Multifunctional Surface Construction Of Carbon Fiber Reinforced Polyether Ether Ketone And Its Investigation In Bone Defect Repair

Bones play a role in supporting and protecting of organism,the storage of trace elements and minerals in the body,the normal operation of the hematopoietic system,and various activities of the body all rely on a healthy bone system.Natural bones with high strength and elasticity show the ability to self-repair after being damaged.However,for large-scale bone defects caused by trauma,infection,tumors,genetic diseases,etc.,only relying on the self-healing ability is difficult to heal and clinically requires bone transplantation treatment.In recent years,based on traditional autologous/allogeneic bone transplantation techniques,bone tissue engineering technology has become a research hotspot.An ideal bone tissue engineering implant needs not only good mechanical properties and biocompatibility,but also various biofunctions required for different healing stages,including immune regulation,angiogenesis,and osteoanagenesis.Bone tissue engineering materials in clinical practice commonly include metals,bio-ceramics,polymers and their composite materials.Inside polymer-based composite materials,carbon fiber reinforced polyether ether ketone（CFRPEEK）composite materials,have many excellent properties,such as elastic modulus matching with human cortical bone,x-ray penetrability,non-cytotoxicity,high temperature resistance,solvent resistance,impact resistance,wear resistance,fatigue resistance,etc.However,CFRPEEK lacks bioactivity and has poor integration ability with surrounding bone tissue,which severely limits the application of CFRPEEK implants in the field of bone tissue engineering.Meanwhile,bone repair is a complex dynamic process that includes three overlapping and interdependent stages:the immune response of macrophages,the angiogenesis of endothelial cells,and the osteogenic differentiation of osteoblasts.Focusing on the three stages of the bone defect repair process,this paper designs the sustained-release system of simvastatin,rosmarinic acid,and zinc ions and constructs them on the surface of CFRPEEK through carboxyl,hydroxyl,and amino groups,thereby developing three multifunctional CFRPEEK implants to satisfy different requirements of immunoregulation,angiogenesis,and osteogenesis in the bone defect repair process.These studies provide new ideas for the preparation of multifunctional CFRPEEK implants and a scientific basis for the clinical application of CFRPEEK materials.1.In order to solve the problem of untimely vascularization and poor osteogenic ability of CFRPEEK implants in bone defect repair,a sustained-release system of simvastatin（SIM）was designed and constructed on the surface of CFRPEEK.Prepared the carboxylated CFRPEEK with pore structure on the surface through sulfonation reaction and Friedel-Crafts reaction;adsorbed SIM onto the pore structure of the carboxylated CFRPEEK surface;covalently grafted chitosan and amino terminated polyethylene glycol（CPN）as biocoating onto the carboxylated CFRPEEK surface loaded with SIM through amide bonds.This study fully utilizes the special morphology and active groups of the sample surface,cleverly achieving hydrophobic adsorption and fixation of SIM through carboxylated CFRPEEK surface pore structure and carboxyl groups,and covalently grafting CPN biocoating to prevent rapid diffusion of SIM.This study successfully prepared a simvastatin sustained-release system modified carboxylated CFRPEEK implants（SCP/SIM/CPN）.The hydrophilic CPN coating has a significant promoting effect on cell adhesion,diffusion,and proliferation;the release of SIM in the initial stage can induce angiogenesis,while the release of SIM in the osteogenic stage can promote osteoanagenesis;and the synergistic effect between CPN and SIM can further enhance the angiogenic and osteogenic abilities of CFRPEEK.The SIM sustained-release system constructed on the surface of CFRPEEK is generally suitable for various hydrophobic drugs and can effectively solve the limitations of bioinertness in the clinical application of CFRPEEK implants.The results indicate that the SIM release efficiency of SCP/SIM/CPN within 21 days was 57.31%,and the degradation of the surface CPN biocoating was more than 14 days.Meanwhile,SCP/SIM/CPN also exhibited good hydrophilicity（water contact angle of 63.88±1.38°）,in vitro mineralization ability,and biocompatibility.In vitro angiogenesis assays showed that human umbilical vein endothelial cells（HUVECs）formed the densest vascular network structure in the SCP/SIM/CPN group,the branch length of tubes increased by 90.85%compared to the CFRPEEK（CP）group;in vitro osteogenesis assays showed that,compared with the CP group,the ALP activity of bone marrow mesenchymal stem cells（BMSCs）on the surface of SCP/SIM/CPN increased by51.55%,and calcium deposition increased by 293.42%.Cell experiments confirmed that SCP/SIM/CPN has angiogenic and osteogenic activities.The rat subcutaneous implantation assays showed that the immunofluorescence intensity of vascular growth factors VEGF and CD31 in the surrounding tissues of SCP/SIM/CPN implants,respectively,increased by 65.27%and 59.87%in compared to the CP group;the rat skull defect assays showed that the bone mineral density and bone volume fraction of newly formed bone around the SCP/SIM/CPN implants,respectively,increased by84.11%and 58.35%in compared to the CP group.Animal experiments further confirmed that SCP/SIM/CPN can induce angiogenesis in vivo and accelerate bone defect healing.2.In order to solve the problem of excessive inflammatory response in the early stage of CFRPEEK implants affecting osteogenesis in anaphase,a sustained-release system of rosmarinic acid（RA）was designed and constructed on the surface of CFRPEEK.At present,the relevant reports on the application of RA in the field of bone tissue engineering only discuss the effect of adding different concentrations of RA to the culture medium on osteoblast differentiation without related research on modifying RA onto the surface of bone implant materials.Therefore,constructing a RA sustained-release system on the surface of CFRPEEK can broaden the application of RA in the field of bone implantation.Considering the negatively charged nature of RA molecules,this study attempts to construct a RA sustained-release system through layer-by-layer self-assembly of polyelectrolytes with opposite charges onto the surface of CFRPEEK based on electrostatic interactions.Prepared the hydroxylated CFRPEEK through sulfonation reaction and sodium borohydride reduction reaction;immersed poly-l-lysine onto the surface of hydroxylated CFRPEEK;based on electrostatic interactions,carboxymethyl cellulose sodium and chitosan loaded with rosmarinic acid alternating deposited onto the hydroxylated CFRPEEK surface immersed with poly-l-lysine through layer-by-layer self-assembly technology,thereby endowing CFRPEEK implants with anti-inflammatory and osteogenic ability.The rapid release of RA in the initial stage can create a favorable bone immune regulatory microenvironment in the early stage,the sustained release of RA in the osteogenic stage can promote the differentiation of osteoblasts;the self-assembly layer also has anti-inflammatory and osteogenic properties,and the chitosan in the components can synergistically activate CFRPEEK with RA.This study successfully prepared a rosmarinic acid sustained-release system modified hydroxylated CFRPEEK implant（SCPP/CC₅@RA）.The results indicate that the RA cumulative release of SCPP/CC₅@RA within 21 days was0.23μg/m L,and the degradation of the self-assembly layer was more than 14 days.Meanwhile,SCPP/CC₅@RA had excellent hydrophilicity（water contact angle of 43.9±4.9°）and biocompatibility.In vitro anti-inflammatory assays indicated that compared with sulfonated CFRPEEK（SCP）,the secretion of pro-inflammatory factor TNF-αof macrophages（RAW 264.7）was down regulated by 35.31%and the secretion of anti-inflammatory factor IL-10 was up regulated by 150.07%in the SCPP/CC₅@RA group;in vitro osteogenesis assays showed that compared with the SCP group,the ALP activity of BMSCs on the surface of SCPP/CC₅@RA increased by 43.28%and calcium deposition increased by 222.50%.Cell experiments confirmed that SCPP/CC₅@RA has anti-inflammatory and osteogenic properties.The rat subcutaneous implantation assays showed that the ratio of immunofluorescence intensity of M1 and M2 macrophage marker factors in the surrounding tissue of the SCPP/CC₅@RA implant decreased by51.49%compared to the SCP group;the rabbit tibia defect assays showed that the maximum pushing force of the SCPP/CC₅@RA implant and the bone trabecular thickness of the surrounding newly formed bone,respectively,increased by 167.13%and 35.73%compared to the SCP group.Animal experiments further confirmed that SCPP/CC₅@RA can regulate the immune inflammatory response,thereby accelerating bone regeneration.3.In order to solve the problems of excessive inflammatory response,untimely vascularization,and poor osteogenic ability caused by the bioinertness of CFRPEEK implants,a zinc ion(Zn²⁺)sustained-release system was designed and constructed on the surface of CFRPEEK.Prepared the aminated CFRPEEK through nitration reaction and hydrazine hydrate reduction reaction;grafted carboxylated graphene oxide（GC）onto the surface of aminated CFRPEEK through amide bonding andπ-πinteractions;loaded Zn²⁺onto the aminated CFRPEEK surface grafted with GC based on coordination,cation-π,and electrostatic interactions;and immersed the chitosan onto the surface of aminated CFRPEEK loaded with Zn²⁺to prevent the rapid diffusion of Zn²⁺.GC in the zinc ion sustained-release system can significantly improve the surface loading capacity of Zn²⁺;CS can achieve multi-level release of Zn²⁺.Meanwhile,the synergistic effect of CS and Zn²⁺further enhances the anti-inflammatory and osteogenic ability of CFRPEEK implants,promoting bone defect repair.This study successfully prepared a zinc ion sustained-release system modified aminated CFRPEEK implant（CP/GC@Zn/CS）.The results indicate that the Zn²⁺multi-level release behavior of CP/GC@Zn/CS within 14 days was consistent with the bone regeneration process,including rapid release in the early stage（7.27μM,immune regulation）,sustained release in the middle stage（3.75μM,angiogenesis）,and slow release in the later stage（2.80μM,osteoanagenesis）.In vitro anti-inflammatory,angiogenic,and osteogenic assays confirmed that compared with aminated CFRPEEK（NCP）,the secretion of pro-inflammatory factor TNF-αof RAW 264.7 decreased by 28.41%and the secretion of anti-inflammatory factor IL-10 increased by 140.12%in the CP/GC@Zn/CS group;the branching length of HUVECs increased by 122.90%;the ALP activity of BMSCs increased by 32.46%,and calcium deposition increased by 75.02%.Cell experiments confirmed that CP/GC@Zn/CS can effectively regulate the immune inflammatory response,create a favorable bone immune microenvironment in the early stage,promote vascular network remodeling,and be favorable for transporting oxygen and nutrients to the injured site in the middle stage;induce osteoblast differentiation,and promote osteoanagenesis in the later stage.In vivo experiments further indicated that the maximum pushing force of the CP/GC@Zn/CS implant and the bone trabecular thickness of the surrounding newly formed bone,respectively,increased by 105.09%and 32.43%compared to the NCP group,suggesting that the long term sustained release of Zn²⁺on the surface of CP/GC@Zn/CS implants can accelerate the recovery of bone homeostasis around the implant and improve osseointegration.In summary,this paper is based on chemical modification and combined with various surface modification methods to prepare multifunctional carbon fiber reinforced polyether ether ketone implants,promoting bone defect repair by enhancing immune regulation,angiogenesis,and osteogenic ability.This paper provides a theoretical basis and technical support for the bioactivation of CFRPEEK bone implant materials and provides new strategies for the development of multifunctional bone repair materials in the future.