Exosomes of adult human fibroblasts cultured on 3D silk fibroin nonwovens intensely stimulate neoangiogenesis

Background:Bombyx mori silk fibroin is a biomacromolecule that allows the assembly of scaffolds for tissue engineering and regeneration purposes due to its cellular adhesiveness,high biocompatibility and low immunogenicity.Earlier work showed that two types of 3D silk fibroin nonwovens(3D-SFnws)implanted into mouse subcutaneous tissue were promptly vascularized via undefined molecular mechanisms.The present study used nontumorigenic adult human dermal fibroblasts(HDFs)adhering to a third type of 3D-SFnws to assess whether HDFs release exosomes whose contents promote neoangiogenesis.Methods:Electron microscopy imaging and physical tests defined the features of the novel carded/hydroentangled 3D-SFnws.HDFs were cultured on 3D-SFnws and polystyrene plates in an exosome-depleted medium.DNA amounts and D-glucose consumption revealed the growth and metabolic activities of HDFs on 3D-SFnws.CD9-expressing total exosome fractions were from conditioned media of 3D-SFnws and 2D polystyrene plates HDF cultures.Angiogenic growth factors(AGFs)in equal amounts of the two groups of exosomal proteins were analysed via doubleantibody arrays.A tube formation assay using human dermal microvascular endothelial cells(HDMVECs)was used to evaluate the exosomes’angiogenic power.Results:The novel features of the 3D-SFnws met the biomechanical requirements typical of human soft tissues.By experimental day 15,3D-SFnws-adhering HDFs had increased 4.5-fold in numbers and metabolized 5.4-fold more D-glucose than at day 3 in vitro.Compared to polystyrene-stuck HDFs,exosomes from 3D-SFnws-adhering HDFs carried significantly higher amounts of AGFs,such as interleukin(IL)-1α,IL-4 and IL-8;angiopoietin-1 and angiopoietin-2;angiopoietin-1 receptor(or Tie-2);growth-regulated oncogene(GRO)-α,GRO-βand GRO-γ;matrix metalloproteinase-1;tissue inhibitor metalloproteinase-1;and urokinase-type plasminogen activator surface receptor,but lesser amounts of anti-angiogenic tissue inhibitor metalloproteinase-2 and pro-inflammatory monocyte chemoattractant protein-1.At concentrations from 0.62 to 10μg/ml,the exosomes from 3D-SFnws-cultured HDFs proved their angiogenic power by inducing HDMVECs to form significant amounts of tubes in vitro.Conclusions:The structural and mechanical properties of carded/hydroentangled 3D-SFnws proved their suitability for tissue engineering and regeneration applications.Consistent with our hypothesis,3D-SFnws-adhering HDFs released exosomes carrying several AGFs that induced HDMVECs to promptly assemble vascular tubes in vitro.Hence,we posit that once implanted in vivo,the 3DSFnws/HDFs interactions could promote the vascularization and repair of extended skin wounds due to burns or other noxious agents in human and veterinary clinical settings.

Neural grafts containing exosomes derived from Schwann cell-like cells promote peripheral nerve regeneration in rats

Background:Schwann cell-like cells(SCLCs),differentiated from mesenchymal stem cells,have shown promising outcomes in the treatment of peripheral nerve injuries in preclinical studies.However,certain clinical obstacles limit their application.Hence,the primary aim of this study was to investigate the role of exosomes derived from SCLCs(SCLCs-exo)in peripheral nerve regeneration.Methods:SCLCs were differentiated from human amniotic mesenchymal stem cells(hAMSCs)in vitro and validated by immunofluorescence,real-time quantitative PCR and western blot analysis.Exosomes derived from hAMSCs(hAMSCs-exo)and SCLCs were isolated by ultracentrifugation and validated by nanoparticle tracking analysis,WB analysis and electron microscopy.A prefab-ricated nerve graft was used to deliver hAMSCs-exo or SCLCs-exo in an injured sciatic nerve rat model.The effects of hAMSCs-exo or SCLCs-exo on rat peripheral nerve injury(PNI)regeneration were determined based on the recovery of neurological function and histomorphometric variation.The effects of hAMSCs-exo or SCLCs-exo on Schwann cells were also determined via cell prolifer-ation and migration assessment.Results:SCLCs significantly expressed the Schwann cell markers glial fibrillary acidic protein and S100.Compared to hAMSCs-exo,SCLCs-exo significantly enhanced motor function recov-ery,attenuated gastrocnemius muscle atrophy and facilitated axonal regrowth,myelin forma-tion and angiogenesis in the rat model.Furthermore,hAMSCs-exo and SCLCs-exo were effi-ciently absorbed by Schwann cells.However,compared to hAMSCs-exo,SCLCs-exo signifi-cantly promoted the proliferation and migration of Schwann cells.SCLCs-exo also significantly upregulated the expression of a glial cell-derived neurotrophic factor,myelin positive regulators(SRY-box transcription factor 10,early growth response protein 2 and organic cation/carnitine transporter 6)and myelin proteins(myelin basic protein and myelin protein zero)in Schwann cells.Conclusions:These findings suggest that SCLCs-exo can more efficiently promote PNI regeneration than hAMSCs-exo and are a potentially novel therapeutic approach for treating PNI.