Effect Of Bone Marrow Adipocytes On The Function Of Osteoblasts And Osteoclasts

Objective: To observe the difference of the endocrine function of adipose cells derived from bone marrow mesenchymal stem cells in different periods;To observe the effect of different sizes of bone marrow adipocytes on the differentiation of osteoblasts and osteoclasts by cell co-culture;To observe the effects of different concentrations of fatty acids on the induced differentiation of osteoblasts.Methods: 1. Differentiation and identification of various types of cells: Select 4 weeks Wistar rats, isolation of bone marrow mesenchymal stem cells(BMSCs) were induced to differentiation into adipocytes and osteoblasts; oil red O staining was used to identify the adipocytes, Alizarin red s taining was used to identify the osteoblasts; Collect Wistar rats marrow liquid, induced Differentiation into osteoclasts, tartrate resistant acid phosphatase staining was used to identify theosteoclasts. 2. Adipocytes derived from bone marrow mesenchymal stem cells were co-cultured with osteoblasts and Osteoclasts: 1) After the BMSCs induced to differentiate into adipocytes, further induction of differentiation, collected once every two days adipocytes culture medium,-20 ℃ cryopreservation(sampling time points: 21 d, 24 d, 27 d, 30 d, 33 d, 36 d, 39 d, 42 d, 45 d, 48 d, 50d), all samples collected to complete the monitoring of inflammatory cytokines; while observing the volume and number of adipocytes, using a ruler measuring cell volume, cell number in 40 × microscope counting six horizons averaged. Based on the above monitoring data, to determine the time point of the fat cells and osteoblast co-culture. 2) Adipocytes inducers P3 generation of BMSCs to differentiate into adipocytes were induced to the 21 d and 40 d, were co-cultured. 3) Non direct contact co-culture group: the culture medium was collected every other day, and the culture medium was replaced by adipose tissue culture medium.And add the osteogenic agents, induced to osteoblasts. 4) Direct contact co-culture group: about 10 cm2 scrape adipocytess in a Petri dish, inoculated fresh BMSCs, medium was replaced with adipocytess maintain medium and add osteogenic agents, induced to differentiate into osteoblasts;The added bone marrow cells were incubated in complete medium for 24 hours in a cell incubator, non-adherent cells were collected, approximately 10 cm2 scrape the adipocytes in a petri dish, in which the non-adherent cells were inoculated,medium was replaced with adipocytess maintain medium and add osteoclast inducing agent, induced to differentiate into osteoclasts. 5) Medium expression of inflammatory cytokines and bone metabolism Factor Levels. ELISA was used to detect inflammatory cytokines, transported by RT-PCR method to detect bone metabolism factor. 3. Different concentrations of FFA on osteoblast differentiation:P3 generation of BMSCs in Wistar rats were divided into osteoblasts, and different concentrations(25m M,50 m M,100 m M and 200 m M) of Palm Acid and oleic acid were added into the osteogenic induction medium to observe the effects of two kinds of fatty acids on the osteogenic differentiation.Results: 1. Wistar rat BMSCs were successfully cultured for 15 days, and the surface molecular markers CD29 and CD44 were positive after three generations.Wistar rats P3 generation BMSCs induced by medium for 21 days to differentiate into adipocytes, oil red O staining were positive.Wistar rats P3 generation BMSCs induced by medium for 21 days to differentiate into osteogenic,alizarin red staining were positive.The whole bone marrow of Wistar rats was divided into osteoclasts under the action of osteoclast culture medium for 28 days,acid phosphatase stain were positive. 2. To induce the differentiation of adipocytes, to induce 21 d adipocytes the number of adipocytes was significantly higher than that of 40 d adipocytes(P < 0.001), while the volume was significantly lower than that of 40 d adipocytes(P < 0.05).with increasing time differentiation, adipocytes secretion of TG,TNF-a and IL-6 expression showed an upward trend, induced to 40 d was significantly higher than that in the 21 d(P< 0.01), in the 40d-50 d basic maintain level unchanged. 3. In the co-culture process,induced to 21 d adipocytes and osteoblasts were co-cultured in vitro,the number of calcium nodules significantly increased than that of the positive control group(P < 0.001), to induce 40 d of adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly decreased thanthat of the positive control group(P< 0.05). 4. The OPG expression was significantly increased(P<0.01), and the expression of RANKL wassignificantly lower(P<0.01) from induction of 21 d of adipocytes group compared with the induction of 40 d of adipocytes group. 5. In osteogenic induction culture medium added with different concentrations of oleic acid(25m M,50 Mm, 100 m M and 200 m M), bone marrow mesenchymal stem cells most differentiation into adipocytes and with the increase of the concentration, the number and volume of adipocytes increased, but were not observed the osteoblasts. Different concentrations of palm acid have toxic effects on cells, cell death.200 m M oleic acid can be used as inducer to induce the differentiation of bone marrow mesenchymal stem cells into adipocytes directly, the number and volume of adipocytes are much larger than those induced by traditional methods(P<0.01).Conclusion: 1. In the bone marrow cavity, small adipocytes and osteoblasts were co-cultured, which can up regulate the expression of OPG and promote the formation of osteoblasts. 2. In the bone marrow cavity, large adipocytes and osteoblasts were co-cultured, which can down regulate the expression of OPG and inhibit the formation of osteoblasts.