Estudio descriptivo de las células madre mesenquimales en la articulación artrósica y su aplicación en terapia celular para reparar cartílago articular humano

[Resumen] La artrosis es una patología articular degenerativa caracterizada por el deterioro y la pérdida del cartílago articular. Dada su sintomatología, se trata de una de las enfermedades reumáticas más dolorosas e incapacitantes en la actualidad. Esta patología afecta a un elevado porcentaje de la población, por lo que se han desarrollado opciones terapéuticas para frenar y/o retardar su progresión y, además, regenerar la superficie del cartílago. No obstante, ninguna de ellas conduce a la obtención de un cartílago articular completamente regenerado y funcionalmente competente. Actualmente, las células madre mesenquimales se consideran un potencial tratamiento para diferentes enfermedades, incluidas las reumáticas. Estas células pueden diferenciarse in vitro en varios tipos celulares, proliferar en cultivo e incluso reparar lesiones del cartílago articular. No obstante, distintos estudios sugieren que la población de CMM comprende subpoblaciones de células con diferentes potenciales de diferenciación y que la capacidad de diferenciación también puede variar en función del tejido del que se aíslan. Las CMM se caracterizan fenotípicamente por la expresión de antígenos de superficie, por lo que es necesario determinar el patrón de expresión de antígenos que define la subpoblación celular con mayor capacidad de diferenciación condrogénica. Sin embargo, no se ha analizado cuantitativamente la expresión de estos marcadores en los tejidos fuente de CMM, en particular, en la membrana sinovial, ni se han determinado diferencias de esta expresión entre el tejido sano y el artrósico. En este estudio se ha demostrado que las células madre mesenquimales aisladas de las diferentes fuentes tisulares reparan in vitro tanto los defectos condrales como las superficies artrósicas. Además, nuestros resultados sugieren que la médula ósea es la mejor fuente de obtención de células madre mesenquimales para aplicación clínica. Por otra parte, se ha puesto de manifiesto que con la artrosis se produce una mayor expresión de marcadores de CMM en membrana sinovial, y que estos marcadores, excepto el antígeno CD105, están presentes en el tejido de reparación espontánea del cartílago articular artrósico. Por último, nuestros resultados sugieren que la subpoblación CD271+ de CMM aporta ventajas en la reparación de defectos de cartílago articular

Alternative protocols to induce chondrogenic differentiation: transforming growth factor-β superfamily

[Abstract] Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFβ) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFβ superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFβ-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFβ-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage.Galicia. Consellería de Sanidade; PS07/84Ministerio de Ciencia e Innovacion; PLE2009-0144Instituto de Salud Carlos III; PI 08/202

Molecular profile and cellular characterization of human bone marrow mesenchymal stem cells: donor influence on chondrogenesis

[Abstract] Background. The use of autologous or allogenic stem cells has recently been suggested as an alternative therapeutic approach for treatment of cartilage defects. Bone marrow mesenchymal stem cells (BM-MSCs) are well-characterized multipotent cells that can differentiate into different cell types. Understanding the potential of these cells and the molecular mechanisms underlying their differentiation should lead to innovative protocols for clinical applications. The aim of this study was to evaluate the usefulness of surface antigen selection of BM-MSCs and to understand the mechanisms underlying their differentiation. Methods. MSCs were isolated from BM stroma and expanded. CD105+ subpopulation was isolated using a magnetic separator. We compared culture-expanded selected cells with non-selected cells. We analyzed the phenotypic profiles, the expression of the stem cell marker genes Nanog, Oct3/4, and Sox2 and the multi-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic). The multi-lineage differentiation was confirmed using histochemistry, immunohistochemistry and/or real-time polymerase chain reaction (qPCR) techniques. Results. The selected and non-selected cells displayed similar phenotypes and multi-lineage differentiation potentials. Analyzing each cell source individually, we could divide the six donors into two groups: one with a high percentage of CD29 (β1-integrin) expression (HL); one with a low percentage of CD29 (LL). These two groups had different chondrogenic capacities and different expression levels of the stem cell marker genes. Conclusions. This study showed that phenotypic profiles of donors were related to the chondrogenic potential of human BM-MSCs. The chondrogenic potential of donors was related to CD29 expression levels. The high expression of CD29 antigen seemed necessary for chondrogenic differentiation. Further investigation into the mechanisms responsible for these differences in BM-MSCs chondrogenesis is therefore warranted. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous human BM-MSCs for articular cartilage repair.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-004

Treatment of Joint Cartilage Lesions with Cell Therapy

Monográfico Osteoartritis[Resumen] Las lesiones del cartílago articular que no afectan a la integridad del hueso subcondral no se reparan espontáneamente. El carácter asintomático de estas lesiones propicia la progresiva degeneración articular y el desarrollo de un proceso artrósico. Para evitar la necesidad de reemplazo protésico, se han desarrollado distintos tratamientos celulares con el objetivo de fomar un tejido de reparación con estructura, composición bioquímica y comportamiento funcional iguales que los del cartílago articular natural. Las técnicas basadas en facilitar el acceso al sistema vascular generan un tejido de reparación fibrocartilaginoso que no reúne las condiciones del cartílago articular. El implante de condrocitos autólogos y la mosaicoplastia autóloga aportan un tejido de reparación de mayor calidad, pero ambas técnicas implican la escisión de cartílago sano, bien para obtener una elevada cantidad de condrocitos, bien para extraer cilindros osteocondrales que se implantan en el defecto. Las células madre mesenquimales constituyen una prometedora herramienta de reparación del cartílago articular en fase de experimentación. Aunque las estrategias actuales de terapia celular producen mejorías clínicas y funcionales, todavía no es posible generar un tejido de reparación resistente a la degeneración y con características de cartílago articular normal.[Abstract] Articular cartilage lesions which do not affect the integrity of subchondral bone, they are not able to repair it expontaneously. The asymptomatic nature of these lesions induces articular cartilage degeneration and development of an arthrosic process. To avoid the necessity to receive joint replacement surgery, it has been developed different treatments of cellular therapy which are focused to create new tissues whose structure, biochemistry composition and function will be the same than native articular cartilage. Approaches used to access the stream produce a fibrocartilaginose tissue which is not an articular cartilage. Implantation of autologous chondrocytes and autologous mosaicplasties induces a quality better articular cartilage. Furthermore both techniques involve damage in the sane cartilage; because of trying to get a big amount of chondrocytes or because of extraction osteochondral cylinder which will be implanted in the injured joint. The stem cells are a promising toll to repair articular cartilage, however they are in a previous experimentation step yet. Although the present studies using cellular therapy improves clinically and functionally, it is not able to regenerate an articular cartilage which offer resistance the degeneration proces

Cell and Tissue Transplant Strategies for Joint Lesions

[Abstract] Articular cartilage lesions that do not disrupt the integrity of subchondral bone are not capable of spontaneous repair. The asymptomatic nature of these lesions leads to articular cartilage degeneration and development of the osteoarthritic process. To avoid joint replacement surgery, several cellular therapies have been developed. These therapies focus on the regeneration of a new tissue, whose structure, biochemistry composition and function should be the same as those of endogenous articular cartilage. Current approaches for interrupting the osteoarthritic process produce a fibrocartilaginous tissue, not articular cartilage. The implantation of autologous chondrocytes and autologous mosaicplasty induces a better quality of articular cartilage; however, both techniques damage the existing cartilage because of the need to harvest large numbers of chondrocytes or to extract an osteochondral cylinder for implantation. While stem cells are a promising tool for repairing articular cartilage, their use is in an early experimental stage at this time. Although studies of cell therapy have shown clinical and functional improvement in joints, the ability to regenerate articular cartilage that resists the degeneration process remains elusive.Xunta Galicia; PGIDIT06PXIC916175PNInstituto de Salud Carlos III; CB06/01/004

Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes

[Abstract] Background. Osteoarthritis (OA) is a multifactorial disease characterized by destruction of the articular cartilage due to environmental, mechanical and genetic components. The genetics of OA is complex and is not completely understood. Recent works have demonstrated the importance of microRNAs (miRNAs) in cartilage function. MiRNAs are a class of small noncoding RNAs that regulate gene expression and are involved in different cellular process: apoptosis, proliferation, development, glucose and lipid metabolism. The aim of this study was to identify and characterize the expression profile of miRNAs in normal and OA chondrocytes and to determine their role in the OA. Methods. Chondrocytes were moved to aggregate culture and evaluated using histological and qPCR techniques. miRNAs were isolated and analyzed using the Agilent Human miRNA Microarray. Results. Of the 723 miRNAs analyzed, 7 miRNAs showed a statistically significant differential expression. Amongst these 7 human miRNAs, 1 was up-regulated in OA chondrocytes (hsa-miR-483-5p) and 6 were up-regulated in normal chondrocytes (hsa-miR-149*, hsa-miR-582-3p, hsa-miR-1227, hsa-miR-634, hsa-miR-576-5p and hsa-miR-641). These profiling results were validated by the detection of some selected miRNAs by qPCR. In silico analyses predicted that key molecular pathways potentially altered by the miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage. Conclusions. We have identified 7 miRNAs differentially expressed in OA and normal chondrocytes. Our potential miRNA target predictions and the signalling cascades altered by the differentially expressed miRNAs supports the potential involvement of the detected miRNAs in OA pathology. Due to the importance of miRNA in mediating the translation of target mRNA into protein, the identification of these miRNAs differentially expressed in normal and OA chondrocyte micropellets could have important diagnostic and therapeutic potential. Further studies are needed to know the function of these miRNAs, including the search of their target mRNA genes, which could lead to the development of novel therapeutic strategies for the OA treatment.Instituto de Salud Carlos III; CIBER BBN CB06-01-004

Effects of severe hypoxia on bone marrow mesenchymal stem cells differentiation potential

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed

Human amniotic mesenchymal stromal cells as favorable source for cartilage repair

[Abstract] Introduction: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. Material and Methods: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. Results: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. Conclusion: HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.Servizo Galego de Saúde; PS07/84Xunta de Galicia; CN2012/142Xunta de Galicia; R2014/050Xunta de Galicia; GPC201

Intra-articular administration of hydrogen sulphide ameliorates severity of experimental osteoarthritis

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Ovine mesenchymal stromal cells for osteochondral tissue engineering

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