| Background:Large areas of bone defects caused by tumor resection,trauma,orthodontics,bone dysplasia or severe fractures require ideal biomaterials to facilitate the bone repair process.3D bioprinting is gradually applied to the field of bone tissue engineering because it can precisely locate cells and biological agents in an automated and highthroughput manner,and realize the refined and personalized manufacturing of target organs and tissues.Hydrogels are the gold standard materials for cell encapsulation systems,but single photocrosslinked hydrogels have disadvantages such as poor printing performance and insufficient mechanical strength.The double photocrosslinked copolymer of Gelatin Methacryloyl(Gel MA)and Hyaluronic acid Methacryloyl(HAMA)can improve the strength of the scaffold.In order to avoid sacrificing the viability of encapsulated cells due to excessive photocrosslinking time,various crosslinking methods or nanofillers can be added to further increase the strength of the scaffold.Alginate,which regulates viscosity through chemical cross-linking,has become a very attractive hydrogel for bioprinting.Graphene oxide(GO)is easily functionalized,provides a large specific surface area,and can be mixed with alginate to improve its mechanical properties and has a high osteogenic ability.The immune system plays a key role in bone tissue regeneration,and proper tissue healing requires a series of well-regulated immune responses.In order to avoid excessive acute inflammatory response after material implantation,most materials are designed as bio-inert materials,such as bio-inert ceramics,but it has no biological activity and will inevitably form a fibrous tissue interface between the bone and the material,hindering the material combination with bones.In recent years,the cross fusion of immunology and bone tissue engineering has become a research hotspot in the scientific community,forming a new discipline-bone immunology,which aims to study the direct interaction between the two key cells in the bone marrow in bone tissue engineering,promoting bone repair through indirect linkage of transcription factors,cytokines and their receptors.Therefore,in the application of 3D bioprinting in bone tissue,a new generation of intelligent bioactive materials that can regulate the immune microenvironment of bone defects can be designed to further induce bone remodeling and regeneration.The relationship between immune response and bone repair should be considered during cell selection.As the first line of defense in immunity,macrophages are important regulators of inflammation,and their depletion impairs wound healing in animal models.It also stimulates osteogenic differentiation of mesenchymal stem cells to increase bone mineralization.Compared with cell line-derived macrophages,Bone Marrow-derived Macrophages(BMMs)are more favored,and they also have a certain osteogenic ability.In order to better mimic the state in vivo,multi-cell printing has become a recent research trend,and Bone Marrow Mesenchymal Cells(BMSCs)have become another option due to their simple availability and strong differentiation ability,they can differentiate into osteoblasts and possess immunomodulatory properties such as promoting the M2-type differentiation of macrophages.When more than two cell types are involved at the same time,if they are mixed in the same channel of bio-inks for printing,it is difficult to achieve uniform distribution of various types of cells,and the loading capacity of cells would also be reduced,so dual-channel 3D bioprinting can be used to simultaneously introduce BMMs and BMSCs two types of primary cells of the same origin to promote the process of bone repair from the perspectives of immune regulation and osteogenic differentiation.Objective:By introducing primary cells BMMs and BMSCs from the same origin,integrating3 D bioprinting and bone immunology repair,a 3D dual-channel bioprinting scaffold is constructed to promote the bone repair process from the perspectives of early immune regulation and late osteogenesis.Methods:1.Extract BMMs and BMSCs from rat bone marrow for cell culture.The extracted BMMs were qualitatively analyzed by flow analysis.2.Determine the printability of the bioinks in the dual channel by rheological performance test(the first channel bioink for encapsulating BMMs: Gel MA+HAMA;the second channel bioink for encapsulating BMSCs: Alginate+GO).Through the naked eye,optical microscope,electron microscope and compression performance test,the adaptability of the dual-channel printing filaments was observed,and the appropriate concentration of bioinks was screened.3.The effects of different concentrations of HAMA in the first-channel bioink on the biological activity of BMMs were further screened by cell live-death staining under a confocal microscope.The living-dead cell staining and cell proliferation activity analysis were used to further screen the activity of BMMs and BMSCs in dual-channel bioprinting.4.Assess the mutual crosstalk between BMMs and BMSCs.The effects of BMSCs on the expression of inflammatory factors and M1 and M2 polarization of BMMs were analyzed by PCR and immunofluorescence;the effects of BMMs on the osteogenic differentiation of BMSCs was evaluated by PCR,Alkaline phosphatase(ALP)analysis and Alizarin red analysis.5.The model of rat calvarial defects was established,and the 3D dual-channel printing scaffolds(0,BMMs group,BMSCs group,and BMMs+BMSCs group)were implanted into the bone defect,and angiogenesis,osteogenic tendency,inflammatory factor expression and macrophage polarization in the calvarial defects were analyzed by hematoxylin-eosin(H&E)staining,Masson staining,and immunohistochemical analysis within 1 week;Micro-CT,H&E staining,Masson staining,and immunohistochemical analysis were used to evaluate the effect of bone repair after 4weeks and 8 weeks.Results:1.The successful extraction of rat BMMs was qualitatively indicated by flow cytometry.2.Both the first-channel bioink(Gel MA+HAMA)encapsulating BMMs and the second-channel bioink(Alginate+GO)encapsulating BMSCs had good shear performance,temperature sensitivity and printability;when the first channel selected 8%Gel MA+1% HAMA and the second channel selected 3% Alginate+0.5 mg/ml GO as bioinks,the scaffolds showed good mechanical properties and were not easy to collapse and deform.3.The cell viability of BMMs encapsulated with 8% Gel MA+1% HAMA was better;when BMMs and BMSCs were simultaneously introduced into the dual-channel printing,it was shown that the growth activity and proliferative activity of both types of cells were good.4.Crosstalk analysis between BMMs and BMSCs.BMSCs could promote the M2-type polarization of BMMs,inhibit the expression of pro-inflammatory factors,and promote the expression of anti-inflammatory factors;BMMs could promote the expression of osteogenic genes and osteogenic differentiation of BMSCs,calcium salt deposits.5.After the dual-channel bioprinted scaffolds were implanted into the rat calvarial defect,the double-cell group(BMMs+BMSCs group),compared with the single-cell group(BMMs group,BMSCs group)and the cell-free group(0 group),attained more effective immune regulation in the early stage of bone defect microenvironment and the M2 polarization of macrophages was promoted better,and the promotion of new bone formation in the late stage also had a better effect.Conclusion:We selected a dual-channel mixed bioinks with good rheological properties and biocompatibility(the first channel bioinks: 8% Gel MA+1% HAMA;the second channel bioinks: 3% Alginate+0.5 mg/ml GO),printed a 3D bioprinted scaffold with good mechanical properties,and innovatively introduced rat BMMs and BMSCs encapsulated in the first and second channels respectively,thereby promoted the poralization of M2 macrophages in the early stage of rat calvarial defect microenvironment,promoted angiogenesis and induced late bone formation.This may provide new ideas and methods for bone immune repair in the field of 3D bioprinting. |
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