The Effects of Cyclic Mechanical Stimuli on the Osteoblast-like Cell Differentiation in a 3-D Scaffold

Kuiwon Choi and J.K. Kim

Biomedical Research Center, Korea Institute of Science and Technology

Seoul, Korea

Objective: Tissue regeneration represents a new direction toward bone regeneration. This is based on the premise that progenitor cells are capable of providing an environment to induce cell migration from the surrounding tissue into the wound site. Subsequently, progenitor cells proliferate, differentiate, and ultimately regenerate the tissue. Nowadays, tissue engineering attempts to create tissue replacement by culturing cells on synthetic three-dimensional scaffolds.  Many researchers have conducted experiments to study the effects of mechanical stimulation on bone cell proliferation and differentiation. However, they have not been well understood since most of the experiments involving mechanical stimuli were performed in two-dimensional cultures and in fluid-induced shear conditions using rotating bioreactors.

The purpose of our study was to examine the effect of applied cyclic compressive strain on the proliferation and differentiation of MC3T3-E1 cell cultured in a three-dimensional chitosan scaffold.

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Materials and Method: MC3T3-E1 cells, a mouse osteoblast-like cell line, were grown in α-minimal essential medium containing 10% fetal bovine serum (FBS) and penicillin streptomycin (100µg/ml). Cells were maintained in a humidified incubator at 37°C with 5% CO₂, 95% air and were subcultured every 3 days. For mechanical stimuli studies, these cells were seeded to three-dimensional chitosan scaffolds at 1× 10⁶ cells/scaffold. A cyclic compressive strain was applied to the specimens using a specially-designed testing apparatus which also allows the cells to be simultaneously visualized using an inverted microscope.  The cyclic compressive strain (2.5%) was applied over a period of 17 days with 150 cycles per day at a frequency of 0.5Hz.  The amount of protein and alkaline phosphatase activity were evaluated for the degree of differentiation and proliferation at the indicated times (day 4, 7, 10, 14, 17).

Total protein was estimated for the degree of cell proliferation using Bredford method and alkaline phosphatase was estimated for the initiation time of cell differentiation using a commercially available kit. Experimental images were acquired from CCD camera using image acquisition board.  Image processing methods were utilized for cell deformation.  In order to prevent the cells from contaminating, all experiments were performed within acryl chamber which was specially made for sterilization using an air circulating device and UV light.

Results:  The total amount of protein produced in the control group was higher than the mechanically stimulated group.  This was due to cell death for the nodule formation of the mechanical stimuli group which resulted from cell differentiation.  During the MC3T3-E1 cell differentiation, alkaline phosphatase activity was used as a mark of osteoblast differentiation. The alkaline phosphatase activity increased slightly in the control group, and statistically significant maximum point was not observed.  In the mechanical stimuli group, the level of alkaline phosphatase activity increased significantly and reached its peak on day 7. After maintaining the peak level of alkaline phosphatase activity for few days, its level dropped sharply on day 14.  This point was statistically significant maximum point (P<0.05), indicated the beginning of cell differentiation. We also acquired images for examining the degree of cell differentiation from inverted microscope. At day 7, the black spots were observed.  They initially became visible on starting cell differentiation in the mechanical stimuli group, however, these spots were not found in the images of control group during the experimental period. Conclusively, it could be noted that the effects of the differentiation in the mechanically stimulated group was significant.

Summary

In this study, an osteoblast-like cell was cultured in three- dimensional chitosan scaffolds and a cyclic compressive strain was applied to the scaffolds. The effects of the mechanical stimuli were significant in the cell proliferation and differentiation process as well as calcification process.

Key words: Cyclic Mechanical Stimuli, Cell Differentiation, 3-D Scaffold, Tissue Engineering, Bone regeneration