Bone tissue engineering in a rotating bioreactor: A quantitative approach.

摘要

In this thesis, we have undertaken a quantitative approach toward the design, construction, and functional evaluation of a new microcarrier scaffold system of osteoblast-like cell culture for in vitro tissue engineering of bone in the High Aspect Ratio Vessel (HARV) rotating bioreactor. Using novel methods of numerical simulation and in situ particle tracking, we showed that microcarriers with density greater than the surrounding fluid exhibit a periodic orbital motion equal to their sedimentation speed, and an outward, radial migration leading to repeated collisions with the bioreactor wall. In contrast, lighter-than-water microcarriers exhibit an inward migration towards the center of the bioreactor, and avoid bioreactor wall collisions and consequent damage to attached cells and tissues.; To exploit this result, we fabricated novel lighter-than-water scaffolds based on hollow microcapsules of biodegradable poly(lactic-co-glycolic acid) (PLAGA). Individual microcapsules were thermally fused in predetermined size ranges to form three-dimensional (3-D) scaffolds with 25–40% internal pore volume and 100 to 300 μm median pore size. Scaffolds exhibited a controlled, collision free trajectory in the bioreactor with velocities in a range from 1 to 100 mm/s depending on scaffold properties. Peak shear stress imparted to cells on the exterior scaffold during culture ranged from 0.22 to 0.26 N/m2. Rates of internal fluid perfusion during scaffold motion were calculated using a mathematical model and ranged from 0.01–1 mm/s. Cells within interior regions of the scaffold experience peak shear stress far less (≈0.03 N/m2) than those on the exterior.; Bone cells cultured on microcarrier scaffolds in the rotating bioreactor attached homogenously to the scaffolds at densities ranging from 10 4–105 cell/cm2. Cells also retained their osteoblastic phenotype and showed significant increases in mineralized bone matrix synthesis after 7 days of dynamic cultivation in the bioreactor as compared to appropriate static culture controls. In addition, cells exhibited a robust expression of key bone marker genes such as alkaline phosphatase collagen I, and early expression of osteocalcin. These results show that the microcarrier scaffold system may be utilized to quantify functional differences in osteoblastic cell function, and to enhance the phenotype development osteoblast-like cells.