Fabrication Of Mg²⁺/ZIF-8 Incorporated Hydrogel-porous Titanium Complex And Its Osteogenic And Antibacterial Activity

BackgroundTitanium alloy has good biocompatibility,excellent corrosion resistance and satisfactory mechanical properties,thus being the most widely used metallic implant materials in clinical.However,titanium alloy is bioinert and lacks osteogenic and antibacterial abilities.As a result,titanium alloy will face two major problems when implanted in human body: aseptic loosening and implant associated infection(IAI).These two postoperative complications pose serious challenges to long-term implantation of titanium alloy.And they are also the main reasons for implant failure.Therefore,it has become a focus in recent years to functionalize titanium alloy implants with enhanced osteogenic and antibacterial ability.The functional modification of titanium alloy is mainly achieved by bioactive substances such as bioactive ions,organic molecules and biochemical factors.Mg2+ is a kind of metal ion with biological activity,which can promote the proliferation and differentiation of osteoblasts and bone tissue regeneration.Zeolitic imidazolate framework-8(ZIF-8)is a kind of bioactive organic molecule,belonging to metal organic framework(MOF),which has the dual function of osteogenesis and anti-infection.The functional modification of titanium alloy also relies on suitable carrier.Hydrogel is a sort of degradable polymer.During its degradation,hydrogel gradually releases loaded bioactive substances to perform specific biological functions.Quarternized chitosan(QCS)and aldehyde Pluronic F127(PF127-CHO)can be combined to form hydrogel(QP),which has good biocompatibility,loading capacity and antibacterial properties,suitable for functional modification of titanium alloy.With the development of functional modification technology,modifying titanium alloy with single biological function has been unable to meet clinical needs.Therefore,in the present study,titanium alloy was functionalized by bioactive ions and organic molecules to synergistically perform multiple biological functions,which is of great significance for improving the osteogenic and antibacterial ability of titanium alloy.Objective 1.Fabricate the Mg2+/ZIF-8 incorporated hydrogel-porous titanium complex(QPMZ-Ti)to fulfill dual functionalization of titanium alloy by the synergistic effect among QP hydrogel,Mg2+ and ZIF-8.Analyze physiochemical properties of the complex via characterization.2.Evaluate the effect of QPMZ-Ti on the proliferation,adhesion and osteogenic differentiation of MC3T3-E1 cell.3.Test in vivo osteogenic effect of QPMZ-Ti via rabbit femoral condyle defect model.4.Assess the effect of QPMZ-Ti on bacterial growth and biofilm formation.5.Inspect in vivo antibacterial effect of QPMZ-Ti by rat subcutaneous infection model.Methods 1.3D-printed porous titanium alloy scaffolds with various size were successfully fabricated via selective laser melting(SLM): scaffolds for mechanical test were Φ10mm×10mm;scaffolds for in vitro test were Φ14mm×3mm;scaffolds for in vivo study were Φ6mm×8mm.The parameters(pore size,porosity,elastic modulus and compressive strength)of the scaffolds were determined by Micro-CT analysis and compression experiments.Hydrogen spectra of QCS and PF127-CHO solutions were performed by nuclear magnetic resonance.QCS/PF127-CHO hydrogel(QP),QCS/PF127-CHO/Mg2+ hydrogel(QPM)and QCS/PF127-CHO/Mg2+/ZIF-8 hydrogel(QPMZ)was synthesized,and their surface morphologies were observed by scanning electron microscopy(SEM).The elemental composition and distribution of the three hydrogels were detected by energy dispersive spectrometer(EDS)elemental mapping.The concentration of Mg2+ and Zn2+ released by QPMZ hydrogel was measured by ICP-AES and their release curve was drawn respectively.The hemolysis rates of QP,QPM and QPMZ hydrogel were determined by hemolysis test.The effect of polydopamine(PDA)on the bonding between hydrogel and titanium was evaluated by universal testing machine and SEM.Three hydrogel-porous titanium alloy complexes(QP-Ti,QPM-Ti,QPMZ-Ti)were constructed based on the synthesized QP,QPM and QPMZ hydrogel,respectively.2.Mouse osteoblastic precursor cells(MC3T3-E1)were co-cultured with Ti,QP-Ti,QPM-Ti and QPMZ-Ti,respectively.Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.The cytotoxicity of materials from each group was determined by live/dead staining.The morphology of cells on the surface of Ti,QP-Ti,QPM-Ti and QPMZ-Ti was observed by scanning electron microscopy(SEM).Cytoskeleton(F-actin)morphology and the expression of Vinculin were observed by fluorescence staining.Alkaline phosphatase(ALP)staining and alizarin red staining were used to detect the expression level of ALP and extracellular matrix mineralization.The expression level of RUNX2 from each group was determined by immunofluorescence staining.RT-q PCR was employed to measure the expression of osteogenesis-related genes(Runx2,Opn,Ocn,Osterix)in each group.3.Rabbit femoral condyle defect model was established,and materials from each group were implanted.Rabbits were sacrificed 4,8,and 12 weeks after implantation respectively and samples were collected.Micro-CT and VG staining were used to measure bone ingrowth in each group to evaluate the in vivo osteogenic effect of Ti,QP-Ti,QPM-Ti and QPMZ-Ti.4.Staphylococcus aureus(S.aureus,ATCC25923)and Escherichia coli(E.coli,ATCC25922)were co-cultured with Ti,QP-Ti,QPM-Ti and QPMZ-Ti,respectively.The number of colony forming unit(CFU)from each group was determined by spread plate method and antibacterial rate was accordingly calculated.Crystal violet staining was used to evaluate inhibition effect on biofilm formation in each group.SEM was employed to observe the morphology of bacteria on the surface of Ti,QP-Ti,QPM-Ti and QPMZ-Ti,respectively.5.Rat subcutaneous infection model was established,and materials from each group were implanted.Rats were sacrificed 7 and 14 days after implantation respectively and samples were collected.HE staining was used to evaluate the in vivo antibacterial effects of each group.Results1.The 3D-printed porous titanium alloy scaffold was successfully fabricated.The parameters of the scaffold were listed as following: pore size was 585.74±21.61μm,porosity was 61.57±2.03%,elastic modulus was 4.84±0.23 GPa,and compression strength was 153.71±5.52 MPa.NMR results showed the characteristic peaks of quaternary ammonium group in QCS and aldehyde group in PF127-CHO,respectively.SEM images demonstrated that QP,QPM and QPMZ hydrogels all had connected porous structures.Mg2+ and ZIF-8 had significant effects on the pore size of the hydrogels,and the pore size of the hydrogels had the trend of QP > QPM > QPMZ.EDS elemental mapping showed that the main elemental compositions of QP,QPM and QPMZ hydrogels were C,O and N,while Mg and Zn were evenly distributed in QPM and QPMZ hydrogels,respectively.ICP-AES indicated that Mg2+ and Zn2+ were released stably and continuously from QPMZ hydrogel.Hemolysis test results manifested that the hemolysis rates of QP,QPM and QPMZ hydrogels were all below 5%,showing their absence of hemolysis.Binding force experiments showed that PDA significantly enhanced the binding force on the hydrogel-titanium alloy interface.SEM images indicated that the interface between the hydrogel and titanium alloy was tightly bonded,with no obvious gaps observed.2.Live/dead staining showed that only a few cells died on the surface of materials in each group,indicating there was no obvious cytotoxicity.CCK-8 and flow cytometry demonstrated that QPM-Ti and QPMZ-Ti could significantly promote cell proliferation but had no significant difference in terms of apoptosis rate.SEM images showed that cells on QPM-Ti and QPMZ-Ti spread better and had more pseudopods,with QPMZ-Ti being the most significant.Immunofluorescence staining demonstrated that cytoskeleton on the surface of QPM-Ti and QPMZ-Ti was more extensive,and the ratio of cell area to nucleus area increased significantly,with the highest value in the QPMZ-Ti group.The expression of Vinculin increased significantly in QPM-Ti and QPMZ-Ti,with that of QPMZ-Ti being the highest.ALP staining and alizarin red staining showed that ALP activity and extracellular matrix mineralization of QPM-Ti and QPMZ-Ti increased significantly,and QPMZ-Ti had the highest level.Immunofluorescence staining showed that QPM-Ti and QPMZ-Ti significantly increased the expression of RUNX2.RT-q PCR test manifested that QPM-Ti and QPMZ-Ti enhanced the expression of osteogenesis-related genes(Runx2,Opn,Ocn,Osterix),with the increase of QPMZ-Ti being the most significant.3.Micro-CT analysis showed that bone ingrowth in all groups increased as time goes by from 4 weeks to 12 weeks.At the same time point,bone ingrowth of QPM-Ti and QPMZ-Ti was significantly increased compared with that of Ti and QP-Ti,and QPMZ-Ti had the most bone ingrowth.VG staining indicated that bone ingrowth of QPM-Ti and QPMZ-Ti increased significantly,and new bone tissue was found to grow into the internal pores of the scaffold.4.Colony counting results showed that the number of bacteria on the surface of QP-Ti,QPM-Ti and QPM-Ti decreased significantly,and the number of bacteria in QPMZ-Ti was the least.There was no significant difference between antibacterial rate of QP-Ti and that of QPM-Ti,while antibacterial rate of QPMZ-Ti increased significantly compared to that of QP-Ti and QPM-Ti.Crystal violet staining indicated that the amount of biofilm on QP-Ti,QPM-Ti and QPMZ-Ti was significantly less than that on Ti.But there was no significant difference between QP-Ti and QPM-Ti,while the amount of biofilm on QPMZ-Ti was the least and significantly different from that in the other three groups.SEM images showed that the number of bacteria on the surface of QP-Ti,QPM-Ti and QPMZ-Ti was significantly reduced compared with Ti.Bacteria on QP-Ti,QPM-Ti and QPMZ-Ti were contracted and had their cell walls disrupted and residual structure of dead bacteria could be detected.5.HE staining showed that 7 days after surgery,a large number of inflammatory cell infiltrated into tissues in Ti group,indicating severe inflammatory response,while the inflammatory response was weaker in QP-Ti,QPM-Ti and QPMZ-Ti group,and the degree of inflammatory cell infiltration in these three groups was milder,with the least inflammatory infiltration in QPMZ-Ti.14 days after surgery,inflammatory cell infiltration was further aggravated in Ti group,and the inflammatory response was continuously enhanced.By contrast,no obvious inflammatory cell infiltration was observed in QP-Ti,QPM-Ti and QPMZ-Ti and the inflammatory response in these three groups was significantly relieved.ConclusionsIn the present study,Mg2+/ZIF-8 incorporated hydrogel-titanium complex(QPMZ-Ti)was successfully fabricated.In the complex,Mg2+,ZIF-8 and QP hydrogel were synergically used for functional modification of titanium alloy,endowing it with osteogenic and anti-infection abilities.Characterization analysis showed that the elastic modulus of 3D-printed porous titanium alloy was close to that of human bone tissue,QPMZ hydrogel had the ability to release Mg2+ and Zn2+ stably and sustainably,and PDA could significantly enhance the binding strength on the interface between hydrogel and titanium alloy.In vitro experiments confirmed that QPMZ-Ti had good biocompatibility and promoted the proliferation,adhesion and osteogenic differentiation of MC3T3-E1 cells.In vivo study demonstrated that QPMZ-Ti had excellent osteogenic ability.In vitro bacterial experiments showed that QPMZ-Ti could significantly inhibit the growth of S.aureus and E.coli and reduce their biofilm formation.The study of rat subcutaneous infection model showed that QPMZ-Ti had superior antibacterial ability in vivo.In conclusion,the Mg2+/ZIF-8 incorporated hydrogel-titanium complex in the present study had both osteogenic and anti-infection capabilities,which could serve as a new approach to improving the bio-inertness of titanium alloy and had good prospect for clinical application.