Acute Inflammation

Acute inflammation is of relatively short duration, lasting from minutes to days, depending on the extent of injury. The main characteristics of acute inflammation are the exudation of fluid and plasma proteins (edema) and the emigration of leukocytes (predominantly neutrophils). Neutrophils and other motile white cells emigrate or move from the blood vessels to the perivascular tissues and the injury (implant) site (Fig. 3) (29-31).

The accumulation of leukocytes, in particular neutrophils and monocytes, is the most important feature of the acute and chronic inflammatory reactions, respectively. Leukocytes accumulate through a series of processes including margination, adhesion, emigration, phagocytosis, and extracellular release of leukocyte products (32). Increased leukocytic adhesion in inflammation involves specific interactions between complementary "adhesion molecules" present on the leukocyte and endothelial surfaces (33,34). The surface expression of these adhesion molecules is modulated by inflammatory agents; mechanisms of interaction include stimulation of leukocyte adhesion molecules (C5a, LTB4), stimulation of endothelial adhesion molecules [interleukin (IL)-1], or both effects [tumor necrosis factor (TNF)-a]. Integrins comprise a family of transmembrane glycoproteins that modulate cell-matrix and cell-cell relationships by acting as receptors to extracellular protein ligands and also as direct adhesion molecules (35). An important group of integrins (adhesion molecules) on leukocytes include the CD11/CD18 family of adhesion molecules. Leukocyte-endothelial cell interactions are also controlled by endothelium-leukocyte adhesion molecules [(ELAMs) E-selectins] or intracellular adhesion molecules [(ICAMs) ICAM-1, ICAM-2, and VCAMs] on endothelial cells (36).

White cell emigration is controlled in part by chemotaxis, which is the unidirectional migration of cells along a chemical gradient. A wide variety of exogenous and endogenous substances have been identified as chemotactic agents (13,29-41). Important to the emigration or movement of leukocytes is the presence of specific receptors for chemotactic agents on the cell membranes of leukocytes. These and other receptors may also play a role in the activation of leukocytes. Following localization of leukocytes at the injury (implant) site, phagocytosis and the release of enzymes occur following activation of

FIGURE 3 Acute inflammatory response of a swellable, bioresorbable polymer matrix at three days' implantation. (A) Low-magnification view showing fragmentation of the polymer matrix with polymorphonuclear leukocyte acute inflammatory response (arrows) predominantly to polymer particle surfaces. (B) High-magnification view of the polymorphonuclear leukocytes present between particles of the swellable, resorbable polymer matrix (P). Hematoxylin and eosin stain.

FIGURE 3 Acute inflammatory response of a swellable, bioresorbable polymer matrix at three days' implantation. (A) Low-magnification view showing fragmentation of the polymer matrix with polymorphonuclear leukocyte acute inflammatory response (arrows) predominantly to polymer particle surfaces. (B) High-magnification view of the polymorphonuclear leukocytes present between particles of the swellable, resorbable polymer matrix (P). Hematoxylin and eosin stain.

neutrophils and macrophages. The major role of the neutrophils in acute inflammation is to phagocytose microorganisms and foreign materials. Phagocytosis is seen as a three-step process in which the injurious agent undergoes recognition and neutrophil attachment, engulfment, and killing or degradation. With regard to biomaterials, engulfment and degradation may or may not occur depending on the size and properties of the biomaterial.

Although biomaterials are not generally phagocytosed by neutrophils or macrophages because of the size disparity (i.e., the surface of the biomaterial is greater than the size of the cell), certain events in phagocytosis may occur. The process of recognition and attachment is expedited when the injurious agent is coated by naturally occurring serum factors called opsonins. The two major opsonins are IgG and the complement-activated fragment C3b. Both of these plasma-derived proteins are known to adsorb to biomaterials, and neutrophils and macrophages have corresponding cell membrane receptors for these opsonization proteins. These receptors may also play a role in the activation of the attached neutrophil or macrophage. Because of the size disparity between the biomaterial surface and the attached cell, "frustrated phagocytosis" may occur (37,38). This process does not involve engulfment of the biomaterial but does cause the extracellular release of leukocyte products in an attempt to degrade the biomaterial. Neutrophils adherent to complement-coated and immunoglobulin-coated nonphagocytosable surfaces may release enzymes by direct extrusion or exocytosis from the cell (37-39). The amount of enzyme released during this process depends on the size of the polymer particle, with larger particles inducing greater amounts of enzyme release. This suggests that the specific mode of cell activation in the inflammatory response in tissue is dependent on the size of the implant and that a material in a phagocytosable form (e.g., powder or particulate) may provoke a degree of inflammatory response different from that of the same material in a nonphagocytosable form (e.g., film).

In considering protein or peptide delivery systems, the shape and size of the carrier may play a significant role in mechanisms by which proteins and peptides may be modified. Nanoparticles and microparticles, no larger than 5 mm in diameter, may be phagocytosed where the intracellular lysosomal and phagosomal compartments may play a direct role in protein/peptide modification. Microparticles with a diameter larger than 10 mm do not normally undergo phagocytosis, but cells at the material surface may undergo frustrated phagocytosis. Both phagocytosis and frustrated phagocytosis involve three general categories of reactive chemicals that can modify proteins and peptides, thus leading to significant changes in their bioavailability, efficacy, and phar-macokinetics. These general categories are acids, enzymes, and reactive oxygen species (ROS).

Chronic Inflammation

Chronic inflammation is histologically less uniform than acute inflammation. In general, chronic inflammation is characterized by the presence of monocytes and lymphocytes (11,12,42,43) (Fig. 4). It must be noted that many factors modify the course and histologic appearance of chronic inflammation.

Persistent inflammatory stimuli lead to chronic inflammation. Although the chemical and physical properties of the biomaterial may lead to chronic

FIGURE 4 Chronic inflammatory response of a swellable, bioresorbable polymer matrix at three days' implantation. (A) Low-magnification view showing initial swelling and fragmentation of the surface of the polymer matrix at the tissue/implant interface with focal chronic inflammation (arrows). (B) High-magnification view of the implant/tissue interface of the swellable bioresorbable polymer (P) showing two interfacial responses: initial swelling and fragmentation at the surface of the polymer matrix and a focal chronic inflammatory response (arrows). A zone of resolving inflammation with macrophages and exudate is present between the polymer matrix (P) with a focal chronic inflammatory response (arrows) and SM. Hematoxylin and eosin stain. Abbreviation: SM, skeletal muscle.

FIGURE 4 Chronic inflammatory response of a swellable, bioresorbable polymer matrix at three days' implantation. (A) Low-magnification view showing initial swelling and fragmentation of the surface of the polymer matrix at the tissue/implant interface with focal chronic inflammation (arrows). (B) High-magnification view of the implant/tissue interface of the swellable bioresorbable polymer (P) showing two interfacial responses: initial swelling and fragmentation at the surface of the polymer matrix and a focal chronic inflammatory response (arrows). A zone of resolving inflammation with macrophages and exudate is present between the polymer matrix (P) with a focal chronic inflammatory response (arrows) and SM. Hematoxylin and eosin stain. Abbreviation: SM, skeletal muscle.

inflammation, motion in the implant site by the biomaterial may also produce chronic inflammation. The chronic inflammatory response to biomaterials is confined to the implant site. Inflammation with the presence of mononuclear cells, including lymphocytes and plasma cells, is given the designation chronic inflammation, whereas the foreign body reaction with granulation tissue development is considered the normal wound-healing response to implanted biomaterials (i.e., the normal foreign body reaction). Chronic inflammation with biocompatible materials is usually of very short duration, that is, a few days.

The presence of chronic inflammation with lymphocytes and monocytes beyond the first two weeks following injection or implantation usually indicates an adverse reaction secondary to infection or cytotoxicity of the bioactive agent that is being released. Figure 5 demonstrates a response to cytotoxic naltrexone released from a polylactic acid-glycolic acid (PLGA) matrix. Figure 5B indicates that the placebo polymer alone does not initiate focal chronic inflammation, whereas focal chronic inflammation is seen with the naltrexone-containing PLGA matrix in Figure 5A (7,44).

Lymphocytes and plasma cells are involved principally in immune reactions and are key mediators of antibody production and delayed hypersensi-tivity responses. Their roles in nonimmunologic injuries and inflammation are largely unknown. Little is known regarding humoral immune responses and cell-mediated immunity to synthetic biomaterials. The role of macrophages and dendritic cells must be considered in the possible development of immune responses to synthetic biomaterials. Macrophages and dendritic cells process and present the antigen to immunocompetent cells and are thus key mediators in the development of immune reactions.

Chronic inflammation is also seen in injection sites where cell-mediated immune reactions are elicited by the bioactive agent, which functions as an antigen to initiate this response. Figure 6 demonstrates the expected response when an antigen, recombinant human growth hormone, is released into another

FIGURE 5 Tissue responses to a naltrexone-containing PLGA matrix and placebo matrix at 14 days' implantation. (A) Marked focal chronic inflammation composed of monocytes and lymphocytes (arrows) is present at the naltrexone-containing bead surface and surrounds the bead in a well-demarcated zone (arrow). (B) The placebo PLGA bead shows a minimal foreign body reaction at its surface and slight fibrous capsule formation (arrows) with no focal chronic inflammation. Hematoxylin and eosin stain. Abbreviation: PLGA, polylactic acid-glycolic acid.

FIGURE 5 Tissue responses to a naltrexone-containing PLGA matrix and placebo matrix at 14 days' implantation. (A) Marked focal chronic inflammation composed of monocytes and lymphocytes (arrows) is present at the naltrexone-containing bead surface and surrounds the bead in a well-demarcated zone (arrow). (B) The placebo PLGA bead shows a minimal foreign body reaction at its surface and slight fibrous capsule formation (arrows) with no focal chronic inflammation. Hematoxylin and eosin stain. Abbreviation: PLGA, polylactic acid-glycolic acid.

FIGURE 6 Chronic inflammatory response secondary to release of a bioactive agent from PLGA microparticles at 14 days' implantation. (A) Marked focal chronic inflammatory response (arrows) with monocytes and lymphocytes at the tissue/particle aggregate interface of recombinant human growth hormone-containing (Nutropin®) PLGA microparticles. (B) Focal macrophage and foreign body giant cell responses to placebo PLGA microparticles. Hematoxylin and eosin stain. Abbreviation: PLGA, polylactic acid-glycolic acid.

FIGURE 6 Chronic inflammatory response secondary to release of a bioactive agent from PLGA microparticles at 14 days' implantation. (A) Marked focal chronic inflammatory response (arrows) with monocytes and lymphocytes at the tissue/particle aggregate interface of recombinant human growth hormone-containing (Nutropin®) PLGA microparticles. (B) Focal macrophage and foreign body giant cell responses to placebo PLGA microparticles. Hematoxylin and eosin stain. Abbreviation: PLGA, polylactic acid-glycolic acid.

species and results in an expected immune reaction because of cross-species reactivity. Neutralizing immune responses have been identified after 14 to 16 days of infusing human growth factors into rats (45).

Therapeutic proteins and peptides released from implanted delivery systems must be considered as potential antigens capable of initiating immune reactions (46). Because of the potential antigenicity of proteins and peptides in implantable delivery systems, immunotoxicity testing is a requirement in the overall biocompatibility and tissue response evaluation of protein/peptide delivery systems. Immune responses to injected cytokines and growth factors are well documented in the literature (47-49).

The macrophage is probably the most important cell in chronic inflammation because of the great number of biologically active products it produces.

Important classes of products produced and secreted by macrophages include neutral proteases, chemotactic factors, arachidonic acid metabolites, reactive oxygen metabolites, complement components, coagulation factors, growth-promoting factors, and cytokines.

Growth factors such as PDGF, fibroblast growth factor (FGF), TGF-b, TGF-a/endothelial growth factor (EGF), and IL-1 or TNF are important for the growth of fibroblasts and blood vessels and the regeneration of epithelial cells. Growth factors, released by activated cells, stimulate production of a wide variety of cells; initiate cell migration, induce cellular differentiation, initiate tissue remodeling, and may be involved in various stages of wound healing (19,50-54). It is clear that there is a lack of information regarding interaction and synergy among various cytokines and growth factors and their abilities to exhibit chemotactic, mitogenic, and angiogenic properties.

Granulation Tissue

Within one day following implantation of a biomaterial (i.e., injury), the healing response is initiated by the action of monocytes and macrophages, followed by proliferation of fibroblasts and vascular endothelial cells at the implant site, leading to the formation of granulation tissue, the hallmark of healing inflammation. Granulation tissue derives its name from the pink, soft granular appearance on the surface of healing wounds, and its characteristic histologic features include the proliferation of new small blood vessels and fibroblasts. Depending on the extent of injury, granulation tissue may be seen as early as three to five days following implantation of a biomaterial.

The new small blood vessels are formed by budding or sprouting of preexisting vessels in a process known as neovascularization or angiogenesis (55-57). This process involves proliferation, maturation, and organization of endothelial cells into capillary tubes. Fibroblasts also proliferate in developing granulation tissue and are active in synthesizing collagen (fibrous tissue) and proteoglycans. In the early stages of granulation tissue development, proteo-glycans predominate; later, however, collagen—especially type I collagen— predominates and forms the fibrous capsule.

LATE, PERSISTENT TISSUE/MATERIAL RESPONSES Foreign Body Reaction: Macrophages and Foreign Body Giant Cells

Two factors that play a role in monocyte/macrophage adhesion and activation and FBGC formation are the surface chemistry of the substrate onto which the cells adhere and the protein adsorption that occurs before cell adhesion. These two factors have been hypothesized to play significant roles in the inflammatory and wound-healing responses to biomaterials, medical devices, and drug delivery systems.

Macrophage interactions with biomaterials are initiated when blood-borne monocytes in the early, transient responses migrate to the implant site and adhere to the blood protein-adsorbed biomaterial through monocyte-integrin interactions. Following adhesion, adherent monocytes differentiate into macrophages, which may then fuse to form FBGCs (Fig. 2).

Figure 7 shows monocyte and macrophage infiltration into a PLGA microcapsule aggregate at 14 days of implantation. Infiltration of monocytes and

FIGURE 7 Monocyte, macrophage, FBGC infiltration into polylactic acid-glycolic acid microcapsule aggregates at 14 days' implantation. (A) Low-magnification view showing cellular infiltration and the foreign body response (arrows) within the particle aggregate with fibrous capsule formation encapsulating the microcapsule aggregate. (B) High-magnification view showing macrophages and FBGCs at the microcapsule surfaces in a typical foreign body response within the microcapsule aggregate. Hematoxylin and eosin stain. Abbreviation: FBGC, foreign body giant cell.

FIGURE 7 Monocyte, macrophage, FBGC infiltration into polylactic acid-glycolic acid microcapsule aggregates at 14 days' implantation. (A) Low-magnification view showing cellular infiltration and the foreign body response (arrows) within the particle aggregate with fibrous capsule formation encapsulating the microcapsule aggregate. (B) High-magnification view showing macrophages and FBGCs at the microcapsule surfaces in a typical foreign body response within the microcapsule aggregate. Hematoxylin and eosin stain. Abbreviation: FBGC, foreign body giant cell.

FIGURE 8 FBGC response and fibrous capsule formation to an injected aggregate of PLGA microcapsules at 42 days. (A) Low-magnification view. The PLGA microcapsule aggregate has been completely infiltrated by monocytes, macrophages, FBGCs, and fibroblasts. Resolution of the acute and chronic inflammatory responses has occurred. A thin fibrous capsule (arrows) completely encapsulates the PLGA microcapsule aggregate. (B) A high-magnification view of the core of the PLGA microcapsule aggregate showing monocytes, macrophages, and FBGCs at the surfaces of the PLGA microcapsules. FBGCs (arrows) are seen. The interstitial space between PLGA microcapsules is sufficient to provide for fibroblast infiltration with subsequent collagen deposition. Hematoxylin and eosin stain. Abbreviations: FBGC, foreign body giant cell; PLGA, polylactic acid-glycolic acid.

FIGURE 8 FBGC response and fibrous capsule formation to an injected aggregate of PLGA microcapsules at 42 days. (A) Low-magnification view. The PLGA microcapsule aggregate has been completely infiltrated by monocytes, macrophages, FBGCs, and fibroblasts. Resolution of the acute and chronic inflammatory responses has occurred. A thin fibrous capsule (arrows) completely encapsulates the PLGA microcapsule aggregate. (B) A high-magnification view of the core of the PLGA microcapsule aggregate showing monocytes, macrophages, and FBGCs at the surfaces of the PLGA microcapsules. FBGCs (arrows) are seen. The interstitial space between PLGA microcapsules is sufficient to provide for fibroblast infiltration with subsequent collagen deposition. Hematoxylin and eosin stain. Abbreviations: FBGC, foreign body giant cell; PLGA, polylactic acid-glycolic acid.

macrophages is a time-dependent process, and it is clear that days to weeks are necessary for complete cellular infiltration into the interstices of microcapsule aggregates. Figure 8 shows complete macrophage infiltration into an injected aggregate of PLGA microspheres at 42 days. FBGCs are seen on the surfaces of the microcapsules within the aggregate, and a fibrous capsule surrounds the entire aggregate.

The foreign body reaction is composed of FBGCs and the components of granulation tissue, which consist of macrophages, fibroblasts, and capillaries in varying amounts, depending on the form and topography of the implanted material. Relatively flat and smooth surfaces, such as those found on breast prostheses, have a foreign body reaction that is composed of a layer of macrophages one to two cells in thickness. Relatively rough surfaces, such as those found on the outer surfaces of expanded poly(tetrafluoroethylene) (ePTFE) vascular prostheses or polymethyl-methacrylate bone cement, have a foreign body reaction composed of several layers of macrophages and FBGCs at the surface. Fabric and porous materials generally have a surface response composed of macrophages and FBGCs with varying degrees of granulation tissue subjacent to the surface response. The foreign body reaction consisting mainly of macrophages and/or FBGCs may persist at the tissue-implant interface for the lifetime of the implant (1-10,58-61).

As previously discussed, the form and topography of the surface of the biomaterial determines the composition of the foreign body reaction. With biocompatible materials, the composition of the foreign body reaction in the implant site may be controlled by the surface properties of the biomaterial, the form of the implant, and the relationship between the surface area of the biomaterial and the volume of the implant. For example, high surface-to-volume implants such as fabrics, porous materials, or microparticle aggregates (Figs. 7 and 8) will have higher ratios of macrophages and FBGCs in the implant site than smooth-surface implants, which will have fibrosis as a significant component of the implant site.

Cytoskeletal and adhesive structure studies of in vitro macrophages and FBGCs have demonstrated that podosomal structures, and not focal contacts, are the major adhesive structures present within macrophages and FBGCs on surfaces. The podosomal structures are present at the ventral periphery of the FBGCs and contain vinculin, talin, and paxillin in a ring-like structure surrounding an F-actin core. These podosomal adhesion structures are similar to those identified for osteoclast adhesion. The podosomal structure present at the ventral and peripheral macrophage and FBGC surface implies a functional polarization and suggests the presence of frustrated phagocytosis via the formation of a closed compartment between the macrophage or FBGC and the underlying substrate where acid, degradative enzymes, reactive oxygen intermediates, and/or other products are secreted.

The adhesion of macrophages and FBGCs at the surfaces of biomaterials, prostheses, medical devices, and controlled-release systems to produce a closed compartment, that is, privileged microenvironment that exists between the cell membrane and the surface of the material, has significant implications in regard to the biodegradation of the biomaterial as well as the chemical modification and potential degradation of bioactive agents released from controlled-release systems. In a process described by Henson as frustrated phagocytosis, macrophages and FBGCs can release mediators of degradation such as acid, degradative enzymes, and ROS into this privileged zone between the cell membrane and the biomaterial surface so that immediate buffering or inhibition of these mediators is delayed or reduced. Phagolysosomes in macrophages can have acidity as low as a pH of 4, and direct microelectrode studies of this acid environment have determined pH levels as low as 3.5. Moreover, only several hours are necessary to achieve these acid levels following adhesion of macrophages (62-66). Proteins and peptides released from controlled-release systems would immediately encounter this highly acid environment. Conformational

TABLE 3 Macrophage and Neutrophil Degradation Products

Enzymes

Reactive oxygen species

Acid hydrolases

Superoxide anion

Neutral hydrolases

Hydrogen peroxide

Proteases

Hydroxyl radical

DNases

Nitric oxide

Lipases

Peroxynitrite

Cathepsins

Hypochlorous acid

Elastase

Chloramines

Phosphatases

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