Anna K. Blakney

The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)-based hydrogels.

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG-RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F-actin and localized α(V) integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater α(V) integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF-α, IL-1β, and IL-6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications.

Student award winner in the undergraduate category for the society of biomaterials 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)-based hydrogels

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG-RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F-actin and localized α(V) integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater α(V) integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF-α, IL-1β, and IL-6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications.

Immunomodulation by mesenchymal stem cells combats the foreign body response to cell-laden synthetic hydrogels

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro, murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo, hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome.

Temporal progression of the host response to implanted poly(ethylene glycol)‐based hydrogels

Poly(ethylene glycol) (PEG) hydrogels hold great promise as in vivo cell carriers for tissue engineering. To ensure appropriate performance of these materials when implanted, the host response must be well understood. The objectives for this study were to characterize the temporal evolution of the foreign body reaction (FBR) to acellular PEG-based hydrogels prepared from PEG diacrylate precursors when implanted subcutaneously in immunocompentent c57bl/6 mice by (immuno)histochemical analysis and gene expression. Compared with a normal FBR elicited by silicone (SIL), PEG hydrogels without or with a cell adhesion ligand RGD elicited a strong early inflammatory response evidenced by a thick band of macrophages as early as day 2, persisting through two weeks, and by increased interleukin-1β expression. PEG-only hydrogels showed a slower, but more sustained progression of inflammation over PEG-RGD. Temporal changes in gene expression were observed in response to PEG-based materials and in general exhibited, elevated expression of inflammatory and wound healing genes in the tissues surrounding the implants, while the expression patterns were more stable in response to SIL. While a stabilized FBR was achieved with SIL and to a lesser degree with PEG-RGD, the PEG-only hydrogels had not yet stabilized after 4 weeks. In summary, PEG-only hydrogels elicit a strong early inflammatory reaction, which persists throughout the course of the implantation even as a collagenous capsule begins to form. However, the incorporation of RGD tethers partially attenuates this response within 2 weeks leading to an improved FBR to PEG-based hydrogels.

Linking the foreign body response and protein adsorption to PEG-based hydrogels using proteomics.

Poly(ethylene glycol) (PEG) hydrogels with their highly tunable properties are promising implantable materials, but as with all non-biological materials, they elicit a foreign body response (FBR). Recent studies, however, have shown that incorporating the oligopeptide RGD into PEG hydrogels reduces the FBR. To better understand the mechanisms involved and the role of RGD in mediating the FBR, PEG, PEG-RGD and PEG-RDG hydrogels were investigated. After a 28-day subcutaneous implantation in mice, a thinner and less dense fibrous capsule formed around PEG-RGD hydrogels, while PEG and PEG-RDG hydrogels exhibited stronger, but similar FBRs. Protein adsorption to the hydrogels, which is considered the first step in the FBR, was also characterized. In vitro experiments confirmed that serum proteins adsorbed to PEG-based hydrogels and were necessary to promote macrophage adhesion to PEG and PEG-RDG, but not PEG-RGD hydrogels. Proteins adsorbed to the hydrogels in vivo were identified using liquid chromatography-tandem mass spectrometry. The majority (245) of the total proteins (≥300) that were identified was present on all hydrogels with many proteins being associated with wounding and acute inflammation. These findings suggest that the FBR to PEG hydrogels may be mediated by the presence of inflammatory-related proteins adsorbed to the surface, but that macrophages appear to sense the underlying chemistry, which for RGD improves the FBR.

Delivery of multipurpose prevention drug combinations from electrospun nanofibers using composite microarchitectures

Background Electrospun drug-eluting fabrics have enormous potential for the delivery of physicochemically diverse drugs in combination by controlling the underlying material chemistry and fabric microarchitecture. However, the rationale for formulating drugs at high drug loading in the same or separate fibers is unknown but has important implications for product development and clinical applications. Methods Using a production-scale free-surface electrospinning instrument, we produced electrospun nanofibers with different microscale geometries for the co-delivery of tenofovir (TFV) and levonorgestrel (LNG) – two lead drug candidates for multipurpose prevention of HIV acquisition and unintended pregnancy. We investigated the in vitro drug release of TFV and LNG combinations from composites that deliver the two drugs from the same fiber (combined fibers) or from separate fibers in a stacked or interwoven architecture. For stacked composites, we also examined the role that fabric thickness has on drug-release kinetics. We also measured the cytotoxicity and antiviral activity of the drugs delivered alone and in combination. Results Herein, we report on the solution and processing parameters for the free-surface electrospinning of medical fabrics with controlled microarchitecture and high drug loading (up to 20 wt%). We observed that in vitro release of the highly water-soluble TFV, but not the water-insoluble LNG, was affected by composite microarchitecture, fabric thickness, and drug content. Finally, we showed that the drug-loaded nanofibers are noncytotoxic and that the antiviral activity of TFV is preserved through the electrospinning process and when combined with LNG. Conclusion Electrospun fabrics with high drug loading create multicomponent systems that benefit from the independent control of the nanofibrous microarchitecture. Our findings are significant because they will inform the design and production of composite electrospun fabrics for the co-delivery of physicochemically diverse drugs that may be useful for multipurpose prevention.

Intramyocardial Injection of a Synthetic Hydrogel with Delivery of bFGF and IGF1 in a Rat Model of Ischemic Cardiomyopathy

It is increasingly appreciated that the properties of a biomaterial used in intramyocardial injection therapy influence the outcomes of infarcted hearts that are treated. In this report the extended in vivo efficacy of a thermally responsive material that can deliver dual growth factors while providing a slow degradation time and high mechanical stiffness is examined. Copolymers consisting of N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and degradable methacrylate polylactide were synthesized. The release of bioactive basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF1) from the gel and loaded poly(lactide-co-glycolide) microparticles was assessed. Hydrogel with or without loaded growth factors was injected into 2 week-old infarcts in Lewis rats and animals were followed for 16 weeks. The hydrogel released bioactive bFGF and IGF1 as shown by mitogenic effects on rat smooth muscle cells in vitro. Cardiac function and geometry were improved for 16 weeks after hydrogel injection compared to saline injection. Despite demonstrating that left ventricular levels of bFGF and IGF1 were elevated for two weeks after injection of growth factor loaded gels, both functional and histological assessment showed no added benefit to inclusion of these proteins. This result points to the complexity of designing appropriate materials for this application and suggests that the nature of the material alone, without exogenous growth factors, has a direct ability to influence cardiac remodeling.

Understanding the host response to cell-laden poly(ethylene glycol)-based hydrogels

Poly(ethylene glycol) (PEG)-based hydrogels are promising in situ cell carriers for tissue engineering. However, their success in vivo will in part depend upon the foreign body reaction (FBR). This study tests the hypothesis that the FBR affects cells encapsulated within PEG hydrogels, and in turn influences the severity of the FBR. Fibroblasts were encapsulated within PEG hydrogels containing RGD to support cell attachment. Macrophages were seeded on top of cell-laden hydrogels to mimic in vivo macrophage interrogation and treated with lipopolysaccharide to induce an inflammatory phenotype. The presence of activated macrophages reduced fibroblast gene expression for extracellular matrix molecules and remodeling, but stimulated VEGF and IL-1β gene expression. Fibroblasts impacted macrophage phenotype leading to increased iNOS, IL-1β and TNF-α expressions. Syngeneic cell-laden and acellular hydrogels were also implanted subcutaneously into C57bl/6 mice for 2, 7 and 28 days. Encapsulated fibroblasts secreted collagen type I during the first week, but tissue deposition and cellularity decreased by 28 days. The presence of encapsulated fibroblasts led to greater acute inflammation, but did not influence the fibrotic response. In summary, this work emphasizes the importance of the host response in tissue engineering, and the potentially deleterious impact it may have on cell-laden synthetic scaffolds.

Nanoparticle-Based ARV Drug Combinations for Synergistic Inhibition of Cell-Free and Cell–Cell HIV Transmission

Nanocarrier-based drug delivery systems are playing an emerging role in human immunodeficiency virus (HIV) chemoprophylaxis and treatment due to their ability to alter the pharmacokinetics and improve the therapeutic index of various antiretroviral (ARV) drug compounds used alone and in combination. Although several nanocarriers have been described for combination delivery of ARV drugs, measurement of drug-drug activities facilitated by the use of these nanotechnology platforms has not been fully investigated for topical prevention. Here, we show that physicochemically diverse ARV drugs can be encapsulated within polymeric nanoparticles to deliver multidrug combinations that provide potent HIV chemoprophylaxis in relevant models of cell-free, cell-cell, and mucosal tissue infection. In contrast to existing approaches that coformulate ARV drug combinations together in a single nanocarrier, we prepared single-drug-loaded nanoparticles that were subsequently combined upon administration. ARV drug-nanoparticles were prepared using emulsion-solvent evaporation techniques to incorporate maraviroc (MVC), etravirine (ETR), and raltegravir (RAL) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We compared the antiviral potency of the free and formulated drug combinations for all pairwise and triple drug combinations against both cell-free and cell-associated HIV-1 infection in vitro. The efficacy of ARV-drug nanoparticle combinations was also assessed in a macaque cervicovaginal explant model using a chimeric simian-human immunodeficiency virus (SHIV) containing the reverse transcriptase (RT) of HIV-1. We observed that our ARV-NPs maintained potent HIV inhibition and were more effective when used in combinations. In particular, ARV-NP combinations involving ETR-NP exhibited significantly higher antiviral potency and dose-reduction against both cell-free and cell-associated HIV-1 BaL infection in vitro. Furthermore, ARV-NP combinations that showed large dose-reduction were identified to be synergistic, whereas the equivalent free-drug combinations were observed to be strictly additive. Higher intracellular drug concentration was measured for cells dosed with the triple ARV-NP combination compared to the equivalent unformulated drugs. Finally, as a first step toward evaluating challenge studies in animal models, we also show that our ARV-NP combinations inhibit RT-SHIV virus propagation in macaque cervicovaginal tissue and block virus transmission by migratory cells emigrating from the tissue. Our results demonstrate that ARV-NP combinations control HIV-1 transmission more efficiently than free-drug combinations. These studies provide a rationale to better understand the role of nanocarrier systems in facilitating multidrug effects in relevant cells and tissues associated with HIV infection.

In pursuit of functional electrospun materials for clinical applications in humans

Electrospinning is a simple, low-cost and versatile approach to fabricate multifunctional materials useful in drug delivery and tissue engineering applications. Despite its emergence into other manufacturing sectors, electrospinning has not yet made a transformative impact in the clinic with a pharmaceutical product for use in humans. Why is this the current state of electrospun materials in biomedicine? Is it because electrospun materials are not yet capable of overcoming the biological safety and efficacy challenges needed in pharmaceutical products? Or, is it that technological advances in the electrospinning process are needed? This review investigates the current state of electrospun materials in medicine to identify both scientific and technological gaps that may limit clinical translation.