Aim of work was to locate a simple, reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited, namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size, pore orientation, porosity, and pore distribution were produced using two different techniques, such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity, seeding efficiency, and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method, as these conditions successfully improved the cellularization of polymeric patches. Furthermore, the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results, it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover, the protocol turned out to be simple, repeatable, and reproducible.
Keywords: electrospinning; patch engineering; porcine mesenchymal stem cells; temperature induced precipitation.