Large-scale Expansion Of Osteoblasts In A Novel Bioreactor

Osteoblast is an important seeding cell of bone tissue engineering with many virtues, such as extensive source, high ability of differentiation and proliferation in vitro. Therefore, it is very important to expand and culture osteoblasts in vitro.Cells from cranium of Sprague-Dawley (SD) rats were used for primary and passaged cultures. The biological morphology and properties were assayed, and the cultured cells were purified and determined to be osteoblasts without pollution of other cells. The osteoblasts could provide sufficient seed cells for bone tissue engineering for later researches and ensure the accuracy and repetitiveness of the experiments.A novel rotating wall hollow fiber membrane bioreactor (RWHFMB) was developed based on the characteristics of low shear stress, great deals of a three-dimensional (3D) communications among cells provided by rotating wall vessel bioreactor and the good biocompatibility possessed by acetyl cellulose hollow fiber membranes. The purpose was to set up an ideal 3D culture system for osteoblasts.By using the hollow fiber membranes as the carrier, large-scale expansion of osteoblasts was studied in the RWHFMB to provide seeding cells for bone tissue engineering. The fourth passaged osteoblasts with lxlO5 cells/ml inoculated density were cultivated in the 3D RWHFMB and in T-flasks as the controls. After five days culture the cells were harvested and assayed with inverted microscope, scanning electron microscope (SEM), and the cell specimens were stained with hematoxylin and eosin for the qualitative analysis of histological sections. Moreover, alkaline phosphatase (ALP), von-Kossa and Alizarin Red S staining on mineralized nodule formation were also performed. The results show that in RWHFMB, the cells present better morphology and vitality and secrete much more extracellular matrix. It is concluded that the RWHFMB combines the advantages of rotating wall vessel and hollow-fiber membrane bioreactors. The hydrodynamic stimulation within it accelerates the metabolism of osteoblast and mass transfer, which is propitious to the cell differentiation and proliferation.In conclusion, the proper techniques and operating parameters for expanding seed cells of bone tissue engineering in RWHFMB were determined. The novel bioreactor can provide culture environment with low shear stress and necessary 3D interactions among cells and are suitable for osteoblasts expansion in vitro, and the hollow fiber membranes can be regarded as a modified carrier to expand seed cells in large-scale in vitro. Moreover, with the stressstimulation inside the fluid in the RWHFMB, the ALP expression can be increased and the formation of mineralized nodules can be accelerated. The rapid proliferation and differentiation of osteoblasts are possible.