Experimental Study Of Sheep ADM Loaded With HucMSC-exos On The Repair Of Full-layer Skin Defect Wounds In Mice

Objective To prepare and characterize human umbilical cord mesenchymal stem cell exosomes（huc MSC-exos）;to prepare huc MSC-exos-loaded sheep decellular dermal matrix（s ADM）and examine its physicochemical properties;to use huc MSC-exos-loaded s ADM to repair traumatic skin defects on the back of mice,to observe the trauma repair and explore the relevant pro-repair mechanisms,and to provide a basic theoretical basis for trauma repair.To provide a basic theoretical basis for wound repair.Methods 1.human umbilical cord mesenchymal stem cells:（provided by the Experimental Center of Baogang Hospital,Inner Mongolia）umbilical cord mesenchymal stem cells were isolated from fresh umbilical cord tissues of healthy newborns and morphologically characterized,and the expression of surface markers CD34 and CD45 were observed.2.huc MSC-exos extraction and identification:huc MSC-exos was obtained by PEG precipitation（polymer precipitation method）combined with differential centrifugation.huc MSC-exos,transmission electron microscopy to observe huc MSC-exos morphology;particle size analysis instrument to analyze huc MSC-exos particle size and concentration;Western blot to detect huc MSC-exos surface marker CD63;3,s ADM preparation:enzymatic digestion to decellularize,HE staining to observe the degree of decellularization of s ADM;4 Preparation and physicochemical properties of s ADM loaded with huc MSC-exos:huc MSC-exos was loaded on s ADM,and its morphology was observed by scanning electron microscope（SEM）,and protein release was detected by protein release assay;5,animal experiments:SPF（Specified pathogen free）grade s ADM was taken.Specified pathogen free)grade BALB/c mice,54 males,were randomly divided into blank control group（n=18）,s ADM group（n=18）,and s ADM group loaded with huc MSC-exos（n=18）.（1）Establishment of a mouse model of total skin defect trauma:fasting water for 12 h before operation,each mouse was anesthetized by intraperitoneal injection of 0.3%sodium pentobarbital（0.1-0.2 ml/10 g）.After satisfactory anesthesia,a circular skin defect trauma model with a diameter of 1.5 cm,deep to the fascial layer,was prepared on the back of the mice;（2）treatment protocol:mice were given different treatments according to the experimental grouping method,and the medication was changed on alternate days;（3）index detection:photographs of the trauma were taken at each medication change to observe the trauma healing and calculate the trauma healing rate.The skin specimens of each group were collected at 7d,14d and 21d postoperatively,and the histopathological changes of each group were detected by HE staining,and the serum levels of tumor necrosis factor-α（TNF-α）and transforming growth factor-β（TGF-β）were detected by Elisa.The expression ofα-smooth muscle actin（α-SMA）was measured by Western blot in the control and s ADM groups loaded with huc MSC-exos at 7d,14d and 21d.Immunohistochemical staining was performed to detect the expression of CD34 protein in the traumatic tissues of each group at 7d,14d and21d.Results 1.huc MSC-exos was successfully extracted:huc MSC-exos vesicle-like structures were visible under transmission electron microscopy,with a bilayer-like circular shape and a diameter of about 100 nm.The diameters of the obtained huc MSC-exos were mostly concentrated around 120 nm,which was consistent with the size of huc MSC-exos;the concentration of huc MSC-exos was(1×10¹⁰Particles/m L);The extracted huc MSC-exos was enriched with transmembrane protein CD63,which was consistent with the characteristics of huc MSC-exos surface markers;2.Successful preparation of s ADM:after cell removal by enzymatic digestion,HE staining observed that the matrix was free of cellular components,no skin attachment,no blood vessels,and collagen fibers were structurally intact and neatly arranged;3.Successful preparation of loaded huc MSC-exos s ADM:SEM detection and comparison revealed that the morphology and pores did not change significantly after s ADM loaded with huc MSC-exos,but the surface had roughness and gravelly feeling,and a large number of spherical and sphere-like huc MSC-exos were seen to exist in s ADM.huc MSC-exos loaded with s ADM showed a significant sudden release of the protein in which the release amount on the first day was The release of protein was 74.99%on the first day and95.81%on the third day;4.The mouse model of total skin defect was successfully established:the visual photograph showed that the original trauma of the mouse back was a circular total skin defect with a diameter of 1.5 cm;5.At 7 d,14 d,and 21 d postoperatively,the wound healing rates in the control group were 9.06±0.90,44.98±0.52,and 68.45±0.39,respectively,while at 7 d,14 d,and 21 d postoperatively,the wound healing rates in the s ADM group loaded with huc MSC-exos were 47.5±0.44,64.96±0.38,and 98.12±0.45,respectively.The healing rates were 21.51±0.50,52.26±0.36,and 79.55±0.57,respectively;at 7d,14d,and 21d postoperatively,the wound healing rates in the s ADM group loaded with huc MSC-exos were significantly higher than those in the control group,and also higher than those in the s ADM group,with statistically significant differences（p<0.05）;（2）HE staining results showed that the inflammatory cell infiltration was relatively lighter in the s ADM group loaded with huc MSC-exos compared with the control group at 7 d,14 d and 21 d postoperatively;the follicular hyperplasia was significantly higher in the s ADM group loaded with huc MSC-exos compared with the control group;（3）The results of Elisa assay for TNF-αand TGF-βin serum showed that at 7d,14d and 21d postoperatively,the serum levels of TNF-αin the control group were 580.21±5.44,488.28±12.76 and 438.97±11.52,respectively,and at 7d,14d and21d postoperatively,the serum levels of TNF-αin the s ADM group loaded with huc MSC-exos TNF-αlevels in the s ADM group were 512.11±3.98,447.08±5.61,and357.79±8.70 at 7d,14d,and 21d postoperatively,respectively,and 550.89±5.07,479.85±8.98,and 394.57±3.64 at 7d,14d,and 21d postoperatively,respectively;at 7d,14d,and 21d postoperatively,the s ADM group loaded with huc MSC-exos At 7d,14d and 21d postoperatively,the serum levels of TNF-αin mice in the s ADM group loaded with huc MSC-exos showed a gradual decrease,and the differences were statistically significant when compared with the control group as well as the s ADM group（p<0.05）.At 7d,14d and21d postoperatively,the serum levels of TGF-βin the control group were 57.66±1.08,63.67±1.56 and 88.81±2.80,respectively,and at 7d,14d and 21d postoperatively,the serum levels of TGF-βin the s ADM group loaded with huc MSC-exos were 72.17±1.11,79.81±1.94,108.15±1.53,and at 7d,14d and 21d postoperatively,the serum TGF-βlevels in the s ADM group were 62.31±1.09,70.55±2.09 and 93.60±2.86,respectively;at 7d,14d and 21d postoperatively,the serum TGF-βlevels in mice in the s ADM group loaded with huc MSC-exos showed a gradually increasing The differences were statistically significant（p<0.05）when compared with the control group as well as the s ADM group;（4）Western blot results showed that the amount ofα-SMA protein expression was significantly lower in the s ADM group loaded with huc MSC-exos compared with the control group at 7d,14d and 21d after surgery;（5）Immunohistochemical staining results showed that CD34 protein was expressed in the control,s ADM and s ADM groups loaded with huc MSC-exos at all time points after injury.At 7 d,14 d,and 21 d postoperatively,CD34-positive vessels showed brownish-yellow luminal,striated,or individually stained CD34-positive vascular endothelial cells under the microscope,and the s ADM group loaded with huc MSC-exos had more CD34protein expression than the other two groups.Conclusions Loading human umbilical cord MSC exosomes onto sheep decellularized dermal matrix for the treatment of total skin defects in mice modulated the inflammatory response,promoted the regeneration of blood vessels in the traumatic area,improved re-epithelialization,and effectively promoted wound healing.