| BackgroundPolymethyl methacrylate(PMMA)bone cement(PBC),an injectable material,has been commonly used in orthopedic surgery for a long time.PBC was first applied in stomatology and then used in orthopedics in 1945.As a routine filling material,PBC has been used to fill bone defects,grout joint prostheses,and to achieve vertebroplasty and to augment the trajectory of pedicle screws.Meanwhile,it also functioned as the gold standard antibiotic delivery in the treatment of bone infection.Antibiotic-loaded PBC has been applied to fill infected bone defects,to revise the infected joint replacement,and in the technique of membrane-guided tissue regeneration to deal with traumatic bone defects.Although PBC is the most commonly applied filling material and has achieved good clinical outcomes in orthopedic surgeries,many drawbacks still merit further consideration.For example,polymerization volume shrinkage-related cracks formed at the PBC-bone interface,and subsequent biomechanical instability was regarded as the reason for prosthesis loosening and vertebral augmentation failure.In addition,exothermic injury,toxicity,low bioactivity,lack of osteogenesis and osteointegration prevent PBC from being an ideal material.However,there are no effective ways to solve these bottleneck problems of PBC due to its intrinsic properties,and there are currently no materials that can replace PBC.Therefore,modification of PBC or developing a substitute material to overcome the aforementioned drawbacks deserves consideration and has profound clinical implications.To count against the polymerization volume shrinkage of PBC,a self-expandable P(MMA-AA-St)was developed in our previous work,which overcame the volume shrinkage by volume expansion during water absorption.In addition,the maximum temperature of polymerization was also decreased,which could reduce exothermic injury.However,the direct effect of P(MMA-AA-St)volume expansion after water absorption was the deterioration of its biomechanics,which was only 58 MPa and did not satisfy the requirement of Standard ISO 5833.ObjectiveTo improve the biomechanics of the developed P(MMA-AA-St)while preserving expandable ability and improving its osteointegration ability.Graphene oxide(GO),a hydrophilic material with good biomechanics and osteogenic ability,was applied to optimize the biomechanics and bioactivity of P(MMA-AA-St),and the mechanisms of GO’s modification effect relating to biomechanics and bioactivity were further elucidated.Methods(1)Materials preparation and characterization:P(MMA-AA-St)-GO was synthesized using the method of dispersing polyermzation,and the final product of the reaction was characterized using Raman spectroscopy,transmission electron microscopy(TEM)and scanning electronic microscopy(SEM).The experiment was divided into three groups according to the GO incorporation:○1 PBC group:commercial PBC,○2 PGBCs group:P(MMA-AA-St synthesized with GO and○~3PGBCm group:P(MMA-AA-St)mixed with GO.First,the rate of water absorption and volume expansion was evaluated.Thereafter,the biomechanics and setting parameters were tested according to Standard ISO 5833.(2)Cytotoxicity evaluation:primary osteoblasts were isolated from neonatal rat calvaria and characterized using alkaline phosphatase(ALP)staining,Alizarin Red S staining and Von Kossa staining.Then,the primary osteoblasts were cultured with the extracts of PBC,PGBCs and PGBCm to assess the cytotoxicity of each material using the CCK-8 test and live-dead cell staining and to detect the effect of the extracts on the apoptosis rate of osteoblasts in each group.(3)Osteogenesis evaluation:the primary osteoblasts were seeded on the samples of PBC,PGBCs and PGBCm for 4 days and 7 days,and then the samples were pre-treated and scanned using SEM to observe attachment,morphology and proliferation of the attached osteoblast.In addition,the attached osteoblasts were stained by Rhodamine-phalloidin to detect the characteristics of cytoskeletons in each group.Thereafter,osteoblasts were cocultured with the materials to detect and compare the expression levels of osteogenic-related genes(Alp,Opn and Smad 5)by real-time quantitative polymerase chain reaction(q RT–PCR).In addition,the protein expression levels of OPN and Smad 5 were further detected and compared using Western blot assays among the groups.(4)Osteointegration and biosafety evaluation:PBC,PGBCs and PGBCm were implanted into bone defects in the femoral condyles of rabbits to assess the biosafety and osteointegration.○1 The postoperative X-ray examination was carried out to check the injectability and the local reaction of bone tissue after material implantation.○2 Blood samples were taken pre-and 1,2,3 and 4 weeks after surgery to perform hematological tests and biochemical tests,and samples of vital organs,including the lung,liver,kidney,heart and material surrounding muscle,were sectioned to perform hematoxylin&eosin(HE)staining to evaluate the biosafety of PBC,PGBCs and PGBCm.○~3The microarchitecture of the femoral condyle was analysed using micro-CT scanning at 4 and 16 weeks after material implantation to detect bone formation around the implanted material.○4 The 4-week femoral condyle samples were decalcified for HE staining and Masson staining to compare the number,morphology and collagen maturity of the material-surrounding trabecular bone.○5 The undecalcified 16-week femoral condyle samples were sectioned for Van Gieson(VG)staining to detect the bone-material interface and osteointegration.Results(1)Materials preparation and characterization:○~1Raman spectral analysis revealed that the characterized D peak and G peak of GO were detected in the spectra of P(MMA-AA-St)-GO,and the I_D:I_G ratio of P(MMA-AA-St)was 0.020,while that of P(MMA-AA-St)-GO increased to 0.902,which indicated that P(MMA-AA-St)was successfully synthesized onto the GO sheet.○2 TEM images showed that the P(MMA-AA-St)particles were wrapped by GO sheets,which was in accordance with the results of Raman spectral analysis.○3 The SEM results showed that PBC,PGBCs and PGBCm were all regular spheral particles with diameters of 20.80±6.20μm,222.97±52.49 nm and 341.83±52.49nm,respectively,and the diameters of each material were normally distributed.○4Biomechanical tests indicated that the compressive strengths of PGBCs before or after PBS immersion all surpassed the standard requirement(70 MPa)and were better than those of PGBCm.Meanwhile,the corresponding elastic modulus of PGBCs were significantly reduced when compared to PBC(P<0.05).○5 After GO optimization,PGBCs possessed excellent water absorption(95.14%)and volume expansion(101.18%).○6 Setting parameters indicated that the maximum setting temperature was significantly decreased(P<0.05),which could reduce the exothermic injury after implantation,while the dough time was lengthened to allow a longer handling time intraoperation.(2)Cytotoxicity evaluation:primary osteoblasts were successfully isolated from neonatal rat calvaria and verified by ALP,Alizarin Red S,and Von Kossa staining.The CCK-8 assay performed after osteoblasts were cultured with material extracts for 1,4,and7 days indicated that the cytotoxicity of PGBCs was significantly lower than that of PBC(P<0.05).○3 In addition,live-dead cell staining revealed that the number of dead cells was less than that of PBC when observed under the same magnified field.○~4The 4-day and 7-day apoptosis rates of osteoblasts in the PGBCs group were significantly higher than those in the PBC group(P<0.05).(3)Osteogenesis evaluation:○~1SEM scanning demonstrated that PGBCs and PGBCm had coral-like rough surfaces that benefited cell attachment,while PBC had a regular smooth surface.Furthermore,osteoblasts attached to the surface of PBC exhibited elongation,while osteoblasts on the surface of PGBCs and PGBCm were evenly distributed with a larger spreading area.○2 Rhodamine-phalloidin staining indicated that osteoblast attached on PGBCs exhibited better spreading than the other two groups and with the best morphology of cytoskeleton.○3 The levels of Alp,Opn and Smad 5 gene expression in the PGBCs and PGBCm groups were relatively higher than those detected in the PBC group with significance(P<0.05).○4 Higher expression levels of OPN and Smad 5 protein in PGBCs and PGBCm were discovered by Western blot when compared to PBC.(4)Osteointegration and biosafety evaluation:○~1PBC,PGBCs and PGBCm could be well prepared and injected into bone defects successfully during the operation,and the postoperative X-ray indicated that they were all good radiopaque materials.No leakage or adverse effects were detected after implantation.○~2No obvious disparities were detected by hematological tests,biochemical tests and HE staining of vital organs,which indicated no difference in biosafety among PBC,PGBCs and PGBCm.○~3The microarchitecture of the material-surrounded bones in the three groups was qualified using micro-CT,and the results indicated that better bone formation was discovered in the PGBC group than in the PGBCm and PBC groups(P<0.05).○~4HE staining of decalcified femoral condyles revealed thicker trabecular bone in the PGBC group,while the least bone formation was detected in the PBC group.Masson staining revealed that collagen synthesis was most obvious in PGBCm.○~5Obvious line transparency was detected at the interface of bone and PBC,while this was not observed in the other two groups,which indicated that the osteointegration ability of PGBCs and PGBCm was superior to that of PBC.ConclusionThe biomechanics of self-expandable PGBCs could be significantly improved by synthesizing GO with P(MMA-AA-St),with its compressive strength satisfying the standard ISO 5833 and its elastic modulus significantly reduced.PGBCs preserved an excellent ability of water absorption and volume expansion to count against the volume shrinkage of PBC.In addition,the polymerized temperature of PGBCs was significantly decreased,which could reduce exothermal injury,and the dough time was lengthened,which could improve the intraoperative manipulation.Furthermore,lower cytotoxicity,better osteogenic ability,and excellent biosafety and osteointegration of PGBCs were detected by in vitro and in vivo experiments.Above all,GO modification improved the physiochemical properties,cytocompatibility,and osteogenic ability of PGBCs and could overcome the drawbacks of PBC,allowing its material derivatives to serve as effective implantable biomaterials. |
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