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Description
Introduction
In pathologies of esophagus the restoration of digestive continuity after the ablation of a portion of the organ is currently ensured by the interposition of a colon segment or by the tubulation of the stomach. The development of a tissue-engineered esophageal substitute is part of the improvement of the surgical treatment of esophageal pathologies. The first objective of this study is to develop a decellularized porcine esophageal matrix able to be cellularized. Then, objectives are to characterize biological and mechanical properties of the decellularized matrix (DM) and the immunogenic potential of mesenchymal stem cells of Wharton jelly (MSC-WJ) using for cellularization.
Methodology
Esophagi were collected from pigs; several steps were required to design a sterile DM: a decontamination process with antibiotics/antimycotics solutions, a decellularization process with a successive perfusion of chemical and enzymatic solutions, a detoxification process, and a sterilization process using peracetic acid (DM-PA).
Biological and mechanical characterizations
Histological analysis
DM (n=6) and DM-PA (n=6) were embedded in paraffin, cross-sectioned and stained with hematoxylin eosin saffron (HES). The efficacy of the decellularization process and the histological structure were then analyzed.
Cytotoxicity analysis
BALB/3 T3 cells were seeded in direct contact with the DM (n=3) and DM-PA (n=3) following ISO 10993-5 standard. Viable cells were counted after neutral red staining.
Mechanical tests
Suturability retention and tensile failure were evaluated with the Shimadzu system (Autograph, AGS-X series) for native esophagus (NE) (n=15), DM (n=30) and DM-PA (=9).
Immunogenicity analysis of MSCs-WJ
Human leucocyte antigens (HLA) of class II (HLA-DR) of MSCs-WJ were immuno-labeled and cells were analyzed by flow cytometry (Canto II Thor). Then, a co-culture analysis with peripheral blood mononuclear cells (PMBCs) was carried out to determine the activation of T cells when in contact with MSCs-WJ.
Results
No remaining cells were observed in HES, and the cohesion of the tissue was preserved after the decellularization and sterilization. The non-cytotoxic character of the DM and DM-PA were confirmed with more than 70% of cells viable when in contact with the DM and DM-PA. For traction tests, there was no significant difference observed for tensile failure between DM (n=30) and DM-PA (n=9), which respectively required a mean force of 33.22N ± 10.90N and 35.12N ± 7.45N. Tensile failure for native esophagus (NE) was obtained for a maximum value of 17.57N ± 6.84N (n=15). For suturability tests, there was no significant difference; DM (n=20) and DM-PA (n=7) had respectively a mean suture retention force of 6.25N ± 1.56N and 5.77N ± 1.03N, for which compared to the NE data (6.89N± 1.77N). Phenotyping of MSCs-WJ demonstrated that only 0.3% of cells expressed HLA-DR. PMBCs expressed a very weakly T cell markers, such as CD69 and CD25, which confirms the immuno-privileged character of these MSCs-WJ for the cellularization of the DM.
Conclusion
We obtained a decellularized and non- cytotoxic biological matrix, able to be recellularized by MSCs-WJ with low immunogenic potential. This matrix preserves the mechanical properties of the native organ, which will promote its tissue and functional integration.
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