| Background and objectivesBone tissue engineering(BTE)is a cross-discipline that develops rapidly along with the progress of biotechnology,material science,and regenerative medicine.With the advantages of unlimited donor sources and ideal repair effect,it is in the research focus for bone defect treatment.The performances of scaffold materials are crucial for the BTE technology.Therefore,the preparation of scaffold materials with an effective loading and slow release of growth factors,good biocompatibility,and degradability,and construction of the microenvironment of the extracellular matrix(ECM)with efficient osteoconductivity and osteoinductivity are the purpose and focus of this experimental study.Nanofiber scaffolds prepared by electrostatic spinning have a three-dimensional porous cross-linked structure,which can effectively mimic the ECM microenvironment and enable the effective loading of drugs.Another drug-carrying method is based on protein nanoparticles with diameters of 10-1000 nm,which have a good biocompatibility,degradability,drug delivery properties and bone induction performance.In the design of BTE drug loaded scaffolds,if drug loaded nanospheres are embedded in electrospun fiber scaffolds,the drug loading performance of electrospinning can be improved.The drug release has undergone two stages:the degradation of electrospun fibers and the degradation of nanoparticles,which not only guarantees the biological activity of drugs,but also realizes the sustained and slow release of drugs.Aspirin,also known as Acetylsalicylic acid(ASA),is a widely used analgesic,antipyretic and anti-inflammatory drug,which can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and human dental pulp stem cells,increase bone density and improve bone formation.Abaloparatide was approved by the Food and Drug Administration(FDA)in 2017 and is currently the latest clinical medication for the treatment of osteoporosis.It promotes the differentiation of mesenchymal stem cells into osteoblasts by selectively activating the signaling pathway of the parathyroid hormone type I receptor.Compared to its predecessor Teriparatide,Abaloparatide can more effectively promote the increase in bone mineral density and reduce the occurrence of hypercalcemia.In the design of BTE scaffold materials,ASA and abaloparatide can be combined in order to play the synergistic role of abaloparatide and ASA in promoting osteogenesis and build a good bone immune microenvironment.In the scaffold design,the advantages of various materials are combined to form a BTE composite scaffold with excellent performance.Polycaprolactone(Poly(ε-caprolactone),PCL)can be fabricated into nanofiber scaffolds by the electrostatic spinning technology to provide sites for cell adhesion,proliferation,and differentiation.Nanohydroxyapatite(n-HA)is the main inorganic component of human bone,which has been widely used as a bioceramic material for bone grafting.Chitosan can enhance the cell adhesion and proliferation,and promote osteoblast differentiation and mineralization.The above materials are excellent matrix materials for BTE scaffolds.Based on the above understanding,this study aimed to design a nanoparticle/electrospinning composite scaffold with a sequential controlled release of ASA and abaloparatide.Abaloparatide-loaded bovine serum albumin(BSA)nanoparticles were constructed by desolventization,and then a chitosan stabilization layer was prepared by electrostatic self-assembly on the outer layer of the BSA nanoparticles,which ensured the activity and slow release of the drug.Subsequently,Abaloparatide-loaded nanoparticles were compositely loaded with ASA and n-HA into PCL electrostatically spun fiber scaffolds,and a novel dual-barrier drug sustained-release scaffold was prepared.ASA is in the carrier of electrospinning,and abaloparatide is in the dual carrier of nanoparticles and electrospinning.The drug is released in different carriers.The release of ASA was earlier and faster,and the release of abaloparatide was relatively slow and sustained.The sequential controlled release of ASA and abaloparatide is realized,in order to play the anti-inflammatory role of ASA in the early stage of bone formation and build a good bone immune microenvironment.Abaloparatide and ASA play a synergistic role in promoting bone formation during bone formation,providing an efficient ECM microenvironment for bone regeneration.The composite scaffold combines a variety of materials with good biocompatibility and biodegradability,which is more in line with the needs of tissue engineering scaffolds.Objective to explore the bone regeneration promoting ability of the composite scaffold material through physical and chemical property tests,in vitro experiments and in vivo experiments,so as to provide a new choice of drug delivery methods for the design of BTE scaffold material,and provide valuable ideas and methods for the clinical treatment of bone defects.Methods1.Construction of the nanoparticle/electrospinning composite scaffolds with a sequential controlled release of aspirin and abaloparatideAbaloparatide-loaded BSA nanoparticles were prepared using the desolventization method.A layer of chitosan was adsorbed on the surface of the nanoparticles by the principle of electrostatic self-assembly to form a chitosan protective layer.Abaloparatide-loaded nanoparticles(Chi/ANPs),ASA,and n-HA were loaded into the PCL matrix together for electrostatic spinning to prepare nanoscale electrostatically spun fibrous composite scaffolds,i.e.,dual-drug-loaded nanoparticle electrostatically spun dual-barrier drug retarded release scaffolds.The fibrous scaffolds were divided into five groups,blank fibrous scaffolds(PCL/HA),ASA-loaded fibrous scaffolds(ASA/PCL/HA),non-drug-loaded nanoparticle fibrous scaffolds(NPs/PCL/HA),Abaloparatide-loaded nanoparticle fibrous scaffolds(ANPs/PCL/HA),and ASA-loaded and Abaloparatide-loaded nanoparticle fibrous scaffolds(ANPs/ASA/PCL/HA).The morphology,structure,surface characteristics,mechanical properties,in vitro degradability,and other properties of the nanoparticles and fibrous scaffolds were investigated by physicochemical property tests to analyze the drug encapsulation rate of nanoparticles and drug release characteristics of ASA and Abaloparatide in fibrous scaffolds.2.In vitro osteoinductive activity of the nanoparticle/electrospinning composite scaffoldsEach group of scaffold materials was cocultured with MC3T3-E1 cells.The osteoconductive properties of the scaffold materials were investigated by a scanning electron microscopy observation of cell adhesion,cell adhesion rate assay,fluorescence staining,live and dead cell staining,and Cell Counting Kit-8(CCK-8)assay.The osteoinductive properties of the scaffold material were studied by alizarin red staining of cells,alkaline phosphatase staining,alkaline phosphatase activity assay,reverse transcription polymerase chain reaction(RT-PCR),and protein immunoblotting(Western blot)methods.3.In vivo bone defect repair capability of the nanoparticle/electrospinning composite scaffoldsThe in vivo experiments were divided into two parts.The aim of the first part was to establish a subcutaneous implantation model of rat scaffold material and detect the in vivo local host response of the scaffold material by HE staining,Masson staining,and immunohistochemical staining methods to investigate its biosafety.In the second part,a rat cranial bone defect repair model was established,and the ability of the scaffold material to repair bone defects was analyzed by micro-computed tomography(Micro-CT),hematoxylin-eosin staining(HE)staining,Masson staining,and immunohistochemical staining methods.Results1.Successful preparation of the abaloparatide-loaded nanoparticles with a chitosan stabilizing layerIn the experiments,first,we prepared BSA nanoparticles with a chitosan stabilizing layer,Abaloparatide-loaded nanoparticles(Chi/ANPs),and non-drug-loaded nanoparticles(Chi/NPs).Transmission electron microscopy(TEM)results showed that the nanoparticles were morphologically regular and round,with smooth surfaces and good particle dispersion.A dynamic light scattering(DLS)analysis showed that the average diameter of Chi/NPs was 330±39 nm,while that of Chi/ANPs was 289± 34 nm.The smaller average diameters of Chi/ANPs indicated that the nanoparticles had a higher stability after loading with Abaloparatide.Zeta potential results showed that the prepared nanospheres had the best stability when the pH of the BSA solution was 8.5.Attenuated total reflection Fourier-transform infrared(ATR-FTIR)spectroscopy confirmed that the nanoparticles were encapsulated with chitosan on the surface without material changes of BSA and chitosan.The encapsulation rate of Abaloparatide in the nanoparticles was 85.602 ± 3.927%.2.Successful preparation of the nanoparticle/electrospinning composite scaffolds with a sequential controlled release of aspirin and abaloparatideThe nanoparticle/electrospinning composite scaffolds were prepared by loading Chi/ANPs into the PCL matrix containing ASA and n-HA.SEM showed that the five groups of fiber scaffolds were all overlapped and interwoven by nano-scale filamentous structures,showing a three-dimensional network structure,forming irregular pores and communicating with each other.The surface of the nanoparticle-containing fibrous scaffolds was slightly rough.Small nodule-like projections could be observed.The TEM images showed that the nanoparticles were encapsulated and dispersed in the fibrous matrix.A scattered distribution of n-HA particles could be observed in the fibrous matrix.The ATR-FTIR results confirmed that the nanoparticles were structurally stable during the electrostatic spinning.The hydrophilicity and tensile strength experiments indicated that the loading of ASA and nanoparticles well promoted the hydrophilicity for PCL but led to a decrease in tensile strength.The results of in vitro degradation experiments showed that the loading of n-HA,ASA,and nanoparticles could regulate the degradation time of the materials.The loading of n-HA could effectively counteract the acidification of the solution caused by the degradation of the materials.3.The nanoparticle/electrospinning composite scaffolds enabling a sequential controlled release of aspirin and abaloparatideThe sequential release of ASA and Abaloparatide was achieved in the ANPs/ASA/PCL/HA group.The drug release of ASA involved two phases:an initial rapid release and late sustained release,with most of the ASA being released in the first eight days,followed by a smaller increase in the amount of release.Abaloparatide,due to the dual-barrier protection of the nanoparticles and electrically spun fibers,exhibited a slow release pattern similar to the zero-stage release,releasing slightly in the first few days but at a low level,and then exhibiting a slightly accelerated but constant release with a slow release time of up to 30 days or more.The drug release profiles of the single-drug scaffolds ASA/PCL/HA and ANPs/PCL/HA were similar to those of the dual-drug scaffolds ANPs/ASA/PCL/HA,but with a lower release rate,which was related to the excellent hydrophilicity and degradability of the ANPs/ASA/PCL/HA scaffolds.4.Good biocompatibility of the nanoparticle/electrospinning composite scaffoldsSEM results of the cell adhesion showed that MC3T3-E1 cells were well attached and stretched out on the surface of the scaffolds,extending pseudopods and connecting with each other.Fluorescence staining results showed that the cells were visible at different levels of the scaffolds,with a three-dimensional distribution.The cells embedded inside the fibrous scaffolds had good morphology and spreading.The fluorescence staining of live/dead cells confirmed that all groups of fibrous scaffolds had a good biocompatibility.5.Good in vitro osteoconduction and osteoinduction properties of the nanoparticle/electrospinning composite scaffoldsThe results of CCK-8 experiment showed that the cell proliferation of the five groups of scaffold materials was better than that of the negative control group,and the ANPs/ASA/PCL/HA group had the best cell proliferation effect,which began to surpass other material groups on the 3rd day and surpassed the positive control group on the 5th day,with statistical significance(P<0.01).The sudden release of ASA in the early stage has a short-term adverse effect on cell proliferation,and the appropriate concentration of ASA in the later stage can promote cell proliferation,and the sequential release of ASA and Abaloparatide can promote cell proliferation.ALP staining showed that MC3T3-E1 cells on the scaffolds in each group had undergone osteogenic differentiation.The trend of NPs/PCL/HA and PCL/HA groups was similar,and the ALP activity in ANPs/ASA/PCL/HA group had been in the best state since the 14th day.The results of ARS were similar to those of ALP.There were more red calcified nodules on the fiber scaffolds in the five groups than in the control group,and the number and area of red nodules in ANPs/ASA/PCL/HA group were the largest.The results of osteogenesis-related gene and osteogenesis-related protein expression assays showed that both ASA/PCL/HA group loaded with ASA and ANPs/PCL/HA group loaded with Abaloparatide were able to highly express the genes and proteins of runt-related transcription factor 2(Runx2),osteocyte specific transcription factor(Osterix,Osx),Osteopontin(OPN),and Osteocalcin(OCN)genes and proteins,with statistically significant differences from the positive control group(p<0.05),whereas the double-loaded ASA and Abaloparatide ANPs/ASA/PCL/HA group expressed the highest amount of Runx2,Osterix,OPN,and OCN genes and proteins.This suggests that the dual-loaded ANPs/ASA/PCL/HA fibrous scaffolds could promote the osteogenic differentiation of MC3T3-E1 via Runx2,Osterix,OPN,and OCN pathways,and that the two drugs had a synergistic promotional effect in conducting osteogenic differentiation.6.Good biosafety and in vivo bone defect repair performances of the nanoparticle/electrospinning composite scaffolds(1)In vivo subcutaneous material implantation experimentLocal host response was observed after implantation of in vivo subcutaneous fibrous scaffold materials.There was a small amount of inflammatory cell infiltration in all groups of scaffolds at two weeks,dominated by macrophages,which were mainly distributed at the junction of the fibrous scaffolds and tissue envelope.The composite scaffolds ASA/PCL/HA and ANPs/ASA/PCL/HA had low degree of inflammatory cell infiltration.The anti-inflammatory effect of ASA inhibited the expression of inflammatory factors,and the early slow release of ASA greatly controled the early inflammatory cell infiltration of the materials.(2)Experiment on bone defects at the cranial in ratsMicro-CT results showed that the blank control group could not complete the repair of bone defects.The five groups of scaffold material implantation after the repair effect were better than the control group.The repair effect order was drug-carrying group>scaffolding group alone>control group.The dual-drug ANPs/ASA/PCL/HA group exhibited the best bone volume(BV),bone volume/total volume(BV/TV),trabecular number(TbN),and bone mineral density(BMD)(P<0.01).At 12 weeks,the repair of bone defect in ANPs/ASA/PCL/HA group tended to be complete,the defect was filled with new callus,and the bone defect almost disappeared.The results of HE staining and Masson staining showed that the ANPs/ASA/PCL/HA group had the best bone defect repair effect,with obvious new bone formation and neovascular structure.The bone defect repair in the 12 w ANPs/ASA/PCL/HA group was basically completed,with abundant trabeculae and almost complete cortical bone continuity.The results of immunohistochemical staining showed that there were more positive expressions of OCN and Runx2 in all the drug-loaded composite materials,and the expressions of OCN and Runx2 in ANPs/ASA/PCL/HA group were higher than those in other groups(P<0.01).In the early stage of bone development,ASA played an anti-inflammatory role to inhibit the inflammatory reaction in the bone repair environment,while ASA and abaloparatide played a synergistic role in promoting bone formation in the process of bone formation,thus effectively playing the role of bone tissue engineering scaffold in promoting bone regeneration.Conclusion1.In the experiment,abaloparatide-loaded nanoparticles with a chitosan stabilizing layer were successfully prepared.Abaloparatide-loaded nanoparticles were loaded into the PCL matrix containing ASA and n-HA for electrostatic spinning.The nanoparticle/electrospinning composite scaffolds with a sequential controlled release of aspirin and abaloparatide were prepared.The fiber scaffolds have good hydrophilicity and degradability.2.This composite scaffolds enable a sequential controlled release of ASA and Abaloparatide,which provides a new choice of drug delivery methods for the design of BTE scaffold material.3.In vitro experiments confirmed that the composite scaffolds have a good biocompatibility,efficient osteoconductivity,and osteoinductive properties.The composite scaffolds can promote the adhesion and proliferation of MC3T3-E1 cells and osteogenic differentiation of MC3T3-E1 cells through Runx2,Osterix,OPN,and OCN pathways.The two drugs have a synergistic promotional effect in contributing to the osteogenic differentiation.4.In vivo experiments confirmed that the composite scaffolds have good biosafety and in vivo bone defect repair performance.The composite scaffolds exerted the anti-inflammatory effect of ASA in the early stage of bone formation to inhibit the inflammatory reaction in the bone repair environment,and exerted the synergistic effect of abaloparatide and ASA in promoting bone formation in the process of bone formation,thus effectively exerting the role of BTE scaffolds in promoting bone regeneration,and providing valuable ideas and methods for the clinical treatment of bone defects. |
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