| Background Application of implants in orthopedic surgery has been demonstrated to restore the structure and function of bones and joints that have been damaged by trauma,arthritis,bone tumors,and infections or medication-related osteonecrosis.Various amounts of materials,such as metals,ceramics,polymers and composites,are commonly applied during the preparation of orthopedic implants.Metals?e.g.,stainless steel and titanium alloys?are widely used as prostheses owing to their high mechanical strength and excellent friction resistance.However,they release harmful metal ions and are radiopaque,thus limiting the applications of metal alloys.Ceramics?e.g.,Al2O3,HA and bio-glass?exhibit excellent biocompatibility and good corrosion resistance,but their mechanical properties,fracture toughness and ductility are too low to meet the demands of load-bearing applications.Polymers such as polytetrafluoroethylene?PTFE?and polylactide?PLA?are widely used as biomedical implants.However,because of their poor mechanical properties and instability during sterilization processes,only a few are used as orthopedic implants.Polyetheretherketone?PEEK?,a thermoplastic polymer,remains stable during sterilization processes because of its stable chemical and physical properties.Carbon fiber-reinforced PEEK?CFR-PEEK?is one type of PEEK composite that is composed of layers of carbon fiber sheets.In contrast to metal alloys,CFR-PEEK is radiolucent,and the modulus of CFR-PEEK can be controlled to match the modulus of bone more accurately by changing the orientation and number of carbon fiber layers.This ability can reduce the stress shielding and makes CFR-PEEK popular for a number of orthopedic implants.However,CFR-PEEK possesses a hydrophobic surface;it cannot promote bone apposition because of its bioinert chemical properties.Currently,two main strategies – surface modification and composite preparation – have been proposed to increase the bioactivity of PEEK-based hybrid materials to improve the bone-implant interface.Reinforcements such as Ti O2,Zr O2,and nanohydroxyapatite?n-HA?improve the biological properties of CFR-PEEK,but they usually impair the mechanical properties of CFR-PEEK,thus limiting their clinical applications.In this respect,surface modification is an effective method to tailor the surface mechanical and biological properties while preserving the favorable bulk characteristics of the materials.Graphene,isolated by Novoselov and Geim in 2004,is an atomically thick sheet.Over the last decade,it has received unprecedented attention owing to its high mechanical strength,large surface area,low mass density and excellent thermal and electrical conductivity.Graphene has also shown good performance in medical applications,such as disease diagnosis,drug/gene delivery,cancer treatments,biosensor,and antibacterial materials.In2010,a study by Kalbacova et al.indicated that graphene has osteogenic potential for human mesenchymal stem cells?h MSCs?.One of our recent studies using graphene-coated polyethylene terephthalate?PET?artificial ligaments demonstrated that graphene has the ability to promote the adhesion,proliferation,and osteogenic differentiation of MSCs.Because of the excellent mechanical and biological properties of graphene,our study provided a new type of coating material for implants and scaffolds.To improve the bioactivity of CFR-PEEK,in this study,graphene was transferred onto the surface of CFR-PEEK.We hypothesize that graphene could be stably coated onto the surfaces of the CFR-PEEK and promote the proliferation and osteogenic differentiation of bone marrow stromal cells?BMSCs?on the graphene-coated CFR-PEEK?G-CFR-PEEK?.Part ? The fabrication and characterization of graphene surface modified CFR-PEEKOBJECTIVE:The CFR-PEEK samples by graphene modification were prepared using the chemical etching method.The surface morphologies and the wettability properties were applied to determine whether graphene was stably coated on the surface of CFR-PEEK.METHOD:The large-scale graphene used in this study was synthesized via the chemical vapor deposition method on copper foils.The CFR-PEEK samples by graphene modification were prepared by etching the copper with FeCl3 solution.Afterward,the G-CFR-PEEK was obtained by dissolving the PMMA with acetone.The fine structures of graphene on the copper were characterized via Raman spectroscopy.After coating with gold,the surface morphologies of the CFR-PEEK and G-CFR-PEEK samples were investigated via SEM.The wettability of the scaffolds with respect to water was examined by measuring the contact angle at three different locations on each sample.RESULTS:The large-scale CVD-grown graphene on the copper foils was characterized via SEM.The G(1580 cm-1)and 2D(2670 cm-1)bands are the most prominent features in the graphene samples.These features were used to identify graphene and determine its layers via the Raman spectroscopy.Graphene was transferred onto and stably conjoined to the surface of the CFR-PEEK samples.The two systems had comparable local surface morphologies,but they looked very different on a large scale.The G-CFR-PEEK samples consisted of many ripples and wrinkles at the micrometer scale,whereas the surface of the CFR-PEEK consisted instead of a large number of valleys.The G-CFR-PEEK samples had a significantly lower water contact angle?74.96±1.65°?than the CFR-PEEK samples?91.26±1.53°,P<0.05?,which indicated that the graphene coating effectively modified the wettability of the CFR-PEEK scaffolds.CONCLUSIONS:The graphene was successfully and stably transferred onto the surface of CFR-PEEK.And graphene surface modification can significantly improve the hydrophilic of CFR-PEEK without changing the mechanical and physical properties of CFR-PEEK.Part ?The enhanced surface bioactivity of graphene modificated CFR-PEEK in vitroOBJECTIVE:Investigate the enhancement of the surface bioactivity of graphene modified CFR-PEEK by prompting the adhesion,proliferation and differentiation of BMSCs in vitro.METHOD:Bone marrow was harvested from the tibia and femur of euthanized,male,3-week-old Sprague Dawley?SD?rats under sterile conditions.The marrow cavity was gently and repeatedly rinsed with?-MEM medium??-MEM,HyClone,Logan,UT?to obtain a single-cell suspension.The BMSCs were used in the experiments before passage five.Flow cytometry was used to confirm surface antigen marker CD29,CD34,CD44 and CD45expression of the BMSCs.The BMSCs were seeded on the G-CFR-PEEK and CFR-PEEK samples in the 24-well plates with the density of 1.5×104 cells per well.After the samples were seeded and cultured for 1 day and 3 days,the samples were observed via SEM after sputter coating with gold.The CCK-8 assay was employed to quantitatively evaluate the cell cytotoxicity on the samples at 1,3,5,and 7 days after seeding.The BMSCs were seeded on the samples?three replicates?at a density of 3×104 cells per well.After 1,3,5,and 7 days,fluorescein diacetate?FDA,Sigma?and propidium iodide?PI,Sigma?were simultaneously added to each well to measure the number of living cells and dead cells.The BMSCs were seeded on the samples at a density of 1.5×104 cells per well.At 1 and 3 days after seeding,the cells that adhered to the samples were fixed,permeabilized,and then blocked with 1%BSA.Afterwards,the G-CFR-PEEK and CFR-PEEK samples were incubated with primary anti-vinculin antibody?10?g/ml?,fluorescein isothiocyanate?FITC?-labeled goat anti-rabbit IgG antibody,and TRITC-labeled phalloidin to stain the actin cytoskeletons of the cells.Afterward,DAPI was used to stain the cell nuclei.The scaffolds were mounted on coverslips and examined using a confocal laser scanning microscope.The ALP staining was performed using a BCIP/NBT ALP staining kit according to the instructions,and the staining was then observed using stereomicroscopy.The alkaline phosphatase activity was assayed using an alkaline phosphatase assay kit.Calcium mineralization was measured from the selective binding of alizarin red S to calcium salts.The pictures were obtained using stereomicroscopy.The osteogenic differentiation properties were assessed using a western blot analysis for the typical osteogenesis-related protein accumulation after 7 and 14 days,including COL1A1,Runx2,BMP2 and OSX.RESULTS:The SEM images revealed that the BMSCs were firmly adhered to the G-CFR-PEEK scaffolds and presented more cellular pseudopods than those on the CFR-PEEK scaffolds after 24 h of seeding,and the cells on the G-CFR-PEEK formed a spindle morphology,in contrast with the star-like morphology observed on the CFR-PEEK scaffolds.After 3 days of culturing,a greater amount of extracellular matrix?ECM?was observed covering the G-CFR-PEEK scaffolds than was observed for the CFR-PEEK scaffolds.To further investigate the biocompatibility of the scaffolds,the morphology of the BMSCs was observed after 24 and 72 h of seeding.The immunoassay results for vinculin?green?,F-actin?red?,DNA?blue?and the merged results were obtained for the CFR-PEEK and G-CFR-PEEK scaffolds.After 24 h of culturing,the BMSCs were well distributed on the G-CFR-PEEK scaffolds with a projected cell area of 1589.3±178.7?m2,whereas a star-like morphology was observed on the CFR-PEEK scaffolds,and the cell area decreased to 692.0±68.2?m2.After 72 h of culturing,the projected cell areas of the G-CFR-PEEK and CFR-PEEK scaffolds were 2336.5±132.3?m2and 1915.5±195.8?m2,respectively.There was a significant difference between the projected cell areas of the pure and graphene-coated CFR-PEEK scaffolds?P<0.05?at all time points.As shown in Figure 4,the integral optical density?IOD?values for the vinculin immunostaining were calculated to quantitatively evaluate the BMSC adhesion,and the results revealed that the cells on the G-CFR-PEEK scaffolds exhibited significantly higher levels of focal adhesion compared with the CFR-PEEK scaffolds regardless of the culturing time?P<0.05?.BMSC proliferation on the CFR-PEEK and G-CFR-PEEK scaffolds was evaluated via a CCK-8assay at 1,3,5 and 7 days after seeding.The OD values of the CFR-PEEK and G-CFR-PEEK scaffolds increased over time,with significantly higher OD values observed for the G-CFR-PEEK scaffolds than for the CFR-PEEK scaffolds at all time points?P<0.05?.The number of live cells increased over time in all groups.The number of live cells on the G-CFR-PEEK scaffolds was significantly greater than that on the CFR-PEEK scaffolds at all time points?Figure 5B,P<0.05?.As an early marker,ALP was used to evaluate the osteoblastic differentiation ability of the BMSCs.After 4 and 7 days of culturing,the intracellular ALP activity was measured,and histochemical detection measurements were performed to determine the osteogenic differentiation ability.The results suggested that the ALP expression on the G-CFR-PEEK scaffolds was significantly up-regulated after culturing for 4 and 7 days.Moreover,the ALP activities of the cells in the G-CFR-PEEK scaffold group were 1.82±0.07 and 3.03±0.09,respectively,at 4 and 7 days after culturing;these values were significantly greater than those of the CFR-PEEK group,which were 0.91±0.03 and 1.61±0.03,respectively,at the indicated time points?P<0.05?.After 21 and 28days of induction,alizarin red S staining was performed?Figure 6B?.Obvious calcium-salt sedimentation was observed in the G-CFR-PEEK group.The osteogenic differentiation properties were assessed using a western blot analysis for the typical osteogenesis-related protein accumulation after 7 and 14 days,including COL1A1,Runx2,BMP2 and OSX.At two time points,the BMSCs on G-CFR-PEEK exhibited higher protein accumulation of COL1A1,Runx2 and OSX than CFR-PEEK after 7 and 14 days culturing?P<0.05?.Conversely,the BMP2 accumulation on the CFR-PEEK scaffolds was slightly greater than that on the G-CFR-PEEK at day 14,although the difference was not statistically significant.CONCLUSIONS:Graphene surface modified CFR-PEEK can promote the adhesion and proliferation of BMSCs.Furthermore,graphene can also effectively stimulate osteogenic differentiation of BMSCs.Part ?The enhanced surface bioactivity on CFR-PEEK via graphene surface modificated in vivoOBJECTIVE:Investigate the enhancement of the surface bioactivity of graphene modified CFR-PEEK by prompting the osteointegration between the CFR-PEEK scaffolds and the bone interface in vivo.METHOD:Thirty-six mature New Zealand rabbits?male,12 weeks old,3.2±0.3 kg?were subjected to an operative extra-articular graft-to-bone healing procedure.A G-CFR-PEEK graft was implanted into the condyle of the femur,and a CFR-PEEK graft was implanted into the contralateral femoral condyle as the control.At 4,8 and 12 weeks after surgery,the rabbits were humanely euthanized for the subsequent examinations.Two fluorochromes,i.e.tetracycline?50 mg/kg,Sigma?at 14 days and calcein?8 mg/kg,Sigma?at 4 days,respectively,were administered via intramuscular injection before euthanizing the rabbits at the 4-week time point to assess the osteogenic activity.The femoral condyles were carefully dissected and placed in a sample holder and scanned using a Micsron X-ray 3D Imaging System.For each specimen,a 4×10 mm cylindrical region in the middle of the bone tunnel along its longitudinal axis was selected as the region of interest?ROI?.The BV/TV?%?,BS/BV?1/mm?,Tb.Th?mm?,Tb N?1/mm?,and Tb.Sp?mm?were calculated.The push-out test was applied to investigate the weld strength of the implant-bone interface via the universal mechanical testing machine.The maximum failure load was recorded based on the load-displacement curves,and then the shear strength between bone tissues and the implant was calculated.Sections prepared from animals euthanized at 4 weeks were observed using a fluorescence microscope to measure the mineral apposition rate?MAR?of the new bone formation by monitoring the length between the two labels over time??m/d?.Then,all of the bone sections were stained with Van Gieson stain,and a light microscope was used to observe the bone ingrowth and integration with the host tissue.RESULTS:At all time points,there was more bone trabecula around the scaffolds in the G-CFR-PEEK group than in the CFR-PEEK group.At the 4-week time point,the G-CFR-PEEK group exhibited a greater bone-volume fraction?BV/TV?and Tb.Th,0.32±0.01 and 0.17±0.05,respectively,than those of the CFR-PEEK group?P<0.05?,which were 0.22±0.02 and 0.11±0.03,respectively.The BS/BV in the G-CFR-PEEK group was12.21±3.16%,which was less than that in the CFR-PEEK group?18.85±5.59%,P<0.05?.The BV/TV values of the G-CFR-PEEK scaffolds?0.34±0.02?were significantly greater than those of the CFR-PEEK scaffolds?0.26±0.01,P<0.05?at 8 weeks.However,there was no statistically significant difference in the BV/TV,BS/BV,Tb.Th,Tb.Sp and TbN between the G-CFR-PEEK scaffolds and the CFR-PEEK scaffolds at 12 weeks.The maximum failure load of both groups increased over time.However,at each time point,the maximum failure load of the G-CFR-PEEK group was greater than that of the CFR-PEEK group.At 4 weeks,the strength values of the G-CFR-PEEK group?3.37±0.11 MPa?were significantly greater than that of the CFR-PEEK group?2.33±0.10 MPa,P<0.01?.At the8-weeks,the strength of the G-CFR-PEEK group was 3.92±0.18 MPa,which was higher than that in the CFR-PEEK group?3.25±0.09 MPa,P<0.05?.Although,the strength of the G-CFR-PEEK group was still higher than that of the CFR-PEEK group at the 12 weeks,the difference was not significant?P>0.05?.The MAR was evaluated by measuring the interval distance between the centers of the yellow and green bands?Figure 10?.The MAR in the G-CFR-PEEK scaffolds was 3.57±0.18?m/d,which was significantly greater than the value of 2.11±0.11?m/d for the CFR-PEEK scaffolds?P<0.05?.After 4 weeks,the newly formed bone on G-CFR-PEEK implants was continuous and bonded tightly to the implant surface,whereas new bone was sparsely distributed around CFR-PEEK implants.The new bone area rate and bone-implant contact ratio were significantly higher for G-CFR-PEEK implants?30.1±1.7%and 74.7±4.7%,respectively?than CFR-PEEK implants?23.1±1.9%and 63.3±6.5%,respectively,P<0.05?,indicating graphene promoted early stage bone-implant osseointegration.At 8 weeks post-implantation,a larger new bone area and bone-implant contact were observed for G-CFR-PEEK implants?31.9±2.0%and83.6±5.3%,respectively?than CFR-PEEK implants?27.3±3.3%and 73.8±7.2%,respectively,P<0.05?.Moreover,there was a significantly greater amount of soft tissue between newly formed bone and the CFR-PEEK implants than the G-CFR-PEEK implants.After prolonging the healing time to 12 weeks,the volume of the newly formed bone around the G-CFR-PEEK scaffolds increased significantly to form a continuous and thick bone layer,indicating better osteointegration.In contrast,osteointegration was weaker between the CFR-PEEK implants and newly formed bone.However,while the new bone area rate and bone-implant contact ratio were higher for G-CFR-PEEK implants than CFR-PEEK implants at 12 weeks,these differences were not significant?P>0.05?.CONCLUSIONS:Graphene surface modified CFR-PEEK can promote new bone formation and the osteointegration of the CFR-PEEK scaffolds-bone interface. |
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