Intermittent axial compressive stress promotes adhesion, proliferation and osteogenic differentiation of seed cells in tissue engineered bone

Abstract

Objective To investigate the effect of intermittent axial compressive stress on adhesion, proliferation and osteogenic differentiation of seed cells in tissue engineered bone. Methods: Rabbit bone marrow mesenchymal stem cells that expressed green fluorescent protein were used as seed cells, a rotation training instrument was used to co-culture cancellous bone scaffold and seed cells together for 7 days to obtain tissue engineered bone (TEB), then the TEB were divided into two groups. One group was subjected to cyclic uniaxial compressive stress stimulation of a magnitude of 10 N, frequency of 1 Hz, and duration of 4 hours per day from days 7 ~ 14, the other group had no stress stimulation. Finally, two groups of seed cells were obtained by trypsinization, and their ability of adhesion, proliferation and osteogenic differentiation were compared. Results (1) Flow cytometry identification showed that rBMSCs were successfully isolated. (2) Inverted fluorescence microscope and scanning electron microscopy showed that the seed cells of TEB had good compatibility with the scaffold. (3) Results obtained from in vivo fluorescence imaging system and scanning electron microscopy showed that the growth of seed cells in stress-stimulation group was better than that in non-stress stimulation group. The average fluorescence density and cell number / 500-fold visual field of the former were larger than the latter, and the difference was statistically significant (average fluorescence density:(3.75 ± 0.34)×108vs (2.91 ± 0.22)×108, t = 2.90, P = 0.04; cell number / 500-fold visual field: 30.50 ± 4.43 vs 21.00 ± 5.13, t = 3.14, P = 0.01). (4) Cell adhesion experiments showed that cell attachment time of 75% seed cells in stress-stimulation group. With the time of (3.00 ± 0.41) h and (13.33 ± 1.70) h respectively, the difference was statistically significant (t = 8.20, P < 0.01). Besides, the final cell adherence rate of the former was significantly higher than the latter (99.97%± 0.34% vs 85.83% ± 1.18%), and the difference was statistically significant (t = 11.31, P < 0.01). (5) CCK-8 assay showed that the seed cells of the stress-stimulation group proliferated more rapidly than those of the non-stress stimulation group during the 48 th - 96 th h, and the 450 nm absorbance values were as follows: at the 48 th h, the values were 0.49 ± 0.02 and 0.40 ± 0.02 respectively, at the 72 th h, the values were 0.76 ± 0.07, 0.64 ± 0.04, at the 96 th h, the values were 1.58 ± 0.07, 1.34 ± 0.13, and the difference was statistically significant (t = 5.15, 2.57, 2.86, P < 0.05). (6) After 14 days since osteogenic induction, the positive rate of ALP and Ca nodule staining in seed cells of stress-stimulation group was significantly higher than that in non-stress stimulation group. The positive rates of ALP staining in the two groups were 26.73%± 4.56%and 16.68%± 3.89%respectively, and the difference was statistically significant (t = 3.33, P = 0.03). The positive rate of Ca nodule staining in the two groups were 41.81%± 3.56% and 27.40% ± 2.35% respectively, and the difference was statistically significant (t = 3.68, P = 0.02). Conclusion Intermittent axial compressive stress stimulation could accelerate adhesion, proliferation and osteogenic differentiation of seed cells in TEB. Key words: Intermittent; Axial stress; Bone marrow mesenchymal stem cells; Adhesion; Proliferation; Osteogenic differentiation