| Along with the social aging, osteoporosis and other bone related symptoms and diseases have caused extensive concern of society and attention. Bone health has become a focus of social attention. At the same time, due to physical damage, such as a car accident, and postoperative bone defect caused by bone tumor, a reasonable diet is required to promote human body bone tissue regeneration and repairing. Astragalus has been as a part of the diet formula coming into people’s life. Its main ingredient, astragalus polysaccharide(AP), has been proved to be possessed the immunomodulatory avtivity, antioxidant activity, antitumor activity, antidiabetic activity, antiviral activity, hepatoprective activity, anti-inflammatory activity and promoting hematopoietic activity. In this study, we aim to evaluate the biological effects of astragalus polysaccharides on bone cells growth and differentiation through in vitro cell replacement methods.In the first part, we chose the mice osteoblast cell line MC-3T3-E1 as in vitro cell model for the evaluation. It was found that the half inhibitory concentration(IC50) of AP on MC-3T3-E1 cells was 12.22 mg/m L detected by 24 h cytotoxicity test. Then according to the cell IC50, the concentration gradient of AP in this study was 0.001, 0.01, 0.1 and 1 mg/m L. Afterwards, the cell proliferation and differentiation in two-dimensional(2D) culture were determined via CCK-8 test, calcium deposition, alkaline phosphatase(ALP) activity, quantitative real-time polymerase chain reaction(RT-PCR) analysis and confocal laser immunofluorescence observations. The results demonstrated that AP, ranged from 0.001 to 0.1 mg/m L, showed the effect of enhancing the cell proliferation with the increase of its concentration. Meanwhile, AP also performed the effect of promoting the osteogenic differentiation of MC-3T3-E1 cells, indicated by ALP activity and calcium deposition. The gene expressions of Runx2, osteopontin(OPN) and osteocalcin(OCN) were also improved in AP groups, which further demonstrated that AP could promote the osteogenic differentiation of MC-3T3-E1 cells. Furthermore, AP could also promote the secretion of Col-I with the observation of immunofluorescence staining. Above all, AP showed the effects of promoting MC-3T3-E1 osteoblasts proliferation and osteogenic differentiation, which indicated that AP could promote the regeneration of the bone tissue.In the second part, we want to try some three-dimensional(3D) evaluation methods based on microcarriers. At first, the microcarriers mainly made of gelatin were fabricated with the assist of alginate via a microsphere generating device. The concentration of Ca Cl2, the concentration of gelatin/alginate solution, voltage and bio-electrospraying parameters of nozzle size were systematically investigated in order to allow stable production of size-controlled microcarriers. And the final fabricated conditions in the subsequent studies were that the concentration of Ca Cl2 was 0.1 M, the concentration of gelatin/alginate solution was 12%(w/v), the voltage was 10 Kv, the distance between nozzle and Ca Cl2 was 10 cm and bio-electrospraying parameters of nozzle was 0.6 mm.In the third part, the 3D cell culture using gelatin/alginate microcarriers was employed. MC-3T3-E1 osteoblasts were seeded on the surface of the microcarriers(cell/microcarriers) and then the cell/microcarriers method was used to determine the biological effect of AP on the MC-3T3-E1 osteoblasts. The cell proliferation and differentiation were also determined via CCK-8 test, ALP activity and RT-PCR analysis using the same concentration of AP as 2D cell culture. After 1 d culture, AP had not performed the promotion of cell proliferation. And the promotion of cell proliferation was observed in AP groups after 3 d culture. However, when the concentration of AP increased to 1 mg/m L, little promotion of cell proliferation was observed. The cells cultured on the surface of the microcarriers grew well and performed long spindle shape. In this 3D culture condition, AP also performed the effect of promoting the osteogenic differentiation of MC-3T3-E1 cells, indicated by ALP activity and osteogenic gene expression. It was concluded that AP possessed the effects of promoting MC-3T3-E1 osteoblasts proliferation and osteogenic differentiation in the 3D cell/microcarriers culture. Meanwhile, as the 3D cell/microcarriers culture was conducive to the mutual contact between the cells and the stability of the microenvironment, it was indicated that the 3D cell/microcarriers culture was of great value for the biological evaluation of nutrients as an in vitro cell replacement method.In the fourth part, another 3D culture method, cell encapsulated gelatin/alginate microcarriers(cell@microcarriers), was employed to determine the biological effect of AP on the MC-3T3-E1 osteoblasts. The cell proliferation and differentiation were also determined via CCK-8 test, ALP activity and RT-PCR analysis using the same concentration of AP as 2D cell culture. After 1 d culture, AP had not performed the promotion of cell proliferation, either. And the promotion of cell proliferation was also observed in AP groups after 3 d culture. In this 3D culture condition, AP also performed the effect of promoting the osteogenic differentiation of MC-3T3-E1 cells, indicated by ALP activity and osteogenic gene expression. It was concluded that AP possessed the effects of promoting MC-3T3-E1 osteoblasts proliferation and osteogenic differentiation in the 3D cell@microcarriers culture. And in this 3D cell@microcarriers culture, the distribution and the state of the cells were closer to the condition in the tissue. So we speculated that the 3D cell@microcarriers culture was of great value for the biological evaluation of nutrients as another in vitro cell replacement method.In the fifth part, the MC-3T3-E1 cell proliferation and differentiation in different culture methods was compared. It was found that the cell proliferation was more obvious in 2D culture after 1 d culture. However, the cell proliferation was more obvious in 3D cell/microcarriers culture after 7 d culture, which indicated that the 3D cell/microcarriers culture was more suitable for the later evaluation of cell proliferation. Although, relatively weak cell proliferation rate was found in 3D cell@microcarriers culture, the 3D cell@microcarriers culture could also reflect the biological effects of AP on the proliferation of MC-3T3-E1 cells. The ALP activity and osteogenic gene expression were observed higher in the two 3D culture conditions, especially in the 3D cell@microcarriers culture, than in 2D culture condition, which indicated that the 3D culture conditions were more conducive to the evaluation of AP on promoting the osteogenic differentiation of MC-3T3-E1 cells. Above all, it was concluded that the three culture methods are all applied to evaluate the biological effects of AP as in vitro cell replacement methods. And the 2D culture condition is more suitable for early evaluation of cell proliferation, while 3D cell/microcarriers culture is more suitable for the long-term evaluation. What’s more, 3D cell@microcarriers culture is of great potential in the evaluation of cell differentiation. |
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