[Application of human adipose-derived stromal cells in bone tissue engineering].

Human adipose-derived stromal cells (hASCs) can be obtained from adipose tissues that offer an abundant and easily accessible pool of stem cells. Thus, hASCs have become a highly attractive source of seed cells in bone tissue engineering and have promising prospects in bone regeneration. Since 2002, our research group has performed a series of experiments on hASCs and its application in bone tissue engineering, including: to substitute dexamethasone by 1,25 (OH)2 vitamin D3 to induce osteogenic differentiation of hASCs; to explore the effect of epigenetic regulation and to inflammation on the osteogenic differentiation of hASCs; to construct a novel and simple tissue engineered bone system by hASCs and human platelet-rich plasma (hPRP) and to investigate the bone formation capability of this tissue engineered bone and the stimulatory effect of simvastatin. Our results suggested that 1,25 (OH)2 vitamin D3 could replace dexamethasone to induce the osteogenic differentiation of hASCs; retinoblastoma binding protein 2 (RBP2), as one of histone demethylases, could regulate the osteogenic differentiation of hASCs epigenetically while tumor necrosis factor α (TNFα), as a inflammatory factor, could also influence the osteogenic differentiation of hASCs. Moreover, we found that in vivo bone formation could be detected by our novel tissue engineered bone composed with hASCs and hPRP; simvastatin could enhance the bone formation capability of this tissue-engineered structure.

[Effects of rhTNF-alpha on the secretion of angiogenesis-related growth factors of human adipose tissue-derived stromal cells before and after osteogenic differentiation].

OBJECTIVE: To investigate the proliferation and the secretion of vascular endothelial growth factor(VEGF), fibroblast growth factor-2(FGF-2) and insulin-like growth factor-1(IGF-1) of human adipose tissue-derived stromal cells(hADSCs) before and after osteogenic differentiation under the stimuli of recombinant human tumor necrosis factor alpha (rhTNF-alpha). METHODS: hADSCs were obtained from human lipoaspirates. All the cells used were at passage four. The proliferation of hADSCs was measured with MTT assays 48, 72, 96 hours after being treated with 0, 1, 5, 10, 50 or 100 microg/L rhTNF-alpha respectively. The secretion of VEGF, FGF-2 and IGF-1 of the undifferentiated hADSCs under stimuli of rhTNF-alpha with the above 5 concentration grades was observed and the secretion of these 3 growth factors of hADSCs at different stages of osteogenic differentiation under stimuli of 10 microg/L rhTNF-alpha was also observed. All the supernatants were harvested for measuring after 24 hours' incubation with rhTNF-alpha. The secretion of VEGF, FGF-2 and IGF-1 was measured with ELISA, and the values were normalized to the cell number of the corresponding wells. RESULTS: The effect of rhTNF-alpha on the proliferation of hADSCs varied with the concentration and time. Compared with the control(0 microg/L), 10 microg/L rhTNF-alpha showed no suppression or acceleration on proliferation of hADSCs at hour 48, but significantly promoted the proliferation at hour 96 (0.903+/-0.042 vs 0.810+/-0.011, P<0.01), 100 microg/L rhTNF-alpha seemed to suppress the proliferation at hour 48 (0.317+/-0.024 vs 0.458+/-0.046, P<0.01), but appeared to promote it (0.956+/-0.030 vs 0.810+/-0.011, P<0.01) at hour 96. rhTNF-alpha(1, 5, 10, 50 and 100 microg/L) significantly increased VEGF, FGF-2 and IGF-1 production of hADSCs versus the control (0 microg/L) (P<0.01). After osteogenic differention, the secretion of the three growth factors of hADSCs (without rhTNF-alpha treated) was elevated with the days increasing. Under the stimulus of 10 microg/L rhTNF-alpha, the hADSCs after 1 day of osteogenic differentiation significantly increased the secretion of VEGF (P<0.01) compared with the group without rhTNF-alpha treated; after 3 and 7 days of osteogenic differentiation, the hADSCs significantly increased the secretion of VEGF (P<0.01), FGF-2 (P<0.05)and IGF-1 (P<0.05). However, after 14 days of osteogenic differentiation, 10 microg/L rhTNF-alpha appeared to suppress the production of VEGF (P<0.01), FGF-2 (P<0.05) and IGF-1 (P<0.05) of the differentiated hADSCs. CONCLUSION: Within certain concentration range, rhTNF-alpha can promote the proliferation of hADSCs and the production of VEGF, FGF-2 and IGF-1. The effect of 10 microg/L rhTNF-alpha on the production of VEGF, FGF-2 and IGF-1 of the differentiated hADSCs varied at different stages of osteogenic differentiation.

[Comparison of biological characteristics of human, rabbit and rat adipose tissue-derived stromal cells in vitro].

OBJECTIVE: This study aims to investigate the difference of proliferation patterns and osteogenic and adipogenic differentiation capability of adipose-derived stromal cells (ADSCs) obtained from human lipoaspirates, rat and rabbit inguinal subcutaneous adipose tissues in vitro. METHODS: Adipose tissues of healthy adults were obtained by liposuction. Human ADSCs were isolated from these adipose tissues and cultured in DMEM containing 10% fetal bovine serum (FBS). Rat and rabbit ADSCs were obtained from inguinal subcutaneous adipose tissues and cultured with the same methods. These cells were observed under inverted microscope each day and cell growth was measured with MTT assay. Adipogenic differentiation was induced by culturing ADSCs for 1 or 2 weeks in adipogenic medium (AM) containing 1 micromol/L dexamethasone, 10 micromol/L insulin, 200 micromol/L indomethacin, 0.5 mmol/L isobutyl-methylxanthine (IBMX), and assessed by Oil Red O staining as an indicator of intracellular lipid accumulation. Osteogenic differentiation was induced by culturing ADSCs in osteogenic medium (OM) containing 0.1 micromol/L dexamethasone, 50 micromol/L ascorbate-2-phosphate, 10 mmol/L beta-glycerophosphate, and examined via alkaline phosphatase (AP) activity and extracellular matrix (ECM) calcification by alizarin red S staining and quantification of matrix calcification. RESULTS: Fibroblast-like cells were digested from both inguinal subcutaneous adipose tissues of rabbit or rat and human lipoaspirates obtained from subcutaneous adipose tissues. Lipid-filled droplets were accumulated in human, rat and rabbit ADSCs upon treatment with adipogenic medium and were stained by Oil Red O. No lipid droplets were observed in the control undifferentiated ADSCs. After exposure to osteogenic differentiation medium, human and rat ADSCs were found to possess greater osteogenic potentials than cells isolated from rabbit inguinal subcutaneous adipose tissues, which was evidenced by significantly different osteogenic markers including alkaline phosphatase and mineral deposition. CONCLUSION: Rabbit ADSCs obtained from inguinal subcutaneous adipose tissues poorly possess osteogenic potentials compared with ADSCs of human lipoaspirates obtained from subcutaneous adipose tissues or ADSCs of rat from inguinal subcutaneous adipose tissues, although they all possess comparable adipogenic capacity.