The Effect Of Growth Differentiation Factor-5 On Osteogenesis Of Human Adipose-derived Stromal Cells

Bone defects caused by wound, tumour and malformation are common in dental clinic. The main treatment methods, such as autograft, allograft, and bone-grafting materials, couldn't completely meet the clinical needs. The bone tissue engineering provided novel methods for restoring bone defects. The content of bone tissue engineering includes three elements:seed cells, scaffolds and growth factors. It is hypothesized that human adipose tissue is an abundant reservoir of adult stem cells, which have potent to differentiate into many mature cell lineages varying from adipogenic precursors, osteogenic precursors, chondrogenic precursors to myogenic precursors. ADSCs are easier to obtain, carry relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Growth factors play important role in the ADSCs'osteogenic differentiation. The common growth factors were BMP2, BMP7, GDF-5, TGF-βPDGF, IGF and so on. Growth differentiation factor-5 (GDF-5) is a divergent member of the bone morphogenetic proteins(BMPs) subfamily which belongs to the transforming growth factor-β(TGF-β) superfamily. It plays a very important role in the chondrogenesis and long bone development. GDF-5 can promote the osteochondrogenic differentiation of mesenchymal progenitor cells in vitro and in vivo. In order to further elucidate the biological activities of GDF-5 and to expend its applications in bone tissue engineering. Our investigations were presented as followings:Part 1:Experimental study of the isolation, culture and biological characteristics of hADSCs.Objective:To investigate methods for the isolation, culture and biological characteristics of hADSCs in order to provide seed cells for bone tissue engineering. Methods:Human adipose tissues from liposuction were excised and digested with 0.1% collagenase I, and then the cells were cultured in L-DMEM with 20% FBS. The morphology of hADSCs was observed under inserted microscope. The proliferation of the 3rd,5th and 10th passage cells were examined using MTT. The antigens of CD29, CD44, CD 14, CD45, CD 105, the markers of the mesenchymal stem cells, on the 2nd passage cells were examined using flow cytometric methods. The antigen of vimentin was detected by immunofluorescence. ALP, OPN, Col I and calcified nodules of the 4th passage cells were assessed by cytochemical and immunocytochemical staining. Adipogenic differentiation of the 4th passage cells was assessed by Oil Red O staining.Results:The primary hADSCs had long or short spindle-shape, later fibroblast-like. The proliferation curve showed that the 3rd,5th,10th passage cells proliferated rapidly, there were no differences between them. The 4th cells expressed CD29, CD44, CD 105 and vimentin. ALP, OPN and collagen I were positively expressed with histological and immunohistochemic staining. Calcified nodules formed were formed based on the alizarin red stainning. After induction for 14 days, the lipid droplets were presented in the differentiated cells by Oil Red O staining.Conclusions:The hADSCs have the characters of stromal cells and can be used as a source of seed cells for bone tissue engineering.Part II:The effects of GDF-5 on osteogenic differentiation of hADSCs in vitroObjective:To explore the effects of GDF-5 on osteogenic differentiation potential of hADSCs in vitro and make preparations for further research about bone tissue construction in vivo.Methods:Human adipose-derived stromal cells were divided into 5 groups by different concentration of GDF-5 (0,1,10,100,500 ng/ml), MTT, ALP activity and Alizarin red S were used to determine the appropriate concentration. The 4th passage cells were divided into three groups:control group, mineral group and GDF-5 group, their osteogenic differentiation was assessed by MTT, ALP activity respectively. The mRNA expressions of Runx2, OPN, Col I and VEGF were examined by RT-PCR. The protein expressions of Runx2, OPN, Col I and VEGF were examined by Western-blot.Results:The 100ng/ml GDF-5 was the optimal concentration. MTT value in the GDF-5 were significantly lower than the other groups, but ALP activity, mRNA expression and protein expression in the GDF-5 were significantly higher than other groups(P<0.05).Couclusions:The hADSCs could be induced into osteoblast by GDF-5 in vitro.Part III:The effects of GDF-5 on bone formation of hADSCs/nHAC/PLA construct in vivo.Objective:To explore the effects of GDF-5 on bone formation of hADSCs/nHAC/PLA construct in vivo.Methods:The 4th passage hADSCs were seeded onto nHAC/PLA in vitro to construct cells-scaffold composites. They were divided into the control group, mineral group and GDF-5 group respectively. Then the composites were implanted into the back of immunodeficiency disease (SCID) mice.4 weeks and 12 weeks later. The implantation were observed using HE, Goldner's staining and scanning electron microscope.Results:The hADSCs could adhere and grow on the surfaces of the implants. Bone-like tissues were found to form in the constructs at 4 weeks and 12 weeks in comparison to the control group. The pencentage of newly-formed bone areas in GDF-5 group was signficently higher than those in the other groups.Conclusions:The nHAC/PLA is an ideal scaffold; GDF-5 could enhance 'bone formation of hADSCs/nHAC/PLA in vivo.