| Objective: Using the whole bone marrow cell culture system and stromal cell-depleted bone marrow cell culture system, to explore the effect of caffeic acid on osteoclast cell in vitro. In order to provide a new preparation of prevention and treatment for destructiveness paroxysm diseases due to osteoclast activity , and a foundation for further research of osteoclast in future.Method: Harvesting the whole marrow culture system and stromal cell- depleted bone marrow culture system from a 4-week-old SD male rat(weight 80-120g),then add 10-8 M 1α, 25(OH)2 D3 and osteoclast differentiation factor sRANKL, and carry on cell culture in vitro, to observe the influence of caffeic acid to osteoclast on formation and differentiation.1 Cell culture and medicine adds1.1 The whole marrow culture systemUnder ether anaesthesia, the tibia and femur were gained from a 4-week-old SD male rat with sterilization. Cut away epiphysis and expose bone cavity, then flush the cavity by 10ml a-MEM culture fluid. Extract the whole marrow cells under 1500 turn/min centrifugalization for 5 minutes, and discard the supernatant liquid, then add 20ml the a-MEM culture fluid containing 15% (FBS; fetal bovine serum) , blow and mix enough to form cell suspension. Prepaer the 2×106cells/ml cell suspension 13 ml after compute a cell number by FACS and calculate the concentration of this original cell liquid. Add 1α, 25(OH)2 D3 and osteoclast differentiation factor (sRANKL), and make sure it's eventually concentration are 10-8 Ms/ml and 20ng/ml respectively. Then seed with 24 bores cell plate, 2×106 cells/ml cell suspension 0.5 ml was added each bore(1×106 cells). Put this cell plate into CO2 incubator after seeding, culture cells under the 37℃, 5% CO2 condition. Change culture fluid on the 4th day for one time, and the totally culture procedure is 7 days.1.2 Stromal cell-depleted bone marrow culture system: The whole marrow cell culture fluid was condensed into 2ml after withdrawing, mesenchymal cell was removed by Sephadex G10. Then collect cell original liquid 10-12ml, prepaer the 2×106cells/ml cell suspension 13 ml after compute a cell number by FACS and calculate the concentration of this original cell liquid. Add 1α, 25(OH)2 D3 and osteoclast differentiation factor (sRANKL), and make sure it's eventually concentration are 10-8Ms/ml and 20ng/ml respectively. Then seed with 24 bores cell plate, 2×106 cells/ml cell suspension 0.5 ml was added each bore(1×106 cells). Put this cell plate into CO2 incubator after seeding, culture cells under the 37℃, 5% CO2 condition. Change culture fluid on the 4th day for one time, and the totally culture procedure is 7 days.1.3 The medicine adds: Caffeic acid was added in 24 bores cell plate containing two kinds of cells(the whole bone marrow cell and cell-depleted bone marrow cell). The final concentrations are 0, 0.5, 1, 10, 50, 100 ug/ml respectively. 4 rows 6 arranges were concluded, each row contains 6 kinds of concentration, and each concentration contains 4 bores(n=4). The first row 0 is the control group .2 Tartrate Resistant Acid Phosphatase (TRAP) stainings The two kinds of cell culture system were stained by TRAP after being cultured for 7 days respectively. TRAP positive cells were observed and calculated under the microscope (≥3 nucleus in the whole marrow cell culture system). The inhibting effect of caffeic acid on osteoclast and preosteoclas was observed .3 The statistics analysis: By SPSS16.0 software , the data processing was performed by mean±SD examination analysis, each concentration was adopted by student's t-test. All differrent medicine concentrations were compared with the control group, statistics were carried on under the same condition. The influence of caffeic acid on formation and differentiation in osteoclast and preosteoclas was observed. Result:1 The characteristic of osteoclast1.1 The whole marrow cell culture systemA great many of maturity osteoclasts were found after the TRAP staining, which were characteristiced by big cell volume, oval form, linear form, funnel form, fry in oil egg form or irregular form. OC contains plenty of red cytoplasm with clear vacuole structure, several to two or three dozens of cell nucleuses with significant nucleoli, and reachs out many lamellipodia.fig(1-4).1.2 Stromal cell-depleted bone marrow culture systemA great deal of preosteoclasts was clearly found after the TRAP staining, which characteri -stic is similar to maturity osteoclast, quadrilateral form, oval form, linear form or irregular form, but its volume is smaller. Preosteoclast mostly has only one nucleus, but which has two nucleus once in a while. Fig(5-6).2 The inhibiting effect of caffeic acid on formation of osteoclast In the whole marrow cell culture system, the numbers of TRAP positive cells in 0.5, 1, 10,50ug/ml groups are 60.30 %,27.27%,12.73% and 4.85% of the control group respectively. statistics analysis showed the groups of adding medicine could inhibit formation of osteoclast. Comparing with control group, the most remarkable repression to osteoclast was observed in the 50 ug/ml group(p﹤0.01). table 1and Fig.7.In the Stromal cell-depleted bone marrow culture system, the numbers of TRAP positive cells in 0.5, 1, 10,50 ug/ml groups are 75.0 8 %,38.65%,16.74% and 7.53% of the control group respectively, statistics analysis showed the groups of adding medicine have the inhibiting effect on the preosteoclast formation, when the medicine concentration is 50 ug/ml, comparing with control group, a remarkable repression to the preosteoclast was showed. (p﹤0.01). table 2 and Fig.8.Conclusion:1 Caffeic acid could effect on osteoclast directly, and repressed its formation and differentiation. 2 The inhibiting effect of caffeic acid on mature osteoclast and preosteoclast depended on its concentration.
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