Preparation And Modification Of Poly L-lactic Acid Porous Scaffold And Manufacture Of Tissue Engineering Cartilage

The preparation, modification, and biocompatibility of poly-L-lactic acid (PLLA) scaffold for cartilage tissue engineering were studied. The PLLA scaffolds with porous, highly interconnective 3-D structure were fabricated via thermally induced phase separation (TIPS) and gelatin porogen leaching methods. The scaffolds were then modified by layer-by-layer assembly method, gelatin coating by porogen leaching method or hydrogel-filled method to enhance their biocompatibility. Finally, these modified scaffolds were implanted into the nude mice subcutaneously to determine their effect on cartilage regeneration.In TIPS two different coarsening protocols, i.e. normal coarsening and multi-step coarsening were compared in consideration of phase separation and domain growth. A normal coarsening route produced scaffolds with pore size from several micrometers to 150nm depending on the coarsening time after phase separation, accompanying with the emergence of isolated pores at long time coarsening. Scaffolds with large pores with size up to ～300μm were fabricated by the two-step coarsening technique, e.g. the PLLA-solvent (dioxane/water) system was coarsened at a temperature after phase separation for a period, followed by coarsened at a lower temperature for another period. In parallel with formation of the large pores, the interconnectivity between pores was also improved, which was evidenced by scanning electron microscopy, gelatin solution pervasion and collagen entrapment.PLLA scaffolds with well-controlled interconnected round or irregular pores were also fabricated by a porogen-leaching technique using gelatin spheres or particles as the porogen. The gelatin spheres or particles were bonded together through a treatment in alcohol solution or saturated water vapor condition at 70℃ to form a three-dimensional assembly in a mold. PLLA was dissolved in dioxane and was cast onto the gelatin assembly. The mixtures were then freeze-dried or dried at room temperature, followed by removal of the gelatin particles to yield the porous scaffolds. The microstructure of the scaffolds was characterized by scanning electron microscopy with respect to the pore shape, inter-pore connectivity, and pore wall morphology. Compressing measurements revealed that scaffolds fabricated by freeze-drying exhibited better mechanical performance than that by room temperature-dying. Along with the increase of polymer concentration, the porosity of the scaffolds decreased, while the compressive modulus increased.Layer-by-layer (LBL) assembly of cytocompatible chondroitin sulfate (CS) and collagen type I (Col) onto PLLA scaffolds were performed aiming at enhancement of cell-material interaction. To introduce charges onto the hydrophobic and neutral PLLA surface so that the electronic assembly can be processed, the PLLA was aminolyzed in hexane diamine solution to obtain free amino groups that can be positively charged at neutral pH. UV-Vis spectroscopy and ninhydrin analysis verified the consecutive deposition of CS/Col on the aminolyzed PLLA membranes. Chondrocyte culture found that the existed CS and Col greatly improved the cytocompatibility of PLLA with respect to cell attachment, proliferation and MTTviability. CLSM observations showed that the chondrocytes adhered and spread well, and distributed evenly in the entire PLLA scaffolds assembled with 1 bilayers of CS/Col.The biological performance of the scaffolds prepared via gelatin particles method was assessed by in vitro chondrocyte culture and in vivo implantation. In comparison with the scaffold fabricated with NaCl particles as porogens at the same conditions, the experimental scaffold has better biological performance because the gelatin molecules are stably entrapped into the pore surfaces. A larger number of cells in the experimental scaffolds were observed under confocal laser scanning microscopy (CLSM) after the viable cells were stained with fluorescein diacetate. Cells show more spreading morphology. Similar results were observed by scanning electron microscopy. Moreover, higher cytoviability and GAG secretion were determined in the scaffolds too.Polymer porous scaffolds and hydrogels have been separately employed as analogues of the native extra-cellular matrix (ECM). However, both of these two kinds of materials have their own advantages and shortcomings. An attempt to combine the advantages of these two kinds of materials is carried out. PLLA scaffolds with good mechanical properties were prepared by thermally induced phase separation, which were then filled with hydrogel aiming at entrapment of cells within a support of predefined shape. Agar, which has a function to promote chondrogenesis, was selected to entrap chondrocytes, acting as analogues of native ECM. A straight forward merit of this construct is that both mechanical strength and macroscopic shape, and analogous ECM can be simultaneously achieved. The morphology and distribution of the chondrocytes were studied by CLSM and scanning electron microscopy. The cell growth behaviors were determined by MTT assay and collagen and glycosaminoglycan (GAG) secretion. After culture for 7 and 14d, the cells in the construct were round and surrounded by the hydrogel. The MTT viability and the cell secretion in the chondrocytes/agar/scaffold construct were also higher than that of the chondrocytes/scaffold construct. Gelatin was further introduced into the construct, yielding improved GAG secretion and cytoviability.Finally, two kinds of scaffolds were selected for animal test because of their good comprehensive properties. In the case of hydrogel-filled polymer scaffold, after implantation in the subcutaneous dorsum of nude mice for 4 weeks, cartilage-like specimens maintaining their original rectangular shapes were harvested. Histological examination showed that new cartilage was regenerated and a large quantity of collagen and GAG were secreted, while the cells in the control PLLA scaffold turned to be fibroblast-like with less secretion of extracellular matrices. The method provides a useful pathway of scaffold preparation and cell transplantation, which can achieve suitable mechanical properties and good cell performance. In the case of scaffold prepared via gelatin particles porogen, after implantation of the chondrocytes/PLLA scaffold construct to the subcutaneous dorsum of nude mice for 30-120 days, cartilage-like specimens were harvested. Histological examination showed that cartilages containing a large quantity of collagen and GAG were regenerated. These features indicate that these kinds of PLLA scaffolds have potential application intissue engineering, especially for cartilage regeneration.