Experimental Study On Porous Se@SiO₂ Nanocomposites Combined With Decellularised Extracellular Matrix To Enhance The Myogenic Differentiation Of Adipose-derived Mesenchymal Stem Cells

Background Volumetric muscle loss(VML)is a pathological disease associated with traumatic skeletal muscle injury.Patients affected by VML are unable to restore muscle mass due to impaired muscle fiber regeneration,resulting in permanent muscle loss or even permanent disability.The current clinical treatment has limited ability to regenerate damaged muscles and restore tissue function.Therefore,seeking to develop a new method of VML treatment is of great significance to human health and social development.Purpose The aim of this study was to investigate the effects of porous Se@SiO2 nanocomposites(SeNPs)and decellularised extracellular matrix(dECM)on inducing proliferation,myogenic differentiation and antioxidant stress of adipose mesenchymal stem cells(ADSCs)compared with traditional myoblast inducer 5-azacytidine(5-aza).Methods1 Preparation of materials1.1 Preparation of porous selenium nanocomposites:2ml Cu2-xSe nanocrystals synthesized in nitrogen were completely mixed with 20ml n-hexane,20ml nhexanol,2ml TritonX-100,0.6ml deionized water and 0.08ml ethyl orthosilicate.Then,0.1ml ammonia was added,orthosilicate was hydrolyzed,and silica was encapsulated in Se quantum dots prepared by oxidation to form solid Se@SiO2 nanospheres.Finally,they were dispersed in polyvinylpyrrolidone solution of 10mg/ml and etched in hot water to form porous structure.1.2 Preparation of decellularised extracellular matrix:ADSCs was cultured in a petri dish pretreated with 0.2%gelatin,and 100-μM L-ascorbic acid was added to the culture medium for 7 days.After that,adipose mesenchymal stem cells were incubated in 37℃ extraction buffers(0.5%TritonX-100,20mM NH4OH,PBS,pH7.4)for 5 minutes.Then treated with 100U/mL DNaseI at 37℃ for 1 hour.After washing with PBS for three times,the residual extraction buffer was removed and the acellular matrix was obtained.2 Experimental groups:group A:blank control;group B:5-Aza;group C:5Aza+dECM;D group:5-Aza+SeNPs;E group:5-Aza+dECM+SeNPs.Cultured in DMEM medium,the concentration of 5-Aza was 10 μM and the concentration of SeNPs was 40 μg/mL.3 Detection index:XRD diffraction pattern,scanning electron microscope analysis of dECM and SeNPs characterization;live cell staining to analyze the effect of Se@SiO2&dECM on ADSCs cell viability;immunofluorescence staining to analyze the expression levels of dECM-related components type Ⅰcollagen,type Ⅲ collagen,fibronectin and laminin,as well as the expression levels of MHC,Myosin and Desmin related to myogenic differentiation.Western-blot test was used to analyze the expression of myoblast differentiation-related protein MYOD and antioxidant-related protein SDO1;ELISA test was used to analyze the expression of dECM-related components IGF-1 and TGF-β,and mitochondrial respiratory function in ADSCs.Mitochondrial tracer technique was used to observe the changes of mitochondrial number and reactive oxygen species in cells.Results XRD diffraction pattern analysis showed that the structure of the prepared SeNPs phase showed hexagonal phase,which was the standard selenium phase.The scanning electron microscope results showed that the SeNPs diameter was about 55nm,and the dECM structure was reticular,and the lattice was composed of nanofibers and small bundles of collagen fibers.the results of living dead cell staining showed that Se@SiO2&dECM could promote the activity of ADSCs cells.The results of immunofluorescence staining showed that the prepared dECM contained type Ⅰ collagen,type Ⅲ collagen,fibronectin and laminin,Se@SiO2&dECM could increase the expression of MHC,Myosin and Desmin in ADSCs,and Western-blot test showed that Se@SiO2&dECM could increase the expression of MYOD and SDO1 in ADSCs.The results of ELISA test showed that the prepared dECM contained cytokines IGF-1 and TGF-β,and the respiratory function of mitochondria in ADSCs treated with SeNPs and dECM increased,and the number of mitochondria in ADSCs treated with SeNPs and dECM increased and the level of reactive oxygen species decreased.Conclusion Both SeNPs and dECM can promote the proliferation of ADSCs;SeNPs has a positive effect on myogenic differentiation and intracellular mitochondrial function of ADSCs,and can effectively reduce the effect of reactive oxygen species on ADSCs;the combined use of SeNPs and dECM can significantly promote the myogenic differentiation of ADSCs,reduce the negative effect of 5-Aza on myogenic induction of ADSCs,and enhance the antioxidant capacity and mitochondrial respiratory function of ADSCs.The effect of SeNPs combined with dECM is much better than that of 5-azacytidine,a traditional myogenic inducer.