Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers

Abstract

Growth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH = 4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH = 5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH = 4 and pH = 5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH = 4 assembly condition had higher cells counts, while the PEM assembled at pH = 5 and PEM with no FGF2 showed increased spreading. Overall, the half-life model and cell culture study provide optimal concentration ranges for fibroblast proliferation and a framework for understanding how temporal FGF2 concentration may affect other cell types.

Figure 1

(a) ELISA-detectable FGF2 at high (500 ng/mL) and low (250 ng/mL) initial starting concentrations added exogenously to PBS and unmodified DMEM, measured over the course of 4 days. Error bars represent standard deviation (n = 5). (b) Natural logarithm of the same average concentration values with a linear fit from 8 to 96 h. The fit line is used to determine the degradation constant to calculate the half-life.

Figure 2

Cumulative FGF2 release over 4 days from FGF2-(PMAA/PLH)₅ PEMs. Released data was obtained by immersing 1 cm² substrates into 1 mL of PBS. 1 mL aliquots were taken and replaced over the course of 4 days. Error represents standard deviation (n = 5).

Figure 3

PEM mass during assembly obtained from QCM-D results for (PLH/PMAA)_5.5 PEMs assembled at a pH range from pH = 4 to pH = 8. Polyelectrolyte and wash solutions were fed into the QCM-D at 50 μL/min for 15 min and 10 min, respectively. The final wash continued for an additional 10 min to observe any changes to the PEM.

Figure 4

(a) Modeled concentration of FGF2 released from PEMs. pH values refer to PEM assembly conditions. Model assumes a 1.9 cm² surface area and 600 μL of media. (b) Modeled FGF2 concentrations assuming exogenous supplementation of FGF2 at 4 ng/mL, and 8 ng/mL, at both t = 0 and t = 2 days. Both figures assume FGF2 degradation based on an ELISA-detectable half-life of 22.75 h.

Figure 5

Representative images of cells stained with Hoechst 33342 (blue) and phalloidin coupled with Alexa Fluor 488 (green) at (a) 2 days and (b) 4 days of culture time. pH values refer to PEM assembly conditions. Cells were seeded at an initial density of 5000 cells/cm² in IMDM with 1% FBS.

Figure 6

Comparison between 2-day and 4-day results for (a) cell counts obtained from Hoechst 33342 nuclear staining and (b) average cell surface areas from phalloidin labeling. Error bars represent standard error (n = 12). *Represents statistical differences from PEM-only control of the same time point (p < 0.05). **Represent statistical differences from all other conditions at the same time point (p < 0.05).