Study On Organic Polymer Hybrid Thixotropic Magnesium Phosphate-based Gel Bioink And Its Application In 3D Bioprinting

The 3D bioprinting technology uses cells and bioink as printing raw materials,and accurately constructs cell-like tissue/organ models in vitro.It is currently the cutting-edge research direction in the field of 3D printing.Therefore,it has great research and application value in biomedical fields such as tissue engineering,regenerative medicine,drug high-throughput screening,disease mechanism exploration and precision medicine.The biggest challenge facing 3D bioprinting is how to protect and improve the bioactivity of sensitive cells.Besides the optimal selection of printing techniques and cells,the physical and chemical properties of a bioink play a key role in protecting and maintaining cell activity and function.At present,researchers mainly focused on the application research of 3D bioprinting using natural hydrogel bioinks such as alginate and gelatin.These natural hydrogel bioinks often have problems such as insufficient mechanical properties and limited bioactivity.Therefore,it is necessary to develop a novel bioink that can protect,maintain and improve cell activity.However,due to the lack of suitable printability of bioink,the damage to cells after printing is greater,and the existing bioink often does not have bioactivity,which limits the development of 3D bioprinting technology.The specific challenges are that the existing bioinks difficulting to simultaneously meet the requirements of good printability?injectability and moldability?,mechanical support,biocompatibility and bioactivity.Thixotropic biomaterial is a kind of material which can reduce its viscosity under shearing force and recover its viscosity after removing shearing force,which can ensure that the material has good injectivity and moldability at the same time.Organic hydrogel materials have the advantages of good biocompatibility and high mechanical controllability,such as sodium alginate and gellan gum.Inorganic materials often have excellent bioactivity.The families of magnesium phosphate have been applied in bone tissue engineering due to its high biactivity.Therefore,this thesis aims to design and study a novel functional organic/inorganic composite bioink based on extrusion3D bioprinting,which can protect,maintain and improve the bioactivity of cells.In order to solve the problems of solid-liquid separation and blockage of printer nozzles when existing inorganic bioactive materials and organic gels are used as composite biological inks.Firstly,a bioactive nano inorganic thixotropic magnesium phosphate-based gel?TMP-BG?gel material was developed to improve the printability and bioactivity of bioink and the thixotropy and preparation mechanism of inorganic gel was revealed.Aiming at the problem of poor mechanical support of the existing hydrogel bioink,it is proposed to use rigid Gellan Gum?GG?and flexible Sodium alginate?SA?as organic framework hybrid magnesium phosphate-based gel to prepare ternary composite bioink to improve the mechanical support controllability of the bioink.Finally,the printability of the composite bioink and its ability to promote bone/cartilage repair were studied.The main contents and conclusions of this thesis are as follows:?1?TMP-BG was prepared by sol-gel method using H₃PO₄,Mg?OH?₂ and NaOH as raw materials.By adjusting the ratio of reactants,three TMP-BG preparation parameters that can form stable gel were obtained.The analysis results of X-ray diffraction?XRD?and Fourier Transform Infrared spectroscopy?FTIR?showed that the main component of TMP-BG was MgNa₃H?PO₄?₂,and some samples contain a small amount of Mg₃?PO₄?₂·8H₂O.The chemical reaction equation for preparing TMP-BG was determined.SEM,TEM and laser particle size analysis results showed that TMP-BG was composed of a large number of 5-10 nm nano MgNa₃H?PO₄?₂particles,forming a three-dimensional aggregate in space.Rheological results showed that TMP-BG not only has good shear thinning behavior,but also has good thixotropy and good rheological properties,ensuring that TMP-BG can have good injectivity and moldability at the same time.Based on the fact that the surface of MgNa₃H?PO₄?₂particles may have uneven positive and negative charge distribution at the same time,it was speculated that a reversible typical"house of cards"structure is formed between particles through electrostatic attraction,which is the mechanism of TMP-BG gelation and thixotropy.The extrusion 3D printing results showed that TMP-BG has good printability,its injection rate can reach 93%,and it can print large-size grids?45×45×5 mm?and complex bionic 3D structures such as joint head and ear contour models,with good formability.TMP-BG was co-cultured with MG-63 cells.The results showed that the activity of MG-63 cells was higher than 85%after co-culture for 7 days.MG-63 cells adhered and spread well on the surface of the material,indicating that TMP-BG had good biocompatibility.?2?The ratio of SA to GG was optimized based on the gelling property and mechanical properties of hydrogel.The results showed that SA and GG have complementary properties:SA improves the toughness of GG,GG makes up for the lack of mechanical strength of SA and reduces the volume shrinkage deformation of SA after gelling.Secondly,four kinds of ternary composite bioink?SA-GG/TMP-BG?were obtained by hybridization of SA and GG with TMP-BG by organic/inorganic hybridization method.Based on the rheological properties,mechanical strength,printability and cell compatibility of the composite ink,the ratio of the four composite bioinks was further optimized.Rheological results showed that the composite ink still has good shear thinning behavior.The compressive modulus of SA-GG/TMP-BG scaffolds after gelling ranges from 118 kPa to 299 kPa.SEM results of the scaffold showed that TMP-BG uniformly distributed inside the network pores of SA-GG hydrogel,reducing the pore size of hydrogel.FTIR results showed that there was no strong interaction between GG-SA and TMP-BG.Through biomimetic mineralization experiments in vitro,it was found that SA-GG/TMP-BG scaffold can promote the formation of osteoid apatite and finally transform into hydroxyapatite?HA?.Cell experiments showed that SA-GG/TMP-BG scaffold can promote the proliferation,spreading and adhesion of MG-63 cells,indicating that SA-GG/TMP-BG has certain bioactivity.The results of 3D printing experiments showed that SA-GG/TMP-BG composite bioink has good uniformity,injectability and formability,and can print more complex bionic structures.After uniformly mixing with MG-63 cells,the cells were subjected to 3D bioprinting and then cultured for 7 days.The results showed that the printed MG-63 cells were uniformly distributed in the 3D scaffold.After 5 days of normal cell proliferation,the highest cell proliferation rate was 150%.Compared with the non-printing group,the printed MG-63 cells had higher cell activity.?3?To study the effect of TMP-BG on the physicochemical properties and biological activity of SA-GG hydrogel.SA-GG/TMP-BG composite scaffolds with different contents of TMP-BG were prepared.SEM results showed that the porosity and pore size of the composite scaffolds gradually decreased with the increase of tmp-BG,accordingly,the compressive strength of the composite scaffolds gradually decreased too.When TMP-BG was 60%?v/v?,the compressive modulus of SA-GG/TMP-BG composite hydrogel scaffolds was 110 kPa.In addition,SA-GG/TMP-BG can up-regulate the expression of osteoblast-related genes?Col-I,RunX2,ALP,OPN and OCN?in co-cultured MG-63 cells.Animal experiments showed that the SA-GG/TMP-BG composite scaffold can repair cartilage with higher maturity after 12 w repair compared with SA-GG and control groups.Correspondingly,the rate of subchondral bone formation was about 35.27%,which was 1.5 and 2 times that of SA-GG and control groups,respectively.This indicates that SA-GG/TMP-BG composite scaffold has the ability to promote bone/cartilage repair.On a conclusion,this thesis utilized rigid gellan gum and flexible sodium alginate as organic framework hybrid thixotropic magnesium phosphate-based gel to prepare a novel functionalized SA-GG/TMP-BG composite bioink.The bioink has good injectability and moldability,and also has certain controllable mechanical support,and can be used for co-printing tissue/organs with cells in vitro.Cytological and animal experiments showed that SA-GG/TMP-BG composite scaffold has good biological activity and is expected to promote the repair and regeneration of bone/cartilage and other tissues,which provides research ideas for designing new functional bioink and provides a material basis for in vitro printing of functional three-dimensional bionic cell-loaded structures.