339 research outputs found
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 signiïŹcant
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 deïŹnitions,
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 diversiïŹed 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 cutanĂ©e ouvre la voie Ă des applications fascinantes (1). Ainsi, depuis plus de 15 ans, le gĂ©nie tissulaire a permis de produire des substituts cutanĂ©s pour le traitement des grands brĂ»lĂ©s, car câest dans ce domaine que cette technique rĂ©colte les rĂ©sultats les plus significatifs sur la morbiditĂ© et la mortalitĂ© de ces patients.
Il existe bien sĂ»r dâautres perspectives cliniques : nous ne les aborderons pas directement dans le prĂ©sent article; il sâagit, par exemple, des greffes cutanĂ©es consĂ©cutives Ă une postchirurgie « mutilante » (Ă©tendue) en oncologie ou des transplantations Ă des ïŹns esthĂ©tiques sur des sites affectĂ©s dâune 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) compoâ
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 difâ
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 patâ
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 tisâ
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 musâ
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 alignâ
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 pecuâ
liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excepâ
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 physiologâ
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 eiâ
ther by birefringence measurements [24, 25], mechanical testing in different axis [26], immuâ
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, electroâ
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
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
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