A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells' morphogenesis, and they range in a three-dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffnes

Cell Sources for Tissue Engineering Strategies to Treat Calcific Valve Disease

Cardiovascular calcification is an independent risk factor and an established predictor of adverse cardiovascular events. Despite concomitant factors leading to atherosclerosis and heart valve disease (VHD), the latter has been identified as an independent pathological entity. Calcific aortic valve stenosis is the most common form of VDH resulting of either congenital malformations or senile “degeneration.” About 2% of the population over 65 years is affected by aortic valve stenosis which represents a major cause of morbidity and mortality in the elderly. A multifactorial, complex and active heterotopic bone-like formation process, including extracellular matrix remodeling, osteogenesis and angiogenesis, drives heart valve “degeneration” and calcification, finally causing left ventricle outflow obstruction. Surgical heart valve replacement is the current therapeutic option for those patients diagnosed with severe VHD representing more than 20% of all cardiac surgeries nowadays. Tissue Engineering of Heart Valves (TEHV) is emerging as a valuable alternative for definitive treatment of VHD and promises to overcome either the chronic oral anticoagulation or the time-dependent deterioration and reintervention of current mechanical or biological prosthesis, respectively. Among the plethora of approaches and stablished techniques for TEHV, utilization of different cell sources may confer of additional properties, desirable and not, which need to be considered before moving from the bench to the bedside. This review aims to provide a critical appraisal of current knowledge about calcific VHD and to discuss the pros and cons of the main cell sources tested in studies addressing in vitro TEHV

Development of a Novel Hierarchically Biofabricated Blood Vessel Mimic Decorated with Three Vascular Cell Populations for the Reconstruction of Small-Diameter Arteries

The availability of grafts to replace small-diameter arteries remains an unmet clinical need. Here, the validated methodology is reported for a novel hybrid tissue-engineered vascular graft that aims to match the natural structure of small-size arteries. The blood vessel mimic (BVM) comprises an internal conduit of co-electrospun gelatin and polycaprolactone (PCL) nanofibers (corresponding to the tunica intima of an artery), reinforced by an additional layer of PCL aligned fibers (the internal elastic membrane). Endothelial cells are deposited onto the luminal surface using a rotative bioreactor. A bioprinting system extrudes two concentric cell-laden hydrogel layers containing respectively vascular smooth muscle cells and pericytes to create the tunica media and adventitia. The semi-automated cellularization process reduces the production and maturation time to 6 days. After the evaluation of mechanical properties, cellular viability, hemocompatibility, and suturability, the BVM is successfully implanted in the left pulmonary artery of swine. Here, the BVM showed good hemostatic properties, capability to withstand blood pressure, and patency at 5 weeks post-implantation. These promising data open a new avenue to developing an artery-like product for reconstructing small-diameter blood vessels

Reconstruction of the swine pulmonary artery using a graft engineered with syngeneic cardiac pericytes

The neonatal heart represents an attractive source of regenerative cells. Here, we report the results of a randomized, controlled, investigator-blinded preclinical study, which assessed the safety and effectiveness of a matrix graft cellularized with cardiac pericytes (CPs) in a piglet model of pulmonary artery (PA) reconstruction. Within each of five trios formed by 4-week-old female littermate piglets, one element (the donor) was sacrificed to provide a source of CPs, while the other two elements (the graft recipients) were allowed to reach the age of 10 weeks. During this time interval, culture-expanded donor CPs were seeded onto swine small intestinal submucosa (SIS) grafts, which were then shaped into conduits and conditioned in a flow bioreactor. Control unseeded SIS conduits were subjected to the same procedure. Then, recipient piglets were randomized to surgical reconstruction of the left PA (LPA) with unseeded or CP-seeded SIS conduits. Doppler echocardiography and cardiac magnetic resonance imaging (CMRI) were performed at baseline and 4-months post-implantation. Vascular explants were examined using histology and immunohistochemistry. All animals completed the scheduled follow-up. No group difference was observed in baseline imaging data. The final Doppler assessment showed that the LPA’s blood flow velocity was similar in the treatment groups. CMRI revealed a mismatch in the average growth of the grafted LPA and contralateral branch in both treatment groups. Histology of explanted arteries demonstrated that the CP-seeded grafts had a thicker luminal cell layer, more intraparietal arterioles, and a higher expression of endothelial nitric oxide synthase (eNOS) compared with unseeded grafts. Moreover, the LPA stump adjacent to the seeded graft contained more elastin and less collagen than the unseeded control. Syngeneic CP engineering did not accomplish the primary goal of supporting the graft’s growth but was able to improve secondary outcomes, such as the luminal cellularization and intraparietal vascularization of the graft, and elastic remodeling of the recipient artery. The beneficial properties of neonatal CPs may be considered in future bioengineering applications aiming to reproduce the cellular composition of native arteries

Tubular scaffolds and proangiogenic 3D matrices as alternative strategies in vascular tissue engineering.

The cardiovascular disease (CDV) is a group of disorders which is the main cause of death worldwide. In the western world, it is the main cause of morbidity and mortality and its rate, compared to the other cause of death, is constantly growing. Coronary heart disease (CHD) and peripherical artery disease (PAD) are two of the principal conditions among the CVD. Today, these disorders are treated with strategies of risk-factors management, pharmaceutical therapies and surgical operation but, the mortality index is still too high. In this work, two different tissue-engineered strategies are implemented for the vascular regeneration. On one side, a device and a tubular scaffold are designed to obtain a vascular graft prototype with an endothelial cell coating on the internal surface. On the other side, a biodegradable planar scaffold is developed to deliver pro-angiogenic cells on the vascular stenosis site and induce collateralization. Both the approaches were involved in in-vivo experiments. The results a cell attachment rate, a homogeneous cell distribution over the scaffold internal surface and a 90% viability rate after 5 days of culturing, regarding the first method showed. The planar scaffolds underwent a proper cell colonization and had a good viability, density and proliferation rates at 7 days