| Background:Clinically,bone defects of various causes usually require surgical treatment.Currently,bone transplantation is the treatment of choice for bone defects.However,it has disadvantages of limited supply of donors,donor site pain,and potential risks of infection and rejection.To meet the growing demand for bone repair,bone tissue engineering is a promising method to induce bone regeneration.Titanium and its alloys,such as Ti6Al4 V,have been widely used as implants in orthopedic surgery owing to their good corrosion resistance and mechanical strength.Compared to the conventional solid titanium alloy scaffold,a 3D-printed porous titanium alloy scaffold prepared by electron beam melting(EBM)has high performance;it can effectively alleviate the nonphysical elastic modulus and inadequate compatibility of the conventional solid titanium alloy scaffolds.In addition,the regular pore structure of the porous titanium alloy scaffold obtained with 3D printing provides abundant space for loading of growth factors(GFs)and bone growth,so the porous Ti6Al4 V scaffold obtained with 3D printing has a stronger effect on bone defect repair.However,porous titanium alloys manufactured with EBM are bioinert.To ensure that implants have better bioactivity and bone promoting ability in vivo,it is necessary to introduce GFs into the surface and pores of the implants.Bone morphogenetic protein(BMP)was initially introduced as a bone-inducing component in bone extracts.It plays a role in the growth and development of various organs and tissues,including bone,cartilage,muscle,kidney,and blood vessels.It is a member of the transforming GF beta family and has been proved to promote the proliferation of stem cells and induce osteogenic differentiation.The gene for BMP9,also known as growth differentiation factor 2(GDF2),was first identified in a developing fetal rat liver c DNA library.It had been shown to play a role in the induction and maintenance of cholinergic phenotypes in embryonic basal forebrain cholinergic neurons.Further,it could inhibit hepatic glycogen production and the expression of key enzymes of lipid metabolism as well as regulate endothelial function and angiogenesis.Therefore,it had been considered to be the BMP with the highest osteoinductive differentiation ability,with more bone regeneration potential than BMP2.Moreover,it is the main regulator of angiogenesis and chondrogenesis.The combination of porous scaffolds and carrier materials for controlled release of GFs has potential to impart biocompatibility to implants.This method can improve the biological activity of implants and allow sustained release of GFs.As a carrier material for controllable release of GFs,absorbable thermosensitive collagen has injectability,plasticity,low toxicity,and good biocompatibility and can provide a suitable living environment for cells.Moreover,it is maintained in the liquid form at4°C and gelatinized at 37°C,which makes its application to biological tissues more convenient.Therefore,it has garnered attention in tissue engineering.However,the mechanical properties of thermosensitive collagen are poor,hindering effective support for bone defects.The excellent mechanical strength and internal porous structure of porous titanium alloys can help to provide an ideal cavity to fill BMP9 and thermosensitive collagen.In this study,we used 3D-printed porous titanium implants prepared with the EBM technology as scaffolds and implanted thermosensitive collagen mixed with recombinant human(rh)BMP9 into the scaffold pores,which served as a carrier for controlled release of rh BMP9.We found that with the slow degradation of thermosensitive collagen,rh BMP9 was gradually released from the porous structure,which promoted osteogenesis in and around the porous titanium structure.In vitro,bone marrow mesenchymal stem cells(BMSCs)were used to verify the cytotoxicity and osteogenic effect of the scaffolds.In vivo,a rabbit femoral defect model was used to analyze the effects of bone ingrowth and integration at 6 and 12 weeks.Method:1.3D printing porous titanium alloy scaffolds were prepared by EBM,and the mixture of thermosensitive collagen and rhbmp9 was injected into the pores of 3D printing porous titanium alloy scaffolds.2.Scanning electron microscope was used to observe the 3D printing porous titanium alloy scaffold,thermosensitive collagen and composite scaffold.3.Enzyme linked immunosorbent assay(ELISA)was used to evaluate the release of rhbmp9 from thermosensitive collagen.4.In vitro experiments,the effects of the composite scaffold on the cell activity of bone marrow mesenchymal stem cells were detected by phalloidin / DAPI staining,live/ dead staining and CCK-8 toxicity test.Alizarin red staining and real-time quantitative PCR were used to verify the effect of the composite scaffold on the osteogenic differentiation of bone marrow mesenchymal stem cells.5.In vivo experiment,the rabbit femoral defect model was established.The experimental group was divided into three groups: 3D printing porous titanium alloy stent(e Ti),porous titanium alloy stent loaded with thermosensitive collagen(c Ti),porous titanium alloy stent loaded with thermosensitive collagen and rh BMP9composite(rh BMP9/c Ti).The osseointegration of the composite scaffold and the bone ingrowth in the pores of the scaffold were studied by means of histological evaluation,immunohistochemistry and mechanical push out test.Result:1.Two kinds of titanium alloy scaffolds: disk-shaped(φ10 mm × L3 mm)and cylindrical(φ4 mm × L8 mm)were prepared.The parameters of the two kinds of scaffolds were the same: porosity = 70%,pore size = 500 μ m.Under electron microscope,thermosensitive collagen has collagen fiber structure,which could provide a good living environment for cells,and thermosensitive collagen can wrap and fill the pores of the scaffold.2.ELISA assay showed that the release rate of rh BMP9 reached 50% in the first day,and decreased continuously in the next 14 days.3.After phalloidin / DAPI staining,live / dead staining and CCK-8 toxicity test,it was found that compared with e Ti and c Ti groups,BMSCs in rh BMP9 / c Ti group could adhere to the scaffold and had good activity,and BMSCs showed spindle shape and attached to the matrix surface.Alizarin red staining showed that rh BMP9 / c Ti group produced more calcified nodules,and osteogenic markers Runx2,ALP,OPN and BMP-2 were up-regulated.4.A rabbit femoral defect model was established,and e Ti,c Ti and rh BMP9/c Ti groups were successfully implanted into the defect site.Micro-CT showed that the amount of bone tissue in the rh BMP9/c Ti group was greater than that in the other two groups,and the trabecular separation degree(Tb.Sp)was the lowest among the three groups.Bone volume fraction(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Tb.N)were all the highest.The results of Masson and VG staining in hard tissue sections showed the same trend as that of micro-CT.Immunohistochemistry confirmed the highest expression of type I collagen in rh BMP9/c Ti group.The mechanical rollout test confirmed that the osseointegration strength of rh BMP9/c Ti was higher than that of e Ti and c Ti groups.Conclusion:In this study,we used EBM to prepare 3D printed porous titanium alloy scaffolds with excellent mechanical properties,and injected rh BMP9 / thermosensitive collagen into the scaffolds to form porous titanium alloy composite scaffolds with biological activity.In vitro and in vivo experiments have shown that the composite scaffold has good biocompatibility,can slowly and continuously release growth factors,promote proliferation and osteogenic differentiation of BMSCs in vitro,and can enhance the bone volume,bone integration and bone growth capacity of bone defect in vivo. |
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