| Background and objective:In microgravity environment, there are bone loss, bone density decreased and bone mechanical properties decreased, showing the obvious symptoms of osteoporosis. To eliminate this effect, the measures commonly used are physical exercise and drug treatment, but these measures cannot completely eliminate the effects of microgravity on osteoporosis. With the development of manned space technology, there will be more and more astronauts getting into space, therefore, to find effective measures against osteoporosis induced by microgravity environments is very meaningful. As a negative regulator of bone formation, CKIP-1 knockout can lead to bone mineral density and bone mass increased significantly. Therefore, we can try to take method of CKIP-1 gene knockout against the effects of microgravity on bone metabolism. Although the spaceflight can be equipped with experiments of microgravity on biology, there have great limitations. Consequently, we commonly use animal models of tail suspension to simulated microgravity on the ground. In addition, the osteoblasts responsible for bone formation are derived from BMSCs. In the microgravity environment, how do the BMSCs directional osteogenic differentiation? Is there any relationship between BMSCs and CKIP-1 knockout leading to increased bone mass? Therefore, we also conducted a study on the osteogenic differentiation of BMSCs by using RCCS to simulate microgravity environment.This study used mouse tail suspension model established to deal with WT mice and KO mice for 2 and 4 weeks. The symptoms of osteoporosis in different groups were compared to reveal the effects of CKIP-1 on the balance of bone ossification and breaking into ossification under microgravity environment, providing a new idea for the treatment of osteoporosis induced by microgravity. In addition, the research of BMSCs osteogenic differentiation under microgravity environment can be helpful to further study of CKIP-1 knockout on BMSCs osteogenic effect, and fully reveal the process of bone reconstruction under microgravity condition. Contents and Methods:(1) The establishment of tail suspension mice model to simulated microgravity.Reference the rat tail suspended model, we improved suspension cage structure, optimized suspension condition, and finally established the mice tail suspension model to simulate microgravity. Select the 2-3 month old C57BL/6J mice, male and female were randomly, using medical adhesive tape fixed mouse tail to suspension cage. Height adjustment to ensure the mice hind limbs off the ground, the horizontal angle between the fore limbs and the trunk is about 30 degrees, and they can drink water and feed freely. Selecting mice matched with the WT mice in the model groups to feed under normal condition, as the control groups. Comparison the concentrations of serum calcium, ALP and BGP of different mice groups; Micro-CT scan the femurs, to measure the changes of bone microstructure parameters; three point bending test on the femurs by material testing machine, to compare the changes of mechanical parameters.(2) The effects of CKIP-1 knockout on osteoporosis under microgravity.The tail suspension test of WT mice and KO mice for 2 and 4 weeks was carried out by using the model established. The concentrations of serum ALP and BGP in different mice groups were detected by spectrophotometry and ELISA methods; Micro-CT scan the bone microstructure of femurs, to compare the differences of trabecular parameters of bone microstructure, such as BV/TV, Tb.Pf, Tb.Th, Tb.N and Tb.Sp; HE staining and immunohistochemical comparison of the femur section shape to detect the changes of mechanical properties; three point bending test compared fracture displacement, fracture strain, fracture load and fracture energy; ELISA method was used to detect the serum bone formation marker Pâ… NF and bone resorption marker CTX-â… ; RT-PCR and western blot were used to compare the expression levels of osteogenic genes and proteins.(3) The effects of microgravity on osteoblastic differentiation of BMSCs.Using density gradient centrifugation, the primary mouse BMSCs were extracted, and their purity were identified by surface antigen flow cytometry. Simulated microgravity environment was got in RCCS cell culture, adding osteogenic inducer for 14 days in culture to investigate BMSCs direction changes. Spectrophotometry was used to detect the changes of ALP concentration in culture supernatant, differences of the cell ALP staining kit levels, and western blot was used to detect the expression of bone related proteins. Results:(1) Establish the mice tail suspension model to simulated microgravity.Compared with the WT mice, the concentrations of serum calcium in TS mice significantly increased, and concentrations of ALP and BGP were significantly decreased; Micro-CT scanning results showed that the BV/TV decreased, Tb.Pf increased, Tb.Th decreased, Tb.N decreased and Tb.Sp increased; three point bending test proved that the fracture displacement, fracture strain, fracture load and fracture energy of the femurs were all decreased. Compared with the WT mice, TS mice regardless of biochemical indexes, microstructure, or mechanical properties, showed obvious symptoms of osteoporosis, proving that the microgravity model was successfully established which layed the foundation for the subsequent research.(2) The research of CKIP-1 knockout on osteoporosis under microgravity.Compared with the untreated mice, TS groups showed obvious symptoms of osteoporosis, both WT and KO mice, and the symptoms of osteoporosis become more obvious with the suspension of TS time, further proved that our model was successful. The concentrations of mice serum ALP and BGP of TS groups decreased; the parameters of bone trabecular decreased and bone microstructures were more loosely; femur sections with HE staining and BGP immunohistochemical staining showed that the ossification degree of femurs to be weaker; mechanical properties decreased; the concentration of serum P â… NF, a bone formation markers, decreased, but the concentration of CTX-â… , an osteoclast marker, remained unchanged; the expression levels of bone related genes and proteins, such as BMP-2, Col- â… and Runx2, decreased significantly.But under the same TS conditions, compared with the WT mice, the KO mice had higher concentrations of serum ALP and BGP; Micro-CT scanning showed that BV/TV increased, Tb.Pf decreased, Tb.Th increased, Tb.N increased, and Tb.Sp decreased, the bone microstructures were more compact; femur sections with HE staining and BGP immunohistochemical staining showed that the ossification degree was much stronger; the three point bending test showed that the related parameters of mechanical properties were higher with better mechanical performance; the concentration of bone formation marker Pâ… NF was higher, but the concentration of bone resorption marker CTX-â… remained unchanged; the expression levels of osteogenic related genes and proteins, such as BMP-2, Col-â… and Runx2, were significantly increased. All the experimental results showed that, under the same TS conditions, compared with the WT mice, the osteoporosis symptoms of KO mice were lighter, fully proved that the effects of CKIP-1 knockout on microgravity, and CKIP-1 knockout had some certain antagonism to osteoporosis caused by microgravity. At the same time, it also proved that CKIP-1 only affected bone ossification and had almost no effect on bone resorption.(3) The research of microgravity on osteoblastic differentiation of BMSCs.In the induction of osteoblastic induction medium, BMSCs had obvious osteogenic differentiation. In simulated microgravity of RCCS culture condition, the concentration of ALP in BMSCs culture solution was decreased; the degree of ALP staining was shallow; the expression of steoblastic related proteins BMP-2, BGP and Col- â… decreased, proved that BMSCs reduced the osteoblastic differentiation under the microgravity environment, and microgravity could inhibit osteogenic differentiation of BMSCs. This research provides a reference for the further study of CKIP-1 knockout and high expression to the osteogenic differentiation of BMSCs under microgravity environment. Conclusions:We successfully established the mouse model of tail suspension to simulate microgravity. Under the same TS conditions, many studies have found that KO mice compared with WT mice showed mild symptoms of osteoporosis, demonstrated that CKIP-1 knockout could offset partly the mice osteoporosis induced by microgravity, and it provided a new idea for the treatment of osteoporosis. But this effect only was in the bone ossification, and almost had no effect on bone resorption. In addition, we found that microgravity can affect the osteogenic of mice BMSCs, and microgravity could inhibit the differentiation of BMSCs into osteoblasts, laiding the foundation for the subsequent research of CKIP-1 knockout and high expression to osteogenic differentiation of BMSCs. |
