Effect of marrow adipocytes on osteogenic ability and apoptosis of bone marrow stromal cells

AIM To observe the effects of adipocyte in bone marrow cavity on osteogenic potential of bone marrow stromal cells and if adipocyte could induce apoptosis of bone marrow stromal cell. METHODS This experiment was conducted at the laboratory of Department of Orthopaedics Tongji Hospital Tongji Medical College Huazhong University of Science and Technolohy from July 2001 to June 2003. Bone marrow stromal cells were inoculated in culture plate and Petri dish with the concentration of 109$L . DMEM culture medium containing 0.1 volume fraction of fetal bovine serum 100 U$mL penicillinum and 100 mg$L streptomycin was added. The above-mentioned cells were randomly divided into blank control group 106,107,108,109 L-1 adipocytes groups  in which some adipocytes with concentration of 0 106 107 108 109$L were added respectively. All the cells were cultured in the saturated humidity culture box with 0.05 volume fraction of CO2 at 37 ℃. The co-culture system of adipocyte and bone marrow stromal cells was established. After 4 8 12 days alkaline phosphatase ALP activities of the samples were detected the mRNA expression of collagen type I was detected with in situ hybridization method the collagen synthesis ability with 3H-proline in corporation test. Cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP mick end-labeling TUNEL and flow cytometer. RESULTS ① Cellular morphological observation after establishing co-culture system After 48 hours cells began to adhere to the wall. 72 hours later the adhered bone marrow stromal cells mainly presented short-shuttle shape some were polygon and mutations stretched out and the volume was enlarged possessing typical characteristics of fibroblasts. The adhered adipocytes presented polygon or round shape and good cellular transparence. ②Effect of adipocytes on ALP activities of bone marrow stromal cells as compared with blank control group with the increase of concentration of adipocytes in the co-culture system ALP activities of bone marrow stromal cells all had decrease at different degrees at the 4th 8th and 12th days of the culture (P 0.05 or P 0.01). ③ Effect of adipocytes on mRNA expression of collagen type I in bone marrow stromal cells The staining was the darkest in blank control group that was to say the expression was the strongest. With the increase of adipocytes in the co-culture system the staining gradually became from dark to light that was to say collagen I mRNA expression was gradually weakened. ④ 3H-proline in corporation test in blank control group counts per minute was increased on the 4th day . With the elongation of time counts per minute had a little decrease. With the increase of concentration of adipocytes counts per minute had decrease at different degrees in each group with concentration-time dependent manner. ⑤ TUNEL detection of apoptotic cells In the co-culture system containing adipocytes nuclear chromatin of brown-labeled apoptotic cells condensed into dense spot shape karyopycnosis even cataclasm appeared. At the same time apoptotic cells were significantly more in the 106,107,108,109 L-1 adipocytes group than in blank control group P 0.01 there was seldomly apoptotic cells in blank control group. ⑥ Detection result of cellular apoptosis by flow cytometer Through detecting suspended cells by flow cytometer in blank control group cells gathered left down quadrant apoptotic cells of co-culture system could be found in the right down quadrant. CONCLUSION Adipocytes in bone marrow cavity interfere osteogenic potential of bone marrow stromal cell and induce apoptosis of bone marrow stromal cell. It might be related to the morbidity of primary osteoporosis.