Patterned PLG substrates for localized DNA delivery and directed neurite extension

Abstract

Tissue engineering strategies that enable nerve regeneration will require methods that can promote and direct neurite extension across the lesion. In this report, we investigate an in vitro combinatorial approach to directed neurite outgrowth using gene delivery from topographically patterned substrates, which can induce expression of neurotrophic factors to promote neurite extension and direct the extending neurites. Poly(lactide-co-glycolide) (PLG), which has been used to fabricate conduits or bridges for regeneration, was compression molded to create channels with 100, 150, and 250 microm widths. DNA complexes were immobilized to the PLG, and cells cultured on the substrate were transfected with efficiencies dependent on channel width and DNA amount. A co-culture model consisting of primary neurons and accessory cells was employed to investigate neurite outgrowth within the channels. Localized secretion of nerve growth factor (NGF) by the accessory cells promoted neuron survival and neurite extension. Neurons cultured in channels with NGF expression exhibited longer primary neurites than in the absence of channels. Neurons cultured in smaller width PLG microchannels exhibited a greater degree of directionality and less secondary sprouting than larger channels. Finally, surface immobilization allowed for the delivery of distinct plasmids from each channel, which may enable channels to be tailored for specific nerve tracts. This approach demonstrates the ability to combine gene delivery with physical guidance, and can be tailored to target specific axonal populations with varying neurotrophic factor requirements.

Multiple Channel Bridges for Spinal Cord Injury: Cellular Characterization of Host Response

“…41,42 Recently, in vitro studies indicated that 100 mm channel patterns are able to attain linear and guided neuronal extensions more effectively than 250 mm channel patterns. 21 Further, the 150 mm channel design allowed for an increase in number of channels from 7 channels (250 mm channel design) to 20 channels. By providing more channels, the sprouting axons may have a higher chance of entering a channel that will guide them through the bridge.…”

“…More recently, bridges with multiple channels have been developed with the objective of maintaining a path for regrowing axons, which may also orient axonal elongation. 6,[18][19][20][21][22] Channels with diameters ranging from 40 to 600 mm have been fabricated for spinal cord regeneration, [18][19][20] though the correlation between channel diameter and regeneration has not previously been determined.…”

Electrostatic immobilization of DNA polyplexes on small intestinal submucosa for tissue substrate-mediated transfection

“…The activity of reporter b-galactosidase was evaluated by X-gal staining. In this staining assay, transfected HT-1080 cells on SIS were fixed (0.25% glutaraldehyde at 4°C for 5 min) and stained with a 1.25 mg ml À1 X-gal solution containing 1 mM MgCl 2 , 0.5 mM K 4 Fe(CN) 6 on SIS. Blank SIS seeded with HT-1080 cells was used as control in X-gal staining.…”

“…This strategy is particularly useful for implementing physiological cues, such as growth factor-encoding genes, in the tissue substrate to stimulate tissue regeneration. Promising results using combined tissue-engineering therapy and gene therapy have been reported in nerve regeneration, cutaneous wound healing and bone graft healing [6][7][8][9][10][11].…”

“…One potential solution is implanting tissue-engineering constructs loaded with DNA vectors for localized transgene delivery [1][2][3][4][5][6]. Compared to delivery of free vectors, using immobilized vectors could probably delay clearance of vectors in vivo, provide longer periods of exposure of target cells to vectors, and result in higher localized vector concentrations.…”