Rachael A. Oldinski

Chitosan-based nanofibrous membranes for antibacterial filter applications

Nanofibrous membranes have drawn considerable interest for filtration applications due to their ability to withstand high fluid flux while removing micro- and nano-sized particulates from solution. The desire to introduce an antibacterial function into water filter applications presents a challenge to widespread application of fibrous membranes because the addition of chemicals or biocides may produce harmful byproducts downstream. Here, we report the development of chitosan-polycaprolactone (PCL) nanofibrous membranes to utilize the natural antibacterial property of chitosan for antibacterial water filtration. Chitosan-PCL fibers with diameters of 200-400 nm and chitosan contents of 25, 50 and 75 wt% were prepared by electrospinning. In a series of bacterial challenge tests, chitosan-PCL fibrous membranes significantly reduced Staphylococcus aureus adhesion compared to PCL fibrous membranes. In water permeability and particulate size removal tests, fibrous membranes with 25% chitosan supported the greatest water flux (∼7000 L/h/m(2)) with 100% removal of 300-nm particulates, while maintaining the membrane integrity. This study demonstrates the potential of chitosan-PCL nanofibrous membranes as pre-filters for water filtration systems that demonstrate combinatorial filtration and intrinsic antibacterial advantages.

Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration

Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell-extracellular matrix interactions. We have established effective methods for efficient de and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼ 1-3 cm(3)) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold.

Integrated bi-layered scaffold for osteochondral tissue engineering.

Osteochondral tissue engineering poses the challenge of combining both cartilage and bone tissue engineering fundamentals. In this study, a sphere-templating technique was applied to fabricate an integrated bi-layered scaffold based on degradable poly(hydroxyethyl methacrylate) hydrogel. One layer of the integrated scaffold was designed with a single defined, monodispersed pore size of 38 μm and pore surfaces coated with hydroxyapatite particles to promote regrowth of subchondral bone while the second layer had 200 μm pores with surfaces decorated with hyaluronan for articular cartilage regeneration. Mechanical properties of the construct as well as cyto-compatibility of the scaffold and its degradation products were elucidated. To examine the potential of the biphasic scaffold for regeneration of osteochondral tissue the designated cartilage and bone layers of the integrated bi-layered scaffold were seeded with chondrocytes differentiated from human mesenchymal stem cells and primary human mesenchymal stem cells, respectively. Both types of cells were co-cultured within the scaffold in standard medium without soluble growth/differentiation factors over four weeks. The ability of the integrated bi-layered scaffold to support simultaneous matrix deposition and adequate cell growth of two distinct cell lineages in each layer during four weeks of co-culture in vitro in the absence of soluble growth factors was demonstrated.

Osteogenic differentiation of human mesenchymal stem cells through alginate-graft-poly(ethylene glycol) microsphere-mediated intracellular growth factor delivery.

The intracellular delivery of growth factors increases opportunities for controlling cell behavior and maintaining tissue homeostasis. Recently, VEGFA was reported to enhance osteogenic differentiation of mesenchymal stem cells (MSCs) through an intracrine mechanism, suggesting a new strategy to promote bone tissue formation in osteoporotic patients. The goal of this study was to design and fabricate ligand-conjugated alginate-graft-poly(ethylene glycol) microspheres for intracellular delivery and release of VEGFA in primary human MSCs to enhance osteogenic differentiation as a potential therapeutic. Three types of microspheres were synthesized and characterized by scanning electron microscopy, in vitro drug release kinetics, MSC uptake and internalization: alginate alone (Alg), alginate-graft-poly(ethylene glycol) (Alg-g-PEG) and alginate-graft-poly(ethylene glycol)-S-S-arginine-glycine-aspartic acid (Alg-g-RGD). Each of the different microsphere formulations successfully transported bioactive VEGFA into primary human MSCs within 48h of culture, and significantly enhanced osteogenic differentiation compared to control treatments with empty microspheres (intracellular control) or non-encapsulated VEGFA (extracellular control). Adipogenic differentiation was not affected by the presence of VEGFA intracellularly or extracellularly. These results demonstrating the internalization of alginate-based microspheres and intracellular delivery of VEGFA support the efficacy of using this drug delivery and intracrine mechanism to control the fate of human MSCs and enhance osteogenic differentiation.

Self-Healing and Thermoresponsive Dual-Cross-Linked Alginate Hydrogels Based on Supramolecular Inclusion Complexes

β-Cyclodextrin (β-CD), with a lipophilic inner cavity and hydrophilic outer surface, interacts with a large variety of nonpolar guest molecules to form noncovalent inclusion complexes. Conjugation of β-CD onto biomacromolecules can form physically cross-linked hydrogel networks upon mixing with a guest molecule. Herein, the development and characterization of self-healing, thermoresponsive hydrogels, based on host-guest inclusion complexes between alginate-graft-β-CD and Pluronic F108 (poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol)), are described. The mechanics, flow characteristics, and thermal response were contingent on the polymer concentration and the host-guest molar ratio. Transient and reversible physical cross-linking between host and guest polymers governed self-assembly, allowing flow to occur under shear stress and facilitating complete recovery of the materials properties within a few seconds of unloading. The mechanical properties of the dual-cross-linked, multi-stimuli-responsive hydrogels were tuned as high as 30 kPa at body temperature and are advantageous for biomedical applications such as drug delivery and cell transplantation.

Visible light crosslinking of methacrylated hyaluronan hydrogels for injectable tissue repair

Tissue engineering hydrogels are primarily cured in situ using ultraviolet (UV) radiation which limits the use of hydrogels as drug or cell carriers. Visible green light activated crosslinking systems are presented as a safe alternative to UV photocrosslinked hydrogels, without compromising material properties such as viscosity and stiffness. The objective of this study was to fabricate and characterize photocrosslinked hydrogels with well-regulated gelation kinetics and mechanical properties for the repair or replacement of soft tissue. An anhydrous methacrylation of hyaluronan (HA) was performed to control the degree of modification (DOM) of HA, verified by (1) H-NMR spectroscopy. UV-activated crosslinking was compared to visible green light activated crosslinking. While the different photocrosslinking techniques resulted in varied crosslinking times, comparable mechanical properties of UV and green light activated crosslinked hydrogels were achieved using each photocrosslinking method by adjusting time of light exposure. Methacrylated HA (HA-MA) hydrogels of varying molecular weight, DOM, and concentration exhibited compressive moduli ranging from 1 kPa to 116 kPa, for UV crosslinking, and 3 kPa to 146 kPa, for green light crosslinking. HA-MA molecular weight and concentration were found to significantly influence moduli values. HA-MA hydrogels did not exhibit any significant cytotoxic effects toward human mesenchymal stem cells. Green light activated crosslinking systems are presented as a viable method to form natural-based hydrogels in situ.

Physically crosslinked polyvinyl alcohol and gelatin interpenetrating polymer network theta-gels for cartilage regeneration

Theta-gels are hydrogels that form during the solidification and phase separation of two dislike polymers, in which a low molecular weight polymer behaves as a porogen and is removed through dialysis. For this study, interpenetrating polymer network (IPN) hydrogels were formed between polyvinyl alcohol (PVA) and gelatin using theta-gel fabrication techniques, i.e., in the presence of a porogen. The addition of gelatin to a PVA theta-gel, formed with a porogen, polyethylene glycol (PEG), created macro-porous hydrogels, and increased shear storage moduli and elastic moduli, compared to PVA-gelatin scaffold controls. A reduction in PVA crystallinity was verified by Fourier transform infrared (FTIR) spectroscopy in hydrogels fabricated using a porogen, i.e., PVA-PEG-gelatin, compared to PVA, PVA-PEG, or PVA-gelatin hydrogels alone. Van Geison staining confirmed the retention of gelatin after dialysis. A range of hydrogel moduli was achieved by optimizing PVA concentration, molecular weight, and gelatin concentration. PVA-gelatin hydrogels maintained primary human mesenchymal stem cell (MSC) viability. Soft (∼10 kPa) and stiff (∼100 kPa) PVA-gelatin hydrogels containing type II collagen significantly increased glycosaminoglycan (GAG) production compared to controls. PVA-gelatin hydrogels, formed using theta-gel techniques, warrant further investigation as articular cartilage tissue engineering scaffolds.

Dynamic mechanical analysis and biomineralization of hyaluronan–polyethylene copolymers for potential use in osteochondral defect repair

Treatment options for damaged articular cartilage are limited due to its lack of vasculature and its unique viscoelastic properties. This study was the first to fabricate a hyaluronan (HA)-polyethylene copolymer for potential use in the replacement of articular cartilage and repair of osteochondral defects. Amphiphilic graft copolymers consisting of HA and high-density polyethylene (HA-co-HDPE) were fabricated with 10, 28 and 50 wt.% HA. Dynamic mechanical analysis was used to assess the effect of varying constituent weight ratios on the viscoelastic properties of HA-co-HDPE materials. The storage moduli of HA-co-HDPE copolymers ranged from 2.4 to 15.0 MPa at physiological loading frequencies. The viscoelastic properties of the HA-co-HDPE materials were significantly affected by varying the wt.% of HA and/or crosslinking of the HA constituent. Cytotoxicity and the ability of the materials to support mineralization were evaluated in the presence of bone marrow stromal cells. HA-co-HDPE materials were non-cytotoxic, and calcium and phosphorus were present on the surface of the HA-co-HDPE materials 2 weeks after osteogenic differentiation of the bone marrow stromal cells. This study is the first to measure the viscoelastic properties and osseocompatibility of HA-co-HDPE for potential use in orthopedic applications.

Dual-Cross-Linked Methacrylated Alginate Sub-Microspheres for Intracellular Chemotherapeutic Delivery

Intracellular delivery vehicles comprised of methacrylated alginate (Alg-MA) were developed for the internalization and release of doxorubicin hydrochloride (DOX). Alg-MA was synthesized via an anhydrous reaction, and a mixture of Alg-MA and DOX was formed into sub-microspheres using a water/oil emulsion. Covalently cross-linked sub-microspheres were formed via exposure to green light, in order to investigate effects of cross-linking on drug release and cell internalization, compared to traditional techniques, such as ultraviolet (UV) light irradiation. Cross-linking was performed using light exposure alone or in combination with ionic cross-linking using calcium chloride (CaCl2). Alg-MA sub-microsphere diameters were between 88 and 617 nm, and ζ-potentials were between -20 and -37 mV. Using human lung epithelial carcinoma cells (A549) as a model, cellular internalization was confirmed using flow cytometry; different sub-microsphere formulations varied the efficiency of internalization, with UV-cross-linked sub-microspheres achieving the highest internalization percentages. While blank (nonloaded) Alg-MA submicrospheres were noncytotoxic to A549 cells, DOX-loaded sub-microspheres significantly reduced mitochondrial activity after 5 days of culture. Photo-cross-linked Alg-MA sub-microspheres may be a potential chemotherapeutic delivery system for cancer treatment.

Synthesis and characterization of a hyaluronan-polyethylene copolymer for biomedical applications

Hyaluronan (HA)-based biomaterials are of interest for bone and cartilage tissue engineering because HA plays an important role in orthopedic tissue development, function, and repair. The goal of this project was to develop a biomaterial that incorporated the constituents of both a hydrogel and a hydrophobic polymer for biomedical applications. A series of amphiphilic graft copolymers consisting of HA, a glycosaminoglycan, and high-density polyethylene (HDPE), that is, HA-co-HDPE, were fabricated. The chemical characteristics, physical and viscoelastic properties, and cytocompatibility of novel HA-co-HDPE materials were characterized via Fourier Transform infrared (FTIR) spectroscopy, solid state nuclear magnetic resonance (ssNMR) spectroscopy, differential scanning calorimetry (DSC), dynamic shear testing, and an in vitro human osteoblast cell study. The esterification reaction between HA and functionalized HDPE resulted in semicrystalline, insoluble powder. The dynamic shear properties of HA-co-HDPE concentrated solutions were more like natural proteoglycans than the HA control. HA-co-HDPE was successfully compression molded into disks that swelled upon hydration. Osteoblasts were viable and expressed the osteoblast phenotype after 7 days of culture on HA-co-HDPE materials. These HA-co-HDPE materials may have several biomaterial applications in saline suspension or molded form, including orthopedic tissue repair.