Two in one: use of divalent manganese ions as both cross-linking and MRI contrast agent for intrathecal injection of hydrogel-embedded stem cells

Cell therapy is a promising tool for treating central nervous system (CNS) disorders; though, the translational efforts are plagued by ineffective delivery methods. Due to the large contact surface with CNS and relatively easy access, the intrathecal route of administration is attractive in extensive or global diseases such as stroke or amyotrophic lateral sclerosis (ALS). However, the precision and efficacy of this approach are still a challenge. Hydrogels were introduced to minimize cell sedimentation and improve cell viability. At the same time, contrast agents were integrated to allow image-guided injection. Here, we report using manganese ions (Mn2+) as a dual agent for cross-linking alginate-based hydrogels and magnetic resonance imaging (MRI). We performed in vitro studies to test the Mn2+ alginate hydrogel formulations for biocompatibility, injectability, MRI signal retention time, and effect on cell viability. The selected formulation was injected intrathecally into pigs under MRI control. The biocompatibility test showed a lack of immune response, and cells suspended in the hydrogel showed greater viability than monolayer culture. Moreover, Mn2+-labeled hydrogel produced a strong T1 MRI signal, which enabled MRI-guided procedure. We confirmed the utility of Mn2+ alginate hydrogel as a carrier for cells in large animals and a contrast agent at the same time.This research was funded by The National Centre for Research and Development, grant number 12/EuroNanoMed/2016

Mn-based methacrylated gellan gum hydrogels for MRI-guided cell delivery and imaging

This work aims to engineer a new stable injectable Mn-based methacrylated gellan gum (Mn/GG-MA) hydrogel for real-time monitored cell delivery into the central nervous system. To enable the hydrogel visualization under Magnetic Resonance Imaging (MRI), GG-MA solutions were supplemented with paramagnetic Mn2+ ions before its ionic crosslink with artificial cerebrospinal fluid (aCSF). The resulting formulations were stable, detectable by T1-weighted MRI scans and also injectable. Cell-laden hydrogels were prepared using the Mn/GG-MA formulations, extruded into aCSF for crosslink, and after 7 days of culture, the encapsulated human adipose-derived stem cells remained viable, as assessed by Live/Dead assay. In vivo tests, using double mutant MBPshi/shi/rag2 immunocompromised mice, showed that the injection of Mn/GG-MA solutions resulted in a continuous and traceable hydrogel, visible on MRI scans. Summing up, the developed formulations are suitable for both non-invasive cell delivery techniques and image-guided neurointerventions, paving the way for new therapeutic procedures.Sílvia Vieira acknowledges the FCT Ph.D. scholarship (SFRH/BD/102710/2014). J. Miguel Oliveira and J. Silva-Correia acknowledge the FCT grants under the Investigator FCT program (IF/01285/2015 and IF/00115/2015, respectively). The authors also acknowledge the funds provided under the project NanoTech4ALS, funded under the EU FP7 M-ERA.NET program, and ESF (POWR.03.02.00-00-I028/17-00)

MRI-guided intracerebral convection-enhanced injection of gliotoxins to induce focal demyelination in swine.

Demyelinating disorders such as multiple sclerosis (MS) or transverse myelitis are devastating neurological conditions with no effective cure. Prevention of myelin loss or restoration of myelin are key for successful therapy. To investigate the disease and develop cures animal models with good clinical relevance are essential. The goal of the current study was to establish a model of focal demyelination in the brain of domestic pig using MRI-guided gliotoxin delivery. The rationale for developing a new myelin disease model in the domestic pig was based on the fact that the brain in pigs is anatomically and histologically much more similar to that of humans compared to the rodent brain. For MRI-assisted gliotoxin injection, eight 30 kg pigs were subjected to treatment with lysolecithin (20, 30 mg/ml); or with ethidium bromide (0.0125, 0.05, 0.2 mg/ml). Animals were placed in an MRI scanner for intraparenchymal targeting of gliotoxin into the corona radiata (250 μl over 1h), with real-time monitoring of toxin distribution on T1 scans and monitoring of lesion evolution over seven days using both T1 and T2 scans. After the last MRI, animals were transcardially perfused and brains were processed for histological and immunofluorescent analysis. Gadolinium-enhanced T1 MRI during injection demonstrated biodistribution of the contrast (as a surrogate marker for toxin distribution) and its diffusion through the brain parenchyma. Lesion induction was confirmed on T2-weighted MRI and histopathology, thus enabling the establishment of optimal doses of gliotoxins. To conclude, MRI-guided focal demyelination in swine is accurate and provides real-time confirmation of gliotoxin, thus facilitating placement of focal lesions with high precision. This new model of focal demyelination can be used for further investigation and development of novel therapeutic approaches

Advances in bioinks and in vivo imaging of biomaterials for CNS applications

Due to increasing life expectancy incidence of neurological disorders is rapidly rising, thus adding urgencyto develop effective strategies for treatment. Stem cell-based therapies were considered highly promisingand while progress in this field is evident, outcomes of clinical trials are rather disappointing. Suboptimalengraftment, poor cell survival and uncontrolled differentiation may be the reasons behind dismal results.Clearly, new direction is needed and we postulate that with recent progress in biomaterials and bioprint-ing, regenerative approaches for neurological applications may be finally successful. The use of biomate-rials aids engraftment of stem cells, protects them from harmful microenvironment and importantly, itfacilitates the incorporation of cell-supporting molecules. The biomaterials used in bioprinting (thebioinks) form a scaffold for embedding the cells/biomolecules of interest, but also could be exploited asa source of endogenous contrast or supplemented with contrast agents for imaging. Additionally, bioprint-ing enables patient-specific customization with shape/size tailored for actual needs. In stroke or traumaticbrain injury for example lesions are localized and focal, and usually progress with significant loss of tissuevolume creating space that could be filled with artificial tissue using bioprinting modalities. The value ofimaging for bioprinting technology is advantageous on many levels including design of custom shapesscaffolds based on anatomical 3D scans, assessment of performance and integration after scaffold implan-tation, or to learn about the degradation over time. In this review, we focus on bioprinting technologydescribing different printing techniques and properties of biomaterials in the context of requirementsfor neurological applications. We also discuss the need forin vivoimaging of implanted materials and tis-sue constructs reviewing applicable imaging modalities and type of information they can provide.This work was supported by NanoTech4ALS (ref. ENMed/0008/2015, 13/EuroNanoMed/2016), funded under the EU FP7 M-ERA.NET program and Strategmed 1/233209/12/ NCBIR/2015

Rabbit Model of Human Gliomas: Implications for Intra-Arterial Drug Delivery - Fig 4

<p>Identification of implanted tumor with histology. H&E staining (A,B) shows a cellular astrocytic tumor with large necrotic central regions (red arrowhead). Scale bars 50 μm in A,C and 100μm in B.</p

Immunohistochemical characterization of the tumor microenvironment.

<p>(A) Staining against IBA-1 detected microglial activation throughout the tumor. (B) Immunohistochemistry against the T cell marker, CD3, detected very low infiltration, with only single cells observed inside or at the periphery of the tumor. (C) GFAP immunoreactivity was detected in the tumor with all cells expressing high levels of this protein, as well as in the area around the tumor, consistent with activation of surrounding endogenous astrocytes.</p

MRI of GBM-1-implanted rabbits.

<p>(A) T2-weighted scan showing hyperintense tumor unilaterally near the thalamus (red arrowhead). T1-weighted scan before (B) and after gadolinium injection (C) showing a lack of enhancement within the tumor.</p

Body weight measurements over the course of immunosuppression.

<p>Body weight measurements over the course of immunosuppression.</p