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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 B6 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.

Abstract 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.

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.

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.

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.

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.

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.

Abstract 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.

Abstract 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

Abstract 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

The use of Ti6Al4V in bone engineering is limited, due to the biological inertia of the surface. In this study, a porous Ti6Al4V scaffold with mechanical properties similar to cancellous bone was designed and 3D-printed. Under physiological conditions, the scaffold was immersed firstly in a 5-fold-concentrated simulated body fluid, then in a supersaturated CaP solution containing BMP-2, to form a bone-like porous micro/nano structured biomimetic coating on the surface. Scaffolds were implanted in the muscle pouches created in six beagle dogs and were retrieved four weeks later for histologic and histomorphometric analysis. Results showed that BMP-2 integrated biomimetic CaP coating induced ectopic bone formation, which was absent in other two groups. Soft tissue infiltrated the scaffold's outside 1 mm layer, while the new-formed bone was evenly distributed in the longitudinal and horizontal directions within the rest of the scaffold based on BA/TA, BIC and BA measurements. In conclusion, the BMP-2 incorporated biomimetic CaP coating creates a micro/nano surface structure on the Ti6Al4V scaffold, which helps to increase biocompatibility. The integrated BMP-2 is capable of inducing ectopic bone formation in vivo. The proposed combination may have the potential for bone reconstruction, but further studies are needed to explore its clinical applicability.

Abstract 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.

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.

Many critically ill patients infected with SARS-CoV-2 have been submitted to an intensive care unit (ICU). Patients with a SARS-CoV-2 infection that survive critical illness are confronted with months of physical impairments. To maximize recovery, it is important to understand the musculoskeletal involvement in critically ill patients infected with SARS-CoV-2. The aim of the present study was to assess the myocellular changes in SARS-CoV-2 patients that occur throughout the first week of ICU admission. In n = 22 critically ill patients infected with SARS-CoV-2, a biopsy sample from the vastus lateralis muscle was obtained at day 1–3 and day 5–8 following ICU admission. Fluorescence microscopy was used to assess type I and type II muscle fiber size and distribution, myonuclear content, and muscle tissue capillarization. Transmission electron microscopy was used to support quantitative data at an ultrastructural level. Changes in type I and type II muscle fiber size showed large inter-individual variation. The average change in type I fiber size was +309 ± 1834 µm2, ranging from −2129 µm2 (−31%) to +3375 µm2 (+73%). The average change in type II fiber size was −224 ± 1256 µm2, ranging from −1410 µm2 (−36%) to +2592 µm2 (+48%). Ultrastructural observations showed myofibrillar and hydropic degeneration, and fiber necrosis. This study shows that ICU patients admitted with SARS-CoV-2 suffer from substantial muscle fiber damage during ICU admission. These results are a call for action towards more specialized rehabilitation programs for patients admitted to the ICU with SARS-CoV-2 infection.

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.

Abstract Background Human hematopoietic progenitor cells (HPCs) are important for cell therapy in cancer and tissue regeneration. In vitro studies have shown a transient association of 40 nm polystyrene nanoparticles (PS NPs) with these cells, which is of interest for intelligent design and application of NPs in HPC-based regenerative protocols. In this study, we aimed to investigate the involvement of nanoparticles’ size and membrane-attached glycan molecules in the interaction of HPCs with PS NPs, and compared it with monocytes. Human cord blood-derived HPCs and THP-1 cells were exposed to fluorescently labelled, carboxylated PS NPs of 40, 100 and 200 nm. Time-dependent nanoparticle membrane association and/or uptake was observed by measuring fluorescence intensity of exposed cells at short time intervals using flow cytometry. By pretreating the cells with neuraminidase, we studied the possible effect of membrane-associated sialic acids in the interaction with NPs. Confocal microscopy was used to visualize the cell-specific character of the NP association. Results Confocal images revealed that the majority of PS NPs was initially observed to be retained at the outer membrane of HPCs, while the same NPs showed immediate internalization by THP-1 monocytic cells. After prolonged exposure up to 4 h, PS NPs were also observed to enter the HPCs’ intracellular compartment. Cell-specific time courses of NP association with HPCs and THP-1 cells remained persistent after cells were enzymatically treated with neuraminidase, but significantly increased levels of NP association could be observed, suggesting a role for membrane-associated sialic acids in this process. Conclusions We conclude that the terminal membrane-associated sialic acids contribute to the NP retention at the outer cell membrane of HPCs. This retention behavior is a unique characteristic of the HPCs and is independent of NP size.

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 (

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.

Background and PurposeUp 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 MethodsAssays 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.ResultsExposure 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.ConclusionPre-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.

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.

Summary: Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation. : In this article, García-León JA and colleagues demonstrate the generation of functional oligodendrocytes (OLs) from human pluripotent stem cells in a rapid and efficient manner by the single overexpression of SOX10. Generated OLs resemble primary OLs at the transcriptome level and can myelinate neurons both in vivo and in vitro. Neuron-OL co-cultures, adapted to high-throughput screening formats, responded to drugs affecting myelination. Keywords: oligodendrocyte, induced pluripotent stem cells (iPSCs), multiple sclerosis, amyotrophic lateral sclerosis, myelination, disease modeling, drug screening

Abstract The aim of this Letter to the Editor was to report some methodological shortcomings in the recently published article “Application of red light phototherapy in the treatment of radioactive dermatitis in patients with head and neck cancer” by Zhang et al. There are some issues regarding the incomplete photobiomodulation (PBM) parameters, the chosen outcome measures, and some missing reference articles. In conclusion, the results of this study should be interpreted with caution and further research is necessary.

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 (r2* = 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.

Stroke is the second most common cause of death and permanent disability. It is characterized by loss of neural tissue in which inflammation plays a crucial role in both the acute contribution to ischemic damage as in the late-stage impact on post-ischemic tissue regeneration. Microglia play a key role in the inflammatory stroke microenvironment as they can adapt a disease-promoting pro-inflammatory- or pro-regenerative phenotype thereby contributing to the exacerbation or alleviation of ischemic damage, respectively. Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor which in the central nervous system is mainly expressed on microglia. This receptor has been shown to play an essential role in microglial phagocytosis and function but its contribution in stroke pathobiology remains unclear. TREM2 was shown to be activated by nucleotides and lipid mediators, key factors that are secreted in the extracellular stroke environment by apoptotic neurons and cell/myelin debris. These factors in turn stimulate TREM2 signaling which mediates microglial migration toward- and phagocytosis of myelin debris and apoptotic cells. Moreover, microglial TREM2 appears to counteract the toll-like receptor response, thereby decreasing the production of pro-inflammatory cytokines. Finally, TREM2 is involved in microglial migration, survival, and is suggested to directly stimulate pro-regenerative phenotype shift. Therefore, this receptor is an attractive target for microglial modulation in the treatment of ischemic stroke and it provides additional information on microglial effector functions. This short review aims to elaborate on these TREM2-mediated microglial functions in the pathobiology and resolving of ischemic stroke.

Despite the high prevalence and devastating outcome, only a few treatment options for cerebral ischemic stroke exist. Based on the nitric oxide (NO)-stimulating capacity of Non-pulsed Sinusoidal Electromagnetic Field (NP-SEMF) and the possible neuroprotective role of NO in ischemic stroke, we hypothesized that NP-SEMF is able to enhance survival and neurological outcome in a rat model of cerebral ischemia. The animals, in which ischemic injury was induced by occlusion of both common carotid arteries, received 20 min of NP-SEMF of either 10 or 60 Hz daily for 4 days. NP-SEMF dramatically increased survival, reduced the size of the infarcted brain area and significantly improved the neurological score of the surviving rats. Corresponding to previous reports, NP-SEMF was able to induce NO production in vitro. The importance of NO as a key signaling molecule was highlighted by inhibition of the NP-SEMF beneficial effects in the rat stroke model after blocking NO synthase (NOS). Our results indicate for the first time that NP-SEMF exposure (13.5 mT at 60 and 10 Hz) improves the survival and neurological outcome of rats subjected to cerebral ischemia and that this effect is mediated by NO, underlining the great therapeutic potential of NP-SEMF as a therapy for ischemic stroke.

Pathologies of the central nervous system are characterized by loss of brain tissue and neuronal function which cannot be adequately restored by endogenous repair processes. This stresses the need for novel treatment options such as cell-based therapies that are able to restore damaged tissue or stimulate repair. This study investigated the neuroregenerative potential of the conditioned medium of human dental pulp stem cells (CM-hDPSCs) on neural stem cell (NSC) proliferation and migration as well as on neurite outgrowth of primary cortical neurons (pCNs). Additionally, the effect of leukocyte- and platelet-rich fibrin (L-PRF) priming on the neuroregenerative potential of the hDPSC secretome on NSCs and pCNs was evaluated. L-PRF contains factors that enhance stem cell-induced regeneration, but its effect on hDPSC-mediated neuroregeneration is unknown. This study demonstrated that CM-hDPSCs enhanced neuritogenesis. Moreover, CM-hDPSCs had a chemoattractant effect on NSCs. Although priming hDPSCs with L-PRF increased brain-derived neurotrophic factor secretion, no additional effects on the paracrine-mediated repair mechanisms were observed. These data support the neuroregenerative potential of hDPSCs, and although priming had no additional effect, the potential of L-PRF-primed hDPSCs on distinct regenerative mechanisms remains to be clarified.

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.

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.

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

Adequate vascularization, a restricting factor for the survival of engineered tissues, is often promoted by the addition of stem cells or the appropriate angiogenic growth factors. In this study, human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) were applied in an in vivo model of dental pulp regeneration in order to compare their regenerative potential and confirm their previously demonstrated paracrine angiogenic properties. 3D-printed hydroxyapatite scaffolds containing DPSCs and/or SCAPs were subcutaneously transplanted into immunocompromised mice. After twelve weeks, histological and ultrastructural analysis demonstrated the regeneration of vascularized pulp-like tissue as well as mineralized tissue formation in all stem cell constructs. Despite the secretion of vascular endothelial growth factor in vitro, the stem cell constructs did not display a higher vascularization rate in comparison to control conditions. Similar results were found after eight weeks, which suggests both osteogenic/odontogenic differentiation of the transplanted stem cells and the promotion of angiogenesis in this particular setting. In conclusion, this is the first study to demonstrate the successful formation of vascularized pulp-like tissue in 3D-printed scaffolds containing dental stem cells, emphasizing the promising role of this approach in dental tissue engineering.

Periodontal ligament stem cells (PDLSCs) represent a good source of multipotent cells for cell-based therapies in regenerative medicine. The success rate of these treatments is severely dependent on the establishment of adequate vasculature in order to provide oxygen and nutrients to the transplanted cells. Pharmacological preconditioning of stem cells has been proposed as a promising method to augment their therapeutic efficacy. In this study, the aim was to improve the intrinsic angiogenic properties of PDLSCs by in vitro pretreatment with deferoxamine (DFX; 100μM), fibroblast growth factor-2 (FGF-2; 10ng/mL) or both substances combined. An antibody array revealed the differential expression of several proteins, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). ELISA data confirmed a 1.5 to 1.8-fold increase in VEGF for all tested conditions. Moreover, 48 hours after the removal of DFX, VEGF levels remained elevated (1.8-fold) compared to control conditions. FGF-2 and combination treatment resulted in a 5.4 to 13.1-fold increase in PlGF secretion, whereas DFX treatment had no effect. Furthermore, both PDLSCs as pretreated PDLSCs induced endothelial migration. Despite the significant elevated VEGF levels of pretreated PDLSCs, the induced endothelial migration was not higher by pretreated PDLSCs. We find that the observed induced endothelial cell motility was not dependent on VEGF, since blocking the VEGFR1-3 with Axitinib (0.5nM) did not inhibit endothelial motility towards PDLSCs. Taken together, this study provides evidence that preconditioning with DFX and/or FGF-2 significantly improves the angiogenic secretome of PDLSCs, in particular VEGF and PlGF secretion. However, our data suggest that VEGF is not the only player when it comes to influencing endothelial behavior by the PDLSCs.

Within the field of tissue engineering, natural tissues are reconstructed by combining growth factors, stem cells, and different biomaterials to serve as a scaffold for novel tissue growth. As adequate vascularization and innervation are essential components for the viability of regenerated tissues, there is a high need for easily accessible stem cells that are capable of supporting these functions. Within the human tooth and its surrounding tissues, different stem cell populations can be distinguished, such as dental pulp stem cells, stem cells from human deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and periodontal ligament stem cells. Given their straightforward and relatively easy isolation from extracted third molars, dental stem cells (DSCs) have become an attractive source of mesenchymal-like stem cells. Over the past decade, there have been numerous studies supporting the angiogenic, neuroprotective, and neurotrophic effects of the DSC secretome. Together with their ability to differentiate into endothelial cells and neural cell types, this makes DSCs suitable candidates for dental tissue engineering and nerve injury repair.

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.

Pancreatic islets (PIs) transplantation is an alternative approach for the treatment of severe forms of type 1 diabetes (T1D). To monitor the success of transplantation, it is desirable to follow the location of engrafted PIs non-invasively. In vivo magnetic resonance imaging (MRI) of transplanted PIs is a feasible cell tracking method; however, this requires labeling with a suitable contrast agent prior to transplantation. We have tested the feasibility of cationic magnetoliposomes (MLs), compared to commercial contrast agents (Endorem and Resovist), by labeling insulinoma cells and freshly isolated rat PIs. It was possible to incorporate Magnetic Ressonance (MR)-detectable amounts of MLs in a shorter time (4 h) when compared to Endorem and Resovist. MLs did not show negative effects on the PIs’ viability and functional parameters in vitro. Labeled islets were transplanted in the renal sub-capsular region of healthy mice. Hypointense contrast in MR images due to the labeled PIs was detected in vivo upon transplantation, while MR detection of PIs labeled with Endorem and Resovist was only possible after the addition of transfection agents. These findings indicate that MLs are suitable to image PIs, without affecting their function, which is promising for future longitudinal pre-clinical and clinical studies involving the assessment of PI transplantation.

Demyelination is one of the pathological hallmarks of multiple sclerosis (MS). To date, no therapy is available which directly potentiates endogenous remyelination. Interleukin-11 (IL-11), a member of the gp130 family of cytokines, is upregulated in MS lesions. Systemic IL-11 treatment was shown to ameliorate clinical symptoms in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. IL-11 modulates immune cells and protects oligodendrocytes in vitro. In this study, the cuprizone-induced demyelination mouse model was used to elucidate effects of IL-11 on de- and remyelination, independent of the immune response. Prophylactic-lentiviral- (LV-) mediated overexpression of IL-11 in mouse brain significantly limited acute demyelination, which was accompanied with the preservation of CC1+ mature oligodendrocytes (OLs) and a decrease in microglial activation (Mac-2+). We further demonstrated that IL-11 directly reduces myelin phagocytosis in vitro. When IL-11 expressing LV was therapeutically applied in animals with extensive demyelination, a significant enhancement of remyelination was observed as demonstrated by Luxol Fast Blue staining and electron microscopy imaging. Our results indicate that IL-11 promotes maturation of NG2+ OPCs into myelinating CC1+ OLs and may thus explain the enhanced remyelination. Overall, we demonstrate that IL-11 is of therapeutic interest for MS and other demyelinating diseases by limiting demyelination and promoting remyelination.

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.

Abstract Introduction Auto-transplantation of third molars is frequently undertaken in order to restore a perfect occlusion and to improve mastication following a substantial loss of molars. However, little is known about the precise role of the periodontal membrane during this procedure. Therefore, we investigated if the epithelial rests of Malassez persist in the periodontal ligament of auto-transplanted teeth and, if so, whether these may show signs of a neuro-epithelial relationship. Case presentation We report a case of a 21-year-old Caucasian woman who underwent an auto-transplantation of two third molars. After two years, renewed progressive caries of the auto-transplanted teeth led to the removal of the auto-transplanted elements. The periodontal ligament was removed and studied with a light and transmission electron microscope. Conclusion In this report we examined the ultrastructure of the periodontal ligament after auto-transplantation in order to see if the periodontal ligament recovers completely from this intervention. We observed fully developed blood vessels and a re-innervation of the epithelial rests of Malassez which were proliferating following auto-transplantation. This proliferation might be critical in the remodelling of the alveolar socket in order to provide a perfect fit for the transplanted tooth. In order to minimalise the damage to the epithelial rests of Malassez, the extraction of the tooth should be as atraumatic as possible in order to provide an optimal conservation of the periodontal ligament which will be beneficial to the healing-process.