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The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal's heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

The construction of the high biocompatible biomaterials pretreated with MSC offers a promising strategy to improve the effects of stem cell therapy for the myocardial infarction (MI). However, assembling vascularized three-dimensional (3-D) myocardial tissues remains an enormous challenge. In this study, we optimized the decellularization protocol with the umbilical artery to construct microporous 3-D scaffold which is suitable for the stem cells (SC) proliferation. The SD rats underwent proximal left coronary ligation and a 5-mm diameter microporous SC patch was implanted directly on the infarct area (SC patch group). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 4 weeks after patch implantation. The MSC patch integrated to the local heart tissue and the neo-vessels have been observed in the MSC patch. The vessels in the MSC patch were positive for the CD31 (marker for the mature endothelial cells). The left ventricle wall was thicker in the MSC patch group than the control group (p<0.05 vs. empty patch group). And the LVEF has been improved in the MSC patch group than empty patch group (59±6.7% vs. 31±4.5%, p<0.05)., Conclusions: Our results showed that the implantation of the MSC patch improved cardiac contractile function in heart infarction rat model. The construction of artificial tissue from the decellularized umbilical artery and the MSC may open a promising perspective for the tissue therapy for MI.

In tissue engineering approaches, the quality of substitutes is a key element to determine its ability to treat cartilage defects. However, in clinical practice, the evaluation of tissue-engineered cartilage substitute quality is not possible due to the invasiveness of the standard procedure, which is to date histology. The aim of this work was to validate a new innovative system performed from two-photon excitation laser adapted to an optical macroscope to evaluate at macroscopic scale the collagen network in cartilage tissue-engineered substitutes in confrontation with gold standard histologic techniques or immunohistochemistry to visualize type II collagen. This system permitted to differentiate the quality of collagen network between ITS and TGF-β1 treatments. Multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) at macroscopical scale represent an innovative and non-invasive technique to monitor the quality of collagen network in cartilage tissue-engineered substitutes before in vivo implantation.

Liver transplantation is the definitive treatment for patients with end-stage liver diseases (ESLD). However, it is hampered by shortage of liver donor. Liver tissue engineering, aiming at fabricating new livers in vitro, provides a potential resolution for donor shortage. Three elements need to be considered in liver tissue engineering: seeding cell resources, scaffolds and bioreactors. Studies have shown potential cell sources as hepatocytes, hepatic cell line, mesenchymal stem cells and others. They need scaffolds with perfect biocompatiblity, suitable micro-structure and appropriate degradation rate, which are essential charateristics for cell attachment, proliferation and secretion in forming extracellular matrix. The most promising scaffolds in research include decellularized whole liver, collagens and biocompatible plastic. The development and function of cells in scaffold need a microenvironment which can provide them with oxygen, nutrition, growth factors, et al. Bioreactor is expected to fulfill these requirements by mimicking the living condition in vivo. Although there is great progress in these three domains, a large gap stays still between their researches and applications. Herein, we summarized the recent development in these three major fields which are indispensable in liver tissue engineering.

Background: Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering., Methods: In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC., Results: After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC., Conclusions: Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use.During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction…).

Objectives: In present study, we plan to produce a decellularization protocol from rat liver to generate a three-dimensional whole organ scaffold., Methods: A combination of 1% SDS and 1% tritonX-100 were used orderly to decellularize rat livers. After about 6 h of interactive antegrade/retrograde perfusion, a decellularized whole translucent liver scaffold with integrated blood vessel networks was generated. The decellularized livers are charactered by light microscopy, scanning electron microscopy, and biochemical analysis (DNA quantification) for preservation of the three-dimension of extracellular matrix architecture., Results: The decellularization protocol was verified by observation of the whole translucent liver organ with intact vascular trees under macroscopy, in conjunction with the hematoxylin-eosin staining that showed no cells or nuclear material remained. Additionally, the Masson's stain indicted that the extracellular proteins were well kept and scanning electron microscopy (SEM) revealed a preserved decellularized matrix architecture. Compared to normal livers, DNA in the decellularized livers was quantified less than 10% at the same mass., Conclusions: The current method of decellularization protocol was feasible, simple and quick, and was verified by an absence of residual cells. The decellularized extracellular matrix had preserved integrate vascular network and a three-dimensional architecture.

The shortage of organ resource has been limiting the application of liver transplantation. Bioartificial liver construction is increasingly focused as a replacement treatment. To product a bioartificial liver, three elements must be considered: seeding cells, scaffold and bioreactor. Recent studies have shown that several methods can successfully differentiate MSC (mesenchymal stem cells) derived from Wharton's jelly into hepatocyte, such as stimulating MSC by cytokines and growth factors, direct and indirect co-culture MSC with hepatocytes, or promote MSC differentiation by 3-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases. In this review, the characterization of MSC from Wharton's jelly, their potential of application in liver tissue engineering on base of decellularized scaffold, their status of banking and their preclinical work performed will be discussed.

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on cartilage tissue engineering combining cells, scaffold materials and environmental factors. This paper focuses on the main parameters in tissue engineering and in particular, on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. We discussed the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarized the advantages and disadvantages of these cells in articular cartilage engineering.

Human tissues don't regenerate spontaneously, explaining why regenerative medicine and cell therapy represent a promising alternative treatment (autologous cells or stem cells of different origins). The principle is simple: cells are collected, expanded and introduced with or without modification into injured tissues or organs. Among middle-term therapeutic applications, cartilage defects, bone repair, cardiac insufficiency, burns, liver or bladder, neurodegenerative disorders could be considered.

Tissue engineering aims at developing functional substitutes for damaged tissues by mimicking natural tissues. In particular, tissue engineering for bone regeneration enables healing of some bone diseases. Thus, several methods have been developed in order to produce implantable biomaterial structures that imitate the constitution of bone. Electrospinning is one of these methods. This technique produces nonwoven scaffolds made of nanofibers which size and organization match those of the extracellular matrix. Until now, seldom electrospun scaffolds were produced with thickness exceeding one millimeter. This article introduces a new kind of electrospun membrane called 3D scaffold of thickness easily exceeding one centimeter. The manufacturing involves a solution of poly(ε-caprolactone) in DMF/DCM system. The aim is to establish parameters for electrospinning in order to characterize these 3D scaffolds and, establish whether such scaffolds are potentially interesting for bone regeneration.

Vascular tissue engineering aims to develop implantable blood-vessels, exhibiting biological and biomechanical characteristics close to those of the native vessels. The ultimate goal of our group is to engineer suitable blood vessel substitutes which could be stored for a long time in vascular bank conditions.First attempts tried to develop coating procedures allowing endothelial cells (EC) differentiation, adhesion and retention on current vascular substitutes but the weak in vivo patency of these grafts was related. Since 2003, our group have been evaluated a new surface modification of internal surface of blood vessels based on polyelectrolyte films coating. The layer-by-layer self-assembly and the resulting polyelectrolyte multiplayer films (PEM) is a simple and versatile way to engineer surfaces with highly specific properties. Previous studies indicated that the poly(sodium-4 styrene sulfonate)/poly (allylamine hydrochloride) PSS/PAH multilayered films when ended by PAH, induce strong adhesion and retention of mature EC which spread and keep their phenotype as well on glass, on expanded polytetrafluoroethylene ePTFE and on cryopreserved arteries. The mechanical properties (compliance), leading to early intimal hyperplasia and graft failure, were lost after artery cryopreservation. We have demonstrated that the compliance and elasticity restoration of PEM treated cryopreserved arteries close to native arteries.In other respect, the use of the circulating progenitor which could be differentiated into matures vascular cell offers new opportunities in vascular engineering. Currents protocols, expend at least 1 month to observe both smooth muscle (SMCs) and endothelium (ECs)-like morphology and about two months for confluent monolayer cells. The progenitor cells cultivated on PEM treated glass showed mature and functional vascular cells (SMCs and ECs) development after only 14 days of culture. The morphological appearance, mature and healthy phenotype markers expression and repartition of differentiated cells are close to mature cells.Challenge now is to build up in less a month, an autologous cellularized vascular graft using patient peripheral stem cells.

Mesenchymal stem cells (MSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into endothelial-like cells when seeded on nonmodified cover glasses. This absence of removable surface, preventing recovery of cell sheet, constitutes a critical obstacle to predict an application in tissue engineering. It remains unknown whether MSCs differentiation could be realized when the cells are cultivated on a scaffold that could be used in vascular engineering. In this study, we propose to differentiate human MSCs into endothelial-like cells on surfaces coated with polyelectrolyte multilayer film (PMF) and fibronectin (control surfaces). We quantified Platelet Endothelial Cell Adhesion Molecule (PECAM) and von Willebrand Factor (vWF) expressions (endothelial cell specific markers) and nitric oxide (NO) production, which is representative of the cell functionality. After only two weeks of differentiation, we showed, on PMF, that MSCs expressed PECAM and vWF, exhibiting a differentiation into endothelial-like cells, which functionality was explored by a significant production of nitrites. These results highlight the importance of PMF to get human MSCs differentiation and suggest that this film of nanometer thickness opens a new route for vascular bioengineering by pre-seeding hMSCs directly into a vascular graft functionalized by a removable coating., (2010 Wiley Periodicals, Inc.)

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.

Cartilage tissue engineering gives the ability to product adaptable neocartilage to lesion with autologous cells. Our work aimed to develop a stratified scaffold with a simple and progressive spraying build-up to mimic articular cartilage environment. An Alginate/Hyaluronic Acid (Alg/HA) hydrogel seeded with human Mesenchymal Stem Cells (hMSC) was construct by spray. First, cells repartition and actin organization were study with confocal microscopy. Then, we analyzed cells viability and finally, metabolic activity. Our results indicated a homogenous cells repartition in the hydrogel and a pericellular actin repartition. After 3 days of culture, we observed about 52% of viable cells in the scaffold. Then, from day 7 until the end of culture (D28), the proportion of living cells and their metabolic activity increased, what indicates that culture conditions are not harmful for the cells. We report here that sprayed method allowed to product a scaffold with hMSCs that confer a favorable environment for neocartilage construction: 3D conformation and ability of cells to increase their metabolic activity, therefore with few impact on hMSCs.

In 1997, the Joint Accreditation Committee ISCT & EBMT (JACIE) was created. The following year, it approved the first edition of standards for haemopoietic progenitor cell collection, processing and transplantation. The purpose of the standards is to ensure a minimal level of quality, alertness and homogeneity in the implementation of autologous and allogeneic haemopoietic stem cell transplantation (HSCT) programme in onco-hematology. The acquisition of accreditation is based upon the system of examination by trained medical professionals according to countries endorsements with the national regulation obligations applicable to HSCT. In 2008, the fourth edition has been launched. The range of application of the standards comprises both donors and recipients, and all phases of collection, processing, storage and administration of haemopoietic progenitor cells. Among the accredited processing facilities, a few have been integrated JACIE standards into their existing management quality system which is inspected by national health authority. In France, the comparison between JACIE standards and the good manufacturing practices of cellular therapy product reveals some common points and some differences to apply.

Background: recent studies in bio-engineering have showed the influence of Polyelectrolyte Multilayer (PEM) films on endothelial cells (ECs), especially poly(sodium-4-styrene-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). They were tested either with human mature ECs or rabbit immature endothelial progenitor cells (EPCs), but never on human EPCs. In view to obtain an EC covered surface, human cord blood (HCB) EPCs were cultivated on PSS/PAH films., Material and Methods: PEMs were obtained by 7 alternate depositions of cationic PAH and anionic PSS layers. HCB mononuclear cells were isolated by centrifugation through density gradient. 7 days after seeding on PEM, unattached cells were removed and adherent EPCs were cultivated in endothelial specific medium until P6. Appearance of CD31 and vWF was evaluated by confocal microscopy., Results: EPCs not only successfully adhered on PEM, but also spread and proliferated. Moreover, cells differentiated into a typical endothelial cobblestone monolayer within 2 weeks. Immunostaining of CD31 and vWF confirmed the formation of an EC-like confluent monolayer. Furthermore, these cells showed after 6 passages a good phenotypic stability while reseeded on the PEM film., Conclusion: these results show an easy way to obtain mature ECs from human stem cells, which may open new applications for a scaffold cellularization in tissue bio-engineering.

In the last years, there were many studies based on the use of human bone marrow mesenchymal stem cells (hMSCs) in cell therapy and tissue engineering. Although hMSCs can be easily obtained and expanded in culture, a large number of cells are often needed. The expansion of hMSCs depends on the culture conditions, such as media, cell density or culture flasks. Moreover, growth factors are often added to improve cell proliferation. In this study, we compared the effect of two culture media (DMEM and alpha-MEM), two culture flasks (75 or 25 cm2) and two different mononuclear cell seeding densities (1 x 10(4) or 5 x 10(4) MNC/cm2) on the isolation of hMSCs from bone marrow samples and analyzed if the isolation conditions affected the expansion of these cells in the first two passages. Experiments were performed without the addition of exogenous growth factors. Our results showed that alpha-MEM is the optimal culture medium for both, isolation and expansion of mesenchymal stem cells. Moreover, the cell seeding density of 50,000 MNC/cm2 in 25 cm2 culture flasks seems to be the best condition for the isolation step.

Complex three-dimensional structures can "a priori" be built layer-by-layer with a large number of different components, including various cell types, polyelectrolytes, drugs, proteins, peptides or DNA. Our approach is based on the spraying of such elements in order to form a highly functionalized and structured biomaterial. The proposed route will allow the control at the surface and in depth the distribution of the different included elements (matrix and cells).The main objective of this work concerns the buildup of biomaterials aimed to reconstruct biological tissue. The proposed ways are highly innovative and consist in a simple and progressive spraying of all the elements constituting finally the biomaterial.We report here that it is possible (i) to build an alginate gel by alternate spraying of alginate and Ca(2+); (ii) to spray active alginate gel and cells; (iii) to build layer-by-layer an active reservoir under and on the top of this sprayed gel and cells; (iv) to follow the activity of these sprayed cells with time; (v) to propose a three-dimensional sprayed structure for tissue engineering application.

In tissue engineering, surface characteristics of a biomaterial are one of most important factors determining the compatibility with the environment. They influence attachment and growth of cells onto the material. In many cases, the surface should to be modified and engineered in the desired direction. The modification of non-adhesive surfaces with polyelectrolyte multilayer films (PMF) was recently depicted as a powerful technique to promote the growth of different cell lines. In this study, we evaluated the possible use of two different PMF as surface modification for the culture of mesenchymal stem cells (MSC). We used two types of PMF which differed by the nature of the initial anchoring layer which was poly(ethylenimine) (PEI) or poly(allylamine hydrochloride) (PAH). This initial polyelectrolytes adsorption was followed by the alternated deposition of poly(sodium 4-styrenesulfonate) (PSS) and (PAH) in order to obtain a PEI-(PSS-PAH)(3) film or a PAH-(PSS-PAH)(3) film. In order to control the behaviour of MSC, the cell viability was evaluated by Alamar Blue assay and the actin cytoskeleton was labelled and visualised in a confocal microscope. The behaviour of cells on the two PMF was compared to cells cultivated on surfaces treated with fibronectin. The results showed that PAH-(PSS-PAH)(3) PMF improve the growth of cells, inducing a higher cell viability compared to PEI-(PSS-PAH)(3) PMF and fibronectin at 2, 3 and 7 days of culture. Moreover, those cells showed a well-organized actin cytoskeleton. In conclusion, PAH-(PSS-PAH)(3) polyelectrolyte multilayer film seems to constitute an excellent material for MSC seeding.

Tissue engineering requires the response of the cells to different stimuli inducing the synthesis of the extracellular matrix (ECM). It was been shown that mechanical and biochemical stimuli acted on the synthesis of ECM, particularly type I and III collagens. Growth factors implied in transduction pathways are multiple, but the main is TGF-beta. Member of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of SMADs proteins. Nevertheless, the effects of mechanical stress of this pathway remain unknown. The aim of this work was to study the pathway of TGF-beta via the SMADs proteins under mechanical (stretching) and biochemical (TGF-beta) stimulations. Endogenous SMADs expression and its modulation by biochemical and mechanical stimulations were evaluated by both flow cytometry and confocal microscopy. Our results demonstrate that 10 ng of TGF-beta and stretching (5%, 1 Hz) applied during 15 min induced a negative feed back loop which blocks the signalling pathway to control TGF-beta activity. This inhibition effect was raised after 1 h of stimulation. Nevertheless, these preliminary studies should be continued by study of expression and localization of inhibitory SMADs (SMAD7).

To investigate whether the application of alginate culture and mechanical stimulation will improve the synthesis of cartilaginous matrix in dedifferentiated chondrocytes, rat chondrocytes underwent dedifferentiation upon serial monolayer culture up to passage 6, and then were encapsulated in 2% alginate gel and subject to static culture. After 28 days culture in static, the beads were exposed to 48 h of mechanical stimulation with continuous agitation. The sGAG content in alginate bead was measured by alcian blue staining. The expression of collagen protein was detected using immunofluorescence. After 28 days culture in alginate bead, the dedifferentiated chondrocytes remained round in shape and re-synthesized the chondrocyte-specific matrix. Compared with static culture, mechanical stimulation induced statistically increases in the production of glycosaminoglycan (p< or =0.01), as well as in the synthesis of collagen type II protein (p< or =0.05). On the contrary, no positive expression of collagen type I protein was observed at the end of culture. Our results demonstrated that both of alginate culture and mechanical stimulation help to restore chondrocyte phenotype and promotes the synthesis of cartilaginous matrix.

Although autogenous vessels are useful in surgery, often patients cannot furnish suitable vessels. If there are not available, two possible alternatives for vessel replacements are to use vascular synthetic prostheses such as Dacron((R)) and polytetrafluoroethylene (PTFE) or cryopreserved allografts. However, their success has been limited to replace small-diameter (<6 mm) arterial vessel because of their high thrombogenicity and compliance mismatch. On account of a clear clinical need for a functional arterial substitute, tissue engineering techniques have been developed. This review encompasses the use of mature endothelial, endothelial progenitor and bone marrow cells combined with natural or synthetic scaffolds whose surface has been modified with multiple origin matrices.

In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml(-1). The exogenous TGF-beta1 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.

Decellularized allograft tissues have been identified as a potential extracellular matrix scaffold for tissue-engineered vascular substitutes. In order to improve the thromboresistance, it is necessary to pre-coat the intra-luminal vessel surface. Recently a new surface modification technique appeared, based on the alternate adsorption of positive and negative charged polyelectrolytes. Our objective was to develop an alternative vascular scaffold made of decellularized human umbilical arteries treated with a PAH/PSS polyelectrolyte multilayered film. The vessels luminal surfaces covered with the multilayer film were observed by electronic scanning microscopy. Our observations showed that the luminal surface is completely devoid of ECs following treatment with trypsin. A top view of the coated artery indicated that the multilayer uniformly covered internal surface of the vessels. The successful of the multilayer correct deposition and retention on the arterial wall were controlled by confocal microscopy using a fluorescent polyelectrolyte (rhodamine-PAH). The data suggest that decellularized cryopreserved arteries represent a potential scaffold for further vascular tissue engineering efforts. Moreover, the multilayer films can be used to coat biological surfaces and following the terminated layer (PAH or PSS), favour the cell adhesion or cell resistance.

Most human tissues do not regenerate spontaneously; this is why cell therapies and tissue engineering are promising alternatives. The principle is simple: cells are collected in a patient and introduced in the damaged tissue or in a tridimentional porous support and harvested in a bioreactor in which the physico-chemical and mechanical parameters are controlled. Once the tissues (or the cells) are mature they may be implanted. In parallel, the development of biotherapies with stem cells is a field of research in turmoil given the hopes for clinical applications that it brings up. Embryonic stem cells are potentially more interesting since they are totipotent, but they can only be obtained at the very early stages of the embryo. The potential of adult stem cells is limited but isolating them induces no ethical problem and it has been known for more than 40 years that bone marrow does possess the regenerating functions of blood cells. Finally, the properties of foetal stem cells (blood cells from the umbilical cord) are forerunners of the haematopoietic system but the ability of these cells to participate to the formation of other tissues is more problematic. Another field for therapeutic research is that of dendritic cells, antigen presenting cells. Their efficiency in cell therapy relies on the initiation of specific immune responses. They represent a promising tool in the development of a protective immune response against antigens which the host is usually unable to generate an efficient response (melanomas, breast against cancer, prostate cancer, ..). Finally, gene therapy, has been nourishing high hopes but few clinical applications can be envisaged in the short term, although potential applications are multiple (haemophilia, myopathies, ..). A large number of clinical areas stand as candidates for clinical applications: leukaemia and cancers, cardiac insufficiency and vascular diseases, cartilage and bone repair, ligaments and tendons, liver diseases, ophthalmology, diabetes, neurological diseases (Parkinson, Huntington disease, ..), .. Various aspects of this new regenerative therapeutic medicine are developed in this work.

Cartilage is a hydrated connective tissue that withstands and distributes mechanical forces within joints. Chondrocytes utilize mechanical signals to maintain cartilaginous tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Some mechanotransduction mechanisms are known, while many others no doubt remain to be discovered. Various aspects of chondrocyte mechanobiology have been applied to tissue engineering, with the creation of replacement tissue in vitro from bioresorbable or non-bioresorbable scaffolds and harvested cells. The tissues are maintained in a near-physiologic mechanical and biochemical environment. This paper is an overview of both chondrocyte mechanobiology and cartilage tissue engineering

The surface modification using thin polyelectrolyte multilayered films was proposed as a new scaffold material for different cell lines. In this study, we evaluated the possible use of polyelectrolyte multilayers as surface modification for the development of endothelial cells. In order to control the behaviour of endothelial cells, cell viability by MTT assay was studied. Moreover, the endothelial cell phenotype was checked and the expression of a leukocyte adhesion molecule (ICAM-1) was quantified. The behaviour of the cells on two polyelectrolyte multilayers was compared to cells on polystyrene, and two polyelectrolyte monolayers (terminating the multilayer architectures). The results have shown a better cell viability on the polyelectrolyte multilayers, inducing a higher cell number compared to polyelectrolyte monolayers after 1 and 3 days of culture. Moreover, the cells showed a normal morphology of cytoskeleton. The phenotype of the endothelial cells was kept and a low level of leukocyte adhesion molecules was observed. In conclusion, the polyelectrolyte multilayers can be considered as a potential surface modification procedure to enhance the development of endothelial cells on hydrophobic substrate and which can be applied to vascular tissue engineering.

The promising concept of cell transplantation and cardiac tissue engineering has been developed in the last few years and focused on strategies attempting to replace dysfunctional, necrotic, and/or apoptotic cardiomyocytes with new cells of mesodermal origin. Transplantation of autologous cells minimizes the risk of neoplasia and avoids immune rejection associated with allogenic or xenogenic cells and recent data hold enormous hopes for short term clinical practices. Tissue engineering represents another promising approach that makes possible the creation of new functional tissues to replace the lost or failing one. Three-dimensional polymeric scaffolds provide the mechanical support for the candidate cells until the formation of cardiac-like tissue prior to surgical repair of the infarcted myocardium. For ultimate clinical applications, further investigations have to select the appropriate cell types, to determine the sufficient number of grafted cells and to provide the long term evaluation of these strategies in the global improvements of cardiac function (neoangiogenesis, synchronous contraction and extracellular matrix remodelling).

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix., (Copyright 2001 John Wiley & Sons, Inc.)

Vascular engineering developed through advancing knowledge of the physiological and pathological processes operating in cells and tissues subjected to environmental mechanical stress. We review briefly the more relevant cellular and general mechanism characteristic of vascular tissue examining the performances obtained with prosthetic materials (allografts, synthetic grafts) and current research and development of new vessel substitutes (biohybrids or biovessels).