Response Of Cells To Substrate Topography And Fabrication Of PLGA Porous Scaffolds For Cartilage Repair

Cartilage repair is a classic yet still challenging topic in regenerative medicine. Tissue engineering provides a new strategy to repair the cartilage. Cartilage damage is often accompanied by bone damage, and the articular cartilage and underlying subchondral bone have different intrinsic structures and physiological functions. So, bilayered scaffolds have recently been suggested for osteochondral regeneration. Yet, some basic questions such as appropriate pores and porosity distributions still need to be explored.The development of new biomaterials relies on understanding of cell-material interactions. Various material cues have been revealed to influence cell behaviors. Among those cues, the surface topography is very important due to universality of this phenomenon and feasibility of the pertinent application. None of materials is not microscopically rough; the topography modification could be free of change of chemical composition, and thus the modified biomaterials are relative easy to be approved into clinical applications.This thesis is focused upon the effect of surface topography on cell responses and associated porous scaffolds with modified topography for cartilage tissue engineering. The popular biodegradable polyester poly(lactide-glycolide)(PLGA) was employed as the basic matrix. Firstly, we studied the cell response to microscale topography on2D films and obtained the basic rules; Secondly, we moved from2D to3D, and developed approaches to adjust the microscale topography on the pore wall of PLGA porous scaffolds; PLGA bilayered scaffolds were also tried to repair the osteochondral defects in rabbits in cooperation with surgeon groups. The research might help better understanding of the physical cues of biomaterials and stimulate designing and fabricating of new biomaterials for regenerative medicine. The main innovative achievements are listed as follows:1. Examination of cell responses to PLGA micropillars or micropits of a series of height or depth, and finding of appropriate dimensions to enhance cell adhesion on2D films. We fabricated PLGA micropillars or micropits of a series of height or depth, and then cultured menenchyma stem cells derived from rat bone marrow (BMSC) on these films. The micropillars or micropits with1μm height or depth improved the cell viabilities signficantly. The results trigger the fabrication of microscale topography on the interior pore surfaces of3D porous scaffolds.2. Finding of the phenomenon of serious self-deformation of cell nucleus on micropillars, and achieving of the control of cell nucleus shapes via designing micropillar patterns. We unexpectedly observed self deformation of the nuclei of BMSC in a case of micropillars, and made further investigation of this abnormal phenomenon. A series of micropillar arrays were fabricated, and dimensions for the occurrence of nucleus deformation were determined. The deformation on micropillars was not due to gravity, and might be generated by the cytoskeleton stress during cell adhesion. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. The shapes of cell nuclei could be controlled by designing the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes have been achieved for the first time, such as square, cross, dumbbell, and asymmetric sphere-protrusion. We further confirmed that the cell nucleus deformation occurred in several cell types besides BMSCs.3. From2D to3D:Preperation of novel porogens with surface topography and fabrication of large three dimensional porous scaffolds with interior pores templated by porogen surfaces. Under the porogen-leaching strategy, we modified the soft porogens of paraffin by colliding with small and hard salt particles, which generated micropits on the surfaces of paraffin spheres. The eventual PLGA scaffolds after leaching the modified porogens had thus interior surfaces of microscale roughness imprinted by those micropits. The microrough scaffolds were confirmed to benefit adhesion of BMSC and meanwhile did not hamper the proliferation and osteogenic differentiation of the cells. Moreover, we prepared a new porogen called as sugar-glued spherical salt by us and obtained the porous scaffold with spherical pores, excellent inter-connectivity, an interior surface of microroughness. The leaching time by water was found to be much shortened, and growth of BMSC was enhanced in the improved scaffold without any harmful effects to cell adhesion and osteogenic differentiation. The insight and technique might be helpful for biomaterial designing in tissue engineering and regenerative medicine.4. Fabrication of PLGA bilayered scaffolds, and in a cooperative research, examination of the porosity effect and pore size effect on in vivo repairing of the osteochondral defect in a comparative way. We first evaluated the basic biocompatibility and degradation behaviors of PLGA porous scaffolds in vitro or in vivo according to the GB standard protocols. Then, we fabricated bilayered scaffolds with different porosities and pore sizes in the two layers and examined in vivo efficacy to repair osteochondral defect. The constructs of scaffolds and allogenic BMSC were implanted into pre-created osteochondral defects (4mm in diameter and5mm in depth) in the femoral condyle of New Zealand white rabbits. After12weeks, all of experimental groups exhibited good cartilage repairing according to macroscopic appearance, cross-sectioned view, hematoxylin and eosin staining, toluidine blue staining, immunohistochemical staining, and real-time PCR of characteristic genes. The group of92%porosity in the cartilage layer,77%porosity in the bone layer and100-200μm pore size in the cartilage layer,300-450μm pore size in the bone layer exhibited the best result.During the short visiting time in the University of Michigan, I synthesized two-component polyanhydride of sebacic acid (SA) and1,3-bis(p-carboxyphenoxy) propane (CPP) with surface erosion property, and then designed delivery device to achieve pulsatile release of drug.