| Background & Objectives: Tissue-engineered tracheas could serve as amazing restorative materials for surgical reconstruction of tracheal defects after extensive resection,improving the prognosis and quality of life of patients with long-segmental tracheal defects.However,there are still lots of questions in engineering trachea with electrospun scaffolds and mesenchymal stem cells.For instance,the chondrogenic incubation in vitro induced by TGF-?3 is a time-consuming process,and the phenotype of formed chondrocytes is unstable.In the present research,to optimize the chondrogenic differentiation of human umbilical cord mesenchymal stem cells(h UCMSCs),kartogenin(KGN)preconditioning was performed prior to TGF-?3induction.And core-shell electrospun nanofilms encapsulating TGF-?3 were fabricated as scaffolds to optimize the engineering process of tracheal patches.Methods: The primary h UCMSCs was obtain by tissue explant attachment culture method,and identified by flow cytometry.After h UCMSCs were preconditioned with1 ?M of KGN for different periods,the proliferation ability and apoptosis levels were detected by CCK-8 and Annexin/PI methods,and expression of specific marker of precartilaginous stem cells was detected by Western blotting assay.h UCMSCs were preconditioned with 1 ?M of KGN for 3 days,sequentially pelleted,and incubated with TGF-?3 for 28 days.Then,the expression of chondrogenesis/ossification-related genes was evaluated by immunohistochemistry and RT-PCR.The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphokinase screening and validated in vitro and in vivo using JNK inhibitor(SP600125)and?-catenin activator(SKL2001).Electrospun nanofilms encapsulating TGF-?3 with different compositions(PLCL : collagen 75:25,50:50,25:75)were fabricated by coaxial electrospinning technology.The structure surface morphology and degradation of nanofilms were evaluated by scanning electron microscope(SEM),transmission electron microscope(TEM),and laser scanning confocal microscope(LSCM).The hydrophilic performance and tensile mechanical properties of different scaffolds was evaluated by water contact angle testing and elongation testing.The cumulative release profiles of TGF-?3 were evaluated by enzyme-linked immuno sorbent assay(ELISA)method in vitro.The biocompatibility of different scaffolds was comparatively evaluated by CCK-8 and immunofluorescence of cytoskeleton methods.After the preferable compositional ratio was determined,cell-scaffold-construct was engineered with KGN-preconditioned h UCMSCs and electrospun nanofilms encapsulating TGF-?3 by Sandwich model,and then embedded subcutaneously in nude mice for maturation in vivo.The obtained tracheal patches were applied to restore tracheal defects in rabbit models.Results: The apoptotic rate of KGN preconditioned h UCMSCs increased gradually with prolonged exposure but without significant difference.While the proliferation tended to decline,and the proliferative ability of h UCMSCs preconditioned for 4 days was significantly lower than the other groups.After KGN preconditioning for 3 days,expression of FGFR3,a marker of precartilaginous stem cells,was up-regulated in h UCMSCs.Furthermore,the KGN preconditioned h UCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene(SOX9,aggrecan,and collagen II)expression and reduced expression of ossific genes(collagen X and MMP13)compared with h UCMSCs treated with TGF-?3 only.Phospho-kinase screening indicated the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of ?-catenin levels.Blocking/activating tests revealed that the pro-chondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/RUNX1 pathway,and anti-ossific effects were imparted by suppressing the ?-catenin/RUNX2 pathway.The biocompatibility of different scaffolds with different compositional ratios was all satisfactory,and core-shell scaffolds with a PLCL/collagen proportion of 75:25 were eligible for tracheal patches,due to their superior mechanical properties,ideal release behavior and proper degradation speed.The stable and sustained release of TGF-?3 from scaffolds could efficiently promote the chondrogenic differentiation of MSCs and shorten the incubation time.Eventually,tracheal patches,engineered by KGN preconditioned h UCMSCs and TGF-?3 encapsulated electrospun nanofilms,were successfully used for restoring tracheal defects in rabbit models.Tracheal integrity and phenotype of formed chondrocytes were well maintained for 2 months after restoration;meanwhile,re-epithelialization also accomplished,ensuring the structural and functional reconstruction of airway.Conclusion: KGN preconditioning likely improves the chondrogenic differentiation of h UCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed ?-catenin,and endochondral ossification of the formed cartilage tissue is simultaneously attenuated.This novel tissue-engineered trachea can be rapidly fabricated by KGN-preconditioned MSCs and electrospun scaffolds encapsulating TGF-?3,and applied for restoring tracheal defects in rabbit models. |
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