| IntroductionPorous titanium(Ti) alloys have beneficial uses as orthopedic implants. However, they do have shortcomings that they do not integrate very well with surrounding bone tissue, and bone ingrowth into the implants is not substantial. The reasons for the unsatisfactory integration and poor bone ingrowth may be that(1) although the apparent elastic modulus is reduced by adopting a porous structure, the whole wall elastic modulus, determined by material properties, is still much greater than natural bone, and(2) the absence of the electromagnetic field formed by the piezoelectric effects of normal spongy bone may also play an important role in bone health.To solve these problems, we examined several alternatives. A new implant alloy, Ti-24Nb-4Zr-7.9Sn(Ti2448), that has lower elastic modulus compared with existing Ti alloys was developed in our country and it reduces the whole wall elastic modulus. Porous Ti2448 implants are fabricated to reduce the apparent elastic modulus to further match the bone. Meanwhile, to rebuild the normal electromagnetic milieu that is essential for bone repair and remodeling, we have proposed using exogenous pulsed electromagnetic fields(PEMFs). Our previous study have demonstrated the positive effects of PEMF on osteoblasts cultured on the surface of Ti-6Al-4V scaffolds. However, the biological effects of the combination of PEMF and porous Ti2448 scaffolds have not been reported yet. Thus, the biological effects of PEMFs on porous Ti2448 scaffolds were evaluated both in vitro and in vivo in this study. Methods1. The fabrication and examination of porous Ti2448 scaffoldsThe 3D models of the two porous Ti2448 scaffolds were created in Materialise software, and then models were transferred into an EBM system to fabricate the porous Ti2448 scaffolds as expected. The surface topography and elements of scaffolds were examined by scanning electron microscopy(SEM) and energy dispersive spectrum(EDS).2. The construction of PEMF stimulation systemThe PEMF stimulation system comprised a PEMF generator of GHY- Ⅲand solenoids. The PEMF generator can produce a particular open-circuit output waveform PEMF which was demonstrated to have positive effects on osteogenesis. Solenoids used in vitro study were comprised of two Helm Holz solenoids, and in vivo study there were advanced three Helm Holz solenoids to produce enough extending of well-distributed PEMFs. The magnetic fields in the solenoids and the surrounding environment were determined using a gaussmeter.3. In vitro studyThe osteoblast-like cell line MC3T3-E1 was cultured in the absence(control) or presence of PEMF stimulation on porous Ti2448 disc surfaces, and the adhesion and proliferation of the cells was investigated by SEM, live/dead cell imaging kit and CCK-8 assay. Furthermore, the expression of osteogenesis-related genes were also examined by quantitative real-time PCR.4. In vivo animal studyThe porous Ti2448 scaffolds for animal were implanted into the lateral femoral epicondyle of female New Zealand white rabbits with an average weight of 2.8-3.3 kg. All of the 24 rabbits were then randomly divided equally into two groups, one group with PEMF stimulation for 2 h everyday and the other with no stimulation as control. After 4 weeks and 12 weeks, 12 rabbits(6 in each group) were sacrificed for histological analysis at each time point. ResultsThe porous Ti2448 scaffolds were observed to have rough surfaces with a pore size of 710±42 μm and a porosity of 68±5.3%. The porous Ti2448 disc had a diameter of 12 mm and a height of 3 mm, and the porous Ti2448 scaffold with a diameter of 6 mm and a height of 10 mm.The PEMF stimulation system for cell culture and animal study was successfully constructed. The magnetic flux densities in the solenoids and in the surrounding circumstances were determined to be 2 m T and 0.05±0.002 m T, separately.In cell study, CCK-8 test showed no significant difference in cell adhesion and proliferation after 1 day’s culture between two groups. However, the cell proliferation, and, therefore, the number of viable cells in the PEMF group was significantly higher than the control group on day 4 and day 7. The confocal microscopy images of MC3T3-E1 cells stained with live/dead cell imaging kit after 48 h in culture showed that in both the control and PEMF groups, most cells with green fluorescence(live) had an osteoblast-like shape and extended well. Occasional spots of red fluorescence, which represented dying or dead cells, were detected among the green cells. Compared with the control group, more cells with green fluorescence and fewer cells with red fluorescence were observed in the PEMF group. Greater numbers of cell clusters were formed in the PEMF group as well. The SEM findings in both control and PEMF groups after 48 h in culture showed the MC3T3-E1 cells covering the surface with healthy levels of proliferation and cytoplasmic extensions. Even deep inside the porous Ti2448 disc, cells were attaching to the surface of the struts and spreading into an osteoblast-like shape. Furthermore, greater numbers of cells on the available surface and the formation of cell clusters were observed in the PEMF group, compared to the controls. As to the expression levels of osteogenesis-related genes, including ALP, Col-1, OCN, OPN, Runx2, and BMP-2, PEMF stimulation generally up-regulated m RNA levels of all the genes of interest. On day 4, the expression levels of ALP and BMP-2 were not significantly different between the control and PEMF groups, but levels of Col-1, OCN, OPN, and Runx2 m RNAs were up-regulated by 1.3-fold, 3.3-fold, 1.7-fold, and 1.4-fold, respectively, after PEMF stimulation. On day 7, ALP, Col-1, OCN, Runx2, and BMP-2 were up-regulated by 4.2-fold, 1.9-fold, 2.0-fold, 1.3-fold, and 4.0-fold, respectively, after PEMF stimulation, while no significant difference in OPN expression was found between the control and PEMF groups.In animal study, fluorescent labeling results revealed that the growth rate of new bone around the implant in PEMF group was significantly higher than that in control group. The histomorphologic(Van Gieson staining) results also showed that the percentage of regeneration bone in the implant of PEMF group was significantly higher than that in control group at 4 weeks and 12 weeks. ConclusionThe porous Ti2448 scaffolds fabricated by EBM technique have low apparent elastic modulus and better micromechanical properties compared with existing Ti alloys to match the bone. PEMF have positive effects both on the functions of osteoblast-like cells culture on the surface of porous Ti2448 scaffolds in vitro and on the osseointegration and bone ingrowth of porous Ti2448 implants in animal study in vivo. Our results demonstrate that the combination of PEMFs and porous Ti2448 implants may constitute a new therapeutic mode to treat bone disorders. |
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