Surface Entrapment of Fibronectin on Electrospun PLGA Scaffolds for Periodontal Tissue Engineering

Abstract

Nowadays, the challenge in the tissue engineering field consists in the development of biomaterials designed to regenerate ad integrum damaged tissues. Despite the current use of bioresorbable polyesters such as poly(l-lactide) (PLA), poly(d,l-lactide-co-glycolide) (PLGA), and poly-ɛ-caprolactone in soft tissue regeneration researches, their hydrophobic properties negatively influence the cell adhesion. Here, to overcome it, we have developed a fibronectin (FN)-functionalized electrospun PLGA scaffold for periodontal ligament regeneration. Functionalization of electrospun PLGA scaffolds was performed by alkaline hydrolysis (0.1 or 0.01 M NaOH). Then, hydrolyzed scaffolds were coated by simple deposition of an FN layer (10 μg/mL). FN coating was evidenced by X-ray photoelectron analysis. A decrease of contact angle and greater cell adhesion to hydrolyzed, FN-coated PLGA scaffolds were noticed. Suitable degradation behavior without pH variations was observed for all samples up to 28 days. All treated materials presented strong shrinkage, fiber orientation loss, and collapsed fibers. However, functionalization process using 0.01 M NaOH concentration resulted in unchanged scaffold porosity, preserved chemical composition, and similar mechanical properties compared with untreated scaffolds. The proposed simplified method to functionalize electrospun PLGA fibers is an efficient route to make polyester scaffolds more biocompatible and shows potential for tissue engineering.

Keywords: biomaterials; proteins; tissue engineering.

FIG. 1.

SEM micrographs of electrospun PLGA scaffolds and treated groups: (A) PLGA, (B) PLGA PBS, (C) PLGAH01, (D) PLGAH001, (E) PLGAH01FN, and (F) PLGAH001FN. Scale bar, 50 μm. Asterisk (*) corresponds to hydrolyzed collapsed fibers; black arrows correspond to deposited structures between PLGA fibers. FN, fibronectin; PBS, phosphate buffered saline; PLGA, poly(d,l-lactide-co-glycolide); SEM, scanning electron microscopy.

FIG. 2.

(A–F) SEM micrographs, contact angle water drop images, and fiber orientation (arrows) of electrospun PLGA groups: (A) PLGA, (B) PLGA PBS, (C) PLGAH01, (D) PLGAH001, (E) PLGAH01FN, and (F) PLGAH001FN. SEM scale bar, 500 μm. (G) Fiber diameter and (H) scaffold porosity of untreated and treated PLGA fibers measured by image treatment. Data are expressed as mean±SD. *Significant difference between groups (p<0.05).

FIG. 3.

Chemical characterization of untreated, hydrolyzed, and hydrolyzed-and-coated PLGA scaffolds by (A) ATR-FT-IR and (B) XPS spectra. Control groups: PLGA scaffold immersed in PBS (PLGA PBS) and in fibronectin solution (PLGA FN) for 24 h. ATR-FT-IR, attenuated total reflectance Fourier transform infrared; XPS, X-ray photoelectron spectroscopy.

FIG. 4.

Mechanical characterization of untreated, hydrolyzed, and hydrolyzed-and-coated PLGA scaffolds: (A) Tensile stress (MPa) versus strain (%) curves and (B) tensile modulus (MPa). Data are expressed as mean±SD. *Significant difference between groups (p<0.05).

FIG. 5.

Study of PLGA scaffold degradation in PBS at 37°C up to 28 days. (A–E) SEM micrographs of PLGA group weekly observed. Degradation behavior of PLGAH001FN samples up to (F) 7, (G) 14, and (H) 21 days. (J i–iii) Details of fiber agglutination (black arrows), appeared pores (white arrows), and pore dimensions of PLGAH001FN samples up to 21 days of PBS immersion. Scale bar: (A–E) 100 μm; (F–H) 1 mm; (J i–iii) 20 μm. (I) Changes of remaining weight and (K) variations of pH values during in vitro degradation up to 28 days.

FIG. 6.

Biocompatibility observations of PDL cells cultured on untreated PLGA and PLGAH001FN samples. (A,B) SEM micrographs of PDL-like fibroblasts cultured on (A) untreated PLGA [scale bar (i) 100 μm and (ii) 30 μm] and (B) PLGAH001FN [scale bar (i) 100 μm and (ii) 30 μm] up to 7 days. (C,D) Confocal images of PDL cells cultured on (C) untreated PLGA surface and (D) PLGAH001FN surfaces up to 24 h; (i) and (ii) correspond to superior and inferior surfaces of samples, respectively. Scale bar (C,D) 100 μm. (E) Quantitative resazurin results of PDL cells cultured on PLGA and PLGAH001FN samples up to 24 h. Data are expressed as mean±SD (n=3). PDL, periodontal ligament.