Karim Oudina

Tissue-engineered bone regeneration.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling.

Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCα vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCα signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCα pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells.

In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye.

Recent advances in the field of tissue engineering have culminated in new tissue substitutes that combine a biomaterial and precursor cells. The effectiveness of these materials is generally assessed in animals, but few studies explore the fate of the transplanted cells in vivo, despite its paramount importance for understanding the function of the engineered tissues. Current methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a new method for irreversible labeling of cells to track their fate in vivo. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 microM CM-Dil in 30 min. The CM-Dil was not cytotoxic and did not affect cell proliferation in vitro. Cells could be monitored for up to 30 days when placed in a coral scaffold and implanted intramuscularly or in a bony site. However, the fluorescence intensity decreased roughly in parallel with the number of cell divisions. This fact needs to be taken into account during the design and interpretation of experiments. We believe that this technique is also of interest for other cell types.

Proangiogenic and Prosurvival Functions of Glucose in Human Mesenchymal Stem Cells upon Transplantation

A major limitation in the development of cellular therapies using human mesenchymal stem cells (hMSCs) is cell survival post‐transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. We demonstrated that hMSCs could endure sustained near‐anoxia conditions only in the presence of glucose. In this in vitro cell model, the protein expressions of Hif‐1α and angiogenic factors were upregulated by the presence of glucose. Ectopically implanted tissue constructs supplemented with glucose exhibited four‐ to fivefold higher viability and were more vascularized compared to those without glucose at day 14. These findings provided the first direct in vitro and in vivo demonstration of the proangiogenic and prosurvival functions of glucose in hMSC upon transplantation and identified glucose as an essential component of the ideal scaffold for transplanting stem cells. STEM CELLS2013;31:526–535

Desferrioxamine-driven upregulation of angiogenic factor expression by human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are the subject of intense research because of their biological properties and potential use for the repair of damaged tissues. Success of BMSC‐based therapies, however, relies on a number of methodological improvements, including the establishment of a vascular network providing nutrients and oxygen to the transplanted cells and ensuring their immediate survival and long‐term functionality. We described a method to enhance the autocrine expression of angiogenic factors by BMSCs. For this purpose, human BMSCs were treated with desferrioxamine (DFX). No PDGF‐BB, VEGF‐R1 or ‐R2 mRNA expression was detected under any of the conditions tested. mRNA and protein expression levels of TGFβ1 were similar in BMSCs, whether they were exposed to DFX (50 µM) or to control conditions under normoxia for 48 h. In comparison with the results obtained with control conditions under normoxia, exposure of BMSCs to DFX for 48 h resulted in upregulation of bFGF at the protein (26‐fold) but not at the mRNA levels and VEGF at both the mRNA (1.5‐fold) and protein levels (4.5‐fold). In comparison with the results obtained with control conditions under hypoxia, DFX induced a 50% increase in VEGF secretion but led to the same level of hypoxia inducible factor‐1α protein expression (a transduction factor involved in angiogenic factor expression and known to be activated by DFX). Exposure of BMSCs to DFX resulted in oversecretion of angiogenic factors, suggesting that DFX‐treated BMSCs could be used to supply angiogenic factors. Copyright

Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation

Mesenchymal stem cells (MSCs) hold considerable promise in tissue engineering (TE). However, their poor survival when exogenously administered limits their therapeutic potential. Previous studies from our group demonstrated that lack of glucose (glc) (but not of oxygen) is fatal to human MSCs because it serves as a pro‐survival and pro‐angiogenic molecule for human MSCs (hMSCs) upon transplantation. However, which energy‐providing pathways MSCs use to metabolize glc upon transplantation? Are there alternative energetic nutrients to replace glc? And most importantly, do hMSCs possess significant intracellular glc reserves for ensuring their survival upon transplantation? These remain open questions at the forefront of TE based‐therapies. In this study, we established for the first time that the in vivo environment experienced by hMSCs is best reflected by near‐anoxia (0.1% O2) rather than hypoxia (1%–5% O2) in vitro. Under these near‐anoxia conditions, hMSCs rely almost exclusively on glc through anerobic glycolysis for ATP production and are unable to use either exogenous glutamine, serine, or pyruvate as energy substrates. Most importantly, hMSCs are unable to adapt their metabolism to the lack of exogenous glc, possess a very limited internal stock of glc and virtually no ATP reserves. This lack of downregulation of energy turnover as a function of exogenous glc level results in a rapid depletion of hMSC energy reserves that explains their poor survival rate. These new insights prompt for the development of glc‐releasing scaffolds to overcome this roadblock plaguing the field of TE based‐therapies. Stem Cells 2018;36:363–376

Noninvasive bioluminescent quantification of viable stem cells in engineered constructs.

Bioluminescence from murine stem cells tagged with the luciferase gene reporter and distributed within three-dimensional scaffolds of two different materials is quantified in vitro and in vivo. The luminescence emitted from cells adhering to the scaffolds tested is monitored noninvasively using a bioluminescence imaging system. Monitoring the kinetics of luciferase expression via bioluminescence enables real-time assessment of cell survival and proliferation on scaffolds both in vitro and in vivo over prolonged (8 weeks) periods of time.

A New, Segmental, Critical-Size Defect Model for Long Bones: A Sheep Study

In the present study, we (i) established a large animal model in sheep of a two-step surgical procedure for successful reconstruction of large segmental bone defects, based on a concept that proved successful in humans and (ii) explored the possibility of using skeletal stem cells loaded onto a coral scaffolds to repair such defects.

Microfluidique des milieux poreux : application au génie tissulaire osseux

En orthopedie, l’utilisation d’un implant hybride constitue d’un substrat poreux dans lequel sont cultivees des cellules osseuses provenant du patient est une technique de greffe actuellement developpee. La culture des cellules est effectuee au sein d’un bioreacteur, ou l’implant est perfuse pendant plusieurs semaines. La perfusion permet l’apport d’oxygene necessaire aux cellules pour leur developpement. La maitrise des phenomenes de transport au sein de l’implant est donc essentielle a la reussite d’une telle culture cellulaire. L’etude ici presentee, a abouti a la mise en place d’un modele simplifie permettant de resoudre par simulation numerique un probleme de transport d’oxygene stationnaire en milieu poreux. Ce modele prend en compte l’influence de la geometrie complexe de l’implant sur les vitesses locales du fluide nutritif perfuse et un terme « puits » non lineaire impose par la specificite de la consommation cellulaire. La resolution numerique faiblement couplee de l’equation de convection-diffusion et celles de Navier Stokes est alors necessaire. Une longueur de penetration de l’oxygene dans le milieu poreux est calculee, l’heterogeneite locale chimique et mecanique de l’environnement de culture mise en evidence, et l’influence de differents parametres tels que la vitesse de perfusion ou la concentration initiale en oxygene du fluide nutritif est analysee de maniere theorique. Bien qu’il soit necessaire de completer ce modele, nous pouvons ainsi envisager de choisir une taille d’implant adaptee et de definir des conditions de culture optimales prealables a la realisation d’un processus de culture performant.