A neuroinductive biomaterial based on dopamine

Abstract

Chemical messengers such as neurotransmitters play an important role in cell communication, differentiation, and survival. We have designed and synthesized a bioactive biomaterial that derived its biological activity from dopamine. The resultant biodegradable polymer, PCD, has pendent groups bearing dopamine functionalities. Image analysis demonstrated that nerve growth factor-primed rat pheochromocytoma cells (PC12) and explanted rat dorsal root ganglions attached well and displayed substantial neurite outgrowth on the polymer surface. Furthermore, PCD promoted more vigorous neurite outgrowth in PC12 cells than tissue culture polystyrene, laminin, and poly(d-lysine). The histogram of neurite length of PC12 cells showed distinctive patterns on PCD that were absent on the controls. A subset of PC12 cells displayed high filopodium density on PCD. The addition of dopamine in culture medium had little effect on the differentiation of PC12 cells on tissue culture polystyrene. Tyrosine, the precursor of dopamine, did not exhibit this ability to impart specific bioactivity to an analogous polymer. Thus, the dopamine functional group is likely the origin of the inductive effect. PCD did not cause nerve degeneration or fibrous encapsulation when implanted immediately adjacent to the rat sciatic nerves. This work is a step toward creating a diverse family of bioactive materials using small chemical messengers as monomers.

Fig. 1.

PCD was synthesized by using dopamine as a monomer. This synthesis strategy can be applied to a large number of diglycidyl esters and biomolecules containing primary amines.

Fig. 2.

The degradation of PCD in vitro. Shown is degradation of the polymer expressed as a decrease of mass with time. Data are expressed as mean ± SD.

Fig. 3.

NGF-primed PC12 cells grew longer neurites and exhibited higher differentiation rates on PCD versus PCY, PDL, laminin, TCPS, and TCPS with free dopamine (DA). (A) The number of metabolically active cells increased on all four materials as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (absorption measured at 570 nm). (B) Cells displayed the highest differentiation level on PCD, and it increased significantly with time. The control materials did not induce more differentiation as the cell culture time increased. (C) The median neurite length increased substantially on PCD over the culture period. In comparison, the change on the control surfaces was statistically insignificant over the entire culture period. ∗, P < 0.05 with respect to the changes of values on PCD surface with time. #, large standard deviation caused by the presence of a few cells with neurites up to 37 μm amid >89% of the cells with little neurites (Fig. 11). &, Data not representative of the whole cell population because of large numbers of cell aggregates on TCPS surfaces at day 7. The addition of dopamine appeared to further reduce the number of adherent cells.

Fig. 4.

The histogram of neurite length showed very different patterns on PCD (A) versus PCY (B), PDL (C), and laminin (D). The neurite length appeared to group into clusters on PCD, suggesting that the PC12 cell could be more differentiated. The weight of longer neurites increased sharply after day 3 on PCD. Cells in certain areas reached confluence on day 7, which likely contributed to the apparent decrease of longer neurites on PCD, PDL, and laminin surfaces.

Fig. 5.

A significant number of PC12 cells differentiated and exhibited typical neuronal morphology on PCD. The cells are more differentiated and displayed longer neurites on PCD (A) than on PCY (B), PDL (C), and laminin (D). Data are from day 7. (Magnification: ×200. Scale bar: 100 μm.)

Fig. 6.

Scanning electron micrographs of PC12 cells grown on PCD (A) and PCY (B) surfaces with cell bodies partially visible on the top of the image. The cells exhibited growth cone-like structures at the tip of their neurites on both materials. However, the filopodia density along the neurites was higher on PCD surfaces, indicating potential inductive effects of the dopamine moieties. (Magnification: ×5,000. Scale bar: 5 μm.)

Fig. 7.

Extensive neurite outgrowth from rat DRG explants (postnatal day 3) cultured on PCD (A) and PDL (B) surfaces. Data are from day 9. (Magnification: ×200. Scale bar: 100 μm.)

Fig. 8.

Biocompatibility of PCD in vivo. (A) Hematoxylin and eosin staining showed brown residues of a degraded PCD implant (arrows) at 8 weeks after implantation. Cavities were formed inside the material with phagocytic inflammatory infiltrates. The implants caused little fibrosis, and no nerve degeneration was observed. (B) Mason's trichrome staining confirmed that collagen (blue fibers) deposition surrounding the polymer residues was minimal. (C and D) Hematoxylin and eosin staining (C) and Mason's trichrome staining (D) of the sham surgery site at 8 weeks after implantation. N, nerve. (Magnification: ×100. Scale bar: 200 μm.)