Novel fabrication method of porous poly(L-lactic acid) scaffolds using liquid?solid extraction

Abstract

Porogen leaching is the most widely used method to prepare porous scaffolds for tissue engineering. Several porogen materials have been used until now, such as salt [1], hydrocarbon [2], glucose [3], gelatin [4], paraffin [5, 6], sugar [7], ammonium bicarbonate [8–10] and ice particulates [11, 12]. Among them, ice particulates may be the best. It can be removed completely, while other porogens were usually residual. Moreover, the process by freeze-drying to leach out the solvent needs much time. In this paper, PLLA scaffolds were fabricated by liquid–solid extraction method with ice particulates as porogen instead of freeze-drying. Ice particulates were firstly prepared by spraying distilled water into liquid nitrogen from a nozzle, and then ice particulates with special sizes were achieved by sieving. PLLA was dissolved in chloroform, and the mixed solution was pre-cooled to −30 ◦C. Then ice particulates were added to the pre-cooled PLLA solution and stirred to gain uniform mixture. The mixture was poured into a copper mold and subsequently put into liquid nitrogen. To remove the solvent, the solidified mixture was taken out from the mold and subjected to liquid–solid extraction with alcohol at −60 ◦C for 12 h. Vacuum drying was used to remove the ice and alcohol for another 12 h, finally the material was held at room temperature to evaporate residual alcohol. In contrary, a PLLA scaffold was prepared by freezedrying. The morphologies of PLLA scaffolds coated with a thin film of gold by vacuum-deposition were observed by scanning electron microscope (SEM, SIRION, 5 kV). The porosity of the scaffolds was determined by Archimedes method. The SEM morphologies of the scaffolds are shown in Figs 1 and 2. The scaffolds in Fig. 1 were prepared by liquid–solid extraction using ice particles with different sizes. Fig. 1e and 1f show the morphologies of the scaffolds as shown in Fig. 1c and 1d at a higher magnification. Fig. 2 shows the scaffold prepared by freeze-drying with the same amounts and sizes of materials as the one shown in Fig. 1d. From Fig. 1, it can be seen clearly that the scaffolds fabricated by liquid–solid extraction had two kinds of pores with different sizes. The sizes of the bigger pores correspond to the ones of the ice particulates, while the sizes of the smaller one are less than 100 μm. Thus the bigger pores may be formed by ice particles while the smaller ones were created by chloroform. That is to say, the size of the bigger pores can be controlled by using ice particulates with different sizes. It also can be seen that all scaffolds had an interconnected pore structure, which is needed by tissue engineering. Comparing Fig. 1d with Fig. 2, no obvious difference appeared between the scaffolds prepared by liquid–solid extraction and the ones using freeze-drying. According to the morphology of scaffolds, the formation process of scaffolds is as follows. At first, ice particulates existed in the solution. During freezing in liquid nitrogen, phase separation happened in chloroform solution and a porous structure with big and small pores generated. During liquid–solid extraction, chloroform was extracted and its space was occupied by absolute alcohol. During the vacuum-drying, ice sublimated and absolute alcohol evaporated, the spaces originally occupied by ice and alcohol became porous. The space where chloroform occupied had smaller pores, while the space occupied by ice particulate had bigger pores. In order to guarantee formatting scaffold, the extractant must have several characters: firstly, it cannot