Voltage-gated Calcium Channels Effect The Osteogenic Differentiation Of BMMSC In Senile Osteoporosis Model

Senile osteoporosis is a special manifestation for organism's aging in bone tissues. With the aging process, the ratio of bone mineral composition to bone matrix is decreasing. In addition, the volume and intensity of trabecular bone in a unit volume also decreases. With the deterioration of global aging, senile osteoporosis led pain, bone fracture and expiratory dyspnea are becoming an important healthy and social issues. The zmpste24-/-mouse showing symptoms of aging is a good aging model. As a kind of precursor cell, bone marrow mesenchymal stem cells(BMMSCs) played an critical role in bone development and remodeling. BMMSCs have the multi differentiation capacity which could generate new bone tissues to replace old ones to realize a self newel process. As a result, BMMSCs become popular in the research of tissue engineering. However, it is not clear would the capacity of proliferation and differentiation of BMMSCs be affected by aging process and what are the internal molecular regulating mechanisms. Besides that, few researches focused on the bio-behavior of BMMSCs and regeneration capacity of bone tissues under aging condition. It is well known that many signaling pathways regulate the osteogenic process of BMMSCs while calcium pathway is one of them. In recent years, many literatures reported that L type voltage-gated calcium channel affected the osteogenic differentiation of osteoblasts and BMMSCs. Thus author of this study hypothesis that L type voltage-gated calcium channel might regulate the osteogenic differentiation of BMMSCs under senile osteoporosis conditions. Aims of this study1 To observe the bone volume changes of premature senility mouse.2 To test the capacity changes of osteogenic differentiation of BMMSCs of premature senility mouse.3 To test the calcium signaling changes of BMMSCs in premature senility mouse.4 To confirm the effect of L type voltage-gated calcium channel toward the osteogenic differentiation of BMMSCs in premature senility mouse. Methods of this study1 Three premature senility mouse(zmpste 24-/-, KO) and three wild type mouse(WT) were randomly chosen. Micro CT scanning was performed with a 360 o and 10 um scanning resolution for the proximal end of humeri. Targeting area was chosen to reconstruct the 3 dimensional images of bone trabecular to further analyze the difference of bone volume fraction?trabecular bone volume and Bone mineral density between the two groups.2 Six mouse from KO group or WT group were sacrificed to get primary passage of BMMSCs and cells in Passage 3(P3) were used in further study. After osteogenic induction for 14 days, alizarin red staining was taken to observe the formation of calcium nodules in each group. Besides this, RT-qPCR and western blot were also performed to analyze osteogenic differentiation related genes and proteins of mouse from the two groups.3 Laser scanning confocal microscope was used to observe the concentration difference of calcium in the 3rd passage of BMMSCs from KO and WT mouse. RT-qPCR was taken to test the expression difference of voltage-gated calcium channel of BMMSCs from two groups.4 Cells in the experimental group were induced by calcium channel inhibitor and osteogenic induction while cells in the control group were induced by the osteogenic induction. The effect of calcium inhibitor toward the osteogenic differentiation capacity of BMMSCs was observed. Then BMMSCs from KO mouse were transfected by calcium channel Cav 1.2. After 14 days of osteogenic induction, the osteogenic differentiation capacity BMMSCs was further tested. Results of this study1 Results of micro CT scanning indicated that the bone volume of the metaphyseal region of humerus of KO mouse was thinner. BV/TV, Tb.N and BMD of KO mouse were all lower than that of WT mouse with statistical significance(P<005).2 BMMSCs from the two groups were all had long polygon shapes and grew in an adherent way. Alizarin red staining showed that calcium nodules formed in both groups while the semi quantitative results indicated that number of the positive nodules was lower in KO group than that in WT groups(P<0.05). Results of RT-qPCR also demonstrated that the mRNA expressions of ALP, Runx2 and OPN were weaker in KO group(P<0.05) which were in accordance of the western blot results.3 Observing of laser scanning confocal microscope found that the intensity of calcium fluorescence was weaker in KO group with statistical significance(P<0.05). RT-qPCR test indicated that the expression of Cav 1.1, 1.2, 2.1 and 2.2 of BMMSCs were all higher in the WT group(P<0.05).4 Alizarin red staining confirmed that the number of positive calcium nodules was lower in BMMSCs from Cav 1.2 inhibitor WT group(P<0.05). Western blot test also showed that the protein expressions of Runx 2 and OPN of BMMSCs in Cav 1.2 group were lower. However, these results could all be up regulated after transfecting Cav 1.2 to BMMSCs from KO mouse. Conclusions of this study1 Zmpste 24 KO mouse has the symptoms of bone aging with decreasing of number of trabecular bone and bone volume fraction.2 Bio-behavior of BMMSCs from KO mouse changed; cells exhibited down regulated osteogenic differentiation capacity.3 Calcium signaling pathway of BMMSCs from KO mouse also changed. Cells had a lower concentration of calcium and the expression of Cav 1.2 was down regulated.4 L type voltage-gated calcium channel Cav 1.2 might participat regulating the osteogenic differentiation capacity of BMMSCs from KO mouse.