Effects Of Pulsed Electromagnetic Field On The Functions Of Osteoblasts On Titanium Surfaces

Fixing titanium implants into bone faster and more firmly has been a persistent challengeand is necessary to expand the therapeutic indications of these devices, reduce patientmorbidity and improve the success rate of such implants in reconstructive dental andorthopedic treatments. A critical factor necessary for implant fixation is rapid andsuccessful osseointegration. The desire to accelerate and improve osseointegration drivesmany implantology research and development efforts, particularly for patients whosebones have been compromised by disease or age. Implant surface modifications, drugdelivery systems and adjuvant therapies have been considered to achieve betterosseointegration. Biophysical stimulation represents a non-invasive and locally appliedstrategy to enhance bone regeneration around implants among these methods.Pulsed electromagnetic field (PEMF) therapy, a safe biophysical adjunct treatment,has been demonstrated to have beneficial therapeutic effects on a variety of bone-andcartilage-related disorders. This treatment has reported success rates of70-95%forconditions such as non-union bone fractures, congenital pseudoarthrosis and failed jointfusions. The use of electromagnetic-field stimulation has gained credibility as a therapy following observations that placing physical stress on bones promotes the formation ofvery small electric currents that are related to bone formation. Additionally, the smallmagnetic field in tissue created by a PEMF has also been shown to promote bone healingand relieve pain and inflammation. PEMF stimulation promotes bone repair by increasingthe activity of osteoblasts. Although considerable research on the osteogenic effects ofPEMF has been reported, few studies have evaluated the osteogenic effects of PEMF ontitanium implant surfaces or the underlying mechanism. Recently, several studies haveinvestigated the effect of PEMF on implant osseointegration, and the positive outcomes ofthese studies indicate that this type of biophysical stimulation has a potential role inimplantology. However, studies published thus far have focused only on the positiveeffects on bone formation around implants in vivo, or on various types of parametersunder various experimental conditions. A number of questions remain to be answeredincluding (1) whether and to what extent PEMF influence the adhesion, spread,proliferation and differentiation of osteoblasts on titanium surfaces,(2) whether PEMFhave different effects on osteoblast function when the cells are on a polished flat surface, amicro-topographical surface or a nano-topographical surface, and (3) whether PEMFaffect the gene expression and proteins of osteoblasts growing on implant surfaces.1.The selection of pulsed electromagnetic field parameters on the functions ofosteoblasts on implant surfaces with different topographies.Primary osteoblast cultures under the no stimulation (Control) and PEMF stimulationwere evaluated on implant surfaces in this work. Three representative implant surfacetopographies, namely an SLA micro-structured surface (Micro), an anodizednanotubular-structured surface (Nano), and a polished surface (Flat) were used assubstrates. Frequency parameter (1Hz,15Hz) and intensity parameter (0.1mt，0.5mt，1mt and5mt) were compared to select the best group of pulsed electromagnetic fieldparameters. The results indicated that the effect windows‘are different in the Flat, Microand Nano surfaces. A group of parameters (15Hz，1mt) was choosen due to the positiveeffect on all the three surfaces.2.The effects of pulsed electromagnetic field on the functions of osteoblasts on implant surfaces with different topographies.Protein adsorption, adhesion, proliferation and differentiation of osteoblasts as wellas the spread of cytoskeletal with or without PEMF stimulation were investigated. Ratosteoblasts were cultured on three types of titanium surfaces (Flat, Micro and Nano) underPEMF stimulation or control conditions. Protein adsorption was significantly increased bythe PEMF. Although the cell numbers on all three surfaces in the PEMF-stimulated groupappeared to be higher than those for the control group at0.5h, the differences were notstatistically significant. However, at1h and1.5h, the numbers of adherent cells on eachsurface in the PEMF group were significantly higher than those for the control group.PEMF stimulation oriented the osteoblasts perpendicular to the electromagnetic field linesand increased the number of microfilaments and pseudopodia formed by the osteoblasts.The cell proliferation on the implant surfaces was significantly promoted by the PEMF.Significantly increased ECM mineralization nodules were observed under PEMFstimulation. The cell viabilities on all surfaces in the PEMF-stimulated group wereobviously higher than those for the no-stimulated group on days4and7. On day4, theOD value for the Nano/PEMF samples increased significantly to235%of that for theNano/Control samples. PEMF stimulation significantly increased the level of ALP activityonly on the Nano surface on day7. However，Matrix mineralization was dramaticallyaffected by PEMF stimulation on day14. Our findings suggest that the stimulation ofosteoblasts using a PEMF accelerates cellular proliferation during the active proliferationstage and enhances cellular differentiation during the differentiation stage3.The effects of pulsed electromagnetic field on the osteogenic genes and proteinson implant surfaces with different topographiesFurthermore, to define the possible molecular mechanism associated withosteoblastic differentiation activated by the PEMF, the expression profile of alkalinephosphatase (alp), bone morphogenetic protein-2(bmp2), osteocalcin (ocn), type1collagen (col-1), runt-related transcription factor2(runx2) and Osterix (osx) transcriptionfactors were also investigated. The expression of osteogenesis-related genes includingbmp2, ocn and col-1, alp, runx2and osx were upregulated on all the surfaces by PEMF stimulation. BMP-2was most responsive to the applied PEMF, with maximum increasesin expression of1.9-fold (Flat),2.6-fold (Micro) and2.7-fold (Nano) at day4and2.6-fold(Flat),2.9-fold (Micro) and4.1-fold (Nano) at day7over the corresponding controls in thecontrol group Elisa results showed that BMP2secretion was promoted by the PEMFstimulation. Compared to the control group, a significant (p <0.05) increase in BMP2secretion was observed from osteoblasts cultured on Micro/PEMF and Nano/PEMFsurfaces on both day4and day7and on Flat/PEMF surface at day7. No significantdifference in secretion of BMP2was detected between the Flat/Control and Flat/PEMF atday4. The key proteins in BMP2/Smad pathway were tested by western blot. PEMFstimulation upregulated the expression of p-Smad1/5/8，the downstream proteins of thispathway (Runx-2and OSX) were also promoted by PEMF. After adding the inhibitor(Noggin), the expression of p-Smad1/5/8, Runx-2and OSX decreased. Taking the aboveinto consideration, we speculated that BMP2signal pathway may play an important role inpromoting osteoblast functions under the PEMF stimulation.Our findings suggest that PEMF enhances the osteoblast compatibility on titaniumsurfaces but to different extent with regard to implant surface topographies. Theexpression of osteogenesis-related genes was upregulated on all the surfaces by PEMFstimulation. PEMF stimulation upregulated the expression of p-Smad1/5/8， thedownstream proteins of this pathway (Runx-2and OSX) were also promoted by PEMF.Taking the above into consideration, we speculated that BMP2signal pathway may playan important role in promoting osteoblast functions under the PEMF stimulation.