PCL nerve conduit fabrication

All chemicals were analytical grade or purer and were purchased from commercial suppliers. Poly(vinyl alcohol) (average M _w 9000–10,000, 80% hydrolyzed), poly( dl -lactide-co-glycolide) (lactide:glycolide (50:50), mol wt 40,000–75,000 units), poly(caprolactone) ( M _w 65,000), Dichloromethane, and Ethyl Acetate.

To create double-walled microspheres, a 17.5% poly(lactic-co-glycolic acid) (PLGA) solution was created with 150 mg PLGA in dichloromethane. In a separate glass scintillation vial, a 10% solution of poly(lactide) (PLLA) of equal polymer mass was prepared. After both polymers were fully dissolved, to encapsulate glial cell line-derived neurotrophic factor (GDNF), a solution of 40 μl (0.1 mg/mL) of GDNF, 100 mg of docusate sodium salt and 7 mg human serum albumin was prepared in 0.5 mL sterile water over ice added to the PLGA solution and vortexed for ∼30 s to achieve a homogenous mixture. The PLGA solution was then combined with the PLA solution and vortexed for an additional 60 s. This oil-in-oil emulsion was added drop-wise through a Pasteur pipette to 200 mL of aqueous 0.5% poly(vinyl alcohol) solution stirring at 900 rpm for 3 h. Then, the polymer microspheres were collected through centrifugation (1500 g for 10 min) and washed three times. Finally, the microspheres were lyophilized using a Labconco freeze dry system (without a cryoprotectant) and stored in a desiccant at −20 °C.

PCL nerve guides were fabricated as follows. Glass capillary mandrels 1.5 mm in diameter were coated with a 17% w/v% aqueous solution of poly(vinyl alcohol) (PVA), air dried and then immersed into the polymer slurry creating NaCl/PCL mandrel coatings. The ethyl acetate was allowed to evaporate for a minimum of 10 min between successive mandrel immersions into the polymer slurry. After the completion of the dip-coating process, the resulting polymer conduits were submerged in distilled water to allow for salt and PVA dissolution, and the guides were removed from the glass mandrels. The final wall thickness after 6 successive immersions of the mandrels into the polymer solutions was 600–700 μms.

To incorporate double-walled microspheres into the inner half of the nerve guide wall, 15 mg of microspheres were evenly spread onto a drawn grid on parchment paper. After the first immersion of the glass mandrel into the PCL slurry, the ethyl acetate was allowed to evaporate for only 30 s leaving a semi-hardened polymer layer on the mandrel. This was then smoothly rolled across the microspheres on parchment paper. The PCL with embedded microspheres was allowed to dry for 10 min and then repeatedly coated with additional layers of polymer as done in nerve guides without microspheres.