Tissue engineering of the vascular system : from capillaries to larger blood vessels

Tissue engineering is a novel approach to the repair of wounded tissues. Application of this technology to the vascular system is important because of the fundamental nutritional role of the vasculature. This perspective is currently being applied to the first tissue-engineered organ: the skin. Knowledge of capillary constitution and factors inducing their formation has led to attempts to induce their formation in reconstructed skin. Such vascular conduits grown in vitro could also benefit the nutrition of tissues and organs in vivo. The paper reviews recent progress in thein-vitro development of vascularised skin and tissue-engineered blood vessels. It points out the necessity of obtaining pure and well-characterised cultures of the different cell populations that are the basic building blocks of the reconstructions. The importance of an adequate cell-culture environment (nutrients and bi-or tri-dimensional scaffolds for cells) for success in elaborating a reconstructed living tissue able to replace the original is emphasised. Engineered tissues can serve not only as tissue replacements but also asin-vitro models for research in organ physiology and physiopathology. These tissues are also attractive vehicles for gene therapy, one of the more promising new methods of disease treatment

The self-assembly approach for organ reconstruction by tissue engineering

One must not forget that tissue engineering was first introduced as a life saving procedure for burn patients (1). The successful engraftment of autologous epidermal sheets was the first proof of concept of the powerful technology that we know today (2-4). However this very interesting initial approach fell into some disrepute because of perceived drawbacks and limitations (5, 6). The subsequent efforts in the field followed essentially three "schools" of thought. The first approach consists in the seeding of cells into various gels, which are then reorganized, by the incorporated cells (7-14). Alternatively, a second approach is to seed cells into a scaffold where they will thrive and secrete extracellular matrix (15-17). The scaffold materials are bioresorbable over a wide range of time periods depending on their chemical structures (18-25). A third approach is different since it uses the principle of a tissue template that allows, after implantation, the ingress of cells into the appropriately organized scaffold. Thus, these grafts are acellular and must stimulate the regenerative potential of the tissue wherever they are implanted (26-31). Our group has developed a different and original method for the reconstruction of soft tissues. It takes full advantage of the various intrinsic properties of cells when appropriately cultured. This entails particular media composition and appropriate mechanical straining of these threedimensional structures

A full spectrum of functional tissue-engineered blood vessels : from macroscopic to microscopic

Tissue engineering has created several original and new avenues of investigation in biology (Auger et al., 2000). This new domain of research in biotechnology was introduced in the l980$ as a life-saving procedure for burn patients. The successful engrai‘tment of autologous living epidermis was the first proof of concept of this powerful approach. From the efforts in this field, two schools of thought emerged. A first one is the seeding of cells into various gels or scatTolds in which the cells secrete and/or reorganize the surrounding extracellular matrix (ECM), and a second one, the coaxing of cells onto the secretion of an abundant autologous ECM, thus creating their own environment in the absence of any exogenous material. This latter methodology, which we called the “self assembly approach,” takes advantage of the ability of cells to recreate in vitro tissue-like structures when appropriately cultured (Auger et al., 2000). The conditions entail particular media composition and adapted mechanical straining ol‘ these three-dimensional structures. Our own experience with the culture of autologous epidermal sheets gave us some insight in the property of cells to recreate such in rim: tissue-like structures. This expertise led us to develoP tissue-engineered structures on the basis ol‘ the following two concepts: the living substitutes that we created have no artificial biomaterial, and the ECM is either a biological one repopulated by the ceiis or an ECM neosynthesized by the cells themselves. Such living substitutes have distinct advantages because of their cellular composition that confer to them superior physiologicai characteristics when implanted into the human body, that is, their ability to renew themselves over time and their healing property if they are damaged. Moreover, the presence of autologous cells in the living reconstructed tissue should facilitate its interactions with the surrounding host environment. Here, we describe our own experience in the reconstruction of a full spectrum of blood vessels by tissue engineering: macroscopic and microscopic. We applied the self-assembly approach with some impressive results to the reconstruction of a small-diameter blood vessel and the use of a cell-seeded scaffold leading to the formation of capillary-like structures in a full-thickness skin. The following highlights the major points for the generation of these organs

A truly new approach for tissue engineering : the LOEX self-assembly technique

Tissue engineering has created several original and new avenues in the biomedical sciences. There is ongoing progress, but the tissue-engineering field is currently at a crossroads in its evolution; the validity of this technique is weIl established. Thus, new clinical applications must appear rapidly, within a few years, so that it will have a true impact on patient care. The self-assembly approach of the Laboratoire d'Organogénèse Expérimentale (LOEX) should be at the forefront

Recent optimization of a tissue engineered blood vessel : the LOEX experience

Creating a blood vessel by tissue engineering is one of the most demanding goals in tissue engineering. Our laboratory developed, using the self-assembly approach, the first completely biological tissue engineered blood vessel (TEBV) constituted of living human cells in the absence of any synthetic or exogenous material. The phenotypic and functional variations of smooth muscle cell (SMC) are of paramount importance in TEBV reconstruction. Thus, the phenotype and extracellular matrix (ECM) production of SMC were studied along the whole sequence of TEBV production. The functional and mechanical properties can be greatly enhanced by active cell orientation in the ECM. Accordingly, the method of preparing living tissue engineered sheets was modified to obtain an optimal alignment of SMC before rolling them into a tubular form. These results have allowed us to create a better TEBV

Endothelium properties of a tissue-engineered blood vessel for small-diameter vascular reconstruction

Purpose: A tissue-engineered blood vessel (TEBV) produced in vitro by the self-assembly method was developed in our laboratory for the replacement of small-diameter blood vessels. The interior of this vessel is covered by an endothelium. The aim of the present study was to evaluate whether the endothelial layer would make a favorable contribution at the time of implantation of the TEBV by investigating in vitro the hemocompatible properties of the endothelial cells covering its interior. Methods: The secretion of the von Willebrand factor (vWF) and expression of thrombomodulin by the endothelium were assessed, and the adhesive molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) were quantified as a function of maturation time. To evaluate the functional response of the endothelium on injury, the cellular response to physiological stimulatory factors (thrombin and lipopolysaccharide [LPS]) was analyzed. Results: The endothelial cells formed a confluent monolayer displaying favorable hemocompatible properties (78% 10% of cells expressing thrombomodulin with only 12 3 mU/106 cells of vWF secreted over a 2-hour period), which acquired their full expression after a culture period of 4 days. Moreover, pro-adhesive properties toward inflammatory cells were not observed. The cells were also able to respond to physiological-stimulating agents (thrombin and LPS) and demonstrated a statistically significant overexpression of the corresponding molecules under the conditions tested. Conclusions: These results indicate that the endothelium of the tissue-engineered blood vessel produced by the self-assembly approach displays advantageous qualities with regard to the vessel’s future implantation as a small-diameter vascular prosthesis

Mesenchymal cell culture : blood vessels

The replacement of small diameter vascular prostheses by nonautologous veins is the subject of intensive research. Attempts to produce synthetic, hybrid artificial or biological conduits are numerous and dependent on progress in technology and comprehension of the mechanisms implicated in their reconsrruction. The production of a completely biological vascular prosthesis is described in this chapter. Exclusively composed of human cells embedded in their own extracellular matrix, this graft could be produced autologously and offers advantages related to its biological composition. The steps to produce such a living structure are detailed, and some important areas, such as the purity and the phenotype of cells, and the application of mechanical strain to the construct, are pointed out as essential factors for the success of our tissue engineered blood vessel (TEBV). The main characteristics obtained with this prosthesis are presented, as well as directions future research could take to improve this human living prosthesis

Evaluating poly(Acrylamide-co-Acrylic Acid) hydrogels stress relaxation to direct the osteogenic differentiation of mesenchymal stem cells

The aim of this study is to investigate polyacrylamide-based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross-linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein-2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star-shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine-tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro