探究间歇式轴向压应力对组织工程骨种子细胞黏附、增殖与成骨分化能力的影响。

构建表达绿色荧光蛋白的兔骨髓间充质干细胞（rBMSCs）作为示踪种子细胞，运用旋转细胞培养仪将松质骨支架和种子细胞共培养7 d获得组织工程骨（TEB），实验组在第7 ~ 14天施加大小10 N、频率1 Hz、4 h/d的间歇式轴向压应力刺激，对照组常规培养，14 d后胰酶消化法获取两组种子细胞并比较其黏附、增殖和成骨分化能力。采用两组独立样本t检验进行统计学分析。

（1）流式细胞术显示rBMSCs被成功提取分离。（2）倒置荧光显微镜及扫描电镜显示TEB中种子细胞与支架相容性良好。（3）活体荧光成像系统及扫描电镜显示应力刺激组种子细胞的生长状况要优于非应力刺激组，前者平均荧光密度及细胞数/500倍视野均大于后者，差异均具有统计学意义（平均荧光密度：（3.75±0.34）×10⁸ vs （2.91±0.22）×10⁸，t = 2.90，P = 0.04；细胞数/500倍视野：30.50±4.43 vs 21.00±5.13，t = 3.14，P = 0.01）。（4）细胞黏附实验显示，应力刺激组种子细胞的75%细胞贴壁时间短于非应力刺激组，两组时间分别为（3.00±0.41）h、（13.33±1.70）h，差异具有统计学意义（t = 8.20，P < 0.01），前者的最终细胞贴壁率高于后者（99.97%±0.34% vs 85.83%±1.18%），差异具有统计学意义（t = 11.31，P < 0.01）。（5）CCK-8检测显示，在培养第48 ~ 96 h，应力刺激组种子细胞的增殖能力优于非应力刺激组，将两者的450 nm吸光度值在第48小时（0.49±0.02、0.40±0.02）、72 h（0.76±0.07、0.64±0.04）和96 h（1.58±0.07、1.34±0.13）分别进行比较，差异均具有统计学意义（t = 5.15、2.57、2.86，P均< 0.01）。（6）在成骨诱导14 d后，应力刺激组种子细胞的ALP和Ca结节染色阳性率要强于非应力刺激组：两组ALP染色阳性率分别为26.73%±4.56%、16.68% ± 3.89%，差异具有统计学意义（t =3.33，P = 0.03）；两组Ca结节染色阳性率分别为41.81%±3.56%、27.40% ± 2.35%，差异具有统计学意义（t = 3.68，P = 0.02）。

间歇性轴向压应力可促进组织工程骨种子细胞的黏附、增殖与成骨分化。

To investigate the effect of intermittent axial compressive stress on adhesion, proliferation and osteogenic differentiation of seed cells in tissue engineered bone.

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.

(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)×10⁸ vs (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).

Intermittent axial compressive stress stimulation could accelerate adhesion, proliferation and osteogenic differentiation of seed cells in TEB.