Benjamin G. Keselowsky

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH2-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH3 groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against β1 integrin inhibited matrix mineralization on the OH and NH2 surfaces, whereas function-perturbing antibodies specific for β3 integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.

The control of cell adhesion and viability by zinc oxide nanorods

The ability to control the behavior of cells that interact with implanted biomaterials is desirable for the success of implanted devices such as biosensors or drug delivery devices. There is a need to develop materials that can limit the adhesion and viability of cells on implanted biomaterials. In this study, we investigated the use of zinc oxide (ZnO) nanorods for modulating the adhesion and viability of NIH 3T3 fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells. Cells adhered far less to ZnO nanorods than the corresponding ZnO flat substrate. The few cells that adhered to ZnO nanorods were rounded and not viable compared to the flat ZnO substrate. Cells were unable to assemble focal adhesions and stress fibers on nanorods. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Time-lapse imaging revealed that cells that initially adhered to nanorods were not able to spread. This suggests that it is the lack of initial spreading, rather than long-term exposure to ZnO that causes cell death. We conclude that ZnO nanorods are potentially useful as an adhesion-resistant biomaterial capable of reducing viability in anchorage-dependent cells.

Contributions of surface topography and cytotoxicity to the macrophage response to zinc oxide nanorods.

Macrophages associated with implanted biomaterials are primary mediators of chronic inflammation and foreign body reaction to the implant. Hence, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate inflammatory responses. Nanostructured materials possess unique surface properties, and nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. Additionally adherent macrophage number on ZnO flat substrate was reduced as compared to glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. Cell death was apparently not apoptotic, given the lack of activated caspase-3 immunostaining. A decrease in viable macrophage numbers when ZnO substrates were present in the same media verified the role of ZnO substrate dissolution, and dissolved levels of Zn in culture media were quantified. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 d. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. In conclusion, although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent.

Adhesive substrate-modulation of adaptive immune responses

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4(+) T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4(+) T-cell proliferation and T(h)1 type response in terms of IFN-gamma producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T(h)2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.

Microparticle surface modifications targeting dendritic cells for non-activating applications

Microparticulate systems for delivery of therapeutics to DCs for immunotherapy have gained attention recently. However, reports addressing the optimization of DC-targeting microparticle delivery systems are limited, particularly for cases where the goal is to deliver payload to DCs in a non-activating fashion. Here, we investigate targeting DCs using poly (d lactide-co-glycolide) microparticles (MPs) in a non-stimulatory manner and assess efficacy in vitro and in vivo. We modified MPs by surface immobilizing DC receptor targeting molecules - antibodies (anti-CD11c, anti-DEC-205) or peptides (P-D2, RGD), where anti-CD11c antibody, P-D2 and RGD peptides target integrins and anti-DEC-205 antibody targets the c-type lectin receptor DEC-205. Our results demonstrate the modified MPs are neither toxic nor activating, and DC uptake of MPs in vitro is improved by the anti-DEC-205 antibody, the anti-CD11c antibody and the P-D2 peptide modifications. The P-D2 peptide MP modification significantly improved DC antigen presentation in vitro both at immediate and delayed time points. Notably, MP functionalization with P-D2 peptide and anti-CD11c antibody increased the rate and extent of MP translocation in vivo by DCs and MΦs, with the P-D2 peptide modified MPs demonstrating the highest translocation. This work informs the design of non-activating polymeric microparticulate applications such as vaccines for autoimmune diseases.

Multifunctional dendritic cell-targeting polymeric microparticles: Engineering new vaccines for type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by T-cell mediated destruction of insulin-producing β-cells.1 Approximately 13,000 new cases of T1D are diagnosed each year in US children, with a prevalence of about 2 cases per 1,000 individuals, and associated treatment and care costs are large.2 Following clinical diagnosis, many T1D patients maintain functional pancreatic β-cell mass for some time.3 This therapeutic window has motivated attempts to halt autoimmune inflammation of pancreatic islets before too many islets are destroyed to maintain glucose regulation. Schemes involving broad immunosuppression4 or antigen non-specific immunomodulation5 have been explored for T1D treatment, however, only antigen-specific approaches hold the unique promise of long-lasting disease remission in the absence of adverse side effects.6 Therefore, efforts have focused on manipulating the body’s most efficient antigen-presenting cell, dendritic cells (DCs), in T1D and other autoimmune diseases.7 Early and ongoing efforts involving DCs therapeutically focus on the exogenous generation of DCs for administration as a cellular vaccine.7,8 In fact, an exogenously manipulated DC-based vaccine is currently being investigated in clinical trials for application in T1D.9 However, it is generally accepted that cell-based vaccines for T1D are primarily intended to provide proof-of-concept, as several factors limit this approach.10 In particular, dissemination and lymph node homing of exogenously delivered DCs is inefficient11 and treatment involves isolation and storage of DCs under stringent manufacturing standards, amounting to high costs which prohibit widespread application.12,13 An attractive alternative strategy involves the in vivo targeting of DCs with injectable polymeric, biodegradable microparticles delivering a payload of vaccine components and immunomodulatory factors. Microparticle systems may be easily administered in a single injection to patients with extended delivery of both prime and boost doses using timed-release materials.14,15 Furthermore, polymeric microparticle strategies greatly simplifies issues related to manufacturing, storage and shipping, as microparticle encapsulation affords stability for off-the-shelf availability.16,17 Microparticles can be engineered to be multifunctional and modular, where features of particular interest are: (1) targeting to DCs, (2) providing a depot for antigen, (3) providing controlled delivery and subcellular targeting of adjuvants, immunosuppressants, chemokines or other conditioning factors. Conceptually, microparticle systems can thus be designed to attract DCs and precursors into an injection site, provide signals to promote differentiation into DCs, promote uptake of antigen and provided immunosuppressive/tolerogenic conditioning of DCs to induce specific tolerance. In practice, the amalgamation of these design principles are just beginning to be assembled for application toward T1D. The goal of this review is in part to showcase recent microparticle-based efforts toward immunosuppression and T1D mitigation in particular, but also to draw on the broader literature of DC-targeting of microparticles, largely applied to infection or cancer, as instructive examples to highlight the repertoire of tools available for the scientist/clinician investigating T1D therapies.

A high-throughput microparticle microarray platform for dendritic cell-targeting vaccines.

Immunogenomic approaches combined with advances in adjuvant immunology are guiding progress toward rational design of vaccines. Furthermore, drug delivery platforms (e.g., synthetic particles) are demonstrating promise for increasing vaccine efficacy. Currently there are scores of known antigenic epitopes and adjuvants, and numerous synthetic delivery systems accessible for formulation of vaccines for various applications. However, the lack of an efficient means to test immune cell responses to the abundant combinations available represents a significant blockade on the development of new vaccines. In order to overcome this barrier, we report fabrication of a new class of microarray consisting of antigen/adjuvant-loadable poly(D,L lactide-co-glycolide) microparticles (PLGA MPs), identified as a promising carrier for immunotherapeutics, which are co-localized with dendritic cells (DCs), key regulators of the immune system and prime targets for vaccines. The intention is to utilize this high-throughput platform to optimize particle-based vaccines designed to target DCs in vivo for immune system-related disorders, such as autoimmune diseases, cancer and infection. Fabrication of DC/MP arrays leverages the use of standard contact printing miniarraying equipment in conjunction with surface modification to achieve co-localization of particles/cells on isolated islands while providing background non-adhesive surfaces to prevent off-island cell migration. We optimized MP overspotting pin diameter, accounting for alignment error, to allow construction of large, high-fidelity arrays. Reproducible, quantitative delivery of as few as 16+/-2 MPs per spot was demonstrated and two-component MP dosing arrays were constructed, achieving MP delivery which was independent of formulation, with minimal cross-contamination. Furthermore, quantification of spotted, surface-adsorbed MP degradation was demonstrated, potentially useful for optimizing MP release properties. Finally, we demonstrate DC co-localization with PLGA MPs on isolated islands and that DCs do not migrate between islands for up to 24 h. Using this platform, we intend to analyze modulation of DC function by providing multi-parameter combinatorial cues in the form of proteins, peptides and other immuno-modulatory molecules encapsulated in or tethered on MPs. Critically, the miniaturization attained enables high-throughput investigation of rare cell populations by reducing the requirement for cells and reagents by many-fold, facilitating advances in personalized vaccines which target DCs in vivo.

VEGF neutralization can prevent and normalize arteriovenous malformations in an animal model for hereditary hemorrhagic telangiectasia 2

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface. Understanding why and how AVMs develop is crucial for developing therapies to inhibit the formation, growth, or maintenance of AVMs in HHT patients. Previously, we have shown that secondary factors such as wounding are required for Alk1-deficient vessels to develop skin AVMs. Here, we present evidences that AVMs establish from nascent arteries and veins rather than from remodeling of a preexistent capillary network in the wound-induced skin AVM model. We also show that VEGF can mimic the wound effect on skin AVM formation, and VEGF-neutralizing antibody can prevent skin AVM formation and ameliorate internal bleeding in Alk1-deficient adult mice. With topical applications at different stages of AVM development, we demonstrate that the VEGF blockade can prevent the formation of AVM and cease the progression of AVM development. Taken together, the presented experimental model is an invaluable system for precise molecular mechanism of action of VEGF blockades as well as for preclinical screening of drug candidates for epistaxis and gastrointestinal bleedings.

Integrin-directed modulation of macrophage responses to biomaterials

Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials.

Combinatorial delivery of immunosuppressive factors to dendritic cells using dual-sized microspheres

Microparticulate systems are beginning to show promise for delivery of modulatory agents for immunotherapeutic applications which modulate dendritic cell (DC) functions. Co-administration of multiple factors is an emerging theme in immune modulation which may prove beneficial in this setting. Herein, we demonstrate that localized, controlled delivery of multiple factors can be accomplished through poly (lactic-co-glycolic acid) (PLGA) microparticle systems fabricated in two size classes of phagocytosable and unphagocytosable microparticles (MPs). The immunosuppressive ability of combinatorial multi-factor dual MP systems was evaluated by investigating effects on DC maturation, DC resistance to LPS-mediated maturation and proliferation of allogeneic T cells in a mixed lymphocyte reaction. Phagocytosable MPs (~2 μm) were fabricated encapsulating either rapamycin (RAPA) or all-trans retinoic acid (RA), and unphagocytosable MPs (~30 μm) were fabricated encapsulating either transforming growth factor beta-1 (TGF-β1) or interleukin-10 (IL-10). Combinations of these MP classes reduced expression of stimulatory/costimulatory molecules (MHC-II, CD80 and CD86) in comparison to iDC and soluble controls, but not necessarily to single factor MPs. Dual MP-treated DCs resisted LPS-mediated activation, in a manner driven by the single factor phagocytosable MPs used. Dendritic cells treated with dual MP systems suppressed allogeneic T cell proliferation, generally demonstrating greater suppression by combination MPs than single factor formulations, particularly for the RA/IL-10 MPs. This work demonstrates feasibility of simultaneous targeted delivery of immunomodulatory factors to cell surface receptors and intracellular locations, and indicates that a combinatorial approach can boost immunoregulatory responses for therapeutic application in autoimmunity and transplantation.