Justin L. Brown

Polymeric nanoparticles: the future of nanomedicine

Polymeric nanoparticles (NPs) are one of the most studied organic strategies for nanomedicine. Intense interest lies in the potential of polymeric NPs to revolutionize modern medicine. To determine the ideal nanosystem for more effective and distinctly targeted delivery of therapeutic applications, particle size, morphology, material choice, and processing techniques are all research areas of interest. Utilizations of polymeric NPs include drug delivery techniques such as conjugation and entrapment of drugs, prodrugs, stimuli-responsive systems, imaging modalities, and theranostics. Cancer, neurodegenerative disorders, and cardiovascular diseases are fields impacted by NP technologies that push scientific boundaries to the leading edge of transformative advances for nanomedicine.

Polyphosphazene/Nano-Hydroxyapatite Composite Microsphere Scaffolds for Bone Tissue Engineering

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.

Biomimetic, bioactive etheric polyphosphazene-poly(lactide-co-glycolide) blends for bone tissue engineering.

The long-term goal of this work is to develop biomimetic polymer-based systems for bone regeneration that both allow for neutral pH degradation products and have the ability to nucleate bonelike apatite. In this study, the etheric biodegradable polyphosphazene, poly[(50%ethyl glycinato)(50%methoxyethoxyethoxy)phosphazene] (PNEG(50)MEEP(50)) was blended with poly(lactide-co-glycolide) PLAGA and studied their ability to produce high-strength degradable biomaterials with bioactivity. Accordingly, two blends with weight ratios of PNEG(50)MEEP(50) to PLAGA 25:75 (BLEND25) and 50:50 (BLEND50) were fabricated using a mutual solvent approach. Increases in PNEG(50)MEEP(50) content in the blend system resulted in decreased elastic modulus of 779 MPa when compared with 1684 MPa (PLAGA) as well as tensile strength 7.9 MPa when compared with 25.7 MPa (PLAGA). However, the higher PNEG(50)MEEP(50) content in the blend system resulted in higher Ca/P atomic ratio of the apatite layer 1.35 (BLEND50) when compared with 0.69 (BLEND25) indicating improved biomimicry. Furthermore, these blends supported primary rat osteoblast adhesion and proliferation with an enhanced phenotypic expression when compared with PLAGA. These findings establish the suitability of PNEG(50)MEEP(50)-PLAGA biodegradable blends as promising bioactive materials for orthopedic applications.

Substrate curvature sensing through Myosin IIa upregulates early osteogenesis

Topographical cues mimicking the extracellular matrix (ECM) have demonstrated control over a diverse range of cellular behaviours including: initial adhesion, migration, cell growth, differentiation and death. How cells sense, and in turn translate, the topographical cues remains to be answered, but likely involves interactions through interfacial forces that influence cytoskeletal structure and integrin clustering, leading to the downstream activity of intracellular signalling cascades. Electrospun fibers have shown significant success as a biomimetic topography for bone tissue engineering applications, but mechanisms by which osteoprogenitor cells translate the fiber geometry into intracellular signalling activity is only recently being examined. We hypothesized that increased cellular differentiation observed on fibrous topography is due to acto-myosin contractility and cellular stiffness via the small GTPase RhoA. In order to evaluate this hypothesis, MC3T3-E1 osteoprogenitor cells were grown on poly(methyl methacrylate) (PMMA) fibers of 1.153 ± 0.310 μm diameter. The elastic modulus of the cell surface was measured by atomic force microscopy (AFM) with a colloidal probe. Overall cellular stiffness was found to increase more than three-fold in osteoprogenitors adhered to a fiber, as opposed to those grown on a flat substrate. Pharmacological inhibition of RhoA signalling activity decreased cellular stiffness and cytoskeletal integrity of osteoprogenitors growing on fibrous substrates. Finally, we demonstrated not only RhoA activity through its effector Rho-associated coiled coil kinase II (ROCKII), but also Myosin IIa promotes early osteogenic differentiation, as shown by alkaline phosphatase (ALP) staining. Previous studies have demonstrated the importance of ROCKII on early differentiation. Our results shed light on mechanisms underlying geometry sensing by highlighting the role of Myosin IIa in addition to ROCKII and could ultimately contribute to scaffold design strategies.

Composite scaffolds: Bridging nanofiber and microsphere architectures to improve bioactivity of mechanically competent constructs

Tissue engineering often benefits from the use of composites to produce an ideal scaffold. We present the focused development of a novel structure that combines the biomimetic properties of nanofibers with the robust mechanical aspects of the sintered microsphere scaffold to produce a composite scaffold that demonstrates an ability to mimic the mechanical environment of trabecular bone while also promoting the phenotype progression of osteoblast progenitor cells. These composite nanofiber/microsphere scaffolds exhibited a mechanical modulus and compressive strength similar to trabecular bone and exhibited degradation resulting in a mass loss of 30% after 24 weeks. The nanofiber portion of these scaffolds was sufficiently porous to allow cell migration throughout the fibrous portion of the scaffold and promoted phenotype progression through focal adhesion kinase-mediated activation of the transcription factor Runx2, control scaffolds not containing nanofibers did not demonstrate extensive cell migration or phenotype progression. Ultimately, the focal adhesion kinase activity on the composite nanofiber/microsphere scaffolds demonstrated causality over the production of the mature osteoblast marker, osteocalcin, and the development of a calcified matrix.

Synthesis and characterization of biomimetic citrate‐based biodegradable composites

Natural bone apatite crystals, which mediate the development and regulate the load-bearing function of bone, have recently been associated with strongly bound citrate molecules. However, such understanding has not been translated into bone biomaterial design and osteoblast cell culture. In this work, we have developed a new class of biodegradable, mechanically strong, and biocompatible citrate-based polymer blends (CBPBs), which offer enhanced hydroxyapatite binding to produce more biomimetic composites (CBPBHAs) for orthopedic applications. CBPBHAs consist of the newly developed osteoconductive citrate-presenting biodegradable polymers, crosslinked urethane-doped polyester and poly (octanediol citrate), which can be composited with up to 65 wt % hydroxyapatite. CBPBHA networks produced materials with a compressive strength of 116.23 ± 5.37 MPa comparable to human cortical bone (100-230 MPa), and increased C2C12 osterix gene and alkaline phosphatase gene expression in vitro. The promising results above prompted an investigation on the role of citrate supplementation in culture medium for osteoblast culture, which showed that exogenous citrate supplemented into media accelerated the in vitro phenotype progression of MG-63 osteoblasts. After 6 weeks of implantation in a rabbit lateral femoral condyle defect model, CBPBHA composites elicited minimal fibrous tissue encapsulation and were well integrated with the surrounding bone tissues. The development of citrate-presenting CBPBHA biomaterials and preliminary studies revealing the effects of free exogenous citrate on osteoblast culture shows the potential of citrate biomaterials to bridge the gap in orthopedic biomaterial design and osteoblast cell culture in that the role of citrate molecules has previously been overlooked.

Nanofiber diameter-dependent MAPK activity in osteoblasts.

The major challenge for bone tissue engineering lies in the fabrication of scaffolds that can mimic the extracellular matrix and promote osteogenesis. Electrospun fibers are being widely researched for this application due to high porosity, interconnectivity, and mechanical strength of the fibrous scaffolds. Electrospun poly methyl methacrylate (PMMA, 2.416 ± 0.100 μm) fibers were fabricated and etched using a 60% propylene glycol methyl ether acetate (PGMEA)/limonene (vol/vol) solution to obtain fiber diameters ranging from 2.5 to 0.5 μm in a time-dependent manner. The morphology of the fibrous scaffolds was evaluated using scanning electron microscopy and cellular compatibility with etchant-treated scaffold was assessed using immunoflurescence. Mitogen-activated protein kinases (MAPK) activation in response to different fiber diameter was evaluated with western blot as well as quantitative in-cell western. We report that electrospun micro-fibers can be etched to 0.552 ± 0.047 μm diameter without producing beads. Osteoblasts adhered to the fibers and a change in fiber diameter played a major role in modulating the activation of extracellular signal-regulated kinase (ERK) and p38 kinases with 0.882 ± 0.091 μm diameter fibers producing an inverse effect on ERK and p38 phosphorylation. These results indicate that nanofibers produced by wet etching can be effectively utilized to produce diameters that can differentially modulate MAPK activation patterns.

The role of substrate topography on the cellular uptake of nanoparticles.

Improving targeting efficacy has been a central focus of the studies on nanoparticle (NP)-based drug delivery nanocarriers over the past decades. As cells actively sense and respond to the local physical environments, not only the NP design (e.g., size, shape, ligand density, etc.) but also the cell mechanics (e.g., stiffness, spreading, expressed receptors, etc.) affect the cellular uptake efficiency. While much work has been done to elucidate the roles of NP design for cells seeded on a flat tissue culture surface, how the local physical environments of cells mediate uptake of NPs remains unexplored, despite the widely known effect of local physical environments on cellular responses in vitro and disease states in vivo. Here, we report the active responses of human osteosarcoma cells to fibrous substrate topographies and the subsequent changes in the cellular uptake of NPs. Our experiments demonstrate that surface topography modulates cellular uptake efficacy by mediating cell spreading and membrane mechanics. The findings provide a concrete example of the regulative role of the physical environments of cells on cellular uptake of NPs, therefore advancing the rational design of NPs for enhanced drug delivery in targeted cancer therapy.

3D Near‐Field Electrospinning of Biomaterial Microfibers with Potential for Blended Microfiber‐Cell‐Loaded Gel Composite Structures

This paper describes the development of a novel low-cost and efficient method, 3D near-field electrospinning, to fabricate high-resolution, and repeatable 3D polymeric fiber patterns on nonconductive materials with potential use in tissue engineering. This technology is based on readily available hobbyist grade 3D printers. The result is exquisite control of the deposition of single fibers in an automated manner. Additionally, the fabrication of various fiber patterns, which are subsequently translated to unique cellular patterns, is demonstrated. Finally, poly(methyl methacrylate) fibers are printed within 3D collagen gels loaded with cells to introduce anisotropic properties of polymeric fibers within the cell-loaded gels.

Avian and human influenza virus compatible sialic acid receptors in little brown bats

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.