Engineering three-dimensional collagen-IKVAV matrix to mimic neural microenvironment

Abstract

Engineering the cellular microenvironment has great potential to create a platform technology toward engineering of tissue and organs. This study aims to engineer a neural microenvironment through fabrication of three-dimensional (3D) engineered collagen matrixes mimicking in-vivo-like conditions. Collagen was chemically modified with a pentapeptide epitope consisting of isoleucine-lysine-valine-alanine-valine (IKVAV) to mimic laminin structure supports of the neural extracellular matrix (ECM). Three-dimensional collagen matrixes with and without IKVAV peptide modification were fabricated by freeze-drying technology and chemical cross-linking with glutaraldehyde. Structural information of 3D collagen matrixes indicated interconnected pores structure with an average pore size of 180 μm. Our results indicated that culture of dorsal root ganglion (DRG) cells in 3D collagen matrix was greatly influenced by 3D culture method and significantly enhanced with engineered collagen matrix conjugated with IKVAV peptide. It may be concluded that an appropriate 3D culture of neurons enables DRG to positively improve the cellular fate toward further acceleration in tissue regeneration.

Figure 1

¹H NMR (500 MHz) spectrum analysis of collagen (A) and collagen-IKVAV peptide (B).

Figure 2

Light microscopy photographs (A) and cross-sectional SEM photographs of collagen-IKVAV peptide matrix (B).

Figure 3

Light microscopic photographs of a collagen-IKVAV matrix (A) and unmodified collagen matrix (B) after cross-linking with 0.4 wt % glutaraldehyde solution in 0.2 vol % acetic acid for 24 h at 4 °C.

Figure 4

Cell attachment for the 3D collagen matrix with and without VVIAK, IKVAV, RGD, and RGD-IKVAV modification. *, p < 0.05; significant relative to 2D culture method. †, p < 0.05; significant relative to 3D unmodified collagen matrix. ‡, p < 0.05; significant relative to 3D modified collagen-VVIAK matrix. §, p < 0.05; significant relative to 3D modified collagen-RGD matrix. #, p < 0.05; significant relative to 3D modified collagen-IKVAV matrix.

Figure 5

Proliferative profile of cells on 2D culture (●), 3D collagen matrix without modification (■), 3D collagen matrix with VVIAK modification (□), 3D collagen matrix with RGD modification (○), 3D collagen matrix with IKVAV modification (▲), and 3D collagen matrix with RGD-IKVAV modification (△). *, p < 0.05; significant relative to 2D culture method. †, p < 0.05; significant relative to 3D unmodified collagen matrix. ‡, p < 0.05; significant relative to 3D modified collagen-VVIAK matrix. §, p < 0.05; significant relative to 3D modified collagen-RGD matrix. #, p < 0.05; significant relative to 3D modified collagen-IKVAV matrix.

Figure 6

(A) Real-time PCR study of gene expression of Nestin related to DRG cells grown in nerve differentiation (■) and standard medium (□) on 2D and 3D collagen matrix with and without VVIAK, IKVAV, RGD, and RGD-IKVAV modification. *, p < 0.05; significant relative to level of gene expression compared to DRG cells cultured in standard medium. †, p < 0.05; significant relative to level of gene expression compared to DRG cells cultured in 2D culture method in standard medium. (B) Real-time PCR study of gene expression of Map-2 related to DRG cells grown in nerve differentiation (■) and standard medium (□) on 2D and 3D collagen matrix with and without VVIAK, IKVAV, RGD, and RGD-IKVAV modification. *, p < 0.05; significant relative to level of gene expression compared to DRG cells cultured in standard medium. †, p < 0.05; significant relative to level of gene expression compared to DRG cells cultured in 2D culture method in standard medium. ‡, p < 0.05; significant relative to level of gene expression of DRG cells cultured in nerve differentiation comapred with level of gene expression of DRG cells cultured in standard medium.