Preparation of gelatin and gelatin/hyaluronic acid cryogel scaffolds for the 3D culture of mesothelial cells and mesothelium tissue regeneration

Mesothelial cells are specific epithelial cells that are lined in the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a three-dimensional (3D) scaffold for tissue engineering applications. Towards this end, we fabricated macroporous scaffolds from gelatin and gelatin/hyaluronic acid (HA) by cryogelation, and elucidated the influence of HA on cryogel properties and the cellular phenotype of mesothelial cells cultured within the 3D scaffolds. The incorporation of HA was found not to significantly change the pore size, porosity, water uptake kinetics, and swelling ratios of the cryogel scaffolds, but led to a faster scaffold degradation in the collagenase solution. Adding 5% HA in the composite cryogels also decreased the ultimate compressive stress (strain) and toughness of the scaffold, but enhanced the elastic modulus. From the in vitro cell culture, rat mesothelial cells showed quantitative cell viability in gelatin (G) and gelatin/HA (GH) cryogels. Nonetheless, mesothelial cells cultured in GH cryogels showed a change in the cell morphology and cytoskeleton arrangement, reduced cell proliferation rate, and downregulation of the mesothelium specific maker gene expression. The production of key mesothelium proteins E-cadherin and calretinin were also reduced in the GH cryogels. Choosing the best G cryogels for in vivo studies, the cell/cryogel construct was used for the transplantation of allograft mesothelial cells for mesothelium reconstruction in rats. A mesothelium layer similar to the native mesothelium tissue could be obtained 21 days post-implantation, based on hematoxylin and eosin (H&E) and immunohistochemical staining.