Effects Of Tunneling Nanotubes On MSC/HUVEC Spheroids Activating Osteogenesis Via Mitochondria-regulated ?-catenin

Introduction: Tunneling nanotubes(TNTs)are a kind of intercellular tubular structures that physically connect two cells to share organelles,viruses,calcium ions,proteins,and other cytoplasmic components,thus allowing direct cell-to-cell communication and ultimately changing the fate of the connected cells.Previous studies have focused on the delivery role of TNTs and their significance in physiological and pathological processes,but there is still a lack of deeper research on how to apply TNTs practically based on their function.Mesenchymal stem cells(MSCs),as one of the classical seed cells in the field of bone tissue engineering,require sufficient energy supply during the differentiation process,and the energy metabolism of the cells is mainly achieved through mitochondria.Previous studies have shown that TNTs could act as one of the channels for transcellular transport to deliver exogenous mitochondria into MSCs intracellularly.Endothelial cells(ECs)will possess increased mitochondrial biogenesis during proliferation and more frequent mitochondrial fusion and division,and thus ECs have sufficient intracellular mitochondrial content to serve as an ideal source of exogenous mitochondria.In summary,we propose a hypothesis that transferring mitochondria from endothelial cells to MSCs using TNTs and thus improving their cellular function may be a potential method to stimulate osteogenesis of MSCs.Objective: 1.To construct tunneling nanotubular expressway between MSCs and ECs using MSC/ECs co-culture cellular spheroids;2.To verify that endogenous mitochondrial deliver can be performed based on TNTs between heterogeneous cells;3.To evaluate the effects of TNTs-mediated mitochondrial transfer on cellular functions of MSCs and ECs,and the potential mechanisms underlying these phenomena.Methods: MSC/human umbilical vein endothelial cell(MSC/HUVEC)cellular spheroids were used as a study model to investigate the application and potential mechanisms of TNTs.1.Characterization of TNTs:Phalloidin staining was used to label the intercellular TNTs structure.Laser Scanning Confocal Microscopy(LSCM)was used for three-dimensional reconstruction of TNTs;Field emission scanning electron microscopy(FESEM)was used for direct observation of these intercellular junctions;CD31 immunofluorescence staining was used to specifically label HUVECs,Phalloidin staining labeled TNTs,and LSCM observed their accurate localization within cell spheroids;PKH26 cell membrane staining was used to label MSCs and recombinant GFP adenovirus infection was used to label HUVECs,and 3D reconstruction technique was used to further verify the precise localization of TNTs.2.Mitochondrial transfer between MSCs and HUVECs: using mitochondria-localized recombinant adenovirus to infect one kind of cells,and recombinant GFP adenovirus was used to label another kind of cells for the observation of bidirectional transfer of mitochondria between HUVECs and MSCs under LSCM;mitochondrial membrane potential assay was used to detect the effect of TNTs-mediated mitochondrial transfer on cellular state;MSCs were cocultured with HUVECs that had taken up small molecule biomaterials,and LSCM was used to verify the ability of TNTs to deliver biomaterials.3.Validation of the effects of TNTs on cellular functions and the preliminary studies of potential mechanisms: the expression of osteogenesis-related genes,collagen type I(COL-I)and Runt-associated transcription factor 2(RUNX2),in MSCs was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR);alkaline phosphatase staining was applied to detect the effect of TNTs on osteogenesis of cell spheroids.Matrigel angiogenesis assay was performed to detect the angiogenic ability of HUVECs;the total amount and sub-localization of the osteogenic proteins ?-catenin and RUNX2 in cell spheroids were further detected using Western Blot(WB)and immunofluorescence staining.Results: 1.In this experiment,MSC/HUVEC co-cultured cell spheroids were successfully constructed,and the LSCM 3D reconstruction and FE-SEM results demonstrated the existence of a stereoscopic TNTs network within this co-culture spheroids.2.The LSCM fluorescence colocalization results showed that mitochondrial translocation between MSCs and HUVECs could occur in both directions,and this TNTsmediated transfer phenomenon has a significant effect on the maintenance of intracellular mitochondrial membrane potential,while the mitochondrial membrane potential decreased significantly when TNTs were blocked(p <0.05);fluorescence co-localization results showed that exogenous biomaterials took up by HUVECs were found to be partially present in the cytoplasm of MSCs,demonstrating that TNTs could serve as a channel for direct delivery of biomaterials,but that co-localization between MSCs and biomaterials was significantly weakened when TNTs were disturbed.3.The results of Real-time PCR showed that osteogenesis-related genes COL-I and RUNX2 were significantly upregulated in the presence of TNTs(p < 0.05);ALP staining results also demonstrated that the existence of TNTs could effectively maintain the osteogenic activity of cells;the results of Matrigel angiogenesis assay showed that the effect of TNTs on maintaining the proliferative activity of HUVECs and promoting their angiogenic effect;Western Blot results showed that the total protein amount of ?-catenin and RUNX2 in co-culture cell spheroids decreased significantly when the formation of TNTs was blocked(p<0.05);immunofluorescence staining showed that the subcellular localization of ?-catenin and RUNX2 in co-culture cell spheroids also changed when the formation of TNTs was interrupted.Conclusion: This experiment successfully constructed MSC/HUVEC co-culture cell spheroids and TNTs between MSCs and HUVECs,which built an "expressway" for mitochondrial transfer from HUVECs to MSCs.These TNTs-mediated mitochondrial transfer improved the osteogenic ability of MSCs and the angiogenic ability of HUVECs.We also preliminarily explored that the underlying mechanism of TNTs-mediated mitochondrial transfer on cell function may be through the ?-cateninrelated signaling pathway.All the results provide some ideas for bone tissue engineering and regenerative medicine research.