combining-3d-printing-electrospinning-to-manufacture-biomimetic-heart

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds ...

Technical University Munich

Covered stents have been demonstrated to reduce restenosis; however, the membrane's limited biocompatibility can still lead to thrombus formation. To obtain optimal surface hemocompatibility, endothelialization of the luminal surface has been proposed. However, the effect of delivery procedures, such as crimping and balloon dilatation, on the endothelial layer has not been quantified. This study investigated the impact of such procedures on endothelialized covered stents in vitro.

In Vitro Quantification of Luminal Denudation After Crimping and Balloon Dilatation of Endothelialized Covered Stents.

In vitro tissue engineering of vascular grafts requires dynamic conditioning in a bioreactor system for in vitro tissue maturation and remodeling to receive a mechanically adequate and hemocompatible implant. The goal of the current work was to develop a bioreactor system for the conditioning of vascular grafts which is (i) able to create a wide range of flow, pressure and frequency conditions, including physiological ones; (ii) compact and easy to assemble; (iii) transportable; (iv) disposable. The system is driven by a small centrifugal pump controlled via a custom-made control unit, which can also be operated on batteries to allow for autonomous transportation. To show the potential of the newly developed bioreactor system small-caliber vascular composite grafts (n = 5, internal diameter = 3 mm, length = 12.5 cm) were fabricated using a fibrin scaffold embedding human umbilical artery smooth muscle cells and a polyvinylidene fluoride warp-knitted macroporous mesh. Subsequently, the vascular grafts were endothelialized and mounted in the bioreactor system for conditioning. The conditioning parameters remained within the predefined range over the complete conditioning period and during operation on batteries as tested for up to 25 h. Fabrication and pre-conditioning under arterial pressure and shear stress conditions resulted in robust and hemocompatible tissue-engineered vascular grafts. Analysis of immunohistochemical stainings against extracellular matrix and cell-specific proteins revealed collagen I and collagen III deposition. The luminal surface was confluently covered with endothelial cells. The developed bioreactor system showed cytocompatibility and pH, pO, pCO, glucose and lactate stayed constant. Sterility was maintained during the complete fabrication process of the vascular grafts. The potential of a versatile and mobile system and its functionality by conditioning tissue-engineered vascular grafts under physiological pressure and flow conditions could be demonstrated.

VascuTrainer: A Mobile and Disposable Bioreactor System for the Conditioning of Tissue-Engineered Vascular Grafts.

Elastin is a key extracellular matrix (ECM) protein that imparts functional elasticity to tissues and therefore an attractive candidate for bioengineering materials. Genetically engineered elastin-like recombinamers (ELRs) maintain inherent properties of the natural elastin (e.g. elastic behavior, bioactivity, low thrombogenicity, inverse temperature transition) while featuring precisely controlled composition, the possibility for biofunctionalization and non-animal origin. Recently the chemical modification of ELRs to enable their crosslinking via a catalyst-free click chemistry reaction, has further widened their applicability for tissue engineering. Despite these outstanding properties, the generation of macroporous click-ELR scaffolds with controlled, interconnected porosity has remained elusive so far. This significantly limits the potential of these materials as the porosity has a crucial role on cell infiltration, proliferation and ECM formation. In this study we propose a strategy to overcome this issue by adapting the salt leaching/gas foaming technique to click-ELRs. As result, macroporous hydrogels with tuned pore size and mechanical properties in the range of many native tissues were reproducibly obtained as demonstrated by rheological measurements and quantitative analysis of fluorescence, scanning electron and two-photon microscopy images. Additionally, the appropriate size and interconnectivity of the pores enabled smooth muscle cells to migrate into the click-ELR scaffolds and deposit extracellular matrix. The macroporous structure together with the elastic performance and bioactive character of ELRs, the specificity and non-toxic character of the catalyst-free click-chemistry reaction, make these scaffolds promising candidates for applications in tissue regeneration. This work expands the potential use of ELRs and click chemistry systems in general in different biomedical fields.

Macroporous click-elastin-like hydrogels for tissue engineering applications.

Fibrosis is a life-threatening pathological condition resulting from a dysfunctional tissue repair process. There is no efficient treatment and organ transplantation is in many cases the only therapeutic option. Here we review tissue engineering and regenerative medicine (TERM) approaches to address fibrosis in the cardiovascular system, the kidney, the lung and the liver. These strategies have great potential to achieve repair or replacement of diseased organs by cell- and material-based therapies. However, paradoxically, they might also trigger fibrosis. Cases of TERM interventions with adverse outcome are also included in this review. Furthermore, we emphasize the fact that, although organ engineering is still in its infancy, the advances in the field are leading to biomedically relevant in vitro models with tremendous potential for disease recapitulation and development of therapies. These human tissue models might have increased predictive power for human drug responses thereby reducing the need for animal testing.

Fibrosis in tissue engineering and regenerative medicine: treat or trigger?

Directed differentiation of induced pluripotent stem cells (iPSCs) towards specific lineages remains a major challenge in regenerative medicine, while there is a growing perception that this process can be influenced by the three-dimensional environment. In this study, we investigated whether iPSCs can differentiate towards mesenchymal stromal cells (MSCs) when embedded into fibrin hydrogels to enable a one-step differentiation procedure within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like phenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, faint expression of CD73 and CD105, and reduced differentiation potential towards osteogenic and adipogenic lineage. Transcriptomic analysis demonstrated that characteristic genes for MSCs and extracellular matrix were upregulated on flat substrates, whereas genes of neural development were upregulated in 3D culture. Furthermore, the 3D culture had major effects on DNA methylation profiles, particularly within genes for neuronal and cardiovascular development, while there was no evidence for epigenetic maturation towards MSCs. Taken together, iPSCs could be differentiated towards MSCs on tissue culture plastic or on a flat fibrin hydrogel. In contrast, the differentiation process was heterogeneous and not directed towards MSCs when iPSCs were embedded into the hydrogel.

Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels.

Pulmonary valve replacement is performed with excellent resultant hemodynamics in patients that have underlying congenital or acquired heart valve defects. Despite recent advancements in right ventricular outflow tract reconstruction, an increased risk of developing infective endocarditis remains, which has a more common occurrence for conduits of bovine jugular vein (BJV) origin compared with cryopreserved homografts. The reason for this is unclear although it is hypothesized to be associated with an aberrant phenotypic state of cells that reendothelialize the graft tissue postimplantation. The aim of this study was to develop an  model that enables the analysis of endothelial cell (EC) attachment to cardiac graft tissues under flow. In the experiments, EC attachment was optimized on bovine pericardium (BP) patch using human umbilical vein ECs. Different biological coatings, namely gelatin, fibronectin, plasma, or a combination of fibronectin and plasma were tested. After cell adaptation, graft tissues were exposed to laminar flow in a parallel-plate flow chamber. Cell retention to the tissue was analyzed after nuclear staining with YO-PRO-1 and a membranous localization of VE-cadherin. Experiments showed that combined coating with fibronectin and blood plasma together with a two-phased shear pattern resulted in a relevant cell monolayer on BP patch and cryopreserved homograft. For BJV tissue, no adherent cells under both static and shear conditions were initially observed. In conclusion, having established the new flow chamber system we could obtain EC layers on the surface of BP patch and cryopreserved pulmonary homograft tissues. The presented  system can serve as a competent model to study cell phenotypes on cardiac grafts in the close-to-physiologic environment. Moreover, this approach allows broad applications and enables further development by testing more complex conditions.

An  Model to Study Endothelialization of Cardiac Graft Tissues Under Flow.

Melt electrowriting (MEW) is a high-resolution fiber-forming technology for the digital fabrication of complex micro-structured scaffolds for tissue engineering, which has convincingly shown its potential in in vitro and in vivo animal studies. The clinical translation of such constructs to the patient requires the capability to visualize them upon implantation with clinically accepted methods such as magnetic resonance imaging (MRI). To this end, this work presents the modification of polycaprolactone (PCL) scaffolds with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to render them visualizable by MRI. Composite scaffolds containing up to 0.3 weight % USPIOs were 3D printed by MEW and could be sensitively detected in vitro using T2- and T2*-weighted MRI. At the same time, USPIO incorporation did not affect the usability of PCL for tissue engineering applications as demonstrated by the mechanical and cytocompatibility evaluation. Concentrations up to 0.2% caused small to no decrease in the ultimate tensile strength and Young's modulus. Cytocompatibility tests resulted in excellent cell viability, with proliferating cells adhering to all the scaffolds. This work contributes to the materials library for MEW and opens the possibility of using MRI for longitudinal monitoring of MEW grafts.

Visualization of USPIO-labeled melt-electrowritten scaffolds by non-invasive magnetic resonance imaging.

Medical meshes are used as structural reinforcement both in clinical surgery and tissue engineering. However, complex loading conditions often found in such applications result in a non-homogenous stress distribution, for which the uniform reinforcement provided by the meshes is not optimal. This study aims to design a textile reinforcement with a spatially heterogeneous, load-tailored fiber architecture. To this end, we developed a simple method of manipulating a standard uniform mesh by stretching in warp and weft directions to various extents in order to control fiber orientation and fiber volume fraction. Subsequent thermal treatment locked the manipulated configurations allowing further use of the meshes. Firstly, samples in five uniform configurations (two manipulated longitudinally (warp direction), two manipulated transversely (weft direction), one non-manipulated) were obtained and analyzed regarding their morphology as well as their mechanical properties under cyclic uniaxial loading. Significant effects of the manipulation on key characteristics of the pores such as angles, side lengths, aspect ratios, and fiber volume fraction were shown. Tensile testing demonstrated the range of tensile properties achievable with the simple manipulation of the mesh, not only in magnitude but also in the shape of the stress-strain response curve. Finally, local manipulation combining different mesh configurations was exemplarily applied to create a spatially heterogeneous load-tailored reinforcement to match local strain directions in tissue-engineered tubular heart valves. The proposed method enables the use of well-established uniform medical meshes to produce load-tailored non-uniform mesh reinforcement for many applications in an easy-to-implement manner.

Article	Year
In Vitro Quantification of Luminal Denudation After Crimping and Balloon Dilatation of Endothelialized Covered Stents. Cardiovascular and interventional radiology\| PubMed ID: 28523446	2017
VascuTrainer: A Mobile and Disposable Bioreactor System for the Conditioning of Tissue-Engineered Vascular Grafts. Annals of biomedical engineering\| PubMed ID: 29340931	2018
Macroporous click-elastin-like hydrogels for tissue engineering applications. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 29636129	2018
Fibrosis in tissue engineering and regenerative medicine: treat or trigger? Advanced drug delivery reviews\| PubMed ID: 31295523	2019
Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels. Scientific reports\| PubMed ID: 31666572	2019
An Model to Study Endothelialization of Cardiac Graft Tissues Under Flow. Tissue engineering. Part C, Methods\| PubMed ID: 33544046	2021
Visualization of USPIO-labeled melt-electrowritten scaffolds by non-invasive magnetic resonance imaging. Biomaterials science\| PubMed ID: 34096938	2021
Mesh manipulation for local structural property tailoring of medical warp-knitted textiles. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 35182915	2022

Petra Mela

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