The pivotal role of vascularization in tissue engineering.

Vascularization is one of the great challenges that tissue engineering faces in order to achieve sizeable tissue and organ substitutes that contain living cells. There are instances, such as skin replacement, in which a tissue-engineered substitute does not absolutely need a preexisting vascularization. However, tissue or organ substitutes in which any dimension, such as thickness, exceeds 400 µm need to be vascularized to ensure cellular survival. Consistent with the wide spectrum of approaches to tissue engineering itself, which vary from acellular synthetic biomaterials to purely biological living constructs, approaches to tissue-engineered vascularization cover numerous techniques. Those techniques range from micropatterns engineered in biomaterials to microvascular networks created by endothelial cells. In this review, we strive to provide a critical overview of the elements that must be considered in the pursuit of this goal and the major approaches that are investigated in hopes of achieving it.

Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

Regeneration of lymphatic vessels is important for treatment of various disorders of lymphatic system and for restoration of lymphatic function after surgery. We have developed a method for generating a human 3D lymphatic vascular construct. In this system, human lymphatic endothelial cells, co-cultured with fibroblasts, spontaneously organized into a stable 3D lymphatic capillary network without the use of any exogenous factors. In vitro-generated lymphatic capillaries exhibited the major molecular and ultra-structural features of native, human lymphatic microvasculature: branches in the three dimensions, wide lumen, blind ends, overlapping borders, adherens and tight junctions, anchoring filaments, lack of mural cells, and poorly developed basement membrane. Furthermore, we show that fibroblast-derived VEGF-C and HGF cooperate in the formation of lymphatic vasculature by activating ERK1/2 signaling, and demonstrate distinct functions of HGF/c-Met and VEGF-C/VEGFR-3 in lymphangiogenesis. This lymphatic vascular construct is expected to facilitate studies of lymphangiogenesis in vitro and it holds promise as a strategy for regeneration of lymphatic vessels and treatment of lymphatic disorders in various conditions.

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.

Production of a Bilayered Self-Assembled Skin Substitute Using a Tissue-Engineered Acellular Dermal Matrix.

Our bilayered self-assembled skin substitutes (SASS) are skin substitutes showing a structure and functionality very similar to native human skin. These constructs are used, in life-threatening burn wounds, as permanent autologous grafts for the treatment of such affected patients even though their production is exacting. We thus intended to shorten their current production time to improve their clinical applicability. A self-assembled decellularized dermal matrix (DM) was used. It allowed the production of an autologous skin substitute from patient's cells. The characterization of SASS reconstructed using a decellularized dermal matrix (SASS-DM) was performed by histology, immunofluorescence, transmission electron microscopy, and uniaxial tensile analysis. Using the SASS-DM, it was possible to reduce the standard production time from about 8 to 4 and a half weeks. The structure, cell differentiation, and mechanical properties of the new skin substitutes were shown to be similar to the SASS. The decellularization process had no influence on the final microstructure and mechanical properties of the DM. This model, by enabling the production of a skin substitute in a shorter time frame without compromising its intrinsic tissue properties, represents a promising addition to the currently available burn and wound treatments.

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.

Making recombinant proteins in animals--different systems, different applications.

Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.

Porcine Niemann Pick-C1 protein is expressed in steroidogenic tissues and modulated by cAMP.

Niemann-Pick C-1 (NPC-1) protein is essential for trafficking of low density lipoprotein-derived cholesterol in mammalian cells. The low density lipoprotein pathway is a major route for supply of cholesterol for steroidogenesis in the adrenals and gonads of many species. We investigated the occurrence and regulation of NPC-1 in porcine tissues, with emphasis on the corpus luteum and on granulosa cells undergoing luteinization in vitro. The porcine open reading frame for NPC-1 predicted a protein of 1278 amino acids (aa). It displayed a domain structure consistent with the human protein, and overall homologies were 89% and 86% with the deduced human and mouse aa sequences, respectively. The mRNA for NPC-1 comprised two transcripts, migrating at 5.0 and 2.2 kb, respectively. Transcripts were detected in a variety of pig tissues and were in highest abundance in steroid-producing organs. NPC-1 mRNA abundance increased with the differentiation of the corpus luteum in vivo and with luteinization of granulosa cells in vitro. Actinomycin D blockade of transcription in luteinized granulosa cells resulted in reduced NPC-1 mRNA and provided a half-life estimate of 20 h. Cycloheximide treatment increased NPC-1 transcript abundance in excess of 5-fold over 24 h. Treatment of luteinized granulosa cells with 1 mM (Bu)(2)cAMP increased the abundance of the NPC-1 message by 2- to 4-fold. The 5'-flanking region of the pig sequence displayed consensus sequences for binding transcription factors, including specificity protein-1, cAMP response element-binding protein/activating transcription factor-1, activating protein-1, GATA, modified zinc finger protein-1, transcription factor-11 and a CpG island in the first 400 bp upstream of the ATG transcription initiation site. Transient transfection of 1.86 kb of the 5'-flanking region coupled to the luciferase reporter into three steroidogenic cell lines resulted in constitutive transcription. Treatment with (Bu)2cAMP for 24 h increased the luciferase signal in all three lines. Thus, three types of evidence indicate that cAMP regulates pig NPC-1 expression. These are the presence of consensus binding sites for cAMP-induced transcription factors (cAMP response element-binding protein/activating transcription factor-1) in the proximal 5'-flanking region of the gene, increases in transcription by the NPC-1 promoter, and increases in NPC-1 mRNA abundance induced by (Bu)2cAMP. We conclude that NPC-1 is expressed in the steroidogenic tissues of the pig and is regulated by the principal pathway of stimulation of steroidogenesis in the gonads and adrenal, the cAMP-PKA pathway.

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.