Construction And Biological Study Of Platelet-rich Plasma Self-assembly Gel Coating On Modified Surface Of Polyetheretherketone Implants

Background:Polyetheretherketone(PEEK)has the advantages of stable physical and chemical properties,close to the elastic modulus of human bone,radiation penetration,etc.Thus,it is a promising bone implant material.However,the biological inertia of PEEK material itself has hindered its further clinical application.In addition,similar to bone implants made of other polymers,metals and other materials,PEEK implants can cause inflammation around the prosthesis after implantation in vivo,leading to irreversible bone loss and implant failure.Therefore,improving the bioinertness of PEEK materials and developing modification methods to inhibit periprosthesis inflammation are hot research directions in the field of biological modification with PEEK materials.One of the main methods to improve the bioinertness of PEEK materials is to fix bioactive substances on its surface by physical or chemical means,and to select suitable bioactive substances to achieve the purpose of regulating the immune microenvironment around the prosthesis.However,the application of exogenous bioactive substances on PEEK surface faces the problems of biosafety(immune rejection)and high cost.Therefore,it is necessary to search for a human bioactive substance and develop innovative combination strategies to overcome and improve the bioinertness of PEEK materials.Platelet rich plasma(PRP)is a platelet-rich liquid bioactive substance extracted from autologous whole blood.After being activated by an activator such as thrombin,the fibrin in PRP can quickly cross-link and convert the liquid PRP into a gel state.Activated PRP gel can release a variety of growth factors,including transforming growth factor(TGF),platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),etc.,to promote tissue repair.This synergistic effect of multiple growth factors on tissue repair is superior to that of any growth factor alone.Because PRP has good osteogenic,angiogenic and immunomodulatory effects,it can play an important role in wound healing,bone defect repair and arthritis.Theoretically speaking,PRP is a suitable autobioactive substance to improve the bioinertness of PEEK materials.However,how to effectively incorporate PRP onto the surface of PEEK materials remains a challenge.Therefore,this study explored the common modified interface of PEEK that is most suitable for PRP adhesion,innovatively prepared the self-assembled gel coating of PRP on the frosted modified surface of PEEK,and completed the biological study of the gel coating.Methods:1.The PEEK surface was modified with concentrated sulfuric acid,gaseous sulfur trioxide and 220-mesh sandpaper to prepare PEEK samples with different surface microstructures.Next,the PRP derived from Sprague Dawley(SD)rats was prepared and incubated on PEEK materials with different surface microstructures.Lactate dehydrogenase(LDH)test,scanning electron microscopy(SEM)and enzyme-linked immunosorbent assay(Elisa)were used to evaluate the adhesion effect of PRP.2.Bone forming ability of PRP coatings on different PEEK modified surfaces was further evaluated by Cell Counting KIT-8(CCK-8 Kit),alkaline phosphatase(ALP)staining,alizarin red(ARS)staining,quantitative and reverse transcriptional fluorescence quantitative polymeric chain reaction(RT-q PCR)and other cell experiments.Finally,the tibial defect model of SD rats was established,and the new bone was evaluated by micro-computed tomography(Micro-CT),hematoxylin-eosin(HE)staining,Sirius red staining and immunofluorescence staining.3.N,N’-dissuccinyl carbonate(DSC)was used as the medium to graft thrombin onto the surface of frosted PEEK material.PRP was activated by thrombin on the surface of PEEK samples and self-assembled into gel.The morphology,secondary structure,thrombin grafting amount and thrombin activity of thrombin grafted on PEEK surface under different grafting concentrations were evaluated by SEM,FT-IR,Lowery protein concentration test and thrombin hair color substrate.After selecting the optimal grafting concentration of thrombin,PRP with different platelet enrichment was activated to form biogel coating on the surface of PEEK material.Finally,SEM and Elisa kits were used to evaluate the microstructure and growth factor release ability of PRP biogels with different platelet enrichment on PEEK surface.4.Mouse embryonic osteoblast precursor cells(MC3T3-E1)were used to perform CCK-8 assay,ALP staining,ALP quantification,ARS staining and RT-q PCR to evaluate the osteogenic ability of PRP biogel coating on PEEK surface.Human umbilical vein endothelial cells(HEUVC)were used for immunofluorescence staining,cell invasion assay,tubule formation assay and RT-q PCR to evaluate the vasogenic ability of PRP biogel coating on PEEK surface.Mouse mononuclear macrophage leukemia cells(RAW264.7)were used for fluorescence staining of M1/M2 polarization-related markers and RT-q PCR to evaluate the immunomodulatory ability of PRP biogel coating on PEEK surface.Finally,the tibial defect model of SD rats was established.Micro-CT,HE staining and immunofluorescence staining were used to evaluate the in vivo osteogenesis,angiogenesis and immunoregulation ability of self-assembled PRP biogel coating on the surface of PEEK.Results:1.SEM results showed that the surface of PEEK polished with 220-mesh sandpaper was most suitable for platelet adhesion.LDH platelet count test and Elisa growth factor release test confirmed this point,that is,the frosted group PEEK samples had the most platelets adhesion and released the most growth factors.2.The results of cell and animal experiments showed that the bone formation ability of PEEK samples modified by different methods was significantly improved after incubation with PRP,and the the samples of sandpaper group of had the strongest bone formation ability after cultured with PRP.3.SEM,EDS and FT-IR results confirmed that thrombin was successfully grafted on the surface of PEEK samples,and FT-IR peak fitting results showed that the grafted thrombin retained the protein secondary structure required for enzyme activity.According to the results of thrombin grafting amount and enzyme activity after grafting,when the thrombin concentration reached 100 U/m L,the thrombin grafting amount and enzyme activity were the highest,and both did not increase with the increase of thrombin grafting concentration.PRP with different platelet enrichment was incubated on PEEK surfaces grafted with thrombin,and the PRP solution could be activated to form self-assembled biofunctional hydrogels.The SEM results showed that the PRP self-assembled hydrogel on PEEK surface has a porous structure.Elisa experiments showed that the growth factor release time of PRP self-assembled hydrogel on PEEK surface could last for 16 days,and the cumulative release amount of growth factor increased with the increase of platelet enrichment ratio in PRP.4.In vitro cell experiment results showed that the PRP self-assembled biogel coating on PEEK surface could promote the proliferation and osteogenic differentiation of MC3T3-E1,promote HUVEC cell migration and angiogenesis,and promote M2 polarization while inhibit M1 polarization of RAW264.7 cells.The results of in vivo bone defect model experiments showed that thrombin-activated PRP biogel on PEEK surface could effectively promote bone formation,angiogenesis and immune regulation,and these biological effects were platelet dose-dependen.Conclusion:1.Because it carries the most PRP and has the strongest in vitro/in vivo osteogenic ability,compared with modified PEEK surface with sulfuric acid and sulfur trioxide,frosted PEEK surface is considered to be the most suitable surface for loading PRP bioactive coating in this study.2.When DSC is used as the linker,100U/m L is considered to be the best graft concentration to graft thrombin on PEEK surface.The grafted thrombin on PEEK surface could activate PRP to self-assemble and form a gel coating,which has the ability to release growth factors for a long time.3.The PRP biogel coating on PEEK surface has played a good role in osteogenesis,angiogenesis and immune regulation both in vivo and in vitro.Therefore,the thrombin-activated PRP self-assembly gel coating on PEEK surface is a promising strategy for biological functionalization of PEEK implants.