Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

From Pluripotent Stem Cells to Organoids and Bioprinting: Recent Advances in Dental Epithelium and Ameloblast Models to Study Tooth Biology and Regeneration.

Ameloblasts are the specialized dental epithelial cell type responsible for enamel formation. Following completion of enamel development in humans, ameloblasts are lost and biological repair or regeneration of enamel is not possible. In the past, in vitro models to study dental epithelium and ameloblast biology were limited to freshly isolated primary cells or immortalized cell lines, both with limited translational potential. In recent years, large strides have been made with the development of induced pluripotent stem cell and organoid models of this essential dental lineage - both enabling modeling of human dental epithelium. Upon induction with several different signaling factors (such as transforming growth factor and bone morphogenetic proteins) these models display elevated expression of ameloblast markers and enamel matrix proteins. The advent of 3D bioprinting, and its potential combination with these advanced cellular tools, is poised to revolutionize the field - and its potential for tissue engineering, regenerative and personalized medicine. As the advancements in these technologies are rapidly evolving, we evaluate the current state-of-the-art regarding in vitro cell culture models of dental epithelium and ameloblast lineage with a particular focus toward their applicability for translational tissue engineering and regenerative/personalized medicine.

Extracellular vesicle-associated cholesterol supports the regenerative functions of macrophages in the brain.

Macrophages play major roles in the pathophysiology of various neurological disorders, being involved in seemingly opposing processes such as lesion progression and resolution. Yet, the molecular mechanisms that drive their harmful and benign effector functions remain poorly understood. Here, we demonstrate that extracellular vesicles (EVs) secreted by repair-associated macrophages (RAMs) enhance remyelination ex vivo and in vivo by promoting the differentiation of oligodendrocyte precursor cells (OPCs). Guided by lipidomic analysis and applying cholesterol depletion and enrichment strategies, we find that EVs released by RAMs show markedly elevated cholesterol levels and that cholesterol abundance controls their reparative impact on OPC maturation and remyelination. Mechanistically, EV-associated cholesterol was found to promote OPC differentiation predominantly through direct membrane fusion. Collectively, our findings highlight that EVs are essential for cholesterol trafficking in the brain and that changes in cholesterol abundance support the reparative impact of EVs released by macrophages in the brain, potentially having broad implications for therapeutic strategies aimed at promoting repair in neurodegenerative disorders.

Soluble guanylyl cyclase: A novel target for the treatment of vascular cognitive impairment?

Vascular cognitive impairment (VCI) describes neurodegenerative disorders characterized by a vascular component. Pathologically, it involves decreased cerebral blood flow (CBF), white matter lesions, endothelial dysfunction, and blood-brain barrier (BBB) impairments. Molecularly, oxidative stress and inflammation are two of the major underlying mechanisms. Nitric oxide (NO) physiologically stimulates soluble guanylate cyclase (sGC) to induce cGMP production. However, under pathological conditions, NO seems to be at the basis of oxidative stress and inflammation, leading to a decrease in sGC activity and expression. The native form of sGC needs a ferrous heme group bound in order to be sensitive to NO (Fe(II)sGC). Oxidation of sGC leads to the conversion of ferrous to ferric heme (Fe(III)sGC) and even heme-loss (apo-sGC). Both Fe(III)sGC and apo-sGC are insensitive to NO, and the enzyme is therefore inactive. sGC activity can be enhanced either by targeting the NO-sensitive native sGC (Fe(II)sGC), or the inactive, oxidized sGC (Fe(III)sGC) and the heme-free apo-sGC. For this purpose, sGC stimulators acting on Fe(II)sGC and sGC activators acting on Fe(III)sGC/apo-sGC have been developed. These sGC agonists have shown their efficacy in cardiovascular diseases by restoring the physiological and protective functions of the NO-sGC-cGMP pathway, including the reduction of oxidative stress and inflammation, and improvement of vascular functioning. Yet, only very little research has been performed within the cerebrovascular system and VCI pathology when focusing on sGC modulation and its potential protective mechanisms on vascular and neural function. Therefore, within this review, the potential of sGC as a target for treating VCI is highlighted.

The Effect of Leukocyte- and Platelet-Rich Fibrin on Central and Peripheral Nervous System Neurons-Implications for Biomaterial Applicability.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient's own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Nanomaterials for Periodontal Tissue Regeneration: Progress, Challenges and Future Perspectives.

Bioactive nanomaterials are increasingly being applied in oral health research. Specifically, they have shown great potential for periodontal tissue regeneration and have substantially improved oral health in translational and clinical applications. However, their limitations and side effects still need to be explored and elucidated. This article aims to review the recent advancements in nanomaterials applied for periodontal tissue regeneration and to discuss future research directions in this field, especially focusing on research using nanomaterials to improve oral health. The biomimetic and physiochemical properties of nanomaterials such as metals and polymer composites are described in detail, including their effects on the regeneration of alveolar bone, periodontal ligament, cementum and gingiva. Finally, the biomedical safety issues of their application as regenerative materials are updated, with a discussion about their complications and future perspectives. Although the applications of bioactive nanomaterials in the oral cavity are still at an initial stage, and pose numerous challenges, recent research suggests that they are a promising alternative in periodontal tissue regeneration.

Volumetry of ovine incisors dental pulp for further regenerative therapy.

Mesenchymal stem cells (MSCs) are used for regenerative therapy. Dental pulp MSCs make extracted wisdom teeth a useful resource in humans. Preclinical validation of regenerative therapies requires large animal models such as the sheep. Since stem cells can be retrieved from the dental pulp of ovine incisors, the best age to extract a maximal volume of dental pulp needs to be defined. The objective of this ex vivo study was to quantify incisors dental pulp volume, in sheep of various age. Three jaws were dedicated to histology (one per age group); the others were imaged with a computed tomography scanner [3 years-old (n = 9), 4 (n = 3) and 6 (n = 5)]. The incisors dental pulp volume was measured after 3D reconstruction. Multiple linear regression showed that dental pulp volume of ovine incisors decreases with age (ß-estimate = -3.3; p < 0.0001) and teeth position from the more central to the more lateral (ß-estimate = -4.9; p = 0.0009). Weight was not a relevant variable in the regression model. The dental pulp volume ranged from 36.7 to 19.6 mm3 in 3-year-old sheep, from 23.6 to 11.3 in 4-year-old sheep, and from 19.4 to 11.5 in 6-year-old sheep. The pulp volume of the most central teeth (first intermediate) was significantly higher than the most lateral teeth (corner). Haematoxylin-Eosin-Safran of the whole incisors, and of isolated dental pulps demonstrated a similar morphology to that in humans. The first intermediate incisor of 3-year-old sheep should be selected preferentially in preclinical research to retrieve the highest volume of dental pulp.

Biomimetic Periodontal Ligament Transplantation Activated by Gold Nanoparticles Protects Alveolar Bone.

Stem cell therapy might be a promising method to stimulate alveolar bone regeneration, which is currently a major clinical challenge. However, its therapeutic features largely depend on pretreatment and transplantation preparation. Herein, a novel biomimetic periodontal ligament transplantation composed of human periodontal ligament stem cells (hPDLSCs) pretreated with gold nanocomplexes (AuNCs) and embedded in a type-I collagen hydrogel scaffold is developed to protect alveolar bone from resorption. AuNCs are readily absorbed by primary hPDLSCs, with limited cytotoxicity, and promote osteogenic differentiation of hPDLSCs effectively in vitro. In addition, the AuNCs-induced hPDLSCs are encapsulated with type-I collagen hydrogel scaffold to mimic their native physiological niche, and then are transplanted into a rat model of alveolar bone resorption. Both micro-computed tomography (micro-CT) and immunohistochemical assays demonstrate that alveolar bone loss is significantly prevented. Furthermore, the underlying therapeutic mechanism is elucidated, in which transplantation-activated osteogenesis is associated with autophagy, which enables bone remodeling and regeneration. This study provides critical insight into the role of PDLSCs in bone homeostasis and proposes an innovative AuNCs-based strategy for stem cell therapy in bone regeneration.

Organoids from mouse molar and incisor as new tools to study tooth-specific biology and development.

Organoid models provide powerful tools to study tissue biology and development in a dish. Presently, organoids have not yet been developed from mouse tooth. Here, we established tooth organoids (TOs) from early-postnatal mouse molar and incisor, which are long-term expandable, express dental epithelium stem cell (DESC) markers, and recapitulate key properties of the dental epithelium in a tooth-type-specific manner. TOs display in vitro differentiation capacity toward ameloblast-resembling cells, even more pronounced in assembloids in which dental mesenchymal (pulp) stem cells are combined with the organoid DESCs. Single-cell transcriptomics supports this developmental potential and reveals co-differentiation into junctional epithelium- and odontoblast-/cementoblast-like cells in the assembloids. Finally, TOs survive and show ameloblast-resembling differentiation also in vivo. The developed organoid models provide new tools to study mouse tooth-type-specific biology and development and gain deeper molecular and functional insights that may eventually help to achieve future human biological tooth repair and replacement.

Nectins and Nectin-like molecules drive vascular development and barrier function.

Angiogenesis, barriergenesis, and immune cell migration are all key physiological events that are dependent on the functional characteristics of the vascular endothelium. The protein family of Nectins and Nectin-like molecules (Necls) is a group of cell adhesion molecules that are widely expressed by different endothelial cell types. The family includes four Nectins (Nectin-1 to -4) and five Necls (Necl-1 to -5) that either interact with each other by forming homo- and heterotypical interactions or bind to ligands expressed within the immune system. Nectin and Necl proteins are mainly described to play a role in cancer immunology and in the development of the nervous system. However, Nectins and Necls are underestimated players in the formation of blood vessels, their barrier properties, and in guiding transendothelial migration of leukocytes. This review summarizes their role in supporting the endothelial barrier through their function in angiogenesis, cell-cell junction formation, and immune cell migration. In addition, this review provides a detailed overview of the expression patterns of Nectins and Necls in the vascular endothelium.

Manufacture of extracellular vesicles derived from mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising therapy for various diseases ranging from ischemic stroke to wound healing and cancer. Their therapeutic effects are mainly mediated by secretome-derived paracrine factors, with extracellular vesicles (EVs) proven to play a key role. This has led to promising research on the potential of MSC-EVs as regenerative, off-the-shelf therapeutic agents. However, the translation of MSC-EVs into the clinic is hampered by the poor scalability of their production. Recently, new advanced methods have been developed to upscale MSC cultivation and EV production yields, ranging from new cell culture devices to priming procedures. This review gives an overview of these innovative strategies for manufacturing MSC-EVs.

The Dark Side of an Essential Amino Acid: L-Arginine in Spinal Cord Injury.

L-arginine is a semi-essential amino acid involved in a variety of physiological processes in the central nervous system (CNS). It is essential in the survival and functionality of neuronal cells. Nonetheless, L-arginine also has a dark side; it potentiates neuroinflammation and nitric oxide (NO) production, leading to secondary damage. Therefore, modulating the L-arginine metabolism is challenging because both detrimental and beneficial effects are dependent on this semi-essential amino acid. After spinal cord injury (SCI), L-arginine plays a crucial role in trauma-induced neuroinflammation and regenerative processes via the two key enzymes: nitric oxide synthase (NOS) and arginase (ARG). Studies on L-arginine metabolism using ARG and NOS inhibitors highlighted the conflicting role of this semi-essential amino acid. Similarly, L-arginine supplementation resulted in both negative and positive outcomes after SCI. However, new data indicate that arginine depletion substantially improves spinal cord regeneration after injury. Here, we review the challenging characteristics of L-arginine metabolism as a therapeutic target after SCI.

Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research.

Single-cell (sc) omics has become a powerful tool to unravel a tissue's cell landscape across health and disease. In recent years, sc transcriptomic interrogation has been applied to a variety of tooth tissues of both human and mouse, which has considerably advanced our fundamental understanding of tooth biology. Now, an overarching and integrated bird's-view of the human and mouse tooth sc transcriptomic landscape would be a powerful multi-faceted tool for dental research, enabling further decipherment of tooth biology and development through constantly progressing state-of-the-art bioinformatic methods as well as the exploration of novel hypothesis-driven research. To this aim, we re-assessed and integrated recently published scRNA-sequencing datasets of different dental tissue types (healthy and diseased) from human and mouse to establish inclusive tooth sc atlases, and applied the consolidated data map to explore its power. For mouse tooth, we identified novel candidate transcriptional regulators of the ameloblast lineage. Regarding human tooth, we provide support for a developmental connection, not advanced before, between specific epithelial compartments. Taken together, we established inclusive mouse and human tooth sc atlases as powerful tools to potentiate innovative research into tooth biology, development and disease. The maps are provided online in an accessible format for interactive exploration.

Stretchable printed device for the simultaneous sensing of temperature and strain validated in a mouse wound healing model.

Temperature and strain are two vital parameters that play a significant role in wound diagnosis and healing. As periodic temperature measurements with a custom thermometer or strain measurements with conventional metallic gauges became less feasible for the modern competent health monitoring, individual temperature and strain measurement modalities incorporated into wearables and patches were developed. The proposed research in the article shows the development of a single sensor solution which can simultaneously measure both the above mentioned parameters. This work integrates a thermoelectric principle based temperature measurement approach into wearables, ensuring flexibility and bendability properties without affecting its thermo-generated voltage. The modified thermoelectric material helped to achieve stretchability of the sensor, thanks to its superior mechano-transduction properties. Moreover, the stretch-induced resistance changes become an additional marker for strain measurements so that both the parameters can be measured with the same sensor. Due to the independent measurement parameters (open circuit voltage and sensor resistance), the sensing model is greatly attractive for measurements without cross-sensitivity. The highly resilient temperature and strain sensor show excellent linearity, repeatability and good sensitivity. Besides, due to the compatibility of the fabrication scheme to low-temperature processing of the flexible materials and to mass volume production, printed fabrication methodologies were adopted to realize the sensor. This promises low-cost production and a disposable nature (single use) of the sensor patch. For the first time, this innovative temperature-strain dual parameter sensor concept has been tested on mice wounds in vivo. The preliminary experiments on mice wounds offer prospects for developing smart, i.e. sensorized, wound dressings for clinical applications.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

Non-invasive brain stimulation as therapeutic approach for ischemic stroke: Insights into the (sub)cellular mechanisms.

Although spontaneous recovery can occur following ischemic stroke due to endogenous neuronal reorganization and neuroplastic events, the degree of functional improvement is highly variable, causing many patients to remain permanently impaired. In the last decades, non-invasive brain stimulation (NIBS) techniques have emerged as potential add-on interventions to the standard neurorehabilitation programs to improve post-stroke recovery. Due to their ability to modulate cortical excitability and to induce neuroreparative processes in the brain, multiple studies have assessed the safety, efficacy and (sub)cellular mechanisms of NIBS following ischemic stroke. In this review, an overview will be provided of the different NIBS techniques that are currently being investigated in (pre)clinical stroke studies. The NIBS therapies that will be discussed include transcranial magnetic stimulation, transcranial direct current stimulation and extremely low frequency electromagnetic stimulation. First, an overview will be given of the cellular mechanisms induced by NIBS that are associated with enhanced stroke outcome in preclinical models. Furthermore, the current knowledge on safety and efficacy of these NIBS techniques in stroke patients will be reviewed.

Organoids from human tooth showing epithelial stemness phenotype and differentiation potential.

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue's epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF's epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-ß (TGFß) and abrogated by TGFß receptor inhibition, thereby reproducing TGFß's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGFß receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives.