3D Printed Hydrogel Scaffolds Loading Stem Cells-derived Exosomes Promote Diabetic Wound Healing

Part I The study of high glucose on the biological function of vascular endothelial cellsObjective:Vascular dysfunction caused by hyperglycaemia in diabetic patients is an important factor of diabetic wound healing difficulty.The present study aimed to investigate the effect of high glucose on the biological function of HUVECs and further elucidate the mechanism of hyperglycaemia on vascular dysfunction.Methods:HUVECs were treated with different concentrations of high glucose.The HUVECs viability was detected by CCK-8 assay.The effect of high glucose on the HUVECs proliferation was assessed by EdU staining.Flow cytometry was examined the effect of high glucose on the cell cycle of HUVECs.Western blot was used to detect the protein expression of Cyclin D1 and Cyclin D3 caused by high glucose.The effect of high glucose on the migration ability of HUVECs was assessed by Transwell migration assay and wound healing assay.The effect of high glucose on angiogenic ability of HUVECs was investigated by tube formation assay of HUVECs.Western blot was used to detect the expression of VEGF,an important factor in angiogenesis.Results:The results of CCK-8 showed that high glucose supressed the HUVECs viability.The inhibiting effect of HUVECs viability was more obvious with the increasing concentration of high glucose.The results of EdU staining and flow cytometry showed that high glucose inhibited the HUVECs proliferation and caused HUVECs to stop in S phase.In addition,HG also decreased the expression of cyclins protein,such as Cyclin D1 and Cyclin D3.The result of transwell and wound healing assay indicated that HG reduced the migration ability of HUVECs.We also found that high glucose significantly inhibited the angiogenic ability of HUVECs and reduced the expression of VEGF.Conclusion:High glucose inhibited viability and proliferation of HUVECs,reduced migration and angiogenic capacity of HUVECs,which resulted in vascular dysfunction.Part II The study of exosomes derived from stem cells on high glucose-induced vascular endothelial cell dysfunctionObjective:Exosomes are important secreta of MSCs and regulate intercellular communication.The present study aimed to investigate the role of MSCs-derived exosomes on vascular endothelial cell dysfunction induced by high glucose.Methods:BMSCs were isolated and extracted from bone marrow.The specific surface markers of stem cell was detected by flow cytometry.The osteogenic,adipogenic and chondrogenic differentiation was assessed trilineage differentiation ability of BMSCs.Exosomes were extracted from the culture medium of BMSCs by ultracentrifugation.The morphology of BMSCs-Exos was observed by TEM.Particle size analysis of exosomes was performed by NTA.Western blot was used to detect the exosomal specific markers,such as CD9 and CD63.PKH26 was used to observe BMSCs-Exos uptake by HUVECs.CCK-8 assay was detected the effect of BMSCs-Exos on HUVECs viability induced by high glucose.EdU staining was assessed the effect of BMSCs-Exos on HUVECs proliferation.Flow cytometry was used to investgate the effect of BMSCs-Exos on the cell cycle of HUVECs induced by high glucose.Western blot was used to detect the protein expression of Cyclin D1 and Cyclin D3 caused by BMSCs-Exos.Transwell migration assay and wound healing assay was detected the effect of exosomes on migration ability of HUVECs induced by high glucose.The tube formation assay of HUVECs was evaluated the effect of exosomes on angiogenesis ability of HUVECs induced by high glucose.Western blot was used to detect the protein expression of VEGF induced by BMSCs-Exos.Results:We successfully isolated and cultured BMSCs from bone marrow.The BMSCs highly expressed surface markers,including CD73 and CD90,and expressed less CD34 and CD45,having the capacity of osteogenic,adipogenic and chondrogenic differentiation.The results of TEM showed that BMSCs-Exos our extracted had a spherical and vesicular structure.The results of NTA indicated that the particle size of BMSCs-Exos was conforming the characteristics of exosomes.In addition,BMSCs-Exos could express CD9 and CD63.These results indicated that BMSCs-Exos our extracted conformed exosomal characteristics.The results of CCK-8 showed that BMSCs-Exos reduced the inhibitory effect of high glucose on cell viability.EdU staining and flow cytometry showed that BMSCs-Exos relieved the inhibitory effect of high glucose on HUVECs proliferation and reduced the arrest of HUVECs in S phase.And BMSCs-Exospromoted the cyclins protein expression of Cyclin D1 and Cyclin D3.The result of transwell and wound healing assay indicated that BMSCs-Exos reduced the inhibitory effect of high glucose on the migration ability of HUVECs.The results of tube formation assay showed that BMSCs-Exos alleviated the inhibitory effect of high glucose on the tube formation ability of HUVECs and promoted VEGF expression.Conclusion:We successfully extracted exosomes from the culture medium of BMSCs and found that BMSCs-Exos alleviated the inhibitory effect of high glucose on the viability and proliferation of HUVECs.And BMSCs-Exos also reduced the effect of high glucose on the migration and angiogenic ability of HUVECs.Part III Preparation and characterization of cryogenic 3D printed hydrogel scaffolds loaded stem cell-derived exosomesObjective:Cryogenic 3D printing technology was used to construct SIS/MBG composite hydrogel scaffolds as slow release carrier for BMSCs-Exos.The hydrogel scaffolds was further investigated the properties and effects of the hydrogel scaffolds on the biological functions of HUVECs.Methods:The decellularized small intestinal submucosa of the small intestine was prepared by enzymatic digestion.The mesoporous bioactive glass was produced by template self-assembly method.SIS/MBG hydrogel scaffolds was constructed as a slow release carrier for BMSCs-Exos using the cryogenic 3D printing platform.The morphology and elemental analysis of MBG on SIS were examined by TEM and EDS.SEM was used to observe the SIS/MBG hydrogel scaffolds structure.DHR rheometry was detectd the shear viscosity of the SIS/MBG slurry.XRD was assessed the crystal structure of 3D printed SIS/MBG hydrogel scaffolds,and FTIR was analyzed the chemical structure of 3D printed SIS/MBG hydrogel scaffolds.The BCA assay was used to detect the exosomes release from the SIS/MBG@Exos hydrogel scaffolds.DAPI and Calcein-AM/PI staining were used to assess the adhesion and live/death of HUVECs on the SIS/MBG@Exos hydrogel scaffolds.Liver haemostasis model was assessed the haemostatic effect of SIS/MBG@Exos hydrogel scaffold.After SIS/MBG@Exos hydrogel scaffolds was co-cultured with HUVECs,CCK-8 was assessed the effect of SIS/MBG@Exos on the cell viability of HUVECs.Transwell migration assay and wound healing assay was detected the effect of SIS/MBG@Exos on the migration of HUVECs.The tube formation assay of HUVECs was assessed the effect of SIS/MBG@Exos on the vascular formation ability of HUVECs.Results:The result of TEM showed that the MBG our prepared had a highly ordered mesoporous structure.SEM and EDS confirmed that MBG could better adhere to the 3D printed SIS/MBG hydrogel scaffolds.In addition,the results of SEM showed that the 3D printed SIS/MBG hydrogel scaffolds had a macroscopic pore structure and MBG could increased the microscopic porosity and surface roughness of hydrogel scaffolds.DHR rheometry showed that MBG had no effect on the formation of the SIS slurry during 3D printing.XRD showed that the SIS/MBG hydrogel scaffolds had a characteristic peak of Si02.FTIR indicated that the SIS/MBG hydrogel scaffolds retained the active components of SIS and MBG well.In addition,we found that the SIS/MBG@Exos hydrogel scaffolds had a slow release effect on BMSCs-Exos.The results of MTT assay showed that HUVECs could grow and proliferate on the SIS/MBG@Exos hydrogel scaffold.Cell adhesion assays and Calcein-AM/PI staining indicated that HUVECs could adhere to the hydrogel scaffold well and the hydrogel scaffolds had no toxic effect on HUVECs.The hemorrhagic liver model demonstrated that SIS/MBG@Exos hydrogel scaffolds have good hemostasis performance.The results of CCK-8,tube-forming,and transwell and wound healing migration assays showed that SIS/MBG@Exos hydrogel scaffolds and the exosomes released from it enhanced the cell viability,improved angiogenesis and promoted migration in HUVECs.Conclusion:We used cryogenic 3D printing technology to construct SIS/MBG@Exos hydrogel scaffolds and found that SIS/MBG@Exos hydrogel scaffolds had the ability to release BMSCs-Exos.Besides,SIS/MBG@Exos hydrogel scaffolds had good biocompatibility and haemostatic properties,as well as enhancing the biological function of HUVECs.Part IV The study of 3D printing hydrogel scaffolds promote diabetic wound healingObjective:The study of the effect and mechanism of 3D printed SIS/MBG@Exos hydrogel scaffolds on the diabetic wounds healing.Methods:SD rats were used to construct diabetic wound model.The wound was photographed and recorded at 0,3,7,10 and 14 days postoperatively.The wound closure was calculated.Doppler detection was used to detect blood perfusion of wound.HE staining was used to assessed the healing effect of diabetic wounds.Masson staining was used to observe collagen production.Immunohistochemical staining of Collagen I and Collagen III was used to study the collagen of wounds.Immunohistochemical staining of VEGF and CD31 was analyzed the angiogenesis of the wounds.Immunofluorescence of a-SMA was observed the neovascularisation of the wounds.Results:The results showed that 3D printed SIS/MBG@Exos hydrogel scaffolds promoted diabetic wound healing.The healing effect of wound in SIS/MBG@Exos group was significantly higher than that of the SIS/MBG and exosomes alone group.The results of doppler detection indicated that SIS/MBG@Exos promoted blood perfusion of diabetic wounds.HE staining showed that SIS/MBG@Exos hydrogel scaffolds promoted diabetic wound repair as well as epithelialisation compared to SIS/MBG alone and exosomes.Masson staining showed that SIS/MBG@Exos hydrogel scaffolds promoted collagen deposition and enhance extracellular matrix remodelling in wounds.Immunohistochemistry showed that SIS/MBG@Exos hydrogel scaffold increased the expression of collagen I and Collagen III as well as VEGF and CD31.The results of immunofluorescence showed that the SIS/MBG@Exos hydrogel scaffolds increased the expression of α-SMA in trabecular neovascularisation.Conclusion:3D printed SIS/MBG@Exos hydrogel scaffolds promotes diabetic wounds healing by enhancing angiogenesis.