Development and evaluation of biodegradable silk fibroin scaffolds

The nature of in vivo leukocyte adhesion and foreign-body giant cell (FBGC) formation on polyurethanes was studied through theoretical and statistical analyses in terms of cell size distribution, density changes, and kinetics of FBGC formation. The results showed that the size distribution of FBGCs followed a "most probable" distribution. During FBGC formation, the densities of FBGCs changed with time. At an early stage, the number of FBGCs increased with time to a maximum at the expense of macrophages. As more FBGCs were formed and less macrophages were present, the fusion of FBGCs among themselves became significant. This, in turn, caused a gradual decrease of FBGC density with time. The rate of FBGC formation was characterized by a rate constant that represented certain characteristics of cell fusion and FBGC formation and the density of initial FBGC-forming macrophages that were a small fraction of leukocytes adhering to the surface. The direct correlations of surface cracking and pitting and adherent FBGCs demonstrated the influence of phagocytic actions of FBGCs on the biostability of implanted polyurethanes. While the cracking was thought to be caused by oxidative degradation facilitated by oxygen ion/radical release of FBGCs, the pitting appeared to result from the Methacrol 2138F aggregates diffusing out of the polymer in an acidic microenvironment under FBGCs, which in turn could be enhanced by the surface degradation and cell phagocytosis. The added Santowhite powder in polyurethane had a significant influence on FBGC formation: It reduced FBGC density and rate of FBGC formation by reducing leukocyte adhesion and the number of macrophages participating in FBGC formation.

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.

Due to its antimicrobial, anti-inflammatory and pro-healing properties, the application of cold atmospheric plasma (CAP) has emerged as a new and promising therapeutic strategy in various fields of medicine, including general medicine and dentistry. In this light, the aim of the present study was to investigate the effects of a homemade plasma jet on the cellular behaviour of two important cell types involved in gingivitis, namely gingival fibroblasts (HGF-1 cell line) and macrophages (RAW 264.7 cell line), by the direct application of CAP in different experimental conditions. The cellular behaviour of the HGF-1 cells was investigated in terms of viability/proliferation (LIVE/DEAD and CCK-8 assays), morphological features (immunofluorescent staining of the actin cytoskeleton) and fibronectin expression (immunocytochemical staining of the fibronectin network), while the macrophages' response was evaluated through the assessment of the cellular survival/proliferation rate (LIVE/DEAD and CCK-8 assays), morphological behaviour (immunofluorescent staining of the actin cytoskeleton) and inflammatory activity (pro-inflammatory cytokine secretion profile (ELISA assay) and foreign body giant cells (FBGCs) formation (immunofluorescent staining of the actin cytoskeleton and multinuclearity index determination)). The in vitro biological assessment revealed an upward trend dependent on treatment time and number of CAP applications, in terms of fibroblasts proliferation (p < 0.0001) and fibronectin expression (p < 0.0001). On the other hand, the macrophages exposed to five consecutive CAP applications for longer treatment times (over 120 s) exhibited a strong pro-inflammatory activity, as evinced by their altered morphology, pro-inflammatory cytokine profile (p < 0.0001) and FBGCs formation. Overall, our results demonstrate that CAP exposure, when used with appropriate operating parameters, has a beneficial effect on the cellular response of HGF-1 and RAW 264.7 cells, thus paving the way for further in vitro and in vivo investigations that will allow the translation of CAP treatment from research to clinic as an alternative therapy for gingivitis.

Foreign Body-Type Multinucleated Giant Cell Formation Is Potently Induced by α-Tocopherol and Prevented by the Diacylglycerol Kinase Inhibitor R59022

Macrophages undergo fusion to form multinucleated giant cells in several pathologic conditions, including the foreign body response (FBR). We detected high levels of matrix metalloproteinase (MMP)-9 during macrophage fusion in vitro and in foreign body giant cells (FBGCs) in vivo. Wild-type (WT) bone marrow-derived macrophages were induced to fuse with IL-4 in the presence of MMP-9 function-blocking antibodies and displayed reduced fusion. A similar defect, characterized by delayed shape change and abnormal morphology, was observed in MMP-9 null macrophages. Analysis of the FBR in MMP-9 null mice was then pursued to evaluate the significance of these findings. Specifically, mixed cellulose ester disks and polyvinyl alcohol sponges were implanted s.c. in MMP-9 null and WT mice and excised 2-4 weeks later. Histochemical and immunohistochemical analyses indicated equal macrophage recruitment between MMP-9 null and WT mice, but FBGC formation was compromised in the former. In addition, MMP-9 null mice displayed abnormalities in extracellular matrix assembly and angiogenesis. Consistent with a requirement for MMP-9 in fusion, we also observed reduced MMP-9 levels in MCP-1 null macrophages, previously shown to be defective in FBGC formation. Collectively, our studies show abnormalities in MMP-9 null mice during the FBR and suggest a role for MMP-9 in macrophage fusion.

The inflammatory responses evoked by artificial organs and implantation of devices like biosensors and guide wires can lead to acute and chronic inflammation, largely limiting the functionality and longevity of the devices with negative effects on patients. The present study aimed to reduce the inflammatory responses to biomaterials by covalent immobilization of glycosaminoglycans (GAGs) on amino-terminated surfaces used as model biomaterials here. Water contact angle (WCA) and zeta potential measurements showed a significant increase in wettability and negative charges on the GAG-modified surfaces, respectively, confirming the successful immobilization of GAGs on the amino-terminated surfaces. THP-1-derived macrophages were used as a model cell type to investigate the efficacy of GAG-modified surfaces in modulating inflammatory responses. It was found that macrophage adhesion, macrophage spreading morphology, foreign body giant cell (FBGC) formation, as well as β1 integrin expression and interleukin-1β (IL-1β) production were all significantly decreased on GAG-modified surfaces compared to the initial amino-terminated surface. This study demonstrates the potential of covalent GAG immobilization to reduce the inflammatory potential of biomaterials in different clinical settings.

To probe the role of human plasma fibronectin in modulating human blood-derived macrophage adhesion and fusion to form multinucleated foreign-body giant cells (FBGC), a series of biomimetic oligopeptides based on the functional structure of fibronectin was designed and synthesized. Peptides incorporated the RGD and PHSRN integrin-binding sequences from FIII-10 and FIII-9 modules, respectively, and the PRRARV sequence from the C-terminal heparin-binding domain, either alone or in combination. Peptides were immobilized onto a polyethyleneglycol-based polymer substrate. The following conclusions were reached. Fibronectin modulated macrophage adhesion and the extent (i.e., size) of FBGC formation on control surfaces in the presence of serum proteins. Macrophages adhered to all substrates with relatively subtle differences between adhesion mediated by RGD, PHSRN, PRRARV, or combinations thereof. β1-integrin subunit was essential in macrophage adhesion to peptide-grafted networks in a receptor-peptide specific manner, whereas β3-integrin subunit was less important. Macrophage adhesion to PRRARV was mediated primarily by the direct interaction with integrins. RGD or PHSRN alone did not provide an adequate substrate for macrophage fusion to form FBGCs. However, the PHSRN synergistic site and the RGD site in a single oligopeptide provided a substrate for FBGC formation that was statistically comparable to that on the positive control material in the presence of serum proteins. This response was highly dependent upon the relative orientation between RGD and PHSRN. PRRARV did not support FBGC formation. These results demonstrate the importance of fibronectin and, specifically, the synergy between RGD and PHSRN domains, in supporting macrophage fusion to form FBGCs. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 79–88, 2001

Osteoclasts and foreign body giant cells (FBGCs) are multi-nuclear cells established by fusion of their mono-nuclear forms. Multi-nucleation via cell-cell fusion is a common characteristic of osteoclasts and FBGCs, and Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) and Osteoclast Stimulatory Transmembrane Protein (OC-STAMP) are both required for the process. Thus, it is thought that DC-STAMP and OC-STAMP interaction likely induces this fusion, but details of these mechanisms are not clear. We crossed DC-STAMP knockout (KO) with OC-STAMP KO mice to obtain DC-STAMP/OC-STAMP doubly deficient (DKO) mice. Osteoclasts and FBGC common progenitors were isolated from wild-type (WT), DC-STAMP KO, OC-STAMP KO or DKO mice. We then set up 4 co-culture systems: (1) WT with DC-STAMP KO cells, (2) WT with OC-STAMP KO cells, (3) WT with DKO cells, and (4) DC-STAMP KO with OC-STAMP KO cells to induce osteoclast or FBGC formation. We evaluated osteoclast and FBGC formation by TRAP and May-Gruenwald Giemsa staining, respectively. Finally, we performed bone morphometric analysis of WT, DC-STAMP KO, OC-STAMP KO, and DKO mice. Cell-cell fusion occurs normally in co-cultures of DC KO with WT cells, OC KO with WT cells, and DKO with WT cells in both osteoclast and FBGC-inducing conditions. By contrast, co-cultures of DC-STAMP KO with OC-STAMP KO cells did not show cell-cell fusion. Bone morphometric analysis revealed enhanced bone formation in DKO mice. DC-STAMP and OC-STAMP cooperate to promote osteoclast and FBGC cell-cell fusion. DC-STAMP and OC-STAMP doubly deficient mice exhibit increased osteogenesis.

A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.

Surface chemistry modulates many critical functions of monocyte/macrophages such as adhesion, fusion, spreading, phagocytosis, and secretion. In this study, we investigated the effect of silicone modification on adhesive structure development and cytoskeletal reorganization of adherent macrophages on polyurethanes. Confocal scanning laser microscopy (CSLM) was used for qualitative and quantitative evaluation of cytoskeletal reorganization of adherent macrophages. Data presented here showed less spreading for adherent cells on silicone-modified materials due to the higher hydrophobicity and protein adsorption profile. This decrease in spreading was accompanied by less F-actin content in adherent cells on silicone-modified polyurethanes and PDMS control, indicating that silicone modification reduces the strength of adhesion. With the addition of interleukin-4 (IL-4) at days 3 and 7 to our culture, adherent cell morphology dramatically changed. The change in morphology led to higher macrophage fusion and foreign body giant cell (FBGC) formation on silicone modified materials after 10 days. In addition, mannose receptor (MR) expression was up-regulated on the silicone-modified polyurethanes and PDMS control in the presence of IL-4. Up-regulation of MR expression suggests an alternatively activated phenotype for adherent macrophages, which is accompanied with an attenuated proinflammatory cytokine production and reactive oxygen secretion. It appears that silicone modification accelerates acquisition of an alternative macrophage and FBGC phenotype, which may then result in increased polyurethane biostability.

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

Nanomaterial-enabled delivery systems have transformed therapeutic strategies for treating inflammatory and degenerative diseases by enabling targeted delivery of small molecules and nucleic acids. However, the clinical translation of microRNA (miR) therapeutics remains limited by instability, enzymatic degradation, and inefficient intracellular delivery in biological environments. Here, we present the design and validation of a next-generation lipid nanoparticle (LNP) platform optimized for the stable and effective delivery of the anti-inflammatory microRNA miR-146a. This LNP system is produced using a scalable lipid injection-based formulation method and yields nanoparticles with uniform size distribution and exceptional physicochemical stability across a wide pH range (2.5–8) and in serum-containing conditions. The four-component lipid architecture enables high miR-146a loading efficiency, efficient endo/lysosomal escape, and robust cellular internalization, resulting in effective tissue uptake and biodistribution both in vitro and in vivo. Importantly, LNP-mediated delivery of miR-146a exhibits excellent biocompatibility and potent anti-inflammatory activity in primary cells and animal models. Collectively, these results suggest this LNP-miR146a platform as a stable, efficient, and translatable approach for modulating inflammation and addressing biomaterial-associated inflammatory responses.

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, α2-macroglobulin, complement factor 3b, fibronectin, IgG, thrombospondin, vitronectin (VN), and von Willebrand factor (vWF) from a 25% serum solution was quantified with 125I-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®, 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. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 435–447, 2000.

Immune response to biomaterials can produce chronic inflammation and fibrosis leading to implant failure, which is related to the surface properties of the biomaterials. This work describes the preparation and characterization of polyelectrolyte multilayer (PEM) coatings that combine the anti-inflammatory activity of heparin as polyanion with the potential release of Naproxen, a nonsteroidal anti-inflammatory drug from polymeric nanoparticles (NP) with cationic surface charge. The polyelectrolyte multilayers were characterized by physical methods to estimate multilayer growth, thickness, zeta potential, and topography. It was found that multilayers with NP had negative zeta potentials and expressed a viscoelastic behavior, while studies of topography showed that nanoparticles formed continuous surface coatings. THP-1-derived macrophages were used to study short-term anti-inflammatory activity (time scale 48 h), showing that PEM that contained heparin reduced cell adhesion and IL1-β secretion, when compared to those with polystyrenesulfonate, used as alternative polyanion in multilayer formation. On the other hand, the presence of NP in PEM was related to a reduced foreign body giant cell formation after 15 days, when compared to PEM that contained chitosan as alternative polycation, which suggests a long-term anti-inflammatory effect of Naproxen-containing nanoparticles. It was also shown that macrophages were able to take up NP from multilayers, which indicates a release of Naproxen by digestion of NP in the lysosomal compartment. These findings indicate that surface coatings composed of heparin and Naproxen-based NP on implants such as biosensors have the potential to attenuate foreign body reaction after implantation, which may improve the long-term functionality of implants.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.