| Articular cartilage has to withstand complex joint loads and repeated frictional movements in the physiological environment of the human body,so it will be worn and degraded constantly.Moreover,once the articular cartilage tissue is defective,it is difficult to achieve regeneration and self-repair.Most of the traditional methods for treating articular cartilage defects have shortcomings and limitations.Cartilage tissue engineering is the most promising method for repairing cartilage defects at present.An ideal cartilage scaffold should have good biocompatibility,mechanical stability and a large number of pore structure characteristics to support cell proliferation and nutrient diffusion.3D printing technology has been widely used to prepare cartilage tissue engineering scaffolds because of its advantages of personalized and precise regulation.However,the traditional articular cartilage scaffolds printed by 3D printing are mostly monolayer or homogeneous,which cannot accurately mimic the highly layered and anisotropic structural characteristics of natural articular cartilage.Therefore,the purpose of this study is to construct a three-layer gradient oriented heterogeneous hydrogel cartilage scaffold based on biological 3D printing to simulate the morphological structure of natural articular cartilage,to realize gradient bionic of components and structure.Firstly,a series of gelatin(Gt)/ sodium alginate(Alg)hydrogel inks were prepared based on the biomimetic concept of "more collagen/less proteoglycan" in the superficial tissues of natural articular cartilage,and the best component 7Gt-3Alg was optimized as the printing ink for the superficial layer cartilage scaffold.Combined with 3D printing and template freezing orientation technology,the superficial horizontally oriented cartilage scaffold was successfully constructed,and its properties were compared with that of non-oriented cartilage scaffold.Scanning electron microscopy results show that the 7Gt-3Alg horizontally oriented scaffold has linear multi-scale microporous structure in the horizontal direction.Physicochemical property tests show that the horizontal orientation structure increases the porosity of superficial cartilage scaffold(88.31%),and decreases the equilibrium-swelling rate and in vitro degradation rate(41.6%).Mechanical property tests show that the horizontally oriented structure significantly improves the tensile resistance and viscoelasticity of the superficial cartilage scaffold.Secondly,considering that the interlayer tissue of natural articular cartilage needs to have high compressive ability,a series of nano-composite hydrogel inks Gt-AlgMMT were prepared by adding nano montmorillonite(MMT)into Gt and Alg solutions,and the best component 2Gt-5Alg-5MMT was optimized as the printing ink for the interlayer cartilage scaffold.Combined with 3D printing and template freezing orientation technology,the interlayer cross-oriented cartilage scaffold was successfully constructed,and its properties were compared with that of non-oriented cartilage scaffold.Scanning electron microscopy results show that the 2Gt-5Alg-5MMT crossoriented scaffold has interlaced multi-scale microporous structure.Physicochemical property tests show that the cross-oriented structure increases the porosity of interlayer cartilage scaffold(96.26%),and decreases the equilibrium-swelling rate and in vitro degradation rate(23.8%).Mechanical property tests show that the cross-oriented structure improves the compression resistance and viscoelasticity of the interlayer cartilage scaffold.Then,in order to simulate the good compression resistance and bone conductivity of the deep tissues of natural articular cartilage,a series of nano-composite hydrogel inks Gt-Alg-HA were prepared by adding nano-hydroxyapatite(n HA)to Gt and Alg,and the best component 5Gt-7Alg-3HA was optimized as the printing ink for the deep layer cartilage scaffold.Combined with 3D printing and template freezing orientation technology,the deep vertically oriented cartilage scaffold was successfully constructed,and its properties were compared with that of non-oriented cartilage scaffold.Scanning electron microscopy results show that the 5Gt-7Alg-3HA vertically oriented scaffold has multi-scale microporous structure in the vertical direction.Physicochemical property tests show that the vertically oriented structure increases the porosity of deep layer cartilage scaffold(94.21%),and decreases the equilibrium-swelling rate and in vitro degradation rate(18.2%).Mechanical property tests show that the vertically oriented structure greatly improved the compression resistance and viscoelasticity of deep layer cartilage scaffold.Subsequently,7Gt-3Alg,2Gt-5Alg-5MMT and 5Gt-7Alg-3HA inks were selected to simulate the superficial layer,middle layer and deep layer of cartilage respectively,and 3D printing and template freezing orientation technology were used to construct a multi-layer gradient oriented biomimetic cartilage scaffold.Several groups of multilayer oriented cartilage scaffolds with different gradient pore shape / pore size structures were designed and prepared,and the best combination of gradient pore shape/pore size structures were optimized.Furthermore,the performance of four kinds of multi-layer cartilage scaffolds(uniform & non-oriented,uniform & oriented,gradient pore shape& oriented,gradient pore size & oriented)was compared.Physicochemical property tests show that the gradient distribution of pore size / pore shape structure appropriately reduces the porosity,swelling rate and degradation rate of the multi-layer cartilage scaffold,while the oriented heterogeneous structure of each layer increases the porosity of the scaffold.Mechanical property tests show that the gradient distribution of the pore size structure and the oriented heterogeneous structure of each layer can effectively improve the compression resistance,creep resistance and self-recovery of the multilayer cartilage scaffold,while the gradient distribution of the pore shape structure has no significant effect on it.Finally,the biological properties of 3D printed single-layer oriented heterogeneous cartilage scaffolds and multi-layer gradient oriented biomimetic cartilage scaffolds were evaluated in vitro.The Live/Dead staining and CCK-8 test were used to characterize the biocompatibility of single-layer(superficial layer,middle layer and deep layer)oriented heterogeneous cartilage scaffolds and the non-oriented group scaffolds.The results showed that the ordered secondary porous structure in singlelayer oriented scaffold is more conducive to promoting the adhesion and proliferation of ADTC5 cells.Through tissue section staining,determination of glycosaminoglycan(s GAG)content and expression of cartilage specific genes,the ability of four kinds of multi-layer biomimetic cartilage scaffolds(uniform & non-oriented,uniform &oriented,gradient pore shape & oriented,gradient pore size & oriented)to induce cartilage tissue differentiation in vitro was explored.The results showed that the directional heterogeneous structure of each layer has great influence on the proliferation and differentiation ability of multi-layer biomimetic cartilage scaffolds,but the gradient pore shape/pore size structure has no significant effect on it. |
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