The Construction Of Polycaprolactone Barrier Membranes Incorporated With Mesenchymal Stem Cell-derived Exosome And S-nitrosoglutathione And Detection Of Its Osteogenic Capacity And Anti-inflammatory Capacity

Objective: To construct PCL/PDA+Exo+GSNO modified barrier membrane and detect its effect on RAW264.7 inflammation regulation and the osteogenic differentiation of h BMSCs,with purpose to provide theoretical reference for new polycaprolactone-based bio-barrier membrane constructing strategy.Methods:(1)Isolation and purification of Exo from h BMSCs using kit extraction method;using electrostatic spinning technique to prepare PCL barrier membranes.(2)PDA coating was used to immobilize Exo and GSNO on PCL barrier membrane to construct four modified composite barrier membranes:(a)PCL/PDA(control group);(b)PCL/PDA + GSNO;(c)PCL/PDA + Exo;(d)PCL/PDA + GSNO + Exo.(3)Inoculation of RAW264.7 cells and h BMSCs cells on the four groups of barrier membranes,respectively.(4)Using q RT-PCR to examine RAW264.7 inflammatory factors(IL-1β,IL-6,TNF-α and i NOS)induction levels caused by the four groups of composite biofilms:(a)PCL/PDA(control);(b)PCL/PDA + GSNO;(c)PCL/PDA +Exo;(d)PCL/PDA + GSNO +Exo.(5)ALP activity of h BMSCs was detected by ALP staining and ALP assay kit,and the expression of h BMSCs osteogenesis-related genes(ALP,OPN,Col-1 and RUNX2)was detected by q RT-PCR.Results:(1)The Exo were extracted and purified from h BMSCs,and results showed that the Exo were oval or cup-shaped under electron microscopy,with peaks in particle size occurring at 50 nm and 120 nm;(2)Electrospinning technique was used to prepare(a)PCL/PDA;(b)PCL/PDA + GSNO;(c)PCL/PDA + Exo;(d)PCL/PDA + GSNO +Exo four sets of biofilms.(3)After inoculation of RAW264.7 cells(after LPS stimulation)on four groups of biofilms for 24 h,the biofilm inflammatory factor levels were detected significantly lower in(b)PCL/PDA + GSNO;(c)PCL/PDA + Exo;(d)PCL/PDA + GSNO + Exo groups compared to that in(a)PCL/PDA(control group),P<0.05.And(d)PCL/PDA + GSNO +Exo group exhibited the most significant decrease in IL-6 gene expression level,which was about 1/5of(a)PCL/PDA(control group);in addition,the expression levels of TNF-α,i NOS and IL-1β in(d)group were about 1/2,1/3 and 1/4 of the control group,respectively,P<0.05.(4)After inoculation of h BMSCs cells on biofilms of four groups(a)PCL/PDA,(b)PCL/PDA+GSNO,(c)PCL/PDA+Exo and(d)PCL/PDA+GSNO+Exo for 3 days,(d)PCL/PDA+GSNO+Exo group showed significantly higher expression levels of the osteogenic gene ALP,which is 2-fold higher than the control group,P<0.001;(d)the expression level of Col-1 gene in the(d)PCL/PDA+GSNO+Exo group was about 1.7-fold higher than that in the control group,P<0.001;(d)the expression level of RUNX2 gene in the(d)PCL/PDA+GSNO+Exo group was about 1.5 times higher than that in the control group,P<0.01;(d)the expression level of BMP-2 gene expression level in the PCL/PDA+GSNO+Exo group was about 2.2-fold higher than that in the control group,P<0.05.(5)After h BMSCs cells were inoculated on biofilms of four groups(a)PCL/PDA,(b)PCL/PDA+GSNO,(c)PCL/PDA+Exo and(d)PCL/PDA+GSNO+Exo for 7 days,(b)PCL/PDA+GSNO,(c)PCL/PDA+Exo and(d)PCL/PDA +GSNO+Exo group had significantly higher ALP activity compared to(a)PCL/PDA group;and(d)PCL/PDA+GSNO+Exo group had the strongest ALP activity,which was about 1.7 times higher than the control group,P<0.01.Conclusion:(1)We constructed(a)PCL/PDA;(b)PCL/PDA + GSNO;(c)PCL/PDA +Exo;(d)PCL/PDA + GSNO + Exo composite barrier membranes;(2)Results showed that PCL/PDA + GSNO + Exo composite barrier membrane has excellent ability to reduce inflammation and boost osteogenesis,and is expected to be a novel membrane material.