The Effects Of Pulse Electromagnetic Fields On The Osseointegration Of Porous Titanium Implant Materials And The Potential Mechanisms

Treatment of osseous defects caused by tumor resection, trauma, infection, or osteo-degenerative diseases imposes tremendous socioeconomic burdens. Despite of extensive clinical use of autograft and allograft as bone substitutes, the tissue availability, donor site morbidity or immunological rejection remains to be their major limitations. Titanium and titanium alloys have exhibited their superiority as load-bearing metal implants because of the excellent biocompatibility and corrosion resistance. However, the mechanical mismatch between titanium implants and surrounding natural bones leads to the stress-shielding effect and accelerates bone resorption, and thus increases the risk of--implant loosening. Titanium alloys with porous structures show structural s imilarity with cancellous bones and also provide favorable pore channels for the transport of nutrients and metabolites. However, titanium-based materials are bioinert which are more easily connected with natural bones in the form of mechanical interlock rather than chemical bonding. Inadequate osseointegration of titanium alloys remains to be a major clinical limitation, which fails to assure the early fixation and secure long-term stability of implants, especially for osteoporotic patients. Thus, developing novel techniques to speed up osseointegration of titanium implants holds great clinical significance for increasing the repair efficiency and quality of bone defects.In the past four decades, substantial and growing evidence has accumulated to show that pulsed electromagnetic fields(PEMF) therapy as an alternative noninvasive method is capable of producing satisfying therapeutic effects on a wide range of bone diseases, such as fresh and nonunion fractures and osteoarthritis. Several in vivo investigations have also demonstrated that PEMF stimulation could inhibit bone loss and improve bone quality in various osteoporotic animals. The anti-osteoporotic efficiency of PEMF was further confirmed by several clinical investigations. PEMF stimulation has been shown to promote proliferation and mineralization of osteoblasts in vitro and also inhibit osteoclastogenesis. Despite of these positive findings of PEMF stimulation, critical questions regarding whether PEMF exposure is able to promote the bone ingro wth and accelerate osseointegration of p Ti in animals with bone defect remain unanswered yet.In the present study, the potential effects of PEMF stimulation on the biological performance of p Ti were systematically evaluated both in vitro and in vivo. First, we investigated the screening of the PEMF parameters, and then we used the better combined parameters to investigate the impacts and underlying mechanisms of PEMF on in vitro osteoblastic activities and functions in p Ti. Then, the promotional effects of PEMF stimulation on the repair of long bone defect by p Ti implants were systematically evaluated via analyses for Micro-CT and histomorphometric parameters. Furthermore, the molecular signaling pathway mechanisms of PEMF on in vivo osteogenesis in p Ti implants were also investigated. O ur findings suggest that p Ti implants accompanied by PEMF exposure exhibit high efficiency and quality in the repair of long bone defects, and might become a clinically applicable treatment modality for osseous defects. The present study mainly divided into four parts:1. The preparation of porous Titanium alloy(p Ti) and selection of pulsed electromagnetic fields(PEMF)parametersBackgrouds: Titanium alloy as a dense material its elastic modulus is far higher than that of cancellous bone. Therefore, the mismatch in the mechanical strength between metallic implants and surrounding natural bone can cause the stress-shielding effect and accelerate bone resorption, and thus increase the risk of implant loosening. Recently developed titanium-based alloys with porous structure were able to effectively decrease the mismatch of elastic modulus between implants and bone tissues. Moreover, the internal porosity forms interconnected pore channels for the transport of nutrient and metabolites. The elastic modulus of p Ti is mainly determined by the aperture size, porosity and other characteristics. EBM can make p Ti in accordance with the requirements. The different results of PEMF on cellular activities are due to the parameters. It is necessary to investigrate better parameters for the following research of the mechanisms of PEMF on bone remodling.Methods: The porous Ti6Al4 V implants were designed using the computer-aided design(C AD) software and fabricated using the EBM system. The prepared p Ti implant was scanned with Micro-CT and SEM for visualizing its microstructure and morpholo gy, respectively. According to the effects of cell activity, the stimulation parameters of PEMF on osteoblast were investigated.Results: Both of cylinder and disc p Ti matched the requirement. The present study evaluated a best group of PEMF parameters. And the parameters, 20 Gs(2 m T) and 2 h/d PEMF exposure, were choosen for the positive effect.Conclusion: EBM is an efficient and highly controlled manufacturing method of the material. The parameters were consistented with our previous study in vivo.2. The effect of PEMF exposure on osteogenesis in vitro and its associated mechanismBackgrouds: Osteoblasts performent main function in bone formation. Some literatures reported that PEMF improved bone remodling depends on the different stage of bone cells. But there is still lack of systematically investigation on phenotypes and mechanisms.Methods: We used CCK-8 and cytoskeleton fluorescence staining to evaluate the activity of osteoblasts in the stage of proliferation. The ALP staining and alizarin red staining were used to evaluate the ability of PEMF on differentiation of osteoblasts. q RT-PCR and Western Blot were used to investigate the potential mechanism.Results: In vitro osteoblastic FITC cytoskeleton staining show that cells in the PEMF group displayed well-developed cytoskeleton with higher fluorescence intensity, more microfilaments and thicker stress fibers. The ALP staining reveals that cells cultured in the osteoinductive medium in the PEMF group were more positive than those in the control group. Quantitative comparison of the ALP staining demonstrates that the ALP activity was significantly increased after PEMF stimulation as compared with the Control group in the differentiation stage(P < 0.05). The results for osteoblastogenesis-related gene expressions in MC3T3-E1 cells at the proliferation stage and differentiation compared with the Control group, PEMF stimulation significantly enhanced proliferation-related gene expressions at the cellular proliferation stage, including Ccnd 1 and Ccne 1(P < 0.05). PEMF exposure also significantly promoted the gene expression of Runx2, whereas PEMF decreased ALP gene expressions in the proliferation stage(P < 0.05). No significant difference of OCN gene expression was observed between the Control and PEMF groups in the proliferation stage. Furthermore, PEMF enhanced the gene expressions of canonical Wnt signaling during the stage of cellular proliferation, including Wnt1, Lrp6 and β-catenin(P < 0.05). PEMF resulted in significant increases in osteogenesis-associated gene expressions in the differentiation stage, including ALP, OCN, Runx2, COL1(P < 0.05). Moreover, gene expressions of canonical Wnt signaling(including Wnt1, Lrp6 and β-catenin) were also significantly higher in the PEMF group than those in the Control group(P < 0.05). The results of in vitro osteoblastogenesis-related protein expressions via western blotting analyses show that in the proliferation stage, ALP protein expression was decreased and OCN protein expression was not obviously changed by PEMF. Runx2, Wnt1, Lrp6 and β-catenin protein expressions were significantly higher in the PEMF group than those in the Control group in the proliferation stage(P < 0.05). In the differentiation stage, significant increases of protein expressions were found after PEMF exposure, including ALP, OCN, Runx2, Wnt1, Lrp6 and β-catenin(P < 0.05). The ECM mineralization via Alizarin Red staining revealed increased area of mineralization with more stained nodules. Q uantification of the solubilized stain demonstrates that the ECM mineralization was dramatically increased by PEMF stimulation as compared with the Control group(P < 0.05).Conclusion: The data in this study reveals that canonical Wnt pathway involved in PEMF regulating biological behavior of the osteoblasts.3. The effects of pulse electromagnetic fields on the osteogenesis of porous titanium implant materials and the potential mechanismsBackgrouds: The p Ti implants with superior structure features have excellent biocompatibility and corrosion resistance what makes it the ideal bone substitute implants. However, the effects of PEMF on osteogenesis and osseointegration of p Ti remain unknown thus far. Herein we systematically evaluated the potential efficiency of PEMF on the biological performance of p Ti in vitro.Methods: DAPI staining was used to observe the early cell adhesion. SEM and CLSM were used to observe the morphology of osteoblasts. CCK-8 was used to evaluate the activity of osteoblasts. q RT-PCR and Western Blot were used to investigate the potential mechanism.Results: The PEMF exposure significantly increased cellular attachment for osteoblasts seeded in p Ti as compared with the Control group via DAPI staining(P<0.05). Representative in vitro osteoblastic FITC cytoskeleton staining images show that cells in the PEMF group displayed well-developed cytoskeleton with higher fluorescence intensity, more microfilaments and thicker stress fibers. In contrast, cells in the Control group showed lower cell number and poorly-organized cytoskeleton. Moreover, statistical comparisons further demonstrate that PEMF stimulation significantly promoted cellular proliferation for osteoblasts seeded in p Ti via CCK-8 analyses(P<0.05). SEM scanning shows that cells were proliferated with more pseudopodia in p Ti under the PEMF stimulation. Images with higher magnification reveal that the PEMF-stimulated group shows more ruffled membranes, and many more lamellipodia and filopodia as compared with the Control group. The results of in vitro osteogenesis-related gene and protein expressions for osteoblasts seeded in p Ti show that, in comparison with the Control group, PEMF stimulation significantly promoted the expressions of osteogenesis-related genes via q RT-PCR analyses, including Runx2, Osx and COL1(P<0.05), and also increased the gene expressions of canonical Wnt signaling, including Wnt1, Lrp6 and β-catenin(P<0.05). The results of western blotting analyses reveal that in vitro osteogenesis-related protein expressions for osteoblasts seeded in p Ti, including Runx2, Wnt1, Lrp6 and β-catenin were significantly higher in the PEMF-stimulated group than those in the Control group(P<0.05).Conclusion: The PEMF is beneficial for osteoblasts in adhesion and mineralization in p Ti. The results reveals PEMF improve osteogenesis and new bone formation in p Ti through a canonical Wnt signaling-associated mechanism.4. Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in the repair of long bone defectsBackgrounds: Although the elastic modulus of p Ti has been improved, the mechanical mismatch between p Ti implants and surrounding natural bones is still exist. As a bioinert material, p Ti is easily in the form of mechanical interlock with surrounding natural bone. Whereas inadequate osseointegration of p Ti poses risks for unreliable long-term implant stability. Therefore, it has great clinical significance to find a clinically applicable treatment modality with high efficiency and quality in the repair of long bone defects.Methods: The bone defect model was constructed by surgical. Micro-CT,histology and histomorphometry were used to evaluate the effects of PEMF promote on bone remodling in p Ti. q RT-PCR was used to investigate the potential mechanism of PEMF on osteogenesis in p Ti.--Results: As indicated by 3-D Micro-CT images, PEMF exposure for 6 weeks and 12 weeks significantly increased the amount of newly formed bone within the implants as compared with the Control group. Images of 2-D mid-coronal and mid-sagittal slices further confirmed that bone ingrowth through the pores of p Ti was significantly promoted by 6-week and 12-week PEMF stimulation. Q uantitative statistical comparisons demonstrate that PEMF exposure for 6 weeks and 12 weeks resulted in significant increase of BV/TV(P<0.05, +78.5% at 6 weeks and +88.0% at 12 weeks). PEMF stimulation for 6 weeks and 12 weeks also significantly decreased the levels of BS/BV(P<0.05,-36.2% and-43.6%) and Tb.Sp(P<0.05,-30.7% and-42.8%). Moreover, PEMF exposure caused higher levels of Tb.N at 6 weeks(P=0.416, +21.7%) and 12 weeks(P<0.05, +16.3%), and also increased Tb.Th at 6 weeks(P=0.254, +41.8%) and 12 weeks(P=0.307, +49.6%). Masson-Goldner trichrome staining demonstrate that PEMF exposure stimulated more new trabecular bone ingrowth through the pores of p Ti in the region of bone defects. Statistical comparisons of the histological analyses further reveal that the levels of BV/TV were significantly higher in the PEMF-stimulated group(P<0.05, +143.1% at 6 weeks and +169.8% at 12 weeks). Dynamic histomorphometric analyses via calcein double-labeling staining show that PEMF stimulation speeded up the new bone formation in the region of bone defects. Quantitative comparisons reveal that 6-week and 12-week PEMF stimulation significantly increa sed MAR(P<0.05, +62.7% and +96.3%), MS/BS(P<0.05, +85.6% and +85.6%) and BFR/BS(P<0.05, +209.3% and +239.9%) as compared with the Control group. The results of in vivo osteogenesis-related gene expressions via q RT-PCR analyses show that PEMF exposure fo r 6 weeks and 12 weeks significantly promoted osteogenesis-related gene expressions as compared with the Control group, including Runx2, BMP2 and OCN(P<0.05). Moreover, m RNA levels in the canonical Wnt signaling pathway(including Wnt1, Lrp6 and β-catenin) in the PEMF group were also significantly higher than those in the Control group(P<0.05).Conclusion: The present study represents that PEMF stimulation promoted bone ingrowth and osseointegration of p Ti implants via obvious anabolic actions in the repair of long bone defects. The PEMF stimulation improved reliable long-term p Ti stability through a canonical Wnt signaling-associated mechanism. The p Ti implants accompanied by PEMF exposure might become a clinically applicable treatment modality for osseous defects.