RESUMEN
Osteoporosis is a disease characterized by low bone mass and an increased risk of fractures. Although several cellular players leading to osteoporosis have been identified, the role of mesenchymal stromal cells (MSC) is still not fully elaborated. The aim of this study was, therefore, to isolate and characterize MSCs from vertebral body of healthy non-osteoporotic and osteoporotic patients, with a particular focus on their osteogenic differentiation potential. Isolated MSCs were characterized by their osteogenic, adipogenic, and chondrogenic differentiation, as well as surface marker expression, proliferation behavior, and immunomodulatory capacity. The mineralization process was confirmed using Alizarin Red S and alkaline phosphatase (ALP) stains and further evaluated by determining ALP activity, mineral deposition, and free phosphate ion release. MSCs from both healthy and osteoporotic patients showed common fibroblast-like morphology and similar proliferation behavior. They expressed the typical MSC surface markers and possessed immunomodulatory capacity. Both groups demonstrated solid trilineage differentiation potential; osteogenic differentiation was further confirmed by increased ALP activity, deposition of inorganic crystals, phosphate ion release, and expression of osteoblast marker genes. Overall, MSCs from osteoporotic and non-osteoporotic patients showed neither a difference in general MSC features nor in the detailed analysis regarding osteogenic differentiation. These data suggest that vertebral body MSCs from osteoporotic patients were not impaired; rather, they possessed full osteogenic potential compared to MSCs from non-osteoporotic patients.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Osteoporosis/metabolismo , Adipogénesis , Anciano , Fosfatasa Alcalina/metabolismo , Médula Ósea/metabolismo , Proliferación Celular/fisiología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Humanos , Inmunomodulación/fisiología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Osteoblastos/metabolismo , Osteogénesis/genética , Osteoporosis/fisiopatologíaRESUMEN
Currently, there is an increasing focus on mesenchymal stromal cells (MSC) as therapeutic option in bone pathologies as well as in general regenerative medicine. Although human MSCs have been extensively characterized and standardized, ovine MSCs are poorly understood. This limitation hampers clinical progress, as sheep are an excellent large animal model for orthopedic studies. Our report describes a direct comparison of human and ovine MSCs from three corresponding sources under the same conditions. All MSCs presented solid growth behavior and potent immunomodulatory capacities. Additionally, we were able to identify common positive (CD29, CD44, CD73, CD90, CD105, CD166) and negative (CD14, CD34, CD45, HLA-DR) surface markers. Although both human and ovine MSCs showed strong osteogenic potential, direct comparison revealed a slower mineralization process in ovine MSCs. Regarding gene expression level, both human and ovine MSCs presented a comparable up-regulation of Runx2 and a trend toward down-regulation of Col1A during osteogenic differentiation. In summary, this side by side comparison defined phenotypic similarities and differences of human and ovine MSCs from three different sources, thereby contributing to a better characterization and standardization of ovine MSCs. The key findings shown in this report demonstrate the utility of ovine MSCs in preclinical studies for MSC-based therapies.
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Antígenos de Superficie , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Expresión Génica , Humanos , Inmunomodulación , Células Madre Mesenquimatosas/inmunología , Modelos Animales , Osteogénesis/genética , Medicina Regenerativa , OvinosRESUMEN
Mesenchymal stromal cells (MSC) harvested in different tissues from the same donor exhibit different phenotypes. Each phenotype is not only characterized by a certain pattern of cell surface markers, but also different cellular functionalities. Only recently were different harvesting and processing techniques found to contribute to this phenomenon as well. This study was therefore set up to investigate proteomic and functional properties of human bone marrow-derived MSCs (hBM-MSC). These were taken from the same tissue and donor site but harvested either as aspirate or bone chip cultures. Both MSC populations were profiled for MSC markers defined by the International Society for Cellular Therapy (ISCT), MSC markers currently under discussion and markers of particular interest. While classic ISCT MSC markers did not show any significant difference between aspirate and outgrowth hBM-MSCs, our additional characterization panel revealed distinct patterns of differentially expressed markers. Furthermore, hBM-MSCs from aspirate cultures demonstrated a significantly higher osteogenic differentiation potential than outgrowth MSCs, which could be confirmed using a transcriptional approach. Our comparison of MSC phenotypes obtained by different harvesting techniques suggests the need of future standardized harvesting, processing and phenotyping procedures in order to gain better comparability in the MSC field.
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Biomarcadores/metabolismo , Células Madre Mesenquimatosas/citología , Proteómica/métodos , Manejo de Especímenes/métodos , Adipogénesis , Biopsia con Aguja , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrogénesis , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , FenotipoRESUMEN
As the percentage of overweight individuals in the population rises, diseases associated with excess weight resulting from poor nutrition are becoming more and more widespread. So far, the influence of weight or nutrition on bone health has shown conflicting results. In the literature, the existing studies disagree about the effect of diet on bones. Therefore, this study investigated the impact of a long-term, high-fat, and high-cholesterol diet on the spine in a mouse model. Wild-type mice were randomly separated into two groups; one group received a high-fat and high-cholesterol diet, and a control group was fed with a regular diet since birth for a duration of 8 months. The first to fifth thoracic vertebrae were extracted and investigated using histology and micro-CT. Samples were analyzed regarding different parameters: percentage of bone structure compared to the whole vertebra and the amount and thickness of the trabeculae. Both methods of the analysis showed similar results. Diet did not have a significant impact on the bone density of the vertebrae. The micro-CT examination showed that the average bone percentage of the examined vertebra was 6% (p = 0.2330) higher in the control group compared to the diet group. The same tendency was demonstrated in histology even though with a smaller difference of only 5%. The results of both methods were comparable and showed trends for the influence of different diets but not significant impacts. In summary, this study showed that a high-fat and high-cholesterol diet has a slightly negative impact on bone density. In order to further analyze the effects of different diets on bone composition, structure, and density, additional long-term studies should be carried out, and more parameters such as movement and genetic factors should be analyzed. Furthermore, other parameters such as exercise and genetic factors that could have a secondary influence on obesity should be investigated.
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Densidad Ósea , Obesidad , Animales , Colesterol , Dieta , Ratones , Columna VertebralRESUMEN
Extracellular vesicles (EVs) comprise an as yet insufficiently investigated intercellular communication pathway in the field of revision total joint arthroplasty (RTJA). This study examined whether periprosthetic joint synovial fluid contains EVs, developed a protocol for their isolation and characterized them with respect to quantity, size, surface markers as well as documented their differences between aseptic implant failure (AIF) and periprosthetic joint infection (PJI). EV isolation was accomplished using ultracentrifugation, electron microscopy (EM) and nanoparticle tracking analysis evaluated EV presence as well as particle size and quantity. EV surface markers were studied by a bead-based multiplex analysis. Using our protocol, EM confirmed the presence of EVs in periprosthetic joint synovial fluid. Higher EV particle concentrations and decreased particle sizes were apparent for PJI. Multiplex analysis confirmed EV-typical surface epitopes and revealed upregulated CD44 and HLA-DR/DP/DQ for AIF, as well as increased CD40 and CD105. Our protocol achieved isolation of EVs from periprosthetic joint synovial fluid, confirmed by EM and multiplex analysis. Characterization was documented with respect to size, concentration and epitope surface signature. Our results indicate various differences between PJI and AIF EVs. This pilot study enables new research approaches and rising diagnostic opportunities in the field of RTJA.
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Accurate diagnosis of periprosthetic joint infections (PJI) is one of the most widely researched areas in modern orthopedic endoprosthesis. However, our understanding of the immunological basis of this severe complication is still limited. In this study, we developed a flow cytometric approach to precisely characterize the immune cell composition in periprosthetic joints. Using high-dimensional multi-parametric data, we defined, for the first time, the local immune cell populations of artificial joints. We identified significant differences in the cellular distribution between infected and non-infected samples, and revealed that myeloid-derived suppressor cells (MDSCs) act as potential regulators of infiltrating immune cells in PJI. Further, we developed an algorithm to predict septic and aseptic samples with high sensitivity and specificity, that may serve as an indispensable addition to the current criteria of the Musculoskeletal Infection Society. This study describes a novel approach to flow cytometrically analyze the immune cell infiltrate of joint fluid that not only improves our understanding of the pathophysiology of PJI, but also enables the development of a novel screening tool to predict infection status. Our data further suggest that pharmacological targeting of MDSCs represents a novel strategy for addressing PJI.