3D scaffolds for tissue engineering produced by microfabrication technology

Rapid prototyping techniques (RP) hold great promise for designing 3-dimensional (3-D) regular and ordered scaffolds for tissue engineering applications. With these techniques, good architecture reproducibility as well as porosity control of the structure can be obtained. This work dealt with the fabrication of tissue engineering scaffolds with regular micrometric geometry by using an in-house built microfabrication system. Poly(D,L-lactic acid) (PDLA), poly(D,L-lactic-co-glycolic acid) (PLGA) and chitosan scaffolds presenting homogeneously distributed 100 µm size pores were fabricated. Fabrication consisted in a layer by layer deposition of filaments of PDLA and PLGA dichloro methane/dimethylformamide (DMC/DMF) solutions and chitosan acetic acid solutions, respectively, on a plate moving with micrometric precision in the x,y,z directions. Additional chitosan scaffolds filled with amorphous calcium phosphate (ACP) particles were also microfabricated, considering the possibility to take advantage of the osteoconductive character of ACP for bone tissue regeneration applications. The in-house built system utilizes highly accurate 3-D micro-positioning slides having a resolution up to 1 µm. Through a microsyringe equipped with a micro-needle having 60 µm inner diameter, an automatic pumping system extrudes a filament of the selected solution on a plate. The plate is connected to three slides moving independently in the x,y,z directions. A computer controls the slides movement so that the filament that deposits on the plate builds layer by layer scaffolds of designed geometry. Rheological tests were used to characterize the polymer solution viscosities while thermal analysis (DSC), ATR-FTIR and dynamic mechanical tests (DMTA) have characterized the produced scaffold. Cast films from the same polymer solutions were used as control. Preliminary biological evaluations were done by seeding on the scaffolds osteoblasts (MG63) and fibroblasts (MRC5) cell lines. SEM and LV-SEM imaging evidenced scaffold morphology and cell adhesion and growth behavior. Surface topography of ACP filled chitosan scaffolds has been determined by atomic force microscopy (AFM) and their surface elemental composition evaluated by energy dispersive spectroscopy (EDS). In addition to the above activity a second part of the work revolves around fused deposition modeling (FDM) scaffolds cell cultured with human osteoblasts over different time. Human osteoblasts, isolated from the tibial sponge bone, were seeded on medical-grade polycaprolactone-tricalcium phosphate (mPCL-TCP 80:20) and poly(D, L lactic acid) - tricalcium phosphate (PDLLA-TCP 90:10) scaffolds. Furthermore, once the cells had reached the confluent stage, osteogenic media was used during cell culture to induce matrix formation. The newly formed matrix could provide a physical support forming an osteoblast sheet layer that was used to wrap the scaffold. Cells attachment, growing and proliferation were measured by imaging analysis techniques. Cells viability was evaluated by confocal laser microscopy after fluorescein diacetate (FDA)/ propidium iodide (PI) staining. The extent of cell proliferation was examined by PicoGreenTM quantification assay through the calculated cell DNA amount profile. In general, the work aimed at investigating how the two techniques, able to produce tissue engineering scaffolds with ordered structure, could assist the cellular growth and tissue regeneration.