Study On Osseointegration Of Titanium Implants In Osteoporotic Rats Based On Titanium-Affinity DOPA-BMP-2 Polypeptide

Part I Characterization and Immunogenicity of Bone Marrow-DerivedMesenchymal Stem Cells upon Osteoporotic ConditionObjective:Mesenchymal stem cells(MSCs)are widely used in regenerative medicine and tissue engineering for their multidirectional differentiation potential and low immunogenicity,whereas MSCs in disease state have not been clearly reported.In this study,we established ovariectomized(OVX)osteoporotic rat model to compare the three-line differentiation potential and immunogenicity of BMSCs derived from healthy and osteoporotic inflammatory microenvironment.Methods:The rat model of osteoporosis was established by ovariectomy,and on the basis of confirming the formation of osteoporosis,BMSCs were isolated from the bone marrow of healthy and osteoporosis rats by whole bone marrow culture.After identification,they were named as H-BMSCs and OP-BMSCs,respectively.(1)The stem cell phenotype was detected by flow cytometry assay(FCA),and the proliferation efficiency of H-BMSCs and OP-BMSCs was detected by CCK-8.(2)After osteogenic induction for 7 days,ALP staining was performed and real-time fluorescence quantitative PCR(qRT-PCR)was used to detect osteogenic related genes of H-BMSCs and OP-BMSCs.The differentiation of efficiency was detected by alizarin red staining after osteogenic induction for 14 days.The expression of Wnt/p-catenin pathway-related proteins was detected by Western blot.(3)H-BMSCs and OP-BMSCs were induced by cartilage induction and adipogenic induction,and then toluidine blue staining,COL-2 immunofluorescence staining and qRT-PCR were used to detect cartilage-related genes to compare the ability of cartilage;oil red-O staining and qRT-PCR was used to detect the ability of adipogenic genes to compare the adipogenicity between H-BMSCs and OP-BMSCs.(4)The expression of costimulatory molecules on the surface of H-BMSCs and OP-BMSCs was detected by FCA,H-BMSCs and OP-BMSCs were co-cultured with CD3 monoclonal antibody-activated T cells of human,and the proliferation of T cells was detected by CCK-8.FCA was used to detect the markers of early activation of T cells,and CBA was used to detect the secretion of cytokines in the supernatant of T cells.Results:(1)OP-BMSCs and H-BMSCs have similar cell morphology.There was no significant difference in the expression of stem cell surface markers CD29 and CD90,but CD34 and CD45 were not expressed.CCK-8 showed that OP-BMSCs had weaker proliferation efficiency than H-BMSCs during normal culture.(2)The results of ALP staining,alizarin red staining showed that,compared with H-BMSCs,OP-BMSCs had weaker osteogenic ability.The expression of osteogenic related genes OPN,OCN and BMP-2 in OP-BMSCs was significantly decreased which was detected by qRT-PCR.The results of Western blotting also showed that the expression of β-catenin and osteoblast-specific protein OCN and Runx2 in OP-BMSCs was significantly decreased under osteogenic induction.(3)Compared with H-BMSCs,toluidine blue staining,immunofluorescence and qRT-PCR showed that OP-BMSCs had weaker chondrogenic ability,while oil red-O staining and qRT-PCR showed that OP-BMSCs were stronger in adipogenic ability.(4)The results of FCA showed that the expression of positive costimulatory molecules CD40,CD80 and CD86 in OP-BMSCs was significantly higher than that in H-BMSCs,while the expression of negative co-suppressor molecule PD-L1 was significantly decreased.CCK-8 also showed that OP-BMSCs could not inhibit the activation of T cells.While the results of CBA showed that OP-BMSCs promoted the secretion of inflammatory factors TNF-a and IL-6 from activated T cells and inhibited the secretion of anti-inflammatory factors IL-4.Conclusion:Compared with H-BMSCs,OP-BMSCs decreased the osteogenic and chondrogenic differentiation,increased adipogenic ability and the immunogenicity.Part Ⅱ Promotion of Osseointegration of Titanium Implants in Osteoporotic Rats Based on Titanium Affinity DOPA-BMP-2 PolypeptideObjective:Improving the osseointegration of implants(such as titanium-based materials)and peripheral tissue is a key issue in the treatment of orthopedic patients.The introduction of bioactive molecules to effectively regulate bone osteointegration is of great significance for improving the bone osseointegration of titanium implants,especially for the special clinical symptoms(such as osteoporosis).In this part,on the basis of finding that the osteogenic ability of OP-BMSCs is weaker than that of H-BMSCs,to study whether titanium affinity DOPA-BMP-2 peptide promotes the osteogenesis of OP-BMSCs in vitro and accelerates the osseointegration of titanium screws in vivo.Methods:Titanium sheets and titanium screws were functionalized by soaking method.After grafting of DOPA,BMP-2 functional bioactive polypeptide was loaded onto the surface of the titanium sheets and titanium screws for experiments.(1)The physicochemical properties among the different groups was studies by using X-ray photoelectron spectroscopy(XPS)to detect the chemical composition,then water contact angle(WCA)was used to examine the hydrophilicity,and atomic force microscopy(AFM)was used to examine the surface morphology.(2)The effects of different concentrations of free BMP-2 polypeptide on the osteogenic ability of OP-BMSCs by using ALP staining and alizarin red staining with the concentration of different free BMP-2 polypeptides.(3)Different polypeptide concentrations were set and the optimal concentration of DOPA-BMP-2 polypeptide modified surface on OP-BMSCs to promote bone formation was chosen by the methods of ALP staining and alizarin red staining.(4)The osteogenesis-related genes were detected by qRT-PCR.The effects of different proportions of different proportions of DOPA-BMP-2 polypeptide and DOPA-RGD polypeptide(promoting cell adhesion)were studied,which also used ALP staining and alizarin red staining.(5)Titanium screw coated with DOPA-BMP-2 polypeptide and DOPA-RGD polypeptide were implanted into the osteoporotic rats to comprehensively evaluate the osseointegration.Micro-CT was used to analyze the parameters of osseointegration.Histological examination was analyzed by toluidine blue staining for bone-implant contact(BIC).The anchorage force is tested by biomechanical test.Results:(1)The increase of N content in XPS proved the successful grafting of bioactive peptides.WCA data showed that the titanium became hydrophilic after grafting DOPA,which was beneficial to the grafting of peptides.AFM results also showed that the surface roughness of the titanium was increased.All the results showed that the DOPA and BMP-2 polypeptides were successfully grafted onto the titanium surface.(2)Both ALP staining and alizarin red staining showed that 100 ng/mL of free BMP-2 polypeptide had the strongest bone-formation ability on OP-BMSCs.(3)According to the morphological staining results,ALP staining and alizarin red staining positive areas were the most abundant in the 0.01 mg/mL DOPA and 0.1 mg/mL BMP-2 polypeptide group.(4)ALP staining and alizarin red staining results showed that the ratio of BMP-2 and RGD at 3:1(BMP-2:RGD=3:1)had the strongest bone-promoting of OP-BMSCs.qRT-PCR results showed that the expression of bone-related genes OPN and BMP-2 was significantly higher than other groups.(5)Quantitative results of Micro CT showed that the Bone volume/tissue volume(BV/TV),trabecular bone number(Tb.N),trabecular bone thickness(Tb.Th)of dual-functionalized group were higher than other groups,trabecular bone separation(Tb.Sp)and trabecular pattern factor(Tb.Pf)were decreased.The toluidine blue staining showed that the dual-functionalized group promoted osseointegration.The mechanical properties of the dual-functionalized group were enhanced according to the mechanical results.In vivo results indicated the osseointegration of titanium implants in osteoporotic rats was promoted in BMP-2:RGD=3:1 group.Conclusion:Biomimetic active peptides and dual-functionalized strategies have successfully promoted osseointegration of titanium implants in osteoporotic rats.