Fabrication and surface modification of poly lactic acid (PLA) scaffolds with epidermal growth factor for neural tissue engineering

Figures & data

Figure 1. PLA aminolysis reaction involving (A) polyallylamine (PAAm) and (B) ethylenediamine (EtDA).

Figure 2. SEM imaging of (A) pristine and (B-D) PAAm-treated PLA nanofibers. PAAm grafting was carried out at pH 12.5 and 60°C for (B) 1 h, (C) 3 h, and (D) 20 h.

Table 1. Properties of pristine and aminolysed PLA nanofibers.

	Treatment	pH	Reaction time	Reaction temperature (°C)	Average Fiber Diameter (AFD, nm, n = 10)	Porosity (%, n = 3)	Amine group density (µmol/g, n = 3)
A	Pristine				408 ± 77	87
B		12.5	1 h	60	407 ± 62	N/D	109.8 ± 23.7
C		12.5	3 h	60	474 ± 69	N/D	120.3 ± 26.3
D	PAAm	12.5	20 h	60	461 ± 94	N/D	N/A
E		11.5	1 h	50	465 ± 114	89	88.6 ± 22.7
F		12.5	1 h	50	472 ± 59	90	133.2 ± 16.3
G	PVAm	12.5	1 h	50	414 ± 119	88	76.7 ± 9.8
H	Control (water + dioxane)		1 h	60	428 ± 68	N/D	0.3 ± 0.1
I	Control (water + dioxane)		1 h	50	420 ± 85	85	0.4 ± 0.1
J			10 min	50		78	0.3 ± 0.1
K	EtDA		20 min	50		82	0.4 ± 0.1
L			30 min	50		83	0.3 ± 0.1
M	Control (MeOH only)		20 min	50		86	0.2 ± 0.1

N/A: Not Applicable. The mats were too fragile for accurate Orange II measurements.

N/D: Not Determined.

The porosity values were only calculated for the mats that were used in cellular assays, i.e., mats treated at 50°C. These values correspond to the average of measurements performed on 3 distinct mats. Based on the precision of the procedure, we estimate that the standard deviation of these values is about 2%.

Figure 3. Characterization of the EGF grafting on PLA nanofibers by direct ELISA (n = 4). Optical densities (O.D.) corresponding to PAAm-covered mats treated with (A) PEG linker, EGF and ethanolamine (for deactivation of unreacted PEG), (B) Mats covered with PEG linkers that were deactivated before EGF incubation (C) or without any EGF incubation (D) were used as negative controls. As reference, the O.D. value obtained on (D) unmodified PAAm-covered mats is presented. Statistical differences are noted * (p < 0.05).

Figure 4. NSLC proliferation on pristine, PAAm-grafted, EGF-grafted and laminin-coated PLA mats (n = 3). Cells were cultured in basal medium (denoted “-”) or basal medium supplemented with FGF only (denoted FGF) or a mix of FGF and EGF (denoted F + EGF) for 2 d (D2, light gray), 6 d (D6, medium gray) or 10 d (D10, dark gray). Error bars correspond to standard deviation. Statistical differences noted * correspond to p < 0.05.

Figure 5. Representative fluorescence imaging (SYTOX) for NSLCs cell proliferation after 10 d of culture on pristine (A, B), PAAm-grafted (C, D), EGF-grafted (E, F, G) and laminin-coated PLA mats (H). Cells were cultured in basal medium (denoted “-”) or basal medium supplemented with FGF only (denoted FGF) or a mix of FGF and EGF (FGF/EGF). Scale bars correspond to 400 µm.

Figure 6. Representative Fluorescent imaging of NSLCs cultured after 5 d (A-D) or 14 d (E-H) of culture on pristine and EGF-grafted mats. Cells were positively stained for ßIII-Tubulin (A, E, C, G) and nestin (B, F, D, H) under continued proliferation conditions. Cell nuclei were labeled with SYTOX green (B, F, D, H). The scale bars correspond to 100 μm. When present, insets correspond to a 4x higher magnification of the corresponding image.