| Objective: To find the safe and osteogenic concentration of Graphene Oxide Quantum Dots(GOQDs)for Dental pulp stem cells(DPSCs).And to construct a composite in which GOQDs play a role as the bioactive factor,DPSCs as seed cells and hydrogel as the scaffold.To investigate the effect of the composite on repairing bone defects.Methods: Human primary DPSCs were isolated and cultured,and their osteogenic,lipogenic and chondrogenic differentiation abilities were examined respectively,and their immunophenotype was examined by flow cytometry to check whether they were mesenchymal stem cells(MSCs).The effect of different concentrations of GOQDs on the cell viability and proliferation was detected via CCK-8,and the influence of osteogenic medium(OM)with various doses of GOQDs on osteogenic differentiation of DPSCs was determined through alkaline phosphatase(ALP)staining,ALP activity assay,alizarin red S(ARS)staining and Western blot.The optimal concentration of GOQDs for promoting osteogenic differentiation of DPSCs without cytotocxity was confirmed according to the above two experiments(recorded as OM+GOQDs).Gelatin methacryloyl(Gel MA)hydrogel were used as scaffolds to construct DPSCs/hydrogel complexes(DPSCs/Gel),and they were cultured in medium with or without apoptosis-inducing drugs,then the growth status of cells within the hydrogel was observed by Calcein AM/PI staining.To determine the osteogenic differentiation of DPSCs within hydrogel,DPSCs/Gel composites were divided into DPSCs/Gel group and GOQDs/DPSCs/Gel group.For the former,the hydrogel did not contain GOQDs and was incubated in OM;for the latter,the hydrogel contained GOQDs and was incubated in OM+GOQDs.And ALP staining was performed eight days after the induction.In vivo study was also divided into DPSCs/Gel group and GOQDs/DPSCs/Gel group,and these DPSCs/hydrogel composites were implanted into the calvarial defect of rats after 8 days of in vitro incubation.The skulls of rats was removed three weeks after the operation,and the reparation level of skulls was scanned by micro-CT,analyzed by HE staining,Masson staining and immunohistochemical staining of paraffin slices.Results: ALP staining and ARS staining showed that DPSCs had the potential of osteogenic differentiation.And Oil Red O staining showed DPSCs was induced into adipocytes-like cells,suggesting they own the capacity of adipogenic differentiation.And the results of Alcian blue staining showed that DPSCs had the ability of chondrogenic differentiation.Flow cytometry showed that the primary human DPSCs obtained in this study expressed surface marker molecules of mesenchymal stem cells including CD29,CD105 and CD146,but not that of hematopoietic stem cells including CD14 and CD45.CCK-8 results showed that GOQDs at 0.5,1 and 5 μg/m L had little negative effect on the viability of DPSCs,but GOQDs at 10 and 20 μg/m L inhibited the proliferation of DPSCs in a concentration-dependent manner.The results of ALP staining,ALP activity assay,ARS staining,mineralized nodule quantification and Western blot showed that GOQDs at 5,10 μg/m L could significantly promote DPSCs to differentiate into osteoblasts.Based on the above results,GOQDs at 5 μg/m L can enhance the osteoblastic differentiation of DPSCs without cellular toxicity.Therefore,GOQDs at 5 μg/m L were chosen for in vivo investigation.Calcein AM/PI staining showed that cells in the control group grew morphologically well in the hydrogel,while in the experimental group,apoptosis-inducing drugs in the culture medium successfully promoted apoptosis in the cells in the hydrogel,suggesting that nutrients or drugs of the medium could effectively act on the cells inside the hydrogel scaffold.ALP staining results showed that GOQDs/DPSCs/Gel group had higher ALP expression than DPSCs/Gel group,indicating that the addition of GOQDs to medium and hydrogel promoted the osteogenic differentiation of DPSCs within the hydrogel,which was consistent with the experimental results obtained with two-dimensional cultured cells.The above two experiments demonstrated that the hydrogel preparation strategy that we used allowed cells within hydrogels for growth and differentiation status.The results of micro-CT,HE staining,Masson staining and immunohistochemical staining showed that GOQDs/DPSCs/Gel was more effective in repairing rat calvarial defects than DPSCs/Gel.Conclusion: GOQDs at 5 μg/m L could effectively promote the osteogenic differentiation of DPSCs without cytotoxicity,and the cell/hydrogel composite GOQDs/DPSCs/Gel significantly promoted the repair of rat calvarial defects. |
