关键词: 人骨髓间充质干细胞, 成骨诱导, 电针

Abstract:

BACKGROUND: Present studies have reported many methods of osteogenic induction, and provided many new strategies and methods of osteogenic induction of bone marrow mesenchymal stem cells (BMSCs). However, it remains unclear whether electroacupuncture can induce the differentiation of BMSCs into osteoblasts.

OBJECTIVE: To try to induce the differentiation of human BMSCs into osteoblasts using electroacupuncture therapeutic apparatus, and to assess the feasibility of BMSCs as bone tissue engineered seed cells.

METHODS: Bone marrow was collected form posterior superior iliac spine of patient. After identified as isolated and cultured BMSCs, the third passages of BMSCs were cultured. When spreading > 90% of the culture flask bottom, cells were digested by trypsin, and then incubated in a 6-well culture plate at 3.0×10³/cm². This study contained three groups. In the blank control group, cells were incubated in 2 mL L-DMEM/F12 containing 10% volume fraction fetal bovine serum. In the chemical induction group, cells were incubated in 2 mL L-DMEM supplemented with 10% fetal bovine serum. When cells grew 60%-70% confluency, bone inducer was added. In the electroacupuncture stimulation group, cells were incubated in 2 mL L-DMEM/F12 containing 10% volume fraction fetal bovine serum, subjected to electroacupuncture. Using continuous wave output, fundamental wave pulse frequency was 50 Hz, and fundamental wave pulse width was 0.5 ms, for 30 consecutive minutes, totally for 28 days. Phase-contrast inverted microscope was used to observe morphological changes. Alizarin red staining results and alkaline phosphatase activities following induction of 14 days and 28 days in cells were measured. Reverse transcription-polymerase chain reaction was utilized to determine osteocalcin mRNA expression in cells. Western blot assay was employed to detect osteocalcin protein contents in cells.

RESULTS AND CONCLUSION: During 28 days of induction, cells were confluent into a single layer at 5-7 days; cell processes connected each other, showed overlapping growth, without connection inhibition phenomenon in the chemical induction group. In the electroacupuncture stimulation group, cell volume became large, showing triangle, polygonal or squame shape at 9 or 10 days. In the blank control group, cells presented spindle shape. In the chemical induction and electroacupuncture stimulation groups, mineralized nodes appeared under an inverted phase contrast microscope at 28 days. Cells showed positive reaction to alizarin red. However, cells in the blank control group were negative for alizarin red. During in vitro induction of BMSCs, activities of alkaline phosphatase were greater in the chemical induction and electroacupuncture stimulation groups than the blank control group at 14 and 28 days (P < 0.05). Moreover, activities of alkaline phosphatase were higher in the chemical induction group than the electroacupuncture stimulation group at 14 days (P < 0.05). However, no significant difference was determined at 28 days (P > 0.05). Osteocalcin mRNA and protein contents were lower in the blank control group than in the chemical induction and electroacupuncture stimulation groups (P < 0.05). Above-mentioned results have suggested that electroacupuncture can induce differentiation of human BMSCs into osteoblasts.