Effect Of Smoke On Proliferation And Osteoblast Differentiation In Human Alveolar Bone Marrow Mesenchymal Stem Cells

Smoke is an essencial risk factor for dental implants. Many studies haveconfirmed that tobacco can negatively affect the success rate of implant. Smokenot only affect the success rate of dental implants, but also affect the peri-implantosseointegration. Smoke can affect wound healing,reduce collagenproduction,reduce the peripheral blood circulation and damage the function ofwhite blood cells and macrophages. In addition, a series of basic studies haveshown that the negative impact of tobacco or nicotine to bone healing aroundtitanium implant.The cellular mechanisms of smoke affect on implant bone healthrough the establishment of the smoke model,however Smoke stimulation andinjection of nicotine can not fully simulate the long-term smoking on the bodycells.The experimental isolation and identification of human alveolar bonemarrow mesenchymal stem cells from non-smokers and smokers, comparision ofhABMMSCs (human alveolar bone marrow mesenchymal stemcells)proliferation and osteogenic differentiation ability between non-smokergroup and smoker group. These can Provide the theoretical basis of smoke onimplant osseointegration. The main findings are as follows: Experiment one: isolation and identification of human alveolar bone marrowmesenchymal stem cellsIn this study, we successfully isolated and cultured a hABMMSCs use thedensity gradient centrifugation, adherent method and limited dilution method Thesame culture conditions, the cell growth rate of the smoker group wassignificantly slower than non-smoker group. Flow cytometry results show that themolecular phenotype of non-smoker group and smoker group show the similarcharacteristic.Two group of cells were positive for expression of the STRO-1,CD146, CD105, CD90, CD29and CD44, these are the mesenchymal stemcell-specific expression markers, and negative expression Cultured cells of themolecular phenotype of CD31and molecular phenotype of hematopoietic cellsfor CD34, of CD45.Non-smoker group the STRO-1, CD146expression was11.5%,5.9%greater than the smoker group was11.3%,3.5%.This part of the experiment, we successfully isolation and identification ofhuman alveolar bone marrow mesenchymal stem cells. smoker alveolar bonemarrow mesenchymal stem cells has more slower growth rate than thenon-smoker, the key cell receptor of stem cells are STRO-1and CD146,whichexpressed less than non-smoker.Experiment two: the comparision of hABMMSCs proliferation abilitybetween non-smoker group and smoker group,In this study, MTT assay, colony formation experiments and flow cytometrycycle detection are three ways to analysis for non-smoker group and smokergroup’s alveolar bone marrow mesenchymal stem cells ability.MTT assay showedthat non-smoker groups and smoker group have a significant difference on proliferation, the s-hABMMSCs showed poor in vitro proliferative capacity, theirproliferation activity was significantly weaker than that of n-hABMMSCs. Sincestarting the third day after inoculation culture, this difference is more obvious;colony formation assay also confirmed, the s-hABMMSCs the clone-formingability is worse than the n-hABMMSCs flow cytometry cycle test results with thefirst two test results. s-hABMMSCs, growth and proliferation capacity ofn-hABMMSCs a statistically significant difference between the two group.These results suggest that smoke affect the alveolar bone marrowmesenchymal stem cells proliferation.Indicate that the tobacco long-term highconcentrations of stimulating cells, thus inhibiting cell proliferation.Experiment three: non-smoker group and smoker group hABMMSCsosteogenic differentiation abilityThrough ALP activity detection experiments, the vitro mineralization abilitycontrast to non-smoker group and smoking the group hABMMSCs,bonedifferentiation can be analysised by detect real-time PCR. ALP activities ofexperimental results show that the upward trend of the two groups of cells weretested within14days of ALP activity. The growth rate of the smoker group wassignificantly slower than non-smoker group. On the3d,7d and14d three timepoints, s-hABMMSCs’s ALP activity were lower than n-hABMMSCs.Mineralization in vitro test results showed that the two cell osteogenic threeweeks, n1, n2, n3and s1, s2, s3are red-brown mineralized nodules, from amorphological point of view, smoker group of mineralized nodule area, lowdensity and the arrangement of scattered, rather than the area of mineralizednodules of the smoker group, and high density. The Mineralized area in the two sets of pictures shows that the mineralized nodule area of the smoking group wassignificantly less than non-smoker group. Quantitative analysis results provedthat the smoking group osteogenesis Ming lower than in non-smoker group whichhas the same trendency with the upper results. To expressthe boneformationrelated genes that detected from the RNA level by RT-PCR,we foundthe osteogenic differentiation genes associated with the ALP, COL-1, BSP andCON smokers should be lower than non-smoker.A number of studies have reported the relationship of smoke andosteoporosis, the occurrence of the alveolar bone of smokers in general thannon-smoker during the same time lasts, smoker bone implant success rate is alsolower than the non-suction smoke, and smoking cessation can rise to the successrate of smokers. Our study demonstrates that the s-hABMMSCs has more lowerthe osteogenic differentiation capacity than n-hABMMSCs. This shows that theharmful substances in tobacco regulates the expression of osteoblast-relatedgenes, reduce the formation of hABMMSCs minerals bracket and mineraldeposition, decrease the quantity and quality of new bone formation、theextracellular matrix of the hABMMSCs cellsthe degree of calcification, whichaffects implant osseointegration.