In Vitro Construction And In Vivo Regeneration Of Esophageal Muscle Tissue

Objective: A polyurethane(PU) scaffold with micro-channels on both sides is prepared through the research of esophageal muscular structure. The scaffold can guide the directional growth of endocircular and exolongitudinal muscle tissue, and be acted as a substitute for the repair and/or regeneration of the injured esophagus tissue. The results can provide the foundation for the treatment of the esophageal diseases.Method: The primary smooth muscle cells(SMC) obtained by tissue explants method from New Zealand white rabbits was used as the seeding cells for the follow-up experiments. A silicon wafer with two predetermined micro-channel prototypes was prepared, i.e. prototype1(P1) and prototype2(P2). P1 had micro-channels of 200 ?m width, continuous walls with 30 ?m thickness and 30 ?m height. P2 had micro-channels of 100 ?m width, discontinuous walls with 30 ?m thickness, 30 ?m height, and 30 ?m gap at regular interval. PU without micro-channels(P3) was prepared and used as the negative control. The PU1 and PU2 scaffolds were prepared by PU solution in 1,4-dioxane which was poured on the daughter PDMS mold. The PU1-SF and PU2-SF were prepared via grafting silk fibroin(SF) onto PU1 and PU2 scaffolds, aiming at improving scaffolds’ biocompatibility. SMCs were seeded on these prepared scaffolds in vitro and tested their growth characteristics using methods like methyl thiazolyl tetrazolium(MTT) assay, immunofluorescence(IF) technique, scanning electron microscope(SEM), and western blotting. MTT assay was used for detecting cell growth rate. Immunofluorescence and SEM were used for observing the morphology and phenotype of cells on the scaffolds. Western blotting was used for testing the expression quantitatively of smooth muscle actin. The results revealed that scaffolds had good cytocompatibility and could guide the growth of SMC. SMC on PU2-SF had higher cell growth rate and better SMC morphology than cells on PU1-SF. For promoting the angiogenesis, vascular endothelial growth factor(VEGF) was coated on the SF grafted scaffolds. In order to remit the mechanical tension when tabulation, scaffold with PU1 on the exterior and PU2 in the lumen was prepared through being bonded with 1, 4-dioxane. The substitute was implanted into the muscle-defected rabbits to detect the function of repairing and regeneration using H&E staining.Result: The primary SMCs were gained from the muscle tissue of rabbits. Cells with passage 2~4 were used as the seeding cells. The scaffolds with two kinds of prototypes were prepared. The SF grafted PU scaffolds had good cytocompatibility and promoted the growth of SMC in vitro. The micro-channels on the PU guided the direction of cell growth. In vivo tests demonstrated that scaffolds fabricated with the present protocol could guide the growth of muscle tissue and repair/regenerate the esophageal muscle with endocircular and exolongitudinal architecture.Conclusion: The prepared scaffold with micro-channels on both sides could direct cells’ growth. It is possible to be used as a substitute to repair and/or regenerate the esophageal muscle.