| The core of tissue engineering is a new field which involves research and development of viable body tissue or organ lesions to repair, maintain and improve the morphology and function of related tissues and organ by utilizing principles of engineering and life sciences. So far, both domestic and foreign tissue engineering research has achieved remarkable results and demonstrates great prospect for the application of surgical repair and reconstruction.Whether or not function alternative, seed cell selection, processing and application can be achieved after tissue-engineered tissue or organ transplantation became key issues within the field tissue engineering technology. Large amount of seed cells are required for construction of tissue-engineered products. The specific requirement is needed for the amplification of seed cell: Improve amplification efficiency by obtaining sufficient number of cells within a short time period. Due to these requirements, there is a demand for the high quality of the amplification technique. Shortage of seed cells is a major constraint of industrialization of tissue-engineering products. In order to address this problem, the major solutions and their inadequacies are: (1) Use of growth factors: high risk of inducing genetic mutations and the dose-effect relationship is unclear. (2) Improve the seed culture system by applying micro-carrier technology: difficulty in simulating in vivo microenvironment due to many technical flaws and it is expensive. (3) Transgenic technology: low transfection efficiency and a short in vivo expression period. All of the solutions exists their respective disadvantages and imperfections, therefore more and more researchers are seeking a more effective technical means.The doses of Low-Intensity Pulsed Ultrasound (LIPUS) can be as low 30mw/cm2 or lower in pulse emission mode. Currently, LIPUS has been widely used in bone and soft tissue healing as it is safe and efficient. In recent years, with more and more studies on LIPUS, the horizon of LIPUS application are widening with particular interest in the important role it plays in cell biology. LIPUS could promote the in vitro fibroblasts, osteoblasts and other cells'proliferation and secretion of extracellular matrix. This paper combines LIPUS and Bone Mesenchymal Stem Cells (BMSC) to explore the possibility of identifying in vitro amplification of seed cells for tissue-engineering.Objectives1. Observe the influence of LIPUS on BMSCs proliferation in different culture conditions (normal stem cell culture system and the low serum culture system) to induce the feasibility of LIPUS amplifying seed cells in tissue engineering.2. To explore mechanism of LIPUS'affection on the proliferation of BMSCs and provide a theoretical basis for LIPUS promoting the proliferation of BMSCs.Methods1. The third generation of BMSCs were seeded in 16 6-well plates and were divided into 4 groups according to their density: 3×103/ml, 5×103/ml, 1×104/ml and 5×104/ml. Holes numbered1, 2 and 3 of each plate were ultrasonic irradiation group (LIPUS group) and holes numbered4, 5 and6 holes were the control group. Each well plate was exposed to 20min of pulsed ultrasound daily using the following parameters: ultrasound power was 100mw; pulse width of 800ms and pulse interval of 200ms. Four well plates were processed during one LIPUS session, and cell growth was observed daily microscopically. Cell proliferation was determined by MTT pre-irradiation and 1d, 3d, 5d post-irradiation.2. (1) The third generation of BMSCs were seeded in 16 6-well plates with the density of 1×104/ml. These plates were randomly divided into LIPUS group and the control group (n=8). Ultrasound exposure and testing method used were detailed above; (2) the third generation of BMSCs was seeded in disposable culture flasks. These flasks were divided into LIPUS group and control group. The daily ultrasound irradiation time was 20min per flask. Cell cycles were determined pre-irradiation and 1d, 2d, 3d, 4d, 5d, 6d, 8d and 10d post-irradiation using flow cytometry.3. (1) Third generation BMSCs were cultured using culture medium containing 1% fetal bovine serum. The majority of the BMSCs were arrested at G0 stage of the cell cycle. (2) The BMSCs were seeded in 16 6-well plates with the density of 1×104/ml and these plates were randomly divided into LIPUS group and the control group (n=8). Ultrasound exposure and testing method used were detailed above. (3) Culture BMSCs in disposable culture flasks. These flasks were randomly separated divided into LIPUS and control groups with the ultrasound exposure and testing method used were detailed as above. Cell cycles were determined pre-irradiation, 1d, 3d and 5d post-irradiation with flow cytometry.4. BMSCs of the common stem cell culture systems and the low serum culture system were seeded in disposed culture flasks. Ultrasound exposure and testing method used were detailed above. RT-PCR was used to determine mRNA expression of the BMI-1 gene in pre-irradiation, 0d, 1d, 3d, 5d and 7d post-irradiation. BMI-1 gene mRNA expression were in primary BMSCs were simultaneously detected.Results1. Morphology: Cells of each density group showed long spindle, polygonal and strong refraction. 3×103 group: microscopic observation could not find any significant differences between the LIPUS group and the control group. Cells of each group were scattered and sparse colony; 5×103 group: Cells of LIPUS group were formed cluster hyperplasia lesions 1d post irradiation, while the control group continued to show a scattered single cell growth. 3d, 5d post-irradiation the LIPUS group and the control group showed a clustered cell growth, but LIPUS group colony is more intensive. 1×104 group: the cell proliferation of LIPUS group was more active than the control group, and the cell colonies were more intensive in the LIPUS group. 5×104 group: Cell proliferation in the LIPUS-group was more active than the control-group's at 1d, 3d post-irradiation, but there was no significant difference on 5d. MTT:3×103 group: No significant difference was observed in the cell proliferation between the two groups (P>0.05); 5×103 group: there was no significant difference between the two groups on 0d and 5d (P>0.05), but on 1d and 3d the cell proliferation of LIPUS group was higher (P<0.05); 1×104 group: the cell proliferation of LIPUS group was significant higher than the control group (P<0.05); 5×104 group: rapid cell growth was observed and there were no significant difference between two groups(P>0.05). Observed the cell proliferation activity curves, the proliferation curve of 1×104 group was more stable, so it was as the best plank density in the follow-up study.2. Morphology: Cell proliferation in the LIPUS group was significantly more active when compared with the control-group. LIPUS group also had more intensive cell colonies; MTT test showed that cell proliferation in the LIPUS group on 1d, 3d and 5d post-irradiation were significantly higher than the control group (P <0.05); the cell cycle tested by flow cytometry showed that the cell proliferation rate (S + G2%) of the LIPUS-group were significantly higher than the control group after irradiation (P <0.05); the highest rate of cell proliferation which was higher than the control group could reach to 113.2% and the lowest was 21.1%. In the LIPUS group, the average value-added rate was 40.9% higher than the control group.3. Morphology observations showed that the cells of two groups were large flat and translucent enhancement. The cell-growth of LIPUS group was more active than the control group. MTT showed that after ultrasonic irradiation, the cell proliferation in the LIPUS-group was significantly more active when compared with the control-group(P<0.05); the cell cycle tested by flow cytometry also showed that the cell proliferation rate (S + G2%) in the LIPUS group were significantly higher than the control group on 2d, 4d and 6d post-irradiation(P <0.05).4. Under different culture systems, mRNA expression of BMI-1 gene of LIPUS group was higher than the control group (P <0.05); the mRNA expression of LIPUS group under normal culture conditions was significantly higher than the primary BMSCs and LIPUS group of the low serum culture condition (P <0.05). BMI-1 gene mRNA expression in LIPUS group under low serum culture condition was also higher than in primary BMSCs (P <0.05).Conclusions1. The cell inoculation density of 1×104/ml was more convenient for long-term studies and observation.2. The results suggested that LIPUS could promote the proliferation of the BMSCs and also effectively promote the BMSCs in quiescent state to enter cell division.3. LIPUS also promoted the proliferation of the BMSCs under low serum culture condition.4. LIPUS promoted BMSCs proliferation by regulating the expression of the BMI-1 gene.
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