Ameliorated healing of biliary anastomosis by autologous adipose-derived stem cell sheets

Abstract

Introduction: Cell sheets consisting of adipose-derived stem cells (ADSCs) have been reported to be effective for wound healing. We conducted this study to clarify the efficacy of ADSC sheets in wound healing at the duct-to-duct biliary anastomotic site in pigs.

Methods: Eleven female pigs (20-25 kg) were divided into two groups: biliary anastomosis with an ADSC sheet (n = 6) or without an ADSC sheet (n = 5). To follow the transplanted ADSCs, PKH26GL-labeled sheets were used in one of the ADSC pigs. Two weeks prior to laparotomy, ADSCs were isolated from the lower abdominal subcutaneous adipose tissue. After three passages, ADSCs were seeded on temperature-responsive culture dishes and collected as cell sheets. ADSC sheets were gently transplanted on the anastomotic site. We evaluated specimens by PKH26GL labeling, macroscopic changes, infiltration of inflammatory cells, and collagen content.

Results: Labeled ADSCs remained around the bile duct wall. In the no-ADSC group, more adhesion developed at the hepatic hilum as observed during relaparotomy. Histopathological examination showed that the diameter and cross-sectional area of the bile duct wall were decreased in the ADSC group. In the no-ADSC group, a large number of inflammatory cells and more collagen fibers were identified in the bile duct wall.

Conclusions: The present study demonstrated that autologous ADSC sheet transplantation reduced hypertrophic changes in the bile duct wall at the anastomotic site. A long-term follow-up is required to evaluate the efficacy of this mechanism in prevention of biliary anastomotic strictures.

Keywords: ADSC, adipose-derived stem cell; APC, allophycocyanin; Adipose-derived stem cell; Anastomotic healing; BAS, biliary anastomotic strictures; BMSC, bone marrow stem cells; Biliary anastomosis; CBD, common bile duct; Cell sheet; FBS, fetal bovine serum; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; MSC, mesenchymal stem cell; VEGF, vascular endothelial growth factor.

Fig. 1

Flow cytometry analysis of ADSC primary cultures showing expression of MSC-specific markers (CD90 and CD44) and no expression of hematopoietic markers (CD31 and CD45).

Fig. 2

Preparation and transplantation of autologous ADSC sheets. ADSCs were harvested as cell sheets using temperature-responsive culture dishes (a). End-to-end biliary anastomosis was performed, and then an ADSC sheet was transplanted (the area indicated by a dotted line) (b). Two weeks after the surgery, severe adhesion around the liver hilum developed in the no-ADSC group, which required sharp dissection (arrowhead) (c). In contrast, adhesiolysis was not required in the ADSC group (d). Evaluating the severity of adhesion using a previously published classification (grade 0: none, grade 1: film-like with no neovascularization, grade 2: moderately thick with partial neovascularization, grade 3: thick, solid adhesion with neovascularization), we noted grade 2 or higher in 17% (1/6) of the ADSC group and in 80% (4/5) of the no-ADSC group, though the difference was not statistically significant (P = 0.07) (e).

Fig. 3

The levels of serum bilirubin, aspartate transaminase, alanine transaminase, alkaline phosphatase, and γ-glutamyltranspeptidase were not significantly different between the groups.

Fig. 4

Macroscopic findings of the anastomotic site on postoperative day 14. The diameter of the bile duct in the no-ADSC group was significantly larger than in the ADSC group (a). The cross-section of the luminal area was not different between the groups (b). The area of the bile duct wall was significantly larger in the no-ADSC group than in the normal bile duct and ADSC group (c).

Fig. 5

Microscopy findings of the normal bile duct without manipulation, anastomotic site without an ADSC sheet on postoperative day 14, and anastomotic site with an ADSC sheet on postoperative day 14. H&E (a–c) and Masson's trichrome (d–f). A large number of inflammatory cells and increased collagen content were identified in the no-ADSC group (b, e). In contrast, fewer inflammatory cells and many small vessels were observed in the ADSC group (c, f).

Fig. 6

To follow the presence of transplanted ADSCs, cells were labeled with PKH26GL (a). Fluorescence microscopy of frozen sections revealed the presence of PKH26GL-positive ADSCs around the bile duct wall on postoperative day 7 (arrow) (b).