Reducing neuroinflammation by delivery of IL-10 encoding lentivirus from multiple-channel bridges.

The spinal cord is unable to regenerate after injury largely due to growth-inhibition by an inflammatory response to the injury that fails to resolve, resulting in secondary damage and cell death. An approach that prevents inhibition by attenuating the inflammatory response and promoting its resolution through the transition of macrophages to anti-inflammatory phenotypes is essential for the creation of a growth permissive microenvironment. Viral gene delivery to induce the expression of anti-inflammatory factors provides the potential to provide localized delivery to alter the host inflammatory response. Initially, we investigated the effect of the biomaterial and viral components of the delivery system to influence the extent of cell infiltration and the phenotype of these cells. Bridge implantation reduces antigen-presenting cell infiltration at day 7, and lentivirus addition to the bridge induces a transient increase in neutrophils in the spinal cord at day 7 and macrophages at day 14. Delivery of a lentivirus encoding IL-10, an anti-inflammatory factor that inhibits immune cell activation and polarizes the macrophage population towards anti-inflammatory phenotypes, reduced neutrophil infiltration at both day 7 and day 28. Though IL-10 lentivirus did not affect macrophages number, it skewed the macrophage population toward an anti-inflammatory M2 phenotype and altered macrophage morphology. Additionally, IL-10 delivery resulted in improved motor function, suggesting reduced secondary damage and increased sparing. Taken together, these results indicate that localized expression of anti-inflammatory factors, such as IL-10, can modulate the inflammatory response following spinal cord injury, and may be a key component of a combinatorial approach that targets the multiple barriers to regeneration and functional recovery.

In vivo capture and label-free detection of early metastatic cells.

Breast cancer is a leading cause of death for women, with mortality resulting from metastasis. Metastases are often detected once tumour cells affect the function of solid organs, with a high disease burden limiting effective treatment. Here we report a method for the early detection of metastasis using an implanted scaffold to recruit and capture metastatic cells in vivo, which achieves high cell densities and reduces the tumour burden within solid organs 10-fold. Recruitment is associated with infiltration of immune cells, which include Gr1(hi)CD11b(+) cells. We identify metastatic cells in the scaffold through a label-free detection system using inverse spectroscopic optical coherence tomography, which identifies changes to nanoscale tissue architecture associated with the presence of tumour cells. For patients at risk of recurrence, scaffold implantation following completion of primary therapy has the potential to identify metastatic disease at the earliest stage, enabling initiation of therapy while the disease burden is low.

Sonic hedgehog and neurotrophin-3 increase oligodendrocyte numbers and myelination after spinal cord injury.

Spinal cord injury (SCI) results in loss of sensory and motor function below the level of injury and has limited available therapies. Multiple channel bridges have been investigated as a means to create a permissive environment for regeneration, with channels supporting axonal growth through the injury. Bridges support robust axon growth and myelination. Here, we investigated the cell types that myelinate axons in the bridges and whether over-expression of trophic factors can enhance myelination. Lentivirus encoding for neurotrophin-3 (NT3), sonic hedgehog (SHH) and the combination of these factors was delivered from bridges implanted into a lateral hemisection defect at T9/T10 in mice, and the response of endogenous progenitor cells within the spinal cord was investigated. Relative to control, the localized, sustained expression of these factors significantly increased growth of regenerating axons into the bridge and enhanced axon myelination 8 weeks after injury. SHH decreased the number of Sox2(+) cells and increased the number of Olig2(+) cells, whereas NT3 alone or in combination with SHH enhanced the numbers of GFAP(+) and Olig2(+) cells relative to control. For delivery of lentivirus encoding for either factor, we identified cells at various stages of differentiation along the oligodendrocyte lineage (e.g., O4(+), GalC(+)). Expression of NT3 enhanced myelination primarily by infiltrating Schwann cells, whereas SHH over-expression substantially increased myelination by oligodendrocytes. These studies further establish biomaterial-mediated gene delivery as a promising tool to direct activation and differentiation of endogenous progenitor cells for applications in regenerative medicine.

Long-term characterization of axon regeneration and matrix changes using multiple channel bridges for spinal cord regeneration.

Spinal cord injury (SCI) results in loss of sensory and motor function below the level of injury and has limited available therapies. The host response to SCI is typified by limited endogenous repair, and biomaterial bridges offer the potential to alter the microenvironment to promote regeneration. Porous multiple channel bridges implanted into the injury provide stability to limit secondary damage and support cell infiltration that limits cavity formation. At the same time, the channels provide a path that physically directs axon growth across the injury. Using a rat spinal cord hemisection injury model, we investigated the dynamics of axon growth, myelination, and scar formation within and around the bridge in vivo for 6 months, at which time the bridge has fully degraded. Axons grew into and through the channels, and the density increased overtime, resulting in the greatest axon density at 6 months postimplantation, despite complete degradation of the bridge by that time point. Furthermore, the persistence of these axons contrasts with reports of axonal dieback in other models and is consistent with axon stability resulting from some degree of connectivity. Immunostaining of axons revealed both motor and sensory origins of the axons found in the channels of the bridge. Extensive myelination was observed throughout the bridge at 6 months, with centrally located and peripheral channels seemingly myelinated by oligodendrocytes and Schwann cells, respectively. Chondroitin sulfate proteoglycan deposition was restricted to the edges of the bridge, was greatest at 1 week, and significantly decreased by 6 weeks. The dynamics of collagen I and IV, laminin, and fibronectin deposition varied with time. These studies demonstrate that the bridge structure can support substantial long-term axon growth and myelination with limited scar formation.

Hydrogel macroporosity and the prolongation of transgene expression and the enhancement of angiogenesis.

The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37 °C. The macropores were interconnected, as evidenced by homogeneous cell seeding in vitro and complete cell infiltration in vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications.