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.

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.

3,4-Dihydroxy-L-Phenylalanine as a Novel Covalent Linker of Extracellular Matrix Proteins to Polyacrylamide Hydrogels with a Tunable Stiffness.

Cells acquire mechanical information from their surrounding and convert this into biochemical activity. The concept and mechanism behind this cellular mechanosensing and mechanotransduction are often studied by means of two-dimensional hydrogels. Polyacrylamide hydrogels (PAAMs) offer chemical, mechanical, and optical advantages but due to their inert surface do not allow protein and cell adherence. Several cross-linkers have been used to functionalize the surface of PAAMs with extracellular matrix (ECM) proteins to enable cell culture. However, the most commonly used cross-linkers are either unstable, expensive, or laborious and often show heterogeneous coating or require PAAM modification. Here, we introduce 3,4-dihydroxy-l-phenylalanine (L-DOPA) as a novel cross-linker that can functionalize PAAMs with ECM without the above-mentioned disadvantages. A homogenous collagen type I and fibronectin coating was observed after L-DOPA functionalization. Fibroblasts responded to differences in PAAMs' stiffness; morphology, cell area, and protein localization were all affected as expected, in accordance with literature where other cross-linkers were used. In conclusion, L-DOPA can be used as a cross-linker between PAAMs and ECM and represents a novel, straightforward, nonlaborious, and robust method to functionalize PAAMs for cell culture to study cell mechanosensing.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

PURPOSE: The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. METHODS: A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor ß (TGF-ß [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. RESULTS: MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-ß1 and TGF-ß2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. CONCLUSIONS: Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

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.

The antitumor activity of hydrophobin SC3, a fungal protein.

The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.

Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components.

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFNγ) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages.

Endotoxin contamination delays the foreign body reaction.

Biomaterials are at continuous risk of bacterial contamination during production and application. In vivo, bacterial contamination of biomaterials delays the foreign body reaction (FBR). Endotoxins such as lipopolysaccharides (LPS), major constituents of the bacterial cell wall, are potent stimulators of the immune system in vitro and in vivo. In vitro, biomaterials contaminated with LPS induce the production of proinflammatory cytokines by adherent macrophages. This suggests that the presence of endotoxins on biomaterials will intensify the FBR. The effects of LPS on the course of the FBR have never been studied in vivo. In this study, the influence of LPS contamination on the FBR to subcutaneously implanted Puramatrix-loaded hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) disks was studied in rats. During the onset phase of the FBR, a massive influx of granulocytes was detected in LPS-contaminated disks, while their presence was prolonged. IL-10 production inside LPS-contaminated disks was increased at days 10 and 21. Macrophage densities were not affected by the presence of LPS. However, macrophage functionality was altered: giant cell formation and biomaterial degradation were delayed by LPS-contamination up to 21 days. On the basis of these results, we conclude that LPS delays the FBR. This finding indicates that endotoxin contamination has significant implications for the in vivo function of biomaterials and medical devices and emphasizes the importance of endotoxin testing.