The Effect Of 3d-printed Tantalum Scaffolds With Various Pore Sizes And Porosities On Osteogenesis And Osseointegration

Tantalum(Ta)has been used for orthopedic surgery as a biophile metal in recent years due to its good stability,high friction coefficient,and antibacterial effect.However,with the high elastic modulus of the dense material structure,a significant effect of stress shielding will be produced after implantation,eventually leading to bone resorption,implant loosening or even dislocation.A porous design of the material can effectively avoid the above defects and meet the mechanical requirements of perfectly matching with the natural bone tissue.Several studies have shown that the pore characteristics of porous scaffolds,such as pore size,porosity,strcut diameter,topology,and interconnectivity,are key factors affecting the performance of bone ingrowth and osteointegration.Nevertheless,views on the optimal pore size and porosity and its biological mechanism are still not consistent.Furthermore,the related researches on porous Ta are not enough.3D-printed technology was utilized to precisely control the structural parameters of scaffolds in our study.On the basis of the same design configuration,four Ta scaffolds with various pore sizes and porosities were prepared.At the same time,the effects of scaffolds with various pore sizes and porosities on osteogenesis and osteointegration were compared in vivo and in vitro experiments.This study is mainly divided into the following three parts:PART Ⅰ FABRICATION AND CHARACTERIZATION OF3D-PRINTED POROUS TA SCAFFOLDSThree-dimensional models with pore sizes of 100-200μm,200-400μm,400-600μm and 600-800μm(porosities of 25%,55%,75% and 85%,respectively)were created by computer aided design(CAD),and scaffolds with the same design configuration were prepared by selective laser melting(SLM)technology.The morphological characteristics and pore size of the scaffolds were evaluated by scanning electron microscopy(SEM).All scaffolds had regular hexahedron configurations,interconnected pores,uniform and smooth surfaces.The actual pore sizes were 173.2±39.4μm,376.9±68.3μm,534.3±64.6μm and 726.9±63.6μm respectively,and the corresponding porosities were 23%,53%,69% and 79% by gravimetric methods.The values of average roughness 21.9±3.4μm,17.2±3.1μm,31.0±5.7μm and 25.4±5.2μm were obtained using a laser scanning confocal microscopy(LSCM)and there was no statistical difference between the four groups(P>0.05).All these results were approximately consistent with the parameters of the designed model.Although SLM technology cannot completely achieve zero-error,it is a reliable method for preparing porous biological scaffolds through precisely controlling the various parameters of the material.PART Ⅱ THE EFFECTS OF POROUS TA SACFFOLDSWITH VARIOUS PORE SIZES AND POROSITIES ON THE BIOLOGICAL BEHAVIOR OF MBMSCS IN VITROSTUDYIn the experimental group,co-cultured of the porous Ta scaffold with mouse bone marrow stromal cells(mBMSCs)was conducted,while a simple cell culture was performed in the control group.The CCK-8 test showed that all scaffolds had no cytotoxicity.After 7 days of culture,the relative growth rates(RGR)were 138.5%,153.9%,179.3% and 167.9%,respectively.The absorbance value(OD)of the 400-600μm group was higher than that of other groups(P<0.05).There were almost no dead cells attaching on all scaffolds from Live-Dead staining for their good biocompatibility.Meanwhile,in the same three-dimensional space(X-axis1200μm,Y-axis 1200μm,Z-axis 200μm),400-600μm group showed a more obvious distribution of fluorescence intensity compared with other groups.Cells adhesion in four groups increased with time,and gradually covered the surfaces and pores of the material with interconnection of many filopodia extensions.The phalloidin/DAPI staining also indicated that the 400-600μm group showed a stronger ability in promoting cell adhesion and proliferation than other groups.The alizarin red staining analysis revealed that the performance of osteogenic differentiation in 400-600μm group and 600-800μm group was better than other groups(P<0.05),but the data between the two groups was not statistically different(P>0.05).The similar trend was found in the expression levels of the ALP and COL-I in qPCR test.However,the expression levels of VEDF and VWF in 600-800μm group were slightly higher than those in other groups after 7 days ofinduction(P<0.05).The overall effective permeability in 600-800μm group was about 5-25 times higher than that of other three groups by computational fluid dynamics(CFD)analysis.On the other hand,the surface area of the material gradually reduced as the pore size and porosity increased.The above results indicate that porous Ta scaffolds with too large or too small pores are not conducive to promoting the adhesion and proliferation of mBMSCs.The biological behavior can obtain an ideal equilibrium state in a limited pore size range of 400-600μm(porosity of75%).The scaffolds with pore sizes ranging from 400-600μm(porosity of75%)and 600-800μm(porosity of 85%)have more obvious advantages in inducing osteogenic differentiation of mBMSCs cells,and the process of large pore scaffolds tends to be performed by direct vascularization.PART Ⅲ THE EFFECTS OF POROUS TA SACFFOLDSWITH VARIOUS PORE SIZES AND POROSITIES ONBONE FORMATION AND OSTEOINTEGRATION FEMORAL DEFECT OF RABBIT MODELIn the experimental group,the scaffolds were implanted into the cylindrical defect in femoral condyles of rabbits,while a simple model of bone defect was established in the control group.At 2,4,and 8 weeks after implantation,X-ray scans,high-energy CT scans,H&E staining of hardtissue sections,and elemental analysis were performed to evaluate the effect of newly formed bone and osteointegration.After 4 weeks of implantation,the performance of bone volume fraction in 400-600μm group were better than those in other groups(P<0.05),and ultimately no less than the control group(P>0.05).Histological analysis showed that the bone penetration of the 200-400μm group and 400-600μm group were deeper than those of another two groups(P<0.05)and the bone area fraction of the 400-600μm group showed a similar trend with the CT results after 4 weeks of implantation.In the early stage of implantation,the bone contact rate was higher in the 400-600μm group than in the other groups(P<0.05),but there were no significant differences in bone contact rate,interface bonding strength,and new bone maturity between four groups after 4 weeks of implantation(P>0.05).Finally,the elastic modulus of the bone tissue around the femoral condyles measured by nanoindentation,combined with finite element analysis further verified the authenticity and validity of the mechanical tests.The above results fully demonstrate that the porous Ta scaffolds with a pore size of 400-600μm(porosity of 75%)can obtain more new bone ingrowth and ideal osseointegration interface earlier.In summary,the 3D-printed technology is a reliable method for manufacturing porous Ta scaffolds.More importantly,the pore size and porosity are key factors affecting the progress of osteogenesis and osseointegration.Porous Ta scaffolds with a pore size of 400-600μm(porosity of 75%)can obtain a better performance in bone formation and bone-implant interface.These will help provide a feasible direction for future research and application of porous implants to repair bone defects.