| Melt electrohydrodynamic printing,also known as melt electrowritten printing,is a micro-nano scale 3D printing technology that has garnered significant attention in recent years.The melt electrohydrodynamic printed scaffold exhibits high precision,a large specific surface area and excellent cell permeability,creating a favorable microenvironment for cell growth and attachment.Nonetheless,further investigation is required to understand the impact of melt electrohydrodynamic printed scaffolds on bone defect repair.Polycaprolactone（PCL）stands out for its biocompatibility,appropriate melting temperature,and quick solidification properties,making it the most commonly utilized material in melt electrohydrodynamic printing.However,the hydrophobic nature of PCL materials hinders cell adhesion and proliferation,thereby limiting its practical application in the realm of bone tissue engineering to some extent.Calcium phosphate（CaP）contains calcium and phosphorus components similar to those of human bone tissue.It is commonly used as a coating material for modifying material surfaces to enhance their physical and chemical properties,such as hydrophilicity,roughness and surface characteristics.The CaP coating exhibits significant biological activity and plays a crucial role in promoting the integration of the material with bone tissue.Current research primarily focuses on investigating the osteogenic impact of CaP coatings,yet further comprehensive studies are needed to elucidate how these coatings exert their osteogenic effects through the physical and chemical properties of the material.Moreover,as osteoimmunology advances,the significance of the local immune microenvironment in bone tissue regeneration is becoming increasingly apparent.Nevertheless,the immunomodulatory properties of CaP coatings,particularly their influence on macrophage polarization,require further exploration.This study utilized melt electrohydrodynamic printing technology to fabricate a high-precision PCL scaffold,followed by the successful application of a CaP coating using a bionic mineralization method.The study then examined the impact of the CaP coating on the physical and chemical properties of the PCL scaffold.Based on the physicochemical properties of PCL scaffolds,this study preliminarily explored the molecular mechanisms of CaP coating-modified PCL scaffolds in enhaning osteogenic differentiation of bone marrow mesenchymal stem cell（BMSC）and regulating M2 polarization of macrophages.The ultimate goal was to establish a theoretical foundation for the application of high-precision printed scaffolds and CaP coating in bone tissue engineering.The study was divided into five parts.PartⅠThe fabrication and characterization of calcium phosphate coating modified melt electrohydrodynamic 3D printed PCL scaffoldsObjective:To investigate the effect of calcium phosphate（CaP）coating on the physical and chemical properties of melt electrohydrodynamic 3D printed PCL scaffoldsMethods:High-precision PCL scaffolds were fabricated using melt electrohydrodynamic printing technology.The surface of the PCL scaffolds was activated with a sodium hydroxide（Na OH）solution and then immersed in modified simulated body fluid（SBF）.The CaP coating was applied to the surface of the PCL scaffold using a bionic mineralization method.Subsequently,the surface morphology,hydrophilicity,roughness,and mechanical properties of the scaffolds were analyzed using a scanning electron microscopy,a water contact angle tester,an atomic force microscope,and a universal testing machine.X-ray diffraction（XRD）was employed to examine the structure of the CaP coating.The release of calcium ions from the CaP coating was measured using high-resolution inductively coupled plasma mass spectrometry（ICP-MS）.The PCL scaffolds were divided into three groups in this study:the control group（PCL）,the Na OH-treated group（Na OH-PCL）,and the calcium phosphate coating modified group（CaP-PCL）.Results:The CaP coating was effectively applied to the surface of PCL scaffolds using a biomimetic mineralization method.The results of water contact angle indicated that the CaP coating enhanced the hydrophilicity of the PCL scaffolds.The results of atomic force microscopy analysis revealed that the CaP coating led to an increase in the roughness of the PCL scaffold.ICP-MS testing confirmed that the CaP coating enabled the stable release of calcium ions.Mechanical property assessments demonstrated that the CaP coating significantly enhanced both the tensile and compressive mechanical properties of the PCL scaffolds.Conclusion:The CaP coating could not only effectively enhance the physical properties of PCL scaffolds,such as hydrophilicity and roughness,but also facilitate stable release of calcium ions.PartⅡThe study on the mechanism of calcium phosphate coating modified melt electrohydrodynamic 3D printed PCL scaffold to promote osteogenic differentiationObjective:To investigate the role and molecular mechanism of the CaP-PCL scaffold in promoting osteogenic differentiation of BMSC.Methods:The mouse BMSC were co-cultured with three different groups of scaffolds to evaluate the biocompatibility of the CaP-PCL scaffolds using CCK-8 and live-dead staining experiments.The expression levels of osteogenic differentiation markers in BMSC were detected through qRT-PCR and immunofluorescence staining.Alizarin red staining and alkaline phosphatase staining were utilized to observe calcium nodule deposition and alkaline phosphatase activity in BMSC.Furthermore,transcriptome sequencing technology was employed to analyze the molecular mechanism underlying the regulation of osteogenic differentiation of BMSC by CaP-PCL scaffolds.Results:The CCK-8 analysis demonstrated that the CaP-PCL scaffold enhanced the proliferation activity of BMSC.Furthermore,qRT-PCR and immunofluorescence staining analyses indicated that the CaP-PCL scaffold upregulated the expression levels of osteogenic differentiation markers in BMSC.The results of alizarin red and alkaline phosphatase staining also confirmed that the CaP-PCL scaffold promoted the deposition of calcium nodules and alkaline phosphatase activity in BMSC.GO enrichment analysis highlighted that the differently expressed genes（DEGs）were primarily associated with osteoblast differentiation,membrane region,action cytoskeleton,intracellular calcium homeostasis and response to calcium.Furthermore,KEGG enrichment analysis demonstrated that the DEGs were significantly enriched in the focal adhesion and MAPK signaling pathways.Conclusion:The CaP-PCL scaffold might regulate the focal adhesion signaling pathway and the MAPK signaling pathway through both physical factors（surface wettability and roughness）and chemical factors(Ca2+releasing),working together to enhance the osteogenic differentiation of BMSC.PartⅢThe study on the mechanism of calcium phosphate coating modified melt electrohydrodynamic 3D printed PCL scaffold in regulating macrophage polarizationObjective:To investigate the molecular mechanism of the CaP-PCL scaffold in regulating macrophage polarization.Methods:The macrophage RAW264.7 cell line was co-cultured with three different groups of scaffolds to assess the impact of the CaP-PCL scaffold on macrophage proliferation activity using CCK-8 experiments.Immunofluorescence staining and Western Blot experiments were conducted to evaluate the effect of CaP-PCL scaffolds on macrophage polarization.Additionally,ELISA analysis was employed to observe the impact of CaP-PCL scaffolds on the secretion levels of macrophage-related factors.Lastly,transcriptome sequencing technology was utilized to analyze the molecular mechanism through which CaP-PCL scaffolds regulated macrophage polarization.Results:The CCK-8 assays demonstrated that the CaP-PCL scaffold enhanced the proliferation activity of macrophages.The results of immunofluorescence staining and Western Blot confirmed that the CaP-PCL scaffold upregulated the expression of macrophage M2 markers（Arg-1 and CD206）while downregulating the expression of M1markers（i NOS and CD86）.Additionally,ELISA analysis indicated that the CaP-PCL scaffold enhanced the secretion of anti-inflammatory factors（IL-4 and IL-10）and suppressed the secretion of pro-inflammatory factors（IL-6 and TNF-α）.Transcriptome sequencing analysis revealed 129 differentially expressed genes（DEGs）between the PCL and CaP-PCL groups.GO enrichment analysis highlighted that these genes were primarily associated with cytoskeleton,tropomyosin binding,cellular ion channels,and intracellular calcium homeostasis.Furthermore,KEGG enrichment analysis demonstrated that the DEGs were notably enriched in the PI3K-AKT and c AMP signaling pathways.Conclusion:The CaP-PCL scaffold might regulate the PI3K-AKT signaling pathway and the c AMP signaling pathway through both physical factors（surface wettability and roughness）and chemical factors(Ca2+releasing),working together to facilitate M2polarization in macrophages.PartⅣThe study on the impact of calcium phosphate coating modified melt electrohydrodynamic 3D printed PCL scaffold in regulating macrophage polarization for influencing osteogenesisObjective:To investigate the immunosteogenesis effect of CaP-PCL scaffolds.Methods:Macrophage conditioned medium（MCM）was obtained by co-culturing macrophages with different scaffolds.Subsequently,MCM was co-cultured with BMSC,and the expression levels of osteogenic differentiation markers in BMSC were detected using qRT-PCR and immunofluorescence staining.Alizarin red and alkaline phosphatase staining were used to observe calcium nodule deposition and alkaline phosphatase activity in BMSC.Furthermore,the impact of BMSC migration ability was assessed through cell scratch assay and Transwell assay.Results:The results of qRT-PCR assay and immunofluorescence staining showed that the MCM in the CaP-PCL group significantly promoted the expression levels of osteogenic differentiation markers in BMSC.The results of alizarin red staining and alkaline phosphatase staining indicated that the MCM in the CaP-PCL group enhanced the deposition of calcium nodules and the activity of alkaline phosphatase in BMSC.The results from both cell scratch and Transwell experiments demonstrated that the MCM in the CaP-PCL group effectively enhanced the migration capability of BMSC.Conclusion:The CaP-PCL scaffolds could enhance osteogenic differentiation and migration of BMSC by promoting M2 polarization of macrophage.PartⅤThe study on the in vivo immune regulation and osteo-promoting effects of calcium phosphate coating modified melt electrohydrodynamic 3D printed PCL scaffoldObjective:To investigate the regulatory impact of CaP-PCL scaffold on macrophage polarization and bone repair in vivo.Methods:Three distinct groups of scaffolds were implanted in the rat calvaria defect models for subsequent analysis.To assess the effects of CaP-PCL scaffolds on macrophage polarization and cytokine secretion,immunohistochemical staining and immunofluorescence staining were conducted.Additionally,micro-CT analysis,double fluorescence labeling of calcein and alizarin red,and histological staining were utilized to observe the reparative effects of CaP-PCL scaffolds on bone defects.Results:The results of immunofluorescence and immunohistochemical staining demonstrated that the CaP-PCL scaffold effectively enhanced the expression of macrophage M2 markers and increased the levels of the anti-inflammatory factor（IL-10）.Furthermore,the results from micro-CT analysis,double fluorescence labeling and histological staining confirmed that the CaP-PCL scaffold facilitated the repair of rat skull defects.Conclusion:It was found that the CaP-PCL scaffolds had the ability to promote the M2 polarization of macrophages and enhance bone regeneration in the rat calvaria defect model... |
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