Label-free mapping of microstructural organisation in self-aligning cellular collagen hydrogels using image correlation spectroscopy

Hydrogels have emerged as promising biomaterials for regenerative medicine. Despite major advances, tissue engineers have faced challenges in studying the complex dynamics of cellmediated hydrogel remodelling. Second harmonic generation (SHG) microscopy has been a pivotal tool for non-invasive visualization of collagen type I hydrogels. By taking into account the typical polarization SHG effect, we recently proposed an alternative image correlation spectroscopy (ICS) model to quantify characteristics of randomly oriented collagen fibrils. However, fibril alignment is an important feature in many tissues that needs to be monitored for effective assembly of anisotropic tissue constructs. Here we extended our previous approach to include the orientation distribution of fibrils in cellular hydrogels and show the power of this model in two biologically relevant applications. Using a collagen hydrogel contraction assay, we were able to capture cell-induced hydrogel modifications at the microscopic scale and link these to changes in overall gel dimensions over time. After 24 h, the collagen density was about 3 times higher than the initial density, which was of the same order as the decrease in hydrogel area. We also showed that the orientation parameters recovered from our automated ICS model match values obtained from manual measurements. Furthermore, regions axial to cellular processes aligned at least 1.5 times faster compared with adjacent zones. Being able to capture minor temporal and spatial changes in hydrogel density and collagen fibril orientation, we demonstrated the sensitivity of this extended ICS model to deconstruct a complex environment and support its potential for tissue engineering research

Time-resolved characterization of the mechanisms of toxicity induced by silica and amino-modified polystyrene on alveolar-like macrophages

Abstract Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.</jats:p

The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction

Rationale: Pluripotent stem cells (PSCs) are being investigated as a cell source for regenerative medicine since they provide an infinitive pool of cells that are able to differentiate towards every cell type of the body. One possible therapeutic application involves the use of these cells to treat myocardial infarction (MI), a condition where billions of cardiomyocytes (CMs) are lost. Although several protocols have been developed to differentiate PSCs towards CMs, none of these provide a completely pure population, thereby still posing a risk for neoplastic teratoma formation. Therefore, we developed a strategy to (i) monitor cell behavior noninvasively via site-specific integration of firefly luciferase (Fluc) and the human positron emission tomography (PET) imaging reporter genes, sodium iodide symporter (hNIS) and somatostatin receptor type 2 (hSSTr2), and (ii) perform hSSTr2-mediated suicide gene therapy via the clinically used radiopharmacon 177Lu-DOTATATE. Methods: Human embryonic stem cells (ESCs) were gene-edited via zinc finger nucleases to express Fluc and either hNIS or hSSTr2 in the safe harbor locus, adeno-associated virus integration site 1. Firstly, these cells were exposed to 4.8 MBq 177Lu-DOTATATE in vitro and cell survival was monitored via bioluminescence imaging (BLI). Afterwards, hNIS+ and hSSTr2+ ESCs were transplanted subcutaneously and teratomas were allowed to form. At day 59, baseline 124I and 68Ga-DOTATATE PET and BLI scans were performed. The day after, animals received either saline or 55 MBq 177Lu-DOTATATE. Weekly BLI scans were performed, accompanied by 124I and 68Ga-DOTATATE PET scans at days 87 and 88, respectively. Finally, hSSTr2+ ESCs were differentiated towards CMs and transplanted intramyocardially in the border zone of an infarct that was induced by left anterior descending coronary artery ligation. After transplantation, the animals were monitored via BLI and PET, while global cardiac function was evaluated using cardiac magnetic resonance imaging. Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed.Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS

Stearoyl-CoA desaturase-1 impairs the reparative properties of macrophages and microglia in the brain

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination

Platelet-rich plasma for regeneration of neural feedback pathways around dental implants: a concise review and outlook on future possibilities

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Label free optical imaging of engineered neural tissue formation by second harmonic signals from collagen type I

A variety of optical microscopy techniques can visualise individual cells in their extracellular matrix (ECM), most of them requiring exogenous dyes. Many labels have been subject of discussion because of phototoxic effects and perturbation of native cellular behavior. Interestingly, some biological molecules and structures can generate intrinsic optical signals, thereby making the use of exogenous dyes redundant. Cellular autofluorescence can be observed by one- or two-photon excitation (TPE) of for example NADH and flavins. Another intrinsic optical effect is Second Harmonic Generation (SHG), where laser light interacting with non-centrosymmetric molecules such as collagen type I generates frequency-doubled light. The resulting images with high contrast and submicron resolution can be further analyzed to obtain specific quantitative information. Despite the advantages of these nonlinear optical microscopy methods, their use in the biomedical field is not widespread. In areas of tissue engineering, these techniques could be of great value for non-invasive characterization of biomaterials. Collagen type I hydrogels have been proposed for many regenerative applications due to their native-like ECM properties, inherent biocompatibility and suitability as carriers for different cell types. When a collagen type I hydrogel solution seeded with dental pulp stem cells (DPSCs) is casted into a mould with tethering bars positioned at each end, the contractile forces generated by DPSCs create a uniaxial tension along the tethered hydrogel (Fig 1a), resulting in longitudinal cell alignment within this 3D matrix (Fig 1b).[1] Although this engineered neural tissue (EngNT) containing DPSCs represents the desired end result for neuroregenerative applications [1], time-lapse experiments monitoring changes of hydrogel architecture are lacking. In order to truly understand ECM remodeling by enclosed cells, it is essential to monitor the interaction of these cells with the 3D construct in time without the use of fluorescent labels. To this end, we performed TPE and second harmonic imaging of live EngNT, which revealed a marked change in collagen type I organization before and after cell alignment (Fig 2). Furthermore, we applied our in house developed image correlation spectroscopy approach [2] to characterize the spatial organization and structural characteristics of collagen type I fibers in time. This research demonstrates the application of nonlinear label free optical techniques for high resolution biomedical imaging

Ultra low-level gamma ray spectrometry of thorium in human bone samples

Following the use of in vivo measurements of 210Pb to estimate retrospectively radon exposure, interest has been expressed in the use of in vivo measurements of 208Tl to estimate thorium intake. To aid with calibration and to determine the optimum part of the body on which in vivo measurements should be made, the distribution of 208Tl and 228Ac amongst different human bones was measured in the underground laboratory HADES. The 208Tl activity was determined by the 2614.5 keV and the 583.2 keV gamma ray lines. The 228Ac activity was determined by the 911.2 keV and the 969.0 keV gamma ray lines. The background under those peaks when measured on the 106% relative efficiency coaxial HPGe detector in HADES is of the order of 1 d-1, resulting in detection limits in the order of 1 mBq for both radionuclides for a typical 10 g bone sample and for a measuring time of 1 week