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[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 wh

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.

[Abstract] 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.

[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 differentiall

[Resumen] La capacidad de reparación del cartílago articular es muy limitada, debido principalmente a que es un tejido avascular. Actualmente no existen tratamientos farmacológicos eficaces para curar la artrosis (osteoarthritis, OA), aunque algunos fármacos podrían retardar su progresión. La mayoría de los esfuerzos realizados hasta la actualidad con la finalidad de reparar una lesión de cartílago articular van encaminados a superar las limitaciones que posee este tejido para cicatrizar las lesiones, implantar nuevas células con capacidad condrogénica y facilitar el acceso al sistema vascular. Pero el problema es que se genera un tejido de reparación fibrocartilaginoso que no reúne las condiciones del cartílago articular hialino natural. Para evitar la necesidad de reemplazo protésico, se desarrollaron diferentes tratamientos celulares basados en el empleo de células multipotentes mesenquimales estromales (CME), con el objetivo de formar un tejido de reparación con estructura, composición bioquímica y comportamiento funcional biomecánico iguales que los del cartílago articular natural. En la actualidad, las CME son consideradas como una potencial Terapia Celular para el tratamiento de un amplia variedad de enfermedades, incluída la OA. La médula ósea es, por excelencia, la fuente tisular empleada para la obtención de CME adultas, pero presentan una serie de desventajas. Por ello, la identificación de fuentes alternativas de CME con fines terapéuticos sería muy beneficioso. En este estudio se ha demostrado la localización y aislamiento de dos poblaciones de CME procedentes de membrana amniótica humana (MA), las células mesenquimales estromales amnióticas humanas (CMEAh) y las células epiteliales amnióticas humanas (CEAh). Ambas poblaciones celulares mostraron similares perfiles de expresión de marcadores de superficie de progenitores mesenquimales. Sin embargo, mostraron diferencias en cuanto a su rendimiento celular y su potencial de diferenciación hacia los princi

[Resumen] La capacidad de reparación del cartílago articular es muy limitada, debido principalmente a que es un tejido avascular. Actualmente no existen tratamientos farmacológicos eficaces para curar la artrosis (osteoarthritis, OA), aunque algunos fármacos podrían retardar su progresión. La mayoría de los esfuerzos realizados hasta la actualidad con la finalidad de reparar una lesión de cartílago articular van encaminados a superar las limitaciones que posee este tejido para cicatrizar las lesiones, implantar nuevas células con capacidad condrogénica y facilitar el acceso al sistema vascular. Pero el problema es que se genera un tejido de reparación fibrocartilaginoso que no reúne las condiciones del cartílago articular hialino natural. Para evitar la necesidad de reemplazo protésico, se desarrollaron diferentes tratamientos celulares basados en el empleo de células multipotentes mesenquimales estromales (CME), con el objetivo de formar un tejido de reparación con estructura, composición bioquímica y comportamiento funcional biomecánico iguales que los del cartílago articular natural. En la actualidad, las CME son consideradas como una potencial Terapia Celular para el tratamiento de un amplia variedad de enfermedades, incluída la OA. La médula ósea es, por excelencia, la fuente tisular empleada para la obtención de CME adultas, pero presentan una serie de desventajas. Por ello, la identificación de fuentes alternativas de CME con fines terapéuticos sería muy beneficioso. En este estudio se ha demostrado la localización y aislamiento de dos poblaciones de CME procedentes de membrana amniótica humana (MA), las células mesenquimales estromales amnióticas humanas (CMEAh) y las células epiteliales amnióticas humanas (CEAh). Ambas poblaciones celulares mostraron similares perfiles de expresión de marcadores de superficie de progenitores mesenquimales. Sin embargo, mostraron diferencias en cuanto a su rendimiento celular y su potencial de diferenciación hacia los princi

[Abstract] Objective. To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial mem- branes from human osteoarthritic (OA) and healthy joints. Methods. Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. Results. Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. Conclusion. Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

[Abstract] The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM-derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion-derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem-cell markers SSEA-4 and STRO-1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy.