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Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Individuals with type 2 diabetes mellitus (T2DM) are at an increased risk for heart failure, yet preventive cardiac care is suboptimal in this population. Pyridoxamine (PM), a vitamin B 6 analog, has been shown to exert protective effects in metabolic and cardiovascular diseases. In this study, we aimed to investigate whether PM limits adverse cardiac remodeling and dysfunction in rats who develop T2DM. Male rats received a standard chow diet or Western diet (WD) for 18 weeks to induce prediabetes. One WD group received additional PM (1 g/L) via drinking water. Glucose tolerance was assessed with a 1 h oral glucose tolerance test. Cardiac function was evaluated using echocardiography and hemodynamic measurements. Histology on left ventricular (LV) tissue was performed. Treatment with PM prevented the increase in fasting plasma glucose levels compared to WD-fed rats ( p < 0.05). LV cardiac dilation tended to be prevented using PM supplementation. In LV tissue, PM limited an increase in interstitial collagen deposition ( p < 0.05) seen in WD-fed rats. PM tended to decrease 3-nitrotyrosine and significantly lowered 4-hydroxynonenal content compared to WD-fed rats. We conclude that PM alleviates interstitial fibrosis and oxidative stress in the hearts of WD-induced prediabetic rats.

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., (© 2024. The Author(s).)

Background: Dysregulation of the endo-lysosomal-autophagy pathway has been identified as a critical factor in the pathology of various demyelinating neurodegenerative diseases, including peripheral neuropathies. This pathway plays a crucial role in transporting newly synthesized myelin proteins to the plasma membrane in myelinating Schwann cells, making these cells susceptible to lysosome-related dysfunctions. Nevertheless, the specific impact of lysosomal dysfunction in Schwann cells and its contribution to neurodegeneration remain poorly understood., Methods: We aim to mimic lysosomal dysfunction in Schwann cells using chloroquine, a lysosomal dysfunction inducer, and to monitor lysosomal leakiness, Schwann cell viability, and apoptosis over time. Additionally, due to the ethical and experimental issues associated with cell isolation and the culturing of human Schwann cells, we use human dental pulp stem cell-derived Schwann cells (DPSC-SCs) as a model in our study., Results: Chloroquine incubation boosts lysosomal presence as demonstrated by an increased Lysotracker signal. Further in-depth lysosomal analysis demonstrated an increased lysosomal size and permeability as illustrated by a TEM analysis and GAL3-LAMP1 staining. Moreover, an Alamar blue assay and Caspase-3 staining demonstrates a reduced viability and increased apoptosis, respectively., Conclusions: Our data indicate that prolonged lysosomal dysfunction leads to lysosomal permeability, reduced viability, and eventually apoptosis in human DPSC-SCs.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling., (Creative Commons Attribution license.)

Endurance exercise training is a promising cardioprotective strategy in type 2 diabetes mellitus (T2DM), but the impact of its intensity is not clear. We aimed to investigate whether and how isocaloric moderate-intensity exercise training (MIT) and high-intensity interval exercise training (HIIT) could prevent the adverse cardiac remodeling and dysfunction that develop T2DM in rats. Male rats received a Western diet (WD) to induce T2DM and underwent a sedentary lifestyle ( n = 7), MIT ( n = 7) or HIIT ( n = 8). Insulin resistance was defined as the HOMA-IR value. Cardiac function was assessed with left ventricular (LV) echocardiography and invasive hemodynamics. A qPCR and histology of LV tissue unraveled underlying mechanisms. We found that MIT and HIIT halted T2DM development compared to in sedentary WD rats ( p < 0.05). Both interventions prevented increases in LV end-systolic pressure, wall thickness and interstitial collagen content ( p < 0.05). In LV tissue, HIIT tended to upregulate the gene expression of an ROS-generating enzyme (NOX4), while both modalities increased proinflammatory macrophage markers and cytokines (CD86, TNF-α, IL-1β; p < 0.05). HIIT promoted antioxidant and dicarbonyl defense systems (SOD2, glyoxalase 1; p < 0.05) whereas MIT elevated anti-inflammatory macrophage marker expression (CD206, CD163; p < 0.01). We conclude that both MIT and HIIT limit WD-induced T2DM with diastolic dysfunction and pathological LV hypertrophy, possibly using different adaptive mechanisms., Competing Interests: The authors declare no conflicts of interest.

The use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-β1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy.

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., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who have been admitted to the intensive care unit (ICU) often face months of physical disability after discharge. To optimize recovery, it is important to understand the role of musculoskeletal alterations in critically ill patients infected with SARS-CoV-2. The main aim of the present study was to describe the presence and morphology of nemaline bodies found in the skeletal muscle tissue from critically ill patients infected with SARS-CoV-2. In n = 7 patients infected with SARS-CoV-2, ultrastructural characteristics of vastus lateralis muscle obtained on days 1-3 and days 5-8 following ICU admission were investigated in more detail with electron microscopy. Those muscle biopsies consistently showed variable degrees of myofiber necrosis and myofibrillar disorganization. In 4/7 (57%) patients on days 5-8, the Z-line material accumulated into nemaline bodies with a typical lattice-like appearance at higher magnification, similar to that found in nemaline myopathy. This study is the first to describe the disintegration of myofibrils and the accumulation of Z-line material into nemaline bodies in the skeletal muscle tissue obtained from critically ill coronavirus disease-19 patients following ICU admission, which should be interpreted primarily as a non-specific pathological response of extreme myofibrillar disintegration associated with myofiber necrosis., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Type 1 Charcot-Marie-Tooth disease (CMT1) is the most common demyelinating peripheral neuropathy. Patients suffer from progressive muscle weakness and sensory problems. The underlying disease mechanisms of CMT1 are still unclear and no therapy is currently available, hence patients completely rely on supportive care. Balancing protein levels is a complex multistep process fundamental to maintain cells in their healthy state and a disrupted proteostasis is a hallmark of several neurodegenerative diseases. When protein misfolding occurs, protein quality control systems are activated such as chaperones, the lysosomal-autophagy system and proteasomal degradation to ensure proper degradation. However, in pathological circumstances, these mechanisms are overloaded and thereby become inefficient to clear the load of misfolded proteins. Recent evidence strongly indicates that a disbalance in proteostasis plays an important role in several forms of CMT1. In this review, we present an overview of the protein quality control systems, their role in CMT1, and potential treatment strategies to restore proteostasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

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.

Exercise training is an encouraging approach to treat cardiac dysfunction in type 2 diabetes (T2DM), but the impact of its intensity is not understood. We aim to investigate whether and, if so, how moderate-intensity training (MIT) and high-intensity interval training (HIIT) alleviate adverse cardiac remodeling and dysfunction in rats with T2DM. Male rats received standard chow (n = 10) or Western diet (WD) to induce T2DM. Hereafter, WD rats were subjected to a 12-week sedentary lifestyle (n = 8), running MIT (n = 7) or HIIT (n = 7). Insulin resistance and glucose tolerance were assessed during the oral glucose tolerance test. Plasma advanced glycation end-products (AGEs) were evaluated. Echocardiography and hemodynamic measurements evaluated cardiac function. Underlying cardiac mechanisms were investigated by histology, western blot and colorimetry. We found that MIT and HIIT lowered insulin resistance and blood glucose levels compared to sedentary WD rats. MIT decreased harmful plasma AGE levels. In the heart, MIT and HIIT lowered end-diastolic pressure, left ventricular wall thickness and interstitial collagen deposition. Cardiac citrate synthase activity, mitochondrial oxidative capacity marker, raised after both exercise training modalities. We conclude that MIT and HIIT are effective in alleviating diastolic dysfunction and pathological cardiac remodeling in T2DM, by lowering fibrosis and optimizing mitochondrial capacity.

A hallmark of multiple sclerosis (MS) is the formation of multiple focal demyelinating lesions within the central nervous system (CNS). These lesions mainly consist of phagocytes that play a key role in lesion progression and remyelination, and therefore represent a promising therapeutic target in MS. We recently showed that unsaturated fatty acids produced by stearoyl-CoA desaturase-1 induce inflammatory foam cell formation during demyelination. These fatty acids are elongated by the "elongation of very long chain fatty acids" proteins (ELOVLs), generating a series of functionally distinct lipids. Here, we show that the expression and activity of ELOVLs are altered in myelin-induced foam cells. Especially ELOVL6, an enzyme responsible for converting saturated and monounsaturated C16 fatty acids into C18 species, was found to be up-regulated in myelin phagocytosing phagocytes in vitro and in MS lesions. Depletion of Elovl6 induced a repair-promoting phagocyte phenotype through activation of the S1P/PPARγ pathway. Elovl6 -deficient foamy macrophages showed enhanced ABCA1-mediated lipid efflux, increased production of neurotrophic factors, and reduced expression of inflammatory mediators. Moreover, our data show that ELOVL6 hampers CNS repair, as Elovl6 deficiency prevented demyelination and boosted remyelination in organotypic brain slice cultures and the mouse cuprizone model. These findings indicate that targeting ELOVL6 activity may be an effective strategy to stimulate CNS repair in MS and other neurodegenerative diseases.

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 (β-estimate = -3.3; p < 0.0001) and teeth position from the more central to the more lateral (β-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 mm 3 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., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Introduction: Understanding the healing process of dental pulp after tooth autotransplantation (TAT) and regenerative endodontic treatment (RET) of immature teeth is important both clinically and scientifically. This study aimed to characterize the pattern of dental pulp healing in human teeth that underwent TAT and RET using state-of-the-art imaging techniques., Materials and Methods: This study examined 4 human teeth, 2 premolars that underwent TAT, and 2 central incisors that received RET. The premolars were extracted after 1 year (case 1) and 2 years (case 2) due to ankylosis, while the central incisors were extracted after 3 years (cases 3 and 4) for orthodontic reasons. Nanofocus x-ray computed tomography was used to image the samples before being processed for histological and immunohistochemical analysis. Laser scanning confocal second harmonic generation imaging (SHG) was used to examine the patterns of collagen deposition. A maturity-matched premolar was included as a negative control for the histological and SHG analysis., Results: Analysis of the 4 cases revealed different patterns of dental pulp healing. Similarities were observed in the progressive obliteration of the root canal space. However, a striking loss of typical pulpal architecture was observed in the TAT cases, while a pulp-like tissue was observed in one of the RET cases. Odontoblast-like cells were observed in cases 1 and 3., Conclusions: This study provided insights into the patterns of dental pulp healing after TAT and RET. The SHG imaging sheds light on the patterns of collagen deposition during reparative dentin formation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The progressive nature of demyelinating diseases lies in the inability of the central nervous system (CNS) to induce proper remyelination. Recently, we and others demonstrated that a dysregulated innate immune response partially underlies failure of CNS remyelination. Extensive accumulation of myelin-derived lipids and an inability to process these lipids was found to induce a disease-promoting phagocyte phenotype. Hence, restoring the ability of these phagocytes to metabolize and efflux myelin-derived lipids represents a promising strategy to promote remyelination. Here, we show that ApoA-I mimetic peptide 5A, a molecule well known to promote activity of the lipid efflux transporter ABCA1, markedly enhances remyelination. Mechanistically, we find that the repair-inducing properties of 5A are attributable to increased clearance and metabolism of remyelination-inhibiting myelin debris via the fatty acid translocase protein CD36, which is transcriptionally controlled by the ABCA1-JAK2-STAT3 signaling pathway. Altogether, our findings indicate that 5A promotes remyelination by stimulating clearance and degradation of myelin debris., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Foamy macrophages containing abundant intracellular myelin remnants are an important pathological hallmark of multiple sclerosis. Reducing the intracellular lipid burden in foamy macrophages is considered a promising therapeutic strategy to induce a phagocyte phenotype that promotes central nervous system repair. Recent research from our group showed that sustained intracellular accumulation of myelin-derived lipids skews these phagocytes toward a disease-promoting and more inflammatory phenotype. Our data now demonstrate that disturbed lipophagy, a selective form of autophagy that helps with the degradation of lipid droplets, contributes to the induction of this phenotype. Stimulating autophagy using the natural disaccharide trehalose reduced the lipid load and inflammatory phenotype of myelin-laden macrophages. Importantly, trehalose was able to boost remyelination in the ex vivo brain slice model and the in vivo cuprizone-induced demyelination model. In summary, our results provide a molecular rationale for impaired metabolism of myelin-derived lipids in macrophages, and identify lipophagy induction as a promising treatment strategy to promote remyelination. Abbreviations: Baf: bafilomycin a1; BMDM: bone marrow-derived macrophage; CD68: CD68 antigen; CNS: central nervous system; LD: lipid droplet; LIPE/HSL: lipase, hormone sensitive; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MGLL: monoglyceride lipase; MS: multiple sclerosis; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; ORO: oil red o; PNPLA2: patatin-like phospholipase domain containing 2; PLIN2: perilipin 2; TEM: transmission electron microscopy; TFEB: transcription factor EB; TOH: trehalose.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hermans, Bueds, Hemeryck, Lambrichts, Bronckaers and Vankelecom.)

Blood-based (BB) biomarkers for Aβ and tau can indicate pathological processes in the brain, in the early pathological, even pre-symptomatic stages in Alzheimer's disease. However, the relation between BB biomarkers and AD-related processes in the brain in the earliest pre-pathology stage before amyloid pathology develops, and their relation with total brain concentrations of Aβ and tau, is poorly understood. This stage presents a critical window for the earliest prevention of AD. Preclinical models with well-defined temporal progression to robust amyloid and tau pathology provide a unique opportunity to study this relation and were used here to study the link between BB biomarkers with AD-related processes in pre- and pathological stages. We performed a cross-sectional study at different ages assessing the link between BB concentrations and AD-related processes in the brain. This was complemented with a longitudinal analysis and with analysis of age-related changes in a small cohort of human subjects. We found that BB-tau concentrations increased in serum, correlating with progressive development of tau pathology and with increasing tau aggregates and p-tau concentrations in brain in TauP301S mice (PS19) developing tauopathy. BB-Aβ42 concentrations in serum decreased between 4.5 and 9 months of age, correlating with the progressive development of robust amyloid pathology in APP/PS1 (5xFAD) mice, in line with previous findings. Most importantly, BB-Aβ42 concentrations significantly increased between 1.5 and 4.5 months, i.e., in the earliest pre-pathological stage, before robust amyloid pathology develops in the brain, indicating biphasic BB-Aβ42 dynamics. Furthermore, increasing BB-Aβ42 in the pre-pathological phase, strongly correlated with increasing Aβ42 concentrations in brain. Our subsequent longitudinal analysis of BB-Aβ42 in 5xFAD mice, confirmed biphasic BB-Aβ42, with an initial increase, before decreasing with progressive robust pathology. Furthermore, in human samples, BB-Aβ42 concentrations were significantly higher in old (> 60 years) compared to young (< 50 years) subjects, as well as to age-matched AD patients, further supporting age-dependent increase of Aβ42 concentrations in the earliest pre-pathological phase, before amyloid pathology. Also BB-Aβ40 concentrations were found to increase in the earliest pre-pathological phase both in preclinical models and human subjects, while subsequent significantly decreasing concentrations in the pathological phase were characteristic for BB-Aβ42. Together our data indicate that BB biomarkers reflect pathological processes in brain of preclinical models with amyloid and tau pathology, both in the pathological and pre-pathological phase. Our data indicate a biphasic pattern of BB-Aβ42 in preclinical models and a human cohort. And most importantly, we here show that BB-Aβ increased and correlated with increasing concentrations of Aβ in the brain, in the earliest pre-pathological stage in a preclinical model. Our data thereby identify a novel critical window for prevention, using BB-Aβ as marker for accumulating Aβ in the brain, in the earliest pre-pathological stage, opening new avenues for personalized early preventive strategies against AD, even before amyloid pathology develops., (© 2022. The Author(s).)

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.

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-β (TGFβ) and abrogated by TGFβ receptor inhibition, thereby reproducing TGFβ'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 TGFβ 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., (© 2022. The Author(s).)

Teeth play essential roles in life. Their development relies on reciprocal interactions between the ectoderm-derived dental epithelium and the underlying neural crest-originated mesenchyme. This odontogenic process serves as a prototype model for the development of ectodermal appendages. In the mouse, developing teeth go through distinct morphological phases that are tightly controlled by epithelial signaling centers. Crucial molecular regulators of odontogenesis include the evolutionarily conserved Wnt, BMP, FGF and sonic hedgehog (Shh) pathways. These signaling modules do not act on their own, but are closely intertwined during tooth development, thereby outlining the path to be taken by specific cell populations including the resident dental stem cells. Recently, pivotal Wnt-Shh interaction and feedback loops have been uncovered during odontogenesis, showing conservation in other developing ectodermal appendages. This review provides an integrated overview of the interplay between canonical Wnt and Shh throughout mouse tooth formation stages, extending from the initiation of dental placode to the fully formed adult tooth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hermans, Hemeryck, Lambrichts, Bronckaers and Vankelecom.)

Background: Head and neck cancer (HNC) is one of the most common cancers, associated with a huge mortality and morbidity. In order to improve patient outcomes, more efficient and targeted therapies are essential. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) express tumour homing capacity, which could be exploited to target anti-cancer drug delivery to the tumour region and reduce adverse side-effects. Nevertheless, dental pulp stromal cells (DPSCs), an MSC-like population present in teeth, could offer important clinical benefits because of their easy isolation and superior proliferation compared to BM-MSCs. Therefore, we aimed to elucidate the tumour homing and safe usage of DPSCs to treat HNC., Methods: The in vivo survival as well as the effect of intratumourally administered DPSCs on tumour aggressiveness was tested in a HNC xenograft mouse model by using bioluminescence imaging (BLI), (immuno)histology and qRT-PCR. Furthermore, the in vitro and in vivo tumour homing capacity of DPSCs towards a HNC cell line were evaluated by a transwell migration assay and BLI, respectively., Results: Intratumourally injected DPSCs survived for at least two weeks in the tumour micro-environment and had no significant influence on tumour morphology, growth, angiogenesis and epithelial-to-mesenchymal transition. In addition, DPSCs migrated towards tumour cells in vitro, which could not be confirmed after their in vivo intravenous, intraperitoneal or peritumoural injection under the tested experimental conditions., Conclusions: Our research suggests that intratumourally delivered DPSCs might be used as safe factories for the continuous delivery of anti-cancer drugs in HNC. Nevertheless, further optimization as well as efficacy studies are necessary to understand and improve in vivo tumour homing and determine the optimal experimental set-up of stem cell-based cancer therapies, including dosing and timing., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of multimodal contrast agents can potentially overcome the intrinsic limitations of individual imaging methods. We have validated synthetic antiferromagnetic nanoparticles (SAF-NPs) as bimodal contrast agents for in vitro cell labeling and in vivo cell tracking using magnetic resonance imaging (MRI) and computed tomography (CT). SAF-NP-labeled cells showed high contrast in MRI phantom studies (r 2 * = 712 s -1 mM -1 ), while pelleted cells showed clear contrast enhancement in CT. After intravenous SAF-NP injection, nanoparticles accumulated in the liver and spleen, as visualized in vivo by significant MRI contrast enhancement. Intravenous injection of SAF-NP-labeled cells resulted in cell accumulation in the lungs, which was clearly detectable by using CT but not by using MRI. SAF-NPs proved to be very efficient cell labeling agents for complementary MRI- and CT-based cell tracking. Bimodal monitoring of SAF-NP labeled cells is in particular of interest for applications where the applied imaging methods are not able to visualize the particles and/or cells in all organs.

Objectives: This review aimed to present studies that prospectively investigated biological effects in patients following diagnostic dentomaxillofacial radiology (DMFR)., Methods: Literature was systematically searched to retrieve all studies assessing radiobiological effects of using X-ray imaging in the dentomaxillofacial area, with reference to radiobiological outcomes for other imaging modalities and fields., Results: There is a lot of variability in the reported radiobiological assessment methods and radiation dose measures, making comparisons of radiobiological studies challenging. Most radiological DMFR studies are focusing on genotoxicity and cytotoxicity, data for 2D dentomaxillofacial radiographs, albeit with some methodological weakness biasing the results. For CBCT, available evidence is limited and few studies include comparative data on both adults and children., Conclusions: In the future, one will have to strive towards patient-specific measures by considering age, gender and other individual radiation sensitivity-related factors. Ultimately, future radioprotection strategies should build further on the concept of personalized medicine, with patient-specific optimization of the imaging protocol, based on radiobiological variables.

The apical papilla is a stem cell rich tissue located at the base of the developing dental root and is responsible for the progressive elongation and maturation of the root. The multipotent stem cells of the apical papilla (SCAP) are extensively studied in cell culture since they demonstrate a high capacity for osteogenic, adipogenic, and chondrogenic differentiation and are thus an attractive stem cell source for stem cell-based therapies. Currently, only few studies are dedicated to determining the role of the apical papilla in dental root development. In this review, we will focus on the architecture of the apical papilla and describe the specific SCAP signaling pathways involved in root maturation. Furthermore, we will explore the heterogeneity of the SCAP phenotype within the tissue and determine their micro-environmental interaction. Understanding the mechanism of postnatal dental root growth could further aid in developing novel strategies in dental root regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Driesen, Gervois, Vangansewinkel and Lambrichts.)

Tailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.

Background: Bone-to-implant contact ratio (BIC%) plays a critical role in secondary stability of osseointegrated dental implants. The aim of this study was to identify the correlation of 2D/3D micro-CT images with histology as a gold standard for evaluating BIC% and to investigate the influence of the platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on 3D BIC% following delayed implant placement with delayed loading (DIP+DL)., Methods: Nine beagle dogs were recruited. Following bilateral extraction of mandibular 3rd premolar, 4th premolar, and 1st molar, 54 screw-type titanium implants were inserted and randomly divided into one control and two test groups based on a split-mouth design. The control group involved DIP+DL (n = 18) and both test groups included DIP+DL with local application of PRP (n = 18) and PPP (n = 18). A BIC analysis was performed utilizing 2D histomorphometry and 2D/3D micro-CT. Following identification of correlation between histology and 2D/3D micro-CT images, a 3D micro-CT assessment of the 3D BIC% at three follow-up time-points (1, 3, and 6 months) was carried out for observing the influence of PRP and PPP on BIC., Results: The 2D micro-CT BIC% values revealed a strong positive correlation with histology (r = 0.98, p < 0.001) and a moderate correlation existed with 3D micro-CT (r = 0. 67, p = 0.005). BIC levels at 1 month and combined influence of PPP and PRP irrespective of time-points revealed significantly higher 3D BIC% compared to the control. However, a reduction in 3D BIC% was observed at the 3rd and 6th month. No significant difference was observed between both PRP and PPP., Conclusions: Both 2D and 3D micro-CT demonstrated a potential to be utilized as a complimentary method for assessing BIC compared to the histological gold standard. Overall, both PRP and PPP significantly facilitated bone healing and osseointegration with a higher 3D BIC at follow-up. However, their influence was reduced as the observation period was increased.

Stem cells contained within the dental mesenchymal stromal cell (MSC) population are crucial for tissue homeostasis. Assuring their genomic stability is therefore essential. Exposure of stem cells to ionizing radiation (IR) is potentially detrimental for normal tissue homeostasis. Although it has been established that exposure to high doses of ionizing radiation (IR) has severe adverse effects on MSCs, knowledge about the impact of low doses of IR is lacking. Here we investigated the effect of low doses of X-irradiation with medical imaging beam settings (<0.1 Gray; 900 mGray per hour), in vitro , on pediatric dental mesenchymal stromal cells containing dental pulp stem cells from deciduous teeth, dental follicle progenitor cells and stem cells from the apical papilla. DNA double strand break (DSB) formation and repair kinetics were monitored by immunocytochemistry of γH2AX and 53BP1 as well as cell cycle progression by flow cytometry and cellular senescence by senescence-associated β-galactosidase assay and ELISA. Increased DNA DSB repair foci, after exposure to low doses of X-rays, were measured as early as 30 min post-irradiation. The number of DSBs returned to baseline levels 24 h after irradiation. Cell cycle analysis revealed marginal effects of IR on cell cycle progression, although a slight G 2 /M phase arrest was seen in dental pulp stromal cells from deciduous teeth 72 h after irradiation. Despite this cell cycle arrest, no radiation-induced senescence was observed. In conclusion, low X-ray IR doses (< 0.1 Gray; 900 mGray per hour), were able to induce significant increases in the number of DNA DSBs repair foci, but cell cycle progression seems to be minimally affected. This highlights the need for more detailed and extensive studies on the effects of exposure to low IR doses on different mesenchymal stromal cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Belmans, Gilles, Welkenhuysen, Vermeesen, Baselet, Salmon, Baatout, Jacobs, Lucas, Lambrichts and Moreels.)

Background: Early detection of marginal bone loss is vital for treatment planning and prognosis of teeth and implant. This study was conducted to assess diagnostic accuracy of CBCT compared to intra-oral (IO) radiography for detection, classification, and measurement of peri-implant bone defects in an animal model., Methods: Fifty-four mandible blocks with implants were harvested from nine male health adult beagle dogs with acquisition of IO, CBCT and micro-CT images from all samples. Peri-implant bone defects from 16 samples were diagnosed using micro-CT and classified into 3 defect categories: dehiscence (n = 5), infrabony defect (n = 3) and crater-like defect (n = 8). Following training and calibration of the observers, they asked to detect location (mesial, distal, buccal, lingual) and shape of the defect (dehiscence, horizontal defect, vertical defect, carter-like defect) utilizing both IO and CBCT images. Both observers assessed defect depth and width on IO, CBCT and micro-CT images at each side of peri-implant bone defect via CT-analyzer software. Data were analyzed using SPSS software and a p value of < 0.05 was considered as statistically significant., Results: Overall, there was a high diagnostic accuracy for detection of bone defects with CBCT images (sensitivity: 100%/100%), while IO images showed a reduction in accuracy (sensitivity: 69%/63%). Similarly, diagnostic accuracy for defect classification was significantly higher for CBCT, whereas IO images were unable to correctly identify vestibular dehiscence, with incorrect assessment of half of the infrabony defects. For accuracy of measuring defect depth and width, a higher correlation was observed between CBCT and gold standard micro-CT (r = 0.91, 95% CI 0.86-0.94), whereas a lower correlation was seen for IO images (r = 0.82, 95% CI 0.67-0.91)., Conclusions: The diagnostic accuracy and reliability of CBCT was found to be superior to IO imaging for the detection, classification, and measurement of peri-implant bone defects. The application of CBCT adds substantial information related to the peri-implant bone defect diagnosis and decision-making which cannot be achieved with conventional IO imaging.

Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day., Competing Interests: The authors have declared that no competing interests exist.

Exiting developments in tissue engineering and new insights in stem cell biology have led to new possible strategies for the regeneration of damaged tissues in the oral cavity. The regeneration of the pulp-dentin complex regeneration in particular, has drawn the attention of many researchers because of the high clinical needs. While it is still important to perform in vitro research using a wide variety of cells, scaffolds and growth factors, it is also critical to have a reliable animal model for preclinical trials. In this chapter, we describe a mouse model in which a scaffold resembling a tooth containing dental pulp cells is implanted subcutaneously. We also describe which histological stainings could be used to examine blood vessel formation and the regeneration of the pulp-dentin complex.

Tissue engineering aims to structurally and functionally regenerate damaged tissues, which requires the formation of new blood vessels that supply oxygen and nutrients by the process of angiogenesis. Stem cells are a promising tool in regenerative medicine due to their combined differentiation and paracrine angiogenic capacities. The study of their proangiogenic properties and associated potential for tissue regeneration requires complex in vivo models comprising all steps of the angiogenic process. The highly vascularized extraembryonic chorioallantoic membrane (CAM) of fertilized chicken eggs offers a simple, easy accessible, and cheap angiogenic screening tool compared to other animal models. Although the CAM assay was initially primarily performed for evaluation of tumor growth and metastasis, stem cell studies using this model are increasing. In this review, a detailed summary of angiogenic observations of different mesenchymal, cardiac, and endothelial stem cell types and derivatives in the CAM model is presented. Moreover, we focus on the variation in experimental setup, including the benefits and limitations of in ovo and ex ovo protocols, diverse biological and synthetic scaffolds, imaging techniques, and outcome measures of neovascularization. Finally, advantages and disadvantages of the CAM assay as a model for angiogenesis in tissue engineering in comparison with alternative in vivo animal models are described. Impact statement The chorioallantoic membrane (CAM) assay is an easy and cheap screening tool for the angiogenic properties of stem cells and their associated potential in the tissue engineering field. This review offers an overview of all published angiogenic studies of stem cells using this model, with emphasis on the variation in used experimental timeline, culture protocol ( in ovo vs. ex ovo ), stem cell type (derivatives), scaffolds, and outcome measures of vascularization. The purpose of this overview is to aid tissue engineering researchers to determine the ideal CAM experimental setup based on their specific study goals.

In this study, the effects of moderate intense endurance exercise on heart and kidney function and morphology were studied in a thoracic inferior vena cava constricted (IVCc) rat model of abdominal venous congestion. After IVC surgical constriction, eight sedentary male Sprague-Dawley IVCc rats (IVCc-SED) were compared to eight IVCc rats subjected to moderate intense endurance exercise (IVCc-MOD). Heart and kidney function was examined and renal functional reserve (RFR) was investigated by administering a high protein diet (HPD). After 12 weeks of exercise training, abdominal venous pressure, indices of body fat content, plasma cystatin C levels, and post-HPD urinary KIM-1 levels were all significantly lower in IVCc-MOD versus IVCc-SED rats (P < 0.05). RFR did not differ between both groups. The implementation of moderate intense endurance exercise in the IVCc model reduces abdominal venous pressure and is beneficial to kidney function.

Background/aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases., Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL ) and steady-state inactivation of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy., Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in left ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca 2+ current density was significantly decreased in the HMW-AGEs group.
L-type Ca 2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca 2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function., Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Background and Objective: While osseointegration following various dental implant placement protocols has been extensively investigated, the neurohistological integration has received little attention. The primary aim of this study was to compare the myelinated nerve fibers density in peri-implant bone tissue following various implant placement protocols. The secondary aim assessed the effect of follow-up on peri-implant nerve fibers density., Methods: Ten beagle dogs randomly received 68 commercially pure titanium implants in the mandibular premolar or molar region bilaterally following extraction utilizing one of the six treatment protocols: (a) immediate implant placement (IIP) and immediate loading (IL); (b) IIP and delayed loading (DL); (c) IIP and left unloaded (UL); (d) delayed implant placement (DIP) and IL; (e) DIP and DL; and (f) DIP and UL. Histomorphometric analysis of the peri-implant myelinated nerve fibers was performed in a 300 μm peri-implant zone at the cervical, middle, and apical level following implant placement. The follow-up assessment involved longitudinal observation at 3 months following each implant treatment protocol and at 6 months for IIP+IL and IIP+DL protocols., Results: The influence of different treatment protocols, including the fixed effects of implant groups (IIP+IL, IIP+DL, IIP+UL, DIP+IL, DIP+DL, DIP+UL) and regions (cervical, middle, apical), was examined via a linear mixed model. The IIP+IL group showed a significantly higher myelinated nerve density compared to the IIP+UL and DIP+UL group. Peri-implant nerve re-innervation was significantly higher (P = .002) in the apical region compared to the cervical region. After immediate implant placement, the IL group showed a significantly (P = .03) higher density of myelinated nerve fibers compared to DL. No significant (P = .19) effect of follow-up on nerve density was observed., Conclusion: The immediate implant placement and loading protocol showed most beneficial effect on peri-implant innervation with highest myelinated nerve density in the apical region. A longer loading time had no influence on the peri-implant nerve density., (© 2020 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd.)

Growing evidence supports the role of advanced glycation end products (AGEs) in the development of diabetic vascular complications and cardiovascular diseases (CVDs). We have shown that high-molecular-weight AGEs (HMW-AGEs), present in our Western diet, impair cardiac function. Whether HMW-AGEs affect vascular function remains unknown. In this study, we aimed to investigate the impact of chronic HMW-AGEs exposure on vascular function and structure. Adult male Sprague Dawley rats were daily injected with HMW-AGEs or control solution for 6 weeks. HMW-AGEs animals showed intracardiac pressure overload, characterized by increased systolic and mean pressures. The contraction response to PE was increased in aortic rings from the HMW-AGEs group. Relaxation in response to ACh, but not SNP, was impaired by HMW-AGEs. This was associated with reduced plasma cyclic GMP levels. SOD restored ACh-induced relaxation of HMW-AGEs animals to control levels, accompanied by a reduced half-maximal effective dose (EC 50 ). Finally, collagen deposition and intima-media thickness of the aortic vessel wall were increased with HMW-AGEs. Our data demonstrate that chronic HMW-AGEs exposure causes adverse vascular remodelling. This is characterised by disturbed vasomotor function due to increased oxidative stress and structural changes in the aorta, suggesting an important contribution of HMW-AGEs in the development of CVDs.

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., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Bogie et al.)

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13 , TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.

Background and Purpose: Up to 50-60% of all cancer patients receive radiotherapy as part of their treatment strategy. However, the mechanisms accounting for increased vascular risks after irradiation are not completely understood. Mitochondrial dysfunction has been identified as a potential cause of radiation-induced atherosclerosis., Materials and Methods: Assays for apoptosis, cellular metabolism, mitochondrial DNA content, functionality and morphology were used to compare the response of endothelial cells to a single 2 Gy dose of X-rays under basal conditions or after pharmacological treatments that either reduced (EtBr) or increased (rosiglitazone) mitochondrial content., Results: Exposure to ionizing radiation caused a persistent reduction in mitochondrial content of endothelial cells. Pharmacological reduction of mitochondrial DNA content rendered endothelial cells more vulnerable to radiation-induced apoptosis, whereas rosiglitazone treatment increased oxidative metabolism and redox state and decreased the levels of apoptosis after irradiation., Conclusion: Pre-existing mitochondrial damage sensitizes endothelial cells to ionizing radiation-induced mitochondrial dysfunction. Rosiglitazone protects endothelial cells from the detrimental effects of radiation exposure on mitochondrial metabolism and oxidative stress. Thus, our findings indicate that rosiglitazone may have potential value as prophylactic for radiation-induced atherosclerosis., (Copyright © 2020 Baselet, Driesen, Coninx, Belmans, Sieprath, Lambrichts, De Vos, Baatout, Sonveaux and Aerts.)

The brain's endogenous capacity to restore damaged myelin deteriorates during the course of demyelinating disorders. Currently, no treatment options are available to establish remyelination. Chronic demyelination leads to damaged axons and irreversible destruction of the central nervous system (CNS). We identified two promising therapeutic candidates which enhance remyelination: oncostatin M (OSM), a member of the interleukin-6 family, and downstream mediator tissue inhibitor of metalloproteinases-1 (TIMP-1). While remyelination was completely abrogated in OSMRβ knockout (KO) mice, OSM overexpression in the chronically demyelinated CNS established remyelination. Astrocytic TIMP-1 was demonstrated to play a pivotal role in OSM-mediated remyelination. Astrocyte-derived TIMP-1 drove differentiation of oligodendrocyte precursor cells into mature oligodendrocytes in vitro. In vivo, TIMP-1 deficiency completely abolished spontaneous remyelination, phenocopying OSMRβ KO mice. Finally, TIMP-1 was expressed by human astrocytes in demyelinated multiple sclerosis lesions, confirming the human value of our findings. Taken together, OSM and its downstream mediator TIMP-1 have the therapeutic potential to boost remyelination in demyelinating disorders., Competing Interests: The authors declare no competing interest.

Assessing the possible biological effects of exposure to low doses of ionizing radiation (IR) is one of the prime challenges in radiation protection, especially in medical imaging. Today, radiobiological data on cone beam CT (CBCT) related biological effects are scarce. In children and adults, the induction of DNA double strand breaks (DSBs) in buccal mucosa cells and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and antioxidant capacity in saliva samples after CBCT examination were examined. No DNA DSBs induction was observed in children nor adults. In children only, an increase in 8-oxo-dG levels was observed 30 minutes after CBCT. At the same time an increase in antioxidant capacity was observed in children, whereas a decrease was observed in adults. Our data indicate that children and adults react differently to IR doses associated with CBCT. Fully understanding these differences could lead to an optimal use of CBCT in different age categories as well as improved radiation protection guidelines.

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis., Competing Interests: The authors declare no conflict of interest.

Fibroblast activation protein-α (FAPα) is a membrane protein with dipeptidyl-peptidase and type I collagenase activity and is expressed during fetal growth. At the age of adolescence, FAPα expression is greatly reduced, only emerging in pathologies associated with extracellular matrix remodeling. We determined whether FAPα is expressed in human dental tissue involved in root maturation i.e., dental follicle and apical papilla and in dental pulp tissue. The dental follicle revealed a high concentration of FAPα and vimentin-positive cells within the stromal tissue. A similar observation was made in cell culture and FACS analysis confirmed these as dental follicle stem cells. Within the remnants of the Hertwigs' epithelial root sheath, we observed FAPα staining in the E-cadherin positive and vimentin-negative epithelial islands. FAPα- and vimentin-positive cells were encountered at the periphery of the islands suggesting an epithelial mesenchymal transition process. Analysis of the apical papilla revealed two novel histological regions; the periphery with dense and parallel aligned collagen type I defined as cortex fibrosa and the inner stromal tissue composed of less compacted collagen defined as medulla. FAPα expression was highly present within the medulla suggesting a role in extracellular matrix remodeling. Dental pulp tissue uncovered a heterogeneous FAPα staining but strong staining was noted within odontoblasts. In vitro studies confirmed the presence of FAPα expression in stem cells of the apical papilla and dental pulp. This study identified the expression of FAPα expression in dental stem cells which could open new perspectives in understanding dental root maturation and odontoblast function., (Copyright © 2020 Driesen, Hilkens, Smisdom, Vangansewinkel, Dillen, Ratajczak, Wolfs, Gervois, Ameloot, Bronckaers and Lambrichts.)

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.

Background: Autologous plasma fractions, such as platelet-rich plasma (PRP) and platelet-poor plasma (PPP), contain growth factors that can enhance neural cell survival and are therefore likely to have the ability to promote nerve regeneration. The present study compared the effect of PRP and PPP application on myelinated nerve density and diameter in the peri-implant bone region. In addition, the effect of healing time on nerve regeneration was assessed., Materials and Methods: Nine beagle dogs randomly received 54 dental implants in the bilateral mandible according to a split-mouth design. Each implant was randomly assigned to one of three implant protocols: delayed implant placement with delayed loading (DIP + DL) with local application of PRP, DIP + DL with local application of PPP and DIP + DL without any plasma additive. The animals were euthanized at 1, 3, and 6 months after loading (3 dogs per time point). Block biopsies were prepared for histomorphometry in the peri-implant bone within 500 μm around the implants., Results: Myelinated nerve fibers were identified in the trabecular bone and in the osteons near the implants surface. The nerve fibers in the PRP group (median ± IQR; 2.88 ± 1.55 μm) had a significantly (p < 0.05) greater diameter compared to the PPP (2.40 ± 0.91 μm) and control (2.11 ± 1.16 μm) group. The nerve diameter after 6 months healing (3.18 ± 1.58 μm) was significantly (p < 0.05) greater compared to 1 (2.08 ± 0.89 μm) and 3 (2.49 ± 1.22 μm) months. No significant difference was found for myelinated nerve density between groups and healing time., Conclusions: The present study showed that the healing time significantly influenced the diameter of the myelinated nerve fibers in peri-implant bone. PRP exerted a significant effect on the diameter of the myelinated nerve fibers as compared to PPP. Large-scale animal studies and longer follow-up periods are needed to confirm these findings and to verify whether platelet plasma can facilitate nerve regeneration process.

Objectives: To evaluate the peri-implant trabecular bone volume and architecture changes with 6-month follow-up after local application of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) using high-resolution micro-CT., Material and Methods: Seventy-two dental implants were placed into healed mandibular sites of 9 beagle dogs. Implants were randomly divided into 4 groups following a split-mouth design: control I; control II; PPP; and PRP. Primary and secondary stabilities were assessed using resonance frequency analyses. At 1, 3, and 6 months after implant loading, trabecular structural parameters were evaluated at 0.5, 1, and 1.5 mm away from implants using micro-CT (voxel = 20 μm)., Results: Primary and secondary stabilities were equivalent in all conditions. PPP and PRP groups showed higher bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) but lower trabecular separation (Tb.Sp) and total porosity percentage (Po (tot)) at all 3 time points. A significant decrease in BV/TV and Tb.Th was found for the control groups after 3 months of healing, while this was not observed in both the PPP and PRP groups. However, no distinct difference was found between the PRP and PPP groups over time. Moreover, as the investigated distance from the implant surface increased, BV/TV and Po (tot) within the same group and time point stayed the same, yet Tb.Th and Tb.Sp continued to increase., Conclusions: Platelet-rich plasma and PPP with conventional implant placement lead to similar primary and secondary implant stability, but improved peri-implant bone volume and structural integration. The present research does not seem to suggest a different bone remodeling pattern when using PRP or PPP., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)