The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration.

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2.

Erratum: Rémy et al. Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019, 8, 1485.

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A disulfide based low molecular weight gel for the selective sustained release of biomolecules.

Constructing biocompatible soft materials via supramolecular approaches remains an important challenge for in vivo applications. Substantial efforts have been made to develop biocompatible non-polymeric materials allowing sustained release of biomolecules and/or drugs in vivo. Herein, we introduce disulfide based low molecular weight gels (LMWGs) allowing the in vitro selective sustained release of proteins containing thiol residues. The novel glycosylated nucleoside based bola-amphiphile (GNBA), which features a disulfide bond inserted in the hydrophobic segment, self-assembles to stabilize the resulting hydrogel. Rheological studies show the dominant elastic character and thixotropic properties of the disulfide LMWG demonstrating its injectability. In vitro and in vivo biodegradation investigations reveal that the disulfide LMWG is stable for several weeks. Importantly, disulfide bonds can be cleaved through the thiol-disulfide exchange reactions with small redox molecules such as dithiothreitol (DTT). The disulfide LMWG loaded with a thiol-containing protein (bovine serum albumin) features sustained release in vitro, whereas a dextran of the same molecular weight, lacking a thiol biomolecule, shows quick release. The disulfide GNBA is the first example of a LMWG allowing selective long term sustained release in vitro via a disulfide reshuffling mechanism.

Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties.

: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.

Cell and tissue responses at the interface with a chitosan hydrogel intended for vascular applications: in vitro and in vivo exploration.

AIMS: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo. METHODS AND RESULTS: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype. CONCLUSION: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.

Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

A Peptide Targeting Inflammatory CNS Lesions in the EAE Rat Model of Multiple Sclerosis.

Multiple sclerosis is characterized by inflammatory lesions dispersed throughout the central nervous system (CNS) leading to severe neurological handicap. Demyelination, axonal damage, and blood brain barrier alterations are hallmarks of this pathology, whose precise processes are not fully understood. In the experimental autoimmune encephalomyelitis (EAE) rat model that mimics many features of human multiple sclerosis, the phage display strategy was applied to select peptide ligands targeting inflammatory sites in CNS. Due to the large diversity of sequences after phage display selection, a bioinformatics procedure called "PepTeam" designed to identify peptides mimicking naturally occurring proteins was used, with the goal to predict peptides that were not background noise. We identified a circular peptide CLSTASNSC called "Ph48" as an efficient binder of inflammatory regions of EAE CNS sections including small inflammatory lesions of both white and gray matter. Tested on human brain endothelial cells hCMEC/D3, Ph48 was able to bind efficiently when these cells were activated with IL1ß to mimic inflammatory conditions. The peptide is therefore a candidate for further analyses of the molecular alterations in inflammatory lesions.

The proangiogenic potential of a novel calcium releasing biomaterial: Impact on cell recruitment.

In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications. STATEMENT OF SIGNIFICANCE: In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass - poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation.

BACKGROUND: Vasogenic edema dynamically accumulates in many brain disorders associated with brain inflammation, with the critical step of edema exacerbation feared in patient care. Water entrance through blood-brain barrier (BBB) opening is thought to have a role in edema formation. Nevertheless, the mechanisms of edema resolution remain poorly understood. Because the water channel aquaporin 4 (AQP4) provides an important route for vasogenic edema resolution, we studied the time course of AQP4 expression to better understand its potential effect in countering the exacerbation of vasogenic edema. METHODS: Focal inflammation was induced in the rat brain by a lysolecithin injection and was evaluated at 1, 3, 7, 14 and 20 days using a combination of in vivo MRI with apparent diffusion coefficient (ADC) measurements used as a marker of water content, and molecular and histological approaches for the quantification of AQP4 expression. Markers of active inflammation (macrophages, BBB permeability, and interleukin-1ß) and markers of scarring (gliosis) were also quantified. RESULTS: This animal model of brain inflammation demonstrated two phases of edema development: an initial edema build-up phase during active inflammation that peaked after 3 days (ADC increase) was followed by an edema resolution phase that lasted from 7 to 20 days post injection (ADC decrease) and was accompanied by glial scar formation. A moderate upregulation in AQP4 was observed during the build-up phase, but a much stronger transcriptional and translational level of AQP4 expression was observed during the secondary edema resolution phase. CONCLUSIONS: We conclude that a time lag in AQP4 expression occurs such that the more significant upregulation was achieved only after a delay period. This change in AQP4 expression appears to act as an important determinant in the exacerbation of edema, considering that AQP4 expression is insufficient to counter the water influx during the build-up phase, while the second more pronounced but delayed upregulation is involved in the resolution phase. A better pathophysiological understanding of edema exacerbation, which is observed in many clinical situations, is crucial in pursuing new therapeutic strategies.

Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration.

OBJECTIVES: We investigated proinflammatory M1 and immunomodulatory M2 activation profiles of circulating monocytes in relapsing experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, and tested whether altered M1/M2 equilibrium promotes CNS inflammation. RESULTS: Approaches of MRI macrophage tracking with USPIO nanoparticles and expression patterns of M1/M2 macrophages and microglia in brain and M1/M2 monocytes in blood samples at various disease stages revealed that M1/M2 equilibrium in blood and CNS favors mild EAE, while imbalance towards M1 promotes relapsing EAE. We consequently investigated whether M2 activated monocyte restoration in peripheral blood could cure acute clinical EAE disease. Administration of ex vivo activated M2 monocytes both suppressed ongoing severe EAE and increased immunomodulatory expression pattern in lesions, confirming their role in the induction of recovery. CONCLUSION: We conclude that imbalance of monocyte activation profiles and impaired M2 expression, are key factors in development of relapses. Our study opens new perspectives for therapeutic applications in MS.

Inflammation induced neurological handicap processes in multiple sclerosis: new insights from preclinical studies.

Multiple sclerosis (MS) is described as originating from incompletely explained neuroinflammatory processes, dysfunction of neuronal repair mechanisms and chronicity of inflammation events. Blood-borne immune cell infiltration and microglia activation are causing both neuronal destruction and myelin loss, which are responsible for progressive motor deficiencies, organic and cognitive dysfunctions. MRI as a non-invasive imaging method offers various ways to visualise de- and remyelination, neuronal loss, leukocyte infiltration, blood-brain barrier modification and new sensors are emerging to detect inflammatory lesions at an early stage. We describe studies performed on experimental autoimmune encephalomyelitis (EAE) animal models of MS that shed new light on mechanisms of functional impairments to understand the neurological handicap in MS. We focus on examples of neuroinflammation-mediated inhibition of CNS repair involving adult neurogenesis in the sub-ventricular zone and hippocampus and such experimentally observed inhibitions could reflect deficient plasticity and activation of compensatory mechanisms in MS. In parallel with cognitive decline, organic deficits such as bladder dysfunction are described in most of MS patients. Neuropharmacological interventions, electrical stimulation of nerves, MRI and histopathology follow-up studies helped in understanding the operating events to remodel the neurological networks and to compensate the inflammatory lesions both in spinal cord and in cortical regions. At the molecular level, the local production of reactive products is a well-described phenomenon: oxidative species disturb cellular physiology and generate new molecular epitopes that could further promote immune reactions. The translational research from EAE animal models to MS patient cohorts helps in understanding the mechanisms of the neurological handicap and in development of new therapeutic concepts in MS.

Type 3 deiodinase expression in inflammatory spinal cord lesions in rat experimental autoimmune encephalomyelitis.

BACKGROUND: We have shown substantial expression of type 3 deiodinase (D3, a major enzyme involved in the inactivation of thyroid hormone) in infiltrating leukocytes in several models of inflammation. Recently, thyroid hormone has been shown to improve remyelination in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. As induction of D3 may play an important role in decreasing local bioavailability of thyroid hormone at inflammation sites, we hypothesized that D3 is induced in spinal cord inflammatory lesions in EAE. METHODS: The aim of the study was to evaluate D3 expression in spinal cord inflammatory lesions of EAE Dark Agouti rats and to investigate D3 induction in activated monocytes. RESULTS: Here, we show marked expression of D3 by granulocytes and macrophages in spinal cord inflammatory lesions of EAE rats. We further confirm induction of D3 expression in vitro in monocytes that were activated toward proinflammatory or immunomodulatory phenotypes. CONCLUSIONS: We observed increased D3 expression both in spinal cord inflammatory lesions during EAE and in activated monocytes. Although increased D3 expression theoretically results in decreased triiodothyronine availability, it is unknown at present whether reduced local triiodothyronine concentrations are involved in impaired remyelination as observed during EAE.

Aquaporin 4 correlates with apparent diffusion coefficient and hydrocephalus severity in the rat brain: a combined MRI-histological study.

Hydrocephalus features include ventricular dilatation and periventricular edema due to transependymal resorption of cerebrospinal fluid (CSF). Aquaporin 4 (AQP4), a water channel protein located at the blood-brain barrier, might facilitate the removal of this excess of water from the parenchyma into the blood. First, we hypothesized a link between AQP4 expression and the severity of hydrocephalus. We further hypothesized that movements of water through AQP4 could affect apparent diffusion coefficient (ADC) measurements. Communicating inflammatory hydrocephalus was induced in 45 rats, and at various stages, magnetic resonance imaging (MRI) was used to measure CSF volume and periventricular ADC, with immunostaining being used to determine periventricular AQP4. We found an up-regulation of periventricular AQP4 in hydrocephalic rats that was strongly correlated with both CSF volume (Pearson=0.87, p<0.00001) and periventricular ADC (Pearson=0.85, p<0.00001). AQP4 were first located on astrocyte endfeet, but later on the whole membrane of astrocytes that became hypertrophic in the most severe and chronic hydrocephalic rats. These results show that AQP4 expression follows an adaptative profile to the severity of hydrocephalus, which is probably a protective response mechanism. They also suggest that ADC, on top of informing about cell sizes and interstitial bulk water, might also indirectly reflect quantitative water channel expression.

How to trace stem cells for MRI evaluation?

Applications of imaging techniques to visualize stem cells for monitoring, control and treatment of biological systems, in particular the brain, is at the forefront of investigations. These approaches involve the identification of stem and precursor cells that may be of various origins, but are related to specific clinical conditions, and the choice of the appropriate markers to achieve the required imaging while minimizing the side effects. This article will review examples of the contrast agent design for rational approaches in stem cell imaging. Potential pitfalls or side effects associated with contrast agents, in particular iron oxide nanoparticles, for cell labelling are also discussed.

Magnetic resonance imaging of human brain macrophage infiltration.

Macrophage tracking by magnetic resonance imaging (MRI) with iron oxide nanoparticles has been developed during the last decade for numerous diseases of the CNS. Experimental studies on animal models were confirmed by first clinical applications of MRI technology of brain macrophages for multiple sclerosis, ischemic stroke lesions, and tumors. As activated macrophages act in concert with other immune competent cells, this innovative MRI approach provides new functional data on the immune reaction in these CNS diseases. The MRI detection of brain macrophages defines precise spatial and temporal patterns of macrophage involvement that helps to characterize individual neurological disorders. This approach is being explored as an in vivo marker for the clinical diagnosis of cerebral lesion activity, in experimental models for the prognosis of disease development, and to determine the efficacy of immunomodulatory treatments under clinical evaluation. Comparative brain imaging follow-up studies of blood-brain barrier leakage by MRI with gadolinium-chelates, microglia activation by positron emission tomography with radiotracer ligand PK11195 and MRI detection of macrophage infiltration provide more precise information about the pathophysiological cascade of inflammatory events in cerebral diseases. Such multimodal characterization of the inflammatory events should help in the monitoring of patients, in defining precise time intervals for therapeutic interventions, and in developing and evaluating new therapeutic strategies.

Hexitol nucleic acid-containing aptamers are efficient ligands of HIV-1 TAR RNA.

The transactivation responsive element (TAR) plays a crucial role in the transcription of the HIV-1 genome upon specific binding of the viral protein Tat and cellular proteins. We have previously identified a RNA hairpin aptamer forming a stable and specific kissing complex with TAR RNA (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). We chemically modified this aptamer with hexitol nucleic acid (HNA) residues. We demonstrate that a fully HNA-modified aptamer is a poor ligand but, in contrast, mixmers containing both HNA and unmodified RNA nucleotides display interesting properties. Two HNA-RNA mixmers bind to TAR with an equilibrium dissociation constant in the low-nanomolar range and show a reduced nuclease sensitivity. In addition, they show a moderate dependence on magnesium ions for binding to TAR. These HNA-RNA mixmers are able to inhibit transactivation of transcription in an in vitro assay.

Selection by phage display of peptides targeting the HIV-1 TAR element.

As transcription regulatory element, the HIV-1 TAR RNA element is a promising target to inhibit viral replication; indeed, ligands of TAR RNA could prevent the transcription trans-activation process. Phage display in vitro selection was undertaken to select peptidic ligands of TAR RNA. In preliminary experiments, the selection was performed in a magnesium rich buffer (3 mM), but only phages targeted to plastic wells or streptavidin emerged; in addition, a "super-infectious" phage present in the New England Biolabs library (SVSVGMKPSPRP) selected by others with different targets was cloned, due to a high amplification potential. In contrast, the absence of magnesium or an increasing magnesium concentration (0 to 0.5 mM) led to phage selection with 57 amino acid peptides. K(D)s of 420-550 nM were measured by filter binding assays; a significant specificity was obtained when TAR target was compared with unrelated RNA targets. Surprisingly, the binding of selected peptides does not depend on the magnesium concentration.

In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin.

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.