In vitro differentiation of umbilical cord blood-derived mononuclear cells towards osteoclast-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Orthodontic tooth movement is dependent on reconstruction of periodontium. Osteoclastic bone resorption is the first step of tooth movement. The present study hotspots focus on signal transduction pathway regarding osteoclast differentiation and functional development under stress and on the relationship between periodontal ligament cells and osteoclasts. OBJECTIVE: To set up a simple method to in vitro culture human osteoclast-like cells and to observe the effects of bone resorption-stimulating factors on differentiation, proliferation, and function of osteoclast-like cells, DESIGN, TIME AND STTING A cytological in vitro controUod observation was performed at the Central Laboratory,Stomatology Hospital, Xi'an Jiaotong University between October 2007 and May 2008. MATERIALS Umbilical cord blood was sourced from the healthy puerperae who had not suffered from high-risk pregnancy. Freshly prepared fetal femur provided by Laboratory Animal Center, Xi'an Jiaotong University and were used for preparation of bone flaps at 100-200 μm thickness. 1α ,25-(OH)2D3, macrophage colony-stimulating factor (M-CSF), prostaglandin E2 were purchased from Sigma Company, USA.METHODS: Under aseptic condition, umbilical cord blood was collected. Following Ficoll solution separation and centrifugation, supematant was discarded. Umbilical cord blood-derived mononuclear cells were suspended with o -modified minimal essential medium (α-MEM) solution and then inoculated into a 24-well culture plate, in which, coverslips and femoral slices were pre-placed, at a density of 1×109/L, 1.0 mL per well. Five groups were set, blank control, 108 mol/L 1α ,25-(OH)2D3, 10-7 mol/L 1α ,25-(OH)2D3, macrophage colony stimulating factor (M-CSF), and 1α ,25-(OH)2D3+prostaglandin E2 (PGE2). Each group was cultured for 7 days. MAIN OUTCOME MEASURES: Cellular growth morphology was observed under an inverted microscope; osteoclast-like celt formation was examined by tartrate-resistant acid phosphatase (TRAP) staining; and osteoclastic Howship's lacuna was detected by toluidine blue staining. Any cell with TRAP-positive staining and more than two nuclei was considered osteoclast-like cell and counted. RESULTS: After 3 days of culture, cells from the blank control group did not exhibit apparent changes in morphology and quantity. In the remaining groups, mononuclear cells appeared with confluent tendency. After 7 days of culture, a small number of osteoclast- like cells with 2-3 nuclei were found in the blank control group; a great many of multinucleated osteoclast- like cells with 3-20 nuclei were present in the remaining groups. Through the use of optical microscope, osteoclast-like cells could be found for the presence of red cytoplasm, bright yellow nuclei, and TRAP-positive staining in each inducing factor-treated group, in particular in the 108 mol/L 1α ,25-(OH)2D3 group, which displayed osteoclast- like cells exhibiting 14 nuclei, strong TRAP-positive staining, and a relatively big cell body. But no osteoclastic Howship's lacuna was found in any group. Compared to the blank control group, the numbers of osteoclast-like cells were greater in each inducing factor-treated group (F = 9.78, P < 0.01). There was no significant difference in the numbers of osteoclast-like cells between the 108 mol/L 1α ,25-(OH)2D3 and the 10-7 mol/L 1 a ,25-(OH)2D3 groups (P0.05). The M-CSF group and the 1α ,25-(OH)2D3+PGE2 group exhibited significantly less numbers of osteoclast-like cells than the 108 mol/L 1α ,25-(OH)2D3 group (F= 7.46, P< 0.01). CONCLUSION: After in vitro culture of 1α ,25-(OH)2D3, M-CSF, and PGE2, umbilical cord blood-derived mononuclear cells can differentiate into TRAP-positive multinucleated osteoclast-like cells, the 10-8 mol/L 1α ,25-(OH)2D3 being the most effective.