Introduction: Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility and dysphagia. Silk fibroin (SF) biomaterials derived from Bombyx mori cocoons represent unique platforms for esophageal tissue engineering due their high structural strength and elasticity, diverse processing flexibility, tunable biodegradability, and low immunogenicity. In this study, we hypothesized that a bi-layer silk fibroin (BLSF) scaffold would support constructive remodeling of full thickness esophageal defects in a porcine model of onlay esophagoplasty. Materials and Methods: BLSF matrices were fabricated from aqueous SF solutions by a solvent-casting/salt leaching process in combination with SF film casting. Onlay esophagoplasty (1×3 cm~2 implant) was performed with BLSF matrices (N=6) in adult Yucatan miniature swine (30-40 kg) for up to 3 m of implantation (Figure 1). Animal weight assessments and esophagography (fluoroscopy and X-ray following contrast ingestion) were performed pre-operatively and at 1 m intervals until scheduled euthanasia. At 3 m post-surgery, tubular esophageal segments containing the original implantation site were assessed for functional tissue regeneration by ex vivo contractility/relaxation, histological (H&E and Masson's trichrome) and immunohistochemical (IHC) analyses. Results: All swine receiving BLSF grafts survived with no complications and were capable of solid food consumption following a 1 wk post-op G-tube feeding regime while maintaining weight throughout the study period similar to pre-operative levels. X-ray and fluoroscopy evaluations revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula at any point in the study (Figure 2). Gross tissue evaluations demonstrated host tissue ingrowth within the implantation sites with no evidence of scaffold remnants, iteration, or significant axial contraction of the graft site. Ex vivo tissue bath studies of repaired esophageal segments displayed contractile responses to carbachol and KCI while isoproterenol produced tissue relaxation. Histological and IHC (Figure 3) evaluations of the regenerated tissues demonstrated the formation of a de novo muscularis externa and muscularis mucosa layers positive for contractile proteins [a-smooth muscle actin (a-SMA), fast and slow twitch skeletal myosin heavy chain]. A stratified squamous, epithelium positive for pan-cytokeratin (CK) expression was observed throughout the implantation sites. De novo innervation and vascularization were present in all neotissues with synaptophysin (SYP38)+ boutons and vessels lined with CD31+ endothelial cells. Conclusions: These data demonstrate the ability of BLSF grafts to support the formation of innervated, vascularized esophageal tissues with functional properties in a porcine model. These matrices offer an emerging technology for esophageal repair.
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