Encapsulation of allogeneic mesenchymal stem cells in alginate extends local presence and therapeutic function.

Bone marrow derived mesenchymal stem cells (MSCs) have immunomodulatory and trophic capacities. For therapeutic application in local chronic inflammatory diseases, MSCs, preferably of allogeneic origin, have to retain immunomodulatory properties. This might be achieved by encapsulation of MSCs in a biomaterial that protects them from the host immune system. Most studies investigating the properties of MSCs for therapeutic application use short term cultures of cells in monolayer. Since the physical environment of MSCs can influence their functionality, we evaluated the feasibility of preserving the immunomodulatory properties of MSCs encapsulated in a three-dimensional alginate construct. After 5 weeks of implantation in immunocompetent rats, active allogeneic MSCs encapsulated in alginate were still detectable by Bio Luminescence Imaging and Magnetic Resonance Imaging of luciferase transduced and superparamagnetic iron oxide labelled MSCs. MSCs injected in saline were only detectable up to 1 week after injection. Moreover, the MSCs encapsulated in alginate responded to inflammatory stimuli similarly to MSCs in monolayer culture. In addition, MSC-alginate beads secreted immunomodulatory and trophic factors and inhibited T-cell proliferation after 30 d of in vitro culture. Our data indicate that allogeneic MSCs encapsulated in alginate persist locally and could act as an interactive immunomodulatory or trophic factor release system for several weeks, making this an interesting system to investigate for application in inflammatory disease conditions.

[Study of the role of miRNA in mesenchymal stem cells isolated from osteoarthritis patients]. / Estudio del papel de los miARN en células madre mesenquimales aisladas de pacientes artrósicos.

OBJECTIVE: MiRNAs act as gene silencers that are involved in the regulation of essential cell functions. miR-335 is involved in regulating cell differentiation processes in progenitor cells. Mesenchymal stem cells (MSCs) are progenitor cells of chondrocytes and osteoblasts responsible for homeostatic maintenance of cartilage and bone. The aim of this study was to determine a possible relationship between the expression of miR-335 and osteoarthritis. METHODS: MSCs obtained from the bone marrow of 3 osteoarthritic patients and 3 controls with no clinical signs of osteoarthritis or osteoporosis were cultured and phenotypically and functionally characterised in a 3-step culture. Expression levels of miR-335 and the mesoderm-specific transcript gene -MEST- that controls its expression were determined by quantitative PCR. RESULTS: Differences in the expression levels of miR-335 and MEST (median [interquartile range]: 1.69 [0.85-1.74], and 3.85 [3.20-5.67] were detected between MSCs isolated from patients with osteoarthritis and controls. Although the differences detected did not reach statistical significance (P=.1), a clear trend towards lower expression of miR-335 in osteoarthritis MSCs was observed. CONCLUSIONS: Given that miR-335 has the different genes involved in the Wnt signalling pathway as potential targets, the observed trend may help to ascertain, at least partially, some of the alterations which determine the onset or progression of osteoarthritis, and can therefore serve for the design of future therapeutic targets for the treatment of this disease.

Expression of human endogenous retrovirus HERV-K18 is associated with clinical severity in osteoarthritis patients.

OBJECTIVES: The aim of this study was to evaluate the involvement of human endogenous retrovirus K18 (HERV-K18) in osteoarthritis (OA), by genotyping the HERV-K18 env locus in OA patients and controls, and analysing HERV-K18 RNA expression and its association with OA risk and clinical variables. METHOD: We recruited 558 patients with symptomatic OA and 600 controls. We performed the genotyping by TaqMan assays and the analysis of expression by quantitative real-time polymerase chain reaction (qRT-PCR). Scores on the Western Ontario and McMasters Universities Osteoarthritis Index (WOMAC), the Lequesne index, and the Stanford Health Assessment Questionnaire (HAQ) were analysed with regard to the expression levels of HERV-K18. RESULTS: The 18.3 haplotype tended towards an association with OA risk and concordantly this haplotype was associated with a higher HERV-K18 expression (p = 0.05). We found statistically significant differences when we compared the scores on the WOMAC, the Lequesne index for knee and hip, and the HAQ between OA patients with higher expression [normalization ratio (NR) > 10] and OA patients without HERV-K18 expression (p = 0.0003, 0.0005, 0.002, and 0.05, respectively), and also when the comparison was made between OA patients with higher expression (NR > 10) and OA patients with low expression of HERV-K18 (NR = 1) for the WOMAC and the Lequesne index for knee and hip (p = 0.002, 0.013, and 0.006, respectively). CONCLUSIONS: We found an association between health status measurement systems and severity index for OA and the levels of expression of HERV-K18. These results suggest the possible involvement of HERV-K18 in the aetiopathogenesis of the disease.

Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culture.

OBJECTIVE: Mesenchymal stem cells (MSCs) are promising candidates for osteoarthritis (OA) therapies, although their mechanism of action remains unclear. MSCs have recently been discovered to secrete anti-inflammatory cytokines and growth factors. We studied the paracrine effects of MSCs on OA cartilage and synovial explants in vitro. DESIGN: MSC-conditioned medium was prepared by stimulating primary human MSCs with tumour necrosis factor alpha (TNFα) and (50ng/ml each). Human synovium and cartilage explants were cultured in MSC-conditioned medium or in control medium, containing the same amount of added TNFα and IFNγ but not incubated with MSCs. Explants were analyzed for gene expression and the production of nitric oxide (NO). The presence of the inhibitor of nuclear factor kappa B alpha (IκBa) was assessed by Western blot analysis. RESULTS: Synovial explants exposed to MSC-conditioned medium showed decreased gene expression of interleukin-1 beta (IL-1ß), matrix metalloproteinase (MMP)1 and MMP13, while suppressor of cytokine signaling (SOCS)1 was upregulated. In cartilage, expression of IL-1 receptor antagonist (IL-1RA) was upregulated, whereas a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)5 and collagen type II alpha 1 (COL2A1) were downregulated. MSC-conditioned medium reduced NO production in cartilage explants and the presence of IκBa was increased in synoviocytes and chondrocytes treated with MSC-conditioned medium. CONCLUSIONS: In an inflammatory environment, MSCs secrete factors which cause multiple anti-inflammatory effects and influence matrix turnover in synovium and cartilage explants. Thereby, the presented data encourage further study of MSCs as a treatment for joint diseases.

Tratamiento comparado de las lesiones del manguito rotador mediante el uso de células madre mesenquimales en combinación con membranas de colágeno tipo I / Rotator cuff injury comparative treatment between isolated mesenchymal stem cells or combined with collagen I membranes

Objetivo: Estudiar, en rata, la rotura completa del tendón del m. supraespinoso (SE) y su reparación mediante una membrana de colágeno tipo I con células mesenquimales pluripotenciales (MSCs). Material y método: Seccionamos unilateralmente el tendón SE, en ratas Sprague-Dawley de nueve meses de edad. Un mes después de la intervención, se realizó una segunda cirugía para reparar el tendón según tres grupos de tratamiento: cirugía convencional usando una sutura (n=30); con membrana de colágeno tipo I sin células (n=30) y con membrana de colágeno tipo I con 1x106 MSCs alogénicas (n=30; grupo MSC-Membrana). La reparación se evaluó al mes (n=30), a los dos2 meses (n=30) y a los nueve meses (n=30) mediante criterios biomecánicos e histológicos. Resultados: Las matrices de colágeno con MSC en los defectos creados mejoraron la resistencia y la rigidez del tendón SE a los tres meses comparado con las otras dos cirugías reparadoras. Ningún tratamiento logró un patrón histológicamente organizado. Conclusión: El tratamiento con MSC es seguro en los desgarros del manguito rotador (AU)

Objective: To study, in a rat model, the supraspinatus tendon (SE) rupture with isolated MSCs or combined with collagen membranes Material and method: A chronic rotator cuff tear injury model was developed by unilaterally detaching the SE tendon of aged Sprague-Dawley rats (9 months). One month post-injury, a second surgery was then performed to repair the tears by: 1. single suture (n=30), 2. suture and type I collagen membranes (n=30) and 3. suture and type I collagen membranes with 1x106 allogeneic MSCs; (n=30). Lesion restoration was evaluated at one month (n=30), two months (n=30) and three months (n=30) post-injury by biomechanical, histological and inmunohistological criterias. Results: All experimental conditions were well tolerated and no adverse effects were observed. Implantation of collagen type I membrane with MSCs into surgically created tendon defects improved strength and stiffness of SE tendon three months after treatment compared with the other two repair surgeries. No treatment achieved a histological organized pattern. Conclusion: The therapeutic use of allogenic MSCs in collagen type I membrane in the rotator cuff tears seems to be the most promising treatment in our experimental model (AU)