Costantino Del Gaudio

Numerical simulation of the dynamics of a bileaflet prosthetic heart valve using a fluid-structure interaction approach

The main purpose of this study is to reproduce in silico the dynamics of a bileaflet mechanical heart valve (MHV; St Jude Hemodynamic Plus, 27mm characteristic size) by means of a fully implicit fluid-structure interaction (FSI) method, and experimentally validate the results using an ultrafast cinematographic technique. The computational model was constructed to realistically reproduce the boundary condition (72 beats per minute (bpm), cardiac output 4.5l/min) and the geometry of the experimental setup, including the valve housing and the hinge configuration. The simulation was carried out coupling a commercial computational fluid dynamics (CFD) package based on finite-volume method with user-defined code for solving the structural domain, and exploiting the parallel performance of the whole numerical setup. Outputs are leaflets excursion from opening to closure and the fluid dynamics through the valve. Results put in evidence a favorable comparison between the computed and the experimental data: the model captures the main features of the leaflet motion during the systole. The use of parallel computing drastically limited the computational costs, showing a linear scaling on 16 processors (despite the massive use of user-defined subroutines to manage the FSI process). The favorable agreement obtained between in vitro and in silico results of the leaflet displacements confirms the consistency of the numerical method used, and candidates the application of FSI models to become a major tool to optimize the MHV design and eventually provides useful information to surgeons.

Development of bioengineered human larynx.

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.

Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue

In this study, the obtainment and characterization of decellularized rat tracheal grafts are described. The detergent-enzymatic method, already used to develop bioengineered pig and human trachea scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be used to improve our knowledge on the process of tissue-engineered airway transplantation and regeneration. The results demonstrated that, after 9 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues with strong in vivo angiogenic characteristics, could be obtained. Moreover, to improve the mechanical properties of decellularized tracheae, genipin is here considered as a naturally derived cross-linking agent. The results demonstrated that the treatment increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response.

Structural characterization and cell response evaluation of electrospun PCL membranes: micrometric versus submicrometric fibers.

Electrospinning is a valuable technique to fabricate fibrous scaffolds for tissue engineering. The typical nonwoven architecture allows cell adhesion and proliferation, and supports diffusion of nutrients and waste products. Poly(epsilon-caprolactone) (PCL) electrospun membranes were produced starting from 14% w/v solutions in (a) mixture 1:1 tetrahydrofuran and N,N-dimethylformamide and (b) chloroform. Matrices made up of randomly arranged uniform fibers free of beads were obtained. The average fiber diameters were (a) 0.8 +/- 0.2 microm and (b) 3.6 +/- 0.8 microm. PCL matrices showed the following tensile mechanical properties: tensile modulus (a) 5.0 +/- 0.7 MPa (b) 6.4 +/- 0.2 MPa, yield stress (a) 0.55 +/- 0.06 MPa (b) 0.43 +/- 0.02 MPa, and ultimate tensile stress (a) 1.7 +/- 0.2 MPa and (b) 0.8 +/- 0.1 MPa. The ultimate strain ranged between 300% and 400%. Cytotoxicity of electrospun membranes was continuously evaluated by means of electric cell-substrate impedance sensing technique using human umbilical vein endothelial cells (HUVEC). PCL matrices resulted free of toxic amounts of contaminants and/or process by-products. In vitro studies performed by culturing HUVEC on micrometric and submicrometric fibrous mats showed that both structures supported cell adhesion and spreading. However, cells cultured on the micrometric network showed higher vitality and improved interaction with the polymeric fibers, suggesting an increased ability to promote cell colonization.

Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds

In 2011, the first in-man successful transplantation of a tissue engineered trachea-bronchial graft, using a synthetic POSS-PCU nanocomposite construct seeded with autologous stem cells, was performed. To further improve this technology, we investigated the feasibility of using polymers with a three dimensional structure more closely mimicking the morphology and size scale of native extracellular matrix (ECM) fibers. We therefore investigated the in vitro biocompatibility of electrospun polyethylene terephthalate (PET) and polyurethane (PU) scaffolds, and determined the effects on cell attachment by conditioning the fibers with adhesion proteins. Rat mesenchymal stromal cells (MSCs) were seeded on either PET or PU fiber-layered culture plates coated with laminin, collagen I, fibronectin, poly-D-lysine or gelatin. Cell density, proliferation, viability, morphology and mRNA expression were evaluated. MSC cultures on PET and PU resulted in similar cell densities and amounts of proliferating cells, with retained MSC phenotype compared to data obtained from tissue culture plate cultures. Coating the scaffolds with adhesion proteins did not increase cell density or cell proliferation. Our data suggest that both PET and PU mats, matching the dimensions of ECM fibers, are biomimetic scaffolds and, because of their high surface area-to-volume provided by the electrospinning procedure, makes them per se suitable for cell attachment and proliferation without any additional coating.

Induction of angiogenesis using VEGF releasing genipin-crosslinked electrospun gelatin mats

Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications. This study investigates the possible use of natural extracellular matrix-like scaffolds made of gelatin loaded with human vascular endothelial growth factor (VEGF), as a bioresorbable platform for long-term release and consequent angiogenic boosting. For this aim, gelatin was firstly electrospun and then cross-linked at two different concentrations (0.1% and 0.5% w/v) by using genipin, a low toxic agent, in order to fabricate a suitable substrate to be loaded with VEGF. Collected fibers were homogeneous and free of beads, the fibrous structure was retained after cross-linking. Mechanical properties were deeply affected by the chemical treatment showing a different behavior, depending on the testing conditions (i.e., dry or wet state). VEGF release was assessed by means of ELISA assay: a cumulative release of about 90% (0.1% w/v) and 60% (0.5% w/v) at 28 days was measured. Both VEGF loaded mats induced cell viability, endothelial differentiation and showed chemoattractive properties when tested on human mesenchymal stromal cells (hMSCs). In vitro and in vivo angiogenic assays demonstrated that the VEGF loaded mats induced an angiogenic potential in stimulating new vessel formation similar, if not superior, to fresh VEGF. VEGF retains bioactive and pro-angiogenic potential for up to 14 days. The results demonstrated that genipin cross-linked electrospun gelatin mats loaded with VEGF could be part of a useful strategy to stimulate and induce angiogenesis in tissue engineered applications.

Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Vascular tissue engineering of small-diameter blood vessels: reviewing the electrospinning approach

Vascular tissue engineering is a relevant research field aimed at elaborating and proposing innovative solutions to overcome the drawbacks related to the use of conventional blood vessel substitutes, especially referring to small‐diameter grafts. For this aim, electrospinning can be regarded as a valuable technique to produce novel scaffolds with several functional characteristics that can be usefully tailored for the application discussed here. The reproduction of the natural extracellular matrix obtained by processing bioresorbable polymers, either functionalized or not, is driving the biomedical research towards technical solutions that can lead to an actual therapeutic improvement. In this context, this paper reviews those studies focused on the selection of suitable biomaterials for vascular applications, their microstructure, the cell response to polymeric fibres and the strategies considered so far to modify and therefore enhance the performance of final electrospun scaffolds. Copyright

Poly (L-lactic acid)/calcium-deficient nanohydroxyapatite electrospun mats for bone marrow stem cell cultures

Electrospinning of bioresorbable polymers is a promising and valuable scaffolding technique. To improve its potential applications, the addition of specific fillers has been considered. This paper reports the fabrication of electrospun poly(L-lactic acid)/Ca-deficient-hydroxyapatite (PLLA/dHAp) mats, the content of nanosized d-HAp ranged between 1 and 8 wt%. All samples consisted of micrometric and submicrometric fibers, comprising 2D voids of 8 and 13 µm for PLLA and PLLA/d-HAp mats, respectively. The surface of the electrospun fibers was characterized by an uniform distribution of nanopores. Hybrid mats loaded with 1 wt% d-HAp showed the most homogeneous microstructure, differently from the mats loaded with 4 and 8 wt% d-HAp due to the presence of microagglomerates. The viscoelastic properties of PLLA/d-HAp hybrids were characterized by a decreasing trend of the storage modulus with increases in the nanofiller content. The microstructure, viscoelastic behavior, and cytocompatibility were investigated using murine bone marrow mesenchymal stem cells. On the basis of the biological data, the electrospun PLLA and PLLA/d-HAp mats can be regarded as potential scaffolds for bone marrow mesenchymal stem cells culture.