL2 or MCP-1) is known to surround implanted polyethylene materials (Hu et al., 2001). An array of other inflammatory mediators including IL-1, IL-6, IL-10, IL-12, IL-18, TGF-β, IL-8, and macrophage inflammatory protein (MIP)-1α/β are also produced by monocytes/macrophages (Rot and von Andrian, 2004; Fujiwara and Kobayashi, 2005). Macrophages are also capable of secreting growth and angiogenic factors important in the regulation of fibro-proliferation and angiogenesis (Singer and Clark, 1999). Alternatively, activated macrophages over-express certain ECM proteins, such as fibronectin, and are involved in tissue remodeling during wound healing (Mosser, 2003). The diverse biological functions of activated macrophages play central roles in inflammation and host defense response. A comprehensive discussion of macrophage plasticity and the role of this cell type are discussed in Chapter 6.

Macrophages are professional phagocytes capable of ingesting large amounts of small particles and debris (<5 µm), while larger particle sizes (>10 µm) cannot be internalized. The inability of macrophages to phagocytose supra-cellular sized foreign objects leads to “frustrated phagocytosis” around such large objects (Mosser, 2003), releasing mediators of degradation such as reactive oxygen intermediates (ROIs, oxygen free radicals) or degradative enzymes around the biomaterial surface (Henson, 1971). This inflammatory reaction, prolonged if the foreign body (i.e., biomaterial) resists degradation and phagocytic clearance, also correlates with the formation of multinucleated giant cells known as foreign body giant cells (FBGCs) (Xia and Triffitt, 2006). As discussed in detail in Chapter 2, cell–cell fusion of monocytes and macrophages to form multinucleate FBGCs requires a series of highly orchestrated biochemical and cellular events around the implant (Chen et al., 2007a). FBGCs display an antigenic phenotype similar to monocytes and macrophages formed from the fusion of monocyte-derived macrophages (Athanasou and Quinn, 1990). Formation of these cells is a histological hallmark of the FBR, although the precise role for FBGCs in the FBR is still unresolved. Their presence is generally localized to the implant surface and correlates with increased fibroblast presence around the implant and the encapsulation of the biomaterial (Shive and Anderson, 1997). Further understanding of dynamics and interactions of immune system components with inflammatory cells at implants is crucial for designing controls for these events to improve the host response, tissue integration, safety, biocompatibility, and function of these devices (Anderson et al., 2008).

4.3 Skin inflammation: care or damage

The inflammatory response plays a pivotal role in directing the outcome of the healing response and is intimately linked to the extent of scar formation. Although the mechanisms that orchestrate the differences in the scar outcome are not well understood, they possibly reflect an altered inflammatory and/or cytokine profile (Eming et al., 2007; Harty et al., 2003; Martin and Leibovich, 2005). Pathogens and/or endotoxins prolong the release of proinflammatory cytokines such as IL-1 and TNF-α, leading to an unbalanced inflammatory response (Edwards and Harding, 2004; Guo and DiPietro, 2010; Strbo et al., 2014). These processes contribute to chronicity and healing failure (Edwards and Harding, 2004; Menke et al., 2007).

Some evidence indicates that overactive neutrophil response delays wound healing and/or leads to excessive scar formation as a consequence of an exaggerated release of MMP-8 and neutrophil elastase (Canesso et al., 2014; Catalano et al., 2013; Lobmann et al., 2002; Menke et al., 2007; Yager et al., 1996) , thus generating the idea of their dual role in wound healing. In nonhealing wounds, MMPs are not adequately balanced by their endogenous tissue inhibitor of metalloproteinases (Bullen et al., 1995; Yager et al., 1996).

In pathological healing conditions, it is theorized that macrophages fail to switch to the M2 phenotype (Willenborg and Eming, 2014). In chronic venous leg ulcers the prolonged persistence of M1 macrophages may be tissue destructive (Mahdipour et al., 2011; Rodero et al., 2013; Sindrilaru et al., 2011). Thus minimizing M1 activation and promoting M2 activation in the context of chronic inflammation could represent an effective therapeutic strategy to normalize the pathways.

Excessive inflammatory conditions contribute to a deficient ECM due to the rapid degradation of collagen and other ECM components that overwhelm their synthesis by connective tissue cells. In chronic wounds, several fibroblast functions are altered. These fibroblasts show impaired proliferative and migratory capacities as well as reduced responses to growth factors (Ågren et al., 1999; Brem et al., 2007). This also results in healing failures. Furthermore, the chronic wound fibroblasts produce altered quantities of cytokines responsible for the enlarged scars (Menke et al., 2007).

LL-37 protein levels are generally low in chronic wounds, while mRNA expression levels are elevated.

Inflammation and the host response to interventions and grafts

The inflammatory response to vascular interventions is complex because these patients have systemic inflammation, which may influence the degree and direction of local inflammation. Potent chemoattractants such as complement 5a (C5a) and leukotriene B4 recruit neutrophils to the graft surface where they localize in the fibrin coagulum of the graft’s inner and outer capsule via β2 integrins. Also, IgG binds to the neutrophils’ Fcγ receptors activating neutrophils’ proinflammatory response while inhibiting normal clearance of bacteria. Control of neutrophil response (and Netosis) may have great benefits in vascular tissue engineering applications [47]. Neutrophils also interact with various other deposited proteins, including C3bi and factor X, and they adhere to the ECs in the perianastomotic region through selectin- and integrin-mediated mechanisms. L-selectin is thought to modulate neutrophil/EC interactions by presenting neutrophil ligands to both E- and P-selectin on the vascular endothelium. In addition, selectin–carbohydrate bonds are important for the initial cellular contact while the integrin–peptide bonds are responsible for strengthening this adhesion, as well as the transmigration of neutrophils. Both intercellular adhesion molecular-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the EC surface bind these integrins as well, and ECs upregulate ICAM-1 and express VCAM-1 when stimulated by inflammatory agonists such as interleukin (IL)-1, tumor necrosis factor (TNF), lipopolysaccharide, and thrombin. Further, activated neutrophils release oxygen-free radicals and various proteases, which result in matrix degradation and may inhibit both endothelialization and tissue incorporation of the vascular tissue and grafts [48].

Circulating monocytes/macrophages are also attracted to areas of injured or regenerating endothelium, especially in response to IL-1 and TNF-α. There are many plasma monocyte recruitment and activating factors, including LTB4, platelet factor 4, and platelet-derived growth factor (PDGF). This process is propagated in the presence of these plasma activating factors, driving monocytes to differentiate into macrophages that direct the host’s chronic inflammatory response via the release of proteases and oxygen-free radicals. They also are thought to be critical to promoting arteriogenesis via monocyte chemotractant protein-1.

A variety of cytokines are released from the inflammatory cells activated by vascular grafts. Lactide/glycolide grafts are composed of bioresorbable materials that are phagocytosed by macrophages; in culture these materials stimulate macrophages to release mitogens that stimulate vascular cells. This mitogenic activity appears to be related to the secretion of fibroblast growth factor (FGF)-2 since pretreatment of the culture media with a neutralizing anti-FGF-2 antibody significantly diminishes the stimulatory effect on SMC growth in culture [49]. Cultured monocytes and macrophages incubated with Dacron and ePTFE have been demonstrated to produce different amounts of IL-1β, IL-6, and TNF-α that are biomaterial specific [50]. TNF-α is one of the factors that may contribute to the enhanced proliferation of SMCs caused by leukocyte-biomaterial interactions, while IL-1 may be partly responsible for the increased SMC proliferation caused by leukocyte-EC reaction. IL-1 also induces upregulation of insulin-like growth factor-1 expression in ECs, and coculture of neutrophils with IL-1β-treated ECs dramatically increases PDGF release.

It is attractive to think that the host inflammatory response can be manipulated to promote favorable cellular and protein responses with the goal of promoting autogenous ingrowth of TEVGs. Recently, Shinoka and Breuer have demonstrated that the populating cells can be manipulated by modifying the homing cells with angiogotensin inhibitors [51]. Since the inflammatory reaction elicits a cascade of growth processes, it has also been proposed that approaches attenuating the initial inflammatory reaction may improve long-term graft patency. Over time certain inflammatory and profibrotic responses limit the long-term patency of vein grafts [52–54] and can promote a more aggressive atherosclerosis via endothelial to mesenchymal transition [55]. Excitingly focused antiinflammatory interventions may be able to counter this inflammatory process [56]. Similar inflammatory processes are likely involved in arterial disease. We have recently demonstrated that flow-mediated arterial stiffening occurs through specific and reversible profibrotic pathways in both mice and patients with peripheral arterial disease (PAD) [57], and now for the first time, investigators have demonstrated that antiinflammatory medication can decrease cardiovascular events such as strokes and heart attacks [58]. These pathways are likely also important to AV fistula and biologic graft function [51,59], and clinically available blockade of angiotensin signaling pathways may limit this process [51].

10.3.4.4 Control of Chemotaxis and Cellular Adhesion

The inflammatory response is characterized in part by local vasodilation and the accumulation of white blood cells at the site of insult. White blood cells, in particular, macrophages and neutrophils, accumulate at the site of “insult” via a process known as chemotaxis. Chemotaxis is the directed movement of cells up a concentration gradient toward a site of inflammation. The concentration gradient that it follows is known as a class of molecules known as chemoattractants. Chemokines are a special class of cytokines that induce cell directed movement (chemotaxis). Chemokines such as interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) are released from cells at the site of injury. The leukotrienes belong to a class of molecules (eicosanoids) generated from free arachidonic acid via the 5-LOX enzyme. Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils. In addition, LTB4 will elicit the adhesion of white blood cells to the endothelial cells that form the walls of capillaries.

The control of this series of events is achieved first and foremost by the release of prostaglandins which, as we described previously are responsible for the increase in blood flow and as a consequence for the transport of the immune cells (neutrophils, macrophages) to the site of damage in the skin. Various plant-derived compounds have been shown to counteract the chemoattraction process, and as a result to reduce the severity of the inflammatory reaction.188

Examples of a few plant derived compounds such as caffeine, sucrose, and a class of sugars called Sialyl-Lewis, have been shown to reduce the infiltration of leukocytes into the skin. More specifically, 3′-Sialyl lactose has been shown to inhibit cell adhesion, and is thought to act by binding to the Sialyl-Lewis X which is expressed on the cell membrane of neutrophils.189

3.4.1 Nanorobots as Cellular Assistants in Inflammatory Responses

The inflammatory response is a complex biological reaction of the body which appears when healthy tissues are wounded by physical/chemical stimuli or are invaded by bacteria, viruses, or toxins. The protective response of the injured tissue includes immune cells, blood vessels, and molecular mediators. Selective microscopic robots are investigated to assist the healing process of injured tissues, because they are believed to act quickly and neatly together with the white blood cells in helping the injured tissue to heal without unesthetic scars. The nano dimension advantage of robots allowed their migration with white blood cells across the vessels (Casal et al., 2003; Ager, 2003). Furthermore, the presence of nanorobots will be detected by the immune system and because of the nature of these devices, the risk of being digested by phagocytic cells is higher. The researchers are working to preprogram the robots to avoid macrophages, phagocytes, and lymphocytes or to escape from them (Freitas, 1999). Freitas proposed coating nanorobots with a passive diamond layer. The smoothness and flawlessness of the coating layer give a low reaction from the human immune system. However, if the nanodevices are trapped inside phagocytes, the nanorobots can induce exocytosis of the phagosomal vacuole or inhibit both phagolysosomal fusion and phagosome metabolism (Freitas, 2001). Nanorobots have several advantages over common therapeutic methods because they can be preprogrammed to find and fight infection or can compare the healthy tissue with damaged tissue at a molecular level.

The current nanotechnology methods provide more options for treatments by tailoring manageable nanorobots which can quickly rid the blood of nonbacterial pathogens such as viruses, fungus cells, or parasites. Almost every autonomous molecular machine operating in the human body provides opportunities for “cleaning up” after a disease because the affected genes can be repaired without removing the damaged cell (Saha, 2009). Appropriate properties are presented by hybrid nanodevices containing natural and biodegradable polymers and inorganic nanoparticles with antibacterial properties. Biodegradable collagen derivatives, elastin, keratin, and amino acid derivatives show promising results for engineering nanorobots to assist in inflammatory processes (Mark et al., 2009).

Anti-inflammatory activity

The inflammatory response involves many types of tissues and cells. These cells produce some common modulators like: eicosanoids, cytokines, ROS, and nitrogen intermediates. The eicosanoids are classified into three big groups, prostaglandins and prostacyclins, leukotrienes, and thromboxanes.

The prostaglandins are lipid mediators implicated, not only in inflammation, but also, in other pathological processes, such as edema, fever, hyperalgia, cancer or Alzheimer’s disease. The cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of PGE2, TXB2 and prostacyclines from arachidonic acid.

Leukotrienes are involved in immune-regulation, asthma, inflammation, and various allergic conditions. In the presence of 5-lipooxygenase (5-LOX), free arachidonic acid is converted to 5-HPTE, which is then reduced to 5-HETE or dehydrated to a non-stable intermediate LTA4 [115]. LTA4 is further converted enzymatically to leukotrienes, LTB4 and LTC4 [116, 117]. The LTC4 is a leukocyte chemotaxic that participates in cell adhesion, superoxide production, calcium translocation and the release of different enzymes.

Therefore, there are different targets to attack the inflammatory process. Due to this, the study of the anti-inflammatory activity by the PPGs has been studied following different experimental models, trying to justify their mechanism of action as anti-inflammatory agents.

Verbascoside has shown carrageenan-induced rat paw edema inhibition [105].

Liao and cols.,[36] published that different PPGs, as, angoroside A, calceolarioside, capnoepside, echinacoside, forsythoside, and verbascoside, do not inhibit to COX or 5-LOX on rat peritoneal leukocytes at 50 μg/mL.

However, has recently been studied the effect of PPG on COX enzymes in two different publications. On one hand, Sahpaz and cols. [21] found that arenarioside, forsythoside, and verbascoside, were the strongest COX-2-inhibitors at 100 μM. Moreover, these compounds did not exhibit any significant inhibition on COX-1 at the same concentration. The authors defended the existence of a structure-activity relationship: the possession of two or three sugar units in their structure could contribute to the selective inhibition on COX-2. On the other hand, ballotetroside, with four sugar units, exhibited a weaker activity, and, interestingly is more active over COX-1 than COX-2. They think that the addition of a sugar unit (in this case arabinose on position C-2 of rhamnose) increases the steric hindrance, which prevents the molecule easily getting to the active site of the enzyme.

The study of forsythoside and arenarioside is important because they have the same PPG structure, with the only difference in the kind of third sugar moiety joined to C-6 of glucose.

Comparing the activity showed by each active compound, we also would hypothesize even more that apiose moiety joined to C-6 of glucose seems to contribute to COX-2 inhibition more than xylose moiety. Nevertheless, the presence of xylose on this position exerts the same effect on COX-2 inhibition than verbascoside, a diglycoside PPG.

Díaz and cols. [92] studied more PPGs (angoroside A, C and D, isoverbascoside and verbascoside), as anti-inflammatory compounds, and their effect not only over COX-1 and 2, but also over TX-synthase, and NO generation. Angoroside A, C and D and isoverbascoside exhibited inhibitory activity on COX-1 in A23187-stimulated macrophages. The inhibition was more evident with angoroside C and A. Of all tested compounds, only angoroside A, C and verbascoside showed a significant effect on TXB2-release. All compounds strongly inhibited LPS-induced NO production, being more active angoroside A, D and isoverbascoside. All compounds except angoroside C also inhibited the accumulation of PGE2 that means that they are active inhibiting COX-2. All compounds, except angoroside C, strongly inhibited LPS-induced TNF-α production, being more potent in the case of angoroside D and isoverbascoside.

These authors concluded that verbascoside is the most active compound on TX-synthase inhibition; therefore, caffeoyl moiety is an important function for this activity. The replacement by a feruloyl radical, leads to a complete loss of this activity. The attachment of a caffeate moiety at C-6 of glucose (case of isoverbascoside) appears to be favorable for COX-1 activity and TNF-α release inhibition, although it is detrimental for TX-synthase inhibition activity.

On the other hand, the attachment of arabinose is favorable for NO activity and detrimental for TNF-α activity, but, if in this case, the caffeoyl is replaced by a feruloyl moiety, it is favorable for NO and TNF-α activity.

Comparing these results with those showed by Sahpaz and cols. [21], we also can conclude that, it should be interesting to develop the assays of Díaz and cols [92], on forsythoside and arenarioside as well, and study the effect of the methoxylation (feruloyl radicals) on diglycosides like leucosceptoside and martynoside, not only on triglycosides as angoroside.

Also it might compare the activity of forsythoside with poliumoside and angoroside A, all of them with three sugars moieties, but, differing on the last one, apiose, rhamnose and arabinose, respectively, joined at the same position of the second sugar moiety, rhamnose.

Other authors have been studying other aspects of the inflammatory process. Kimura and cols. [44] assayed different PPGs as forsythiaside, suspensaside, verbascoside and β-hydroxyverbascoside, on the 5-HETE and LTB4 inhibition. They conclude that two adjacent phenolic hydroxyl groups (caffeoyl radicals) are essential for potent inhibition of the formation of 5-HETE and LTB4. The inhibition of 5-LOX enzyme is reversible and non-competitive.

With these results, we also suggest that a hydroxylation in position β could decrease this activity. Besides, it seems that there is no influence of the position of the second sugar moiety (rhamnose on position C-3 or C-6 of the glucose) or the metoxilation of the hydroxyl groups. But, surprisingly, despite the compounds inhibited the formation of 5-HETE at concentrations 10- 6-10- 3 M, at concentrations between 10- 5-10- 4 M they stimulated the formations of TXB2 and 6-keto-PGFα.

Xiong and cols.,[56] also studied the influence on the inhibition of nitric oxide (NO) by PPGs in activated macrophages, because during inflammatory reactions NO is also produced by iNOS in different cells, such as macrophages, hepatocytes and renal cells. NO acts as a defense and regulatory molecule with homeostatic activities [118]. However, it is also pathogenic when it is excessively produced. NO, per se, is a reactive radical, damaging directly to functional normal tissue [119-123].

The PPGs assayed 2’-O-acetylverbascoside, cistanoside A, echinacoside, isoverbascoside, and tubuloside A and B, and verbascoside, could specifically scavenge NO radical at high concentration (200 μM) without attenuation of iNOS RNA-expression or iNOS protein levels or iNOS activity. The compounds that had a disaccharide moiety (2’-O-acetylverbascoside, isoverbascoside, tubuloside B, verbascoside) showed a better inhibitory potency than those with a trisaccharide (cistanoside A, echinacoside, and tubuloside A). This result suggests that an increase in the number of monosaccharide units in glycosylated sugar attenuates the scavenging activity of phenylethanoids for macrophage-generated NO radical [56].

Fetal inflammatory response

Unlike the inflammatory response associated with post-natal wounds, the early fetal immune system ‘fails’ to mount a true inflammatory response following injury.58−60 Fetal platelets fail to aggregate and degranulate. Likewise, the few fetal neutrophils and fetal monocytes/macrophages present are immature, lacking phagocytic and chemotactic potential.

Growth factors found in early fetal wounds, either have different isoforms (from those typically seen in adult wounds), or are at a different level of concentration. Most strikingly, early fetal wounds contain high levels of the TGF-β3 isoform. TGF-β3 downregulates (pro-fibrotic) TGF-β1 and TGF-β2 levels, thereby shifting the healing response away from a scarring pathway and towards more regenerative pathway.58 Differences in levels of PDGF, FGF and VEGF are also observed between early fetal wounds and post-natal wounds.59,60 Although (pro-fibrotic) PDGFs and FGFs are present in early fetal wounds, they dissipate within 24 hours of injury. Elevated levels of VEGF in early fetal wounds are thought to promote a more rapid angiogenic process.