Effects Of Tnf-α On Cell Proliferation And Osteogenic Differentiation Of Bone Mesenchymal Stem Cells And Their Effects On Bone Regeneration Around The Loaded Implantation

Background and ObjectivesImplantology has been made rapid progress while starting late demostic. Its advantage and disadvantage has been familiar with. In the course of the procession, the biomechanics has been studying and made great achievements put into practice, including the implanting time and loading time and so on.The procession of the bone absorption, remodeling and new bone formation displayed and was stronger with the physiological stress stimulation in the interface between the bone and implant. Effection on the bone must be changing with variation of stresses when the different angles of inclination of implant. Increase of unbalanced distribution of stress lead to the absorption of bone around implant. While with the physiological stress stimulation, the osseointegration could come into being better in the condition of loss of excessive stress concentration. The bone remodeling was heavily associated with bone metabolism affected by stress in the implant and bone interface. The stress distribution was changing with the angle of implants. It is unknown that the extent of effection of stress on microenvironment around implant caused by angle of inclination of implant and the bone remodeling. There is seldom reported that the relationship between angular dimension and concentration of TNF-α and both effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implantation.As an inflammatory factor with kinds of biological activity, tumor necrosis factor α (TNF-α) is known by researches to play the dominant role during the development of the periodontal disease. Because of an kind of immunoregulator in the inflammatory reaction, it can make the inflammatory cells enter the infection sites and release the metalloproteinase. The latter can degrade the extracellular matrix protein and promote absorption of alveolar and dstruction of collagenous fiber. At the same time, the proliferation of parodontium and gingiva cells is regulated. TNF-a is in the lower level locus in the controlled or healthy patients while it comes a higher level locus in the developing periodontal patients. The combination of TNF-α and TNFR1inspires the formation of compound of TNFR-associated death domain(TRADD), TNF receptor factor2(TRAF2), receptor integration protein (RIP). TNF-α can enhance osteoclast creation and stimulate its cytoactive to accelerate absorption of bone, simultaneously restraining differentiation of osteoblast. TNF-α could inhibit the preosteoblast differentiation, expression of the osteocalcin, Osteogenesis differentiation genes RUNX2and Type I collagen. Then many researches are concentrating on blocking the expression of TNF-α so as to attain the therapy of associated disease.Recently, researches about TNF-α came to increasing trend and extended into implantology. They mainly concerned peri-implantitis with the way of detection quantity of gingival crevicular fluid. However, the factors of peri-implantitis in clinic was not clear. Preventive actions was hardly effectively prohibit the development of the disease. Some angle existed in the axis of implants and long axis in the clinic which leaded to the changes of press from masticatory muscle. Then what level concentration of TNF-α would be in the affection of such press and it was unclear that the effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation Seldom articles about this problem had been seen in the periodicals.BMSCs is recognized the most fully researched and the most easy to separation and the most widely applied stem cells of all the stem cells. Then BMSCs was separated, cultivated and purified in our research. The aim of the study is to dicuss effects of TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation. We study the effection of the TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation to discuss the effection of inflammatory microenvironment on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells in order to provide the theoretical guidance of application of stem cells in the restoration of inflammatory microenvironment around implants.Methods:1. Patients with implantation was selected and angles of inclination of their implants was caculated. Three teams was divided by0-10°、10°-20°and above20°.After3and6months, the concentration of TNF-α and the PICF was tested with different angles of inclination of implants.2. According to the data from the patients with angles of inclination of implants, the model was made. Implants were placed into the dog alveolar and loded. After1and6months the concentration of TNF-α and the PICF were tested.The osseointegration index(OI) and bone ingrowth fraction,(BIF) were calculated. 3. BMSCs were cultured and loaded by strain. Cells were treated with murine TNF-α at concentrations of0,1,10,15and20ng/ml, respectively. At24,48and72h after the TNF-α treatment, the proliferation/survival of the cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) test;The changes in the expression levels of osteogenic transcription factors (Runx2and Osx) and bone matrix proteins (0C and ALP) during osteogenic differentiation were observed by real-time PCR; The change in ALP activity was determined using PNPP assay; In vitro mineralization of BMSCs treated with TNF-α at concentrations of0,1,15and20ng/ml for four weeks under osteogenic induction was monitored using Alizarin red staining.Results:1. Test and analysis of TNF-a and PCIF around loaded implants in patients.It did not reveal any statistically significant changes about PCIF and the concentrations of TNF-a in the angles of inclination of implants from0to10°while there did show statistically significant changes about PCIF and the concentrations of TNF-α compareing the below-20°angle to the above-20°angle of implants.2. Test and analysis of TNF-α and PCIF around loaded implants and effects of TNF-α on bone regeneration in dogsThere were statistically significance between the the below-20°angle to the above-20°angle of implants in OI and BIF, PCIF and the concentrations of TNF-α. It showed coherence significant result about data from patients and dogs.3.Effects of TNF-α on osteogenic differentiation of BMSCs revealed some statistically significant changes as below:(1) The stem cells were successfully cultured. The ability of colony formation was high, and stem cells had formed mineralized nodules when cultured in osteogenic medium.(2) Real time PCR analysis showed that the mRNA levels of the potent osteogenic transcription factors (Runx2and Osx) and some bone matrix proteins (OC and ALP) were up-regulated in cell cultures treated with TNF-a at lower concentrations (0and1ng/ml). In contrast, the mRNA levels of Runx2, Osx, OC and ALP were dose-dependently down-regulated in cell cultures treated with TNF-a at concentrations above1ng/ml.(3)48h treatment of the BMSCs with TNF-a at lower concentrations (1ng/ml) enhanced ALP activity in these cells when compared with the control cells (treated with Ong/ml TNF-a). Cells treated with TNF-a at higher concentrations (10,15and20ng/ml) showed decreased ALP activity when compared with the control cells.(4) Long term treatment with TNF-α resulted in a dose-dependent decrease in mineral nodule formation after the4-week osteogenic induction.(5) MTT assay did not reveal any statistically significant changes in the proliferation/survival of BMSCs treated with TNF-a at concentrations ranging from0to20ng/ml.Conclusions:(1) Effects of TNF-a on microenvironment around the implants revealed little effect on implant-bone interface when the angle inclination of implants was below-20°with TNF-α at lower concentrations. While the angle was over20°, effects of TNF-a on microenvironment was that the bone around implants had been absorbed.(2) TNF-α treatment at lower concentrations and short time moderately enhances expression of osteogenic genes (RUNX2、OSX、ALP和OC) and ALP activity in BMSCs while TNF-α treatment at higher concentrations level and long time displays inhibitory effect on osteogenic differentiation and mineralization in BMSCs. at higher concentrations it lower expression of these osteogenic genes and reduce mineralized nodule amount.(3) In the physiology strain extent, it did not reveal changes in the proliferation of BMSCs. While manifest changes are revealed when the strain out of physiology range.