Sequential And Sustained Release Of Fibronectin And Bone Morphogenetic Protein-2 From Graphene Oxide-poly-l-lysine Modified Composite Scaffolds For Bone Regeneration

Objective:Bone deformities from congenital deformity,traumatic injury and oncologic resection severely affect patients'physical function and mental health.The treatment of large bone defect in clinic is limited,which has become a great challenge for surgeons.Although autologous,allograft,or xenograft bone transplantations can repair dysfunctional or defect bones,many limitations still need to be addressed:transplanted bone infection or instability,insufficient transplanted volume,even immunological rejection.Thus,the fabrication of bone graft materials with favorable osteoinductive ability is considered as the best method for bone regeneration.Silk fibroin?SF?and?-calcium phosphate??-TCP?have been widely used as bone graft materials in bone tissue engineering,due to their superior physicochemical and biological properties?i.e.excellent biocompatibility and controllable degradability?.Although both SF and?-TCP have been reported supporting mesenchymal stem cell?MSC?attachment,proliferation and extracellular matrix?ECM?deposition,it is insufficient to induce stem cell differentiation,and even repair large bone defects.Growth factors?GFs?,one of three elementary factors in tissue engineering,can directly regulate multiple biological behaviors of bone cells,including migration,adhesion,proliferation and differentiation.Also,they can interact with other functional proteins to synergistically promote bone formation.However,most of GFs are soluble protein,resulting in unstable status in water and rapid degradation,which cannot match the period of bone regeneration.This prompts the need for release technologies capable of the controlled spatiotemporal delivery of GFs.Bone tissue repair is a highly dynamic process that requires a wide range of inflammatory,angiogenic and osteogenic factors.More importantly,GFs precisely interact with cells though matched biological signals,rather than play their roles simultaneously.Therefore,the sequential and controlled release profiles of through supportive scaffolds are important factors for effective bone repair.Our research is aimed to fabricate a porous scaffold with good physicochemical properties and favorable osteoinductive capability via surface modification technologies.Therefore,four SF/?-TCP scaffolds were firstly fabricated to optimize the proportion of SF and?-TCP by evaluating their physicochemical properties and osteoinductive capability.Sequentially,the nanosized poly-l-lysine functionalized graphene oxide?GO-PLL?was synthesized and mixed with bovine serum albumin?BSA?nanoparticles to form a complex compound?GO-PLL/BSA?.Bone morphogenic protein-2?BMP-2?was encapsulated into BSA nanoparticles modified by GO-PLL.The following experiments have verified that sustained release of BMP-2 from GO-PLL/BMP-2 complex compound could significantly promote osteogenic differentiation of BMSCs.After fibronectin and BSA-BMP-2 nanoparticles were incorporated into the GO-PLL modified SF/?-TCP porous scaffolds,the release profile of FN and BMP-2 in the SF/?-TCP scaffolds were therefore investigated.Additionally,biological properties of the GO-PLL modified SF/?-TCP scaffolds with the incorporation of FN and BMP-2 were evaluated through assessing the capabilities of adhesion,proliferation and osteogenic differentiation of BMSCs on the complex compound modified SF/?-TCP scaffolds.The release profile of BMP-2 from was investigated,and BMSCs were used to evaluate the biocompatibility and osteoinductivity of GO-PLL/BMP-2 complex.Fibronectin and BSA-BMP-2 nanoparticles were incorporated into GO-PLL modified SF/?-TCP porous scaffolds.The release profile of FN and BMP-2 in SF/?-TCP scaffolds were investigated.The biological properties of GO-PLL modified SF/?-TCP scaffolds with incorporation of FN and BMP-2 were evaluated by assessing the ability of adhesion,proliferation and osteogenic differentiation of BMSCs.Methods:1.SF/?-TCP porous scaffolds were fabricated according the proportion of SF and?-TCP?1/0,2/1,1/1,1/2?by freeze-drying method.The physicochemical properties?i.e.surface morphology,pore size,porosity,swelling ratio,degradation ratio,compressive strength and biomineralization?were investigated using field emission scanning electron microscope?FESEM?,X-ray diffraction?XRD?and attenuated total reflectance Fourier transformed infrared spectroscopy?ATR-FTIR?.The biocompatibility and osteogenesis of the composite scaffolds were investigated by evaluating the proliferation and differentiation of BMSCs.Optimal proportion of SF and?-TCP was determined for further research.2.Bovine serum albumin nanoparticles?BSA?were firstly synthesized by desolvation method.Different proportion of poly-L-Lysine?PLL?and graphene oxide?GO?were fabricated by oxidation-reduction reaction as PLL functionalized GO?GO-PLL?before GO-PLL formed a complex compound with BSA nanoparticles as GO-PLL/BSA through a self-assembling technology.Microstructure,composition,particle size distribution and zeta potential were optimized through adjusting the proportion of GO and PLL,and characterized via transmission electron microscope?TEM?,field emission scanning electron microscope?FESEM?,attenuated total reflectance Fourier transformed infrared spectroscopy?ATR-FTIR?and dynamic light scattering?DLS?.Biocompatibility of the complexes was investigated by using bone mesenchymal stem cells?BMSCs?.After BMP-2 was encapsulated into BSA modified by GO-PLL,its release profile was studied to verify the osteogenic differentiation of BMSCs by using Alkaline phosphatase and alizarin red staining.3.FN and BMP-2 were incorporated into GO-PLL modified SF/?-TCP porous scaffolds,respectively.The release profile of the FN and BMP-2 from GO-PLL modified scaffolds were investigated.BMSCs were used to examine the biocompatibility and osteogenesis of the GO-PLL modified scaffolds.In particular,Cell morphologies on the scaffolds were observed at 48 h using SEM and confocal microscopy.Cell viability was evaluated using Cell Counting Kit-8 and live/dead staining.Cell cytoskeletal organization of BMSCs was observed via rhodamine-phalloidin staining under laser scanning confocal microscope.RT-PCR was performed evaluate the osteogenesis-related gene,including osteocalcin?OC?and collagen type I?Col I?),expression of BMSCs cultured on the scaffolds on day 7 and 14.Immunofluorescence technology was performed to investigate the expression of Col I on day 21.Results:1.All scaffolds represented similar microporous morphology and good connectivity between the pores.The pore size and compressive strength increased with the increasing of?-TCP proportion.No significant differences were found on porosity among all scaffolds.Biomineralization capability is an essential factor to promote bone-binding competence for bone repair materials.Minerals are sparsely and randomly distributed on the SF scaffold,while there are denser mineral aggregations on the scaffolds with the increasing proportion of?-TCP nanoparticles.All SF/?-TCP scaffolds exhibited good biocompatibilities,which could facilitate cell adhesion and proliferation.However,all the SF/?-TCP scaffolds had a significantly lower ALP activities when compared with the control group.2.Average particle size of GO was about 100 nm.The size of the poly-l-lysine functionalized graphene oxide increased with the increase of the PLL proportion.All the GO-PLL/BSA complexes exhibited well-distributed round morphologies.The GO-PLL?1/1?/BSA and GO-PLL?1/5?/BSA nanoparticles had almost the same particle size,which were significantly smaller than that of GO-PLL?1/10?/BSA.When compared with the control group,Cell viabilities kept continuously more than 95%in the presence of GO-PLL/BSA nanoparticles,indicating GO-PLL/BSA nanoparticles had good compatibility.When BSA-BMP-2 nanoparticles were modified by GO-PLL,a pronounced decrease in BMP-2 burst release was observed,and extended to over 10-day study periods.Among the three GO-PLL/BMP-2 groups,the accumulative release of BMP-2 in the first three days were almost the same,while GO-PLL?1/1?/BMP-2 had a higher release velocity and accumulative release amount from the 4^th day to the 10^th day,when compared with the other two groups.The released BMP-2 from GO-PLL/BMP-2 nanoparticles could significantly promote ALP activity and extracellular matrix mineralization.3.The composite scaffolds with or without GO-PLL modification exhibited same release velocity of FN,whereas more accumulative amount and longer release period were observed in the presence of GO-PLL modification.No significant differences were found on the release profile of BMP-2 encapsulated nanoparticles and BMP-2,which were physically absorbed onto composite scaffolds.Additionally,both FN and BMP-2 showed a burst release profile of FN and BMP-2 on the composite scaffolds physically incorporated with FN and BMP-2,whereas the release profile FN and BMP-2 on the composite scaffolds modified by GO-PLL complexes?GO-PLL/FN+BMP-2?exhibited a rapid initial release of FN during the first few days,followed by slow and sustained release of BMP-2 for as long as 35 days.The synergistic incorporation of FN and BMP-2 could significantly promote BMSCs adhesion and differentiation compared with FN or BMP-2alone.The GO-PLL modified SF/?-TCP composite scaffold with FN and BMP-2 exhibited well-organized cytoskeleton and favorable cell viabilities,more importantly,it could significantly promote the expression of osteogenesis-related genes compared with physical incorporation of FN and BMP-2.Conclusion:1.When the proportion of SF and?-TCP is 1/2,SF/?-TCP scaffolds exhibited good physicochemical properties and biocompatibility,whereas lacked of osteoinductive capability.2.The GO-PLL modified BSA nanoparticles had proper particle morphology and size,and good biocompatibility.The release profile indicated a controlled and sustained release of BMP-2,which could promote BMSCs differentiation.3.The simultaneous incorporation of FN and BMP-2 onto SF/?-TCP scaffolds possessed synergistic effect on the adhesion,proliferation and differentiation of BMSCs compared with GO or?-TCP nanoparticle alone.More importantly,the sequential and sustained profile of FN and BMP-2 from GO-PLL modified SF/?-TCP scaffolds exhibited the superior osteogenesis of BMSCs.