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Ind. Eng. Chem. Res. 2009, 48, 5310–5316
Nanostructured PLLA-Hydroxyapatite Scaffolds Produced by a Supercritical Assisted Technique Ernesto Reverchon,†,‡ Paola Pisanti,† and Stefano Cardea*,† Department of Chemical and Food Engineering, UniVersity of Salerno, Via Ponte Don Melillo 84084, Fisciano, Italy, and NANO_MATES, Research Centre for Nanomaterials and Nanotechnology at the UniVersity of Salerno Via Ponte Don Melillo 84084, Fisciano, Italy
Temporary bone scaffolds can surrogate the extracellular matrix to favor the regeneration of tissues and organs. It is very difficult to obtain the coexistence of the macro-, micro-, and nanostructural properties necessary to mimic the organization of the human bone. To obtain all of these characteristics, we tested a supercritical fluid assisted technique for the formation of PLLA/ceramic scaffolds using hydroxyapatite (HA) nanoparticles loaded at up to 50% by weight of the polymer to improve the biomimethism and the mechanical properties of the scaffolds. We produced poly(L-lactic acid) scaffolds in relatively short time (90%), large internal surface areas, and very large connectivity at micrometeric and nanometric level. In particular, this process assures the coexistence of the micro- and nanostructural characteristics that have been previously described. On the other hand, this method presents also some limitations: (1) it is performed in several steps, such as raw-material dissolution, gelation, solvents substitution and extraction, and freezing and drying, which make this process very timeconsuming (more than 1 week); (2) it uses some organic solvents, which are difficult to be eliminated and which can remain entrapped inside the nanometric network, and can compromise the subsequent growth of cells in the scaffolds; and (3) it is difficult to preserve three-dimensional structure due to the surface tension of the liquid solvents during the drying step. A collapse of the porous structure is very frequent. Until now, the use of supercritical CO2 (SC-CO2) in tissue engineering has been limited to gas-foaming techniques in which it is used as a porogen to produce polymeric foams.13-16 The process is solventless and very efficient in producing a porous structure, but generally closed or partly closed cells structures are generated. Barry et al.15 prepared methyl-methacrylate scaffolds by SC-CO2 foaming, claiming that the degree of porosity and interconnectivity of the scaffolds can be controlled simply by modifying the depressurization rate of the process: scaffolds with a porosity around 89% and with high connectivity (74% open pores) were obtained according to these authors. However, the rough nanofibrous internal structure that should mime the natural ECM is completely absent in this process, and a controlled 3-D structure is also problematic to obtain. SC-CO2 has also been used as an alternative nonsolvent in phase inversion processes to generate polymeric and biopolymeric membranes.17-27 In this case, the structures obtained present good interconnectivity and high porosity, but suffer various limitations; indeed, it is very difficult to obtain complex 3-D structures (flat membranes are usually generated), and the internal surfaces do not contain nanostructures. In a previous work, to overcome the limitations of LPI process and of the previously proposed SC-CO2 techniques, we have demonstrated the feasibility of a supercritical CO2 assisted process for the production of PLLA scaffolds.28 The process is similar to LPI for several aspects and consists of the following steps: (1) formation of a polymeric solution loaded with a solid porogen; (2) formation of a polymeric gel by thermally induced phase separation; (3) drying of the gel using SC-CO2 forming a supercritical mixture with the solvent used; and (4) washing with water to eliminate the porogen. The time-consuming steps of solvents exchange and extraction are substituted by a very fast supercritical process that is performed at zero surface tension; therefore, the collapse of the structure is avoided. The aim of the present work is to attempt a further evolution of this technique, producing PLLA/ceramic scaffolds using hydroxyapatite (HA) nanoparticles, to increase the biomimethism and to improve the mechanical properties of the scaffolds.
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An analysis of the process parameters and of their influence on the fibrous nanostructure is also performed. Materials and Methods Materials. Poly(L-lactic acid) (PLLA) L210 (MW 210 000) and PLLA L209S (MW 140 000) were purchased from Boehringer Ingelheim (Ingelheim, Germany), D-fructose (mp 119-122 °C), dioxane, ethanol (99.8% purity), and hydroxyapatite nanoparticles (particle size < 200 nm) were bought from Sigma Aldrich (St. Louis, MO), and CO2 (99% purity) was purchased form SON (Societa` Ossigeno Napoli - Italy). All materials were used as received. Preparation of the Composite Scaffolds. Scaffolds were prepared according to the following procedure. Solutions with PLLA concentrations of 15% w/w in dioxane were prepared, and then ethanol as the nonsolvent was added at a dioxane/ ethanol ratio of 1.7 as suggested in the literature;29 the solution was stirred and heated at 60 °C until it became homogeneous. Next, we added fructose particles with diameters ranging between 250 and 500 µm and hydroxyapatite nanoparticles (diameter
