| Research background and purpose:Due to the absence of blood vessels,cartilage has extremely limited self-repair ability.So far,the repair of laryngeal cartilage defects caused by laryngeal tumors,trauma,congenital structural abnormalities and other factors is still a difficult problem in otorhinolaryngology head and neck surgery.In recent years,tissue engineering technology has been gradually developed,which brings some hope to the repair and reconstruction of defective laryngeal cartilages because it does have not the disadvantages of traditional repair methods,such as secondary injury,immune rejection,and limited source.However,the successful implementation of tissue engineering technology must meet three basic elements: scaffolds,seed cells and growth factors.According to the sources,scaffold materials are divided into natural materials,synthetic materials and composite materials.Natural materials have good biocompatibility and low immunogenicity.But their mechanical properties are poor,which need to be improved by modification or combination with other materials.In previous studies,most researchers tend to combine synthetic materials to improve their mechanical properties.However,due to their low biological activity and weak cell adhesion,synthetic materials need to make up for their shortcomings by some ways.Therefore,this study aims to make full use of the advantages of natural materials in cartilage tissue engineering,improve the mechanical properties through non-toxic cross-linking,and develop an ideal scaffold material combined with 3D bioprinting technology.Materials and Methods:In this study,sodium alginate,gelatin,tannic acid and calcium chloride were used as raw materials to form bioloinks through cross-linking,which was applied to 3D printing technology to print up the cartilage scaffolds.Bone marrow mesenchymal stem cells with multi-directional differentiation potential were selected as seed cells.Appropriate growth factors were added to promote their differentiation into cartilage during in vitro culture.The scaffold with well-grown cells was implanted into the defect of rabbit thyroid cartilage plate at the appropriate time point.HE staining,toluidine blue staining,Masson staining and collagen type Ⅱ staining were used to observe the repair of the defect at 4 weeks,8 weeks and 12 weeks,respectively.Results:Through macroscopic observation of the appearance of the scaffolds and scanning electron microscopy tests,the lines of the scaffolds with the lowest concentration of gelatin and sodium alginate collapsed,and the holes partially fused or even disappeared after printing due to their low viscosity,which was not conducive to providing enough space for the growth of cells.However,the formability of the scaffolds was improved after cross-linking with tannin acid.In the test results of mechanical properties,the compression modulus of the fresh cartilage was 1.5MPa,which was closest to the compression modulus of 1.386 MPa measured by the scaffold of group B1(10%Gel/5%SA/0.2%TA),which indicating that the mechanical properties of the scaffold of group B1 were most matching with the fresh cartilage.Moreover,The compression modulus of the scaffolds containing tannic acid was higher than that of the scaffolds without tannic acid,and there was significant difference between the groups.FTIR results confirmed the cross-link between sodium alginate,gelatin and tannic acid,which also explained the lower swelling rate and slower degradation rate of the scaffold containing tannin acid.The results of immunochemical fluorescence analysis of rabbits’ BMSCs showed that CD29 and CD44 expression were positive,and the positive rates were more than 90%.The results of cytotoxicity test showed that the absorbance value at 450 nm was not significantly different between the scaffold extract and the control groups,and the scaffold extract had no obvious toxicity to the growth of BMSCs.There was no obvious red light in the Calcein AM /PI fluorescence staining results,indicating that the composite hydrogels had good biocompatibility and no obvious inhibition of cell growth.The results of HE staining,toluidine blue staining,Masson staining and formation of cartilage tissue in the rabbit thyroid cartilage plate defect.Conclusions:1.By crosslinking with sodium alginate and gelatin,tannic acid reduces the swelling rate of composite scaffolds to a certain extent,but improves the mechanical properties of natural scaffolds.2.The composite scaffold composed of gelatin,sodium alginate,tannic acid and calcium chloride has excellent biocompatibility and no cytotoxicity.3.In vitro and in vivo experiments confirmed that this study successfully prepared the natural material scaffolds in cartilage tissue engineering,and further can provide a new idea and basis for clinical application of laryngeal cartilage defect reconstruction. |
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