*Article* **In Vitro Tissue Reconstruction Using Decellularized Pericardium Cultured with Cells for Ligament Regeneration**

**Mika Suzuki <sup>1</sup> , Tsuyoshi Kimura 1,\* , Yukina Yoshida <sup>1</sup> , Mako Kobayashi <sup>1</sup> , Yoshihide Hashimoto <sup>1</sup> , Hironobu Takahashi <sup>2</sup> , Tatsuya Shimizu <sup>2</sup> , Shota Anzai <sup>3</sup> , Naoko Nakamura 1,3 and Akio Kishida <sup>1</sup>**


**Abstract:** Recent applications of decellularized tissues have included the ectopic use of their sheets and powders for three-dimensional (3D) tissue reconstruction. Decellularized tissues are fabricated with the desired functions to employ them to a target tissue. The aim of this study was to develop a 3D reconstruction method using a recellularized pericardium to overcome the difficulties in cell infiltration into tight and dense tissues, such as ligament and tendon tissues. Decellularized pericardial tissues were prepared using the high hydrostatic pressurization (HHP) and surfactant methods. The pericardium consisted of bundles of aligned fibers. The bundles were slightly disordered in the surfactant decellularization method compared to the HHP decellularization method. The mechanical properties of the pericardium were maintained after the HHP and surfactant decellularizations. The HHP-decellularized pericardium was rolled up into a cylindrical formation. Its mechanical behavior was similar to that of a porcine anterior cruciate ligament in tensile testing. NIH3T3, C2C12, and mesenchymal stem cells were adhered with elongation and alignment on the HHP- and surfactantdecellularized pericardia, with dependences on the cell type and decellularization method. When the recellularized pericardium was rolled up into a cylinder formation and cultured by hanging circulation for 2 days, the cylinder formation and cellular elongation and alignment were maintained on the decellularized pericardium, resulting in a layer structure of cells in a cross-section. According to these results, the 3D-reconstructed decellularized pericardium with cells has the potential to be an attractive alternative to living tissues, such as ligament and tendon tissues.

**Keywords:** decellularization; extracellular matrix; porcine pericardium; high hydrostatic pressurization method; surfactant method; 3D fabrication; ligament
