Realizzazione di un patch miocardico bioingegnerizzato mediante impianto di cellule staminali mesenchimali su matrici polimeriche tridimensionali

Background Following injury, the myocardium cannot replicate and is replaced with fibrous tissue, eventually inducing ventricular remodeling and dilation, and ultimately leading to heart failure. Therefore, how to restore the injured myocardium and ameliorate cardiac dysfunction is a major issue. Mesenchymal stem cells (MSC) are pluripotent progenitor cells able to differentiate into vascular endothelial cells and cardiomyocytes. Many researchers have demonstrated that MSC implantation induces myocardial regeneration and improves cardiac function through myogenesis and angiogenesis. Even though implanted cells can survive and differentiate into cardiomyocytes in the injured myocardium, because of the lack of oxygen and adequate nutrients, the infarcted myocardium is not an environment conducive to cell survival. To solve these problems, cardiac tissue engineering has become a promising strategy. It is a relatively new discipine that intends to overcome the obstacles to prolonging patients’ life after myocardial infarction, is continuously improving. It comprises a biomaterial based ‘vehicle’, either a porous scaffold or dense patch, made of either natural or synthetic polymeric materials, to aid transportation of cells into the diseased region in the heart. Biomaterials suggested for this specific tissue-engineering application need to have particular mechanical properties matching those of native myocardium, so that the delivered donor cells integrate and remain intact in vivo. Objective Our objective was to realize an in vitro bioengineered graft developed by culturing mesenchymal stem cells (MSC) from neonatal rats on electrospun, nanofibrous and biodegradable scaffold. Electrostatic fiber spinning, or electrospinning, is a process that produces ultrafine fibers in the form of a non-woven mesh through the action of a high electric field. The resulting fiber diameters are in the submicron range, and are unattainable by other fiber spinning techniques. Due to the small fiber diameters, the meshes have a high specific surface area conjectured to be beneficial for cell attachment and proliferation. Materials and methods Polycaprolactone (PCL) with an average molecular weight of 80 kDa (Sigma Aldrich) was dissolved in a 3:1 mixture of chloroform and dipheniformammide (DMF; Sigma Aldrich) to obtain a 8 wt% solution. The polymer solution was delivered with a syringe pump (Harvard Apparatus, Holliston, MA) to a stainless steel capillary (inner diameter=0.84 mm; Aisi 304) connected to a high voltage power supply (Gamma High Voltage Research, Ormond Beach, FL). At a voltage of 24 kV, a fluid jet was ejected from the capillary, and a non-woven fibrous mesh was created. Degradation rate of the scaffolds was also evaluated in vitro (with Ringer solution) and in vivo (in rats). Bone marrow cells were obtained by flushing femurs and tibias with phosphate-buffered saline (PBS). The obtained bone marrow cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco BRL, USA) supplemented with 10% foetal bovine serum (FBS) and 50 IU/mL penicillin–streptomycin (Gibco BRL) and was kept at 37°C in humidified air with 5% CO2. The non-adherent cells were discarded with the media changes, which were performed every 3 days. When the cultures became nearly confluent, adherent cells were detached with trypsin– EDTA and subsequently passaged. After four to five passages, cells were harvested and then used for cell implantation. Then, immunocitochemical analysis of some specific cardiac differentiative markers was assessed. After that, the cell-polymer constructs were cultured with cardiac supplements for up to 4 weeks. Finally Scanning electron microscopy (SEM) examinations were performed at 1, 3 and 7 days after the seeding. Results The obtained PCL scaffold had an average diameter of 300nm with good quality and did not show any “bead”. The fiber diameter also significantly influenced both in vitro degradation and in vivo biodegradation rates. Mesenchymal stem cells shown at the immunocitochemical analysis de novo expression of some cardiomyocite markers (alpha sarcomeric Actin, tropomyosin). Penetration of cells and abundant extracellular matrix were observed in the cell-polymer constructs after 1 week. SEM showed that the surfaces of the cell-polymer constructs were partially covered with cells after one week. Conclusion We have established a versatile in vitro system for bioengineered graft combining a nanofibrous electrospun biodegradable scaffold and MSC derived cardiomyocites . In the future this graft may be used on an animal model to investigate the effect of mesenchymal stem cells on myocardial infarction.