| 【Background】Degradable bone repair materials are considered as ideal candidates for bone repair,as they can be completely degraded and absorbed by the human body and replaced by bone tissue eventually.Currently,the commonly used degradable bone repair materials include primarily polymers and bioceramics,etc.However,it is difficult to use a single material to build a degradable bone repair scaffold due to performance deficiencies.Polymers(such as polylactic acid,polycaprolactone,etc.)have a controlled rate of degradation,but mechanical strength and osteogenic activity are relatively poor.Ceramic materials(such as hydroxyapatite,tricalcium phosphate,etc.)have relatively high osteogenic activity and mechanical strength,but are brittle and easily fractured during in vivo implantation.Therefore,preparing biodegradable scaffolds for bone repair by mixing different materials can compensate for deficiencies in a single material and enhance the therapeutic effect of the scaffolds.And how to select the ratio of the different components to optimize the osteogenic activity and degradation mode,and achieve simultaneous degradation and bone repair of the scaffold is an important issue in this field.In our previous research,we physically mixed polycaprolactone(PCL)and β-tricalcium phosphate(β-TCP)in a mass ratio of 80:20,and successfully fabricated a degradable scaffold for bone repair using Fused Deposition Modeling(FDM)technique.The PCL/β-TCP scaffolds showed satisfactory repair effects in an anterior cervical decompression and fusion(ACDF)model in sheep,but there were still some problems such as slow degradation rate and inadequate bone tissue in-growth.In this project,the contents of bioceramics were further increased and a series of new PCL/β-TCP scaffolds with different ratios(mass ratios 70:30 to 30:70)have been prepared to optimize degradation process and bone repair effect of scaffolds.However,during the degradation process,the physical and chemical properties(such as mechanical properties,surface morphology and composition,etc.)of the scaffolds change continuously,and are accompanied by the generation of degradation products.All of these factors yield dynamic influence on the local immune responses and bone regeneration process,but the exact effects and mechanisms involved are still unclear.Therefore,it is of great significance to dynamically evaluate the degradation process of the new degradable PCL/β-TCP scaffolds and elucidate the effects and mechanisms of scaffold degradation affecting bone repair to optimize the degradable modes of the degradable bone repair scaffolds and improve the bone repair effects.【Objective】Fabrication of 3D printed PCL/β-TCP scaffolds with different ratios(mass ratios 70:30 to 30:70);Characterization of the physicochemical properties and biocompatibility of different scaffolds;Systematic evaluation of the dynamic changes of the physiochemical properties and the characters of degradation products during scaffolds degradation;Confirmation of the degradation processes,bone regeneration and macrophage responses of the scaffolds in vivo;Verification of the biological effects of scaffolds degradation on regulating macrophage response and immune regulation of osteogenesis;Clarification of the mechanisms involved in scaffolds degradation affecting macrophage responses and immune regulation of bone repair through transcriptome sequencing and functional validation.【Methods】Part Ⅰ: Fabrication of porous PCL/β-TCP scaffolds with different mass ratios by FDM technique(PT scaffolds;pore size: 600 μm;filament diameter: 400 μm);Micro-CT,Scanning electron microscopy(SEM),energy dispersive spectroscopy(EDS),water contact angle measurement and compressive tests were used to investigate the threedimensional structure,surface morphology,element distribution,hydrophilicity and mechanical strength of scaffolds;The phases,chemical bonds and functional group compositions of the scaffolds were detected by X-Ray Diffraction(XRD)and Fourier Transform Infrared Spectroscopy(FTIR);Gel Permeation Chromatography(GPC)was used to detect the molecular weight of the scaffolds;The cytotoxicity and cell adhesion of the scaffolds were evaluated by CCK-8 measurement and SEM observation of L929 fibroblasts.Part Ⅱ: Construction of a biomimetic dynamic degradation device in vitro to accelerate the degradation of PT scaffolds;Evaluation of dynamic changes in surface morphology,weight loss,molecular weight,compression strength,chemical components and three-dimensional structure during scaffold degradation by SEM,GPC,compression testing,XRD,FTIR,and Micro-CT,etc.;The morphology,particle size and structure of the degradation products were analyzed by Micro-CT 3D reconstruction and TEM.Part Ⅲ: Different groups of PT scaffolds were implanted into the rat femoral condylar bone defect model and specimens were obtained at 4,10 and 16 weeks postoperatively;Micro-CT scanning and reconstruction,SEM,fluorescent double-label staining,Masson staining and immunohistochemical staining of CD31 were performed to evaluate the bone repair,vascularization and scaffold degradation at different time points.The polarization evolution of macrophage phenotype inside the scaffold was evaluated by tissue immunofluorescence staining.Part Ⅳ: PT 73,PT 55 and PT 37 scaffolds were selected for accelerated degradation,in vitro and in vivo degradation periods of scaffolds were matched by SEM combined with elemental distribution energy spectroscopy;Macrophages were co-cultured with scaffolds at different degradation stages(D1-D3)and the morphology and polarization of macrophages were observed by scanning electron microscopy(SEM),immunofluorescence staining and Western blotting;Establishment of a rat subcutaneous implantation model of scaffolds with different degradation stages(D1-D3),and the tissue responses and macrophages polarization around the scaffolds were detected;Extraction of macrophage conditioned medium(CM)and determination of the concentrations of different cytokines in CM;The immunoregulatory effects of macrophage CM on osteogenic differentiation and vascularization were detected by alkaline phosphatase(ALP)staining and tube formation assay.Part Ⅴ: The gene expression levels of macrophages co-cultured with PT 55 and PT37 scaffolds at D2 degradation stages were analyzed by transcriptome sequencing(RNASeq);GO and KEGG enrichment analysis of differential function and pathways were performed;Validation of the enrichment results by TEM,western Blot,lyso-tracker staining and flow cytometric analysis,etc.;Specific inhibitors were selected to inhibit the related targets,and the functional changes of macrophage responses and immune regulation were detected.【Results】Part Ⅰ Preparation and characterization of 3D printed PT scaffolds with different ratios.1.PCL/β-TCP porous scaffolds(PT scaffolds)with mass ratios of 70:30-30:70 were successfully prepared using the FDM technique,defined as: PT 73 scaffolds(mass ratio70:30),PT 64 scaffolds(mass ratio 60:40),PT 55 scaffolds(mass ratio 50:50),PT 46scaffolds(mass ratio 40:60),PT 37 scaffolds(mass ratio 30:70).2.SEM observation showed that the pore size of scaffolds in each group was uniform and close.Micro-CT scanning analysis showed that there was no statistical difference in porosity among groups;EDS analysis results showed that the elements in each group are uniformly distributed,and Ca and P elements increased significantly with higher ceramic contents.3.Water contact angle results showed that PT scaffolds with the high ceramic contents are more hydrophilic;According to the results of stress-strain curve and compression modulus,the increase of ceramic contents strengthened the compressive performance of the scaffolds4.CCK-8 results showed that good biocompatibility of scaffolds.The SEM results showed that the fibroblasts were fully spread out on the scaffold surface and tightly adhered to the scaffolds.Part Ⅱ Dynamic degradation evaluation of 3D printed PT scaffolds with different ratios in vitro.1.The biomimetic accelerated degradation device was constructed in vitro for the degradation study of PT scaffolds,and sodium hydroxide solution was determined as the degradation medium in subsequent experiments.The liquid circulation speed of the device was set to 1 m L/min to conduct dynamic degradation observation on different PT scaffolds(diameter 1 cm,height 0.2 cm)for 360 min.2.PT 46 and PT 37 scaffolds degraded rapidly and disintegrated after 180 min degradation,and surface was corroded;the degradation of PT 73,PT 64 and PT 55 scaffolds was relatively stable and only partial degradation occurred after 360 min degradation,and no obvious corrosion was observed on the surface of the scaffolds.3.The weight,molecular weight and compressive strength of the scaffolds decreased gradually with the degradation of the scaffolds,and the decreasing rates of the PT 46 and PT 37 scaffolds with high ceramic contents were significantly higher than those of the PT 73,PT 64 and PT 55 scaffolds.4.XRD and FTIR analysis results showed that some crystalline phase and chemical bond characteristic peaks of PCL disappeared after 60 min of degradation of the PT 46 and PT 37 scaffolds.After 360 min degradation,the PCL characteristic peak of the PT 64 and PT 55 scaffolds also partially disappeared.5.Micro-CT cross-sectional images showed that degradation pore was observed inside the PT 73,PT 55 and PT 37 scaffolds at 180 min after degradation,and high-density degradation products appeared on the surface of the scaffolds;3D reconstruction and TEM results showed that there were differences in the number,shape and size of degradation products in each group.Part Ⅲ In vivo dynamic degradation,bone regeneration and macrophage response of 3D printed PT scaffolds with different ratios.1.Micro-CT three-dimensional reconstruction and region of interest analysis showed that scaffold degradation and bone in-growth occurred synchronously in the PT 73,PT 64 and PT 37 groups,and the volume fraction of new bone increased gradually while the volume fraction of scaffold decreased steadily;The PT 37 and PT 46 scaffolds had higher volume fractions of new bone at 4 weeks,but were lower than that in other groups due to rapid scaffold degradation.2.The SEM results showed that the PT 73 PT 64 and PT 55 scaffolds were well integrated with the surrounding bone tissues,and there was no obvious corrosion on the surfaces of the scaffolds at each time point.The PT 46 and PT 37 scaffolds were well integrated with the surrounding bone tissues at 4 weeks,but the surfaces of the scaffolds were corroded and degraded obviously at 10 and 16 weeks and the scaffolds were wrapped with fibrous tissues.3.Masson tissue section staining showed that the internal bone ingrowth of the scaffolds increased gradually in PT 73,PT 64 and PT 55 groups,connected into a network structure at 16 weeks after surgery,and the bone regeneration was generally completed;The area of new bone formation in the PT 46 and PT 37 scaffolds was larger than that in other groups at 4 weeks,but the bone ingrowth was impaired and the trabecular bone stuctures were resorbed and at 10 and 16 weeks,and multi-nucleated foreign body giant cells(FBGCs)were shown around the scaffolds at high magnification.4.Immunochemical staining showed that the number of CD 31 positive vascular endothelial cells was higher in the PT 46 and PT 37 scaffolds than that in the other groups at 4 weeks,but vascular growth was impaired,and the number of endothelial cells was significantly lower than that in the PT 73,PT 46 and PT 55 scaffolds at 10 and 16 weeks.5.The results of fluorescent double-label staining of bone tissue showed that the bone formation rate/bone surface(BFR/BS)in PT 46 and PT 37 groups was higher than that in other groups but decreased significantly at 10 and 16 weeks and obviously lower than that in PT 73,PT64 and PT 55 groups.6.The immunofluorescence staining results showed that the polarization of macrophages in PT 73 and PT 55 scaffolds gradually transited from M1(proinflammatory)type to M2(pro-regeneration)type,and M2 type macrophages were predominant inside the scaffolds at 10 and 16 weeks;The polarization evolution process of macrophages inside the PT 37 scaffolds was opposite and M1 type(pro-inflammatory)macrophages dominated inside at 10 and 16 weeks,and the M1 macrophage polarization caused by scaffold degradation may be the key factor of the bone regeneration failure of PT 37 scaffolds.Part Ⅳ The effects of dynamic degradation of 3D printed PT scaffolds with different ratios on macrophage polarization and immunomodulation of osteogenesis and vascularization.1.In vitro degradation periods matched with in vivo scaffolds at 4 weeks,10 weeks and 16 weeks were determined by scanning electron microscopy combined with energy dispersive spectrometry,which were defined as early degradation stage(D1),middle degradation stage(D2)and late degradation stage(D3);The time required for different scaffolds to degrade to each period was recorded.2.Macrophages were co-cultured with scaffolds of different degradation stages;SEM results showed that the macrophages exhibit two typical shapes at different degradation stages: Spherical macrophages with multi-pseudopods and elongated flat macrophages.The macrophage shapes varied on the surface of each scaffold at different degradation stages.3.Immunofluorescence and Western blot results showed that macrophage phenotype on the surface of PT 73 and PT 55 scaffolds changed from M1 type at D1 stage to M2-type at D2 and D3 stages.The polarization sequence of macrophages on the surface of the PT 37 scaffolds was just the opposite,and macrophages showed M1 phenotype at the D2 and D3 stages.4.The ELISA results showed that the expression levels of IL-6 and TNF-ɑ of macrophage in PT 73 and PT 55 groups were higher than those of PT 37 group,while the expression levels of IL-10 and TGF-β in PT 37 group were higher than other groups at D1 stage.As scaffolds degraded,the levels of IL-6 and TNF-ɑ were significantly increased in PT 37 group and were obviously higher than that in other groups at D2 and D3 stages.5.The results of ALP and tube formation experiments showed that the osteogenic differentiation of osteoblast precursors and vascularization of endothelial cells were significantly reduced in PT 37 group at D2 and D3 stage.Part Ⅴ The mechanism of dynamic degradation of 3D printed PT scaffolds with different ratios in the regulation of macrophage polarization and bone regeneration.1.RNA-seq results showed that the expression levels of 1503 genes were different between PT 55 and PT 37 groups(fold difference ≥ 2,P <0.05);In PT 37 group,1288 genes were up-regulated and 215 genes were down-regulated;GO and KEGG enrichment annotation results showed that PT 37 degraded scaffolds significantly promoted the formation of macrophage phagosomes,suggesting that phagocytosis may be a key target for scaffold degradation to regulate the response behavior of macrophages.2.Lysosomal probe staining showed that PT 37 degraded scaffolds increased the number of lysosomes in macrophages compared with PT 73 and PT 55 degraded scaffolds at D2 stage;TEM results directly demonstrated that the PT 37 degraded scaffolds promoted the phagosome formation of macrophages and the phagosome diameter of macrophages was significantly higher than that of the other groups at D2 stage.The uneven distribution of degradation products could be directly observed inside the phagosome.3.The results of Immunofluorescence,WB and flow cytometry showed that the degradation products of PT 37 scaffolds caused oxidative stress and increased the level of reactive oxygen species in macrophages at D2 stage;Inhibition of phagocytosis by Latrunculin B significantly reduced the oxidative stress level and M1 polarization of macrophages and alleviated the inhibition of osteogenic differentiation and vascularization process in PT 37 group at D2 degradation stage.【Conclusion】1.PCL/β-TCP(PT)degradable bone regeneration scaffolds with different proportions(mass ratio 70:30-30:70)were prepared by fused deposition 3D printing method.Increasing the ceramic contents effectively improved the properties such as compressive strength,hydrophilicity,calcium and phosphorus ratios of PT degradable scaffolds,but accelerated the degradation and degradation products generation at the same time.2.In vivo bone repair results showed that higher ceramic contents facilitated the bone repair of PT scaffolds at the early stage,but PT scaffolds with high ceramic contents(PT46 and PT 37)hindered the new bone growth and caused bone regeneration failure at the middle and late stages of degradation.3.During PT 73,PT 64 and PT 55 scaffolds degradation,macrophages are polarized in an “M1 pro-inflammatory type to M2 pro-healing type” sequence,which promoted osteogenic differentiation and vascularization;PT scaffolds with high ceramic contents caused M1 polarization of macrophages and inhibited osteogenic differentiation and vascularization.4.PT scaffolds with high ceramic contents produced large-size(>10 μm in diameter)degradation products.Phagocytosis of degradation products triggered oxidative stress and M1 polarization of macrophages,which in turn inhibited osteogenic differentiation and vascularization and resulted in bone regeneration failure eventually. |
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