Tissue Engineering

There is little doubt that tissue engineering is a revolutionary addition to the therapeutic armamentarium of medicine. The dilemma of adequately repairing either failing or traumatized organs has been looming larger as patients either become older or are in dire need of grafts. Compounding some of the intrinsic problems of transplantation is the chronic shortage of tissues and organs. Tissue engineering allows the hope of a regular creation of spare parts for the human body. This is a most significant approach to reconstruct, replace, or repair organs in a way that could not be foreseen 25 years ago. Reconstructive medicine is, in a way, not a very recent concept. If one stays away from punctilious definitions, one of its forms, reconstructive surgery, has been practiced for quite some time, with a surge of development after the Second World War. In 1970s, the development of microsurgery allowed distant tissue transfer and reimplantation.[1-5] Since then, the introduction of various biomaterials has allowed vast and diversified types of reconstruction of the human body. Vascular grafts and prosthetic articulation are two prominent examples.[6] However, tissue engineering does open a radically new chapter in reconstructive medicine, for it is now deemed possible to reconstruct in the laboratory human living tissues and organs for either in-vivo, ex-vivo, and even invitro applications.[7-3] This new domain of biotechnology is remarkably multidisciplinary, bringing together cell and molecular biologists, biochemists, engineers, pharmacologists, physicians, and others. When the aim of tissue engineers is to obtain grafts for in-vivo applications, then the biological and mechanical functions are of utmost importance. In some subdivisions of the field, one can essentially choose between a biological function, as in cell therapy, and a principally mechanical function, as in the use of tissue templates[14] (Fig. 1). Tissue-engineered substitutes are three-dimensional reconstructions that can be implanted into the human body, leading to rapid host integration and acceptance. These substitutes must have at least minimal biological and mechanical functions for such a reparative role

Thérapie génique appliquée au génie tissulaire

La reconstruction tissulaire cutanée ouvre la voie à des applications fascinantes (1). Ainsi, depuis plus de 15 ans, le génie tissulaire a permis de produire des substituts cutanés pour le traitement des grands brûlés, car c’est dans ce domaine que cette technique récolte les résultats les plus significatifs sur la morbidité et la mortalité de ces patients. Il existe bien sûr d’autres perspectives cliniques : nous ne les aborderons pas directement dans le présent article; il s’agit, par exemple, des greffes cutanées consécutives à une postchirurgie « mutilante » (étendue) en oncologie ou des transplantations à des fins esthétiques sur des sites affectés d’une cicatrisation anormale

Alignment of Cells and Extracellular Matrix Within Tissue- Engineered Substitutes

Most of the cells in our body are in direct contact with extracellular matrix (ECM) compo‐ nents which constitute a complex network of nano-scale proteins and glycosaminoglycans. Those cells constantly remodel the ECM by different processes. They build it by secreting dif‐ ferent proteins such as collagen, proteoglycans, laminins or degrade it by producing factors such as matrix metalloproteinase (MMP). Cells interact with the ECM via specific receptors, the integrins [1]. They also organize this matrix, guided by different stimuli, to generate pat‐ terns, essential for tissue and organ functions. Reciprocally, cells are guided by the ECM, they modify their morphology and phenotype depending on the protein types and organization via bidirectional integrin signaling [2-4]. In the growing field of tissue engineering [5], control of these aspects are of the utmost importance to create constructs that closely mimic native tis‐ sues. To do so, we must take into account the composition of the scaffold (synthetic, natural, biodegradable or not), its organization and the dimension of the structure. The particular alignment patterns of ECM and cells observed in tissues and organs such as the corneal stroma, vascular smooth muscle cells (SMCs), tendons, bones and skeletal mus‐ cles are crucial for organ function. SMCs express contraction proteins such as alpha-smoothmuscle (SM)-actin, desmin and myosin [6] that are essential for cell contraction [6]. To result in vessel contraction, the cells and ECM need to be organized in such a way that most cells are elongated in the same axis. For tubular vascular constructs, it is suitable that SMCs align in the circumferential direction, as they do in vivo [7, 8]. Another striking example of align‐ ment is skeletal muscle cells that form long polynuclear cells, all elongated in the same axis. Each cell generates a weak and short contraction pulse but collectively, it results in a strong, long and sustained contraction of the muscle and, in term, a displacement of the member. In the corneal stroma, the particular arrangement of the corneal fibroblasts (keratocytes) and ECM is essential to keep the transparency of this tissue [9-13]. Tendons also present a pecu‐ liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excep‐ tional mechanical properties to the tissue in that axis [14, 15]. Intervertebral discs [16], cartilage [17], dental enamel [18], and basement membrane of epithelium are other examples of tissues/organs that present peculiar cell and matrix organization. By reproducing and controlling those alignment patterns within tissue-engineered substitutes, a more physiolog‐ ical representation of human tissues could be achieved. Taking into account the importance of cell microenvironment on the functionality of tissue engineered organ substitutes, one can assume the importance of being able to customise the 3D structure of the biomaterial or scaffold supporting cell growth. To do so, some methods have been developed and most of them rely on topographic or contact guidance. This is the phenomenon by which cells elongate and migrate in the same axis as the ECM. Topographic guidance was so termed by Curtis and Clark [19] to include cell shape, orientation and movement in the concept of contact guidance described by Harrison [20] and implemented by Weiss [21, 22]. Therefore, if one can achieve ECM alignment, cells will follow the same pattern. Inversely, if cells are aligned on a patterned culture plate, the end result would be aligned ECM deposition [23]. The specific property of tissues or materials that present a variation in their mechanical and structural properties in different axis is called anisotropy. This property can be evaluated ei‐ ther by birefringence measurements [24, 25], mechanical testing in different axis [26], immu‐ nological staining of collagen or actin filaments [23] or direct visualisation of collagen fibrils using their self-fluorescence around 488 nm [27, 28]. Several techniques have been recently developed to mimic the specific alignment of cells within tissues to produce more physiologically relevant constructs. In this chapter, we will describe five different techniques, collagen gel compaction, electromagnetic field, electro‐ spinning of nanofibers, mechanical stimulation and microstructured culture plates

Anticancer properties of chitosan on human melanoma are cell line dependent

Purpose: Chitosan, a natural macromolecule, is widely used in medical and pharmaceutical fields because of its distinctive properties such as bactericide, fungicide and above all its antitumor effects. Although its antitumor activity against different types of cancer had been previously described, its mechanism of action was not fully understood. Materials and methods: Coating of chitosan has been used in cell cultures with A375, SKMEL28, and RPMI7951 cell lines. Adherence, proliferation and apoptosis were investigated. Results: Our results revealed that whereas chitosan decreased adhesion of primary melanoma A375 cell line and decreased proliferation of primary melanoma SKMEL28 cell line, it had potent pro-apoptotic effects against RPMI7951, a metastatic melanoma cell line. In these latter cells, inhibition of specific caspases confirmed that apoptosis was effected through the mitochondrial pathway and Western blot analyses showed that chitosan induced an up regulation of pro-apoptotic molecules such as Bax and a down regulation of anti-apoptotic proteins like Bcl-2 and Bcl-XL. More interestingly, chitosan exposure induced an exposition of a greater number of CD95 receptor at RPMI7951 surface, making them more susceptible to FasL-induced apoptosis. Conclusion: Our results indicate that chitosan could be a promising agent for further evaluations in antitumor treatments targeting melanoma

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. Avail- able options for valve replacement include homograft, allogenic or xenogenic graft as well as the implan- tation 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 physio- logical 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

Grafting on nude mice of living skinquivalents produced using human collagens

Autologous epidermal transplantation for human burn management is an example of a significant breakthrough in tissue engineering. However, the main drawback with this treatment remains the fragility of these grafts during and after surgery. A new human bilayered skin equivalent (hSE) was produced in our laboratory to overcome this problem. The aim of the present work was to study skin regeneration after hSE grafting onto nude mice. A comparative study was carried out over a period of 90 days, between anchored bovine skin equivalent, hSE and hSE+, the latter containing additional matrix components included at concentrations similar to those in human skin in vivo. The addition of a dermal layer to the epidermal sheet led to successful graft take, enhanced healing, and provided mechanical resistance to the grafts after transplantation. In situ analysis of the grafts showed good ultrastructural organization, including the deposition of a continuous basement membrane 1 week after surgery

Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction

Purpose: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. Methods: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2 ). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. Results: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. Conclusions: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency

Impact of cell source on human cornea reconstructed by tissue engineering

Purpose: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. Methods: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. Results: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. Conclusions: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis

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