3D Printed Composite Aerogels To Construct Tracheal Scaffolds For Tracheal Regeneration

The trachea is a hollow-structured organ that plays a vital role in respiration,phonation,and airway protection.The tracheal defects are usually caused by tumor,infection,and trauma,which can significantly affect the quality of life of an individual.When the defected region is more than50 % of the total tracheal length in adults or about 30 % in pediatric patients,it is almost impossible regenerate the defect by the conventional surgical methods,including an end-to-end anastomosis.This necessitates the implantation of tracheal substitutes for airway reconstruction.Tissueengineered scaffolds possessing sufficient mechanical strength and a proper luminal contour have gathered considerable attention of the research community for tracheal regeneration applications.In this study,we have combined three-dimensional（3D）printing,electrospinning,and freezedrying techniques to fabricate tracheal scaffolds.Among them,tubular scaffolds with different strand width（0.5 mm,1.0 mm,1.5 mm）were prepared by 3D printing to strengthen the mechanical properties of tracheal scaffolds;the short nanofibers（SF）dispersion of poly（L-lactide）/gelatin（0.5-1.5 wt%）were prepared by electrospinning followed by homogenization to mimic the tracheal microenvironment.Moreover,the tubular scaffolds（TBS）and the short nanofiber dispersion were poured into the mold,and then freeze-dried and crosslinked to obtain tracheal scaffolds.By evaluating the radial mechanical properties and structural stability of different tubular scaffolds,the TBS with strand width of 1.0 mm were more suitable as a mechanical stent for tracheal scaffolds;the TBS and the short nanofibers of 0.5%（SF0.5）,1.0%（SF1.0）,1.5%（SF1.5）were poured in the mould,freeze-dried,glutaraldehyde crosslinked,and soaked with glutamic acidand rinsed with deionized water.Consequently,we obtained tracheal scaffolds（TSF0.5,TSF1.0,TSF1.5）.To improve the structural stability of the tracheal scaffolds,this study further optimized the treatment of the scaffolds at 60 ℃ for 0.5 h by heating treatment at different temperatures.In terms of the morphology,length,thickness,and diameter,the tracheal scaffolds can replicate the bionics of native trachea.Moreover,the scaffolds characterization results displayed that the short nanofiber aerogels of tracheal scaffolds exhibited low density,high porosity,good water absorption and water uptake,replicating an extracellular matrix（ECM）-like microenvironment of the trachea.In addition,mechanical tests revealed that the elastic recovery rate,maximum stress,and Young’s modulus of the wet TSF1.0 were similar to the native trachea in the radial compression testing.The mechanical properties of longitudinal tensile testing also better better than that of native trachea;the tracheal scaffolds exhibited better structural stability matched the cartilage regeneration cycle in the 12 w in vitro degradation testing.The protein adsorption,hemolysis,cell seeding efficiency,cell proliferation,cell adhesion were carried out to investigate the hemocompatibility and biocompatibility of tracheal scaffolds in vitro,which showed that they possess good protein adsorption,good hemocompatibility,high cell seeding efficiency and good biocompatibility,which may be very beneficial for the proliferation of chondrocytes and the regeneration of cartilage.Chondrocytes-seeded tracheal scaffolds cultured for7 days exhibited good structure stability and cell infiltration in TSF1.0 scaffolds.To further evaluate the structural stability,biocompatibility and angiogenesis in vivo,the TSF0.5,TSF1.0,and TSF1.5stents were subcutaneously implanted into nude mice for 2 w and 4 w.The scaffolds were retrieved after 2 weeks and 4 weeks,and stained histologically by hematoxylin and eosin（H＆E）staining and Masson’s trichrome staining and immunohistochemically by CD31 staining,which further revealed good biocompatibility angiogenic ability.Finally,the tracheal scaffold patches loaded with chondrocytes were subcutaneously implanted into the nude mouse for 4 w and 8 w,which revealed that the samples gradually achieved cartilage regeneration,which even increased further with an increase in the culture time.The H＆E staining,safranin-O staining and type II collagen staining also showed that the tracheal scaffold can effectively achieve the cartilage regeneration and collagen accumulation.In summary,the tracheal scaffolds not only display appropriate mechanical properties to afford a stable structure,but also exhibit a biomimetic ECM-like structure to induce tissue regeneration,which may have significant potential for tracheal regeneration.