Melissa J. Mahoney

Poly(ethylene glycol) (PEG) hydrogels are being developed as cell delivery vehicles that have great potential to improve neuronal replacement therapies. Current research priorities include (1) characterizing neural cell growth within PEG hydrogels relative to standard culture systems and (2) generating neuronal-enriched populations within the PEG hydrogel environment. This study compares the percentage of neural precursor cells (NPCs), neurons, and glia present when dissociated neural cells are seeded within PEG hydrogels relative to standard monolayer culture. Results demonstrate that PEG hydrogels enriched the initial cell population for NPCs, which subsequently gave rise to neurons, then to glia. Relative to monolayer culture, PEG hydrogels maintained an increased percentage of NPCs and a decreased percentage of glia. This neurogenic advantage of PEG hydrogels is accentuated in the presence of basic fibroblast growth factor and epidermal growth factor, which more potently increase NPC and neuronal expression markers when applied to cells cultured within PEG hydrogels. Finally, this work demonstrates that glial differentiation can be selectively eliminated upon supplementation with a γ-secretase inhibitor. Together, this study furthers our understanding of how the PEG hydrogel environment influences neural cell composition and also describes select soluble factors that are useful in generating neuronal-enriched populations within the PEG hydrogel environment.

Developing a mechanism to vascularize tissue‐engineered constructs is imperative for transplant function and integration, particularly when delivering hypoxia‐sensitive tissues, such as pancreatic islets. Previous efforts have focused on bulk modifications of scaffold materials rendering the entire construct permissive to vessel penetration or the formation of a porous structure where vessels can infiltrate the empty spaces. Here, we describe a novel construct composed of large fibrin ribbons encapsulated within a poly(ethylene glycol) (PEG) hydrogel. The PEG/fibrin ribbon composite scaffold facilitates coculture of adhesive and nonadhesive cell types, thus providing closely neighboring environments with distinct material properties specific to the needs of two clinically relevant cell populations. This advantage is demonstrated here by the successful coculture of pancreatic islets in the PEG component and vessel‐forming endothelial cells in entrapped fibrin ribbons. Transplanted endothelial cells can form anastomosies with host vasculature, suggesting that our cocultures may lead to more rapid scaffold vascularization. Additionally, we show that surface‐seeded endothelial cells form multicellular projections that migrate into nonadhesive PEG hydrogels along permissive fibrin ribbons, further demonstrating composite construct vascularization potential. Distribution of large fibrin ribbons throughout PEG hydrogels provide a potential mechanism for vascularization of a well‐established biomaterial without inherently changing its desirable properties. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

Neural precursor cells (NPCs) are a renewable cell source that may be useful for neural cell transplant therapies. Their expansion and differentiation potential have traditionally been explored by culturing them on stiff tissue culture polystyrene. Here we describe advantages of an alternative culture system: bio‐inert poly(ethylene glycol) (PEG) hydrogels. Specifically this work reports the effect that macromer weight percent has on the metabolic and apoptotic activity, proliferation, and cellular composition of a mixed population of neurons and multipotent NPCs grown both on 2D and within 3D PEG hydrogels. In 2D culture, hydrogel properties did not affect metabolic or apoptotic activity but did impact cell proliferation and composition leading to an increase in glial cell reactivity as stiffness increased. In 3D culture, low weight percent hydrogels led to greater metabolic activity and lower apoptotic activity with significant proliferation observed only in hydrogels that closely matched the stiffness of native brain tissue. PEG hydrogels therefore provide a versatile in vitro culture system that can be used to culture and expand a variety of neural and glial cell types simply by altering the material properties of the hydrogel. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

We assessed the ability of a gamma-secretase inhibitor to promote the in vitro differentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.

Hydrogels that degrade at different rates were prepared by copolymerizing slowly degrading macromer poly(ethylene glycol) (PEG) dimethacrylate with a faster degrading macromer poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate. A clinically relevant population of neural cells composed of differentiated neurons and multipotent precursor cells was cultured within hydrogels. Within 2 h after encapsulation, metabolic activity was higher in hydrogels prepared with increasing levels of degradable content. This improvement was accompanied by a reduction in intracellular redox state and an increase in the fraction of glutathione in the reduced state, both of which persisted throughout 7 days of culture and which may be the result of radical scavenging by lactic acid. Importantly, an increase in cellular proliferation was observed in gels prepared with increasing degradable macromer content after 7 days of growth without a shift in the cellular composition of the culture toward the glial cell phenotype. The findings of this study provide additional insight into the growth of neural cells in PEG-based hydrogels. Results suggest that lactic acid released during gel degradation may impact the function of encapsulated cells, a finding of general interest to biomaterials scientists who focus on the development of degradable polymers for cell culture and drug delivery devices.

Purpose Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated. Methods PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. Results In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production. Conclusions These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

(2010). Central nervous system delivery of large molecules: challenges and new frontiers for intrathecally administered therapeutics. Expert Opinion on Drug Delivery: Vol. 7, No. 3, pp. 285-293.

Development of an alternative source of functional, transplantable β-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of β-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective β-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating β-cells and facilitate β-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive’ and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive β-cell population from dissociated precursor cells.

The anti‐inflammatory cytokine interleukin‐10 (IL‐10) shows promise for the treatment of neuropathic pain, but for IL‐10 to be clinically useful as a short‐term therapeutic its duration needs to be improved. In this study, IL‐10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL‐10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL‐10 products for subsequent in vivo testing. PEGylation of IL‐10 by acylation yielded a highly PEGylated product with a 35‐fold in vitro biological activity reduction. PEGylation of IL‐10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of ∼3‐fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL‐10 increased the concentration of IL‐10 in the cerebrospinal fluid over time. Relative to unmodified IL‐10, the 20 kDa PEG‐IL‐10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short‐term treatment or, in conjunction with other approaches, the long‐term treatment of enhanced pain states. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010