Preparation And Biological Properties Of MoS₂ Nanosheets Functionalized Tissue Engineered Bone

In clinical practice,autologous bone transplantation is the gold standard for the treatment of small-area bone defects.However,the treatment of critical-size bone defects caused by infection,tumor and trauma has always been a great challenge for clinicians.Tissue engineering has become a promising bone defect repair technology and achieved remarkable research results^[1,2].In recent years,materials with photothermal effect have been sought after by many researchers,which can be rapidly heated under near infrared light（NIR）irradiation.Mild temperature（about 41°C）caused by NIR can promote the rapid proliferation and osteogenic differentiation of mesenchymal stem cells,so as to achieve the effect of bone regeneration.Part one:Preparation and characterization of MoS₂ functional tissue engineering scaffoldsObjective:To explore the preparation methods and physicochemical properties of porous scaffold BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG based on osteoinductive extracellular matrix（Oi ECM）,and construct photothermal tissue-engineered bone（BTEB/MTEB/PTEB）with rat mesenchymal stem cells（r BMSCs）as seed cells for subsequent biological experimental research.Methods:MoS₂ nanosheets were synthesized by solvent stripping method.The particle size parameters of MoS2 nanoparticles were determined by particle size analyzer.The photothermal properties of MoS₂nanosheets under NIR were detected in real time by electronic thermometer.MoS₂ nanosheets were characterized by X-ray diffraction and Raman spectroscopy.Rat BMSCs were extracted and purified,and the osteogenic differentiation of r BMSCs was induced to prepare decellularized matrix（Oi ECM）,which was characterized by SEM,and the expression of osteogenic genes in Oi ECM was determined by q RT-PCR.Porous MoS₂-BAG scaffolds with photothermal effect were prepared by combining MoS₂ nanosheets with self-assembled BAG scaffolds.CCK-8 was used to determine the cytotoxicity of MoS₂-BAG scaffolds with different proportions of MoS₂.The MoS₂-BAG scaffolds with the best biocompatibility were selected for subsequent experiments,and the Oi ECM-MoS₂-BAG scaffolds were synthesized by wrapping the MoS₂-BAG scaffolds with the best biocompatibility.The properties of BAG/MoS₂-BAG/Oi ECM-MoS₂-BAG scaffolds were characterized by scanning electron microscopy（SEM）,Micro-CT,X-ray diffraction（XRD）,Raman spectroscopy,infrared spectroscopy,porosity,water absorption and water storage rate.In addition,BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG scaffolds were immersed in PBS,and fresh PBS was replaced weekly.After15 days,30 days,45 days and 60 days of immersion,the weight ratios of the scaffolds before and after immersion were measured to determine the in vitro degradation rate of the composite scaffolds.Finally,r BMSCs were planted in BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG scaffolds to jointly construct tissue engineered bone BTEB,MTEB and PTEB.Results:As shown in SEM（A）and TEM（B）scanning results,MoS₂nanoparticles with photothermal effect were successfully prepared,and the particle size distribution parameter was 270±60 nm.The X-ray diffraction（XRD）peak of MoS₂ nanosheets was consistent with the crystal plane（002）at the angle of 2θ=14.3°.Raman spectrum analysis results show that there is a sharp MoS₂ Raman signal at 378 cm-¹,which is assigned to the in-plane E_2g¹ mode,and another sharp Raman signal at 403 cm-¹,which is assigned to the out-plane A_1g vibration mode.Under 808 nm laser irradiation,the temperature rise of MoS₂ nanosheets solution was time-dependent,and the maximum temperature was 46.1°C,indicating that MoS₂ nanosheets had good photothermal properties.In this study,r BMSCs were induced to differentiate into osteoblasts,and white thin film Oi ECM was obtained.DAPI fluorescence experiments before and after decellularization showed that decellularized Oi ECM was successfully prepared.The results of calcium content showed that the content of Ca²⁺increased with the passage of bone induction time,indicating that the mineralization degree of Oi ECM increased gradually with the extension of induction time.The SEM image analysis of Oi ECM showed that the surface of Oi ECM was tough and had a 3D porous structure.q RT-PCR results showed that the expression levels of osteogenic-related genes OCN、OPN和BMP-2 of r BMSCs cultured on Oi ECM were significantly higher than those of the control group at 21 days after osteogenic induction（P<0.05）.The biotin-agarose-gelatin（BAG）scaffold was prepared by chemical crosslinking and freeze drying,and the surface was pale yellow.MoS₂-BAG scaffold,gray black surface.SEM scanning showed that the pore size of MoS₂-BAG scaffolds was about 200-300μm.Compared with BAG scaffolds,the pore size of MoS₂-BAG scaffolds was smaller,about 150–200μm.The pore size of the two groups of scaffolds met the needs of cell growth.Micro-CT scan showed that only Oi ECM-MoS₂-BAG scaffold was covered by mineralized Oi ECM membrane.The scaffolds of BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG showed similar honeycomb structures,and the porosity was 80.44±2.17%,80.96±0.8%and 71.35±2.93%,respectively.The porosity of the scaffolds of Oi ECM-MoS₂-BAG group was significantly lower than that of the other two groups（P<0.05）.In addition,the three groups of composite scaffolds had strong water absorption capacity.The water absorption rates were about 2146.3±219.18%,2766.07±4.62%and 2282.8±4.75,respectively.It can be seen that the MoS₂-BAG group had the highest water absorption capacity（P<0.01）.The Oi ECM-MoS₂-BAG scaffold showed the highest water retention capacity among the three scaffolds,about 93.5%.As the results of X-ray diffraction（XRD）shown,in MoS₂-BAG scaffolds,the spectrum of gelatin exhibits a wide peak at 21°,which can be attributed to itsα-helix structure and triple helix structure.In addition,there are other peaks of MoS₂ in the X-ray diffraction pattern,such as 002,004 and 006.In the Raman spectra of BAG scaffolds,the bands at 680 and1065 cm^-1（C-S）belonged to biotin.In the Raman spectra of MoS₂-BAG scaffolds,the new band at 333 cm^-1（J3）belongs to MoS₂ nanosheets.In the Raman spectra of Oi ECM-MoS₂-BAG scaffold,the bands at 890（CN ring）,1224（-CH）,1234（-CH2）and 1434 cm^-1 Amide III belong to Oi ECM.In the FTIR spectra of BAG and MoS₂-BAG scaffolds,the bands at 1056（C-C）,3261（N-H）,1631（Amide II）,2361（C≡C）and 3500 cm^-1（Amide A）belong to BAG scaffolds.The new bands at 1621（C=O）belong to Oi ECM.The scaffolds in each group have biodegradability in vitro.In contrast,the degradation rate of Oi ECM-MoS₂-BAG can reach 24.66%after 60 days of PBS immersion.It was much higher than that of BAG（20.00%）and MoS₂-BAG（13.45%）groups（P<0.01）.In addition,r BMSCs were used as seed cells,and porous BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG scaffolds were used as the main structures to construct BTEB,MTEB and PTEB tissue engineered bone for subsequent biological characteristics research.Conclusion:The MoS₂ nanosheets have excellent photothermal properties and uniform particle size parameters.The decellularized extracellular matrix Oi ECM derived from BMSCs can highly express osteogenesis-related genes,and have the ability to promote osteogenic differentiation of BMSCs.BAG scaffolds prepared by chemical crosslinking and freeze drying have excellent physical and chemical properties,which can be used together with MoS₂ nanosheets and Oi ECM to prepare biological composite scaffolds to meet the experimental requirements.The BTEB,MTEB and PTEB tissue-engineered bones with ideal biocompatibility were successfully prepared by implanting BMSCs on the scaffolds of each group.Part two:Biological properties of MoS₂ functional tissue engineering scaffolds in vitroObjective:To evaluate the biocompatibility of porous biological scaffolds,including the cytotoxicity,adhesion and effect on cell proliferation.On this basis,the potential effects of composite scaffolds BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG on the osteogenic differentiation of r BMSCs under near-infrared light（NIR）irradiation were further discussed through cell experiments in vitro.Methods:r BMSCs were seeded on BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG scaffolds,and the planting efficiency of r BMSCs was measured by CCK-8.Then the composite scaffolds were incubated with r BMSCs in each group,and the effect of composite scaffolds on cell proliferation rate was measured by CCK-8.The cytotoxicity of tissue engineering scaffolds BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG on cells was evaluated by the cell staining level by Calcein-AM and propidium iodide（PI）double fluorescence staining.The cells were cultured on the scaffolds until the 14th day,and the tissue engineered bone scaffolds of each group were taken out.After gradient dehydration with alcohol and spraying gold on the surface after freeze-drying,SEM scanning was performed to detect the microscopic cell morphology and planting growth of r BMSCs on BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG.Alkaline phosphatase（ALP）detection kit was used to quantitatively analyze the relative activity expression of ALP in the culture medium of each group after co-incubation,and alizarin red staining was performed after 7,14 and21 days of co-incubation of tissue engineering scaffolds with cells to evaluate the effects of scaffolds in each group on cell mineralization and calcium nodule formation.Results:CCK-8 assay showed that the planting efficiencies of r BMSCs on BAG,MoS₂-BAG and Oi ECM-MoS₂-BAG scaffolds were68.41%,66.77%and 78.52%,respectively.The scaffolds in the Oi ECM-MoS₂-BAG group were significantly higher than those in the other two groups（P<0.01）.The results of cell proliferation test showed that r BMSCs could well proliferate and grow on BAG,MoS₂-BAG,Oi ECM-MoS₂-BAG and near-infrared radiation Oi ECM-MoS₂-BAG scaffolds.Among them,the Oi ECM-MoS₂-BAG group under near-infrared radiation showed higher cell proliferation rate than other groups.In addition,the r BMSCs cultured in different scaffolds for 14 days were measured by SEM.Similarly,more living cells adhered to the scaffolds than those in the BAG and MoS₂-BAG groups were observed on the Oi ECM-MoS₂-BAG and near-infrared radiation Oi ECM-MoS₂-BAG scaffolds.The above results showed that Oi ECM-MoS₂-BAG scaffold was more suitable for the adhesion and proliferation of r BMSCs.The ALP determination results of each group after 14 days and 21days of co-culture showed that the ALP activity of the Oi ECM-MoS₂-BAG scaffold group under NIR irradiation was the highest,and its relative activity was about twice that of the control group（P<0.05）.In addition,alizarin red staining results showed that the calcium deposition mineralized nodules in each group increased over time.At 7 and 14 days of differentiation,there was no significant difference in the formation of calcium nodules between Oi ECM-MoS₂-BAG and NIR+Oi ECM-MoS₂-BAG groups.However,on the 21st day,the mineralization effect of r BMSCs in the Oi ECM-MoS₂-BAG group under NIR was the best,and the calcium salt deposition was the most,which was significantly more than that in other groups.Conclusion:The composite porous bioscaffold was beneficial to r BMSCs adhesion and proliferation,and no cytotoxicity,suitable for r BMSCs growth.Oi ECM-MoS₂-BAG with photothermal effect can promote the rapid proliferation of r BMSCs under NIR irradiation,and can greatly improve ALP activity and promote calcium deposition.Part three:Osteogenesis of MoS₂ functionalized tissue engineered bone in vivoObjective:To investigate the repair effect of BTEB/MTEB/PTEB/PTEB+NIR on critical bone defect of rat skull by Micro CT scanning and histological analysis,and to evaluate the toxic effects of tissue-engineered bone on main organs of rats after degrading and metabolizing in vivo.Methods:The photothermal properties of BTEB,MTEB and PTEB implanted into the defect site of rat skull under NIR irradiation were measured by infrared imaging to determine the radiation parameters required for subsequent target temperature（1.5W/cm²,600s）.Tissue-engineered bone in each group was implanted into the defect area of rat skull.The skull samples containing the defect area were taken out at 12weeks after operation for Micro-CT scanning,and the amount of new bone formation BV and BV/TV in each group were quantitatively analyzed.Then,the samples were decalcified,embedded in paraffin,sliced and stained with HE and Masson to determine the formation of new bone in the histological morphology.Finally,tissue sections and HE staining were performed on the heart,liver,spleen,lung and kidney of the important metabolic organs of rats to assess whether the metabolism of scaffold materials in vivo had toxic damage to the visceral system of rats.Results:It can be seen from the results of thermal imaging that PTEB has higher photothermal conversion ability than BTEB.In the PTEB+NIR group,the temperature in the skull defect area of rats increased from the initial 32.7°C to the target temperature of 41.0°C after 120 seconds of NIR irradiation,while in the BTEB group,it only reached 36°C under the same conditions,and the temperature increased to 36.5°C after 600seconds of continuous irradiation.Micro-CT scanning and 3D reconstruction image analysis showed that NIR-irradiated PTEB group had more high-density signal shadows in the skull defect site,and the bone volume（BV）and bone volume/tissue volume（BV/TV）ratio in this group were significantly higher than those in other groups,indicating more new bone formation.The HE staining results of rat skulls showed that only a small amount of light red matrix was formed in the control group at the site of stent implantation in the defect area.Compared with other groups,the matrix in the PTEB+NIR group was the most dense.Masson staining showed that in all groups,PTEB+NIR group had a large number of blue-stained bone collagen formation,followed by PTEB group.In contrast,the control group had the least amount of new bone formation,and there was only a little sparse blue-stained bone matrix at the edge of the defect.The HE staining results of the main organs of heart,liver,spleen,lung and kidney showed that no pathological damage was found in the main physiological function units of the organs of rats in each group,such as cardiac muscle fiber,hepatic sinusoid,spleen medullary structure,alveolar and glomerular structure.Conclusion:PTEB with photothermal effect can rapidly heat up under NIR irradiation,and its mild thermal effect（41°C）can promote bone regeneration in rats.Oi ECM and MoS₂ nanosheets play an important synergistic role in the process of bone regeneration.In addition,the scaffold materials used in the experimental groups have no obvious toxic damage to the main metabolic organs in rats,which could be used for bone tissue engineering research.