| Background:Osteoporosis is a progressive and systemic disease characterized by bone mass reduced,bone microstructure changed and easy to cause fragility fracture.Cemented artificial joint prosthesis,bone transplantation and systemic anti-osteoporosis drugs are commonly used in joint replacement for patients with severe osteoporosis,but the incidence of serious complications such as prosthesis displacement,loosening and periprosthetic fracture remains high.In addition,although the cement interface can play the role of temporary fixation in the early stage,it is easy to aging,rupture,and can not form effective interface osseointegration,which is difficult to ensure the long-term stability of the prosthesis.Bone grafts are also faced with problems such as limited source,donor site complications,pathogen transmission and rejection.In addition,there are many complications in systemic anti-osteoporosis drugs,and the bioavailability of systemic anti-osteoporosis drugs is limited.It is difficult to achieve effective local concentration and can not significantly promote bone formation in the defects.Although various techniques have been invented,osteointegration of osteoporotic bone defects remains a major challenge.Therefore,it is of great clinical significance to develop a new prosthesis bioactive interface with good mechanical properties for osteoporosis and other patients with poor osteogenesis,and to solve the complications of joint replacement in osteoporosis patients.Titanium alloys have great advantages in orthopedic metal implants due to their excellent biocompatibilities and corrosion resistance.However,the stiffness of wrought titanium alloys is much higher than those of cortical and cancellous bone,which primarily leads to consequences of stress-shielding induced osteolysis.A promising approach to alleviate biomechanical mismatch is to fabricate porous structures with significantly reduced modulus.3D printing technology is becoming popular due to the ability to directly print porous scaffolds with the desired shapes and interconnected porosities.Compared with traditional titanium alloys scaffolds,3D porous titanium alloy(pTi)scaffolds manufactured with 3D printed method has superior properties,such as higher surface areas,pore structure and lower stiffness similar to that of cortical bone.Higher surface area in porous structure along with three-dimensional pore connectivity were proven to promote tissue adhesion,growth and vascularization through the transport of oxygen and nutrients,and thereby enhance bone ingrowth.However,Ti implants may fail due to insufficient integration into surrounding bone,owing to the inertness of titanium alloys and the highly complex intrinsic cellular and extracellular cascades of bone generation,especially under pathological conditions such as osteoporosis.Although 3D printed pTi are conducive to the bone ingrowth into scaffolds,but in osteoporosis state,bone regeneration ability is still severely limited.Therefore,it is necessary to reprocess the 3D printed porous scaffolds to stimulate bone regeneration.To achieve a better bone repair,the development of next-generation bone tissue engineering scaffolds include three technologies,namely,topical drug treatment(e.g.,bone morphogenetic protein-2(BMP-2)),cellular therapies(e.g.,bone marrow mesenchymal stem cells(BMSCs)),and optimized scaffolds as well.In our previous work,injectable and self-healing hydrogels were prepared by in situ crosslinking of N-carboxyethyl chitosan(N-chitosan)and dipic acid dihydrazide(ADH)with hyaluronic acid-aldehyde(HA-ALD).These fascinating soft materials with self-healing or self-repairing properties have ability to restore their functionalities and structures after damage,which benefits to maintain the integrity of network structures and mechanical properties of bulk gels.Furthermore,self-healing hydrogels with shear-thinning properties can be used as injectable materials to encapsulate sensitive biological drugs/cells,and to deliver them to target sites.In this work,BMSCs and BMP-2 were selected as model cells and drug to explore the pTi filled with BMSCs and BMP-2 dual-loaded hydrogels as bioactive interface for bone osteointegration effect in osteoporotic bone defects.Methods:1.Prepare injectable and self-repairing hydrogels with good biocompatibility as cell carriers and drug delivery systems,and fabricate 3D printed titanium alloy microporous scaffolds filled with hydrogels with good mechanical properties.2.The appearance of the scaffolds was observed by scanning electron microscopy.Micro-CT and Universal Mechanical Testing Machine were used to explore the pore size,porosity and mechanical properties of porous titanium alloy scaffolds.3.Subcutaneous injection was used to detect the biocompatibility and degradation rate of hydrogels in vivo.The release behavior of porous titanium alloy scaffolds loaded with BMP-2 hydrogel was studied in vitro.4.The proliferation and viability of BMSCs in the composite scaffolds were observed by CCK-8 and Live-Dead cell staining in vitro.Alizarin red staining and realtime quantitative PCR(RT-qPCR)were used to investigate the osteogenic differentiation of BMSCs in the composite scaffold under osteogenic induction in vitro.5.Establishment of osteoporosis rabbit model: 8 months after bilateral ovariectomy,estrogen levels were measured in sham-operated and ovariectomized rabbits,and bone mass changes were examined by micro-CT and histology.6.In vivo experiments were carried out in rabbit model of osteoporosis by bilateral ovariectomy,which were divided into five groups,namely,porous titanium alloy empty scaffolds(S),porous titanium alloy scaffolds filled with hydrogels(SG),porous titanium alloy scaffolds filled with BMSCs loaded hydrogels(SGC),porous titanium alloy scaffolds filled with BMP-2 loaded hydrogels(SGB)and porous titanium alloy scaffolds filled with BMSCs and BMP-2 loaded hydrogels(SGCB).The scaffolds were implanted into the bone defect in distal femur.The effect of osseointegration by these composite scaffolds on osteoporotic bone defect was further studied by micro-CT,histological examination and mechanical push-out experiments.Results:1.The injectable and self-repairing hydrogel was prepared,and the components could gel during 1 minute after mixing.The hydrogel shown a porous structure under the scanning electron microscope and the pore size was 100-200 μm.The porous titanium alloy scaffolds were prepared by 3D printed technology,whose pore size and porosity were 793.4 ± 16.9 μm and 69.2 ± 0.9%,respectively,and compressive strength and elastic modulus were 48.0 ± 2.1 MPa and 1.63 ± 0.2 GPa,respectively.2.The hydrogel was completely degraded in 28 days after subcutaneous injection due to the action of various enzymes in vivo.It did not cause obvious inflammatory reaction of skin and had good biocompatibility.The porous scaffold was filled with BMP-2 loaded hydrogel could sustain release BMP-2 for 42 days in vitro.3.Compared with titanium plate(Con),S and SG groups,BMSCs implanted in SGB system shown a good biocompatibility,and benefited to BMSCs proliferation and survival.After 7 and 14 days of osteogenic induction culture,SGB system promoted BMSCs mineralization and up-regulated the expression of osteogenic related genes,such as ALP,Runx-2,OPN and Col-1.4.Establishment of osteoporosis rabbit model: compared with the sham-operated group,the estrogen level and the bone mass of the ovariectomy group decreased significantly.5.The scaffolds were implanted into the distal femoral bone defect of osteoporotic rabbits and the bone tissues were obtained 3 months after implantation.Micro-CT indicated that SGCB,SGB and SGC shown higher ratio of bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N)and lower trabecular separation(Tb.Sp)than cTi and eTi groups,especially in SGCB group.The results of histological sections were consistent with Micro-CT.Mechanical push-out testing confirmed that SGCB(126.51±13.78 N)had higher integration strength than that of SGB(96.02±9.27 N),SGC(87.72±13.41 N),SG(64.99±11.48 N)and S(57.16±13.60 N).Conclusion:In this study,we successfully prepared porous titanium alloy scaffolds with similar mechanical properties to bone.The scaffolds filled with BMSCs and BMP-2 loaded hydrogel as composite scaffolds can sustained release bioactive BMP-2 and benefit osteogenic differentiation of BMSCs in vitro.As a synergistic treatment,the Dual/cTi system can induce bone growth and promote bone osseointegration in osteoporotic bone defects. |
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