Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Effect of the Pore Structure of an Apatite-Fiber Scaffold on the Differentiation of P19.CL6 Cells into Cardiomyocytes

We previously reported that P19.CL6 cells can be cultured in porous hydroxyapatite ceramics prepared by firing green compacts consisting of apatite fibers and spherical carbon beads (150 μm in diameter). Cells cultured for 20 days in an apatite-fiber scaffold (AFS) proliferated and differentiated into cells expressing troponin T, a cardiomyocyte-specific gene, but the expression level was insufficient to support the functional maturation of cells required for biomedical device applications. In this study, we aimed to optimize the internal AFS environment for cardiomyocytes by mixing two sizes (150-and 20-μm) of carbon beads. P19.CL6 cells were cultured in AFS materials comprising different carbon ratios in the presence of alpha-MEM with (AFS+) or without (AFS-) dimethyl sulfoxide (DMSO), and cell growth and gene expression were assessed. We found that AFS(50, 1:1 ratio) is the most suitable scaffold for the proliferation and differentiation of P19.CL6 cells and the addition of DMSO to the culture medium is necessary for differentiation into cardiomyocytes. We also assessed the culture of P19.CL6 cells in AFS in a radial-flow bioreactor for several days.

Reconstruction of Tissue-Engineered Bone Using an Apatite-Fiber Scaffold, Rat Bone Marrow Cells and Radial-Flow Bioreactor: Optimization of Flow Rate in Circulating Medium

We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the tissue-engineered bone with 3D structure through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB), and examined effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone. Aim in the present work is to establish of the optimal conditions of flow rate in this construction method of 3D tissue-engineered bone. The flow rates were set to 0.4, 1.3, 6.3, 11.5 and 16.5 cm3min-1; tissue-engineered bones cultured by the individual flow rates are defined as bones#1~#5. The level of differentiation of osteoblasts in all the bones#1~#5 was examined by determining the content of two kinds of differentiation maker into osteoblast, alkaline phosphatase (ALP) for initial/middle stage and osteocalcin (OC) for late stage. The ALP activity normalized for DNA content of bone#3 showed the highest value among all of them. Moreover, the OC amount normalized for DNA content of bone#3 also indicated the highest among all the examined samples. These results demonstarate that the flow rate of 6.3 cm3min-1 may promote the differentiation into osteoblast. In conclusion, we determined that this flow rate was the optimal conditions for the bone regeneratrion in RFB.