A New Strategy For Promoting Osseointegration Of Titanium Implants Via Bioorthogonal Click Chemistry Reaction

Titanium base material is the most commonly used medical material.However,titanium material is bioinert which leads to the insufficient osteogenesis and abnormal proliferation of fibrous tissue between the interface of implant and surrounding bone tissues.In particularly,for those osteoporotic patients,the Ti implant is more likely to loosen after implanting into bone tissues.Thus,how to make the bioinert surface into bioactive surface and significantly improve osseointegration of Ti implant is of important significance.Modification of Ti implant surface by mussel inspired biomimetic peptide is a convenient and efficient method which can be achieved by simple one-step immersion into peptide solution.The bioactive molecule within the peptide will play an important role in improving osteogenesis.However,this modification method which is based on bioactive molecule is susceptible of microenvironment and the molecule is easily inactivated and degraded.In addition,the amount of mesenchymal stem cells(MSCs)within the bone marrow is limited,especially those MCSs which can be recruited to the interface of implant and bone are absolutely less.In this study,we designed a new strategy for osseointegration of Ti implant based on bioorthogonal click chemistry.This strategy which does not disturb the physiological function of target cells and is not affected by microenvironment is established on the more stable bioorthogonal reaction than other chemical reactions.By adopting this novel strategy,the azide group(N3)modified BMSCs should be anchored on the DOPADBCO modified Ti implant surface through bioorthogonal click chemistry reaction.Then BMSCs adhere on the implant surface,initiate osteogenic differentiation and modulate immune response.Finally,favorable osseointegration between the interface of Ti implant and surrounding bones can be achieved.Part I Evaluation of the biological function of bone marrow mesenchymal stem cells modified by Ac4ManNAzPurpose:To evaluate the biological function of rat bone marrow mesenchymal stem cells(BMSCs)after surface modification by Ac4ManNAz through cellular glycometabolic engineering.Methods:BMSCs were isolated from whole bone marrow of Sprague-Dawley rats’femurs.Ac4ManNAz was reconstituted to different concentrations in DMSO and PBS solution.(1)BMSCs were treated with different concentration of Ac4ManNAz solution(5μmol/L,25μmol/L,50μmol/L,100μmol/L,500μmol/L)and cellular viability was tested by CCK-8 on Day 1,3,5 and 7.(2)Surface markers CD90,CD29,CD34 and CD45 of BMSCs treated with different concentration of Ac4ManNAz solutions were evaluated by flow cytometry.(3)Immunogenic surface markers(CD40,CD80,CD86 and MHC II)of BMSCs were assessed by flow cytometry to analyze the immunogenicity change of BMSCs treated with different concentration of Ac4ManNAz solutions.(4)Three-lineage differentiation potential of Ac4ManNAz treated BMSCs was analyzed.After osteogenic induction,ALP staining,ALP activity,Alizarin red staining and osteogenic genes expression(Alp and Opn)were tested.Toluidine blue staining and chondrogenic genes(Col2a1 and Sox-9)expression were tested after chondrogenesis induction.Adipogenic differentiation was measured by Oil Red O staining and adipogenic genes expression(Ppar-y and Adiponectin)after adipogenic induction.(5)Fluorescence probe AZDye 488 DBCO was applied and fluorescence intensity of BMSCs treated with different concentration of Ac4ManNAz solutions was tested to verify the introduction and intensity of azide group on cell surface.Results:Rat BMSCs were isolated successfully and identified as MSCs by flow cytometry.(1)According to the results of CCK-8,Ac4ManNAz solution at 25μmol/L was suitable for proliferation of BMSCs and presented no cytotoxicity.(2)Ac4ManNAz treatment did not affect the stem cell surface markers of CD90,CD29,CD34 and CD45 expression which indicated that Ac4ManNAz treatment did not have effect on the stemness of mesenchymal stem cells.(3)Immunogenic surface markers CD40,CD80,CD86 and MHC Ⅱ of BMSCs were also not affected by Ac4ManNAz treatment.Meanwhile,mixed lymphocyte reaction showed low proliferation rate of lymphocytes when BMSCs were treated with different concentration of Ac4ManNAz.These results indicated that the immunogenicity of MSCs was not affected by cellular surface modification.(4)Compared to those cells treated with 25μmol/L Ac4ManNAz,after 7 days of osteogenetic induction,the cells were not treated with Ac4ManNAz did not present significant differences in ALP staining and ALP activity.14 days after osteogenetic induction,results of alizarin red staining and PCR detection of osteogenesis-related genes expression(Alp and Opn)showed no significant differences between the BMSCs treated and untreated with Ac4ManNAz.16 days after chondrogenetic induction,there was no significant difference in toluidine blue staining and the expression of cartilage-related genes Col2al and Sox-9 between the BMSCs.16 days after adipogenic induction,there were also no significant differences in oil red O staining and the expression of adipogenesis-related genes(Ppar-γ and Adiponectin).(5)The AZDye 488 DBCO fluorescent probe successfully detected azide groups on the surface of rat BMSCs after Ac4ManNAz treatment.BMSCs modified by azide group showed green fluorescence,and the intensity of green fluorescence increased with the elevated concentration of Ac4ManNAz.Conclusion:Cellular glycometabolic engineering was an effective method of introducing azide groups onto the surface of BMSCs.The concentration of Ac4ManNAz at 25μmnol/L had no influence on the viability and proliferation of rat BMSCs while ensuring the introduction of sufficient azide groups onto the cellular surface.After the modification of azide groups onto the cell surface,the characteristics of stemness,low immunogenicity and three-lineage differentiation ability of BMSCs were not affected which indicated that this modification method retained the favorable properties of BMSCs as tissue-engineered seed cells.Part Ⅱ Characterization of the titanium surface modified by mussel inspired biomimetic peptidePurpose:To design a DBCO-containing mussel inspired biomimetic peptide.By the coordination reaction between DOPA groups of mussel inspired peptide and TiO2,the DBCO groups are aimed to anchor onto the surface of Ti implant.Methods:Mussel inspired biomimetic peptide containing DOPA and DBCO groups was synthesized by the method of Fmoc solid-phase synthesis.The Ti implant was modified by simple one-step immersion into peptide solution.(1)The chemical composition of the surface of Ti implant was assessed by X-ray photoelectron spectroscopy(XPS)and energy dispersive spectrum analysis(EDS).(2)The morphology of Ti implant surface was evaluated by atomic force microscopy(AFM).(3)The water contact angle(WCA)was assessed to evaluate the hydrophilicity of implant surface.(4)Live-Death cell staining and CCK-8 were performed to evaluate the compatibility of peptide modified Ti implant.(5)Fluorescent probe AZDye 488 N3 was used to detect whether DBCO group was successfully introduced onto implant surface.Results:(1)The results of XPS and EDS presented that the content of N element significantly increased after peptide modification.The atomic ratio of N/Ti increased from 0.75 to 2.46.All these results indicated that the peptide was successfully modified onto the Ti implant surface.(2)The roughness of surface of Ti implant significantly increased after peptide modification.(3)Water contact angle showed that the implant surface became hydrophilic after peptide modification.(4)The results of CCK-8 at day 1,3,5,7 showed that the viability of BMSCs inoculated on Ti implant surface was not affected by modification of peptide.The result of Live-Death cell staining at day 3 presented that there was no significant difference about dead cells on the surface of Ti implant.(5)The fluorescent staining showed that the Ti implant which was modified by DOPA-DBCO peptide presented green fluorescence while those implants which were not modified did not present any fluorescence.Conclusion:The DOPA and DBCO containing biomimetic peptide can be modified onto the Ti implant surface through coordination reaction between DOPA groups and TiO2.The successful introduction of DBCO groups provides the binding site for N3 groups.After modification,the Ti surface becomes hydrophilic and the roughness of surface significantly increases.Part Ⅲ Evaluation of osseointegration of titanium implants promoted by bioorthogonal click chemistry reactionPurpose:To evaluate the bioorthogonal click reaction between N3 modified BMSCs and DOPA-DBCO modified Ti implant and the promotion of osseointegration by bioorthogonal click reaction for Ti implant.Methods:(1)To evaluate the conjugation ability of N3 modified BMSCs and DOPADBCO modified Ti implant in vitro:N3 modified/non-modified BMSCs were inoculated onto DOPA-DBCO modified/non-modified Ti implant surface,fluorescent probe AZDye 488 N3 was used to detect whether the DBCO groups on Ti surface were conjugated by the N3 groups on the surface of BMSCs;N3 modified/non-modified BMSCs were suspended in the solutions of cell culture plate and DOPA-DBCO modified/non-modified Ti implants were placed at the bottom of culture plates.The plates were fixed on the transference decoloring shaker and shook for 3 and 30 minutes,then counted the number of cells anchored on the Ti surface.(2)N3 modified/non-modified BMSCs were inoculated onto DOPADBCO modified/non-modified Ti implant surface,CCK-8 at day 1,3,5 and 7 and LiveDeath cell staining at day 3 were performed to assess the compatibility of BMSCs which anchored onto the Ti surface by bioorthogonal click reaction.(3)N3 modified/non-modified BMSCs inoculated on DOPA-DBCO modified/non-modified Ti implant surface and then osteogenic induction was performed.ALP staining,ALP activity and PCR assessment of osteogenic genes(Alp and Opn)at day 7 after osteogenic induction were performed.Alizarin red staining and quantitative analysis at day 14 and 21 were performed.(4)Ti screws implantation within rat femoral condyles model was established.N3 modified/non-modified BMSCs were injected into the screw channel.Then DOPA-DBCO modified/non-modified Ti screw was placed within the channel.The femur specimens were harvested 4 weeks after surgery and osseointegration of Ti screw was evaluated.①Micro CT was performed to analyze the peri-screw bones and trabecular parameters;②Fluorescent staining was performed to evaluate the inflammatory factors of M1 macrophages surrounding the Ti screw;③Hard tissue slicing containing Ti screw and surrounding tissues was performed to assess the new bone formation and integration of the interface between the screw and surrounding bones;④Biomechanical test about the pullout strength of Ti screw was performed to assess the binding force of screw and surrounding bones.Results:(1)DBCO modified Ti implant presented green fluorescence by the fluorescent probe AZDye 488 N3 while the green fluorescence significantly decreased after inoculating the N3 modified BMSCs onto the DBCO modified Ti surface.This might be attributed to the fact that the N3 binding sites(DBCO groups)was pre-conjugated by the N3 groups on BMSCs surface thus the AZDye 488 N3 probe had no corresponding reactive sites to conjugate.After shaking for 3 and 30 minutes,the N3 modified BMSCs were observed to significantly anchor onto the surface of DOPA-DBCO modified Ti surface than other groups which indicated that N3 modified BMSCs could be anchored by the Ti surface through N3DBCO bioorthogonal click reaction.(2)The viability of N3 modified BMSCs did not decrease while inoculated onto DOPA-DBCO modified Ti surface.The cells could proliferate normally.(3)The ALP staining,ALP activity and PCR assessment of osteogenic genes(Alp and Opn)at day 7 after osteogenic induction showed no statistically significant differences between the groups.Alizarin red staining and quantitative analysis at day 14 and 21 also presented no significant differences among all the groups.(4)Ti screws implantation within rat femoral condyles model was successfully established.①The results of Micro CT showed that N3 modified BMSCs and DOPA-DBCO modified Ti screw implanted into the bone tissue showed that the best result of osseointegration.The parameters of BV/TV,BS/TV,BS/BV,Tb.N,Tb.Th and Tb.Sp were all significantly better in BMSCs-N3+TiO2-DOPADBCO group.② Fluorescent staining showed the numbers of i-NOS positive M1 macrophages significantly decreased around the Ti screw in BMSCs-N3+TiO2-DOPADBCO group than other groups.③Hard tissue slicing containing Ti screw and surrounding bones showed that there were continuous new bone matrix formation on the surface of Ti screw and the BIC was significantly bigger in BMSCs-N3+TiO2-DOPA-DBCO group than the other groups.④The results of biomechanical test presented that the pullout strength of Ti screw was much bigger in BMSCs-N3+TiO2-DOPA-DBCO group than the other groups which indicated that the implanted screw had a stable binding with surrounding new bones and the osseointegration is favorable by using the new strategy.Conclusion:Cellular glycometabolic engineering modified BMSCs(BMSCs-N3)can be captured and stably anchored to the mussel inspired peptide DOPA-DBCO modified Ti implant through bioorthogonal click reaction between the N3 groups on cellular surface and DBCO groups on Ti implant surface.By chemical bond conjugation,the BMSCs are anchored and adhered to the Ti surface,then the BMSCs play an important role in improving osteogenic differentiation and modulating immune response which result in many new bones generated on the interface between Ti implant and surrounding tissues.As a result,favorable osseointegration is achieved by this novel strategy despite the fact that Ti material is bioinert.