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Biomaterial systems such as hydrogels enable localized delivery and post-injection modulation of cellular therapies in a wide array of contexts. Biomaterials as adjuvants has been an active area of investigation, but the study of functionalized biomaterials supporting immunosuppressive cell therapies for tolerogenic applications is still nascent. Here, we developed a 4-arm poly(ethylene-glycol)-maleimide (PEG-4MAL) hydrogel functionalized with interleukin-10 (IL-10) to improve the local delivery and efficacy of a cell therapy against autoimmune disease. The biophysical and biochemical properties of PEG-4MAL hydrogels were optimized to support dendritic cell (DC) viability and an immature phenotype. IL-10-functionalized PEG-4MAL (PEG-IL10) hydrogels exhibited controlled IL-10 release, extended the duration of DC support, and protected DCs from inflammatory assault. After incorporation in PEG-IL10 hydrogels, these DCs induced CD25+FoxP3+ regulatory T cells (Tregs) during in vitro co-culture. These studies serve as a proof-of-concept for improving the efficacy of immunosuppressive cell therapies through biomaterial delivery. The flexible nature of this system enables its widespread application across a breadth of other tolerogenic applications for future investigation.

Biomaterials are known to modulate immune cell functions, which subsequently determine the host inflammatory and immune responses. Poly(lactic-co-glycolic acid) or PLGA, a biodegradable and biocompatible biomaterial, induces a pro-inflammatory, mature phenotype in antigen presentation cells, namely dendritic cells (DCs) in vitro. In vivo, PLGA can boost the humoral immune response to a co-delivered model antigen, a phenomenon known as the PLGA-adjuvant effect. This study elucidates the link between PLGA's effect on the DC phenotype in vitro and its adjuvant effect in vivo using the CD11c-DTR mouse model. These mice undergo conditional ablation of DCs upon treatment with diphtheria toxin. To measure immune activation, the mice were first given ovalbumin (OVA)-reactive T cells from OT-II/OT-I mice. Later, the same mice received subcutaneous OVA-loaded PLGA scaffold implants. In response to the scaffold implants, OVA-reactive OT-II CD4+ T cells showed decreased proliferation in the absence of CD11c+ DCs, indicating an attenuation of the PLGA-adjuvant effect. Furthermore, PLGA may also influence the antigen cross-presentation function of DCs, as evident with the lowered OVA-reactive OT-I CD8+ T-cell response. Understanding the immunomodulatory ability of biomaterials in the context of DCs will aid in designing improved DC-based immunotherapies against infectious diseases and cancer.

Dendritic cells are key players in regulating immunity. These cells both activate and inhibit the immune response depending on their cellular environment. Their response to hyperglycemia, a condition common amongst diabetics wherein glucose is abnormally elevated, remains to be elucidated. In this study, the phenotype and immune response of dendritic cells exposed to hyperglycemia were characterized in vitro and in vivo using the streptozotocin (STZ)-induced diabetes model. Dendritic cells were shown to be sensitive to hyperglycemia both during and after differentiation from bone marrow precursor cells. Dendritic cell behavior under hyperglycemic conditions was found to vary by phenotype, of which tolerogenic dendritic cells were particularly sensitive. Expression of the costimulatory molecule CD86 was found to reliably increase when dendritic cells were exposed to hyperglycemia. Additionally, hydrogel-based delivery of the anti-inflammatory molecule interleukin-10 (IL-10) was shown to partially inhibit these effects in vivo.

Engineering a regulatory phenotype in dendritic cells (DCs) is a potential approach to circumvent an immune response against self-antigens in autoimmunity or alloantigens in allograft rejection. Cell microenvironments influence the differentiation of DC precursors into either proinflammatory/immunostimulatory or tolerogenic/regulatory DCs. Biomaterial-based vehicles can be used to re-engineer cell microenvironments and re-educate the DC phenotype. This study presents the development and validation of a biomaterial-based multicomponent immunomodulatory (MI) scaffold for the purpose of promoting a tolerogenic/regulatory DC phenotype. Glutaraldehyde-crosslinked gelatin microparticles, loaded with specific immunomodulators, were embedded into a porous agarose scaffold. Using the Weibull equation and the Bayesian approach, an empirical mathematical model was derived from the release profile data of "model" molecules. The scaffold design was generated from the model to achieve distinct temporal release profiles of the loaded immunomodulator(s): granulocyte monocyte colony-stimulating factor (GM-CSF), dexamethasone (DEX), and/or peptidoglycan (PGN). The MI scaffold-treated DCs (MI DCs) showed an increase in the expression of tolerogenic markers such as surface immunoglobulin-like transcript 3 (ILT-3) and secreted interleukin-10 (IL-10), with a simultaneous decrease in maturation markers such as CD86 and secreted interferon-γ (IFN-γ). In cell culture studies, these MI DCs were able to suppress T-cell proliferation. This approach is expected to enhance the generation of endogenous regulatory DCs when applied in vivo. This technology serves as a basis for future immunotherapeutic applications in the autoimmunity and allogeneic therapies. It also shows that empirical mathematical modeling can be used to engineer scaffold designs for distinct temporal release of one or more immunomodulators.

In multiple sclerosis (MS), abnormally activated immune cells responsive to myelin proteins result in widespread damage throughout the central nervous system (CNS) and ultimately irreversible disability. Immunomodulation by delivering dendritic cells (DCs) utilizes a potent and rapid MS disease progression driver therapeutically. Here, we investigated delivering DCs for disease severity attenuation using an experimental autoimmune encephalomyelitis preclinical MS model. DCs treated with interleukin-10 (IL-10) (DC10s) were transplanted using in situ gelling poly(ethylene glycol)-based hydrogel for target site localization. DC delivery increased hydrogel longevity and altered the injection site recruited, endogenous immune cell profile within 2 days postinjection. Furthermore, hydrogel-mediated DC transplantation efficacy depended on the injection-site. DCs delivered to the neck local to MS-associated CNS-draining cervical lymph nodes attenuated paralysis, compared to untreated controls, while delivery to the flank did not alter paralysis severity. This study demonstrates that local delivery of DC10s modulates immune cell recruitment and attenuates disease progression in a preclinical model of MS.

Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cells (DCs), supporting pathogen-induced innate immunity and antigen processing, as well as immunosuppressive effects in the tolerance to self-proteins. Additionally, the innate inflammatory response to implanted biomaterials has been hypothesized to be mediated by inflammatory cells interacting with adsorbed proteins, many of which are glycosylated. However, the molecular factors relevant for surface displayed glycoconjugate modulation of dendritic cell (DC) phenotype are unknown. Thus, in this study, a model system was developed to establish the role of glycan composition, density, and carrier cationization state on DC response. Thiol modified glycans were covalently bound to a model protein carrier, maleimide functionalized bovine serum albumin (BSA), and the number of glycans per BSA modulated. Additionally, the carrier isoelectric point was scaled from a pI of ∼4.0 to ∼10.0 using ethylenediamine (EDA). The DC response to the neoglycoconjugates adsorbed to wells of a 384-well plate was determined via a high throughput assay. The underlying trends in DC phenotype in relation to conjugate properties were elucidated via multivariate general linear models. It was found that glycoconjugates with more than 20 glycans per carrier had the greatest impact on the pro-inflammatory response from DCs, followed by conjugates having an isoelectric point above 9.5. Surfaces displaying terminal α1-2 linked mannose structures were able to increase the inflammatory DC response to a greater extent than did any other terminal glycan structure. The results herein can be applied to inform the design of the next generation of combination products and biomaterials for use in future vaccines and implanted materials.

Given the central role of dendritic cells (DCs) in directing T-cell phenotypes, the ability of biomaterial-treated DCs to dictate autologous T-cell phenotype was investigated. In this study, we demonstrate that differentially biomaterial-treated DCs differentially directed autologous T-cell phenotype and polarization, depending on the biomaterial used to pretreat the DCs. Immature DCs (iDCs) were derived from human peripheral blood monocytes and treated with biomaterial films of alginate, agarose, chitosan, hyaluronic acid, or 75:25 poly(lactic-co-glycolic acid) (PLGA), followed by co-culture of these biomaterial-treated DCs and autologous T cells. When autologous T cells were co-cultured with DCs treated with biomaterial film/antigen (ovalbumin, OVA) combinations, different biomaterial films induced differential levels of T-cell marker (CD4, CD8, CD25, CD69) expression, as well as differential cytokine profiles [interferon (IFN)-γ, interleukin (IL)-12p70, IL-10, IL-4] in the polarization of T helper (Th) types. Dendritic cells treated with agarose films/OVA induced CD4+CD25+FoxP3+ (T regulatory cells) expression, comparable to untreated iDCs, on autologous T cells in the DC-T co-culture system. Furthermore, in this co-culture, agarose treatment induced release of IL-12p70 and IL-10 at higher levels as compared with DC treatment with other biomaterial films/OVA, suggesting Th1 and Th2 polarization, respectively. Dendritic cells treated with PLGA film/OVA treatment induced release of IFN-γ at higher levels compared with that observed for co-cultures with iDCs or DCs treated with all other biomaterial films. These results indicate that DC treatment with different biomaterial films has potential as a tool for immunomodulation by directing autologous T-cell responses.

The immunological outcome of dendritic cell (DC) treatment with different biomaterials was assessed to demonstrate the range of DC phenotypes induced by biomaterials commonly used in combination products. Immature DCs (iDCs) were derived from human peripheral blood monocytes, and treated with different biomaterial films of alginate, agarose, chitosan, hyaluronic acid (HA), or 75:25 poly(lactic-co-glycolic acid) (PLGA) and a comprehensive cadre of phenotypic functional outcomes were assessed. Differential levels of functional changes of DC phenotype were observed depending on the type of biomaterial films used to treat DCs. Treatment of DCs with PLGA or chitosan films supported DC maturation with higher levels of DC allostimulatory capacity, pro-inflammatory cytokine release, expression of CD80, CD86, CD83, HLA-DQ and CD44 expression as compared to iDCs, and endocytic ability at a level lower compared to iDCs. Alginate film induced pro-inflammatory cytokine release from DCs at levels higher than iDCs,. Dendritic cells treated with HA film expressed lower levels of CD40, CD80, CD86 and HLA-DR as compared to iDCs. They also exhibited endocytic ability and CD44 expression at levels lower than iDCs, possibly due to an insolublized (cross-linked) form with high molecular weight HA. Interestingly, treatment of DCs with agarose film maintained a DC functional phenotype at levels similar to iDCs except for CD44 expression which was lower than expression levels for iDCs. Taken together, these results can provide selection criteria for biomaterials to be used in immunomodulating applications and can inform potential outcomes of biomaterials within combination products on associated immune responses as desired by the application.

Macrophages (Mϕ) and dendritic cells (DCs) are critical antigen presenting cells that play pivotal roles in host responses to biomaterial implants. Although Mϕs have been widely studied for their roles in the inflammatory responses against biomaterials, the roles that DCs play in the host responses toward implanted materials have only recently been explored. DCs are of significant research interest because of the emergence of a large number of combination products that cross-traditional medical device boundaries. These products combine biomaterials with biologics, including cells, nucleic acids, and/or proteins. The biomaterial component may evoke an inflammatory response, primarily mediated by neutrophils and Mϕs, whereas the biologic component may elicit an immunogenic immune response, initiated by DCs involving lymphocyte activation. Control of Mϕ phenotypic balance from proinflammatory M1 to reparative M2 is a goal of investigators to optimize the host response to biomaterials. Similarly, control of DC phenotype from proinflammatory to toleragenic is of interest in vaccine delivery and tissue engineering/transplantation situations, respectively. This review discusses the interconnection between innate and adaptive immunity, the comparative and contrasting phenotypes and roles of Mϕs and DCs in immunity, their responses to biomaterials and the strategies to modulate their phenotype for applications in tissue engineering and vaccine delivery. Furthermore, the collaboration between and unique roles of DCs and Mϕs needs to be addressed in future studies to gain a more complete picture of host responses toward combination products.

Biomaterials have been shown to differentially support dendritic cell (DC) maturation, a prerequisite for an adjuvant effect. Treatment of DCs with poly(D,L-lactic-co-glycolic acid). (PLGA) films resulted in DC maturation but agarose films did not. In these studies, the biomaterial adjuvant effect was attenuated by material selection (PLGA or agarose scaffolds) or local delivery of an anti-inflammatory/immunosuppressive glucocorticoid, dexamethasone (DX), from PLGA scaffolds. Porous scaffolds (SCs) of PLGA or agarose were produced to deliver equivalent amounts of model antigen, ovalbumin (OVA). Alternatively, PLGA SCs with incorporated OVA were produced with or without DX. These SCs were implanted individually, subcutaneously, and dorsally in C57BL/6 mice. Blood was collected from mice at specific times over a 12-week duration for measurement of antibody production against OVA. Scaffolds were explanted at 12 weeks for histological examination of foreign body response. Scaffolds of PLGA, but not of agarose, were found to elicit higher antibody production against co-delivered OVA, than negative controls. Short term delivery of DX from PLGA SCs delivering OVA temporarily delayed onset of anti-OVA antibody production. More sustained release of DX at an effective dose and with an appropriate time course is expected to extend the effect of DX on the biomaterial adjuvant effect. The immunomodulatory ability of biomaterials to affect the immune response to co-delivered antigen is demonstrated wherein this immunomodulatory ability correlates with the observed in vitro differential effects of biomaterials on DC maturation.

A variety of combination products composed of biomaterials and biologics have been developed for tissue regeneration or vaccine delivery. The host immune response to the immunogenic biological components in such products may be modulated by the biomaterial component. Distinct biomaterials have been shown to differentially affect the maturation of dendritic cells (DCs). DCs are professional antigen-presenting cells (APCs) that bridge innate and adaptive immunity and play a central role in inducing immunity or initiating immune tolerance. However, the biomaterials systems used to study DC response thus far have been insufficient to draw a clear conclusion as to which biomaterial properties are the key to controlling DC phenotype. In this study, we developed a 96-well filter plate-based high-throughput (HTP) methodology to assess DC maturation upon biomaterial treatment. Equivalent biomaterial effects on DC phenotype were measured using the conventional flow cytometric and filter-plate method, which validated the HTP methodology. This methodology will be used to screen a large number of biomaterials simultaneously and to draw correlations between material properties and DC phenotype, thereby providing biomaterial design criteria and immunomodulatory strategies for both tissue engineering and vaccine delivery applications.

Macrophages are central mediators of biomaterial-associated wound healing. Dendritic cells (DCs) link innate and adaptive immunity and are important in the context of the host response to combination products. Starting with human peripheral blood mononuclear cells (PBMCs), DCs were derived from monocytes upon culture with granulocyte macrophage colony-stimulating factor and interleukin-4; macrophages were derived from monocytes upon culture without cytokines. Macrophage or DC cultures were characterized at relevant timepoints in both adherent and nonadherent fractions on control Primaria™ surfaces to characterize and define these inflammatory/immune cells as a prequel to their use in in vitro test biomaterial-host response studies. At day 10 (typical time for harvesting macrophages for subsequent treatment with test biomaterials), macrophages were CD11c+, macrophage mannose receptor (MMR)+, CD14+, and CD64+. At day 6 (typical time for harvesting of DCs after 24-h treatment with test biomaterials), DCs were CD1c+, CD11c+, CD123+, MMR+, CD14+, and CD64−. Furthermore, CD3+ and CD4+ T lymphocytes and CD19+ and CD24+ B lymphocytes were present in both cultures at all timepoints, although to different extents. Immature DCs (∼15 μm), were rounded but presented extensive dendritic processes upon maturation with lipopolysaccharide. Alternatively, adherent macrophages (∼15–20 μm) displayed internalized lipids and exhibited few membrane processes. The characterization and comparison of existing techniques to establish reliable, reproducible primary cultures of DCs or macrophages provides an important basis for examining and interpreting complex macrophage/DC-lymphocyte-orchestrated host responses in future studies with equivalent cell populations on test biomaterials. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin conditional knock-out mice, we demonstrate that plasma fibronectin modulates the foreign body response to polyethylene terephthalate discs implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of plasma fibronectin compared to controls. In contrast, deletion of plasma fibronectin did not alter acute leukocyte recruitment to the biomaterial, indicating that plasma fibronectin modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of plasma fibronectin while macrophage numbers were not different, suggesting that plasma fibronectin regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular fibronectin was present in the capsules of both normal and plasma fibronectin-depleted mice, suggesting that cellular fibronectin could not compensate for the loss of plasma fibronectin. These results implicate plasma fibronectin in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system., Bill & Melinda Gates Foundation, United States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001), Ragon Institute of MGH, MIT and Harvard, Cancer Research Institute (New York, N.Y.) (Irvington Postdoctoral Fellowship), National Institutes of Health (U.S.) (Awards AI104715, CA172164, CA174795, and AI095109)

The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate–protein interactions on a flat surface can be translatable to development of efficacious therapies, such as vaccines, which are delivered in soluble or phagocytosable particles, has not been validated. Thus, a preliminary investigation was performed in which mannose or glucose was conjugated to cationized bovine serum albumin and presented to DCs in soluble, phagocytosable, or non-phagocytosable display modalities. The functional DC response to the glycoconjugates was assessed via a high throughput assay. Dendritic cell phenotypic outcomes were placed into a multivariate, general linear model (GLM) and shown to be statistically different amongst display modalities when comparing similar surface areas. The GLM showed that glycoconjugates that were adsorbed to wells were the most pro-inflammatory while soluble conjugates were the least. DC interactions with mannose conjugates were found to be calcium dependent and could be inhibited via anti-DC-SIGN antibodies. The results of this study aim to resolve conflicts in reports from multiple laboratories showing differential DC profiles in response to similar, if not identical, ligands delivered via different modalities. Additionally, this study begins to bridge the gap between microarray binding data and functional cell responses by highlighting the phenotypes induced from adsorbed glycoconjugates as compared to those in solution or displayed on microparticles.

Microencapsulation of live mammalian cells is one means of creating hybrid artificial organs, like an artificial pancreas or an artificial liver. In addition to creating and developing the methodologies for enclosing cells within the appropriate semipermeable and biocompatible membranes, novel techniques are needed to assess the various features of the resulting capsules. The small size of a capsule or its heterogeneity can lead to additional complexities that go beyond the problem of examining cell behavior in the presence of biomaterials. These problems are illustrated here by comparison of protein release by microencapsulated HepG2 cells within large and small HEMA-MMA (hydroxyethyl methacrylate-methyl methacrylate) capsules, by assessment of the effect of processing conditions on HEMA-MMA microcapsule permeability to horseradish peroxidase at the individual capsule level, and by a confocal microscopy technique for assessing intracapsule cell viability.

Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH2) terminal group at 5.4 or 54 × 104 beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 269–278, 2005

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with 75:25 poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) or film to assess the resultant dendritic cell (DC) maturation as compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The effect of PLGA contact on DC maturation was examined as one possible explanation for the PLGA adjuvant effect we have observed in the enhancement of an immune response to codelivered model antigen, as adjuvants act through the maturation of DCs. Culturing iDCs with PLGA MPs or PLGA film resulted in morphology similar to that of LPS-matured DCs and the association, or possible internalization, of PLGA MPs. Furthermore, biomaterial-treated iDCs demonstrated an increase in MHC class II and costimulatory molecule expression compared to iDCs but to a lower level than that of LPS-matured DCs. Direct iDC contact with PLGA MPs was necessary for maturation. Immature DCs exposed to PLGA MPs were stimulatory of allogeneic T-cell proliferation, whereas cells exposed to PLGA film were not. Further, PLGA MPs supported a moderate delayed type hypersensitivity reaction in mice indicative of in vivo DC maturation. Taken together, these results suggest that PLGA is a DC maturation stimulus and that the form of the biomaterial may influence the extent of DC maturation.

An understanding of the endothelial cell/blood/biomaterial interactions is central to advancing the success of cardiovascular devices that continue to fail because of the lack of nonthrombogenic biomaterials. A simplified endothelial cell/blood cell/biomaterial static model was used to assess these interactions. Human whole blood or isolated blood cells (mononuclear cells, neutrophils, platelets) were pretreated with biomaterial beads with different surface chemistries: polystyrene (PS), PS beads grafted with 3-kDa polyethylene glycol (PEG) with either hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH2) terminal groups at bead concentrations of 5.4 or 54 x 10(4) beads/mL. Leukocyte and platelet activation and microparticle formation was assessed using flow cytometry. Biomaterial-activated whole blood or isolated cells or mononuclear cell fractions were applied to human umbilical cord endothelial cells (HUVEC) for static coculture, and the resultant proinflammatory HUVEC phenotype was characterized. ICAM-1 and E-selectin expression on HUVEC was increased after 4-h static coculture with biomaterial-treated human whole blood or mononuclear cells but not neutrophils or platelets. VCAM-1 expression on HUVEC was similarly increased after 24-h static coculture but not after 4 h of coculture. Increased concentrations of cytokines, IL-6, IL-8, and MCP-1, were detected in the supernatant of cocultures of HUVEC with biomaterial-treated whole blood or mononuclear cells but not neutrophils or platelets, compared with the media control. After 24 h, cytokine release was significantly increased for both IL-8 and MCP-1 but not IL-6 above concentrations after 4 h of coculture. Neither the cell adhesion molecule (CAM) expression nor cytokine release induced by coculture with biomaterial-treated whole blood or isolated cells was dependent on either material surface chemistry or material surface area. The changes in HUVEC CAM expression and cytokine release induced by biomaterial-treated mononuclear cells can be attributed predominantly to adherent cells on beads and nonadherent bulk cells with moderate regulation by the soluble supernatant; however, mononuclear cell-derived microparticles induced no significant changes in CAM expression or cytokine release after static coculture with HUVEC.

Microencapsulation of cells as a means of insulin or other protein delivery (for example, for gene therapy) has not yet realized its potential. Three aspects of this problem are illustrated with reference to the use of poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA–MMA). Conformal coating was used to coat cell aggregates with a very thin layer of a water-insoluble HEMA–MMA membrane that conforms to the shape of the aggregate, and minimizes the polymer’s contribution to the total transplant volume. Cell aggregates were coated at a liquid–liquid interface of a discontinuous density gradient composed of both aqueous and organic liquids. Aggregates of HepG2 cells were coated and remained viable. Immobilization matrices were co-encapsulated in order to control cell phenotype. Ultralow gelling temperature agarose promoted the proliferation of HEK293 cells, while the viability of transfected C2C12 cells was improved in microcapsules that contained Matrigel ® . Rat or human hepatoma cells in HEMA–MMA microcapsules lost viability within a week after implantation into an omental pouch in Wistar rats. The loss of viability was attributed to the tissue reaction, although it is not clear if the cells lost their viability in vivo leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. On the other hand, intraperitoneal implantation of microcapsules containing L929 cells in ‘syngeneic’ C3H mice in a high-strength agarose gel resulted in maintenance of viability of ∼50% of the encapsulated cells. While progress is being made on several fronts, this type of tissue engineering construct is still several years away from routine use in humans.

A tissue-engineered implant is a biologic-biomaterial combination in which some component of tissuehas been combined with a biomaterial to create a device for the restoration or modification of tissue ororgan function. Specific growth factors, released from a delivery device or from co-transplanted cells,would aid in the induction of host paraenchymal cell infiltration and improve engraftment of co-deliveredcells for more efficient tissue regeneration or ameliorate disease states. The characteristic properties ofgrowth factors are described to provide a biological basis for their use in tissue engineered devices. Theprinciples of polymeric device development for therapeutic growth factor delivery in the context of tissueengineering are outlined. A review of experimental evidence illustrates examples of growth factor deliveryfrom devices such as micropaticles, scaffolds, and encapsulated cells, for their use in the applicationareas of musculoskeletal tissue, neural tissue, and hepatic tissue.

Tissue engineering holds great promise for the regeneration of completely natural living tissues and organs, thus addressing the most challenging problem of donor tissue shortage in modern medicine [1, 2]. Traditionally, the tissue engineering concept has been that a relatively small number of cells are obtained from patients, expanded in culture, seeded onto an appropriate biomaterial scaffold, and then transplanted. In addition to autologous differentiated cell sources, current research is investigating the utility of autologous adult stem cells and cells of allogeneic and xenogeneic origin, when autologous differentiated cells are not available [2]. Critical to the success of the engraftment of these cells is the control of the local microenvironment. By engineering the cellular niche, tissue-engineered devices can provide an environment suitable to modify host responses and direct cell differentiation, growth, migration, and survival [3]. Tissue engineering scaffolds have been designed to direct cell function by controlling scaffold material properties and incorporating delivery of cell-instructive biomolecules by immobilization or controlled release [2]. Several tissue-engineered products that incorporate these approaches are already on the market for treating extensive burns [4, 5]. Realizing their potential, new therapeutic strategies resulting from scientific progress in tissue engineering will not only significantly improve the quality of life for the affected individuals, but also save the billions of dollars spent annually to treat patients with lost, damaged, or failing organs.

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the 'capsule eluate') to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not beta-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the beginning of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of 'free-floating' capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, alpha1-antitrypsin, fibronectin, albumin, alpha2-macroglobulin, vitronectin, beta2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.

High resolution carbon, C 1s, X-ray photoelectron spectroscopy (XPS) of the surface of hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75 mol% HEMA) capsules maintained in PBS for 1 week showed that the surface was not pure HEMA-MMA. In these spectra, more carbon was bonded in the C-O form than in the C-C form indicating the presence of the Pluronic surfactant, L101, adsorbed from the precipitation bath to the surface during microcapsule preparation. Capsules maintained in medium containing fetal bovine serum for 1 week showed a nitrogen signal consistent with the presence of adsorbed serum proteins. There was a decrease in the amount of nitrogen on the surface after phosphate buffered saline (PBS) washing, however this did not decrease to zero. These preadsorbed proteins, present on the surface of capsules incubated in serum-containing medium before their implantation, may affect the tissue response to these capsules. Calcium was not detected on freshly-made capsules or capsules maintained in PBS for 1 week but was detected on capsules maintained in medium containing serum. Calcium deposits, if formed in vitro, could act as nucleation sites for calcification of the polymer in vivo.

Given the central role of dendritic cells (DCs) in directing T-cell phenotypes, the ability of biomaterial-treated DCs to dictate autologous T-cell phenotype was investigated. In this study, we demonstrate that differentially biomaterial-treated DCs differentially directed autologous T-cell phenotype and polarization, depending on the biomaterial used to pretreat the DCs. Immature DCs (iDCs) were derived from human peripheral blood monocytes and treated with biomaterial films of alginate, agarose, chitosan, hyaluronic acid, or 75:25 poly(lactic-co-glycolic acid) (PLGA), followed by co-culture of these biomaterial-treated DCs and autologous T cells. When autologous T cells were co-cultured with DCs treated with biomaterial film/antigen (ovalbumin, OVA) combinations, different biomaterial films induced differential levels of T-cell marker (CD4, CD8, CD25, CD69) expression, as well as differential cytokine profiles [interferon (IFN)-γ, interleukin (IL)-12p70, IL-10, IL-4] in the polarization of T helper (Th) types. Dendritic cells treated with agarose films/OVA induced CD4+CD25+FoxP3+ (T regulatory cells) expression, comparable to untreated iDCs, on autologous T cells in the DC-T co-culture system. Furthermore, in this co-culture, agarose treatment induced release of IL-12p70 and IL-10 at higher levels as compared with DC treatment with other biomaterial films/OVA, suggesting Th1 and Th2 polarization, respectively. Dendritic cells treated with PLGA film/OVA treatment induced release of IFN-γ at higher levels compared with that observed for co-cultures with iDCs or DCs treated with all other biomaterial films. These results indicate that DC treatment with different biomaterial films has potential as a tool for immunomodulation by directing autologous T-cell responses.

Dendritic cells (DCs) play a critical role in orchestrating the host responses to a wide variety of foreign antigens and are essential in maintaining immune tolerance. Distinct biomaterials have been shown to differentially affect the phenotype of DCs, which suggested that biomaterials may be used to modulate immune response toward the biologic component in combination products. The elucidation of biomaterial property-DC phenotype relationships is expected to inform rational design of immuno-modulatory biomaterials. In this study, DC response to a set of 12 polymethacrylates (pMAs) was assessed in terms of surface marker expression and cytokine profile. Principal component analysis (PCA) determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an immature DC phenotype. Partial square linear regression, a multivariate modeling approach, was implemented and successfully predicted biomaterial-induced DC phenotype in terms of surface marker expression from biomaterial properties with R prediction 2 = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R prediction 2 = 0.80. These results demonstrated that immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection.

Mammalian cells encapsulated within polymeric membranes is a novel way for in vivo controlled release of therapeutic agents. A permselective polymeric membrane, by acting as a permeability barrier for large molecules (such as antibodies) can protect the encapsulated cells from the cytotoxic components of the host's tissue reaction without immunosuppressants. The high membrane permeability for small molecules (such as nutrients, hormones, etc.), on the other hand, will ensure the maintenance of normal physiological state by the encapsulated cells. We have developed an interfacial precipitation technique for encapsulating mammalian cells in polyacrylate membranes. This technique is based on the co-extrusion of a cell suspension and polymer solution through a concentric needle assembly and subsequent formation of a polymeric membrane around the cells in a precipitation bath. Here, we report a summary of our experience with the performance of the encapsulated cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules.

Dendritic cells (DCs) play pivotal roles in responding to foreign entities during the innate immune response and in initiating effective adaptive immunity as well as maintaining immune tolerance. The sensitivity of DCs to foreign stimuli also makes them useful cells to assess the inflammatory response to biomaterials. Elucidating material property-DC phenotype relationships using a well-defined biomaterial system is expected to provide criteria for immunomodulatory biomaterial design. Clinical titanium (Ti) substrates, including pretreatment (PT), sand blasted and acid etched (SLA), and modified SLA (modSLA), with different roughnesses and surface energies were used to treat DCs and resulted in differential DC responses. PT and SLA induced a mature DC (mDC) phenotype, while modSLA promoted a non-inflammatory environment by supporting an immature DC (iDC) phenotype, based on surface marker expression, cytokine production profiles and cell morphology. Principal component analysis (PCA) confirmed these experimental results, and also indicated that the non-stimulating property of modSLA covaried with certain surface properties, such as high surface hydrophilicity, percent oxygen and percent Ti of the substrates. In addition to previous research that demonstrated superior osteogenic properties of modSLA compared with PT and SLA, the results reported herein indicates that modSLA may further benefit implant osteointegration by reducing local inflammation and its associated osteoclastogenesis.

Biomaterials have the potential to be utilized as immunostimulatory or immunosuppressive delivery agents for biologics. It is hypothesized that this is directed by the ability of a biomaterial to drive dendritic cells (DC) in situ toward an immunostimulatory or an immunosuppressive phenotype, respectively. However, the specific pattern recognition receptors (PRRs) that DCs use to recognize and respond to biomaterials are unknown. From among the many receptors that DCs use to recognize and respond to foreign entities, herein the focus is on integrins. A biomaterial that induces DC maturation, namely poly(lactic-co-glycolic) acid (PLGA), supported increased human monocyte-derived DC adhesion and up-regulation of integrin receptor gene expression, measured via RT-PCR, as compared to culture on tissue culture polystyrene (TCPS). This was not observed for a biomaterial that does not support DC maturation. Through antibody-blocking techniques, the adhesion to both TCPS and PLGA was found to be β(2) integrin dependent and β(1) independent. Significantly, inhibiting β(2)-mediated adhesion to biomaterials via blocking antibodies also lowered the level of maturation of DCs (CD86 expression). β(2) integrins (but not β(1)) were found localized in biomaterial-adherent DC podosomes and also were found in direct contact with the PLGA surface. Therefore, it appeared that β(2) integrin-mediated adhesion is involved in determining the state of DC maturation on the PLGA surface. DC adhesion to biomaterials may be engaged or avoided to manipulate an immune response to biological component delivered with a biomaterial carrier.

Inflammatory responses to implanted biomedical devices elicit a foreign body fibrotic reaction that limits device integration and performance in various biomedical applications. We examined chronic inflammatory responses to microgel conformal coatings consisting of thin films of poly(N-isopropylacrylamide) hydrogel microparticles cross-linked with poly(ethylene glycol) diacrylate deposited on poly(ethylene terephthalate) (PET). Unmodified and microgel-coated PET disks were implanted subcutaneously in rats for 4 weeks and explants were analyzed by histology and immunohistochemistry. Microgel coatings reduced chronic inflammation and resulted in a more mature/organized fibrous capsule. Microgel-coated samples exhibited 22% thinner fibrous capsules that contained 40% fewer cells compared to unmodified PET disks. Furthermore, microgel-coated samples contained significantly higher levels of macrophages (80%) than unmodified PET controls. These results demonstrate that microgel coatings reduce chronic inflammation to implanted biomaterials. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not ad here. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM). In preparation for microscopy, dissolution of the polymer was prevented by the use of frozen sections embedded in a water-soluble compound. Similarly, freeze cleavage of conductively stained capsules permitted SEM observation of the capsule interior along with ultrastructural detail of the cells. In the absence of Matrigel, cells in HEMA-MMA capsules were found to form aggregates in intracapsular pockets with central necrosis occurring at day 7 in large aggregates. The coencapsulation of HepG2 cells with Matrigel, resulted in an initially uniform distribution of essentially individual cells with aggregates appearing later within the Matrigel. Many cells within these capsules had remained viable when examined up to day 14 with only limited cellular necrosis, implying a favorable environment for microencapsulated HepG2 cells. © 1992 John Wiley & Sons, Inc.

Intact and fragmented proteins, eluted from self-assembled monolayer (SAM) surfaces of alkanethiols of different chemistries (CH3, OH, COOH, NH2), following exposure to human plasma (HP) or human serum (HS), were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The SAM surfaces were incubated for 1 h with 10% (v/v) sterile-filtered, heat-inactivated (h.i.) HS or 1% (v/v) sterile-filtered h.i. HP preparations [both in phosphate buffered saline (PBS)]. Adsorbed proteins were eluted using 10% SDS/2.3% dithioerythritol for characterization of protein profiles. The type of incubating medium may be an important determinant of adsorbed protein profiles, since some variations were observed in eluates from filtered versus control unfiltered h.i. 10% HS or 1% HP. Albumin and apolipoprotein A1 were consistently detected in both filtered h.i 10% HS and 1% HP eluates from all SAM surfaces and from control tissue culture-treated polystyrene (TCPS). Interestingly, Factor H and Factor I, antithrombin, prothrombin, high molecular weight kininogen (HMWK), and IgG were present in eluates from OH, COOH, and NH2 SAM surfaces and in eluates from TCPS but not in eluates from CH3 SAM surfaces, following exposure to filtered h.i. 10% HS. These results suggest that CH3 SAM surfaces were the least proinflammatory of all SAM surfaces. Overall, similar trends were observed in the profiles of proteins eluted from surfaces exposed to filtered 10% HS or 1% HP. However, the unique profiles of adsorbed proteins on different SAM surface chemistries may be related to their differential interactions with cells, including immune/inflammatory cells. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

The host response to a biomaterial is characterized by both acute recruitment and attachment of cells as well as chronic encapsulating tissue reaction. The implantation procedure induces production of damage-associated molecular patterns (DAMPs) which may contribute to host recognition of the material. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that bind not only pathogen-associated molecular patterns (PAMPs) but also DAMPs. We sought to investigate whether TLR4/DAMP interactions were involved in the acute and chronic inflammatory response to an implanted biomaterial. When PET discs were implanted intraperitoneally for 16h, no differences were found in the number of leukocytes recruited between TLR4(+) (C57BL/10J) and TLR4(-) (C57BL/10ScNJ) mice. However, a significant shift in the leukocyte profile on the biomaterial surface was observed for TLR4(-) mice. While the total number of adherent cells was the same in both strains, TLR4(+) mice had a profile with equivalent neutrophil and monocyte/macrophage presence on the material surface, and TLR4(-) mice had a profile of predominantly neutrophils with fewer monocyte/macrophages. When implants were placed subcutaneously for 2 weeks, the fibrous capsule thicknesses were not different between TLR4(+) and TLR4(-) mouse strains. These findings illustrate that TLR4 may play a role in the initial recognition of a biomaterial by directing the adhesive cellular profile.

Biomaterial contact triggers dendritic cell (DC) maturation, to an extent depending on the biomaterial, ultimately enhancing an immune response toward associated antigens, implying a role for biomaterials as adjuvants. Self-assembled monolayers (SAM) of alkanethiols on titanium/gold-coated surfaces presenting different chemistries were used to study effects of biomaterial surface chemistry on DC maturation. Although DCs treated with OH, COOH, or NH(2) SAMs showed modest maturation, those treated with CH(3) SAMs were least mature, all based on cytospins, allostimulatory capacity, or maturation marker expression. Surprisingly, DCs treated with CH(3) SAMs secreted highest levels of proinflammatory tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) but were least mature. Secretion of anti-inflammatory mediators by DCs treated with CH(3) SAMs was not responsible for mitigating DC maturation under these conditions. Interestingly, elevated levels of apoptotic markers were measured associated with DCs and T cells upon CH(3) SAMs contact. Since phagocytosis of apoptotic DCs has strong immunosuppressive effects on DCs, more apoptotic DCs on CH(3) SAMs may account for lower DC maturation. Finally, higher expression of cytotoxic T lymphocyte associated antigen receptor-4 (CTLA-4) on T cells may imply a mechanism of T cell inhibition on CH(3) SAMs.

Conserved protein-carbohydrate-lipid pathogen-associated molecular patterns (PAMPs) interact with cells of the innate immune system to mediate antigen recognition and internalization and activation of immune cells. We examined if analogous "biomaterial-associated molecular patterns" composed of proteins, specifically their carbohydrate modifications, existed on biomaterials, which can play a role in mediating the innate immune response to biomaterials. To probe for these carbohydrates in the adsorbed protein layer, as directed by the underlying biomaterial chemistry, self-assembled monolayers (SAMs) presenting -CH(3), -OH, -COOH, or -NH(2) were preincubated with serum/plasma, and the presence of carbohydrate ligands of C-type lectin receptors (CLRs) was investigated using lectin probes in an enzyme-linked lectin assay (ELLA). Presentation of CLR ligands was detected on control tissue culture polystyrene (TCPS). Absorbances of mannose or N-acetylglucosamine increased with decreasing incubating serum concentration, whereas absorbances of sialylated epitopes or fucose remained unchanged. Absorbances of alpha-galactose or N-acetylgalactosamine decreased with decreasing incubating serum concentration; beta-galactose was undetectable. Among SAM endgroups, preincubation with 10% serum resulted in differential presentation of CLR ligands: higher alpha-galactose on COOH SAMs than NH(2) or CH(3) SAMs, highest complex mannose on NH(2) SAMs, and higher complex mannose on OH SAMs than CH(3) SAMs. Least sialylated groups were detected on CH(3) SAMs. In summary, biomaterial chemistry may regulate protein adsorption and hence unique presentation of associated carbohydrates. The ultimate goal is to identify the effects of protein glycosylations associated with biomaterials in stimulating innate immune responses.

The interconnections between innate and adaptive immunity, with a focus on dendritic cells (DCs), in the context of combination products, are discussed. Biomaterials are shown to act as an adjuvant in the enhancement of the adaptive immune response to co-delivered antigen. Biomaterials are considered as agonists for DC maturation and several hypotheses as to the mechanism by which DCs recognize and respond to biomaterials are presented. There is the conceptualization of biomaterials with the idea of 'danger signals'. The goal is to design biomaterials to control DC phenotype and in this way control immune responses to combination products.

Model in vitro culturing systems were developed to analyze roles of biomaterial-induced leukocyte activation on endothelial cell (EC) and smooth muscle cell (SMC) phenotype, and their crosstalk. Isolated monocytes or neutrophils were pretreated with model biomaterial beads and applied directly to "more secretory" (cultured in media containing 5% fetal bovine serum) or forced contractile (serum and growth factor starved) human aortic SMCs (HASMCs), or to the human aortic EC (HAEC) surface of HAEC/HASMC cocultures (HASMC phenotype varied to be "more or less secretory") for 5 or 24 h of static culture. Surface expression of proinflammatory [ICAM-1, VCAM-1, E-selectin], procoagulant (tissue factor), and anticoagulant (thrombomodulin) markers, as well as HAEC proliferation, were assessed by flow cytometry. Incubation of HAEC with biomaterial-pretreated monocytes (and neutrophils to lesser degree) suppressed HAEC proliferation and induced a proinflammatory/procoagulant HAEC phenotype. This HAEC phenotype was amplified in coculture with "more secretory" HASMCs and subdued in coculture with "less secretory" HASMCs. Direct incubation of biomaterial-pretreated monocytes or neutrophils with "more secretory" HASMCs further increased HASMC ICAM-1 and tissue factor expression. Direct incubation of biomaterial-pretreated monocytes or neutrophils with forced contractile HASMCs upregulated ICAM-1, VCAM-1, and tissue factor expression above the presence of serum-containing media alone.

Development of in vitro models of native and injured vasculature is crucial for better understanding altered wound healing in disease, device implantation, or tissue engineering. Conditions were optimized using polyethyleneteraphalate transwell filters for human aortic endothelial cell (HAEC)/smooth muscle cell (HASMC) co-cultures with divergent HASMC phenotypes ('more or less secretory') while maintaining quiescent HAECs. Resulting HASMC phenotype was studied at 48 and 72 h following co-culture initiation, and compared to serum and growth factor starved monocultured 'forced contractile' HASMCs. Forced contractile HASMCs demonstrated organized alpha-smooth muscle actin filaments, minimal interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion, and low intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor expression. Organization of alpha-smooth muscle actin was lost in 'more secretory' HASMCs in co-culture with HAECs, and IL-8 and MCP-1 secretion, as well as ICAM-1, VCAM-1, and tissue factor expression were significantly upregulated at both time points. Alternately, 'less secretory' HASMCs in co-culture with HAECs showed similar characteristics to forced contractile HASMCs at the 48 h time point, while by the 72 h time point they behaved similarly to 'more secretory' HASMCs. These co-culture systems could be useful in better understanding vascular healing, however there remain time constraint considerations for maintaining culture integrity/cell phenotype.

The ability of immature dendritic cells (iDCs) derived from human peripheral blood mononuclear cells to phagocytose poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) as compared to polystyrene MPs and the molecular aspects of this phagocytosis were investigated. Treating iDCs with PLGA or polystyrene fluorospheres of approximately 3 microm in diameter resulted in the internalization of the particles as evidenced by confocal laser scanning micrographs. This uptake of fluorospheres by DCs was decreased by pretreatment of cells with cytochalasin D or by incubation with the fluorospheres at 4 degrees C, and was sensitive to EDTA and trypsin pretreatments in a dose-dependent manner. In agreement with our previous studies, treatment of iDCs with PLGA MPs, but not with polystyrene MPs, led to DC maturation, as measured by increase in release of the autocrine maturation cytokine, tumor necrosis factor-alpha, which was dependent on ratio of PLGA MPs to DCs. Taken together, this work begins to address the role of phagocytosis on PLGA MP-induced DC maturation and the molecular mechanisms involved.

Application of biomaterials in combination products in which the biomaterial is presented to the host with a biological component prompts the need for understanding the biomaterial-associated adjuvant effect in the immune response against antigens associated with such a product. We have previously demonstrated that a polymer commonly used in tissue engineering and vaccine delivery, poly(lactic-co-glycolic acid) (PLGA), exerts an adjuvant effect in vivo, which was supported by PLGA-induced dendritic cell (DC) maturation in vitro. In this study, the effects of agarose and PLGA on DC maturation were compared in vitro to establish differential biomaterial effects. Human monocyte-derived DCs were treated with agarose or PLGA microparticles or films, and their maturation effect was measured as expression of costimulatory and MHC class II molecules, allostimulatory capacity, and proinflammatory cytokine secretion. Direct comparison of DC maturation phenotype indicated that PLGA was a stronger stimulus of DC maturation than agarose, and this maturation was not affected by microparticle phagocytosis. However, agarose-treated DCs showed higher activation of nuclear factor kappaB (NFkappaB) 24 h after the initial stimulation of DCs. Taken together, these results demonstrate differential biomaterial effects on DC maturation, substantiating the maturation effect of PLGA, and provide screening methods for biomaterial adjuvant effect for applications in combination products.

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with films of biomaterials commonly used in combination products (e.g., tissue engineered constructs or vaccines) to assess the resultant dendritic cell (DC) maturation compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The following biomaterials were tested: alginate, agarose, chitosan, hyaluronic acid, 75:25 poly(lactic-co-glycolic acid) (PLGA). The effect of DC culture on these films was undertaken to identify biomaterials which support DC maturation and those biomaterials that did not. Dendritic cells treated with chitosan or PLGA (agarose to a lesser extent) films increased expression levels of CD86, CD40, and HLA-DQ, compared to control iDCs, similar to LPS-matured DCs, whereas DCs treated with alginate or hyaluronic acid films decreased their expression levels of these same molecules. In summary, a differential effect of the biomaterial on which iDCs were cultured was observed as far as the extent of induced DC maturation. The effect of biomaterials on DC maturation, and the associated adjuvant effect, is a novel biocompatibility selection and design criteria for biomaterials to be used in combination products in which immune consequences are potential complications or outcomes.

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

A model shed antigen, ovalbumin (OVA), was incorporated into polymeric biomaterial carriers made of poly(lactic-co-glycolic acid) (PLGA) in the form of microparticles (MP) or scaffolds (SC). These polymeric biomaterial carrier vehicles with incorporated antigen were then injected or implanted into mice and the resulting time-dependent systemic humoral immune response towards the controlled released OVA was assessed by following the OVA-specific IgG concentration and isotypes using ELISA. To assess the differential level of enhancement of the immune response depending on the form of carrier vehicle (MP vs. SC), the total amount of polymer and OVA delivered was kept constant as well as the release rate of OVA for both carrier vehicles. The level of the humoral immune response was higher and sustained for OVA released from PLGA SC which were implanted with associated tissue damage, and lower and transient when the same amount of polymer and OVA were delivered from PLGA MP, which were minimally invasively delivered by injection. This immune response was primarily Th2 helper T cell-dependent, although for the strong adjuvant, CFA, and PLGA SC carriers there was both a Th2 and Th1 response contribution. These results implicate ‘danger signals’ associated with the implantation of the scaffolds due to tissue injury which primed the system for an enhanced immune response.

A model shed antigen, ovalbumin (OVA), was co-delivered with polymeric biomaterial carrier vehicles in C57BL6 mice to test whether the presence of the biomaterial acted as an adjuvant in the immune response towards the associated antigen. The biomaterials tested were non-biodegradable polystyrene microparticles and biodegradable 50:50 or 75:25 poly(lactic-co-glycolic acid) (PLGA) microparticles or scaffolds. For each biomaterial carrier vehicle, to assess the resulting time-dependent systemic humoral immune response towards the co-delivered OVA, the OVA-specific IgG concentration and isotypes (IgG2a or IgG1, indicating a predominant Th1 or Th2 response, respectively) were determined using ELISA. OVA co-delivered with biomaterial carrier vehicles supported a moderate humoral immune response that was maintained for the 18-week duration of the experiment. This humoral immune response was primarily Th2 helper T cell-dependent as indicated by the predominant IgG1 isotype. Furthermore, this humoral immune response was not material chemistry-dependent within the material set tested here. With the presence of the biomaterial resulting in an enhancement of the humoral immune response to co-delivered antigen, it appears that the biomaterial acts as an adjuvant in the development of an adaptive immune response to co-delivered antigen.

Small diameter hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75% HEMA) microcapsules containing an aggregate of viable rat hepatoma H4IIEC3 cells, after implantation into an omental pouch in Wistar rats, contained viable cells at 7 days but not 14 days. A similar transplantation of microencapsulated aggregates of human hepatoma HepG2 cells did not result in viable cells even at 7 days. The loss of viability was attributed to the tissue reaction, because both encapsulated cell types remained viable in vitro. However, it is not clear if the cells lost their viability in vivo, leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. The tissue reactions to microcapsules containing rat or human hepatoma cells at day 1 was one cell layer thick and avascular. At later times, tissue reactions were comprised of three regions: macrophages, fibroblasts, and some foreign body giant cells apposed to the polymer membrane, a dense region of fibroblasts and collagen, and a region of vascularized granulation tissue. Prompt vascularization of the tissue reactions occurred after 4 days and was maintained for up to 14 days. Even at 14 days, immune cells were observed, suggesting a continued immune response toward antigens shed from the encapsulated cells.