| Osteochondral defects threaten people’s health due to population aging,trauma and inflammation.It is limitations a current research hotspot to construct osteochondral tissue engineering scaffolds to repair osteochondral because of drawbacks of traditional treatment methods.However,osteochondral has a complex gradient structure and chemical composition.In addition,osteochondral bone and cartilage have very different structures and properties.Hence,achieving ideal repair by tissue engineering scaffolds,it is necessary to consider the difference between cartilage defects and subchondral bone defects to design scaffolds.Hydrogels have been extensively explored as a promising tissue engineering materials as they are characterized for being a three-dimensional porous network that can perfectly simulate the extracellular matrix(ECM)of human tissues.Among all the hydrogels,poly(vinyl alcohol)(PVA),as a material approved by the US Food and Drug Administration(FDA),has been widely applied in many fields.However,the poor mechanical properties limit their application for osteochondral defects therapying.Furthermore,when the osteochondral damage affects the subchondral bone,it not only requires the repair material to have osteogenic properties,but also requires bionic gradient components to promote tissue regeneration.Thus,it is necessary to construct PVA-based scaffolds with osteogenic properties and gradient components.Herein,firstly,this thesis constructs a PVA-based hydrogel with high strength,high toughness and high elasticity for cartilage repair.Subsequently,a gradient scaffold containing nano-hydroxyapatite(HAp)was prepared by buoyancy density difference to improve the osteogenic properties of PVA and enhance osteochondral repair.Finally,their physical and chemical properties and biological properties were investigated to provide an experimental guidance for design of tissue engineering osteochondral scaffolds.The main research contents and results are as follows.(1)In this thesis,firstly,poly(vinyl alcohol)/polyacrylamide(PVA/PAM)double network(DN)hydrogel was constructed,and then the mechanical properties of PVA/PAM DN hydrogels were further strengthened by Hofmeister effect to obtain osteochondral scaffold(PVA/PAM-2Cit)with high strength,high toughness and high elasticity.Scanning electron microscopy(SEM)results showed that Hofmeister effect can regulate the pore size of PVA/PAM.The results of ATR-FTIR and XRD showed that Hofmeister effect enhanced the intermolecular hydrogen bonding of PVA/PAM and increased the crystallinity of PVA/PAM(11.8%-66.7%).The results of tensile and compression tests showed that the Hofmeister effect could significantly enhance the mechanical properties of PVA/PAM,which increased the tensile strength from 754.6±93.4 k Pa to 9.4±0.2 MPa,the tensile modulus from 132.0±3.7k Pa to 4.8±0.2 MPa and the toughness from 2781.1±59.3 k J/m~3 to 27.7±0.9 MJ/m~3.The compressive strength increased from 3.0±0.1 MPa to 50.1±2.9 MPa,and the compressive modulus increased from 664.7±13.3 k Pa to 4.3±0.1 MPa.The biocompatibility of the scaffolds in vitro was evaluated by chondrocytes.The results of cell proliferation assay(CCK-8)showed that PVA/PAM-2Cit exhibited the best cell proliferation capability.The results of laser confocal microscopy showed that the cells on PVA/PAM-2Cit had abundant actin fibers,indicating that it had the best ability to promote cell spreading.The above results show that it is successful to prepare high strength,high toughness and high elasticity hydrogels by Hofmeister effect,which have excellent biocompatibility and can be as potential cartilage repair scaffolds.(2)Although PVA-based hydrogels with high strength,high toughness and high elasticity were successfully prepared for osteochondral repair,it is difficult to repair subchondral bone due to the lack of osteogenic properties and biomimetic gradient mineralization components of PVA.Therefore,in this thesis,the buoyancy density difference method was used to regulate the viscosity of PVA solution by bacterial cellulose(BC)to fabricate scaffolds with gradient distribution of HAp(GPVA/BC-HAp).SEM results showed that the addition of HAp reduced the pore size of PVA/BC hydrogels.FTIR and XRD results showed that the addition of HAp did not affect the chemical structure and crystal structure of PVA/BC.The porosity test results showed that the addition of HAp reduced the porosity of hydrogels.The tensile test results showed that the addition of HAp significantly strengthened the mechanical properties of PVA/BC hydrogels,and GPVA/BC-HAp scaffolds had the strongest tensile strength(10.1±1.0 MPa).The results of SEM images and nucleus-actin staining showed that MC3T3-E1 cells had better spreading state on GPVA/BC-HAp scaffolds than that of PVA/BC and HPVA/BC-HAp scaffolds,indicating that the gradient structure could promote the spreading capability of MC3T3-E1 cells.The osteogenic properties of PVA/BC,HPVA/BC-HAp and GPVA/BC-HAp were studied by alkaline phosphatase(ALP)activity and alizarin red staining(ARS).Subcutaneous implantation experiments in mice showed that GPVA/BC-HAp had lower inflammation.The above results showed that GPVA/BC-HAp scaffold was successfully constructed by density difference method,and GPVA/BC-HAp scaffold exhibited better mechanical properties,biocompatibility and osteogenic properties,which was expected to be applied as a tissue engineering scaffold to promote osteochondral regeneration.In summary,this thesis fabricated different scaffolds for osteochondral defect.The osteochondral repair scaffolds were prepared with high-strength,high toughness and high-elasticity by Hofmeister effect and dual network structure.The bionic GPVA/BC-HAp osteochondral scaffold was successfully constructed by buoyancy density difference method,which is expected to promote the application of PVA-based composite materials in osteochondral tissue engineering and is expected to provide experimental guidance for the preparation of other gradient osteochondral tissue engineering scaffolds. |
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