Application of Perfusion Culture System Improves in Vitro and in Vivo Osteogenesis of Bone Marrow-Derived Osteoblastic Cells in Porous Ceramic Materials

Abstract

Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue.

Engineering Musculoskeletal Tissues with Human Embryonic Germ Cell Derivatives

“…2J). The upregulation of ALP activity in early stages of osteogenic differentiation is considered a prerequisite for mineralization and subsequent mature bone formation [30,31]. The observed profiles of ALP and calcium production are consistent with previous findings for osteogenesis of MSCs [9,31].…”

.BETA.TCP/Collagen Sponge Composite Enhances the Osteogenic Differentiation of Mesenchymal Stem Cells

“…In the present study, b-TCP/CS enhanced the expression of ALP mRNA and protein compared to CS in the presence of the above differentiation medium. WANG et al 7) supports the findings of our study that b-TCP not only provides a favorable environment for osteoblast attachment and growth, but also acts as a nucleation site for the deposition of calcium and phosphate ions and the formation of apatite crystals so that an apatite layer is easily formed and bone formation improved. On the other hand, a similar tendency was observed between two materials in the expression of OCN mRNA.…”

Gene expression analysis of major lineage‐defining factors in human bone marrow cells: Effect of aging, gender, and age‐related disorders

“…[20][21][22][23][24][25][26] As a means of bone regeneration, a technique for the transplantation of cultured bone [27][28][29][30][31][32][33][34][35][36][37] has been developed to overcome the disadvantages of autologous bone grafts and simple implantation of biomaterial into bone defects. In cultured bone transplantation therapy, cbfa1 is not only a marker of osteoblast differentiation, but also an activator of osteogenesis.…”