Injectable Glycosaminoglycan-Based Cryogels from Well-Defined Microscale Templates for Local Growth Factor Delivery.

Glycosaminoglycan-based hydrogels hold great potential for applications in tissue engineering and regenerative medicine. By mimicking the natural extracellular matrix processes of growth factor binding and release, such hydrogels can be used as a sustained delivery device for growth factors. Since neural networks commonly follow well-defined, high-aspect-ratio paths through the central and peripheral nervous system, we sought to create a fiber-like, elongated growth factor delivery system. Cryogels, with networks formed at subzero temperatures, are well-suited for the creation of high-aspect-ratio biomaterials, because they have a macroporous structure making them mechanically robust (for ease of handling) yet soft and highly compressible (for interfacing with brain tissue). Unlike hydrogels, cryogels can be synthesized in advance of their use, stored with ease, and rehydrated quickly to their original shape. Herein, we use solvent-assisted microcontact molding to form sacrificial templates, in which we produced highly porous cryogel microscale scaffolds with a well-defined elongated shape via the photopolymerization of poly(ethylene glycol) diacrylate and maleimide-functionalized heparin. Dissolution of the template yielded cryogels that could load nerve growth factor (NGF) and release it over a period of 2 weeks, causing neurite outgrowth in PC12 cell cultures. This microscale template-assisted synthesis technique allows tight control over the cryogel scaffold dimensions for high reproducibility and ease of injection through fine gauge needles.

Focal drug administration via heparin-containing cryogel microcarriers reduces cancer growth and metastasis.

Developing drug delivery systems that release anticancer drugs in a controlled and sustained manner remains challenging. We hypothesized that highly sulfated heparin-based microcarriers would allow electrostatic drug binding and controlled release. In silico modelling showed that the anticancer drug doxorubicin has affinity for the heparin component of the microcarriers. Experimental results showed that the strong electrostatic interaction was reversible, allowing both doxorubicin loading and a subsequent slow release over 42 days without an initial burst release. The drug-loaded microcarriers were able to reduce cancer cell viability in vitro in both hormone-dependent and highly aggressive triple-negative human breast cancer cells. Focal drug treatment, of an in vivo orthotopic triple-negative breast cancer model significantly decreased tumor burden and reduced cancer metastasis, whereas systemic administration of an equivalent drug dose was ineffective. This study proves that heparin-based microcarriers can be used as drug delivery platforms, for focal delivery and sustained long-term drug release.

Static and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers.

Neural precursor cells have been much studied to further our understanding of the far-reaching and controversial question of adult neurogenesis. Currently, differentiation of primary neural precursor cells from the mouse dentate gyrus via 2-dimentional in vitro culture yields low numbers of neurons, a major hindrance to the field of study. 3-dimentional "neurosphere" culture allows better 3D cell-cell contact, but control over cell differentiation is poor because nutrition and oxygen restrictions at the core of the sphere causes spontaneous differentiation, predominantly to glial cells, not neurons. Our group has developed macroporous scaffolds, which overcome the above-mentioned problems, allowing long-term culture of neural stem cells, which can be differentiated into a much higher yield of neurons. Herein we describe a method for culturing neural precursor cells on RGD peptide functionalized-heparin containing cryogel scaffolds, either in standard non-adherent well-plates (static culture) or in spinner flasks (dynamic culture). This method includes: •The synthesis and characterization of heparin based microcarriers.•A "static" 3D culture method for that does not require spinner flask equipment.•"Dynamic" culture in which cell loaded microcarriers are transferred to a spinner flask.

Macroporous heparin-based microcarriers allow long-term 3D culture and differentiation of neural precursor cells.

Adult neurogenesis and the neurogenic niche in the dentate gyrus are subjects of much research interest. Enhancing our knowledge of this niche process and the role played by this unique microenvironment would further our understanding of plasticity and its relevance for cognition in health and disease. The complex three-dimensional (3D) nature of the niche microenvironment is poorly recapitulated in current cell culture experimental procedures. Neural precursor cells (NPCs) are cultured either on two-dimensional (2D) surfaces, where cells quickly reach confluency and passaging is required, or as 3D neurospheres, with the limitation of poor diffusion of nutrients and thus partial differentiation of cells over time. Herein, we culture NPCs on microscale scaffolds termed microcarriers, composed of poly(ethylene glycol) and heparin, designed to more closely represent the 3D environment of the neurogenic niche. The interconnected macroporous structure of the microcarriers allows NPCs to attach to their pore walls with subsequent continuous proliferation (analyzed up to 28 days) without formation of a necrotic core. Removal of basic fibroblast growth factor and epidermal growth factor from the culture medium results in differentiation of the NPCs. Unlike 2D culture, a high percentage of neurons was achieved on the microcarriers (22% MAP2 positive cells) indicating that these 3D microscale scaffolds give a more conducive environment for neuronal differentiation. Microcarrier culture of NPCs allows long-term cell expansion and better differentiation, which provides superior culture conditions for studying/modelling the neurogenic niche.

Oxygen producing microscale spheres affect cell survival in conditions of oxygen-glucose deprivation in a cell specific manner: implications for cell transplantation.

This study outlines the synthesis of microscale oxygen producing spheres, which, when used in conjunction with catalase, can raise the dissolved oxygen content of cell culture media for 16-20 hours. In conditions of oxygen and glucose deprivation, designed to mimic the graft environment in vivo, the spheres rescue SH-SY5Y cells and meschymal stem cells, showing that oxygen producing biomaterials may hold potential to improve the survival of cells post-transplantation.

Oxygen-Producing Gellan Gum Hydrogels for Dual Delivery of Either Oxygen or Peroxide with Doxorubicin.

Hypoxic environments in the core of tumors can give rise to resistance against anticancer therapeutics. Oxygen-producing biomaterials may be able to improve chemotherapeutic efficiency by locally disrupting the hypoxic environment. We hypothesized that gellan gum hydrogels could be loaded with both a solid peroxide and the chemotherapeutic drug doxorubicin, to release both oxygen and doxorubicin simultaneously. We show that calcium peroxide physically cross-links gellan gum into a hydrogel, which when loaded with catalase raises the dissolved oxygen content of media for up to 64 h. Additionally, doxorubicin could be loaded into the hydrogel in situ, allowing release in well-defined quantities.

Tackling Cell Transplantation Anoikis: An Injectable, Shape Memory Cryogel Microcarrier Platform Material for Stem Cell and Neuronal Cell Growth.

Highly macroporous semisynthetic cryogel microcarriers can be synthesized for culturing stem cells and neuronal type cells. Growth factors loaded to heparin-containing microcarriers show near zero-order release kinetics and cell-loaded microcarriers can be injected through a fine gauge cannula without negative effect on the cells. These carriers can be applied for cell transplantation applications.