Incorporation of resident macrophages in engineered tissues: multiple cell type response to microenvironment controlled macrophage-laden gelatin hydrogels

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, we developed an in-vitro model of interaction between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, Naive THP-1 monocytes, endothelial cells, and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. In order to mimic resident macrophages, THP-1 cells were encapsulated in the presence or absence of IL-4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage-laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes-laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in secretion of both pro and anti-inflammatory cytokines compared to M2 stimulation. We demonstrated that there is a distinct effect of encapsulated macrophages on the behavior of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation

Electrohydrodynamic printing as a method to micropattern large titanium implant surfaces with photocrosslinkable structures

Metallic implants are widely used in orthopaedic and orthodontic applications. However, generally surface treatment of the metallic surfaces is necessary to render them more biologically active. Herein, we describe a direct write printing method to modify metallic implant surfaces with biocompatible polymers with microscale precision. Application of polymeric micropatterns on metallic implant surfaces can (i) improve their interaction with the host tissue, (ii) enable the delivery of growth factors, antibiotics, anti-inflammatory cytokines etc from the implant surface and (iii) can control the immune responses to the implant via controlling the attachment of immune cells, such as macrophages. Surface patterns with a resolution of less than 50 μm can be created using an electro hydrodynamic (EHD) printing, a template-free and single-step process. We present a revised EHD printing method for the deposition of parallel strips of photocrosslinkable, cell adhesive polymeric composites with spacing of around 20 μm onto medical grade titanium substrates. Optimization of voltage, feeding rate and collection speed resulted in regular structures via very rapid movement of the grounded rotating collector driven to equivalent of the linear surface speed of above 100 cm s−1. In the experimental part a mixture of chemically modified PEG /gelatin was deposited onto a conductive titanium substrate with different surface pretreatments with an area of 400 mm2. Acid etched or UV treated titanium surfaces improved the stability of the printed structures. Polymeric lines induced temporary orientation of human monocytes (THP-1) and induced a thicker cell multilayer formation by 3T3 fibroblasts (p < 0.05). Staining of the monocytes for M1(CD80) and M2 (CD206) macrophage markers on the patterned surface showed mixed populations with higher anti-inflammatory cytokine secretion compared to tissue culture plastic control. The results demonstrate the suitability of this method for the preparation of biomaterials with structured surfaces on large areas and with desired chemical composition

Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation

The function of soft tissues is intricately linked to their connections with the other systems of the body such as circulation, nervous system, and immune system. The presence of resident macrophages in tissues provides a means to control tissue homeostasis and also a way to react to the physical/biological insults and tissue damage. Thus, incorporation of resident macrophage like phenotype-controlled macrophages in engineered tissues can improve their fidelity as model tissues and also improve their rate of integration and facilitate the resolution of inflammation for regenerative medicine applications. Herein, we demonstrate two potential ways to immunoassist the remodeling process of engineered soft tissues in three-dimensional (3-D) gelatin based hydrogels containing fibroblasts and/or endothelial cells: (i) with supplementation of interleukin-4 (IL-4) in the presence of macrophages and (ii) in tri-culture via naive monocytes or differentiated macrophages. The presence of IL-4 had a proliferative effect on fibroblasts, with a significant boosting effect on proliferation and cytokine secretion in the presence of differentiated macrophages with an upregulation of activin, interleukin-1 receptor antagonist (IL-1RA), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β), creating a more stimulating microenvironment. The addition of IL-4 in endothelial cell/macrophage co-culture configuration improved the organization of the sprout-like structures, with a boost in proliferation at day 1 and with an upregulation of IL-6 and IL-1RA at the earliest stage in the presence of differentiated macrophages creating a favorable microenvironment for angiogenesis. In tri-culture conditions, the presence of monocytes or macrophages resulted in a denser tissue-like structure with highly remodeled hydrogels. The presence of differentiated macrophages had a boosting effect on the angiogenic secretory microenvironment, such as IL-6 and IL-8, without any additional cytokine supplementation. The presence of fibroblasts in combination with endothelial cells also had a significant effect on the secretion of angiopoietin. Our results demonstrate that incorporation of macrophages in a resident macrophage function and their phenotype control have significant effects on the maturation and cytokine microenvironment of 3-D multiple cell type-laden hydrogels, which can be harnessed for better integration of implantable systems and for more physiologically relevant in vitro tissue models with an immune component

Development of a Cannabis Assessment Tool (CAT-1) to measure current and lifetime marijuana use among older Veterans.

ObjectiveTo develop a tool to assess current (past 30 days) and lifetime marijuana use in older Veterans.SettingUS Veteran's Affairs Healthcare System.Participants704 older Veterans were screened, 339 completed the initial survey, 100 completed the follow-up.Primary outcome measurePearson's correlation coefficient to assess strength of association between initial and follow-up survey on measures of current and lifetime marijuana use.ResultsBoth a 'gram-month' measure of marijuana smoked in the past 30 days (r=0.83) and a frequency-based measure assessing total number of times smoked in the past 30 days were reliable (r=0.89). Both a simple categorical measure of lifetime use (agreement=85%) and a continuous measure of lifetime use (r=0.82) were reliable.ConclusionsThe Cannabis Assessment Tool offers a reliable assessment of past 30 days and lifetime assessments of smoking cannabis in older adults

Macrophage responses to implants: prospects for personalized medicine

Implants, transplants, and implantable biomedical devices are mainstream solutions for a wide variety of human pathologies. One of the persistent problems around nondegradable metallic and polymeric implants is failure of macrophages to resolve the inflammation and their tendency to stay in a state, named "frustrated phagocytosis." During the initial phase, proinflammatory macrophages induce acute reactions to trauma and foreign materials, whereas tolerogenic anti-inflammatory macrophages control resolution of inflammation and induce the subsequent healing stage. However, implanted materials can induce a mixed pro/anti-inflammatory phenotype, supporting chronic inflammatory reactions accompanied by microbial contamination and resulting in implant failure. Several materials based on natural polymers for improved interaction with host tissue or surfaces that release anti-inflammatory drugs/bioactive agents have been developed for implant coating to reduce implant rejection. However, no definitive, long-term solution to avoid adverse immune responses to the implanted materials is available to date. The prevention of implant-associated infections or chronic inflammation by manipulating the macrophage phenotype is a promising strategy to improve implant acceptance. The immunomodulatory properties of currently available implant coatings need to be improved to develop personalized therapeutic solutions. Human primary macrophages exposed to the implantable materials ex vivo can be used to predict the individual's reactions and allow selection of an optimal coating composition. Our review describes current understanding of the mechanisms of macrophage interactions with implantable materials and outlines the prospects for use of human primary macrophages for diagnostic and therapeutic approaches to personalized implant therapy

Macrophage responses to implants: prospects for personalized medicine

Implants, transplants, and implantable biomedical devices are mainstream solutions for a wide variety of human pathologies. One of the persistent problems around nondegradable metallic and polymeric implants is failure of macrophages to resolve the inflammation and their tendency to stay in a state, named "frustrated phagocytosis." During the initial phase, proinflammatory macrophages induce acute reactions to trauma and foreign materials, whereas tolerogenic anti-inflammatory macrophages control resolution of inflammation and induce the subsequent healing stage. However, implanted materials can induce a mixed pro/anti-inflammatory phenotype, supporting chronic inflammatory reactions accompanied by microbial contamination and resulting in implant failure. Several materials based on natural polymers for improved interaction with host tissue or surfaces that release anti-inflammatory drugs/bioactive agents have been developed for implant coating to reduce implant rejection. However, no definitive, long-term solution to avoid adverse immune responses to the implanted materials is available to date. The prevention of implant-associated infections or chronic inflammation by manipulating the macrophage phenotype is a promising strategy to improve implant acceptance. The immunomodulatory properties of currently available implant coatings need to be improved to develop personalized therapeutic solutions. Human primary macrophages exposed to the implantable materials ex vivo can be used to predict the individual's reactions and allow selection of an optimal coating composition. Our review describes current understanding of the mechanisms of macrophage interactions with implantable materials and outlines the prospects for use of human primary macrophages for diagnostic and therapeutic approaches to personalized implant therapy

Incorporation of resident macrophages in engineered tissues: multiple cell type response to microenvironment controlled macrophage-laden gelatin hydrogels

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, we developed an in-vitro model of interaction between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, Naive THP-1 monocytes, endothelial cells, and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. In order to mimic resident macrophages, THP-1 cells were encapsulated in the presence or absence of IL-4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage-laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes-laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in secretion of both pro and anti-inflammatory cytokines compared to M2 stimulation. We demonstrated that there is a distinct effect of encapsulated macrophages on the behavior of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation

Establishing contact between cell-laden hydrogels and metallic implants with a biomimetic adhesive for cell therapy supported implants

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