Preparation and characterization of microporous poly(D,L-lactic acid) film for tissue engineering scaffold

Abstract

We prepared a series of microporous films based on poly(d,l-lactic acid) (PLA) via phase separation. According to scanning electron microscopy (SEM), a 3-dimensional foamy structure with multimicrometer scale pores on the air surface of film could be observed. As the morphology of PLA film could not be stabilized using solvent-nonsolvent phase separation, we investigated the effect of temperature, air movement, and concentration on the properties of microporous PLA films. The results show that when the temperature was 25°C in a vacuum, it was easy to prepare PLA film with micropores, and it was stable. As the relationship between the morphology and formation factors was clear and the morphology of the PLA film was controllable, we studied the PLA film's potential use for cell culture. SEM results showed that NIH3T3 cell could be adhered on the surface of film well after incubation for 2 days. Meanwhile, in vitro culture experiments revealed the great biocompatibility of the scaffold for adsorption and proliferation of fibroblasts.

Keywords: cell culture; phase separation; poly(d,l-lactic acid); scaffold.

Figure 1

A schematic illustration of preparation of PLA scaffold with microporous structure. Abbreviations: EG, ethylene glycol; GC, THF, tetrahydrofuran; PLA, poly (d,l-lactic acid).

Figure 2

SEM of morphology of PLA film prepared via different methods. A) Volatilization naturally; film prepared via SNS phase separation at the concentration of B) 5%, C) 10%, and D) 15%. All samples were prepared at room temperature without air. Abbreviations: PLA, poly(d,l-lactic acid); SEM, scanning electron microscope; SNS, solvent–nonsolvent.

Figure 3

SEM micrographs of various types of PLA porous scaffold prepared at different temperatures. A) 4°C, B) 25°C, and C) 50°C. Inset: images of corresponding scaffold at higher magnification Abbreviations: PLA, poly(d,l-lactic acid); SEM, scanning electron microscope.

Figure 4

SEM images of PLA scaffolds prepared with various types of ventilation. A) Hermetic environment, B) open environment without air, and C) open environment with ventilated pumping equipment. Abbreviations: PLA, poly(d,l lactic acid); SEM, scanning electron microscope.

Figure 5

SEM images of PLA scaffolds prepared at 25°C with the concentration of 10% (w/v). Morphology observation of PLA film included the air face and cross-section: 1-a) air face of 10% PLA film, 1000×; 1-b) air surface of 10% PLA film, 3000×; 2-a) cross-section of 10% PLA film, 400×; 2-b) cross-section of 10% PLA film, 1000×. Abbreviations: PLA, poly(d,l lactic acid); SEM, scanning electron microscope.

Figure 6

SEM photographs showing morphology of NIH3T3 cells cultured on PLA scaffold. A) Cells cultured for 3 days, 200×; B) Cells cultured for 3 days, 2000×; C) Cells cultured for 12 h, 200×; D) Cells cultured for 12 h, 3500×. Abbreviations: PLA, poly(d,l-lactic acid); SEM, scanning electron microscope.

Figure 7

NIH3T3s proliferation on porous PLA scaffolds. The number of cells was normalized to initial density of seeded cells (1 × 10⁵ cells/well). Mean for n = 3 ± SD. Abbreviations: PLA, poly(d,l-lactic acid); SD, standard deviation.

Figure 8

Fluorescence microscopy images of NIH3T3 cells cultured on A) 1, B) 2, and C) 4 days. The cells were stained by acridine orange stain.