Research On 3D Printed MXene/PCL In Situ Bone Tissue Engineering Scaffold And Osteogenesis Molecular Mechanism

Patients with bone defects need bone substitutes to fill and repair the defect site to restore body function.In clinic,autogenous bone graft has been considered as the"gold standard",with the disadvantages of limited source of autogenous bone graft and secondary trauma.Allogeneic bone grafts carry the risk of immune rejection and infectious diseases.With the rapid development of material science,engineering technology and biotechnology,in situ tissue engineering technology has been widely used to develop bone repair scaffolds.In situ bone tissue engineering scaffolds do not contain cellular components,when implantation of the scaffold into the bone defect,the bioactive molecules are able to induce differentiation of bone marrow mesenchymal stem cells（BMSCs）recruited or migrated to the defect site into osteoblasts,which ultimately lead to the repair of the damaged bone tissue.Bioactive molecules in in situ bone tissue engineering scaffolds mainly include growth factors,chemokines,osteogenic drugs,peptides,bioceramics and rare earth elements,etc.But complex composition and potential biosecurity issues have brought a lot of trouble to large-scale production.Thus,the development of an in situ bone tissue engineering scaffold with safety in vivo use,strong osteogenic differentiation ability,low cost and simple ingredients is a hot topic in this field.Because MXene material has good electrical conductivity,photothermal effect,Immune regulatory ability,low cytotoxicity,antimicrobial ability,and CT/MRI imaging properties,they have been gradually applied in various fields of biomedicine.In this study,polycaprolactone（PCL）and a kind of MXene material（Ti₂AlN）were combined to prepare in situ bone repair scaffolds with different Ti₂AlN contents.Through the physicochemical,in vitro and in vivo characterization of this composite scaffold,the potential of Ti₂AlN materials in the field of bone repair was explored.We used genomic methods to analyse the possible molecular mechanism of bone repair involved in Ti₂AlN materials.Through the above research results,the potential of Ti₂AlN material in the field of in situ bone repair scaffolds has been fully explored.（1）Preparation and physicochemical characterization of Ti₂AlN/PCL composite scaffold for in situ bone repairA small-scale compacting machine was designed and assembled,and composite particles containing 1%（PCL@1#Ti₂AlN）,5%（PCL@5#Ti₂AlN）,10%（PCL@10#Ti₂AlN）and 20%（PCL@5#Ti₂AlN）Ti₂AlN were successfully prepared using this small-scale compacting machine.An FDM-3D printer was designed and assembled to 3D print Ti₂AlN/PCL bone repair scaffolds containing different mass percentages using the granular material as the material.As the Ti₂AlN content increased,the X-ray development of the scaffold was enhanced,the near-infrared irradiation thermal effect of the scaffold increased,and the scaffold became more hydrophilic.The tensile and compression test results of the stents showed the best mechanical properties of the stents in the PCL@5#Ti₂AlN group.FTIR,TGA and XRD results surface different mass percentages of Ti₂AlN were successfully added into PCL.In vitro lipase accelerated degradation experiments of the stent surface with increasing Ti₂AlN content the degradation rate of the stent was accelerated.（2）In vitro characterization and in vivo in situ bone repair studies of Ti₂AlN/PCL scaffoldsIn vitro characterization:The cytocompatibility of the scaffold was evaluated using live/dead cell staining,CCK-8,ghost pen cyclic peptide staining and SEM,and the results showed that the PCL@5#Ti₂AlN group had the strongest cell adhesion and proliferation ability with no significant cytotoxicity.When the Ti₂AlN content was greater than 5%,the scaffolds started to exhibit some cytotoxicity and the cell adhesion and proliferation rates started to decrease.After 7 days of culture,the expression level of Alkaline Phosphatase（ALP）in cells on the scaffold indicated that the PCL@5#Ti₂AlN group was the most favorable for cellular osteogenic differentiation.In vivo characterization:The bone repair ability of the scaffold was evaluated using a rat tibial defect model,and the Micro-CT results after 8W showed that the PCL@5#Ti₂AlN had the best bone repair,and the HE,Masson and Sirius Red histological staining and bone trabecular thickness results also demonstrated that the PCL@5#Ti₂AlN group had the best ability to generate new bone from the scaffold.We then used PCL@5#Ti₂AlN scaffolds to repair rabbit maxillofacial bone defects also obtained satisfactory repair results.（3）Genomics analyzed of the molecular mechanisms and signal pathways involved in the differentiation of cells into osteogenesis involved in Ti₂AlN/PCLThe osteogenesis mechanisms involved in PCL@5#Ti₂AlN versus PCL were analyzed using transcriptomics data.Through the clustering analysis of differentially expressed m RNA,Lnc RNA and mi-RNA target genes,we found that Ti₂AlN was mainly involved in regulating osteogenic differentiation of cells through regulating Wnt/β-catenin signaling pathway and calmodulin,and thus revealed the osteogenic mechanism of Ti₂AlN from the molecular level.In summary,in this study,PCL in situ bone repair composite scaffolds doped with different MXene contents were successfully prepared with the aid of FDM-3D printing technology based on in situ bone tissue engineering technology.Through in vitro and in vivo characterization,the results showed that PCL@5#Ti₂AlN composite scaffolds containing 5%Ti₂AlN had good mechanical properties,the strongest cell adhesion and proliferation capacity,no significant cytotoxicity,the best in vivo bone repair（rat tibial defect model and rabbit mandibular defect model）,and it was found that Ti₂AlN is mainly involved in regulating osteogenic differentiation of cells through regulating Wnt/β-catenin signaling pathway and calmodulin.This study systematically investigated the role of Ti₂AlN as a bioactive molecule in bone repair scaffold in situ,laying a foundation for the application of MXene materials in bone repair in situ.