Linoleic acid supplementation of cell culture media influences the phospholipid and lipid profiles of human reconstructed adipose tissue.

Reconstructed human adipose tissues represent novel tools available to perform in vitro pharmaco-toxicological studies. We used adipose-derived human stromal/stem cells to reconstruct, using tissue engineering techniques, such an adipose tridimensional model. To determine to what extent the in vitro model is representative of its native counterpart, adipogenic differentiation, triglycerides accumulation and phospholipids profiles were analysed. Ingenuity Pathway Analysis software revealed pathways enriched with differentially-expressed genes between native and reconstructed human adipose tissues. Interestingly, genes related to fatty acid metabolism were downregulated in vitro, which could be explained in part by the insufficient amount of essential fatty acids provided by the fetal calf serum used for the culture. Indeed, the lipid profile of the reconstructed human adipose tissues indicated a particular lack of linoleic acid, which could interfere with physiological cell processes such as membrane trafficking, signaling and inflammatory responses. Supplementation in the culture medium was able to influence the lipid profile of the reconstructed human adipose tissues. This study demonstrates the possibility to directly modulate the phospholipid profile of reconstructed human adipose tissues. This reinforces its use as a relevant physiological or pathological model for further pharmacological and metabolic studies of human adipose tissue functions.

Micropatterning of endothelial cells to create a capillary-like network with defined architecture by laser-assisted bioprinting.

Development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. Seeding of endothelial cells in an appropriate environment can give rise to a capillary-like network to enhance prevascularization of bone substitutes. Advances in biofabrication techniques, such as bioprinting, could allow to precisely define a pattern of endothelial cells onto a biomaterial suitable for in vivo applications. The aim of this study was to produce a microvascular network following a defined pattern and preserve it while preparing the surface to print another layer of endothelial cells. We first optimise the bioink cell concentration and laser printing parameters and then develop a method to allow endothelial cells to survive between two collagen layers. Laser-assisted bioprinting (LAB) was used to pattern lines of tdTomato-labeled endothelial cells cocultured with mesenchymal stem cells seeded onto a collagen hydrogel. Formation of capillary-like structures was dependent on a sufficient local density of endothelial cells. Overlay of the pattern with collagen I hydrogel containing vascular endothelial growth factor (VEGF) allowed capillary-like structures formation and preservation of the printed pattern over time. Results indicate that laser-assisted bioprinting is a valuable technique to pre-organize endothelial cells into high cell density pattern in order to create a vascular network with defined architecture in tissue-engineered constructs based on collagen hydrogel.

Cell Seeding on UV-C-Treated 3D Polymeric Templates Allows for Cost-Effective Production of Small-Caliber Tissue-Engineered Blood Vessels.

There is a strong clinical need to develop small-caliber tissue-engineered blood vessels for arterial bypass surgeries. Such substitutes can be engineered using the self-assembly approach in which cells produce their own extracellular matrix (ECM), creating a robust vessel without exogenous material. However, this approach is currently limited to the production of flat sheets that need to be further rolled into the final desired tubular shape. In this study, human fibroblasts and smooth muscle cells were seeded directly on UV-C-treated cylindrical polyethylene terephthalate glycol-modified (PETG) mandrels of 4.8 mm diameter. UV-C treatment induced surface modification, confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis, was necessary to ensure proper cellular attachment and optimized ECM secretion/assembly. This novel approach generated solid tubular conduits with high level of cohesion between concentric cellular layers and enhanced cell-driven circumferential alignment that can be manipulated after 21 days of culture. This simple and cost-effective mandrel-seeded approach also allowed for endothelialization of the construct and the production of perfusable trilayered tissue-engineered blood vessels with a closed lumen. This study lays the foundation for a broad field of possible applications enabling custom-made reconstructed tissues of specialized shapes using a surface treated 3D structure as a template for tissue engineering.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

In Vivo Remodeling of Fibroblast-Derived Vascular Scaffolds Implanted for 6 Months in Rats.

There is a clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are halted by the limited suitability of autologous or synthetic grafts. This study uses the self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold (FDVS) that can be available off-the-shelf. Briefly, extracellular matrix scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and decellularized by immersion in deionized water. The FDVSs were implanted as an aortic interpositional graft in six Sprague-Dawley rats for 6 months. Five out of the six implants were still patent 6 months after the surgery. Histological analysis showed the infiltration of cells on both abluminal and luminal sides, and immunofluorescence analysis suggested the formation of neomedia comprised of smooth muscle cells and lined underneath with an endothelium. Furthermore, to verify the feasibility of producing tissue-engineered blood vessels of clinically relevant length and diameter, scaffolds with a 4.6 mm inner diameter and 17 cm in length were fabricated with success and stored for an extended period of time, while maintaining suitable properties following the storage period. This novel demonstration of the potential of the FDVS could accelerate the clinical availability of tissue-engineered blood vessels and warrants further preclinical studies.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration.

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

The ROVT Elan Valved Biplex Conduits for the Reconstruction of the Right Ventricular Outflow Tract.

The reconstruction of the right ventricular outflow tract (RVOT) system represents a considerable challenge for both manufacturers and surgeons because the patients requiring this type of devices have a very diverse set of anatomical challenges that can lead to complications and subsequent early device failures. We conducted an indepth investigation of a porcine-valve conduit explanted from a patient following an adverse event. A control device was analyzed as a reference. The rapid aging of the porcine valve in the right side of the heart together with major thrombus formation raises several questions. The difficulties encountered with materials used and also the design features of the conduits are once again highlighted. This group of patients continues to increase in number due to success in the surgical outcomes in early childhood. Therefore, there is a greater demand for an appropriate device. However, much work is still needed to achieve this goal, and the best approach to achieving success remains unanswered.

The Triplex BioValsalva Prostheses To Reconstruct the Aortic Valve and the Aortic Root.

The Bentall procedure introduced in 1968 represents an undisputed cure to treat multiple pathologies involving the aortic valve and the ascending thoracic aorta. Over the years, multiple modifications have been introduced as well as a standardized approach to the operation with the goal to prevent long-term adverse events. The BioValsalva prosthesis provides a novel manner to more efficiently reconstruct the aortic valve together with the anatomy of the aortic root with the implantation of a valved conduit. This prosthesis comprises three sections: the collar supporting the valve; the skirt mimicking the Valsalva, which is suitable for the anastomoses with the coronary arteries; and the main body of the graft, which is designed to replace the ascending aorta. The BioValsalva prosthesis allows the Bentall operation to be used in patients whose aortic valve cannot be spared.

Characterization of a corneal endothelium engineered on a self-assembled stromal substitute.

Endothelial dysfunctions are the first indication for allogeneic corneal transplantation. Development of a tissue-engineered posterior cornea could be an alternative to the use of native allogeneic tissues. In this paper, we used the self-assembly approach to form a cellularized stromal substitute that served as a carrier for the engineering of an endothelium. This endothelialized stromal substitute was then characterized using alizarin red staining, histology, scanning and transmission electron microscopy, as well as mass spectrometry and immunodetection of collagens and function-related proteins. We report the engineering of a monolayer of flattened endothelial cells with a cell density of 966 ± 242 cells/mm(2) (mean ± SD). Endothelial interdigitations were present between cells. The stromal fibroblasts deposited a dense and cohesive collagenous matrix. Collagen fibrils had a diameter of 39.1 ± 11.3 nm, and a mean center to center interfibrillar space of 50.9 ± 10.9 nm. The stromal substitute was composed of collagen types I, V, VI and XII, as well as lumican and decorin. Type IV collagen was also present underneath the endothelium. The endothelium expressed both the sodium/potassium (Na(+)/K(-)) ATPase and sodium/bicarbonate (Na(+)/ [Formula: see text] ) cotransporter pumps. These results indicate that the self-assembled stromal substitute is able to support the expression of endothelial cell functionality markers and therefore, is a suitable carrier for the engineering of an endothelium that could be used for the treatment of endothelial dysfunctions.

Potential of Newborn and Adult Stem Cells for the Production of Vascular Constructs Using the Living Tissue Sheet Approach.

Bypass surgeries using native vessels rely on the availability of autologous veins and arteries. An alternative to those vessels could be tissue-engineered vascular constructs made by self-organized tissue sheets. This paper intends to evaluate the potential use of mesenchymal stem cells (MSCs) isolated from two different sources: (1) bone marrow-derived MSCs and (2) umbilical cord blood-derived MSCs. When cultured in vitro, a proportion of those cells differentiated into smooth muscle cell- (SMC-) like cells and expressed contraction associated proteins. Moreover, these cells assembled into manipulable tissue sheets when cultured in presence of ascorbic acid. Tubular vessels were then produced by rolling those tissue sheets on a mandrel. The architecture, contractility, and mechanical resistance of reconstructed vessels were compared with tissue-engineered media and adventitia produced from SMCs and dermal fibroblasts, respectively. Histology revealed a collagenous extracellular matrix and the contractile responses measured for these vessels were stronger than dermal fibroblasts derived constructs although weaker than SMCs-derived constructs. The burst pressure of bone marrow-derived vessels was higher than SMCs-derived ones. These results reinforce the versatility of the self-organization approach since they demonstrate that it is possible to recapitulate a contractile media layer from MSCs without the need of exogenous scaffolding material.

Characterization of tissue-engineered posterior corneas using second- and third-harmonic generation microscopy.

Three-dimensional tissues, such as the cornea, are now being engineered as substitutes for the rehabilitation of vision in patients with blinding corneal diseases. Engineering of tissues for translational purposes requires a non-invasive monitoring to control the quality of the resulting biomaterial. Unfortunately, most current methods still imply invasive steps, such as fixation and staining, to clearly observe the tissue-engineered cornea, a transparent tissue with weak natural contrast. Second- and third-harmonic generation imaging are well known to provide high-contrast, high spatial resolution images of such tissues, by taking advantage of the endogenous contrast agents of the tissue itself. In this article, we imaged tissue-engineered corneal substitutes using both harmonic microscopy and classic histopathology techniques. We demonstrate that second- and third-harmonic imaging can non-invasively provide important information regarding the quality and the integrity of these partial-thickness posterior corneal substitutes (observation of collagen network, fibroblasts and endothelial cells). These two nonlinear imaging modalities offer the new opportunity of monitoring the engineered corneas during the entire process of production.

Mechanical properties of endothelialized fibroblast-derived vascular scaffolds stimulated in a bioreactor.

There is an ongoing clinical need for tissue-engineered small-diameter (<6mm) vascular grafts since clinical applications are restricted by the limited availability of autologous living grafts or the lack of suitability of synthetic grafts. The present study uses our self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold that can then be available off-the-shelf. Briefly, scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and then decellularized by immersion in deionized water. Constructs were then endothelialized and perfused for 1week in an appropriate bioreactor. Mechanical testing results showed that the decellularization process did not influence the resistance of the tissue and an increase in ultimate tensile strength was observed following the perfusion of the construct in the bioreactor. These fibroblast-derived vascular scaffolds could be stored and later used to deliver readily implantable grafts within 4weeks including an autologous endothelial cell isolation and seeding process. This technology could greatly accelerate the clinical availability of tissue-engineered blood vessels.

Understanding the process of corneal endothelial morphological change in vitro.

Corneal endothelial cells often adopt a fibroblastic-like morphology in culture, a process that has been attributed to epithelial- or endothelial-to-mesenchymal transition (EMT or EndMT). Although being extensively studied in other cell types, this transition is less well characterized in the corneal endothelium. Because of their neuroectodermal origin and their in vivo mitotic arrest, corneal endothelial cells represent a particular tissue that deserves more attention. This review article presents the basic principles underlying EMT/EndMT, with emphasis on the current knowledge regarding the corneal endothelium. Furthermore, this review discusses cell culture conditions and major cell signaling pathways that have been identified as EndMT-triggering factors. Finally, it summarizes strategies that have been developed to inhibit EndMT in corneal endothelial cell culture. The review of current studies on corneal and classical EndMT highlights some research avenues to pursue in the future and underscores the need to extend our knowledge of this process in order to optimize usage of these cells in regenerative medicine.

A Floating Thrombus Anchored at the Proximal Anastomosis of a Woven Thoracic Graft Mimicking a Genuine Aortic Dissection.

An aortoesophageal fistula following surgery for a ruptured 6.6-cm thoracic aneurysm in a 69-year-old female was repaired using a 34-mm woven prosthetic graft. A follow-up computed tomography (CT) scan at 10 days postoperatively revealed a dissection-like picture in the region of the graft, which was treated conservatively. The patient eventually died from sepsis and multiorgan failure. At autopsy, the graft was retrieved in situ and studied by detailed gross, microscopy, and scanning electron microscopy (SEM) examination. Gross observation confirmed that the dissection resulted from the rolling of the internal capsule downstream. A massive thrombus anchored at the proximal anastomosis and held by a narrow head was also noted. The thrombus demonstrated reorganization in the area of the anastomosis, with a false lumen in its distal half. The reminder of the thrombus consisted of layered fibrin. After gross examination, the fabric graft was found to be flawless. Additional detailed studies were also done using microscopy, SEM, and gross examination.

Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach.

The aortic heart valve is constantly subjected to pulsatile flow and pressure gradients which, associated with cardiovascular risk factors and abnormal hemodynamics (i.e. altered wall shear stress), can cause stenosis and calcification of the leaflets and result in valve malfunction and impaired circulation. Available options for valve replacement include homograft, allogenic or xenogenic graft as well as the implantation of a mechanical valve. A tissue-engineered heart valve containing living autologous cells would represent an alternative option, particularly for pediatric patients, but still needs to be developed. The present study was designed to demonstrate the feasibility of using a living tissue sheet produced by the self-assembly method, to replace the bovine pericardium currently used for the reconstruction of a stented human heart valve. In this study, human fibroblasts were cultured in the presence of sodium ascorbate to produce tissue sheets. These sheets were superimposed to create a thick construct. Tissue pieces were cut from these constructs and assembled together on a stent, based on techniques used for commercially available replacement valves. Histology and transmission electron microscopy analysis showed that the fibroblasts were embedded in a dense extracellular matrix produced in vitro. The mechanical properties measured were consistent with the fact that the engineered tissue was resistant and could be cut, sutured and assembled on a wire frame typically used in bioprosthetic valve assembly. After a culture period in vitro, the construct was cohesive and did not disrupt or disassemble. The tissue engineered heart valve was stimulated in a pulsatile flow bioreactor and was able to sustain multiple duty cycles. This prototype of a tissue-engineered heart valve containing cells embedded in their own extracellular matrix and sewn on a wire frame has the potential to be strong enough to support physiological stress. The next step will be to test this valve extensively in a bioreactor and at a later date, in a large animal model in order to assess in vivo patency of the graft.

A new construction technique for tissue-engineered heart valves using the self-assembly method.

Tissue engineering appears as a promising option to create new heart valve substitutes able to overcome the serious drawbacks encountered with mechanical substitutes or tissue valves. The objective of this article is to present the construction method of a new entirely biological stentless aortic valve using the self-assembly method and also a first assessment of its behavior in a bioreactor when exposed to a pulsatile flow. A thick tissue was created by stacking several fibroblast sheets produced with the self-assembly technique. Different sets of custom-made templates were designed to confer to the thick tissue a three-dimensional (3D) shape similar to that of a native aortic valve. The construction of the valve was divided in two sequential steps. The first step was the installation of the thick tissue in a flat preshaping template followed by a 4-week maturation period. The second step was the actual cylindrical 3D forming of the valve. The microscopic tissue structure was assessed using histological cross sections stained with Masson's Trichrome and Picrosirius Red. The thick tissue remained uniformly populated with cells throughout the construction steps and the dense extracellular matrix presented corrugated fibers of collagen. This first prototype of tissue-engineered heart valve was installed in a bioreactor to assess its capacity to sustain a light pulsatile flow at a frequency of 0.5 Hz. Under the light pulsed flow, it was observed that the leaflets opened and closed according to the flow variations. This study demonstrates that the self-assembly method is a viable option for the construction of complex 3D shapes, such as heart valves, with an entirely biological material.

Comparison of the direct burst pressure and the ring tensile test methods for mechanical characterization of tissue-engineered vascular substitutes.

Tissue engineering provides a promising alternative for small diameter vascular grafts, especially with the self-assembly method. It is crucial that these grafts possess mechanical properties that allow them to withstand physiological flow and pressure without being damaged. Therefore, an accurate assessment of their mechanical properties, especially the burst pressure, is essential prior to clinical release. In this study, the burst pressure of self-assembled tissue-engineered vascular substitutes was first measured by the direct method, which consists in pressurizing the construct with fluid until tissue failure. It was then compared to the burst pressure estimated by Laplace׳s law using data from a ring tensile test. The major advantage of this last method is that it requires a significantly smaller tissue sample. However, it has been reported as overestimating the burst pressure compared to a direct measurement. In the present report, it was found that an accurate estimation of the burst pressure may be obtained from a ring tensile test when failure internal diameter is used as the diameter parameter in Laplace׳s law. Overestimation occurs with the method previously reported, i.e. when the unloaded internal diameter is used for calculations. The estimation of other mechanical properties was also investigated. It was demonstrated that data from a ring tensile test provide an accurate estimate of the failure strain and the stiffness of the constructs when compared to measurements with the direct method.

Interleukin-10 controls the protective effects of circulating microparticles from patients with septic shock on tissue-engineered vascular media.

During sepsis, inflammation can be orchestrated by the interaction between circulating and vascular cells that, under activation, release MPs (microparticles). Previously, we reported that increased circulating MPs in patients with sepsis play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity. The present study was designed to investigate the effects of MPs from patients with sepsis on the contractile response of TEVM (tissue-engineered vascular media). TEVM that were composed only of a media layer were produced by tissue engineering from human arterial SMCs (smooth muscle cells) isolated from umbilical cords. TEVM was incubated with MPs isolated from whole blood of 16 patients with sepsis. TEVM were incubated for 24 h with MPs and used for the study of vascular contraction, direct measurements of NO and O2- (superoxide anion) production by EPR and quantification of mRNA cytokine expression. MPs from patients with sepsis increased contraction induced by histamine in TEVM. This effect was not associated with inflammation, neither linked to the activation of NF-κB (nuclear factor κB) pathway nor to the increase in iNOS (inducible NO synthase) and COX (cyclo-oxygenase)-2 expression. In contrast, mRNA expression of IL (interleukin)-10 was enhanced. Then, we investigated the effect of IL-10 on vascular hyporeactivity induced by LPS (lipopolysaccharide). Although IL-10 treatment did not modify the contractile response in TEVM by itself, this interleukin restored contraction in LPS-treated TEVM. In addition, IL-10 treatment both prevented vascular hyporeactivity induced by LPS injection in mice and improved survival of LPS-injected mice. These findings show an association between the capacity of MPs from patients with sepsis to restore vascular hyporeactivity induced by LPS and their ability to increase IL-10 in the tissue-engineered blood vessel model.

Human fibroblast-derived ECM as a scaffold for vascular tissue engineering.

The self-assembly approach is based on the capability of mesenchymal cells to secrete and organize their own extracellular matrix (ECM). This tissue engineering method allows for the fabrication of autologous living tissues, such as tissue-engineered blood vessels (TEBV) and skin. However, the secretion of ECM by smooth muscle cells (SMCs), required to produce the vascular media, may represent a long process in vitro. The aim of this work was to reduce the time required to produce a tissue-engineered vascular media (TEVM) and extend the production of TEVM with SMCs from all patients without compromising its mechanical and functional properties. Therefore, we developed a decellularized matrix scaffold (dMS) produced from dermal fibroblasts (DF) or saphenous vein fibroblasts (SVF), in which SMCs were seeded to produce a TEVM. Mechanical and contractile properties of these TEVM (referred to as nTEVM) were compared to standard self-assembled TEVM (sTEVM). This approach reduced the production time from 6 to 4 weeks. Moreover, nTEVM were more resistant to tensile load than sTEVM and their vascular reactivity was also improved. This new fabrication technique allows for the production of a vascular media using SMCs isolated from any patient, regardless of their capacity to synthesize ECM. Moreover, these scaffolds can be stored to be available when needed, in order to accelerate the production of the vascular substitute using autologous vascular cells.