D. Bensoussan

Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Whartons Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to 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 and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organs reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

Designing a three-dimensional alginate hydrogel by spraying method for cartilage tissue engineering

Cartilage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to native zonal organization of articular cartilage. The main objective of our work concerns the buildup of complex biomaterials aimed at reconstructing biological tissue with three dimensional cells construction for mimicking cartilage architecture. In this first step, our strategy is based on structure formation by simple and progressive spraying of mixed alginate and chondrocytes at different pressures. We report the first demonstration of spraying effect on chondrocytes inside an alginate hydrogel at short (i) and long terms (ii) and the mechanical behavior of a sprayed hydrogel by biomechanical tests (plane strain compression tests). Our results indicate clearly that during the first days of culture the cells were influenced by the construction method (spraying or molding, control method) with low viability and higher production levels of nitrite. From day 7, the cell behaviors become similar for both methods. Indeed after 28 days of culture, type II collagen was observed, showing the cartilage gene expression, then a similar behavior for all methods. Finally, we conclude that the mechanical performances of sprayed hydrogels was enhanced compared to the controls. We report here, for the first time, that it is possible to spray mixed alginate and chondrocytes with little damage for cells. Therefore, the sprayed hydrogel keeps not only the mechanical properties needed for cells, but also maintains the chondrocyte phenotype to induce cartilage.

In vitro initial expansion of mesenchymal stem cells is influenced by the culture parameters used in the isolation process

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.

Original approach for cartilage tissue engineering with mesenchymal stem cells.

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.

Introduction to tissue engineering and application for cartilage engineering

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.

Human Stem Cells and Articular Cartilage Tissue Engineering

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.

Quantification of residual DiMethylSulfoxide after washing cryopreserved stem cells and thawing tissue grafts

Dimethylsulfoxide (DMSO) is a cryoprotective substance often used to allow long term storage of stem cells or tissue grafts. However, a high frequency of adverse events is associated with the infusion of thawed cells. These events are in part due to DMSO, leading many cell therapy facilities to introduce a washing step before the delivery of the grafts. The lack of method for evaluating the residual quantities of this substance in the reinfused cells led us to develop a technique, based on capillary zone electrophoresis for assaying DMSO. The cryoprotectant was measured in 55 hematopoietic stem cell grafts, 6 parathyroids and 5 blood vessels immediately after thawing and after washing or bathing in a saline solution. The results showed that DMSO reduction in stem cell grafts reached more than 90% after the washing procedure. Furthermore, this study has shown that 2 washing steps significantly improved DMSO elimination as compared to 1 washing step. For parathyroids and blood vessels, bathing the tissues after thawing in a saline solution allowed more than 95% DMSO reduction. This study demonstrated that the technique of DMSO measurement used here, is simple and feasible on complex matrices such as protein samples after dilution. It is an appropriate method for residual quantification of the cryoprotectant before graft.

Mechanobiology of mesenchymal stem cells: Which interest for cell-based treatment?

Thanks to their immune properties, the mesenchymal stem cells (MSC) are a promising source for cell therapy. Current clinical trials show that MSC administrated to patients can treat different diseases (graft-versus-host disease (GVHD), liver cirrhosis, systemic lupus, erythematosus, rheumatoid arthritis, type I diabetes…). In this case, the most common mode of cell administration is the intravenous injection, and the hemodynamic environment of cells induced by blood circulation could interfere on their behavior during the migration and homing towards the injured site. After a brief review of the mechanobiology concept, this paper will help in understanding how the mechanical environment could interact with MSC behavior once they are injected to patient in cell-based treatment.

The 6th China–France International Symposium “Stem Cells and Regenerative Medicine” and first meeting of the France–China International CNRS Network (GDRI) CeSMeR (Nancy, 10–13 July 2016)

Most human tissues do not regenerate spontaneously. This is why the development of biotherapies with stem cells represents promising alternatives. The principle is simple: cells collected from a patient or a donor are either introduced with or without modifying their properties (by gene transfection for example) into a scaffold primarily made of three-dimensional porous polymers, or cultured in a bioreactor in which physicochemical parameters and mechanical stress are controlled. Once the tissue is fully mature or the number of cells adequate, engraftment may be performed. In vitro preparation of biotissues such as bone, tendon, cartilage, skin, blood vessels, cardiac muscle inspires great hopes for the next decades, with perspectives of new therapies to restore tissue functions or for disabling disorders such as myopathies and degenerative neurological diseases. It is estimated that annual expenses linked to these emerging activities will amount to 100 billion US

173 LONG-TERM STUDY OF STRATIFIED ALGINATE CONSTRUCT FOR CARTILAGE TISSUE ENGINEERING

during the next 10 years. The concept of regenerative medicine is a new emerging multidisciplinary field involving surgery medicine, biology, chemistry, mechanic and engineering. It can be defined as “the way to improve the health and quality of life by restoring, maintaining or enhancing tissue and organ functions”. . . . A large number of experimental differentiation methods has been developed for each tissue or type of therapy. Moreover, mechanical stress influences the differentiation of cells that are used. Such changes are now considered as critical not only for understanding pathological mechanisms (inflammation, atherosclerosis, etc.) but also for tissue reconstruction. For example, the quantities and quality of bone obtained depend on the intensity, magnitude, and frequency of mechanical stress. This fact seems natural since it is well know that prolonged immobilization, implying absence of mechanical stress, weakens bone and muscles and decreases their respective mass.