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. 2012 Feb 13;13(2):358-68.
doi: 10.1021/bm201372u. Epub 2012 Jan 18.

Lubricated biodegradable polymer networks for regulating nerve cell behavior and fabricating nerve conduits with a compositional gradient

Lei Cai  1 , Jie Lu, Volney Sheen, Shanfeng Wang
Affiliations

Lubricated biodegradable polymer networks for regulating nerve cell behavior and fabricating nerve conduits with a compositional gradient

Lei Cai et al. Biomacromolecules. .

Abstract

We present a method of tuning surface chemistry and nerve cell behavior by photo-cross-linking methoxy poly(ethylene glycol) monoacrylate (mPEGA) with hydrophobic, semicrystalline poly(ε-caprolactone) diacrylate (PCLDA) at various weight compositions of mPEGA (ø(m)) from 2 to 30%. Improved surface wettability is achieved with corresponding decreases in friction, water contact angle, and capability of adsorbing proteins from cell culture media because of repulsive PEG chains tethered in the network. The responses of rat Schwann cell precursor line (SpL201), rat pheochromocytoma (PC12), and E14 mouse neural progenitor cells (NPCs) to the modified surfaces are evaluated. Nonmonotonic or parabolic dependence of cell attachment, spreading, proliferation, and differentiation on ø(m) is identified for these cell types with maximal values at ø(m) of 5-7%. In addition, NPCs demonstrate enhanced neuronal differentiated lineages on the mPEGA/PCLDA network at ø(m) of 5% with intermediate wettability and surface energy. This approach lays the foundation for fabricating heterogeneous nerve conduits with a compositional gradient along the wall thickness, which are able to promote nerve cell functions within the conduit while inhibiting cell attachment on the outer wall to prevent potential fibrous tissue formation following implantation.

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Figures

Figure 1
Figure 1
Photo-crosslinking of mPEGA/PCLDA.
Figure 2
Figure 2
Physical properties of uncrosslinked and crosslinked mPEGA/PCLDA samples. (a) Temperature dependence of zero-shear viscosity η0 for uncrosslinked samples. (b) FTIR spectra of mPEGA and crosslinked samples. (c) Swelling ratios of crosslinked samples in CH2Cl2 and water. Inset: Gel fractions of crosslinked samples obtained in CH2Cl2. (d) DSC curves of crosslinked samples. (e) Storage modulus G′ (solid symbols), loss modulus G″ (open symbols) and viscosity η (lines) vs. frequency for crosslinked samples at 37 °C. (f) Tensile stress-strain curves of crosslinked samples at 37 °C.
Figure 3
Figure 3
Surface properties of crosslinked mPEGA/PCLDA disks. (a) Frictional coefficients between a stainless steel plate and hydrated polymer disks. Inset: Frictional force vs. normal force. (b) Water contact angle and protein adsorption. (c) AFM images. Significant difference (p < 0.05) is between any two groups in (a) and (b).
Figure 4
Figure 4
Rat SpL201 cell attachment, proliferation, and differentiation on crosslinked mPEGA/PCLDA disks. (a) Fluorescent images of SpL201 cells stained using rhodamine-phalloidin (RP, red, top row), O4-positive areas (green, bottom row) and nuclei (blue) stained using 4',6-diamidino-2-phenylindole (DAPI) of differentiated SpL201 cells upon forskolin treatment at day 7 post-seeding. Scale bar of 200 µm is applicable to all. (b) SpL201 proliferation at days 1, 4, and 7, compared with cell-seeded tissue culture polystyrene (TCPS) as positive control. (c) Normalized area positive to O4 antibody for SpL201 cells at day 7. *: significantly higher (p < 0.05); #: significantly lower (p < 0.05) than the corresponding data on crosslinked PCLDA disks.
Figure 5
Figure 5
Rat PC12 cell attachment, proliferation, and differentiation on crosslinked mPEGA/PCLDA disks. (a) Fluorescent images stained using RP for PC12 cell growth (top row) and NGF-induced neurites (bottom row) at day 7 post-seeding. Scale bar of 200 µm is applicable to all. (b) PC12 cell attachment at 4 h and proliferation at days 1, 4, and 7, compared with TCPS as positive control. (c) Quantification of PC12 neurites at day 7 using the number of neurites per cell, percentage of differentiated cells, and neurite lengths. *: significantly higher (p < 0.05); #: significantly lower (p < 0.05) than the corresponding data on crosslinked PCLDA disks.
Figure 6
Figure 6
E14 mouse NPC attachment, proliferation, and differentiated lineages on crosslinked mPEGA/PCLDA disks. (a) Fluorescent images of neurospheres stained using DAPI (blue, top row), differentiated astrocytes stained using anti-GFAP (green, middle row), and neurons stained using anti-β-tubulin-III (green, bottom row) at day 7 post-seeding. Scale bar of 200 µm is applicable to all. (b) NPC proliferation at days 1, 4, and 7, compared with TCPS as positive control. (c) Quantification of percentage of differentiated astrocytes and neurons at day 7. *: significantly higher (p < 0.05); #: significantly lower (p < 0.05) than the corresponding data on crosslinked PCLDA disks.
Figure 7
Figure 7
Crosslinked mPEGA/PCLDA nerve tubes with two different structures. (a) SEM images of the tubes and SpL201 cells attached on the inner and outer walls of the tubes at day 1 post-seeding. For the homogeneous tube, ϕm is 5% everywhere. For the compositional-gradient tube, ϕm is 5% on the inner wall while it increased gradually to 30% on the outer wall. (b) FTIR spectra of the cross section of the gradient tube wall at different positions from the inner wall to the outer wall, represented by the percentage of the total wall thickness. (c) The ratios of the absorption at 1106 cm−1 (A1106) for asymmetric C-O-C band to that at 1720 cm−1 (A1720) for the C=O band for indicating the composition of PEG along the wall thickness.
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