Live imaging the foreign body response in zebrafish reveals how dampening inflammation reduces fibrosis.

Implanting biomaterials in tissues leads to inflammation and a foreign body response (FBR), which can result in rejection. Here, we live image the FBR triggered by surgical suture implantation in a translucent zebrafish model and compare with an acute wound response. We observe inflammation extending from the suture margins, correlating with subsequent avascular and fibrotic encapsulation zones: sutures that induce more inflammation result in increased zones of avascularity and fibrosis. Moreover, we capture macrophages as they fuse to become multinucleate foreign body giant cells (FBGCs) adjacent to the most pro-inflammatory sutures. Genetic and pharmacological dampening of the inflammatory response minimises the FBR (including FBGC generation) and normalises the status of the tissue surrounding these sutures. This model of FBR in adult zebrafish allows us to live image the process and to modulate it in ways that may lead us towards new strategies to ameliorate and circumvent FBR in humans.This article has an associated First Person interview with the first author of the paper.

Ultrastructural aspects of foreign body giant cells generated on different substrates.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.

Light- and transmission-electron-microscopic investigations on distribution of CD44, connexin 43 and actin cytoskeleton during the foreign body reaction to a nanoparticular hydroxyapatite in mini-pigs.

Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation.

Cell adhesion on explanted intraocular lenses part 2: experimental study of a surface-modified IOL in rabbits.

PURPOSE: To develop a hydrophobic acrylic IOL with a hydrophilic, anti-cell adhesive surface characteristic. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. DESIGN: Experimental study. METHODS: Hydrophobic acrylic IOLs were coated with hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and implanted in rabbit eyes following lens extraction. Cell adhesion on the IOL surface was histologically compared with that on an uncoated IOL under light microscopy. Specimens were also observed under scanning electron microscopy to examine the effects of MPC coating on cell morphology. RESULTS: Hydrophilic MPC coating reduced cell adhesion on acrylic IOLs at 1 month postoperatively. CONCLUSION: Coating an acrylic IOL with a hydrophilic polymer inhibited cell adhesion on the IOL surface.

Histological study of the left colon of rats after extra-mucosal seromyotomy (continent valves). Evaluation of colonic emptying.

PURPOSE: To elaborate an animal model with the objective of studying the continence of the biological valves surgically performed in the left colon of rats. METHODS: Thirty four rats were operated on and divided into three groups (G). G1 (sham) animals which underwent laparotomy only; G2 (perineal amputation without valves): animals which underwent amputation of the anal sphincter complex combined with a perineal colostomy; G3 (abdominoperineal amputation combined with valves): animals which underwent abdominoperineal amputation combined with three, equidistant and circumferential (360 degrees), extra-mucosal seromyotomies, of the descending colon, which were sutured to create biological valves combined with perineal colostomy. Animals were euthanized in the late postoperative period and surgical valves were saved for histopathological study. RESULTS: Surgical procedure provoked intestinal dilation, as well as segmented chambers along the descending colon. Retained fecalomas between the valves and proximal to them were also noted. Six rats died of intestinal obstruction due to fecal impaction at the surgical site. The sequence of events was: stasis, obstruction, distention, perforation, peritonitis and death. Histopathology showed inflammation due to foreign body type reaction around the sutured colon causing partial concentric stenosis, capable of interfering normal mechanical activity of the distal colon. This process resulted in retardation of the intestinal transit. CONCLUSION: Extra-mucosal seromyotomies, with seromuscular suture, can be used as an operative procedure capable of causing retardation in the intestinal transit of rats.

Histological study of the left colon of rats after extra-mucosal seromyotomy (continent valves): evaluation of colonic emptying / Estudo histológico do cólon esquerdo de ratos após a confecção de seromiotomias extra-mucosas (válvulas continentes): avaliação do grau de esvaziamento do conteúdo colônico

PURPOSE: To elaborate an animal model with the objective of studying the continence of the biological valves surgically performed in the left colon of rats. METHODS: Thirty four rats were operated on and divided into three groups (G). G1 (sham) animals which underwent laparotomy only; G2 (perineal amputation without valves): animals which underwent amputation of the anal sphincter complex combined with a perineal colostomy; G3 (abdominoperineal amputation combined with valves): animals which underwent abdominoperineal amputation combined with three, equidistant and circumferential (360(0)), extra-mucosal seromyotomies, of the descending colon, which were sutured to create biological valves combined with perineal colostomy. Animals were euthanized in the late postoperative period and surgical valves were saved for histopathological study. RESULTS: Surgical procedure provoked intestinal dilation, as well as segmented chambers along the descending colon. Retained fecalomas between the valves and proximal to them were also noted. Six rats died of intestinal obstruction due to fecal impaction at the surgical site. The sequence of events was: stasis, obstruction, distention, perforation, peritonitis and death. Histopathology showed inflammation due to foreign body type reaction around the sutured colon causing partial concentric stenosis, capable of interfering normal mechanical activity of the distal colon. This process resulted in retardation of the intestinal transit. CONCLUSION: Extra-mucosal seromyotomies, with seromuscular suture, can be used as an operative procedure capable of causing retardation in the intestinal transit of rats.

OBJETIVO: Modelo de experimentação, com confecção de válvulas biológicas no cólon esquerdo de ratos com o objetivo de estudar o grau de continência dessas válvulas. MÉTODOS: Trinta e quatro ratos foram operados e distribuidos em três grupos: G1 (grupo simulado) submetido apenas à laparotomia, G2 (grupo amputado sem válvula) submetido à amputação do conjunto esfincteral mais colostomia perineal e G3 (grupo amputado com válvula) submetido à amputação do conjunto esfincteral, confecção de três seromiotomias extra-mucosas, eqüidistantes e circunferenciais (360(0) - válvulas biológicas), no colon descendente mais colostomia perineal. No pós-operatório tardio, os animais dos três grupos foram submetidos à eutanásia para coleta da peça cirúrgica e estudo histopatológico das válvulas. RESULTADOS: Os resultados mostraram que o procedimento culminou em dilatação intestinal, confirmada pela formação de verdadeiras câmaras de segmentação e pela presença de fecalomas retidos entre as válvulas e cranialmente a elas. Seis ratos morreram em decorrência de obstrução intestinal por impacção de fezes no local operado, na seqüência: obstrução, estase, distensão, perfuração, peritonite e morte. As alterações histopatológicas confirmaram o processo inflamatório com reação do tipo corpo estranho, no perímetro do cólon suturado, proporcionando uma estenose parcial concêntrica, levando à alteração da atividade mecânica do cólon distal, resultando no retardo do trânsito intestinal. CONCLUSÃO: As seromiotomia extramucosas, com sutura seromuscular, podem ser utilizadas como técnica operatória para se obter retardo do trânsito intestinal em ratos.

Development of foreign body giant cells in response to implantation of Spongostan as a scaffold for cartilage tissue engineering.

Spongostan, a gelatinous haemostatic sponge, is used in surgery. Moreover, Spongostan may serve as a scaffold for proteins or cells implanted into defects. At the site of biomaterial implantation, foreign body giant cells (FBGCs) may develop which are responsible for Spongostan degradation. The purpose of the present study was to examine whether Spongostan may serve as a scaffold in allogenic grafting of chondrocytes developed from rabbit auricular cartilage. The obtained results indicate that Spongostan fulfils its function as a cell scaffold, induces no inflammatory reaction and involves development of foreign body giant cells which participate in the process of its degradation. Microscopic observation showed that FBGCs manifest presence of cytoplasmic projections and lysosomes, which participate in phagocytosis of the applied scaffold.

Histopathologic examination of conjunctival tophi in gouty arthritis.

PURPOSE: To report a case of conjunctival tophi in a 59-year-old woman with gouty arthritis. DESIGN: Observational case report. METHODS: A 59-year-old woman with a 25-year history of severe gouty arthritis presented with bilateral chalky white conjunctival deposits. Conjunctival biopsies were obtained, and light and electron microscopy studies were performed. RESULTS: Light microscopy confirmed multiple gouty tophi. Transmission electron microscopy revealed intracytoplasmic crystalline deposits within histiocytes. Extracellular aggregates of the crystalline deposits were enclosed by membranous material. CONCLUSIONS: Conjunctival crystals may aid in identifying undiagnosed patients. In addition to conjunctival tophi, patients may also have uveitis, corneal crystals, and band keratopathy.

An in-vivo model to interrogate the transition from acute to chronic inflammation.

This study describes the modulation of the rodent foreign body giant cell (FBGC) response to subcutaneously implanted, biodegradable poly(lactide-co-glycolide)/calcium phosphate (PLGA/CaP) composites by application of a thin surface coat of calcium phosphate. Macroporous PLGA/CaP composite scaffolds, with interconnecting macroporosity, were half coated with a 3mm thick layer of CaP by immersion in simulated body fluid. Half-coated scaffolds were implanted subcutaneously in the dorsum of male Wistar rats for 1, 4 and 8 weeks. Specimens were embedded in paraffin and tissue sections evaluated by light microscopy with particular reference to the FBGC response. Histomorphometry revealed that FBGCs were in contact with 6% (+/- 3.5%) of the uncoated half, at 1 week, but no FBGCs were seen on the coated half. By 4 weeks, FBGCs were seen on both the uncoated and coated halves of the scaffolds with 87% (+/-10%) and 36% (+/-4%) FBGC/polymer contact respectively. By 8 weeks these FBGC contact percentages had risen to 97% (+/-0.45%) in the case of the uncoated halves of scaffolds, but decreased to 22% (+/-4%) in the case of the CaP-coated halves. Thus the CaP coating abrogated the FBGC response to the underlying polymer. Such a model may prove useful in providing an experimental system whereby both the mechanisms of biocompatibility and the transition from acute to chronic inflammation could be interrogated.

Electron microscopy of tissue adherent to explanted electrodes in dystonia and Parkinson's disease.

Deep brain stimulation (DBS) is used to treat a variety of severe medically intractable movement disorders, including Parkinson's disease, tremor and dystonia. There have been few studies examining the effect of chronic DBS on the brains of Parkinson's disease patients. Most of these post mortem studies concluded that chronic DBS caused mild gliosis around the lead track and did not damage brain tissue. There have been no similar histopathological studies on brains from dystonic patients who have undergone DBS. In this study, our objective was to discover whether tissue would be attached to DBS electrodes removed from patients for routine clinical reasons. We hoped that by examining explanted DBS electrodes using scanning (SEM) and/or transmission (TEM) electron microscopy we might visualize any attached tissue and thus understand the electrode-human brain tissue interaction more accurately. Initially, SEM was performed on one control DBS electrode that had not been implanted. Then 21 (one subthalamic nucleus and 20 globus pallidus internus) explanted DBS electrodes were prepared, after fixation in 3% glutaraldehyde, for SEM (n = 9) or TEM (n = 10), or both (n = 2), according to departmental protocol. The electrodes were sourced from two patients with Parkinson's disease, one with myoclonic dystonia, two with cervical dystonia and five with primary generalized dystonia, and had been in situ for 11 and 31 months (Parkinson's disease), 16 months (myoclonic dystonia), 14 and 24 months (cervical dystonia) and 3-24 months (primary generalized dystonia). Our results showed that a foreign body multinucleate giant cell-type reaction was present in all TEM samples and in SEM samples, prewashed to remove surface blood and fibrin, regardless of the diagnosis. Some of the giant cells were >100 microm in diameter and might have originated from either fusion of parenchymal microglia, resident perivascular macrophage precursors and/or monocytes/macrophages invading from the blood stream. The presence of mononuclear macrophages containing lysosomes and sometimes having conspicuous filopodia was detected by TEM. Both types of cell contained highly electron-dense inclusions, which probably represent phagocytosed material. Similar material, the exact nature of which is unknown, was also seen in the vicinity of these cells. This reaction was present irrespective of the duration of implantation and may be a response to the polyurethane component of the electrodes' surface coat. These findings may be relevant to our understanding of the time course of the clinical response to DBS in Parkinson's disease and various forms of dystonia, as well as contributing to the design characteristics of future DBS electrodes.

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.

Histology and electron microscopy of explanted bifurcated endovascular aortic grafts: evidence of early incorporation and healing.

PURPOSE: To report an examination of explanted bifurcated endovascular aortic grafts for histologic evidence of early healing and incorporation. METHOD: Two bifurcated endovascular aortic grafts composed of polycarbonate urethane and Elgiloy wire were explanted 42 and 21 days after successful endovascular exclusion of abdominal aortic aneurysms. Both patients expired from causes unrelated to endograft deployment. The explanted devices were examined using immunohistochemical analysis and electron microscopy. RESULTS: On explantation, both grafts appeared to have excluded the aneurysm with no evidence of endoleak, graft migration, or thrombosis. Histological examination showed numerous inflammatory cells and good ingrowth of tissue into the proximal 2 cm of the graft. Collagen and smooth muscle cells were evident in the proximal portion of the graft with only collagen in the distal segments. Neointimal formation was seen within the proximal 2 cm also, but not at the distal segments. Macrophages were present in the graft. Scanning electron microscopy showed an extensive matrix of fibers that most likely represented collagen. CONCLUSIONS: Bifurcated endovascular aortic grafts show inflammatory and mild foreign body reactions, collagen formation, and intimal ingrowth during healing. These findings are similar to some of the healing properties reported for sutured grafts, as well as other endovascular grafts.

Ultrastructure of multinucleated giant cell apoptosis in foreign-body granuloma.

To elucidate the role of apoptosis in the disappearance of multinucleated giant cells from the granulation tissue in cases of foreign-body granuloma, we induced a foreign-body reaction by implanting a collagen sponge into the dorsum of the rat and observed apoptotic changes within the multinucleated giant cells using electron microscopy. Two types of multinucleated giant cells were identified presenting apoptotic characteristics morphologically. One was characterized by apoptosis of only one nucleus, followed by cytoplasmic changes, rupture of the plasma membrane and necrosis evoking an inflammatory reaction. The other showed typical apoptotic changes in the majority or in all of the nuclei, followed by phagocytosis of the apoptotic syncytia. The results of the present study suggest that apoptosis occurring within only one nucleus might be triggered by overexpression of the p53 protein, because DNA abnormalities are confined to this single nucleus. In contrast apoptosis occurring simultaneously in the majority or all of the nuclei is most probably due to cell death caused by senescence.

Role for interleukin-4 in foreign-body giant cell formation on a poly(etherurethane urea) in vivo.

Interleukin-4 (IL-4) was previously shown to induce extensive macrophage fusion to form foreign-body giant cells (FBGCs) in vitro. In the present study, our goal was to extend these findings to an in vivo test environment on biomaterials. The subcutaneous cage-implant system was modified for mice to elucidate IL-4 participation in mediating FBGC formation in vivo. Exudate leukocyte concentrations from cages containing poly(etherurethane urea) (PEUU A') and empty cage controls indicated a similar inflammatory response that turned toward resolution by 14 days postimplantation, thus confirming the applicability of the cage-implant system in mice. FBGC kinetic analysis showed that the formation of mouse FBGCs occurs through the fusion of adherent macrophages at a constant rate up to 14 days of implantation. Purified goat anti-mouse IL-4 neutralizing antibody (IL4Ab) or normal goat nonspecific control IgG (gtIgG) at various concentrations, or recombinant murine IL-4 (muIL4) was injected into the implanted cages containing PEUU A' every 2 days for 7 days. The injection of IL4Ab significantly decreased the FBGC density on PEUU A' cage-implanted in mice, when compared with the nonspecific IgG or PBS injection controls. Conversely, the FBGC density was significantly increased by the injection of muIL4 when compared with nonspecific IgG and PBS injection controls. Adherent macrophage density, FBGC morphology, FBGC average size, and size distribution were not significantly different among IL4Ab, nonspecific control gtIgG, muIL4, and PBS control groups. Our data suggest that IL-4 participates in FBGC formation on biomaterials in vivo.

Light chain nephropathy with remarkable accumulation of multinucleated giant cells in the kidney.

A case of light chain deposition disease with multinucleated giant cell accumulation in the kidney is described. A 54-year-old man was admitted to out hospital due to moderate renal failure. Complicated with eosinophilic pneumonia two months after admission, his renal function abruptly deteriorated and hemodialysis was started. Three-day methylprednisolone pulse therapy, however, partially recovered his renal function and hemodialysis was discontinued. His renal biopsy specimen revealed kappa light chain deposition disease with nodular mesangial expansion and subendothelial electron dense deposits. The most characteristic finding in this patient was accumulation of foreign body type multinucleated giant cells around atrophic tubules, small arteries and obsolescent glomeruli, probably associated with light chain deposition. No increase in kappa light chain was detected in his serum or concentrated urine. Such disseminated giant cell reaction, other than intra-luminal infiltration in the tubules, has not been reported in the literature and might be related to the physicochemical or structural properties of the deposited protein.

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.

Paramyxovirus-like nuclear inclusions identical to those of Paget's disease of bone detected in giant cells of primary oxalosis.

Nuclear inclusions, identical to those characteristic of Paget's disease of bone, were observed in giant cells in four of eight cases of primary oxalosis. The giant cells containing nuclear inclusions were directly involved in phagocytosis of large oxalate crystals in the context of typical foreign body granulomas in the bone marrow. Cytochemically, all of them exhibited strong tartrate-resistant acid phosphatase activity, and a proportion of them also tartrate-resistant acid ATPase. The inclusions consisted of typical arrays of filamentous material as described in Paget's disease, admixed with variable proportions of electron-dense material closely reminiscent of nucleolar pars fibrillaris and fibrillary centres. These data indicate: (a) the occurrence of Paget-like inclusions in a bone disease unrelated to Paget's disease, not causally related to viral infection, and resulting from an inborn metabolic derangement; and (b) the occurrence of Paget-like inclusions in foreign body giant cells as opposed to osteoclasts. We suggest that the occurrence of paramyxovirus-like nuclear inclusions in either osteoclasts or giant cells may represent an epiphenomenon of cell fusion and giant cell formation whenever appropriate stimuli act on latently infected precursor cells. Furthermore, our data suggest that nucleoli may represent the specific site of virus-like inclusion formation.

[Alloplastic replacement of the anterior cruciate ligament. Comparative studies of ruptured Dacron, Trevira and Ligapro ligaments]. / Alloplastischer Ersatz des vorderen Kreuzbandes. Vergleichende Untersuchungen an rupturierten Dacron-, Trevira- und Ligaprobändern.

Histological investigations were performed on 24 polyethylene terephthalate ligaments (eight Dacron, eleven Trevira and five Ligapro) which had served as substitute for the human anterior cruciate ligament. Because of rupture or loosening they had been explanted six to 48 months after operation. Only in one Trevira ligament a longitudinal direction of collagenous lamellae could be seen, all other ligaments had only few collagenous fibers, mainly orientated in circular direction. In the fibrous tissue of all three kinds of ligaments there were signs of inflammation. Foreign body giant cells have immigrated--they mainly enclose the graft fibres in Dacron and Ligapro ligaments. In Trevira and Ligapro ligaments the foreign body giant cells phagocyte particles of graft fibers after increasing implantation times. The inflammatory reaction to graft fibers and their particles may also damage host tissue. Regarding these results we conclude that the Polyethylene terephthalate ligaments are not qualified as substitutes for the anterior cruciate ligament.