CCL2 and CCR2 are Essential for the Formation of Osteoclasts and Foreign Body Giant Cells.

Osteoclasts are multinucleated cells responsible for bone resorption. They are derived from the fusion of cells in the monocyte/macrophage lineage. Monocytes and macrophages can also fuse to form foreign body giant cells (FBGC). Foreign body giant cells are observed at the interface between a host and a foreign body such as implants during a foreign body reaction. Macrophages are attracted to the site of bone resorption and foreign body reactions by different cytokines. Chemokine (C-C) ligand-2 (CCL2) is an important chemotactic factor and binds to a receptor CCR2. In this study we investigated the importance of CCL2 and the receptor CCR2 in the formation of osteoclasts and FBGC. CCL2 mRNA was more highly expressed in giant cell culture than macrophages, being 9-fold and 16-fold more abundant in osteoclasts and FBGC respectively. Significantly fewer osteoclasts and FBGC were cultured from the bone marrow of CCL2 and CCR2 knockout mice, when compared to wild type. Not only were the number of giant cells reduced but there was a significant reduction in the number of nuclei and the size of these cells in the cultures of CCL2 and CCR2 knockout mice. Formation of osteoclasts and FBGC were recovered in cultures by addition of exogenous CCL2 to the media containing marrow cells from CCL2-/- mice. We conclude that CCL2 and its receptor CCR2 are important for the formation of osteoclasts and FBGC and absence of these genes causes inhibition of osteoclast and FBGC formation.

Dextranomer hyaluronic acid copolymer effects on gastroesophageal junction.

OBJECTIVE: The outcomes of fundoplication for gastroesophageal reflux disease are suboptimal in many children, and alternatives are clearly needed. Dextranomer hyaluronic acid (DxHA) copolymer, an agent with proven efficacy in vesicoureteral reflux, was studied with respect to its effects on the gastroesophageal junction (GEJ). METHODS: Twelve New Zealand white rabbits underwent measurement of lower esophageal sphincter pressure followed by laparotomy and injection into the muscular layer of the GEJ (controls, 1.0 mL saline; low-dose DxHA [0.5 mL]; high-dose DxHA [1.0 mL]). After a 12-week survival period, the animals underwent manometry, sacrifice, and necropsy. Organs were examined histologically by pathologists blinded to the injection delivered. RESULTS: All animals survived. Weight gain was equal in the 3 groups. There was no significant difference in mean lower esophageal sphincter pressure from baseline in any group (control 2.3 mmHg [95% confidence interval, CI -3.3 to 7.9]; low-dose group 3.2 mmHg [95% CI -0.8 to 7.2]; high-dose group -4.0 mmHg [95% CI -18.95 to 10.95]). Histologically, DxHA injection produced an intramural implant, with a foreign body giant cell reaction, and fibroblastic infiltration with collagen deposition. High-dose injection did not consistently result in a qualitative increase in the magnitude of the reaction. There was no mucosal injury or luminal stenosis. CONCLUSIONS: In this first study evaluating the effects of DxHA injection at the GEJ, a histologic bulking effect was observed without obvious functional complications. The agent may have a role in the treatment of gastroesophageal reflux disease.

Cartilage replacement by use of hybrid systems of autologous cells and polyethylene: an experimental study.

This study used porous polyethylene (PE) as a scaffold in an animal model system. The surface of the scaffolds was either modified with collagen II coating or first functionalized by oxygen plasma treatment and then coated with collagen II. The specimens were inoculated with autologous chondrocytes and transplanted into the concha of guinea pigs. Bare scaffolds were used as controls. Periods of 1, 6, and 12 months after implantation, samples of cells containing specimens and control samples were evaluated microscopically. As a result, the pre-seeded specimens were better integrated into the surrounding tissue than cell-free PE-specimens. Also a weaker immune reaction and an improved cartilage generation could be detected in the pre-seeded specimen. Compared to the other surface modifications, no further improvement of cartilage development was observed in the long term in vivo animal experimental study.

The role of osteopontin in foreign body giant cell formation.

Foreign body giant cells (FBGCs) are a hallmark of the foreign body reaction caused by biomaterial implantation and are thought to contribute to biomaterial degradation and the duration of the response. Osteopontin (OPN) is a secreted, acidic matricellular protein with multiple phosphorylation sites that is highly expressed at sites of inflammation. OPN wildtype and knockout mice were implanted with poly(vinyl alcohol) sponges and explanted at 14 days. OPN knockout mice had more foreign body giant cells but fewer macrophages surrounding the implants than their wildtype counterparts. In an in vitro human FBGC assay, addition of soluble OPN was found to reduce macrophage fusion to giant cells. These are the first studies to show a direct inhibitory role of OPN in FBGC formation in response to implantation.

Macrophage behavior on surface-modified polyurethanes.

Adherent macrophages and foreign body giant cells (FBGCs) are known to release degradative molecules that can be detrimental to the long-term biostability of polyurethanes. The modification of polyurethanes using surface modifying endgroups (SMEs) and/or the incorporation of silicone into the polyurethane soft segments may alter macrophage adhesion, fusion and apoptosis resulting in improved long-term biostability. An in vitro study of macrophage adhesion, fusion and apoptosis was performed on polyurethanes modified with fluorocarbon SMEs, polyethylene oxide (PEO) SMEs, or poly(dimethylsiloxane) (PDMS) co-soft segment and SMEs. The fluorocarbon SME and PEO SME modifications were shown to have no effect on macrophage adhesion and activity, while silicone modification had varied effects. Macrophages were capable of adapting to the surface and adhering in a similar manner to the silicone-modified and unmodified polyurethanes. In the absence of IL-4, macrophage fusion was comparable on the modified and unmodified polyurethanes, while macrophage apoptosis was promoted on the silicone modified surfaces. In contrast, when exposed to IL-4, a cytokine known to induce FBGC formation, silicone modification resulted in more macrophage fusion to form foreign body giant cells. In conclusion, fluorocarbon SME and PEO SME modification does not affect macrophage adhesion, fusion and apoptosis, while silicone modification is capable of mediating macrophage fusion and apoptosis. Silicone modification may be utilized to direct the fate of adherent macrophages towards FBGC formation or cell death through apoptosis.

Changes in macrophage function and morphology due to biomedical polyurethane surfaces undergoing biodegradation.

Monocytes are recruited to the material surface of an implanted biomedical device recognizing it as a foreign body. Differentiation into macrophages subsequently occurs followed by fusion to form foreign body giant cells (FBGCs). Consequently, implants can become degraded, cause chronic inflammation or become isolated by fibrous encapsulation. In this study, a relationship between material surface chemistry and the FBGC response was demonstrated by seeding mature monocyte-derived macrophages (MDMs) on polycarbonate-based polyurethanes that differed in their chemical structures (synthesized with poly(1,6-hexyl 1,2-ethyl carbonate) diol, and either (14)C-hexane diisocyanate and butanediol (BD) (referred to as HDI) or 4,4'-methylene bisphenyl diisocyanate and (14)C-BD (referred to as MDI)) and material degradation assessed. At 48 h of cell-material interaction, the FBGC attached to HDI were more multinucleated (73%) compared to MDI or the polystyrene (PS) control (21 and 36%, respectively). There was a fivefold increase in the synthesis and secretion of a protein with an approximate molecular weight of 48 kDa and a pI of 6.1 (determined by two-dimensional gel electrophoresis) only from cells seeded on HDI. Immunoprecipitation confirmed that MSE and CE were synthesized and secreted de novo. Immunoblotting also showed an increase in secreted monocyte-specific esterase (MSE) and cholesterol esterase (CE) from cells seeded on HDI relative to PS and MDI. Significantly more radiolabel ((14)C) release and esterase activity were elicited by MDMs on HDI than MDI (P < 0.05). The material that was more degradable (HDI), elicited greater protein synthesis and esterase secretion as well as more multinucleated MDMs than MDI, suggesting that the material surface chemistry modulates the function of MDM at the site of an inflammatory response to an implanted device.

Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse.

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.

Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation.

An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by beta2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on beta1 integrins is acquired. The combination of anti-beta1 plus anti-beta2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that beta2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, beta1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between beta1 and beta2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation.

In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: the role of RGD and PHSRN domains.

Polymorphonuclear leukocytes, monocytes/macrophages, foreign body giant cells (FBGC), and lymphocytes are central in directing the host foreign body and inflammatory/immune reactions that impact material biostability, biocompatibility, and device efficacy. We employed the functional architecture of fibronectin to probe the mechanisms of protein-cell interaction in modulating leukocyte behavior. We demonstrated previously that the RGD and PHSRN domain of fibronectin and the spatial orientation between the motifs were crucial in regulating macrophage function in vitro. The current study delineates the role of RGD and PHSRN in modulating the host inflammatory response and macrophage behavior in vivo. Oligopeptides containing RGD and/or PHSRN domains were grafted onto a polyethyleneglycol-based substrate and subcutaneously cage implanted into rats. Peptide identity played a minimal role in modulating the host inflammatory response and adherent macrophage density. The RGD motif, either alone or at the terminal position with the PHSRN-containing flanking sequence, elicited a rapid macrophage fusion to form FBGCs at the early stage of implantation. Both the RGD motif and the PHSRN motif were important in mediating FBGC formation at the later implantation time. However, the PHSRN motif, specifically in the configuration of G3 RGDG6 PHSRNG, evoked a larger extent of FBGC coverage. Our results indicate the importance of RGD and PHSRN in modulating macrophage function in a time and orientation dependent fashion in vivo.

Utilizing biomimetic oligopeptides to probe fibronectin-integrin binding and signaling in regulating macrophage function in vitro and in vivo.

Biomimetic oligopeptides were employed to elucidate the molecular mechanisms of fibronectin-integrin interaction in regulating macrophage function. Oligopeptides were designed based on of the functional structure of fibronectin and grafted onto a polymer network containing polyethyleneglycols. Macrophage adhesion was independent of the peptide identity that contained sequence RGD, PHSRN, PRRARV, or combinations thereof in an integrin-dependent fashion in vitro. However, integrin-dependent foreign body giant cell (FBGC) formation in vitro was highly dependent on both RGD and PHSRN in a single peptide formulation and with a specific orientation. In vivo results showed that peptide identity played a minimal role in modulating the host inflammatory response and adherent macrophage density. RGD-containing peptides mediated a rapid FBGC formation by 4 days of implantation by significantly increasing both the number of macrophages that participate in the cell fusion process and the rate of cell fusion. Both RGD and PHSRN domains were important in mediating FBGC formation at later implantation periods. In vitro intracellular signaling studies revealed that the requirement of protein tyrosine kinase and serine/threonine kinase activation and cross-talk compensation for macrophage adhesion dynamically varied with surfaces and culture time. Protein kinase C-dependent adhesion was related to RGD and PHSRN sequences, and to the sequence orientation thereof in a form of GGGRGDGGGGGGPHSRNG. Furthermore, we observed a multiple effect of the mitogen-activated protein kinase/extracellular-signal-regulated kinase signaling factor in mediating macrophage adhesion, which depended on the method of ligand immobilization. These findings represent a mechanistic correlation between the role of substrates and protein functional architectures in ligand-receptor recognition and post-ligation signaling events that control cellular behavior in vitro and in vivo.

Adsorbed serum proteins responsible for surface dependent human macrophage behavior.

Substrate specific cellular responses are the result of a complex biological system that includes protein adsorption, receptor-ligand binding, and signal transduction. This investigation attempted to identify specific proteins adsorbed from human serum that may be responsible for the previously reported in vitro surface dependent behavior of human macrophages and foreign body giant cells (FBGCs). The adsorption of human albumin, alpha(2)-macroglobulin, complement factor 3b, fibronectin, IgG, thrombospondin, vitronectin (VN), and von Willebrand factor (vWF) from a 25% serum solution was quantified with (125)I-labeled protein. Adsorption substrates included clean glass, alkyl-silane modified glass, amino-silane modified glass, poly(ethylene oxide) (PEO)-coupled glass, and the reference biomaterials poly(etherurethane urea), Silastic(R), and poly(tetrafluoroethylene) (PTFE). Following quantification of 2-h adsorption, surfaces were treated with sodium dodecyl sulfate (SDS) and the level of adsorbed proteins remaining was quantified. The pre- and post-SDS adsorption were both compared to previously reported surface dependent in vitro macrophage and FBGC behavior on the same surfaces; however, no correlations could be made. Adsorption strength, defined as the percentage of initially adsorbed protein that remained adsorbed after SDS treatment, correlated well with previously reported in vitro cellular behavior indicating that adsorbed vWF, IgG, and VN may be involved in the modulation of adherent macrophage and FBGC behavior. Those surfaces that strongly adsorbed vWF also inhibited long-term macrophage adhesion, while those surfaces that strongly adsorbed IgG promoted long-term macrophage adhesion. In addition, the highest levels of FBGC formation had been observed only on those surfaces that strongly adsorbed VN. Subsequent human monocyte cultures on protein preadsorbed substrates confirmed the inhibitory effect of adsorbed vWF and the promoting effect of IgG on longterm macrophage adhesion as predicted by adsorption strength correlations. However, preadsorbed VN was not observed to modulate FBGC formation, which is in contrast to the conclusions of the adsorption correlations.

Spatial regulation and surface chemistry control of monocyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces.

A long-standing goal of biomedical device development has been the generation of specific, desired host blood and tissue responses. An approach to meeting this design criteria is precise surface modification that creates micropatterns of distinct physicochemical character to direct cell adhesion and behavior. For this study, poly(ethylene terephthalate) films were coated with poly(benzyl N, N-diethyldithiocarbamate-co-styrene) and sequentially exposed to monomer solutions for photoirradiation. A photomask was placed over different regions to generate micropatterned surfaces with graft polymer stripes of three distinct ionic characters. Human monocytes were cultured on these surfaces to ascertain whether adhesion and fusion of monocytes/macrophages could be controlled. Nonionic polyacrylamide greatly inhibited adhesion and induced clumping of the few monocytes that did adhere. Macrophage adhesion and spreading led to high degrees of interleukin-13 induced foreign body giant cell formation on both the anionic poly(acrylic acid), sodium salt, and benzyl N,N-diethyldithiocarbamate portions of the culture surface. In spite of the highest observed levels of monocyte/macrophage adhesion on cationic poly(dimethylaminopropylacrylamide), methiodide, the adherent cells were not competent to undergo fusion to form foreign body giant cells. These results suggest that inflammatory cell responses may be spatially controlled in a manner that may be ultimately exploited to improve the biocompatibility of medical devices.

Biomaterial particle phagocytosis by bone-resorbing osteoclasts.

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic biomaterials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells.

In vivo biocompatibility study of ABA triblock copolymers consisting of poly(L-lactic-co-glycolic acid) A blocks attached to central poly(oxyethylene) B blocks.

Films of three ABA-block copolymers composed of lactic-co-glycolic acid A blocks and poly(oxyethylene) (PEO) B blocks and one random lactic-co-glycolic acid copolymer (PLG) were studied to investigate the influence of different polymer compositions and molecular weights on the tissue reaction, appearance of toxic degradation products, and swelling behavior in the cage implant system in rats. The inflammatory tissue reaction was followed over a 21-day implantation period by monitoring the leukocyte concentration, the extracellular acid, and alkaline phosphatase activities in a quantitative manner. Size and density of adherent macrophages and foreign body giant cells on the film surfaces were determined. The ABA and PLG implants caused only a minimal inflammatory reaction, as characterized by a low concentration of leukocytes during the implantation period when compared to empty cage controls. The content of PEO had an influence on the density of the adherent cells on the surface of the polymer film. An increase in PEO content and molecular weight decreased the cellular density during the implantation period. As demonstrated by scanning electron microscopy, no degradation was observed for all polymers during the implantation period. Our results demonstrate that the ABA block copolymers and PLG copolymer, are equally well tolerated in the cage implant test system.

Theoretical analysis of in vivo macrophage adhesion and foreign body giant cell formation on strained poly(etherurethane urea) elastomers.

Quantitative description of foreign body giant cell (FBGC) formation on poly(etherurethane urea) (PEUU) surfaces as a function of time can conceivably predict the effects of polymer characteristics on cellular responses in vivo. In the present study, the formation of FBGCs on strained and unstrained PEUUs was quantified with two parameters: the density of adherent macrophages present initially that participate in FBGC formation (d(o)) and the rate constant for cell fusion (k); both kinetic parameters were used to calculate the time-dependent FBGC density (dfc). Relationships were sought between results of the cellular analysis and the extent of environmental stress cracking (ESC), as characterized by scanning electron microscopy. Surface degradation was semiquantified with percent light transmittance. The materials used were: base PEUU, base PEUU with 1% Santowhite antioxidant powder, base PEUU with 5% Methacrol 2138F antifume agent, and base PEUU with both 1% Santowhite and 5% Methacrol 2138F. A comparison of unstrained base PEUU with base PEUU strained to 400% elongation indicated that the rate of cell fusion, but not d(o) and dfc, increased in the presence of strain. In all strained samples, additives that strongly affected the ESC also influenced FBGC kinetic parameters. Strained PEUU containing Santowhite had the lowest d(o), the slowest rate of cell fusion, and lowest dfc, and the least incidence of ESC. The results suggest that the incidence of ESC in PEUU was decreased in the presence of Santowhite, which also lowered the number of adherent macrophages participating in FBGC formation, the rate of FBGC formation and the subsequent FBGC density. These studies also indicate that strain in PEUUs does not directly modulate the adherent macrophage and FBGC density. Further studies are necessary to delineate the relationship between PEUU strain and adherent macrophage and FBGC activation, which leads to the exocytosis of degrading agents and the observed incidence of biodegradation.

Tissue reactions to subcutaneously implanted, surface-modified silicones.

To evaluate the influence of surface properties on tissue reactions, the surface of a medical-grade silicone elastomer was modified without changing the bulk properties. Surface modifications employed were oxidation by corona discharge in air, graft polymerization of water-soluble monomers, coating a polysaccharide through polyion complex formation, and covalent immobilization of cell-adhesive proteins. Surface-modified silicone sheets were implanted in the subcutaneous tissue of rats, and tissue reactions such as infiltration of inflammatory cells, appearance of foreign-body giant cells, and collagenous capsule formation were studied. It was found that the thickness of collagenous capsules formed after 16 weeks' implantation was in the range of 85-95 microns for the nontreated ones and all the modified silicones except the acrylic acid-grafted and the collagen-immobilized silicones.

New aspects of the degeneration of bioprosthetic heart valves after long-term implantation.

Bioprosthetic heart valves removed 76 to 150 months after implantation were morphologically investigated to correlate structural alterations with clinical failure modes. Traditional morphologic methods of evaluating valvular heterografts, such as microradiography and electron microscopy, were complemented by undecalcified ground sections, a new technique for analyzing the distribution of mineral deposits. Apart from well-investigated mechanisms that accelerate tissue degeneration, our observations point to additional facts: (1) phagocytosis of collagen fibrils and elastic material by macrophages and foreign body giant cells in areas near tears and perforations and (2) initial calcification indicated by delicate crystals in the intercellular space arranged in close relation to the periodicity of the cross-striation pattern of collagen fibrils. The present report not only calls attention to degenerative changes that are enhanced by mechanical stress but also underlines phagocytosis as an important mechanism in the destruction of bioprosthetic heart valves.

Phagocytosis and fibronectin of cells observed on intraocular lenses.

The phagocytic activity and distribution of fibronectin in the cells adhering to implanted intraocular lenses (IOLs) were studied in rabbits. IOLs were explanted from the posterior chamber 7 days after implantation. Phagocytosis by the cells from the IOLs was studied by electron microscopy after incubation with polystyrene beads. The distribution of fibronectin was examined immunohistochemically using an anti-fibronectin antibody. Many presumed macrophages and giant cells which were thought to be of macrophagic origin were observed on the IOLs. Adherence of the beads to the surface of the cells, phagocytosis of these beads, and fibronectin immunoreactivity were prominent in these presumed macrophages, whereas giant cells displayed a reduction in these activities. These findings suggest that the adherence activities of the presumed macrophages are less after giant cells are formed, reflecting a reduced production of fibronectin.

The influence of hydrogel functional groups on cell behavior.

This study provides information on the behavior of macrophages on the surface of the subcutaneously implanted hydrogel strips. Hydrogel containing -OH, -CO-NH-, and (CH3)2N- groups induced a spreading of macrophages on the implants. The materials containing -SO3H groups slightly, and materials containing -COOH groups more intensively, inhibited spreading of the macrophages. The fusion of macrophages into multinucleate cells was inhibited on the surface of materials containing acidic groups (-SO3H, -COOH) and increased on the hydrogels containing 30 mol% of alkaline (CH3)2N- groups in comparison with hydrogels containing -OH or -CO -NH- groups. The differences of the behavior of macrophages on the surface of individual types of hydrogels are probably independent on the adsorption of plasma fibronectin onto the hydrogels. The correlation between the macrophages spreading and fusion and surface charge of the hydrogel implant can be hypothetically explained by electrostatic interaction between macrophages cell membrane and implant.

Multinucleated giant cells on Bruch's membrane late in recurrent retinal and subretinal hemorrhaging.

Two cases with a history of recurrent subretinal hemorrhaging are reported. Multinucleated giant cells of macrophage origin were demonstrated on Bruch's membrane in a few stages of their functions of phagocytosis of old blood and storage of blood remnants. Their classification as foreign-body giant cells fails to recognize the important purification, disposal and storage functions of these cells in situations with extensive intraocular hemorrhage, exudation or necrosis.