Regulated Nerve Growth Factor Delivery on Novel Polymers by hNGF-EcR-293-TK Cells

Peripheral nerve defects may require substantial amounts of nerve grafts for significant reconstruction. Nerve tissue engineering seeks to develop a viable nerve substitute by combining transplanted cells, a bioabsorbable polymer scaffold, and growth factors. Growth factors have so far been added to polymer scaffolds only by passive absorption (microspheres). In contrast, NGF production can be regulated by the humann embryonic kidney cell (EcR-293) transfected with NGF and HSV-TK into its genome. The present study examined the compatibility of these cells with various novel polymer(s) to ascertain 1) the polymer combination which can ultimately be used for conduit fabrication needed for cell transplantation in vivo; and 2) whether cells once attached can produce NGF on induction with Ponasterone A.

Polymer combinations of PLA/PCL (%) as 25/75, 40/60, 75/25, 80/20 (1.5%), 10% PLA alone, and 1.5% PLA alone were coated on 600-cm glass plates. Coated plates were sterilized by incubating them overnight in DMEM + 10% FCS + 10 times antibiotics/antimycotics at 37 degrees C. hnGF-EcR-293-TK cells were plated at a concentration of 5 × 104 cells per plate. All plates were incubated at 37 degrees C and 5% humidity. Cells were induced with 2 uM Ponasterone A after 24 hr of plating, and media were harvested every 24 hr post induction to analyze the quantity and quality of NGF produced by ELISA and PC12 bioassay, respectively.

PLA/PCL combination of 80/20 (10 and 1.5%) supported the cell division and growth until week 5 and started to die as the polymer also started to degrade. At week 8, cells were still alive but the population doubling ratio had declined significantly. Similar results were also observed with plates coated with PLA alone (10 and 1.5%), although there were lesser numbers of surviving cells. In contrast, PLA/PCL combination of 40/60% and 25/75% did not support the cell growth in a linear manner and cells died at or before week 5. The polymer degradation ratio at different time points stayed almost the same in all combinations. The cells were able to produce bioactive NGF until post induction in both combinations of 80/20 PLA/PCL alone.

The studies revealed that transfected human embryonic kidney cells can stay alive, divide, and produce NGF when a polymer support system of 80/20 PLA/PCL is used. The authors believe that this polymer combination will be ideal for conduit fabrication and transplantation of these cells in vivo.