Functional microvascularization of human myocardium in vitro.

In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research.

Polysaccharide-Polyplex Nanofilm Coatings Enhance Nanoneedle-Based Gene Delivery and Transfection Efficiency.

Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.

Assessing the impact of silicon nanowires on bacterial transformation and viability of Escherichia coli.

We investigated the biomaterial interface between the bacteria Escherichia coli DH5α and silicon nanowire patterned surfaces. We optimised the engineering of silicon nanowire coated surfaces using metal-assisted chemical etching. Using a combination of focussed ion beam scanning electron microscopy, and cell viability and transformation assays, we found that with increasing interfacing force, cell viability decreases, as a result of increasing cell rupture. However, despite this aggressive interfacing regime, a proportion of the bacterial cell population remains viable. We found that the silicon nanowires neither resulted in complete loss of cell viability nor partial membrane disruption and corresponding DNA plasmid transformation. Critically, assay choice was observed to be important, as a reduction-based metabolic reagent was found to yield false-positive results on the silicon nanowire substrate. We discuss the implications of these results for the future design and assessment of bacteria-nanostructure interfacing experiments.

Nanoceria provides antioxidant and osteogenic properties to mesoporous silica nanoparticles for osteoporosis treatment.

Osteoporosis, a chronic metabolic bone disease, is the most common cause of fractures. Drugs for treating osteoporosis generally inhibit osteoclast (OC) activity, but are rarely aimed at encouraging new bone growth and often cause severe systemic side effects. Reactive oxygen species (ROS) are one of the key triggers of osteoporosis, by inducing osteoblast (OB) and osteocyte apoptosis and promoting osteoclastogenesis. Here we tested the capability of the ROS-scavenger nanoceria encapsulated within mesoporous silica nanoparticles (Ce@MSNs) to treat osteoporosis using a pre-osteoblast MC3T3-E1 cell monoculture in stressed and normal conditions. Ce@MSNs (diameter of 80 ± 10 nm) were synthesised following a scalable two-step process involving sol-gel and wet impregnation methods. The Ce@MSNs at concentration of 100 µg mL-1 induced a significant reduction in oxidative stress produced by t-butyl hydroperoxide and did not alter cell viability significantly. Confocal microscopy showed that MSNs and Ce@MsNs were internalised into the cytoplasm of the pre-osteoblasts after 24 h but were not in the nucleus, avoiding any DNA and RNA modifications. Ce@MSNs provoked mineralisation of the pre-osteoablasts without osteogenic supplements, which did not occur when the cells were exposed to MSN without nanoceria. In a co-culture system of MC3T3-E1 and RAW264.7 macrophages, the Ce@MSNs exhibited antioxidant capability and stimulated cell proliferation and osteogenic responses without adding osteogenic supplements to the culture. The work brings forward an effective platform based for facile synthesis of Ce@MSNs to interact with both OBs and OCs for treatment of osteoporosis.

Distinct macrophage populations and phenotypes associated with IL-4 mediated immunomodulation at the host implant interface.

The host macrophage response to implants has shown to be affected by tissue location and physio-pathological conditions of the patient. Success in immunomodulatory strategies is thus predicated on the proper understanding of the macrophage populations participating on each one of these contexts. The present study uses an in vivo implantation model to analyze how immunomodulation via an IL-4 eluting implant affects distinct macrophage populations at the tissue-implant interface and how this may affect downstream regenerative processes. Populations identified as F4/80+, CD68+ and CD11b+ macrophages at the peri-implant space showed distinct susceptibility to polarize towards an M2-like phenotype under the effects of delivered IL-4. Also, the presence of the coating resulted in a significant reduction in F4/80+ macrophages, while other populations remained unchanged. These results suggests that the F4/80+ macrophage population may be predominant in the early stages of the host response at the surface of these implants, in contrast to CD11b+ macrophage populations which were either fewer in number or located more distant from the implant surface. Gene expression assays showed increased proteolytic activity and diminished matrix deposition as possible mechanisms explaining the decreased fibrotic capsule deposition and improved peri-implant tissue quality shown in previous studies using IL-4 eluting coatings. The pattern of M2-like gene expression promoted by IL-4 was correlated with glycosaminoglycan production within the site of implantation at early stages of the host response, suggesting a significant role in this response. These findings demonstrate that immunomodulatory strategies can be utilized to design and implement targeted delivery for improving biomaterial performance.

Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks.

A major challenge in three-dimensional (3D) bioprinting is the limited number of bioinks that fulfill the physicochemical requirements of printing while also providing a desirable environment for encapsulated cells. Here, we address this limitation by temporarily stabilizing bioinks with a complementary thermo-reversible gelatin network. This strategy enables the effective printing of biomaterials that would typically not meet printing requirements, with instrument parameters and structural output largely independent of the base biomaterial. This approach is demonstrated across a library of photocrosslinkable bioinks derived from natural and synthetic polymers, including gelatin, hyaluronic acid, chondroitin sulfate, dextran, alginate, chitosan, heparin, and poly(ethylene glycol). A range of complex and heterogeneous structures are printed, including soft hydrogel constructs supporting the 3D culture of astrocytes. This highly generalizable methodology expands the palette of available bioinks, allowing the biofabrication of constructs optimized to meet the biological requirements of cell culture and tissue engineering.

Glycosaminoglycan-based biomaterials for growth factor and cytokine delivery: Making the right choices.

Controlled, localized drug delivery is a long-standing goal of medical research, realization of which could reduce the harmful side-effects of drugs and allow more effective treatment of wounds, cancers, organ damage and other diseases. This is particularly the case for protein "drugs" and other therapeutic biological cargoes, which can be challenging to deliver effectively by conventional systemic administration. However, developing biocompatible materials that can sequester large quantities of protein and release them in a sustained and controlled manner has proven challenging. Glycosaminoglycans (GAGs) represent a promising class of bio-derived materials that possess these key properties and can additionally potentially enhance the biological effects of the delivered protein. They are a diverse group of linear polysaccharides with varied functionalities and suitabilities for different cargoes. However, most investigations so far have focused on a relatively small subset of GAGs - particularly heparin, a readily available, promiscuously-binding GAG. There is emerging evidence that for many applications other GAGs are in fact more suitable for regulated and sustained delivery. In this review, we aim to illuminate the beneficial properties of various GAGs with reference to specific protein cargoes, and to provide guidelines for informed choice of GAGs for therapeutic applications.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals.

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

Surface modification of polypropylene for enhanced layer-by-layer deposition of polyelectrolytes.

We have performed three distinct plasma enhanced chemical vapor deposition procedures that can be widely and consistently used in commercially available plasma systems to modify the surface of hydrocarbon-based biomaterials such as polypropylene. In particular, we have evaluated the feasibility of these procedures to provide consistent and stable charged substrates to perform layer-by-layer (LbL) coatings. Surface characterization of both plasma and LbL coatings were done using X-ray photoelectron spectroscopy, attenuated total reflection-Fourier transform infrared spectroscopy, contact angle measurements and surface staining. Results showed successful surface grafting of functional groups in all plasma procedures that led to increased hydrophilicity and uniform LbL coatings with different efficiencies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2078-2085, 2018.

Effects of aging upon the host response to implants.

Macrophage polarization during the host response is now a well-accepted predictor of outcomes following material implantation. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that host responses to materials in aged individuals should differ from those in younger individuals. However, few studies examining the effects of aging upon the host response have been performed. The present work sought to elucidate the impacts of aging upon the host response to polypropylene mesh implanted into 8-week-old and 18-month-old mice. The results showed that there are significant differences in macrophage surface marker expression, migration, and polarization during the early host macrophage response and delayed resolution of the host response in 18-month-old versus 8-week-old mice. These differences could not be attributed to cell-intrinsic defects alone, suggesting that the host macrophage response to implants is likely also dictated to a significant degree by the local tissue microenvironment. These results raise important questions about the design and testing of materials and devices often intended to treat aged individuals and suggest that an improved understanding of patient- and context-dependent macrophage responses has the potential to improve outcomes in aged individuals. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1281-1292, 2017.

Shifts in macrophage phenotype at the biomaterial interface via IL-4 eluting coatings are associated with improved implant integration.

The present study tests the hypothesis that transient, early-stage shifts in macrophage polarization at the tissue-implant interface from a pro-inflammatory (M1) to an anti-inflammatory/regulatory (M2) phenotype mitigates the host inflammatory reaction against a non-degradable polypropylene mesh material and improves implant integration downstream. To address this hypothesis, a nanometer-thickness coating capable of releasing IL-4 (an M2 polarizing cytokine) from an implant surface at early stages of the host response has been developed. Results of XPS, ATR-FTIR and Alcian blue staining confirmed the presence of a uniform, conformal coating consisting of chitosan and dermatan sulfate. Immunolabeling showed uniform loading of IL-4 throughout the surface of the implant. ELISA assays revealed that the amount and release time of IL-4 from coated implants were tunable based upon the number of coating bilayers and that release followed a power law dependence profile. In-vitro macrophage culture assays showed that implants coated with IL-4 promoted polarization to an M2 phenotype, demonstrating maintenance of IL-4 bioactivity following processing and sterilization. Finally, in-vivo studies showed that mice with IL-4 coated implants had increased percentages of M2 macrophages and decreased percentages of M1 macrophages at the tissue-implant interface during early stages of the host response. These changes were correlated with diminished formation of fibrotic capsule surrounding the implant and improved tissue integration downstream. The results of this study demonstrate a versatile cytokine delivery system for shifting early-stage macrophage polarization at the tissue-implant interface of a non-degradable material and suggest that modulation of the innate immune reaction at early stages of the host response may represent a preferred strategy for promoting biomaterial integration and success.