Long-Term Cell Tracking Following Local Injection of Mesenchymal Stromal Cells in the Equine Model of Induced Tendon Disease.

Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10 × 10 6 Molday ION Rhodamine B™-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and histology. Cell tracking based on the hypointense artifacts induced by the superparamagnetic iron oxide (SPIO) labeling agent in MRI as well as based on Rhodamine B fluorescence was feasible. However, Prussian blue staining for assessment of histology was not entirely specific for SPIO. Labeled cells could be traced at their injection site by MRI as well as histology for the whole follow-up period of 24 weeks. Although the numbers of labeled cells within the injected tendon lesions decreased over time, part of the applied cells appeared to remain viable and integrated within the injured tissue. Furthermore, small numbers of labeled cells were identified in peripheral blood within the first 24 h after cell injection and could also be found until week 24 within the contralateral control tendon lesions that had been injected with serum. The present findings unveil details on MSC biodistribution and persistence after their local application, which are of clinical relevance with regard to MSC safety and mechanisms of action

Long-Term Cell Tracking Following Local Injection of Mesenchymal Stromal Cells in the Equine Model of Induced Tendon Disease

Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10106 Molday ION Rhodamine B-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and histology. Cell tracking based on the hypointense artifacts induced by the superparamagnetic iron oxide (SPIO) labeling agent in MRI as well as based on Rhodamine B fluorescence was feasible. However, Prussian blue staining for assessment of histology was not entirely specific for SPIO. Labeled cells could be traced at their injection site by MRI as well as histology for the whole follow-up period of 24 weeks. Although the numbers of labeled cells within the injected tendon lesions decreased over time, part of the applied cells appeared to remain viable and integrated within the injured tissue. Furthermore, small numbers of labeled cells were identified in peripheral blood within the first 24 h after cell injection and could also be found until week 24 within the contralateral control tendon lesions that had been injected with serum. The present findings unveil details on MSC biodistribution and persistence after their local application, which are of clinical relevance with regard to MSC safety and mechanisms of action

In Vivo Magic Angle Magnetic Resonance Imaging for Cell Tracking in Equine Low-Field MRI.

The magic angle effect increases the MRI signal of healthy tendon tissue and could be used for more detailed evaluation of tendon structure. Furthermore, it could support the discrimination of hypointense artefacts induced by contrast agents such as superparamagnetic iron oxide used for cell tracking. However, magic angle MRI of the equine superficial digital flexor tendon has not been accomplished in vivo in standing low-field MRI so far. The aim of this in vivo study was to evaluate the practicability of this magic angle technique and its benefit for tracking superparamagnetic iron oxide-labelled multipotent mesenchymal stromal cells. Six horses with induced tendinopathy in their forelimb superficial digital flexor tendons were injected locally either with superparamagnetic iron oxide-labelled multipotent mesenchymal stromal cells or serum. MRI included standard and magic angle image series in T1- and T2∗-weighted sequences performed at regular intervals. Image analysis comprised blinded evaluation and quantitative assessment of signal-to-noise ratio. The magic angle technique enhanced the tendon signal-to-noise ratio (P < 0.001). Hypointense artefacts were observable in the cell-injected superficial digital flexor tendons over 24 weeks and artefact signal-to-noise ratio differed significantly from tendon signal-to-noise ratio in the magic angle images (P < 0.001). Magic angle imaging of the equine superficial digital flexor tendon is feasible in standing low-field MRI. The current data demonstrate that the technique improves discrimination of superparamagnetic iron oxide-induced artefacts from the surrounding tendon tissue.Peer Reviewe

A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays

Phytochemical Composition, Antioxidant Activity, and the Effect of the Aqueous Extract of Coffee (Coffea arabica L.) Bean Residual Press Cake on the Skin Wound Healing

The world coffee consumption has been growing for its appreciated taste and its beneficial effects on health. The residual biomass of coffee, originated in the food industry after oil extraction from coffee beans, called coffee beans residual press cake, has attracted interest as a source of compounds with antioxidant activity. This study investigated the chemical composition of aqueous extracts of coffee beans residual press cake (AE), their antioxidant activity, and the effect of topical application on the skin wound healing, in animal model, of hydrogels containing the AE, chlorogenic acid (CGA), allantoin (positive control), and carbopol (negative control). The treatments’ performance was compared by measuring the reduction of the wound area, with superior result (p<0.05) for the green coffee AE (78.20%) with respect to roasted coffee AE (53.71%), allantoin (70.83%), and carbopol (23.56%). CGA hydrogels reduced significantly the wound area size on the inflammatory phase, which may be associated with the well known antioxidant and anti-inflammatory actions of that compound. The topic use of the coffee AE studied improved the skin wound healing and points to an interesting biotechnological application of the coffee bean residual press cake