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Combined Application of Mesenchymal Stem Cells and Tissue Fusion Technology in Small Bowel's Anastomosis: An In Vivo Study With Porcine Model
Hong Pan*, Enders K. Ng, Ping Keun Lam, See W. Tong, Kevin K. Leung, Anthony Y. Teoh
Background: Tissue fusion technology is applied to a variety of surgery. Some pilot studies about gut’s sealing with tissue fusion technology got contradictory results. No previous research has assessed the molecular process of anastomotic healing, which induced by tissue fusion technology. Meanwhile, the multi-lineage differentiation property and paracrine function of stem cells have been exploited for clinical applications, especially in regeneration medicine.
Method: Sixteen swine were divided into two groups (survival up to either 7 or 14 days). For each group, the animals were further subdivided to two subgroups, whose anastomoses with or without MSCs reinforcement injection ((MSC and control groups, n=4 for each). Each animal had 5 anastomoses established with Ligasure, whose working foundation is tissue fusion technology. 150μl vehicle solution with or without MSCs was injected by 10 injections along the anastomosis ring. It contained with 0.5 million MSCs for MSC group. General clinical outcomes, anastomotic leakage rate and burst pressure were compared among the 4 subgroups. Molecular assay of the anastomoses were also investigated using HIC, western blot and PCR array (PCR only for samples of day-7) to screen for any potential cytokines or genetic change in anastomotic tissue due to stem cell administration. The immigration and tissue-specific differentiation potency were also investigated by immune-fluorescent technique.
Result: MSC and control group both had one leak at day-7 checkpoint, one and two anastomotic leaks respectively for day-14 checkpoint. No significant differences between the two groups at both checkpoints about anastomoses’ burst pressure (p=1.000 and p=0.441, respectively). Amount of proliferating cell nucleus antigen (PCNA) positive cells and number of micro-vessels in anastomotic fused tissue in MSC group were higher than those of control group at both checkpoints, especially statistical significant for PCNA positive cell counting at day-7 (p=0.021). Labeled MSCs were found in lamina propria of samples in day-14 checkpoint, and some of them were characteristic with positive reaction with smooth muscle antigen (SMA). Western blot revealed acceleration of angiogenesis in MSC group. PCR array found that most of wound healing related genes showed down-regulation of tissue mRNA level in MSC group comparing to control.
Conclusion: (1) Local injection of allogeneic mesenchymal stem cells around tissue-fused anastomosis does not influence clinical outcomes, including leak rate and burst pressure;(2) Grafted MSCs significantly promote epithelial and connective cells proliferation;(3) MSC’s immigration capacity and differentiation potential in fused anastomotic tissue are verified;(4) MSCs could lead to a down-regulation of expression of numerous wound healing related genes in anastomotic tissue at 7 days after graft

Comparison of count of PCNA positive cells

Differentiation of smooth muscle cells from grafted MSCs (400×) at day-14

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