| With the popularity of sports and fitness,more and more people begin to take physical exercises,but due to the aging population and incorrect exercise postures,the incidence of tendon injuries is also increasing year by year.Common tendon injuries include tendinopathy,tendon tear/rupture and so on.The treatment of tendon injury mainly includes non-surgical treatment and surgical treatment.Non-surgical treatment is generally suitable for mild injuries and has limited effect.Surgical treatment includes stump debridement and suture,autologous or allogeneic tendon transplantation,etc.,and has achieved a wide range of curative effects.Due to the special biological properties and healing mechanism of tendons,the result of tendon healing after injury is usually scar tissue formation.Different from the original matrix structure of the tendon,the collagen fibers in the scar tissue matrix are arranged in disorder,with poor biological function and prone to secondary injury.Tissue engineering is an emerging method of tissue repair.Its purpose is to promote the effective repair of the original tissue structure through biomaterials.The research content includes three aspects: seed cells,scaffold material preparation and scaffold-cell complex construction.In tendon repair,tendon stem/progenitor cells(TSPCs)derived from endogenous repair cells play a key role in tendon repair and are commonly used seed cells in tendon tissue engineering.The preparation of scaffold materials is a factor that needs to be considered in tissue engineering.A variety of biological materials are used for tendon repair,which can be divided into natural materials and artificial materials.Natural materials have low biotoxicity but low mechanical strength and poor processability;artificial materials are easy to prepare and have good mechanical properties,but their biocompatibility is poor and have potential toxicity in the body.In recent years,studies have found that the silk fibroin film bio-scaffold has good biocompatibility,plasticity and controllable mechanical strength,and has been applied to the repair of soft tissues such as cornea and nerves with good results.Silk fibroin film is an excellent tissue engineering material in soft tissue repair.It has potential application value in tendon repair,but it has not been studied in depth.To use silk fibroin film for tendon repair,it is necessary to make the silk fibroin film have a microenvironment similar to that of the native tendon in order to effectively improve its biological function and ultimately promote tendon repair.Early studies mostly used bio-scaffolds to cross-link macromolecular active groups(such as growth factors)as the main method,but it has the disadvantages of easy inactivation and instability.In recent years,tissue engineering research has pointed out that when biological scaffolds have similar physical properties(mechanics,hardness,etc.)and microstructures to tissues,they can effectively promote tissue repair.The preparation of the biomimetic physical microstructure on the surface of the silk fibroin film scaffold is an effective method to improve its biological performance and has the characteristics of high stability and precision.Taking the physical properties and microstructure of native tendons as references,biomimetic preparation of microstructure silk fibroin films with biological functions has important application value for tendon repair.This study first analyzed the microstructure and mechanical characteristics of the tendon as a reference for preparing the silk fibroin film bio-scaffold;then biomimetic preparation of the microstructure silk fibroin film and observed its in vivo repair effect on the injured tendon;finally in the in vitro experiment Explore the biological regulation of microstructure silk fibroin film on TSPCs and related molecular pathways,comprehensively evaluate the role of microstructure silk fibroin film in tendon repair and the internal molecular mechanism,and provide new strategies for tendon repair.1.Analysis of the microstructure and mechanical properties of tendons1.1 Method1.1.1 Observe the microstructure of tendon tissue by scanning electron microscope and hematoxylin-eosin staining,and analyze and compare the width of collagen fibers.1.1.2 Test the biomechanical characteristics of the tendon.1.2 Results1.2.1 The appearance of the tendon is bright white,the texture is tight to the touch,and the toughness is strong.It is curled up at rest.It will stretch and elongate after being stressed.The appearance is more slender than at rest,and the stretch is removed.The original shape can be restored after the force.1.2.2 Observe the tendon by SEM.At low magnification(50X),it can be seen that the structure of the surface of the tendon is dense,and the collagen fiber bundles appear fold-like on the surface of the tendon;magnification 100-500 times shows the collagen fiber bundles arranged in parallel with the collagen fibers;magnification 2000X-10000 X can be seen that the collagen fibers are composed of nano-scale filaments.1.2.3 HE staining shows that the collagen fibers are all long cords,and the collagen fibers of different thickness are arranged in parallel in the tendon;the inherent cells of the tendon have similar morphology and arrangement characteristics.1.2.4 The width of collagen fibers is mainly distributed in 5-10?m,and the width of some collagen fibers is less than 5?m or greater than 15?m.1.2.5 As the tensile stress continues to increase,the fiber filaments,collagen fibers and fiber bundles in the tendon are stretched to the ultimate load;as the tendon tissue is further passively stretched,the tendon tissue will separate and break,and the stress value is returned to the initial value.1.3 Conclusion1.3.1 The tendon is a collagen fiber bundle formed by the arrangement of nano-scale filaments,with long and narrow intrinsic cells interspersed in it.The diameter of the fiber bundle is mainly distributed between 5-20?m and the 5-10?m interval accounts for the largest proportion.1.3.2 The mechanical properties of tendon samples are positively correlated with their deformation.1.3.3 The morphology and mechanical data of tendon fibers are used as the reference basis for the preparation of microstructure silk fibroin film materials.2.Preparation,modification and characterization of biomimetic silk fibroin film scaffold2.1 Method2.1.1 Extract the regenerated silk fibroin solution,refer to the results in section 1.3.3 to biomimize the preparation of silk fibroin films with different microstructures,and modify the material by water annealing treatment.2.1.2 Fourier infrared spectrometer(FTIR)detects the protein conformation changes in the silk fibroin film material.2.1.3 Atomic Force Microscope(AFM)detects the physical properties of the silk fibroin solution and silk fibroin film.2.1.4 Scanning electron microscope(SEM)to observe the surface microstructure morphology of silk fibroin film.2.1.5 The tensile test detects the mechanical properties of silk fibroin films with different microstructures after modification.2.2 Results2.2.1 The mass fraction of the silk fibroin solution is 4.53±3.4%.AFM shows that the silk fibroin filaments in the silk fibroin solution are parallel and densely arranged.2.2.2 The silk fibroin films with different surface structures were prepared,and the structure was intact without shrinkage and bubbles.The physical properties of the film materials changed significantly after modification.2.2.3 The conformation of ?-sheet protein inside the silk fibroin film increased significantly after water annealing.2.2.4 AFM found that after the silk fibroin film scaffold was modified by water annealing,nano-scale rough structures appeared on the surface.2.2.5 SEM observed that the surface microstructure of the silk fibroin film is clear and conforms to the biomimetic design size.2.2.6 The mechanical properties of non-microstructure and 5?m microstructure silk fibroin films are lower.The maximum tensile strength of 10,15 and 20?m microstructure silk fibroin films is similar to that of native tendons,but it will reach earlier in the stretching process.Maximum load.2.3 Summary2.3.1 The properties of the regenerated silk fibroin solution are stable,and the silk fibroin film prepared from it as a raw material has a complete shape,and the physical properties of the scaffold are significantly improved after the water annealing modification.2.3.2 After modification,the ?-sheet protein conformation inside the silk fibroin film is significantly increased,and nano-scale rough structures appear on the surface of the material.2.3.3 Silk fibroin films with microstructures of 10,15,and 20 ?m have good mechanical properties.3.The repair effect of microstructure silk fibroin film on injured tendons in vivo3.1 Experimental method3.1.1 Establish rat Achilles tendon injury model: normal group(N),defect group(D),allogeneic tendon replacement group(R),non-microstructure silk fibroin film group(S),microstructure silk fibroin film group(G).3.1.2 MRI,hematoxylin-eosin and immunohistochemical staining were used to analyze the repairing effect of microstructure silk fibroin film on injured tendons in vivo.3.2 Experimental results3.2.1 The width and thickness of the Achilles tendon tissue in group D were significantly higher than those in other groups,the width and thickness of samples in group S and R were lower than those in group D,and the width and thickness of samples in group G were the lowest.3.2.2 MRI fat suppression image shows: in the 4th week after operation,the R group,D group and S group are all mainly inflammatory hyperintensity,R group can see allogeneic tendon low signal,G group has a small amount of discontinuous low signal;8 After weeks,the D group still had inflammatory hyperintensity,and the R group showed inflammatory hyperintensity at both ends of the allogeneic tendon.Continuous low signal was seen in both S and G groups,but the signal continuity of S group was lower than that of G group.3.2.3 Histological staining showed that the fiber morphology of group D was disordered and the expression of regeneration-related markers was low;the fiber structure of grafted tendon in group R was not continuous with the original fibers,and the expression of tendon differentiation markers was low;group S collagen fibers Compared with the D group,the morphology is relatively orderly,the fibers at both ends of the repair area are slightly continuous with the original fibers,and the expression of tenascin C(TNC)and tenasmodulin(TNMD)is slightly increased,but there is no significant difference from the R group;The arrangement of collagen fibers in group G was the most orderly,and the tendon repair markers TNC,TNMD and type I collagen(COLIA1)were significantly higher than those in other groups.3.2.4 Histological comprehensive score: G group was significantly higher than other groups,S group and R group had no significant difference,and D group had the lowest score.3.3 Summary3.3.1 The microstructure silk fibroin film can reduce scar hyperplasia during tendon repair,promote the orderly formation of collagen fibers and the expression of tendon markers.4.The regulation and molecular mechanism of microstructure silk fibroin film on the biological behavior of TSPCs4.1 Regulation of microstructure silk fibroin film on the biological behavior of TSPCs4.1.1 Experimental method4.1.1.1 Extract the primary TSPCs,and identify their stem/progenitor cell characteristics by immunofluorescence and induced differentiation staining.4.1.1.2 After co-cultivation of TSPCs and scaffold materials,live and dead cells were co-stained with fluorescence to observe the cytocompatibility of silk fibroin films,and CCK-8monitored cell proliferation activity.4.1.1.3 Double fluorescence staining of cytoskeleton and nucleus to observe the cell morphology and spatial arrangement of TSPCs.4.1.1.4 Polymerase chain reaction(PCR)analysis of TSPCs in different material groups in the expression of tendon differentiation markers.4.1.2 Results4.1.2.1 The surface markers of TSPCs were negative for CD3 and CD34,and positive for CD44 and CD90;after differentiation induction and culture,TSPCs differentiated into adipose system,bone system and chondrogenic system,respectively.4.1.2.2 The viability of TSPCs on the surface of the microstructured silk fibroin film was good,and there was no significant difference in the proliferation ability of TSPCs between different groups.4.1.2.3 The surface TSPCs of 5?m and 10?m microstructure silk fibroin films showed a narrow morphology and spatial arrangement similar to those in native tendons,and the expression of tendon marker genes was also significantly increased,especially when the microstructure was 10?m.4.1.3 Summary4.1.3.1 Silk fibroin film has good biocompatibility,and the addition of microstructure will not affect the cell viability of TSPCs.4.1.3.2 The 10?m microstructure silk fibroin film can significantly change the cell morphology and spatial arrangement of TSPCs,and promote the differentiation of TSPCs toward tendon lineage through phosphorylation of focal adhesion kinase(FAK).4.2 The inner molecular mechanism of microstructure silk fibroin film regulating TSPCs4.2.1 Experimental method4.2.1.1 m RNA transcriptomics and proteomics high-throughput sequencing to analyze the expression of differential genes and proteins in TSPCs.4.2.1.2 WB comparative analysis of protein expression of key pathway molecules.4.2.2 Experimental results4.2.2.1 Cluster analysis suggests that the differentially expressed genes and proteins are synergistic in function and location.4.2.2.2 GO analysis suggests that the functional differences of differential genes and protein bodies mainly include:(1)Cell physiological function(Biological Process,BP):migration,adhesion and surface protein localization;(2)Gene molecular function(MF):binding muscle Actin filaments and actin;(3)Cellular Component(CC): Actin cytoskeleton,actin filaments,stress fiber filaments.4.2.2.3 KEEG enrichment analysis showed that the difference changes were concentrated in the FAK-Actin signaling pathway and PI3K-AKT signaling pathway.4.2.2.4 10?m silk fibroin film scaffold with microstructure can promote the expression of TSPCs tendon markers SCX,TNC,TNMD and COLIA1.The phosphorylation activation of focal adhesion kinase(FAK)plays a key role;the expression of integrin molecule ?2?1protein in TSPCs The amount was significantly increased;phosphorylated PI3 K and AKT(308/473)and tendon markers TNC and TNMD were significantly increased;after the addition of PI3K-AKT pathway inhibitors,the expression of TNC and TNMD would be inhibited at the same time.4.2.3 Summary4.2.3.1 Integrin ?2?1 serves as the sub-membrane microenvironment sensor of TSPCs,which is responsible for sensing the physical signal in the microstructure silk fibroin film scaffold and transmitting it to the cell.4.2.3.2 The microstructure silk fibroin film synergistically regulates the cytoskeletal protein remodeling of TSPCs through the FAK-Actin and FAK-PI3K-AKT molecular pathways.4.2.3.3 The microstructure silk fibroin film induces the differentiation of TSPCs into tendons through the FAK-PI3K-AKT molecular signaling pathway. |
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