Plasma surface chemical treatment of electrospun poly(L-lactide) microfibrous scaffolds for enhanced cell adhesion, growth, and infiltration

Abstract

Poly(l-lactide) (PLLA) microfibrous scaffolds produced by electrospinning were treated with mild Ar or Ar-NH3/H2 plasmas to enhance cell attachment, growth, and infiltration. Goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold surface chemistry by plasma treatment. AFM and XPS measurements showed that both plasma treatments increased the hydrophilicity without affecting the integrity of the fibrous structure and the fiber roughness, whereas Ar-NH3/H2 plasma treatment also resulted in surface functionalization with amine groups. Culture studies of bovine aorta endothelial cells and bovine smooth muscle cells on the plasma-treated PLLA scaffolds revealed that both Ar and Ar-NH3/H2 plasma treatments promoted cell spreading during the initial stage of cell attachment and, more importantly, increased the cell growth rate, especially for Ar plasma treatment. In vitro cell infiltration studies showed that both plasma treatments effectively enhanced cell migration into the microfibrous scaffolds. In vivo experiments involving the subcutaneous implantation of plasma-treated PLLA scaffolds under the skin of Sprague-Dawley rats also showed increased cell infiltration. The results of this study indicate that surface treatment of PLLA microfibrous scaffolds with mild Ar or Ar-NH3/H2 plasmas may have important implications in tissue engineering. Further modifications with bioactive factors should improve the functions of the scaffolds for specific applications.

FIG. 1.

Scanning electron microscopy (first row) and atomic force microscopy (second and third row) images of untreated and plasma-treated poly(l-lactide) (PLLA) microfibrous scaffolds. UT, Ar, and Ar-NH₃/H₂ indicate untreated, Ar plasma-treated, and Ar-NH₃/H₂ plasma-treated scaffolds, respectively.

FIG. 2.

(A) X-ray photoelectron spectroscopy (XPS) survey spectra and (B) surface concentration of C, O, and N for untreated and plasma-treated PLLA microfibrous scaffolds. Error bars in (B) indicate one standard deviation above and below the corresponding mean value, calculated from at least three measurements.

FIG. 3.

(A) Schematic of trifluoromethyl benzaldehyde (TFBA) labeling of −NH₂ surface groups on Ar-NH₃/H₂ plasma-treated PLLA microfibrous scaffolds, and (B) XPS survey spectra of untreated and plasma-treated PLLA microfibrous scaffolds obtained after treatment with TFBA.

FIG. 4.

Morphologies of bovine aorta endothelial cells (BAECs) (A–C) and bovine smooth muscle cells (BSMCs) (D–F) seeded on untreated and plasma-treated PLLA microfibrous scaffolds obtained after incubation in the serum medium for 5 h. (A, D) Untreated, (B, E) Ar plasma-treated, and (C, F) Ar-NH₃/H₂ plasma-treated scaffold surfaces. (G) Atomic percentage of N indicating the amount of serum protein adsorbed on untreated and plasma-treated PLLA microfibrous scaffolds after incubation in the 10% fetal bovine serum medium for 5 h. Error bars in (G) indicate one standard deviation above and below the corresponding mean value, calculated from at least three measurements.

FIG. 5.

Morphologies of BAECs (A–C) and BSMCs (D–F) seeded on untreated and plasma-treated PLLA microfibrous scaffolds obtained after incubation in the serum medium for 24 h. (A, D) Untreated, (B, E) Ar plasma-treated, and (C, F) Ar-NH₃/H₂ plasma-treated scaffold surfaces.

FIG. 6.

Proliferation rates of BAECs and BSMCs seeded on untreated and plasma-treated scaffold surfaces obtained after incubation in the serum medium for 24 h. The proliferation rate of each treatment differs statistically from the other two treatments for the same cell type (p<0.05, repeated three times). Error bars indicate one standard deviation above and below the corresponding mean value, calculated from at least eight different surface sites of each sample.

FIG. 7.

Cross-sectional images of (A) untreated, (B) Ar plasma-treated, and (C) Ar-NH₃/H₂ plasma-treated scaffolds obtained after in vitro culture with BAECs in the serum medium for 5 days. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). Top and bottom scaffold surfaces are distinguished by dashed lines.

FIG. 8.

Cross-sectional images of (A, D) untreated, (B, E) Ar plasma-treated, and (C, F) Ar-NH₃/H₂ plasma-treated scaffolds obtained after in vivo implantation under the skin of Sprague-Dawley rats for 5 days. Cells in (A–C) were stained with DAPI, while cells in (D–F) were stained with DAPI (blue) and CD68 (green). Top and bottom scaffold surfaces are distinguished by dashed lines.

FIG. 9.

Cross-sectional images of (A, D) untreated, (B, E) Ar plasma-treated, and (C, F) Ar-NH₃/H₂ plasma-treated scaffolds obtained after in vivo implantation under the skin of Sprague-Dawley rats for 14 days. Cells in (A–C) were stained with DAPI (blue) and CD68 (green), while cells in (D–F) were stained with DAPI (blue) and Ki67 (red). Positive Ki67 staining cells in (D–F) are indicated by arrows. Top and bottom scaffold surfaces are distinguished by dashed lines.

FIG. 10.

Cross-sectional images of (A, D) untreated, (B, E) Ar plasma-treated, and (C, F) Ar-NH₃/H₂ plasma-treated scaffolds obtained after in vivo implantation under the skin of Sprague-Dawley rats for 14 days. Cells in (A–C) were stained with DAPI, whereas cells in (D–F) were stained for smooth muscle α-actin. Top and bottom scaffold surfaces are distinguished by dashed lines.