Phenotypic dichotomies in the foreign body reaction

To better understand the relationship between macrophage/foreign body giant cell adhesion and activation on surface-modified biomaterials, quantitative assessment of adherent cell density (cells per mm(2)) and cytokine production (pgs per mL) were determined by ELISA. Further analysis to identify cellular activation was carried out by normalizing the cytokine concentration data to provide a measure of cellular activation. This method of analysis demonstrated that hydrophobic surfaces provided statistically significantly greater adherent cell densities than hydrophilic/neutral surfaces. However, when cell activation parameters were determined by normalization to the adherent cell density, the hydrophilic/neutral surfaces demonstrated statistically significantly greater levels of activation and production of IL-10, IL-1beta, IL-6, IL-8, and MIP-1beta. With increasing time, production of the anti-inflammatory cytokine IL-10 increased, whereas IL-1beta, IL-6, and IL-8 decreased and MIP-1beta was relatively constant over the culture time period. This observed dichotomy or disparity between adhesion and activation may be related to surface-induced adherent cell apoptosis. Further evaluation of macrophage activation on biomaterial surfaces indicated that an apparent phenotypic switch in macrophage phenotype occurred over the course of the in vitro culture. Analysis of cytokine/chemokine profiles with surface-modified biomaterials revealed similarities between the classically activated macrophages and the biomaterial-adherent macrophages early (day 3) in culture, while at later timepoints the biomaterial-adherent macrophages produced profiles similar to alternatively activated macrophages. Classically activated macrophages are those commonly activated by lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and alternatively activated macrophages are those activated by IL-4/IL-13 or IL-10. Surface modification of biomaterials offer an opportunity to control cellular activation and cytokine profiles in the phenotypic switch, and may provide a means by which macrophages can be induced to regulate particular secretory proteins that direct inflammation, the foreign body reaction, wound healing, and ultimately biocompatibility.     

The foreign body reaction in T-cell-deficient mice

The role(s) of T lymphocytes in the foreign body response has not been thoroughly elucidated. Lymphocytes are known to augment macrophage adhesion and fusion in vitro. Furthermore, T lymphocytes are a possible source of the cytokines, IL-4 and IL-13, which induce macrophage fusion. In this study, we used BALB/c mice and BALB/c (nu/nu) nude mice to investigate foreign body giant cell (FBGC) formation in a T-cell-deficient setting. Mice were implanted with Elasthane 80A (PEU), silicone rubber (SR), or poly(ethylene terephthalate) (PET) for 7, 14, or 21 days using the cage implant system. Exudate cells and IL-4 and IL-13 levels in exudate supernatants were analyzed by flow cytometry and a multiplex immunoassay, respectively, at Days 7, 14, and 21. Macrophage adhesion and fusion on material surfaces were analyzed using optical microscopy. T-cell-deficient mice had lower total leukocyte concentrations at the biomaterial implant site at all time points. Adherent cell density was comparable between normal and T-cell-deficient mice except in the PEU group at Day 21. However, percent fusion, average nuclei per FBGC, and FBGC morphology were comparable between normal and T-cell-deficient mice. IL-4 was not detected in any sample, but IL-13 levels were also comparable between normal and T-cell-deficient mice indicating Th2-polarized T-cells are not the sole source of this cytokine. We have shown that there are pathways that do not require thymus-matured T lymphocytes, which lead to a normal foreign body response to biomaterials in a murine model.     

Cellular and molecular dynamics in the foreign body reaction

Intracorporally implanted materials, such as medical devices, will provoke the body to initiate an inflammatory reaction. This inflammatory reaction to implanted materials is known as the foreign body reaction (FBR) and is characterized by 3 distinct phases: onset, progression, and resolution. The FBR proceeds in the creation of a dynamic microenvironment that is spatially well organized. The progression of the FBR is regulated by soluble mediators, such as cytokines, chemokines, and matrix metalloproteinases (MMPs), which are produced locally by tissue cells and infiltrated inflammatory cells. These soluble mediators orchestrate the cascade of cellular processes in the microenvironment that accompanies the FBR, consisting of cellular activation, angiogenesis, extravasation, migration, phagocytosis, and, finally, fibrosis. The nature of the FBR requires that the soluble mediators act in a spatial and temporally regulated manner as well. This regulation is well known for several inflammatory processes, but scarce knowledge exists about the intricate relationship between the FBR and the expression of soluble mediators. This review discusses the key processes during the initiation, progression, and resolution phase, with emphasis on the role of soluble mediators. Besides other sites of implantation, we focus on the subcutaneous implantation model.     

Macrophage phenotypes in the collagen-induced foreign body reaction in rats

Implantation of biomaterials into the body elicits a material-dependent inflammatory response called the foreign body reaction (FBR). Macrophages play a pivotal role in the FBR by orchestrating the pro-inflammatory microenvironment around the biomaterials by secreting cytokines, chemokines and growth factors. When the biomaterial is porous or degradable, macrophages can migrate into the material and continue the generation of a pro-inflammatory microenvironment inside the materials. They also regulate the degradation of biomaterials by secreting proteolytic enzymes and by phagocytosis. We hypothesize that macrophages present in the different microenvironments of the FBR have different phenotypes. Fundamental knowledge of the phenotypes of macrophages and their dynamics during the FBR will contribute to our overall understanding of the mechanisms involved in the FBR, and may provide us with additional tools to modulate the FBR. To investigate the phenotype of macrophages in the FBR, we validated phenotype-specific markers for rat macrophages in vitro by stimulating them with IFNγ/LPS, IL4/IL13 or IL4/dexamethasone to induce classically activated macrophages (M1φ) or alternatively activated macrophages (M2φ). Gene expression analysis, Western blot and immunohistochemistry revealed that iNOS and CD206 are specifically expressed by M1φ and M2φ, respectively. Using these markers, we investigated the distribution of M1φ and M2φ in the FBR induced by subcutaneously implanted hexamethylenediisocyanate cross-linked dermal sheep collagen (HDSC) disks in AO rats. We found that part of the macrophages display an M2 phenotype, whereas the M1phenotype was not detected. Our data suggest that many macrophages in the FBR induced by HDSC do not fit into the classical M1 or M2 dichotomy.     

Biostability and macrophage-mediated foreign body reaction of silicone-modified polyurethanes

In this study, the effect of soft segment chemistry on the phase morphology and in vivo response of commercial-grade poly(ether urethane) (PEU), silicone-modified PEU (PEU-S), poly(carbonate urethane) (PCU), and silicone-modified PCU (PCU-S) elastomers were examined. Silicone-modified polyurethanes were developed to combine the biostability of silicone with the mechanical properties of PEUs. Results from the infrared spectroscopy confirmed the presence of silicone at the surface of the PEU-S and PCU-S films. Atomic force microscopy phase imaging indicated that the overall two-phase morphology of PEUs, necessary for its thermoplastic elastomeric properties, was not disrupted by the silicone modification. After material characterization, the in vivo foreign body response and biostability of the polyurethanes were studied using a subcutaneous cage implant protocol. The results from the cage implant study indicated that monocytes adhere, differentiate to macrophages which fuse to form foreign body giant cells on all of the polyurethanes. However, the silicone-modified surfaces promoted apoptosis of adherent macrophages at 4 days and high levels of macrophage fusion after 21 days. These results confirm that the surface of a biomaterial may influence the induction of apoptosis of adherent macrophages in vivo and are consistent with previous cell culture studies of these materials. This study validates the use of our standard cell culture protocol to predict in vivo behavior and further supports the hypothesis that interleukin-4 is the primary mediator of macrophage fusion and foreign body giant cell formation in vivo. The impact of these findings on the biostability of polyurethanes is the subject of current investigations. Attenuated total reflectance-Fourier transform infrared analysis of explanted specimens provided evidence of chain scission and crosslinking at the surface of all of the polyurethanes. The silicone modification did not fully inhibit the oxidative biodegradation of the polyether or polycarbonate soft segments; however, the rate of chain scission of PEU-S and PCU-S seemed to be slower than the control polyurethanes. To verify this finding and to quantify the rate of chain scission in order to predict long-term biostability, an in vitro environment that simulated the microenvironment at the adherent cell-material interface was used to accelerate the biodegradation of the polyurethanes. Polyurethane films were treated in vitro for up to 36 days in 20% hydrogen peroxide/0.1M cobalt chloride solution at 37 degrees Celsius. Characterization with attenuated total reflectance-Fourier transform infrared and scanning electron microscopy showed soft segment and hard segment degradation consistent with the chemical changes observed after long-term in vivo treatment. The biostability ranking of these four materials based on rate of chain scission and surface pitting was as follows: PEU < PEU-S PCU < PCU-S. The silicone modification increased the biostability of the PEU and PCU elastomers while maintaining the thermoplastic elastomeric properties.     

Hyaluronic acid induced foreign body reaction mimicking neoplastic parotid cytology

Masses near the angle of the mandible containing extracellular matrix or mucin on cytology raise concern for various benign and malignant parotid gland neoplasms. Here a 76‐year‐old female with a history of cosmetic hyaluronic acid (HA) filler injections presented with a painless 6 mm left sided facial mass. Injection of hyaluronidase into the mass had failed to cause regression, raising concern for a neoplastic process. Fine‐needle aspiration (FNA) showed amorphous, mucinous/extracellular matrix‐like material in a background of numerous histiocytes and occasional multinucleated giant cells, consistent with a foreign body giant cell reaction to HA. This uncommon reaction to HA filler creates previously unrecognized diagnostic pitfalls because of its resemblance on FNA to the extracellular matrix or mucin found in many salivary neoplasms.     

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.     

The downmodulation of the foreign body reaction by cytomegalovirus encoded interleukin-10

The foreign body reaction (FBR) is of great importance for the function and turnover of biomaterial scaffolds. The development of biological tools that modulate the FBR will augment scaffold functionality and benefit regenerative medicine. The human cytomegalovirus encodes a functional homolog of the potent anti-inflammatory human cytokine interleukin-10 (cmvIL-10). We hypothesized that cmvIL-10 downmodulates the FBR, impairing degradation of biomaterial. We studied the effect of cmvIL-10 on the FBR to subcutaneously implanted hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) discs in rats. CmvIL-10 impaired macrophage influx, vascularization and ingrowth into the discs up to 21 days. It also impaired the formation of giant cells and the degradation of HDSC. At day 10, deposited fibrin fibers were still present in cmvIL-10 discs. Impaired collagenase activity coincided with the impaired HDSC degradation. These results indicate that cmvIL-10 downmodulates the FBR, impairing the progression of the FBR. This study demonstrates the feasibility of interleukin-10 as a biomolecular tool in biomaterials for regenerative medicine.     

Morphological reaction of uterine wall tissues of the white rat to a foreign body

Summary The author studied the course of aseptic inflammation produced by passing a foreign body (sterile contton thread soaked in carmine) through all the layers of the uterine wall in the white rat.The phagocytic and inflammatory reactions of the uterine wall showed highly distinctive characteristics in the following groups of rats: 12 days old, 1 1/2 months old, sexually mature (in estrus), pregnant (7th–14th day), and immediately postpartum. The rate of formation of the connective tissue capsule was different at various stages of ontogenesis, as was the rate of appearance of giant cells.Of great importance in the regenerative and inflammatory reactions of uterine wall tissues (especially in 12-day-old rats) are erythrocytic extravasations, which cause temporary tissue disintegration with subsequent rapid regeneration of the injured areas.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' éksperimental'noi biologii i meditsiny Vol. 49, No. 2, pp. 116–120, February, 1960.     

Endotoxin contamination in wound dressings made of natural biomaterials

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.     

Material dependent differences in inflammatory gene expression by giant cells during the foreign body reaction

Multinucleated giant cells (GCs) are often observed in the foreign body reaction against implanted materials. The in vivo function of GCs in this inflammatory process remains to be elucidated. GCs degrade collagen implants in rats and may also orchestrate the inflammatory process via the expression and secretion of modulators, such as cytokines and chemokines. In this study, we show that the gene expression of PMN chemoattractants, CXCL1/KC and CXCL2/MIP-2, is high in GCs micro-dissected from explanted Dacron, cross-linked collagen (HDSC), and bioactive ureido-pyrimidinone functionalized oligocaprolactone (bioactive PCLdiUPy). Conversely, the gene expression levels of TGFbeta and pro-angiogenic mediators VEGF and FGF were found to be low in these GCs as compared with the expression levels in total explants. GCs in bioactive PCLdiUPy displayed high cytokine and angiogenic mediator expression compared with GCs isolated from the two other studied materials, whereas chemokine gene expression in GCs isolated form HDSC was low. Thus, GCs adopt their expression profile in response to the material that is encountered.     

The foreign body reaction to a biodegradable biomaterial differs between rats and mice

Before a biomaterial can be applied in the clinic, biocompatibility must be tested in in vivo models, by monitoring the foreign body reaction. In this study, we compared the foreign body reaction (FBR) to the biodegradable biomaterial hexamethylenediisocyanate crosslinked dermal sheep collagen (HDSC) between several strains of rats and mice. HDSC disks were implanted subcutaneously on the backs of AO, BN, F344, LEW, and PVG rats and on the backs of 129 SVEV, BALB/c, and C57BL/6 mice. Materials were explanted after 7, 14, 21, and 28 days and processed for (immuno) light and transmission electron microscopic evaluation. In all rat strains, giant cell formation and phagocytosis of HDSC bundles were comparable. In addition, in the PVG rat, many plasma cells infiltrated the HDSC disks. Only a few T cells were present in AO and PVG rats, whereas, in F344 and LEW rats, the presence of T cells was more pronounced. BN rats showed an intermediate T-cell infiltration. In mice, the FBR to HDSC was comparable between the different strains. Compared with rats, giant cell formation was limited, whereas stroma formation was more abundant. Phagocytosis of HDSC bundles rarely occurred in mice, whereas calcification was observed more often. It is concluded that the FBR to HDSC clearly differs between rats and mice. This has consequences for assessment studies on biocompatibility and also on fundamental biomaterial research.     

Digital pacinian hyperplasia. Report of a case associated with foreign body reaction

Digital Pacinian Hyperplasia is a non tumoral, very rare lesion. To date, only 31 cases have been reported, occasionally named Digital Pacinian Neuroma. Digital pain is usually associated to. Local trauma is reported in about half of the cases. We report a new case occurred in a 62 year-old male, associated with inflammatory and foreign body reaction. No recurrence was observed 6 months after surgical excision. Differential diagnosis and controversies about this lesion are discussed.     

Reduced foreign body reaction to implanted biomaterials by surface treatment with oriented osteopontin

The foreign body reaction (FBR), which leads to the encapsulation of implanted biomaterials, has been implicated in the failure of many medical devices. The protein layer that is nonspecifically adsorbed onto the implant surface immediately after implantation is thought to dictate this reaction. It is hypothesized that biomaterial surfaces having specific proteins with precisely controlled orientations will decrease the FBR. Previously, we have reported that osteopontin (OPN) adsorbed on positively charged surfaces has a preferable orientation for in vitro cell adhesion and spreading as compared to negatively charged surfaces. It is expected that coating a layer of OPN in its preferred orientation on an implant surface will decrease the FBR. In this work, in vivo studies were performed to test this hypothesis. A positively charged polymer (p(HEMA-co-AEMA)) and a negatively charged polymer (p(HEMA-co-CEA)) coated with OPN were implanted subcutaneously in wild-type mice for 7 or 28 days. Uncoated polymers were used as control. For the 7-day implants, cells on OPN-coated p(HEMA-co-AEMA) spread more than cells on the other three materials. Following 28 days of implantation the implants were explanted and the capsule thickness and vascularity around the implants were characterized. Additionally, the macrophage and foreign body giant cells (FBGCs) around the implants were quantified. It was found in this study that the modification of the positively charged polymer surface with OPN in a controlled orientation led to a reduction in the foreign body reaction as determined by capsule thickness. Our finding provides valuable information for designing better biocompatible biomaterials with improved in vivo performance.     

Repetitive subcutaneous implantation of different types of (biodegradable) biomaterials alters the foreign body reaction

In the present study two biodegradable materials (cross-linked collagens) and two non-biodegradable materials (polyurethane and silicone) were applied in a repetitive subcutaneous implantation model in rats. In contrast to the first challenge, the second challenge with the same type of material, but at a different subcutaneous site of the same animal, induced an increase of macrophages and giant cells inside the biodegradable materials. Additionally, only after the second challenge clusters and accumulations of plasma cells were present in the surrounding tissue of each type of material. In the same areas an increase of MHC II expression was measured by immunocytochemistry. Differences in the numbers of macrophages and T cells were not observed around the explants. Undifferentiated B cells or NK cells were not present at any time point. The results indicate that alterations observed after the second challenge did not depend on biodegradation of the materials. Significance of these findings should be considered in view of increased and repetitive use of the same type of biomaterial (possibly for different application sites) for implantation in patients.     

Ongoing foreign body reaction to subcutaneous implanted (heparin) modified Dacron in rats

Dacron-containing heart valve repair devices trigger chronic inflammation characterized by the presence of activated macrophages, foreign body giant cells, and capsule formation. Upon blood contact, proinflammatory proteins adsorb to the material and provide a substrate for monocyte binding and differentiation. Various heparin-coated polymers have been shown to reduce adsorption of proinflammatory proteins in vitro and in vivo. In this study, the effect of knitted, heparin-coated Dacron on the foreign body reaction was tested subcutaneously in rats. We hypothesized that the anti-inflammatory effect of heparin would reduce monocyte recruitment and differentiation and therefore limit the inflammatory reaction. An ongoing foreign body reaction, characterized by the presence of foreign body giant cells and high vascularization, was observed in uncoated as well as (heparin-)coated Dacron at up to 180 days of implantation. Also, a thin capsule was formed around each material up to this time. In conclusion, although heparin coatings might have an effect on the acute inflammatory response, we were not able to show a difference between heparin-coated and uncoated Dacron after 180 days' implantation in rats. Further research needs to be conducted to assess the difference in proinflammatory protein adsorption between the tested materials and the effect this has on the long-term foreign body reaction.     

Endotoxin contamination contributes to the in vitro cytokine-inducing activity of osteopontin preparations.

Recently, both native and recombinant preparations of human osteopontin (OPN) have been shown to be able to induce the production of several proinflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) or purified monocytes. In the present study, we found that commercially available native and recombinant OPNs contain variable amounts of endotoxin (LPS) and that removal of endotoxin by polymyxin B-agarose column abrogated their cytokine-inducing activity. These results suggest the questionable evidence of the ability of OPN to induce several cytokines in human PBMCs and draw attention to the exquisite sensitivity of PBMCs/monocytes to endotoxin contaminants.     

Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 microgram of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 micrograms of lipid A per ml inhibited binding of the enzyme conjugate to adsorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.     

An unusual foreign body in the bladder

In spite of its inaccessibility, every conceivable object has been inserted into the urinary bladder. Such patients may have a psychiatric disorder with a sexual perversion or inquisitiveness (as in children) as the underlying cause. We report a case of an aluminum rod inserted into the urinary bladder by an adult male, which was removed successfully by surgery.