| Titanium(Ti)and its alloys are widely used in various clinical applications including the bone defect reconstruction thanks to the good biocompatibility,strength and corrosion resistance.Currently,nearly all the Ti based bone implants used in clinic are composed of a solid structure,such as the artificial joint and internal fixation plate,which suffer the problems of stress shielding and defect bone-implant bonding that may incur implant failure.Regarding these issues,porous Ti with interconnected porous structure arouses increasing attention.On one hand,the porous Ti possesses decreased elastic modulus and thus better mechanical compatibility to bone.On the other hand,possible bone ingrowth into the porous structure is anticipated to establish a hybrid bone/Ti structure that should have more rigid bone-implant bonding than that on the solid implant surface.The recently quick advance in the 3D printing(or additive manufacturing(AM))techniques,especially those available for Ti,allows us to control the 3D parameters and customize the implants for specific gross shape and microstructure requirements.In the present study,the porous Ti6Al4 V bone implants with controlled pore sizes and good interconnectivity were fabricated via EBM,which were further forwarded to nanotopographical modification as well as Sr incorporation for potentially enhanced bone formation and ingrowth.The study provides insights to the surface modification of porous metal implants.Part Ⅰ Fabrication of Porous Ti6Al4 V Scaffolds and Surface ModificationObjective: To design and fabricate porous Ti6Al4 V scaffolds and test its mechanical properties.To apply titania nanotubes and strontium loaded titania nanotubes onto its surface.Methods: Porous Ti6Al4 V scaffolds with two different pore sizes were fabricated via the Electron Beam Melting technique.The geometric parameters of the as-fabricated implants were obtained by Micro CT scanning,while the mechanical strength was evaluated through the compression test.Titania nanotubes and strontium loaded nanotubes were coated onto samples’ surface via anodization and sequential treatment of anodization and hydrotherm,respectively.FE-SEM,EDS and a water drop instrument were used to test surface properties.The strontium release profile was recorded via ICP-AES.Results: Ti6Al4 V scaffolds with controllable pore sizes were successfully fabricated via EBM.The porosity of the as-manufactured implants is very close to the designed ones,while the surface areas of the as-manufactured implants are a little larger than the designed ones.The structures exhibit good compressive strength and simultaneously low modulus compatible to the natural bone.The surface of the porous implants is not absolutely smooth but has microroughness.Relatively evenly distributed and well-ordered nanotubes were formed on the surface of porous implants after anodization.The tube diameter is approximately 70 nm and the tube wall is pretty thin.After Sr incorporation,the nanotubular structure was well preserved but the nanotube wall becomes obviously thicker.The surface modification of the porous implants leads to significant changes in the wettability.The pristine porous implants are hydrophobic with a water contact angle of about 90 o while the anodization and the further Sr incorporation make the porous implants superhydrophilic where the water drop infiltrates immediately once contacting the sample surface.Compared to the pristine porous implants,the modified ones have increased O contents and decreased Ti contents related to the anodization process.F residuals from the electrolyte are also seen in the modified implants.The presence of Sr element as displayed by EDS indicated the successful Sr incorporation by the hydrothermal treatment.Then the Sr release kinetics was evaluated.Generally,an initial Sr burst release at the first few days followed by a relatively stable Sr release with slight decline during the 30 days.Conclusions: The EBM technique is capable of producing uniformly porous Ti6Al4 V scaffolds with high accuracy and low modulus.Anodization can create well-ordered nanotube arrays,whilst further hydrothermal treatment in strontium solutions can form strontium loaded nanotube coatings,both of which exhibit a superhydrophilic feature.Part Ⅱ In vitro Evaluation of Porous Ti6Al4 V Scaffolds Objective: To test the cell viability and osteogenic differentiation ability of porous Ti6Al4 V scaffolds.Methods: rBMMSCs were isolated from bone marrow tissues of one-week-old Sprague-Dawley rats.WST-8 cell counting kit was used to test cell attachment and proliferation.Cell morphology on samples’ surface was viewed via FE-SEM.GFP-labeled r BMMSCs were applied to analyse cell behaviors on 3D scaffolds.The ALP activity and osteogenic genes expressions were analysed via ALP testing kit and q RT-PCR assays.Results: For the pristine porous implants,the cells cannot easily go into the porous structure but mostly locate on the superficial surface.Such decreasing trend becomes less obvious for the modified samples especially the Sr laden ones.Meanwhile,samples with larger pore size possess slightly better cell infiltration ability comparing to the smaller pore size counterpart.The cell proliferation assay reveals a time dependent cell growth pattern with unmodified samples exhibiting fastest proliferation rate.Consecutive culturing of GFP-labeled cells on 3D samples shows the pores will be gradually covered by cells.The speed and extent of pore covering is related to the pore size as well as the surface modification,with unmodified smaller pores closing first.The cells on unmodified samples have a thin,flat and round shape lack of long extensions.On contrary,the cells on nanotubes assume a dramatically elongated shape with abundant lamellipodia.The higher magnification images disclose that the extracellular matrix(ECM)deposited mainly at the top of nanotube,which broadened the tube wall.Incorporation of Sr into nanotube arrays promotes cell spreading to a polygonal osteoblast-like geometry.It is worth to notice long extended filopodia at high magnification.The ALP activity and genes expression assays reveal that strontium loaded nanotube arrays can induce osteogenic linage commitment of rBMMSCs.Conclusions: The porous Ti6Al4 V scaffolds show no cytotoxic effect.Nanotube arrays and strontium loaded nanotube coating,especially the later one,exhibit a decreased proliferation rate,as well an osteogenic differentiation tendency.Scaffolds with smaller pore size have a stronger tendency on cell proliferation and differetiation,but a weaker ability for cells infiltrating into the inner region of 3D scaffolds.Part Ⅲ In vivo Evaluation of Porous Ti6Al4 V ScaffoldsObjective: To study the bone formation and ingrowth ability of porous Ti6Al4 V scaffolds in vivo with a rabbit femoral model.Methods: The porous Ti6Al4 V scaffolds were implanted into the lateral femoral epicondyle of the hind legs of adult New Zealand white rabbits with a healing period of 4 weeks and 12 weeks,respectively.Micro-CT analysis was used to show bone regeneration capacity among each groups.Sequential fluoresent labeling and VG staining of histological sections were applied to evaluate bone ingrowth abilities.Results: The bone formation ability follows an order of strontium loaded nanotube coating>nanotube arrays>unmodified groups.Under same conditions,samples with smaller pore size exhibit better bone regeneration ability comparing to its larger pore size counterparts.Sequential fluoresent labeling assays show that not only mass of bone formation was elevated,but also the newly formed bone time was advanced by stontium loaded nanotube coating.VG staining,from another aspect,confirmed the aforementioned results.Conclusions: The porous Ti6Al4 V scaffolds with modified surface can improve bone formation and ingrowth abilities.For samples with different pore sizes,the smaller ones promote bone formation and the larger ones promote bone ingrowth ability.Part Ⅳ The mechanism of cell-material interaction on cell decision makingObjective: To study the influence of surface modification on the endoplasmic reticulum.Methods: BMMSCs were cultured on Ti sheets and the cell morphology was viewed by FE-SEM.The cytoskeleton was captured by a confocal microscopy.The endoplasmic reticulum was pictured by a transmission electron microscope.The expression levels of ER-related proteins were analysed by western blotting.Results: The NT and NTSr coatings exhibited a thicker cell conformation.The cytoskeleton was greatly thickened by NTSr coating.The PERK-ATF4 pathway was upregulated by NTSr coating.Conclusions: The NTSr coating caused changes in cell morphology and cytoskeleton.It also induced ER stress and upregulation of ER-related proteins. |
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