RESUMEN
Continuous subcutaneous insulin infusion for Type 1 diabetes relies upon insulin infusion sets (IIS) to reliably deliver insulin to a subcutaneous depot, where it is absorbed into systemic circulation. However, IIS are plagued by short wear times and high failure rates, due in part to inconsistent insulin absorption that can arise over time. While emerging evidence suggests that the local inflammatory response to the IIS cannula may impact both wear times and unreliable insulin adsorption, the mechanisms are poorly understood. Here, we investigated the effects of local infused insulin concentrations on the biomaterial host response to better understand the underlying factors that limit the IIS performance. We first modeled the insulin concentration for a constant basal infusion rate to select a relevant insulin concentration range of 0.1-10 U/mL within the infusion site. We then examined the influence of a commercial insulin analogue (Humulin-N) using an in vitro macrophage-material model, which uses adsorbed fibroblast lysate (containing damage-associated molecular patterns) to activate macrophages and recapitulates macrophage responses on implanted biomaterials. RAW-Blue macrophages cultured on lysate-adsorbed surfaces had increased nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) activity and intracellular reactive oxygen species (ROS) accumulation compared to control surfaces. Humulin-N concentration (0.5-10 U/mL) enhanced the NF-κB/AP-1 activity and ROS accumulation in macrophages on lysate-adsorbed surfaces. However, Humulin-N had no effect on NF-κB/AP-1 or ROS in the absence of the inflammatory stimulus. Additionally, high insulin concentrations arising from therapeutic doses induced macrophage apoptosis with and without adsorbed lysate. This study contributes to emerging evidence that infused insulin affects the tissue response to IIS.
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The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAW-Blue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1ß, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMP-related proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway.
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Activación de Macrófagos , Factor 88 de Diferenciación Mieloide , Receptor Toll-Like 2 , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales , Fibrinógeno , Fibronectinas , Hemostáticos , Histonas , Factor 88 de Diferenciación Mieloide/genética , Proteómica , Transducción de Señal , Receptor Toll-Like 2/genéticaRESUMEN
Implanted biomaterials elicit an immune-mediated foreign body reaction (FBR) that results in the fibrous encapsulation of the implant and can critically impact the performance of some implants. Consequently, understanding the molecular mechanisms that underpin cell-materials interactions that initiate biomaterial-induced inflammation and fibrosis is critical to improving the performance of biomaterial implants negatively impacted by the FBR. Damage-associated molecular patterns (DAMPs) are endogenous mediators of inflammation that are released upon tissue injury and induce sterile inflammation via Toll-like receptors (TLRs). However, the prevalence of DAMPs within the adsorbed protein layer on material surfaces and their role mediating cell-material interactions is unclear. Previously, our group demonstrated that molecules in fibroblast lysates adsorbed to various biomaterials and induced a potent TLR2-dependent inflammatory response in macrophages at 24 h. In this study, we examined the extended response of RAW-Blue reporter macrophages on lysate or serum-adsorbed Teflon™ AF surfaces to understand the potential role of adsorbed DAMPs in macrophage-material interactions at later time points. Lysate-conditioned surfaces maintained increased nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) transcription factor activity and increased expression Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES/CCL5) at 72 h and 120 h, compared to FBS-conditioned surfaces. In contrast, monocyte chemoattractant protein 1 (MCP-1/CCL2) was only elevated at 72 h in lysate conditions. Transforming growth factor beta 1 (TGF-ß1) secretion was significantly increased on lysate-conditioned surfaces, while conditioned media from macrophages on lysate-conditioned surfaces induced alpha smooth muscle actin (αSMA) expression in 3T3 fibroblasts. TLR2 neutralizing antibody treatment significantly decreased NF-κB/AP-1 activity and attenuated TGF-ß1 expression at both time points, and MCP-1 and RANTES at 72 h. Finally, multinucleated cells were observed on lysate-conditioned surfaces at 72 h, indicating adsorbed DAMPs induced a fusion permissive environment for adherent macrophages. This study demonstrates that adsorbed DAMPs continue to influence macrophage-material responses beyond the initial 24-h period and maintain a pro-inflammatory and fibrotic response that models aspects of the early FBR. Furthermore, the transient inhibition of TLR2 continued to exert an effect at these later time points, suggesting TLR2 may be a target for therapeutic interventions in FBR.
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The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-кB/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-кB/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.
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Proteínas Sanguíneas/metabolismo , Receptor Toll-Like 1/aislamiento & purificación , Receptor Toll-Like 1/metabolismo , Adsorción , Células Cultivadas , Humanos , Macrófagos/metabolismo , Transducción de SeñalRESUMEN
The foreign body reaction is a chronic inflammatory response to an implanted biomaterial that ultimately leads to fibrous encapsulation of the implant. It is widely accepted that the host response to implanted biomaterials is largely dependent on the species and conformations of proteins adsorbed onto the material surface due to the adsorbate's role in mediating cellular interactions with the implanted material. While the cellular response to adsorbed serum-derived proteins has been studied extensively, the presence of endogenous, matrix- and cell-derived mediators of inflammation within the adsorbed protein layer and their impact on cell-material interactions is not well-understood. Damage associated molecular patterns (DAMPs) are endogenous ligands released by stressed or damaged tissues to stimulate sterile inflammatory responses via Toll-like receptors (TLRs) and other pattern recognition receptors. The present study investigated the potential role of tissue-derived, pro-inflammatory stimuli in macrophage responses to biomaterials using cell lysate as a complex source of cell-derived DAMPs and poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS) films as model biomaterials. We show that lysate-adsorbed PMMA and PDMS surfaces strongly induced NF-κB/AP-1 transcription factor activity and pro-inflammatory cytokine secretion in the RAW-Blue macrophage cell line compared to serum-adsorbed surfaces. Lysate-dependent NF-κB/AP-1 activation and cytokine expression were strongly attenuated by TLR2 neutralizing antibodies, while TLR4 inhibition resulted in a modest reduction. These data suggest that DAMPs, in their adsorbed conformations on material surfaces, may play a significant role in macrophage activation through TLR signaling, and that TLR pathways, particularly TLR2, merit further investigation as potential therapeutic targets to modulate host responses to implanted biomaterials.
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The development and application of decellularized extracellular matrices (ECM) has grown rapidly in the fields of cell biology, tissue engineering and regenerative medicine in recent years. Similar to decellularized tissues and whole organs, cell-derived matrices (CDMs) represent bioactive, biocompatible materials consisting of a complex assembly of fibrillar proteins, matrix macromolecules and associated growth factors that often recapitulate, at least to some extent, the composition and organization of native ECM microenvironments. The unique ability to engineer CDMs de novo based on cell source and culture methods makes them an attractive alternative to conventional allogeneic and xenogeneic tissue-derived matrices that are currently harvested from cadaveric sources, suffer from inherent heterogeneity, and have limited ability for customization. Although CDMs have been investigated for a number of biomedical applications, including adhesive cell culture substrates, synthetic scaffold coatings, and tissue engineered products, such as heart valves and vascular grafts, the state of the field is still at a relatively nascent stage of development. In this review, we provide an overview of the various applications of CDM and discuss successes to date, current limitations and future directions.
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Materiales Biocompatibles/química , Prótesis Vascular/psicología , Válvulas Cardíacas/fisiología , Medicina Regenerativa/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Matriz Extracelular/química , Válvulas Cardíacas/química , Válvulas Cardíacas/metabolismo , HumanosRESUMEN
The ability to undergo rapid changes in response to subtle environmental cues make stimuli- responsive materials attractive candidates for minimally invasive, targeted and personalized drug delivery applications. This special report aims to highlight and provide a brief description of several of the significant natural and synthetic temperature-responsive materials that have clinical relevance for drug delivery applications. This report examines the advantages and disadvantages of natural versus synthetic materials and outlines various scaffold architectures that can be utilized with temperature-sensitive drug delivery materials. The authors provide a commentary on the current state of the field and provide their insight into future expectations for temperature-sensitive drug delivery, emphasizing the importance of the emergence of dual and multiresponsive systems capable of responding precisely to an expanding set of stimuli, thereby allowing the development of disease-specific drug delivery vehicles.
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Sistemas de Liberación de Medicamentos/métodos , Polímeros/química , Temperatura , Preparaciones de Acción Retardada , Humanos , Preparaciones FarmacéuticasRESUMEN
The expression of native sonic hedgehog (Shh) was significantly increased in poly(methacrylic acid-co-methyl methacrylate) bead (MAA) treated wounds at day 4 compared to both poly(methyl methacrylate) bead (PMMA) treated and untreated wounds in diabetic db/db mice. MAA beads also increased the expression of the Shh transcription factor Gli3 at day 4. Previously, topical application of MAA beads (45 mol % methacrylic acid) improved wound closure and blood vessel density in excisional wounds in these mice, while PMMA beads did not. Gene expression within the granulation tissue of healing wounds was studied to provide insight into the mechanism of vessel formation and wound healing in the presence of MAA beads. In addition to the increased expression of Shh, MAA-treated wounds had increased expression of osteopontin (OPN), IL-1ß and TNF-α, (at day 7) similar to the previously reported MAA response of macrophage-like and endothelial cells in vitro.
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Diabetes Mellitus/patología , Proteínas Hedgehog/metabolismo , Microesferas , Polimetil Metacrilato/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Peso Corporal/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Regulación de la Expresión Génica/efectos de los fármacos , Tejido de Granulación/efectos de los fármacos , Tejido de Granulación/patología , Proteínas Hedgehog/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Cicatrización de Heridas/genética , Proteína Gli3 con Dedos de ZincRESUMEN
Identifying the critical molecules associated with "biocompatibility" is a grand challenge. Poly(methacrylic acid -co- methyl methacrylate) (MAA) beads improve wound closure and wound vascularity in vivo, but the mechanism of this phenomenon is unknown. We used quantitative real-time PCR to identify the subtle changes in the expression of a small selection of molecules involved in wound healing and angiogenesis in a macrophage-like cell (dTHP-1) treated with the MAA beads (45 mol% methacrylic acid). MAA beads decreased the expression of osteopontin (OPN) compared to poly(methyl methacrylate) (PMMA) and untreated cells, and increased the expression of IL-1ß, IL-6 and TNF-α over the 24-96 h of the experiment. Interestingly, molecules associated with angiogenesis, such as bFGF, CXCL12, HIF-1α, PDGF-B, TGF-ß and VEGF, were not significantly affected by MAA beads over the course of the study. MAA beads also increased the gene expression of OPN in HUVEC compared to untreated cells, while PMMA beads did not. MAA beads modified the phenotype (gene expression) of dTHP-1 cells in a subtle yet distinct manner that was different than PMMA. It remains to connect the changes in OPN in dTHP-1 (and HUVEC) and other molecules to the enhanced vascularity seen in vivo with this polymer.
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Cementos para Huesos/farmacología , Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Metilmetacrilato/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Polimetil Metacrilato/farmacología , Cementos para Huesos/química , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Macrófagos/metabolismo , Metilmetacrilato/química , Osteopontina/genética , Polimetil Metacrilato/química , ARN Mensajero/genéticaRESUMEN
This protocol describes the fabrication of a type of micro-tissues called modules. The module approach generates uniform, scalable and vascularized tissues. The modules can be made of collagen as well as other gelable or crosslinkable materials. They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells. The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable. These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues. There are three main steps to making the modules: neutralizing the collagen and embedding cells in it, gelling the collagen in the tube and cutting the modules and coating the modules with endothelial cells.
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Colágeno/química , Células Endoteliales/citología , Ingeniería de Tejidos/métodos , Geles/química , Células Hep G2 , HumanosRESUMEN
We synthesized two thermoresponsive, bioactive cell scaffolds by decorating the backbone of type I bovine collagen with linear chains of poly(N-isopropylacrylamide) (PNIPAAm), with the ultimate aim of providing facile delivery via injection and support of retinal pigment epithelial (RPE) cells into the back of the eye for the treatment of retinal degenerative diseases. Both scaffolds displayed rapid, subphysiological phase transition temperatures and were capable of noninvasively delivering a liquid suspension of cells that gels in situ forming a cell-loaded scaffold, theoretically isolating treatment to the injection site. RPE cells demonstrated excellent viability when cultured with the scaffolds, and expulsion of cells arising from temperature-induced PNIPAAm chain collapse was overcome by incorporating a room-temperature incubation period prior to scaffold phase transition. These results indicate the potential of using PNIPAAm-grafted-collagen as a vehicle for the delivery of therapeutic cells to the subretinal space.