Three-dimensional Coating Of SF/PLGA Coaxial Nanofiber Membranes On The Surface Of Calcium Phosphate Cement For Bone Defect

Background:Bone tissue is one of the most extensive and common organs in the human body.Autologous or allogeneic bone transplantation is the most important treatment method for bone defect at present,but the lack of donor,the greater trauma and high cost of the operation are all difficult problems for patients and clinicians.About two million spine,pelvic and other trunk bone transplants are performed internationally each year.However,about 50 percent of implants fail quickly,either autografts or allografts.Bone and joint related diseases such as osteoporosis,arthritis,obesity,diabetes,cancer,will cause damage to bone tissue,affect the health and function of human bones.Bone tissue engineering has received a lot of attention in order to obtain appropriate bone regeneration.Calcium phosphate(Ca P)bone cement has the potential for minimally invasive surgery in dental,orthopedic and reconstructive surgery due to its biocompatibility and bone conduction properties,as well as its injectability,and is a promising bone replacement material.It has become a commonly used bone defect replacement product in clinical practice.However,the lack of bone induction ability of CPC alone limits its application in bone regeneration and remodeling.Therefore,more and more researches focus on modifying calcium phosphate cement in different ways to obtain calcium phosphate containing bioactive factors.There are several types of bone growth factors,including bone morphogenetic growth factor(BMP),fibroblast growth factor(FGF),platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),and insulin-like growth factor(IGF).In all of these growth factors,BMP is considered to be the most effective way to form bone tissue during embryonic development,growth,healing and throughout adulthood.More and more reports have demonstrated the application of BMP for bone defects.Dexamethasone(DXM)is an effective steroid and has been widely used in various medical and biological applications.It has been reported that DXM can induce osteoporosis or even pathological fractures,DXM also can promote osteoblast differentiation and bone mineralization in vitro.The combination of DXM and BMP2 in tissue engineering scaffolds can play a synergistic role in enhancing the osteoinductive ability of tissue engineering scaffolds.Coaxial electrospinning is a simple and effective method to prepare nanofiber scaffolds,which has been widely used in tissue engineering and drug delivery systems.Coaxial electrospinning scaffolds have a high surface volume ratio,which can promote cell adhesion,realize drug loading,and demonstrate its sustained and controllable drug release capability.Previous studies have shown that some biomolecular molecules can be successfully encapsulated in nanofibers,while the biological activity of biomolecules can be retained.For example,rh BMP2,as a growth factor,can be successfully included in coaxial electrospun nanofiber membrane,achieving continuous drug release and preservation biological activity.Silk fibroin(SF)is a natural structural protein with good biological properties,such as good cell adhesion,controlled degradation,mechanical strength,permeability and resistance to enzyme degradation.Extensive research has been conducted in tissue engineering and drug delivery systems.PLGA has been widely used because of its good biocompatibility and controllable degradation rate.It is approved by the food and drug administration(FDA)for human clinical use.However,despite its biocompatibility,pure PLGA is hindered by poor bone conduction in clinical applications of bone regeneration and has poor mechanical properties when applied to load-bearing sites.Therefore,in this study,SF/PLGA coaxial electrospun nanofiber membranes were used as part of tissue engineering scaffolds.rh BMP2 and DXM were loaded into the core and shell respectively by the good drug loading capability of coaxial nanofiber.A new multi-functional composite scaffold was obtained by using the electrospinning technology to coat SF/PLGA coaxial nanofiber membranes on surfaces of calcium phosphate cement through the self-developed receiving device,which can play a role in the treatment of bone defects.Objectives:In this project,SF/PLGA nanofiber film containing rh BMP2 and DXM was prepared by coaxial electrospinning technology,and a new composite material SF-DXM/PLGA-rh BMP2 /CPC was obtained by using the self-developed receiving device to wrap the nanofiber membrane tightly on the porous calcium phosphate cement through electrospinning technology.Through in vivo and in vitro experimental studies on the new composite material,SF-DXM/PLGA-rh BMP2 /CPC composite material has a better ability to induce osteogenesis,which provides a new research direction for clinical treatment of bone defects.Methods:1.In this project,SF-DXM/PLGA-rh BMP2 nanofiber membranes with different concentrations of rh BMP2 were firstly produced by coaxial electrospinning.The core layer was PLGA with rh BMP2 and the shell layer was SF with DXM.The morphology,mechanical properties,hydrophilic properties and drug sustained-release properties of the nanofiber membrane were evaluated.2.Combined with rat bone marrow mesenchymal stem cells(r BMSCs)in vitro,the ability of dual drug-loaded nanofibers to promote cell proliferation and osteogenic induction was further investigated.3.A new type of receiving device invented by the research group was used to coat the coaxial nanofiber film obtained in the previous experiment on the surface of the porous calcium phosphate cement by means of electrospinning,and the different thickness of the nanofiber film was obtained by controlling the spinning time(20min,40 min and 60min),and its mechanical properties and hydrophilic properties were evaluated.By co-culture of rat bone marrow mesenchymal stem cells(r BMSCs)and composite materials,the in vitro biological properties of composite calcium phosphate scaffolds were evaluated by cell proliferation experiment(CCK-8),staining of live and dead cells,alizarin red staining(ARS),alkaline phosphatase staining(ALP)and real time polymerase chain reaction(PCR).4.We also established a rat skull defect model,implanted the composite material into the bone defect,and conducted Micro CT and HE staining on the rats at 4 weeks and 8 weeks,so as to further evaluate the biological properties of the composite material and promote osteogenesis through in vivo experiments.Results:1.Coaxial nanofiber membrane results indicated that the nanofibers were smooth without beads.the core-shell structure were successfully prepared in this study.The drug release curve shows that both rh BMP2 and DXM contained in coaxial nanofiber with slow release ability,it can release continuously and stably without obvious sudden release effect.In vitro cell experiments showed that SF/PLGA had good biocompatibility,and the daul-loaded nanofiber membrane had a significant role in promoting osteogenesis induction and cell proliferation activity.2.The composite calcium phosphate scaffold obtained by electrospinning and the new receiving device at the spinning time of 40 min hasd better hydrophilic and mechanical properties than pure calcium phosphate and the composite calcium phosphate with other spinning times.SF/PLGA/CPC was verified to have good biocompatibility through CCK8 and staining of living and dead cells,on the other hand,alizarin red staining(ARS),alkaline phosphatase staining(ALP)and real time polymerase chain reaction(PCR)demonstrated that the drug loaded three-dimensional coated calcium phosphate could promote cell proliferation and induce osteogenic differentiation especially SF-DXM/PLGA-rh BMP2/CPC.By implanting compound calcium phosphate into the skull defect model of rats,micro CT,HE staining confirmed that calcium phosphate coated with dual drug-loaded nanofiber membrane(SF-DXM/PLGA-rh BMP2/CPC)had better ability to promote bone regeneration.Conclusion:Coaxial nanofibers containing rh BMP2 and DXM were successfully prepared by electrospinning technique.The nanofibers have good drug sustained-release ability and biocompatibility.A new type of composite calcium phosphate scaffold was obtained by three-dimensional coating of the fiber membrane on the surface of porous calcium phosphate with a newly developed receiving device.This composite calcium phosphate scaffold not only has the ability of drug sustained release,but also has good biocompatibility and promoting bone regeneration.