Different Molecular Features of Epithelioid and Giant Cells in Foreign Body Reaction Identified by Single-Cell RNA Sequencing.

Although macrophage‒epithelioid cell (EPC)‒giant cell (GC) differentiation is acknowledged in foreign body reaction (FBR), the exact molecular features remain elusive. To discover the molecular profiles of EPC and GC, we analyzed mouse sponge and silk FBRs by integrating single-cell RNA sequencing and spatial sequencing, which identified seven cell types, including macrophages and fibroblasts. Macrophages comprised three subsets with a trajectory from M2-like cell to EPC to GC. They were different in many aspects, including cytokine, extracellular matrix organization/degradation, epithelial modules, and glycolysis that were consistent in both sponge and silk FBRs. EPCs exhibited epithelial modules and extracellular matrix organization, and GCs showed glycolysis, extracellular matrix degradation, and cell fusion signatures. Cellular interactions in GCs and M2-like cells were predicted to be higher than that in EPCs. High expression of inflammation or fusion-related (GPNMB, matrix metalloproteinase 12 gene MMP12, DCSTAMP) and glycolysis-related (PGAM1, ALDOA) genes was identified in GCs of human/mouse tissues, suggesting them as GC-specific markers. Our study identified unique signatures of EPCs and GCs in FBR. Importantly, GCs showed strong glycolysis signatures and cellular interactions, suggesting their activation in FBR. Our data on EPC and GC refinement and GC-specific markers enable the understanding of FBR and help to explore preventive and therapeutic management strategies for skin FBRs.

ST2 deletion accelerates inflammatory-angiogenesis and remodeling in subcutaneous implants in mice.

Implantation of biomedical/synthetic devices to replace and/or repair biological tissues very often induces an adverse healing response (scarce angiogenesis, excessive collagen deposition) which is detrimental to implant functionality and integration to host tissue. Interleukin-33/ST2 axis (IL-33/ST2) has been shown to modulate angiogenic and remodeling processes in several types of injuries. However, its effects on these processes after implantation of synthetic matrix have not been reported. Using synthetic matrix of polyether-polyurethane implanted subcutaneously in mice lacking ST2 receptor (ST2/KO), we characterized neovascularization and matrix remodeling in the fibrovascular tissue induced by the implants. Tissue accumulation was increased inside and around the implants in KO implants relative to the wild type (WT). More intense proliferative activity, using CDC 47 marker, was observed in KO implants compared with that of WT implants. Angiogenesis, using two endothelial cell markers, Von Willebrand Factor (VWF) and vascular endothelial cell VE cadherin and hemoglobin content, increased in implants of KO mice relative to control WT. Remodeling of the newly formed fibrovascular tissue (soluble collagen and PicroSirius Red-stained histological sections) showed predominance of type 1 collagen in ST2-KO implants versus type 3 in control implants. The number of positive cells for caspase-3, apoptotic marker, decreased in ST2 group. Our findings evidenced a role of IL-33/ST2 axis in restraining blood vessel formation and regulating the pattern of matrix remodeling in the fibrovascular tissue induced by synthetic implants. Intervention in this cytokine complex holds potential to accelerate integration of biomaterial and host tissue by improving blood supply and matrix remodeling.

Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation.

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

Extent of Inflammation and Foreign Body Reaction to Porous Polyethylene In Vitro and In Vivo.

BACKGROUND/AIM: High-density porous polyethylene (PP) offers possibilities for reconstruction in craniofacial surgery. The purpose of this study was to evaluate the extent of inflammation and foreign body reactions to PP in vitro and in vivo. MATERIALS AND METHODS: Cell attachment, proliferation and expression of inflammatory cytokines were assessed using murine macrophages (RAW 264.7) on two different PP materials in vitro. In vivo, Balb/c mice received PP implants at their dorsum. After sacrifice, samples were analyzed histologically and real-time PCR was used to assess expression of inflammatory cytokines. RESULTS: Cells showed a significantly decreased proliferation (p<0.001) after 48 h and a significantly increased expression of TNF-α (p<0.05) at 24, 48 and 72 h. All animals showed foreign body cell reactions and signs of chronic inflammation. Expression of all but one of the investigated cytokines dropped to non-significant levels after an initial increase. CONCLUSION: Application of porous polyethylene can cause local chronic inflammatory reactions. Although clinical application seems to be immunologically safe, indication and risks should be evaluated carefully when using PP implants.

Biomaterial Encapsulation Is Enhanced in the Early Stages of the Foreign Body Reaction During Conditional Macrophage Depletion in Transgenic Macrophage Fas-Induced Apoptosis Mice.

Macrophages are pivotal cells during the foreign body reaction (FBR), as they orchestrate the proinflammatory microenvironment inside and around biomaterials by secretion of inflammatory mediators. Furthermore, they are responsible for the degradation of biomaterials and are thought to instruct the fibroblasts that generate a fibrous capsule around implanted biomaterials. In this study, we investigated the events during the FBR when macrophages are not present. Hexamethylenediisocyanate crosslinked collagen scaffolds were implanted in "Macrophage Fas-Induced Apoptosis" mice, which allow "on demand" macrophage depletion. We observed that macrophage depletion completely inhibited inflammatory ingrowth into the scaffolds and resulted in an increased capsule size. Quantitative polymerase chain reaction analysis revealed decreased expression levels of proinflammatory mediators such as TNFα and IL1ß, and increased expression levels of collagens and fibroblast-stimulating growth factors such as EGF, FGF1, FGF2, and TGFα. Our results indicate that macrophages are indeed crucial for the generation of a proinflammatory microenvironment inside implanted biomaterials, leading to inflammatory ingrowth. In contrast, macrophages do not appear to be important for the generation of a fibrous capsule around implanted biomaterials. In fact, our data suggest that the macrophages present in the capsule might instruct the surrounding fibroblasts to produce less fibroblast-stimulating factors and less collagens.

Intravascular large B-cell lymphoma associated with silicone breast implant, HLA-DRB1*11:01, and HLA-DQB1*03:01 manifesting as macrophage activation syndrome and with severe neurological symptoms: a case report.

BACKGROUND: Silicone implants have been successfully used for breast augmentation and reconstruction in millions of women worldwide. The reaction to the silicone implant is highly variable; it can lead to local inflammatory symptoms, and sometimes to systemic symptoms and disease. Over 80 cases of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma have been reported in patients with silicone breast implants and have been accepted as a new clinical entity. To the best of our knowledge, an intravascular large B-cell lymphoma associated with a silicone breast implant has not been reported previously. CASE PRESENTATION: A 48-year-old Caucasian woman who presented with high fever was found to have splenomegaly on physical examination. A laboratory diagnosis revealed pancytopenia, hypertriglyceridemia, and hyperferritinemia. She developed signs of altered sensorium, hemiparesis, aphasia, and cauda equina syndrome. On further evaluation, she fulfilled the necessary five out of eight criteria for diagnosis of macrophage activation syndrome/hemophagocytic lymphohistiocytosis. Dexamethasone administration was followed by prompt improvement; however, 3 days later she again manifested high fever, which persisted despite administration of immunoglobulin and cyclosporine A. Her silicone breast implant was considered a possible contributor to her macrophage activation syndrome and was therefore removed. A histological examination of the capsule tissue showed an extensive lymphohistiocytic/giant cell foreign body reaction suggestive of autoimmune/inflammatory syndrome induced by adjuvants. However, the histological examination unexpectedly also revealed an intravascular large B-cell lymphoma. CONCLUSIONS: The genetic background of our patient with silicone breast implants might have predisposed her to three rare and difficult to diagnose syndromes/diseases: macrophage activation syndrome/hemophagocytic lymphohistiocytosis, autoimmune/inflammatory syndrome induced by adjuvants, and intravascular large B-cell lymphoma. The simultaneous manifestation of all three syndromes suggests causal interrelationships. Human leukocyte antigen testing in all women who undergo silicon breast implantation could in the future enable us to better evaluate the risk of potential side effects.

Bone Marrow Mesenchymal Stem Cell-Based Engineered Cartilage Ameliorates Polyglycolic Acid/Polylactic Acid Scaffold-Induced Inflammation Through M2 Polarization of Macrophages in a Pig Model.

UNLABELLED: : The regeneration of tissue-engineered cartilage in an immunocompetent environment usually fails due to severe inflammation induced by the scaffold and their degradation products. In the present study, we compared the tissue remodeling and the inflammatory responses of engineered cartilage constructed with bone marrow mesenchymal stem cells (BMSCs), chondrocytes, or both and scaffold group in pigs. The cartilage-forming capacity of the constructs in vitro and in vivo was evaluated by histological, biochemical, and biomechanical analyses, and the inflammatory response was investigated by quantitative analysis of foreign body giant cells and macrophages. Our data revealed that BMSC-based engineered cartilage suppressed in vivo inflammation through the alteration of macrophage phenotype, resulting in better tissue survival compared with those regenerated with chondrocytes alone or in combination with BMSCs. To further confirm the macrophage phenotype, an in vitro coculture system established by engineered cartilage and macrophages was studied using immunofluorescence, enzyme-linked immunosorbent assay, and gene expression analysis. The results demonstrated that BMSC-based engineered cartilage promoted M2 polarization of macrophages with anti-inflammatory phenotypes including the upregulation of CD206, increased IL-10 synthesis, decreased IL-1ß secretion, and alterations in gene expression indicative of M1 to M2 transition. It was suggested that BMSC-seeded constructs have the potential to ameliorate scaffold-induced inflammation and improve cartilaginous tissue regeneration through M2 polarization of macrophages. SIGNIFICANCE: Finding a strategy that can prevent scaffold-induced inflammation is of utmost importance for the regeneration of tissue-engineered cartilage in an immunocompetent environment. This study demonstrated that bone marrow mesenchymal stem cell (BMSC)-based engineered cartilage could suppress inflammation by increasing M2 polarization of macrophages, resulting in better tissue survival in a pig model. Additionally, the effect of BMSC-based cartilage on the phenotype conversion of macrophages was further studied through an in vitro coculture system. This study could provide further support for the regeneration of cartilage engineering in immunocompetent animal models and provide new insight into the interaction of tissue-engineered cartilage and macrophages.

Evaluation of p53 Polymorphism in Patients with Pannus-Derived Prosthetic Dysfunction.

BACKGROUND AND AIM OF THE STUDY: Prosthetic valve dysfunction (PVD) due to pannus formation is considered to occur due to a bioreaction to prosthetic material. The p53 gene plays a critical role in apoptosis and cell proliferation. p53 Arg72Pro polymorphism has been found to be associated with coronary stent restenosis, but has not yet been studied in prosthetic heart valve dysfunction. The study aim was to evaluate the association between pannus-derived PVD and p53 G72C(Arg72Pro) polymorphism. METHODS: This single-center, prospective study included 25 patients (20 females, five males; mean age 45.6 +/- 12.5 years; group 1) who underwent redo valve surgery due to PVD, and 49 age- and gender-matched control patients (44 females, five males; mean age 47.3 +/- 12.2 years; group 2) with normofunctional prostheses. The prostheses were examined using transthoracic and transesophageal echocardiography. Analyses of p53 G72C(Arg72Pro) polymorphism were performed using Roche LightCyler 2.0 Real-time polymerase chain reaction. RESULTS: The most common location of replaced valves was the mitral position in both groups (88% and 89.8%, respectively). In group 1, normal alleles (GG) were observed in 12 patients (48%), while one patient (4%) showed a homozygous mutation (GC) and 12 patients (48%) showed a heterozygous mutation (CC). In group 2, 21 patients (42.9%) had normal alleles (GG), while four (8.2%) had a homozygous mutation (CC) and 24 (48.9%) had a heterozygous mutation (GC). No significant difference was observed between the groups with regards to p53 Arg72Pro polymorphism (p = 0.769). CONCLUSION: In patients with prosthetic valves, the underlying mechanism behind pannus formation is unrelated to p53 Arg72Pro polymorphism.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Effects of caspase-1 knockout on chronic neural recording quality and longevity: insight into cellular and molecular mechanisms of the reactive tissue response.

Chronic implantation of microelectrodes into the cortex has been shown to lead to inflammatory gliosis and neuronal loss in the microenvironment immediately surrounding the probe, a hypothesized cause of neural recording failure. Caspase-1 (aka Interleukin 1ß converting enzyme) is known to play a key role in both inflammation and programmed cell death, particularly in stroke and neurodegenerative diseases. Caspase-1 knockout (KO) mice are resistant to apoptosis and these mice have preserved neurologic function by reducing ischemia-induced brain injury in stroke models. Local ischemic injury can occur following neural probe insertion and thus in this study we investigated the hypothesis that caspase-1 KO mice would have less ischemic injury surrounding the neural probe. In this study, caspase-1 KO mice were implanted with chronic single shank 3 mm Michigan probes into V1m cortex. Electrophysiology recording showed significantly improved single-unit recording performance (yield and signal to noise ratio) of caspase-1 KO mice compared to wild type C57B6 (WT) mice over the course of up to 6 months for the majority of the depth. The higher yield is supported by the improved neuronal survival in the caspase-1 KO mice. Impedance fluctuates over time but appears to be steadier in the caspase-1 KO especially at longer time points, suggesting milder glia scarring. These findings show that caspase-1 is a promising target for pharmacologic interventions.

Deletion of the chemokine receptor CCR2 attenuates foreign body reaction to implants in mice.

Subcutaneous implantation of synthetic materials and biomedical devices often induces abnormal tissue healing - the foreign body reaction - which impairs their function. Here we investigated the role of the chemokine receptor CCR2 in this reaction to subcutaneous implants in mice. We measured angiogenesis, inflammation and fibrogenesis induced by implantation, for 1, 4, 7 and 14days, of polyether-polyurethane sponges in mice with genetic deletion of CCR2 (KO) and WT mice. Blood flow was determined by dye diffusion and laser Doppler perfusion techniques. Cytokines (VEGF, TNF-α, CCL2, TGF-ß1) were measured by ELISA. Histochemical methods were used to assess collagen deposition and macrophage-derived giant cells in the implants. Skin and implant blood flow was lower in CCR2 KO than in WT mice, as were other aspects of neo-vascularization of the implants. Neutrophil accumulation was increased in KO implants but macrophage accumulation was decreased. Implant content of CCL2 was higher in KO implants, but TGF-ß1, collagen deposition and the number of foreign body giant cells were lower than in WT implants. Deletion of CCR2 decreased blood flow in normal skin and inhibited neo-vascularization, chronic inflammation and fibrogenesis in subcutaneous implants. The chemokine receptor CCR2 plays an important role in both normal skin and in the reaction elicited by subcutaneous implantation of a foreign body.

Gene chip/PCR-array analysis of tissue response to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer surfaces in a mouse subcutaneous transplantation system.

To evaluate the in vivo foreign body reaction to bio-inert 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers, MPC polymer-coated porous substrates, with large surface area, were implanted subcutaneously in mice for 7 and 28 days, and the surrounding tissue response and cells infiltrating into the porous structure were evaluated. The MPC polymer surface induced low angiogenesis and thin encapsulation around the porous substrate, and slightly suppressed cell infiltration into the porous substrate. M1-type macrophage specific gene (CCR7) expression was suppressed by the MPC polymer surface after 7 days, resulting in the suppression of inflammatory cytokine/chemokine gene expression. However, the expression of these genes on the MPC polymer surface was higher than on the non-coated surface after 28 days. These findings suggest that MPC polymer surfaces successfully inhibit inflammatory responses during the early stage of tissue response, and seem to retard its occurrence over time.

Transcriptional switching in macrophages associated with the peritoneal foreign body response.

We previously demonstrated that myeloid cells are the source of fibrotic tissue induced by foreign material implanted in the peritoneal cavity. This study utilised the MacGreen mouse, in which the Csf1r promoter directs myeloid-specific enhanced green fluorescent protein (EGFP) expression, to determine the temporal gene expression profile of myeloid subpopulations recruited to the peritoneal cavity to encapsulate implanted foreign material (cubes of boiled egg white). Cells with high EGFP expression (EGFP(hi)) were purified from exudate and encapsulating tissue at different times during the foreign body response, gene expression profiles determined using cDNA microarrays, and data clustered using the network analysis tool, Biolayout Express(3D). EGFP(hi) cells from all time points expressed high levels of Csf1r, Emr1 (encoding F4/80), Cd14 and Itgam (encoding Mac-1) providing internal validation of their myeloid nature. Exudate macrophages (days 4-7) expressed a large cluster of cell cycle genes; these were switched off in capsule cells. Early in capsule formation, Csf1r-EGFP(hi) cells expressed genes associated with tissue turnover, but later expressed both pro- and anti-inflammatory genes alongside a subset of mesenchyme-associated genes, a pattern of gene expression that adds weight to the concept of a continuum of macrophage phenotypes rather than distinct M1/M2 subsets. Moreover, rather than transdifferentiating to myofibroblasts, macrophages contributing to later stages of the peritoneal foreign body response warrant their own classification as 'fibroblastoid' macrophages.

A culture model to analyze the acute biomaterial-dependent reaction of human primary macrophages.

Macrophages are important in foreign body reactions. We devised a culture model with human primary macrophages to evaluate the acute response of macrophages to biomaterials. First we selected proteins representative for pro-inflammatory (M1) or anti-inflammatory/repair (M2) response of monocytes isolated from blood of healthy human donors by exposing them to LPS+IFNγ or IL-4. A relative M1/M2 index was calculated using IL-1ß, IL-6, tumor necrosis factor (TNF)α, monocyte chemotactic protein (MCP)-3 and macrophage inflammatory protein (MIP)-1α as M1 markers, and IL-1 receptor antagonist (IL-1RA), CCL18, regulated and normal T-cell expressed and secreted (RANTES), and macrophage-derived chemokine (MDC) as M2 markers. Then monocytes were cultured for 3days on 4 materials selected for known different foreign body reactions: Permacol™, Parietex™ Composite, multifilament polyethylene terephthalate and multifilament polypropylene. Macrophages on polypropylene produced high levels of anti-inflammatory proteins with a low M1/M2 index. Macrophages on Parietex™ Composite produced high levels of inflammatory and anti-inflammatory proteins, with a high M1/M2 index. Macrophages on polyethylene terephthalate also resulted in a high M1/M2 index. Macrophages on Permacol™ produced a low amount of all proteins, with a low M1/M2 index. This model with human primary macrophages and the panel of read-out parameters can be used to evaluate the acute reaction of macrophages to biomaterials in vitro to get more insight in the foreign body reaction.

Wear particles promote endotoxin tolerance in macrophages by inducing interleukin-1 receptor-associated kinase-M expression.

Toll-like receptors (TLRs) recognizing pathogen-associated molecular patterns (PAMP) play a role in local immunity and participate in implant-associated loosening. TLRs-mediated signaling is regulated by interleukin-1 receptor-associated kinase-M (IRAK-M). Our previous studies have proved that IRAK-M is induced by wear particles in macrophages from periprosthetic tissues. In this study, the IRAK-M-related mechanisms were further explored by lipopolysaccharide (LPS) and/or titanium (Ti) particles stimulations and small interfering RNAs (siRNAs). The protein level of IRAK-M was studied using western blotting and tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) levels were measured using ELISA. Results showed that in RAW264.7 cells stimulated by LPS after Ti particle pre-exposure, IRAK-M was slightly changed, compared with LPS stimulation. And levels of TNF-α and IL-1ß in cultures stimulated by LPS first after Ti particle pre-exposure were lower than in the other two groups which were stimulated by LPS with or without Ti particles (p < 0.001), whereas there were no statistic differences between the later two (p > 0.05). The cytokines were lowest in Ti particles alone stimulation. After siRNAs silenced, IRAK-M-deficient cells exhibited increased expression of the cytokines in LPS stimulation after Ti particle pre-exposure and when stimulated with Ti particles alone. Our findings suggest that debris-induced IRAK-M decreases foreign body reactions, but at the same time, the over-expression of IRAK-M may also be detrimental on local intrusion of PAMPs or bacteria, negatively regulates the LPS-induced and TLRs-mediated inflammation and results in immunosuppression in periprosthetic tissue, which may predispose to implant-associated infections.

Influence of a new monofilament polyester mesh on inflammation and matrix remodeling.

ABSTRACT Synthetic mesh is widely used for hernia repairs, but mesh-induced chronic inflammatory responses may lead to postoperative complications. We previously showed an elevated response to multifilament polyester (PE) versus monofilament polypropylene (PP) and polytetrafluoroethylene (PTFE) meshes, but it is unclear whether this discrepancy is due to the differences in chemical composition or filament structure. This study compares the influence of a newly available monofilament PE mesh to that of multifilament PE, monofilament PP, and monofilament PTFE on the expression of genes important in inflammation and extracellular matrix remodeling in a rat model. Full thickness abdominal wall defects were corrected with onlay repair or suture repair with no mesh. Explants were harvested 7 or 90 days after repair and divided for histology and mRNA analyses using real-time quantitative polymerase chain reaction arrays to profile expression at the tissue-mesh interface. Monofilament PE elicited a reduced foreign body reaction compared to multifilament PE, corresponding with reduced mRNA expression of important inflammatory cytokines and matrix metalloproteinases (MMPs). Unexpectedly, monofilament PE also resulted in markedly reduced mRNA expression of tumor necrosis factor and MMPs 3 and 9 compared to the widely-used monofilament PP mesh. Findings from this study revealed that both chemical composition and filament structure are important mesh characteristics that may affect a patient's wound healing response and clinical outcome, and should be considered by the surgeon when choosing a particular mesh. Although clinical studies are warranted, results in a rodent model suggest that monofilament PE may be more beneficial than the multifilament form for certain hernia repairs.

Gene expression study of monocytes/macrophages during early foreign body reaction and identification of potential precursors of myofibroblasts.

Foreign body reaction (FBR), initiated by adherence of macrophages to biomaterials, is associated with several complications. Searching for mechanisms potentially useful to overcome these complications, we have established the signaling role of monocytes/macrophages in the development of FBR and the presence of CD34(+) cells that potentially differentiate into myofibroblasts. Therefore, CD68(+) cells were in vitro activated with fibrinogen and also purified from the FBR after 3 days of implantation in rats. Gene expression profiles showed a switch from monocytes and macrophages attracted by fibrinogen to activated macrophages and eventually wound-healing macrophages. The immature FBR also contained a subpopulation of CD34(+) cells, which could be differentiated into myofibroblasts. This study showed that macrophages are the clear driving force of FBR, dependent on milieu, and myofibroblast deposition and differentiation.

Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation.

Fibrous encapsulation of surgically implanted devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA (siRNA) conjugated with branched polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knock-down led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules were observed. The siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants.

Drosophila lamin mutations cause melanotic mass formation and lamellocyte differentiation.

The fruit fly immune system is a valuable model for invertebrate and innate immunity. Cellular immune reactions in Drosophila are of great interest, especially the molecular genetic mechanisms of hemocyte differentiation and the encapsulation of foreign bodies. Here we report that changes in the lamin gene cause melanotic masses. These darkened clusters of cells result from autoimmune-like encapsulation of self-tissue, as shown by the presence in lam larvae of lamellocytes, effector hemocytes that appear in larvae following wounding or parasitization. Lamins thus affect immunity in Drosophila, and lam mutations can serve as genetic tools to dissect cellular immune signaling and effector pathways.

Matrix metalloproteinase-9 deficiency leads to prolonged foreign body response in the brain associated with increased IL-1beta levels and leakage of the blood-brain barrier.

Matrix metalloproteinases (MMPs) are enzymes with specificity towards extracellular matrix (ECM) components. MMPs, especially MMP-9, have been shown to degrade components of the basal lamina and disrupt the blood-brain barrier (BBB) and thus, contribute to neuroinflammation. In the present study we examined the role of MMP-9 in the foreign body response in the brain. Millipore filters of mixed cellulose ester were implanted into the brain cortex of wild type and MMP-9-null mice for a period of 2 d to 8 wks and the response was analyzed by histology and immunohistochemistry. We observed enhanced and prolonged neuroinflammation in MMP-9-null mice, evidenced by persistence of neutrophils, macrophages/microglia, and reactive astrocytes up to 8 wks post-implantation. In addition, blood vessel density around implants was increased in MMP-9-null mice and detection of mouse serum albumin (MSA) indicated that vessels were leaky. Immunohistochemical and western blot analyses indicated that this defect was associated with the absence of tight junction proteins zonula occludens-1 (ZO-1) and ZO-2 from vessels in proximity to implants. Analysis of brain sections and brain protein extracts revealed that the levels of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), which is a substrate for MMP-9, were significantly higher in MMP-9-null mice at 8wks post-implantation. Collectively, our studies suggest that increased levels of IL-1beta and the delayed repair of BBB are associated with prolongation of the FBR in MMP-9-null mice.