Successful muscle regeneration by a homologous microperforated scaffold seeded with autologous mesenchymal stromal cells in a porcine esophageal substitution model

Abstract

Background: Since the esophagus has no redundancy, congenital and acquired esophageal diseases often require esophageal substitution, with complicated surgery and intestinal or gastric transposition. Peri-and-post-operative complications are frequent, with major problems related to the food transit and reflux. During the last years tissue engineering products became an interesting therapeutic alternative for esophageal replacement, since they could mimic the organ structure and potentially help to restore the native functions and physiology. The use of acellular matrices pre-seeded with cells showed promising results for esophageal replacement approaches, but cell homing and adhesion to the scaffold remain an important issue and were investigated.

Methods: A porcine esophageal substitute constituted of a decellularized scaffold seeded with autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed. In order to improve cell seeding and distribution throughout the scaffolds, they were micro-perforated by Quantum Molecular Resonance (QMR) technology (Telea Electronic Engineering).

Results: The treatment created a microporous network and cells were able to colonize both outer and inner layers of the scaffolds. Non seeded (NSS) and BM-MSCs seeded scaffolds (SS) were implanted on the thoracic esophagus of 4 and 8 pigs respectively, substituting only the muscle layer in a mucosal sparing technique. After 3 months from surgery, we observed an esophageal substenosis in 2/4 NSS pigs and in 6/8 SS pigs and a non-practicable stricture in 1/4 NSS pigs and 2/8 SS pigs. All the animals exhibited a normal weight increase, except one case in the SS group. Actin and desmin staining of the post-implant scaffolds evidenced the regeneration of a muscular layer from one anastomosis to another in the SS group but not in the NSS one.

Conclusions: A muscle esophageal substitute starting from a porcine scaffold was developed and it was fully repopulated by BM-MSCs after seeding. The substitute was able to recapitulate in shape and function the original esophageal muscle layer.

Keywords: 3D cell culture; Quantum Molecular Resonance; esophagus; mesenchymal stromal cells; scaffold; tissue engineering.

Figure 1.

The appearance of esophagi. (a) Decellularized esophagus; (b) fresh esophagus.

Figure 2.

Scaffold perforative treatment. (a) Perforative QMR procedure with the needle connected to a Cartesian robot; (b) macroscopic scaffold appearance after QMR perforative treatment in rectangular open shape; (c) macroscopic scaffold appearance after QMR perforative treatment in tubular shape; (d) digital microscope image of the upper surface of a decellularized scaffold after QMR perforative treatment at 118× magnification; (e) SEM image of a decellularized scaffold after QMR perforative treatment, magnification: 33×, scale bar 100 µm; (f) SEM image of a decellularized scaffold after QMR perforative treatment, magnification: 100×, scale bar 100 µm. QMR, Quantum Molecular Resonance®; SEM, scanning electron microscopy.

Figure 3.

SEM image of a decellularized scaffold seeded with MSCs and cell culture outside and inside channels. (a) SEM image of a decellularized scaffold seeded with MSCs: magnification 500×, scale bar 50 μm; (b) cell culture (actina marchers) outside and inside channels. MSC, mesenchymal stromal cell; SEM, scanning electron microscopy.

Figure 4.

Scaffold implantation. (a) Image of surgical procedure with a decellularized scaffold during implantation, with the scaffold wrapping around the esophagus mucosa in a tubular shape with two T-T and one longitudinal anastomosis; (b) image of the decellularized scaffold at the end of surgical procedure. T-T, termino-terminal.

Figure 5.

Histological analyses. (a) NSS retrieved after 3 months from surgery, desmin staining, magnification: 20×; (b) SS scaffold retrieved after 3 months from surgery, actin and desmin staining, magnification: 20×. NSS, non-seeded scaffold; SS, seeded scaffold.