An Experimental Study On Optimization Of Scaffold Microstructure And Cultural Environment For Composition Of Segmental Tissue Engineered Bone In Vitro

A segmental defect of long bone is always recognized as the most difficultly to be repaired in all bone defects in clinic work. However, because a systemic theory and technique was absent, which including cell culture, scaffolds fabrication, cell-scaffolds composition in vitro, et al, the segmental tissue engineered bone cannot be achieved as a transplant made in vitro by tissue engineering techniques to repair a segmental defect of long bone in clinic.Objective To investigate the new theory and methods about composition of a segmental tissue engineered bone in vitro in an approach of optimization of scaffolds microstructure and cultural environment: to modify the isolation and culture of human osteoblasts for acquiring a large amount of cells for bone tissue engineering study; to purpose a novel method for structure design and fabrication of segmental bone tissue engineering scaffolds, to develop a rotating perfusion bioreactor system to produce 3D dynamic culture conditions for segmental tissue engineered bone; to observe adhesion, growth and proliferation of the humanosteoblasts on or in the scaffolds affected by novel scaffolds microstructure and culture system in vitro. Methods1 Modification of the isolation and culture methods of human osteoblasts in vitro: Flank bone tissue was harvested and digested by trypsin for several times to get rid of blood cells and fibroblasts and bone chips were cultured in DMEM for 8-12 hours. Then osteoblasts were isolated and cultured by means of tissue block method. Osteoblasts' biological characters were observed and identified with inverted microscope observation, HE stain, alkaline phosphorase (ALP) and osteocalcin (OCN) activity assaying, particular stain of calcified node and ALP.2 Microstructure design and fabrication of segmental scaffolds for bone tissue engineering: A model of scaffold was designed by means of computer aided design (CAD) software. Data of the CAD model was imported into a stereolithography Rapid Prototyping (RP) machine to fabricate the resin mould. Then the mould was filled with Calcium Phosphate Cement (CPC) or P -tricalcium phosphate (3 -TCP), and then sintered to get rid of the mould and produce the scaffold. The microstructure of mould and scaffold was observed and measured under light microscope.3 Human osteoblasts co-culturing with segmental scaffolds: Osteoblasts were seeded onto the segmental scaffold carriers. 3, 7 and 14 days after seeding of incubation respectively, adhesion, growth, and proliferation of osteoblasts on the surface or inside the scaffolds were observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).4 3D dynamic culture system design and environmental optimization for segmental tissue engineered bone: A rotating perfusion bioreactor system was...