Dissertation > Medicine, health > Oral Sciences

Regulation of Growth and Biomineralization by Nitric Oxide in Mouse Osteoblasts

Author HuangZhan
Tutor LiuHongChen
School PLA Postgraduate Medical School
Course Dental
Keywords Nitric Oxide Pre-osteoblas Bone Sialoprotein Calicification
Type PhD thesis
Year 2006
Downloads 188
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Oral bone loss is the topical outcomes under different systemic and local factors, and it has been found great effects on the clinical success rate of endosseous dental implants.The systemic methabolic condition and local pathologic changes in mandibles could alter the function of osteoblasts,destroy the communication among osteocytes,osteoblasts and osteoclasts,which maynot favour the final osseointegration and long-term stability.More diffuse use of implants for patients with oral bone loss will be obtained with the artificial regulation of bone modelling and remodelling through the application of ideal medicine and design of implants.As there is a relationship between the systemic bone loss and oral osteopenia, researchers have been applying such therapeutics as hormones replacing and bisphosphonates for oral bone loss and have found some of them work.However, there is no ideal medicine or therapeutics in clinical treatment for systemic or oral bone loss.Nitric oxide is a free radical gas that has been indicated as a secondary messenger molecule in many biological pathways.It’s generated by the conversion of the amino acid,L-arginine,catalysed by nitric oxide synthase.Recently the effect of NO on the metabolism and activity of osteocytes are being investigated.It has been shown NO functions as a crucial factor for regulating the bone resorption and formation.And yet, there’s little report the effect of NO in the mandible metabolism.This study is part of our research on medicine administrtation of new dental implant system.A comparison was made here to explore the effect of NO donor, S-nitroso-N-acetyl-dl-penicillamin(SNAP),L-arginine and nitric oxide synthase inhibitor,N omega-nitro-L-arginine methyl ester(L-NAME)on the proliferation, differentiation and biomineralization of primary periodontal osteoblast and pre-osteoblast MC3T3-E1 and the effect on the mandible metabolism in mice with induced acute oral bone loss.The possible ways and mechanisms for NO in mandible growth and differentiation were investigated.It was expected to provide experimental gist for mandible metabolisms and improving ossointegration with new medicine administration system of dental implant.This study was consisted of three parts:PartⅠ:Objective:To isolate,culture and identify the primary osteoblast deriving from the neonatal mouse periodontal mandibles.Methods:Primary osteoblast from the explant culture of neonatal mouse periondontal mandibles after enzyme isolation,were cultured.The morphological and biological features of the primary osteoblasts were studied.Alkaline phosphatase(ALP)activity,Von kossa assay and Alizarin S Red was used to evaluate the phenotype of mineralization,strapping and expanding time assessed,graphs of growth,split index valued.The expression of osteocalcin was detected by immunohistochemisty.Results:Osteoblast cultured gave a characterized steady growth and rapid proliferation and the same biological features with osteoblasts in vivo with high ALP activity and Von Kossa positive.The expression of osteocalcin is positive.The mineralized nodule was tested by Alizarin S Red staining.Conclusion:The opitimized method is proved to assure experiment of authenticity and accuracy and to be applied to the study of bone tissue metabolism as seed cells.PartⅡObjective:To explore the effect of nitric oxide synthase on the biomineralization on primary periondontal osteoblast and pre-osteoblast MC3T3-E1 cells.Methods:1×10-3 mmol/L NO donor S-nitroso-N-acetyl-dl-penicillamine(SNAP)was administered to MC3T3-E1 cells for three weeks.The effect of SNAP on cell proliferation was valuated through DNA content and alkaline phosphatase(ALP)activity,and biomineralization was determined through calcium deposit by Von Kossa Assay,the expression of osteocalcin by immunohistochemistry,and the gene expression of Cbfal by RT-PCR and protein expression of bone sialoprotein(BSP)by Western Blot.Results:Compared with the control group,SNAP significantly increased the DNA content and ALP activity in osteoblasts with the existence ofβ-glycerolphosphate sodium and ascorbic acid.In addition,SNAP promoted formation of calcium nodule and the expression of osteocalcin.The expression of Cbfal reached the climax after 3 days’ culture and the expression of BSP was significantly up regulated after two weeks’ culture.Conclusion:Certain dosage of NO could stimulate the proliferation of both primary periodontal osteoblasts and MC3T3-E1 cells;it may regulate the differentiation and biomineralization through the regulating the expression of Cbfa2 and BSP.PartⅢ:Objective:To explore the function of nitric oxide on mandible metabolism.Methods: Retinoic acid(RA)was used to made mouse systemic osteopenia model.A comparison was made to explore the effect of NO donor L-arginine,NOS inhibitor L-NAME on the bone mineral density of the whole body,femur and mandible.Serum calcium,phosphase,ALP activity and osteocalcin were detected;the bone sialoprotein expression was examined by immunohistochemistry.Results:The bone mineral density of body,femur and mandible,the level of serum osteocalcin and bone sialoprotein were increased by stimulation of NO donor L-arginine.NOS inhibitor L-NAME may aggregate the mandible osteopenia,and NO donor L-Arg may reverse such bone loss caused by retinoic acid.Conclusion:NO may stimulated osteoblast acitivity and thus the bone formation,in which osteocalcin and bone sialoprotein may be involved

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