Multi-factorial nerve guidance conduit engineering improves outcomes in inflammation, angiogenesis and large defect nerve repair.

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.

In Vitro and In Vivo Assessment of PEGylated PEI for Anti-IL-8/CxCL-1 siRNA Delivery to the Lungs.

Inhalation offers a means of rapid, local delivery of siRNA to treat a range of autoimmune or inflammatory respiratory conditions. This work investigated the potential of a linear 10 kDa Poly(ethylene glycol) (PEG)-modified 25 kDa branched polyethyleneimine (PEI) (PEI-LPEG) to effectively deliver siRNA to airway epithelial cells. Following optimization with anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, PEI and PEI-LPEG anti-IL8 siRNA nanoparticles were assessed for efficacy using polarised Calu-3 human airway epithelial cells and a twin stage impinger (TSI) in vitro lung model. Studies were then advanced to an in vivo lipopolysaccharide (LPS)-stimulated rodent model of inflammation. In parallel, the suitability of the siRNA-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. The siRNA nanoparticles were nebulised using an Aerogen® Pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. Only PEI anti-IL8 siRNA nanoparticles were capable of significant levels of IL-8 knockdown in vitro in non-nebulised samples. However, on nebulization through a TSI, only PEI-PEG siRNA nanoparticles demonstrated significant decreases in gene and protein expression in polarised Calu-3 cells. In vivo, both anti-CXCL-1 (rat IL-8 homologue) nanoparticles demonstrated a decreased CXCL-1 gene expression in lung tissue, but this was non-significant. However, PEI anti-CXCL-1 siRNA-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (BAL) fluid. Overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. This work demonstrates the potential of nebulised PEI-PEG siRNA nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development.

The Use of Genipin as an Effective, Biocompatible, Anti-Inflammatory Cross-Linking Method for Nerve Guidance Conduits.

A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs.

Controlling the dose-dependent, synergistic and temporal effects of NGF and GDNF by encapsulation in PLGA microparticles for use in nerve guidance conduits for the repair of large peripheral nerve defects.

Neurotrophic factor delivery via biodegradable nerve guidance conduits may serve as a promising treatment for the repair of large peripheral nerve defects. However, a platform for controlled delivery is required because of their short in vivo half-life and their potential to impede axonal regeneration when used in supraphysiological doses. In this study, we investigated the dose-dependent, synergistic and temporal effects of NGF and GDNF on neurite outgrowth, adult dorsal root ganglia axonal outgrowth, Schwann cell migration and cytokine production in vitro. Using the optimal dose and combination of NGF and GDNF, we developed a PLGA microparticle-based delivery platform to control their delivery. The dose-dependent effects of both NGF and GDNF individually were found to be non-linear with a saturation point. However, the synergistic effect between NGF and GDNF was found to outweigh their dose-dependent effects in terms of enhancing Schwann cell migration and axonal outgrowth while allowing a 100-fold reduction in dose. Moreover, a temporal profile that mimics the physiological flux of NGF and GDNF in response to injury, compared to one that resembles an early burst release delivery profile, was found to enhance their bioactivity. The optimized NGF- and GDNF-loaded microparticles were then incorporated into a guidance conduit, and their capacity to enhance nerve regeneration across a 15 mm sciatic nerve defect in rats was demonstrated. Enhanced nerve regeneration was seen in comparison to non-treated defects and very encouragingly, to a similar level compared to the clinical gold standard of autograft. Taken together, we suggest that this delivery platform might have significant potential in the field of peripheral nerve repair; allowing spatial and temporal control over the delivery of potent neurotrophic factors to enhance the regenerative capacity of biomaterials-based nerve guidance conduits.

A Physicochemically Optimized and Neuroconductive Biphasic Nerve Guidance Conduit for Peripheral Nerve Repair.

Clinically available hollow nerve guidance conduits (NGCs) have had limited success in treating large peripheral nerve injuries. This study aims to develop a biphasic NGC combining a physicochemically optimized collagen outer conduit to bridge the transected nerve, and a neuroconductive hyaluronic acid-based luminal filler to support regeneration. The outer conduit is mechanically optimized by manipulating crosslinking and collagen density, allowing the engineering of a high wall permeability to mitigate the risk of neuroma formation, while also maintaining physiologically relevant stiffness and enzymatic degradation tuned to coincide with regeneration rates. Freeze-drying is used to seamlessly integrate the luminal filler into the conduit, creating a longitudinally aligned pore microarchitecture. The luminal stiffness is modulated to support Schwann cells, with laminin incorporation further enhancing bioactivity by improving cell attachment and metabolic activity. Additionally, this biphasic NGC is shown to support neurogenesis and gliogenesis of neural progenitor cells and axonal outgrowth from dorsal root ganglia. These findings highlight the paradigm that a successful NGC requires the concerted optimization of both a mechanical support phase capable of bridging a nerve defect and a neuroconductive phase with an architecture capable of supporting both Schwann cells and neurons in order to achieve functional regenerative outcome.

The development of non-viral gene-activated matrices for bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds.

The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2µg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2µg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration.