Vasoactive Functional Hydrogel Promotes Angiogenesis And Osseointegration Of 3D Printed Titanium Alloy Implant Interface

Background:Bone has a strong regeneration capacity.In fact,small bone defects are often healed by self-repair.Severe trauma,bone tumor resection,and bone infection,however,lead to a large area of bone defect,which exceeds the ability of self-healing.In these cases,we rely on autologous bone repair after artificial intervention.Moreover,owing to complications like limited amount of autogenous bone,damage of bone graft site,and bone necrosis after transplantation,scientists are continuously searching for effective bone substitutes.In recent decades,the tissue engineering platform used materials science,bioengineering,and stem cell technology to produce bone substitutes.With rising demand in bone repair,bone tissue engineering is an ideal method of promoting bone regeneration and it is gaining ground in clinical practice as well.Blood vessels play an essential role in promoting regeneration and repair of segmental bone defects,and lack of blood vessels can result in severe necrosis of the fracture site.Insufficient blood supply after bone injury is considered to be the main cause of poor fracture healing,affecting rehabilitation in about 10% of fracture patients.Being an important growth factor in angiogenesis,vascular endothelial growth factor(VEGF)is widely used to induce vascular remodeling.VEGF increases vascular permeability and promotes angiogenesis.In addition,it is proved to have a strong chemotaxis effect on the migration of endothelial cells.Relevant studies revealed that VEGF is essential for both bone formation and regeneration.Titanium alloys(Ti6Al4V)have high biocompatibility and mechanical strength,making them attractive bone substitutes in both dental and orthopaedic implants.In addition,the corrosion resistance,chemical inertia,and low Young's modulus of these alloys are additional reasons for their wide application.However,the biological inertia of titanium alloys may inhibit its direct binding to bone tissue after implantation.Nevertheless,titanium alloy,printed by 3D printing technology,can realize private customization and provide ordered porous structure,which can avoid uneven stress caused by uneven pores and reduce risk of scaffolds damage.Pores with sufficient diameter can provide adequate space for cell colonization and metabolite transport.Furthermore,to reduce inertia of the titanium alloy,increase bioactivity,and enhance regeneration of blood vessel and bone within the scaffold,it is necessary to introduce VEGF to the scaffolds.At present,tissue engineering uses hydrogel as a carrier for cells,drugs,and growth factors.Among them,thermosensitive collagen hydrogel is of particular interest,due to its unique ability to alter from a liquid to a gelatinous state,based on temperature.Generally,low temperature liquid hydrogel is used to encapsulate cytokines,whereas thermo coagulation colloid is used for sustained drug release.Compared to the systemic drug usage,local drug administration can reduce the amount of drug used,avoid systemic side effects,and improve regional treatment efficacy.In this study,we combined VEGF,thermosensitive collagen hydrogel,and 3D printing titanium alloy scaffolds to construct biologically active surfaces for localized angiogenesis and osseointegration.We hypothesized that the composite scaffold will offer a sustained release of VEGF post implantation,and the released VEGF will recruit rabbit endothelial cells,and enhance bone and vascular regeneration of rabbit distal femoral bone defects.Method:1.3D printed porous titanium alloy scaffolds were prepared by EBM.The mixture of liquid temperature sensitive collagen and VEGF was injected into the pores of the scaffolds at low temperature,and the composite scaffolds were obtained by standing at37 ?.2.scanning electron microscopy(SEM)was used to evaluate the pore structure of3 D printing scaffolds and the surface morphology of thermosensitive collagen hydrogel.3.Enzyme linked immunosorbent assay(ELISA)was used to evaluate the release rate of VEGF in the composite system.4.In vitro,the effect of composite scaffold on the activity of human umbilical vein endothelial cells(HUVECs)was detected by live/dead staining and CCK-8 assay.Tubule formation experiment and RT-q PCR experiment were used to verify the effect of composite scaffold on the differentiation of HUVECs.5.The bone defect was prepared on the lateral epicondyle of femur in rabbits,and the scaffolds in each group were transplanted into the defect.The experiment was divided into 3 groups: 3D printing titanium alloy scaffold(e Ti),3D printing titanium alloy scaffold filled with thermosensitive collagen hydrogel(c Ti),3D printed titanium alloy scaffold(c Ti/VEGF)loaded with thermosensitive collagen and VEGF complex.The three groups of scaffolds were implanted into rabbit femoral lateral epicondyle respectively.The scaffolds were removed at 6 and 12 weeks,and micro-CT Histological evaluation,immunohistochemistry and mechanical test were used to study the effect of composite scaffold on interfacial angiogenesis and bone integration in bone defect model.Result:1.The disk shape was prepared by 3D printing technology(? 10 mm × L3 mm)and cylindrical support(? 4 mm × L8 mm)two kinds of titanium alloy supports,the parameters are: porosity = 70%,pore diameter = 500 ?m? The results of SEM showed that the titanium alloy scaffolds had rough surface morphology,and the pores of the composite scaffolds were filled with temperature sensitive collagen with porous structure.2.ELISA results showed that VEGF continued to act for more than 15 days after explosive release on the first day.3.After live / dead staining and CCK-8 toxicity test evaluation,it was found that the number and activity of HUVECs in c Ti/VEGF group were higher than those in e Ti and c Ti groups.The results of tubule formation experiment showed that more vascular structures were produced in c Ti/VEGF group,and the vascular marker MMP-2 was upregulated.4.Rabbit femoral defect model was established,e Ti,c Ti and c Ti/VEGF groups were successfully implanted into the defect.Specimens were taken out for detection at6 and 12 weeks after operation.Micro-CT showed that the amount of bone tissue in c Ti/VEGF group was greater than that in the other two groups,and the bone volume fraction(BV / TV),trabecular thickness(TB.Th)and trabecular number(TB.N)were the highest.The results of VG staining in hard tissue sections were consistent with those of micro-CT.Immunohistochemistry confirmed that the expression of type I collagen and CD31 in c Ti/VEGF group was the highest.Mechanical push out test confirmed that the bone integration strength of c Ti/VEGF was higher than that of e Ti and c Ti groups.Conclusion:In this study,we enhanced osseointegration by introducing a surface bioactive coating to the scaffold.This accelerated HUVECs aggregation,blood vessels formation,as well as growth and osseointegration of the porous bone in the 3D printed titanium alloy porous scaffolds.We also evenly coated the thermosensitive collagen hydrogel with VEGF to fill the pores of the titanium alloy scaffolds and form a composite scaffold with good bioactivity and mechanical properties.The slow local VEGF release recruited endothelial cells,and enhanced vascular and bone regeneration and integration at the interface.Our results confirmed that the 3D printed porous titanium scaffold with VEGF/thermosensitive collagen hydrogel coating can provide an effective strategy for promoting angiogenesis and osseointegration.