Fabrication Of 3D-printed Composite PEEK Scaffolds Programmed To Deliver TGF-β1 And Si And Study Of Bone Repair Performance

Background:Regeneration of critical bone defects(CSD)remains an important clinical challenge.To date,many biomaterials have been developed for this application.However,the immune response and the vascular status of the defect area may be critical factors that are overlooked when evaluating bone regeneration outcomes with biomaterials.Considering the spatiotemporal intersection and pathological characteristics of various progressive biological events during bone-implant interactions after biomaterial implantation,the coupling of bone immunomodulation,angiogenesis,and osteogenesis needs to be carefully designed spatiotemporally in order to better exploit the advantages of biomaterial interface modification and ultimately achieve optimal bone repair results.Polyetheretherketone(PEEK)materials are physical and chemical materials that can be used for bone repair.PEEK materials are polymeric materials with stable physicochemical properties and high structural strength.The elastic modulus of PEEK materials is close to that of natural cancellous bone,and they are wear-resistant,corrosion-resistant,fatigueresistant,and biologically safe.Therefore,they are considered as one of the most promising alternatives to titanium.Designing a three-dimensional porous structure for PEEK materials can provide space for bone tissue to grow inward,thus enhancing the strength of the bone-implant bond.Therefore,PEEK scaffolds with a porous structure could prepared by fused deposition modelling(FDM)and sulfonated to obtain sulfonated PEEK scaffolds with a surface pore structure coexisting with scaffold macropores.With the advent of recombinant proteins,there has been increasing interest in the use of growth factors as therapeutic agents for bone regeneration.TGF-β1 has shown pleiotropic effects in modulating cellular behavior,which,in addition to directly promoting bone regeneration through its action on endothelial cells and osteoblasts,indirectly promotes bone repair by modulating the pro-regenerative microenvironment induced by immune cells.In the early inflammatory phase,TGF-β1 is involved in the regulation of various immune cell behaviours and is secreted as an anti-inflammatory and pro-restorative factor in the subsequent bone regeneration process.Poly-dopamine(PDA)coatings are often used as intermediates.PDA coatings are often used as intermediates to firmly anchor biologically functional actives to the surface of PEEK materials,providing an adhesion basis for further activation.Silicate bioceramics not only stimulate osteogenesis of osteoblasts,but also enhance angiogenesis of endothelial cells.In addition,calcium silicate was found in human umbilical vein endothelial cells(Human umbilical vein endothelial cells,HUVECs)and bone marrow mesenchymal stem cells(Bone Marrow Stem Cells(BMSCs)co-culture system can enhance angiogenesis and osteogenesis through the paracrine pathway.Nanohydroxyapatite(nHA)is an inorganic substance that is highly similar to human bone in terms of chemical composition and physical structural characteristics and has good osteoinductive properties.Therefore,the silicon-doped nHA(Si@nHA)obtained by doping silicon into hydroxyapatite has not only excellent osteogenic ability but also the potential to induce angiogenesis Meanwhile,the light-responsive hydrogel gelatin methacrylate anhydride gelatin(Gelatin Methacryloyl,GelMA)also provides a good environment for cell adhesion and proliferation due to its high mimicry of the extracellular matrix,and with its slow degradation,it can achieve a slow-release effect of bioactive ingredients.In summary,the SPEEK-GelMA/Si@nHATGF-β1 composite 3D scaffolds were finally obtained in this study by various techniques such as 3D printing,surface coating method,hydrothermal synthesis method,and light curing method.Subsequently,the physicochemical characterisation was identified,the release and degradation properties of different bioactive components were evaluated,followed by ex vivo and in vivo biocompatibility evaluation,immunomodulation,vascularization ability and osteogenic effect of each group of PEEK scaffolds.Methods:1.In this study,the porous PEEK scaffold was firstly 3D printed by FDM and sulfonated,followed by the construction of a PDA coating on the sulfonated PEEK surface.This SPEEKPDA scaffold has both a connected macroporous structure and a microporous structure on the surface of the scaffold and can be efficiently loaded with recombinant proteins.Subsequently,GelMA or GelMA/Si@nHA was graft-immobilised into the 3D porous structure of the SPEEKPDA scaffold and TGF-β1 was loaded onto the surface thus successfully preparing SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds.The composite PEEK scaffold was also examined for the characterization of the scaffolds by mass-volume method,FT-IR,XPS and SEM,and the mechanical properties and hydrophilicity of the scaffolds were determined by an electronic universal testing machine and a water contact angle analyser,etc.The characterisations of Si@nHA and GelMA were analyzed by FT-IR,XRD,DLS,nuclear magnetic resonance spectrometry(NMRS)and ICP,and the composite PEEK scaffolds were subjected to the tests of XRD,XPS,hydrophilicity and mechanical properties of composite PEEK scaffolds,and the loading and release patterns of the two active components were detected by ICP and ELISA.2.In this study,macrophage RAW264.7,bone marrow mesenchymal stem cells(BMSCs),and human umbilical vein endothelial cells(HUVECs)were used as cell models to establish an in vitro cell model system for the evaluation of immunomodulation,vascular induction,and osteogenic differentiation of SPEEK-GelMA/Si@nHATGF-β1 composite 3D scaffolds by using scratch and transwell migration assays,The biocompatibility,immunomodulation,angiogenesis,and osteogenesis of the scaffolds were evaluated using scratch and transwell migration assay,vascular formation assay,live/dead cell staining assay,cytoskeletal staining assay,CCK-8 assay,RT-PCR,WesternBlot,ELISA,immunofluorescence staining assay,alkaline phosphatase(ALP)and alchemy red staining assay.In addition,BMSCs and HUVECs were co-cultured with composite scaffolds in the present study,and the interaction of the two types of cells through the paracrine pathway in the co-culture environment was evaluated by RT-PCR assay,and whether the two types of cells co-cultured with SPEEKPDA-GelMA/Si@nHA treatment could enhance the osteogenesis of BMSCs and the angiogenesis of HUVECs was assessed by ALP analysis and tube formation assay.3.In the present study,the immunomodulation,angiogenesis and bone repair of SPEEK-GelMA/Si@nHATGF-β1 composite 3D scaffolds were evaluated in vivo using the rat cranial critical bone defect as an in vivo animal model.The in vivo biological evaluation of SPEEK-GelMA/Si@nHATGF-β1 composite 3D scaffolds was performed by Micro-CT qualitative and quantitative analysis of skull specimens and by immunofluorescence staining(iNOS,Arg-1,CD31),hematoxylin-eosin(H&E)staining,Masson staining,and Sirius red staining for inflammation in tissue sections.Immunomodulation,angiogenesis and bone repair effects were observed in the scaffolds,and H&E staining was used to observe the important organs of rats to prove the safety of the bone repair biomaterials.Results:1.The 3D printed PEEK scaffolds have smooth surface,regular structure,uniform pore diameter and good inter-pore connectivity.The average width of the prism was 515.36±11.70μm,the pore diameter was 681.05±16.25 μm,and the average porosity was 64.26%,meanwhile,it had good mechanical properties,and the surface of the scaffolds formed a microporous network with an average pore diameter of 1.15±0.50 μm after sulfonation treatment.The results of the XRD,FT-IR,and ICP tests showed that the Si@nHA was a pure HA phase,and the crystallinity slightly reduced,and the Si doping is about 1.25 wt%,therefore,Si@nHA was successfully synthesised.NMR hydrogen spectroscopy results indicated that methacrylic anhydride was successfully grafted ontoNH2 of the gelatin backbone.Mechanical testing of the composite PEEK scaffolds showed that the compression modulus was further enhanced by the incorporation of Si@nHA,and the XPS and XRD tests demonstrated that the surface modification did not change the original phase structure of PEEK,and Si@nHA was successfully loaded onto the composite PEEK scaffolds with GelMA as the carrier.ELISA showed that the TGF-β1 was efficiently immobilized on the surface of the scaffolds,with a loading rate of 36.12±3.15%.The ELISA test showed that TGF-β1was released in one week,and the release rate reached 95.33%on the 7th day,while the ICP test showed that the release curve of Si was more gentle and long-lasting,and the release rate reached 37.33%on the 28st day,which proved that it had the characteristics of long-lasting and slow-release,and basically fulfilled the requirements of the sequential release of the different functional components in a time-space coordination and throughout the whole bone repair process.2.All groups of porous composite PEEK scaffolds are biocompatible,basically nontoxic,and have no obvious negative effects on cell survival,adhesion,and proliferation.SPEEK-GelMA/Si@nHATGF-β1 composite PEEK scaffolds have obvious recruitment and good wound healing ability for HUVECs and BMSCs,and they have the best performance in terms of the ability of inducing tubularity in vitro.performance.Meanwhile,SPEEK-GelMA/Si@nHATGF-β1 composite PEEK scaffolds significantly increased ALP expression and calcium nodule deposition in BMSCs,and significantly upregulated the expression of osteogenesis-related genes(Runx2,OPN,Col-I,ALP,)and proteins(Runx2,OPN,Col-I,BMP-2,).For RAW264.7,SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds effectively promoted its polarisation to M2 type,inhibited the expression of pro-inflammatory gene iNOS,up-regulated the expression of inflammationsuppressing gene CD206,promoted the expression of Arg-1 protein,decreased the secretion of pro-inflammatory factors(IL-6 and TNF-α),and increased the secretion of inflammation-suppressing factor(IL-4).For HUVECs,SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds upregulated CD31 protein and HIF-1α protein expression,and upregulated the relative expression of VEGF and HIF-1α genes.In addition,Si released from Si@nHA enhanced the relative expression of VEGF gene in BMSCs and BMP-2 gene in HUVECs in the co-culture system,thereby stimulating the angiogenesis and osteogenesis of HUVECs and BMSCs in the co-culture system through the paracrine effect of cellular interactions.3.Micro-CT results of in vivo cranial defect repair experiments showed that SPEEKGelMA/Si@nHATGF-β1 composite scaffolds achieved the best bone repair results compared with other modified scaffolds in each group,and immunofluorescence staining of tissues at 1 week showed that SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds upregulated the expression of the M2-type macrophage marker Arg-1 protein in the area of the implantation of the material,and down-regulated the expression of iNOS protein at 4 weeks.Tissue immunofluorescence staining at 1 week showed that SPEEKGelMA/Si@nHATGF-β1 composite scaffold up-regulated the expression of M2 type protein in the implanted area and down-regulated the expression of iNOS protein,while tissue immunofluorescence staining at 4 weeks showed that the expression of CD31 protein was up-regulated in the defective area by SPEEK-GelMA/Si@nHATGF-β1 composite scaffold.Tissue sections stained at 4 and 8 weeks showed that the new bone was formed clearly around SPEEK-GelMA/Si@nHATGF-β1 composite scaffold.This suggests that the composite PEEK scaffolds designed in this study can synergise with the remodelling of the immune microenvironment and the continuous formation of blood vessel in vivo to ultimately optimize bone repair.Conclusions:1.The SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds prepared by combining 3D printing,sulfonation treatment,construction of PDA coating,and hydrothermal synthesis of Si@nHA have good physicochemical properties and have the effect of spatial-temporal coordination of the ordered release of TGF-β1 and Si,and play a role in the whole stage of bone repair.2.In the early inflammatory stage of bone defects,SPEEK-GelMA/Si@nHATGF-β1 composite scaffolds can form an immune microenvironment conducive to tissue repair by promoting the polarisation of macrophages to the M2 type and the secretion of antiinflammatory factors,and the release of Si can effectively induce the formation of neovasculature over a longer period of time as well as the temporal and spatial coordination of the release of different active ingredients at the different stages of bone repair,which can achieve the temporal and spatial coordination required at different stages of bone repair.The release of Si can effectively induce angiogenesis over a longer period of time,thus releasing different active ingredients in a sequential manner to achieve the temporal and spatial coordination of the different stages of bone repair,and thus synergistically promoting bone repair.This provides a new reference for the design of composite scaffolds for bone tissue engineering.