Neurotrophin releasing single and multiple lumen nerve conduits

Abstract

Tissue engineering strategies for nerve repair employ polymer conduits termed guidance channels and bridges to promote regeneration for peripheral nerve injury and spinal cord injury, respectively. An approach for fabrication of nerve conduits with single and multiple lumens capable of controlled release of neurotrophic factors was developed. These conduits were fabricated from a mixture of poly(lactide-co-glycolide) (PLG) microspheres and porogen (NaCl) that was loaded into a mold and processed by gas foaming. The porosity and mechanical properties of the constructs were regulated by the ratio of porogen to polymer microsphere. The neurotrophin, nerve growth factor (NGF), was incorporated into the conduit by either mixing the protein with microspheres or encapsulating the protein within microspheres prior to gas foaming. A sustained release was observed for at least 42 days, with the release rate controlled by method of incorporation and polymer molecular weight. Released NGF retained its bioactivity, as demonstrated by its ability to stimulate neurite outgrowth from primary dorsal root ganglion (DRG). In vivo results indicate that conduits retain their original architecture, and allow for cellular infiltration into the channels. Polymer conduits with controllable lumen diameters and protein release may enhance nerve regeneration by guiding and stimulating neurite outgrowth.

Fig. 1

Schematics of custom-made molds for conduit fabrication. The single lumen conduit mold has a center rod for creating the lumen (A). The multiple lumen conduit mold has pins that traverse length of the mold for creating multiple channels (B). Pins are positioned by screens located at each end of the mold. These screens are held in position with spacers.

Fig. 2

Single lumen conduits. Conduits were fabricated with HMW PLG and visualized by scanning electron microscopy. Images captured at 25x magnification (A, scale bar=700 µm) and 90x; magnification (B, scale bar=200 µm) for conduits formed without porogen. (C) Conduit fabricated at a porogen to polymer ratio of 10:1 visualized at 90x magnification (scale bar=200 µm).

Fig. 3

Multiple lumen conduits. Conduits were fabricated with HMW PLG containing 18 channels (diameter=250 µm) and visualized by light microscopy. Conduit is visualized from the top (A, scale bar=1 mm) and from the end (B, scale bar=500 µm).

Fig. 4

Transverse compressive strength (S_c) of single lumen conduits. (A) Representative force–displacement curve for a conduit formed at a porogen to polymer ratio of 2:1. The dashed line represents a linear fit through the experimental data at the inflection point. (B) Transverse compressive strength for porogen to polymer ratios of 2:1, 5:1, and 10:1. Conduits were fabricated using HMW PLG. *Indicates statistically significant with p <0.01.

Fig. 5

Elastic modulus of multiple lumen conduits. (A) Representative compression and decompression curves for a conduit with porogen to polymer ratio of 12:1. Linear lines were fit to the compression (solid) and decompression (dashed) curve immediately adjacent to the apex. Elastic modulus for the compression curves (B) and decompression curves (C) of conduits with porogen to polymer ratios of 5 and 12. Conduits were fabricated with HMW PLG and 150 µm channels. *Indicates statistical significance of p <0.01 for the comparison.

Fig. 6

NGF release from single lumen conduits. (A) Release of NGF from conduits fabricated with differing PLG composition and variations in the incorporation method (microsphere encapsulated, mixed with microspheres). □—100% HMW PLG, encapsulated NGF; △—75% HMW/25% LMW PLG, encapsulated NGF; ○— 100% HMW PLG, mixed NGF; ×—75% HMW/25% LMW PLG, mixed NGF. Conduits were fabricated with porogen to polymer ratio of 5:1. A statistically significant difference was observed between encapsulated and mixed NGF for both 100% HMW condition and 75% HMW/25% LMW conditions (p <0.05). No statistical difference was obtained between the 100% HMW and 75% HMW/25% LMW for mixed NGF conditions (p >0.1). However, there is significant difference for the encapsulated NGF conditions ( p <0.05). (B) Release curves for porogen to polymer ratios of 2:1 (□), 5:1 (△), and 10:1 (○). Conduits were fabricated with 75% HMW/25% LMW PLG and encapsulated NGF. No statistical difference was obtained among conditions with various porogen to polymer ratios (p >0.05).

Fig. 7

Bioactivity of released NGF. Conditions tested include: single lumen conduit with encapsulated NGF, single lumen conduit with mixed NGF, and multiple lumen conduit with encapsulated NGF. NGF released at different time points was assayed for the ability to stimulate neurite extension by primary DRG neurons (n ≥ 3). No statistical difference was obtained between the experimental and control conditions (p >0.05).

Fig. 8

Photomicrograph of in vivo retrieved scaffolds. Multiple lumen conduits (porogen to polymer=4:1) with 250 µm channels were implanted subcutaneously for 13 days. Sections (9 µm) were stained with hematoxylin and eosin and imaged under light microscopy (scale bar=100 µm). The labels T and P represent tissue and polymer, respectively.