| Background: Rotator cuff injuries and anterior cruciate ligament injuries are common tendonbone binding site injuries in sports medicine.At present,the treatment of tendon insertion injury is mainly tendon reconstruction surgery.During the reconstruction process,screws need to be implanted into the bone tunnel to fix the graft,and the surgical effect depends on the degree of healing of the tendon-bone interface.The tendon-bone interface is a four-layer structure of tendon/ligament,uncalcified fibrocartilage,calcified fibrocartilage,and bone.Due to the non-renewable nature of tendon tissue,it is generally replaced by scar tissue repair after injury.At this time,whether the fibrocartilage layer and bone layer structure in the tendon-bone interface can be repaired may have greater clinical significance for repairing the damaged tendon-bone junction.The development of bone tissue engineering technology provides a more promising solution for the repair of tendon-bone interface injury.Among the implanted metal materials,titanium alloy is the most widely used orthopedic implant material due to its outstanding physical and chemical properties,such as high strength and high corrosion resistance[1-3].3D printed titanium alloy microporous scaffolds prepared by electron beam melting（EBM）technology have better mechanical properties.The microporous structure can increase the contact area between scaffolds and bone,thus improve the bone integration ability of implants.In addition,microporous structures can be loaded with hydrogels mixed with growth factors.Thermosensitive collagen is in a liquid state at 4°C and turns into a colloid state at 37°C,which makes thermosensitive collagen more convenient to carry growth factors.In the liquid state,growth factors can be loaded and injected into the porous scaffold,and at 37°C,it will turn into a colloidal state and degrade at a certain rate,and the growth factors can be released slowly and continuously,realizing the construction of a stable growth factor sustained release system.The titanium alloy scaffold graft site can be continuously stimulated by growth factors,and the tissue growth between the implant and bone interface is more sufficient,and the bone healing interface is more stable[4-7].Transforming growth factor-β3（TGF-β3）is a growth factor with multiple regulatory functions,which can not only regulate the regeneration process of cartilage,bone,tendon and ligament,but also regulate the secretion of extracellular matrix and reduce scar formation during tissue regeneration[8].In this study,we constructed a complex of thermosensitive collagen filled 3D printed titanium microporous scaffolds loaded with TGF-β3 to construct a "bioactive complex".Thermosensitive collagen carrying TGF-β3 filled 3D printed titanium microporous scaffolds confirmed that TGF-β3 can be released slowly and continuously in the complex structure.It can improve the biological activity of 3D printed titanium alloy microporous scaffolds,effectively promote tendon-bone healing in the long term,and provide a theoretical basis for implant design of patients with tendon-bone healing site injury.Methods: 1.The complex of thermosensitive collagen filled 3D printed titanium alloy microporous scaffolds loaded with TGF-β3 were constructed and its apparent observation.1.1 Preparation of scaffold complex: 3D microporous titanium alloy scaffold was printed by EBM method,thermosensitive collagen was prepared as a carrier loaded with TGF-β3,and filled into 3D printed titanium alloy microporous scaffold to construct scaffold complex loaded with TGF-β3.1.2 Apparent detection of scaffold complex: scanning electron microscope（SEM）was used for microscopic observation of scaffold complex.2.In vitro experiments2.1 Growth factor release ability test: Enzyme-linked immuno sorbent assay（ELISA）was used to determine the sustained release rate of TGF-β3 in thermosensitive collagen.2.2 Cell experiment: The experiment was divided into 3 groups,A,untreated 3Dprinted titanium alloy microporous scaffold（e Ti）;B,thermosensitive collagen filled 3D printed titanium alloy microporous support（c Ti）;C,the 3D printed titanium alloy microporous scaffold was filled with thermosensitive collagen loaded TGF-β3（c Ti/TGF-β3）.1)Biocompatibility experiment: biocompatibility test of bone marrow mesenchymal stem cells（BMSCs）activity,adhesion,proliferation efficiency and cytoskeleton in complex co-culture environment;The cell proliferation ability,cell morphology and cytoskeleton of BMSCs under the influence of complex were observed by cell counting kit-8（CCK-8）,live/dead cell staining,phalloidin and DAPI staining.2)Differentiation potential detection: osteogenesis,alizarin red staining;cartilage formation,oil red O staining;The polymerase chain reaction（PCR）was used to detect the expression of related genes.3.In vivo experimentsIn vivo experimental animal model verification: a tendon-bone interface reconstruction model of rabbit patellar ligament connecting tibial insertion was established,and 48 rabbits in e Ti,c Ti and c Ti/TGF-β3 groups were euthanized 6 and 12 weeks after surgery,and specimens were collected for the relevant examination. HE staining of hard tissue sections: analysis of the healing of tendon-bone junctions at scaffolds,tendons and bone junctions.Results: 1.The complex of thermosensitive collagen filled 3D printed titanium alloy microporous scaffolds loaded with TGF-β3 and its apparent observation were constructed.1.1 Preparation of 3D printed titanium alloy microporous scaffolds with hexagonal honeycomb shape,500 μm aperture size and 70% porosity.Disc-shaped microporous scaffolds with a diameter of 10 mm and a height of 3 mm were used for cell experiments in vitro,and columnar microporous scaffolds with a diameter of 5 mm and a height of 10 mm were used for animal experiments in vivo.thermosensitive collagen was liquid at 4℃,loaded with TGF-β3 and filled with 3D-printed titanium alloy microporous scaffold.Then it was heated to 37℃ and formed a gel state,forming a complex with the scaffold.1.2 Observation by electron microscopy showed that the hydrogel-wrapped scaffold has a uniform collagen fiber structure and well filled the pores of the scaffold,providing an excellent microenvironment for cell adhesion,proliferation and differentiation.2.In vitro experiments2.1 Growth factor release ability test: ELISA test results showed that the sustained release rate of TGF-β3 in the complex reached about 30% on the first day,about 60% on the 7th day,and about 90% on the 14 th day.2.2 Cell experiment: CCK-8 cell proliferation ability test,live/dead cell staining,phalloidin and DAPI staining showed that c Ti/TGF-β3 group had better biocompatibility than e Ti and c Ti group,and the cells could prolifically and adhered to the complex.In addition,c Ti/TGF-β3 was more prominent in osteodifferentiation and chondrogenic differentiation.3.In vivo experimentsThe staining results of hard tissue sections showed that the cells at the tendonbone interface junction in the c Ti/TGF-β3 group were arranged in an orderly manner,with a uniform shape,with bone tissue encapsulating the scaffold,and a large number of muscle fibers were visible.In the e Ti and c Ti groups,the cells were arranged in a disorderly manner,and the morphology was irregular,and the tendon-bone interface was disordered fibrous tissue.Conclusion:In this study,we prepared 3D printed titanium alloy microporous scaffolds with better mechanical properties,and successfully filled thermosensitive collagen loaded TGF-β3 into the microporous scaffolds to form stable scaffold complexes.In vitro experiments,the scaffold complex can slowly and continuously release TGF-β3,and has excellent biocompatibility,which can promote the proliferation,adhesion and differentiation of BMSCs.In vivo experiments,it can promote the orderly proliferation and differentiation of new cells at the tendon-bone interface,and effectively promote the tendon-bone healing. |
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