Melissa J. Mahoney

Intrathecal polymer-based interleukin-10 gene delivery for neuropathic pain

Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy.

Development of porous PEG hydrogels that enable efficient, uniform cell-seeding and permit early neural process extension.

Three-dimensional polymer scaffolds are useful culture systems for neural cell growth and can provide permissive substrates that support neural processes as they extend across lesions in the brain and spinal cord. Degradable poly(ethylene) glycol (PEG) gels have been identified as a particularly promising scaffold material for this purpose; however, process extension within PEG gels is limited to late stages of hydrogel degradation. Here we demonstrate that earlier process extension can be achieved from primary neural cells encapsulated within PEG gels by creating a network of interconnected pores throughout the gel. Our method of incorporating these pores involves co-encapsulating a cell solution and a fibrin network within a PEG gel. The fibrin is subsequently enzymatically degraded under cytocompatible conditions, leaving behind a network of interconnected pores within the PEG gel. The primary neural cell population encapsulated in the gel is of mixed composition, containing differentiated neurons, and multipotent neuronal and glial precursor cells. We demonstrate that the initial presence of fibrin does not influence the cell-fate decisions of the encapsulated precursor cells. We also demonstrate that this fabrication approach enables simple, efficient and uniform seeding of viable cells throughout the entire porous scaffold.

Biocompatibility of poly(ethylene glycol)‐based hydrogels in the brain: An analysis of the glial response across space and time

Poly(ethylene glycol) or PEG-based hydrogels provide a useful methodology for tissue engineering and the controlled-release of drugs within the central nervous system (CNS). To be successful, the local neuroinflammatory response to an implant must be well understood. Toward this end, the focus was to examine the localized recruitment and activation of microglia and astrocytes following implantation of PEG-based hydrogels in the brain. Because they are of clinical relevance and may impact brain tissue differently, hydrogels with different mass loss profiles were examined. At all time points, a needle penetration in sham animals evoked a greater astrocytic response than hydrogel conditions. The astrocyte response that ensued varied with degradation rate. An attenuated response was present in more slowly degrading and nondegrading conditions. Relative to sham, hydrogel conditions attenuated the acute microglial response during the week after implant. By 56 days, microglial levels in shams decreased below the observed response in slowly degrading and nondegradable gels, which remained constant overtime. Although the inflammatory response to PEG-based hydrogels was complex depending on degradation rates, the magnitude of the acute microglia response and the long-term astrocyte response were attenuated suggesting the use of these materials for drug and cell delivery to the CNS.

Environmental cues to guide stem cell fate decision for tissue engineering applications

The human body contains a variety of stem cells capable of both repeated self-renewal and production of specialised, differentiated progeny. Critical to the implementation of these cells in tissue engineering strategies is a thorough understanding of which external signals in the stem cell microenvironment provide cues to control their fate decision in terms of proliferation or differentiation into a desired, specific phenotype. These signals must then be incorporated into tissue regeneration approaches for regulated exposure to stem cells. The precise spatial and temporal presentation of factors directing stem cell behaviour is extremely important during embryogenesis, development and natural healing events, and it is possible that this level of control will be vital to the success of many regenerative therapies. This review covers existing tissue engineering approaches to guide the differentiation of three disparate stem cell populations: mesenchymal, neural and endothelial. These progenitor cells will be of central importance in many future connective, neural and vascular tissue regeneration technologies.

Biocompatibility of PEG-based hydrogels in primate brain.

Degradable polymers have been used successfully in a wide variety of peripheral applications from tissue regeneration to drug delivery. These polymers induce little inflammatory response and appear to be well accepted by the host environment. Their use in the brain, for neural tissue reconstruction or drug delivery, also could be advantageous in treating neurodegenerative disorders. Because the brain has a unique immune response, a polymer that is compatible in the body may not be so in the brain. In the present study, polyethylene glycol (PEG)-based hydrogels were implanted into the striatum and cerebral cortex of nonhuman primates. Four months after implantation, brains were processed to evaluate the extent of astrogliosis and scaring, the presence of microglia/macrophages, and the extent of T-cell infiltration. Hydrogels with 20% w/v PEG implanted into the brain stimulated a slight increase in astrocytic and microglial/macrophage presence, as indicated by a small increase in glial fibrillary acidic protein (GFAP) and CD68 staining intensity. This increase was not substantially different from that found in the sham-implanted hemispheres of the brain. Staining for CD3+ T cells indicated no presence of peripheral T-cell infiltration. No gliotic scarring was seen in any implanted hemisphere. The combination of low density of GFAP-positive cells and CD68-positive cells, the absence of T cells, and the lack of gliotic scarring suggest that this level of immune response is not indicative of immunorejection and that the PEG-based hydrogel has potential to be used in the primate brain for local drug delivery or neural tissue regeneration.

Effect of macromer weight percent on neural cell growth in 2D and 3D nondegradable PEG hydrogel culture

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.

Impact of lactic acid on cell proliferation and free radical-induced cell death in monolayer cultures of neural precursor cells

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic‐acid‐based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices. Biotechnol. Bioeng. 2009;103: 1214–1223.

The administration of BDNF and GDNF to the brain via PLGA microparticles patterned within a degradable PEG-based hydrogel: Protein distribution and the glial response

Tailored delivery of neurotrophic factors (NFs) is a critical challenge that continues to inhibit strategies for guidance of axonal growth in vivo. Of particular importance is the ability to recreate innervation of distant brain regions by transplant tissue, for instance rebuilding the nigrostriatal track, one focus in Parkinsons disease research. Many strategies have utilized polymer drug delivery to target NF release in space and time, but combinatorial approaches are needed to deliver multiple NFs at relevant therapeutic times and locations without toxic side effects. Here we engineered a paradigm of PLGA microparticles entrapped within a degradable PEG-based hydrogel device to locally release two different types of NFs with two different release profiles. Hydrogel/microparticle devices were developed and analyzed for their ability to release GDNF in the caudal area of the brain, near the substantia nigra, or BDNF in the rostral area, near the striatum. The devices delivered their respective NFs in a region localized to within 100 μm of the bridge, but not exclusively to the targeted rostral or caudal ends. BDNF was slowly released over a 56-day period, whereas a bolus of GDNF was released around 28 days. The timed delivery of NFs from implanted devices significantly reduced the microglial response relative to sham surgeries. Given the coordinated drug delivery ability and reduced localized inflammatory response, this multifaceted PEG hydrogel/PLGA microparticle strategy may be a useful tool for further development in combining tissue engineering and drug delivery, and recreating the nigrostriatal track.

Spinal Interleukin‐10 Therapy to Treat Peripheral Neuropathic Pain

Introduction:  Current research indicates that chronic peripheral neuropathic pain includes a role for glia and the actions of proinflammatory factors. This review briefly discusses the glial and cytokine responses that occur following peripheral nerve damage in support of utilizing anti‐inflammatory cytokine interleukin‐10 (IL‐10) therapy to suppress chronic peripheral neuropathic pain.

Long-term control of neuropathic pain in a non-viral gene therapy paradigm.

Traditional approaches to treating chronic neuropathic pain largely focus on manipulations directly altering neuronal activity or neuron-to-neuron communication. Recently, however, it has become clear that glial cells (including microglia and astroglia) play a significant role in pain expression in a variety of neuropathic pain models. Multiple aspects of the inflammatory response of glial cells, commonly observed in neuropathic pain conditions, have been implicated in pain expression. Thus, glial cell inflammation has emerged as a potential therapeutic target in neuropathic pain. Our laboratory has been exploring the use of an anti-inflammatory cytokine, interleukin-10 (IL-10), to control glial inflammatory activation thereby controlling neuropathic pain. IL-10 protein delivery is limited by a short half-life and an inability to cross into the central nervous system from the periphery, making a centrally delivered gene therapy approach attractive. We have recently characterized a non-viral gene therapy approach using two injections of naked DNA to achieve long-term (>3 months) control of neuropathic pain in a peripheral nerve injury model. Timing and dose requirements leading to long-term pain control are discussed in this review, as is recent work using microparticle-encapsulated DNA to achieve long-term therapeutic efficacy with a single injection.