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

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

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

The authors wish to make the following change to their paper [...].

Early childhood caries prevention: non-dental health professionals' viewpoint.

BACKGROUND: Dental caries can develop early in life and have harmful consequences. Objective: To examine non-dental practitioners' knowledge of early childhood caries (ECC). METHODS: A questionnaire on oral health and caries knowledge was emailed to five types of health professional who work with young children: paediatricians, GPs, midwives, paediatric nurses and paediatric healthcare assistants. Questions concerned: when a child should first visit a dentist; at what age toothbrushing should start; aetiopathogenic factors; early diagnosis; and the effect of breastfeeding. RESULTS: 494 health professionals (79 paediatricians, 59 physicians, 217 midwives, 92 paediatric nurses and 47 paediatric healthcare assistants) participated. Although most (89.86%) discussed oral health with parents, responses on when a child should first see a dentist and when toothbrushing should start varied. Almost half of respondents said they could diagnose caries but not all were confident in this. Aetiological factors in ECC mentioned included oral hygiene, bottle feeding, sugar intake, genetics and a lack of fluoride. CONCLUSION: Non-dental practitioners lack knowledge about ECC, so cannot help prevent it. Initiatives including interprofessional training would improve their knowledge of oral health in early childhood.

In vitro comparison of three percutaneous atrial septal defect closure devices for endothelialisation and haemocompatibility.

BACKGROUND: Percutaneous device closure of atrial septal defect (ASD) is the gold-standard treatment, but several delayed complications may occur as a result of incomplete device endothelialisation. AIMS: In this in vitro study, we compared three ASD closure devices [Nit-Occlud® ASD-R (device 1); Hyperion™ ASDO (device 2); and Amplatzer™ Septal Occluder (device 3)] in terms of the endothelialisation process, using human endothelial progenitors cells (EPCs), and haemocompatibility. METHODS: EPCs from umbilical cord blood were extracted, cultured and characterised. Device samples were seeded with 100,000 cells/cm2. EPC adhesion was investigated at 3 and 24hours, and EPC proliferation was monitored, which allowed longitudinal follow-up (days 1-12). Haemocompatibility of device samples was assessed using a complement C3a assay and platelet and coagulation activation. RESULTS: With regard to EPC adhesion and proliferation, no statistically significant differences were found between the three devices. We observed for each device a significant time-dependent EPC proliferation, appearing at day 8 for devices 2 and 3 and day 10 for device 1. No complement or platelet activation occurred within 15minutes of contact with devices. However, there was minimal activation of coagulation for the three devices. CONCLUSIONS: In this in vitro study we showed that, despite the three ASD occluders having different device designs and coatings, adhesion and proliferation of human endothelial cells was similar for all devices. This should be further confirmed by similar studies including shear stress forces and anti-thrombotic treatments.

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

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

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

A Unique Triculture Model to Study Osteoblasts, Osteoclasts, and Endothelial Cells.

IMPACT STATEMENT: In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration.

Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-assisted bioprinting (LAB) was used to pattern Red Fluorescent Protein-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor. LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the 'random seeding' condition and both 'disc' pattern (vr = +203.6%; br = +294.1%) and 'crossed circle' pattern (vr = +355%; br = +602.1%). These results indicate that in vivo LAB is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.

Micropatterning of endothelial cells to create a capillary-like network with defined architecture by laser-assisted bioprinting.

Development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. Seeding of endothelial cells in an appropriate environment can give rise to a capillary-like network to enhance prevascularization of bone substitutes. Advances in biofabrication techniques, such as bioprinting, could allow to precisely define a pattern of endothelial cells onto a biomaterial suitable for in vivo applications. The aim of this study was to produce a microvascular network following a defined pattern and preserve it while preparing the surface to print another layer of endothelial cells. We first optimise the bioink cell concentration and laser printing parameters and then develop a method to allow endothelial cells to survive between two collagen layers. Laser-assisted bioprinting (LAB) was used to pattern lines of tdTomato-labeled endothelial cells cocultured with mesenchymal stem cells seeded onto a collagen hydrogel. Formation of capillary-like structures was dependent on a sufficient local density of endothelial cells. Overlay of the pattern with collagen I hydrogel containing vascular endothelial growth factor (VEGF) allowed capillary-like structures formation and preservation of the printed pattern over time. Results indicate that laser-assisted bioprinting is a valuable technique to pre-organize endothelial cells into high cell density pattern in order to create a vascular network with defined architecture in tissue-engineered constructs based on collagen hydrogel.

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

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

Diagnosis and Management of Natal and Neonatal Teeth: Case Report of Three Newborns.

Natal and neonatal teeth are rare features that can lead to various issues, from traumatic injuries and feeding difficulties to more severe problems, such as risk of aspiration due to excessive mobility of teeth. The purpose of this paper is to discuss the diagnosis and management of natal and neonatal teeth, and describe three cases of newborns with natal teeth. Decision processes and management are detailed regarding the specificity of each case. Periodic follow-up is recommended to maintain oral health and prevent any issues related to premature loss or eruption of primary teeth. A close collaboration between pediatricians and dentists should be considered in order to allow early diagnosis and efficient treatment.

Case Report of a Severe Recurrent Tongue Self-Injury in an Infant With Dystonia.

Dystonia is characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements, postures, or both that are typically patterned, twisting, and sometimes tremulous. It is often initiated or worsened by voluntary action and associated with overflow muscle activation. In this article we report a case of severe oromandibular dystonia, which is a specific form of dystonia characterized by involuntary, action-induced tonic or clonic spasms of the masticatory, lingual, and pharyngeal musculature. Episodes of repeated tongue biting in a 17-month-old girl caused her to stay in the PICU for 4 weeks. These episodes were the consequence of dystonia induced by a perinatal stroke. We highlight the specific dental management that enabled us to treat the child without extractions. Facing this type of complex illness, we insist on the importance of interdisciplinary work with the goal of avoiding outdated techniques. The use of botulinum toxin seemed relevant.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration.

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

Labeling and qualification of endothelial progenitor cells for tracking in tissue engineering: An in vitro study.

PURPOSE: In order to track location and distribution of endothelial cells (ECs) within scaffolds in vitro, we chose lentiPGK-TdTomato transduction of human endothelial progenitor cells (EPCs) isolated and differentiated from cord blood. Because transduction could have a functional impact on cell behavior, we checked different parameters for qualification of labeled- EPCs as well as their use for potential applications in the context of vascular and bone tissue engineering. METHODS: After isolation and expansion, EPCs were classically characterized then transduced with the lentiviral vector containing the TdTomato protein gene under the control of the phosphoglycerate kinase (PGK) promoter. Conventional karyotyping, differentiation capacity, viability, proliferation assays were performed with labeled and unlabeled EPCs. Scaffolds and co-cultures were explored with labeled EPCs, in static or shear stress conditions. RESULTS: Our results show that cell labeling did not affect cell adhesion nor induce cell death. Cell labeling did not induce more chromosomal aberrations. Phenotypical characterization was not affected. In the context of tissue engineering applications, labeled EPCs maintained their ability to line 2D or 3D scaffolds, withstand physiological arterial shear stress, and form tubular networks in co-cultures with human osteoblast progenitor cells. CONCLUSIONS: It is possible to label human EPCs with TdTomato without affecting their behavior by the transduction procedure. This creates an important tool for numerous applications. Our results provide a qualification of labeled EPCs in comparison with unlabeled ones for vascular and bone tissue engineering.

IQ domain GTPase-activating protein 1 is involved in shear stress-induced progenitor-derived endothelial cell alignment.

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m(2)) and AJ proteins (VE-cadherin and ß-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and ß-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with ß-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to ß-catenin. This "switch" was concomitant with the loss of ß-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.

Human progenitor-derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study.

The isolation of endothelial progenitor cells from human peripheral blood generates a great hope in vascular tissue engineering because of particular benefit when compared with mature endothelial cells. We explored the capability of progenitor-derived endothelial cells (PDECs) to line fibrin and collagen scaffolds in comparison with human saphenous and umbilical cord vein endothelial cells (HSVECs and HUVECs): (a) in a static situation, allowing definition of the optimal cell culture conditions with different media and cell-seeding densities to check cell behaviour; (b) under shear stress conditions (flow chambers or tubular vascular constructs), allowing investigation of cell response and mRNA expression on both substrates by oligonucleotide microarray analysis and quantitative real-time PCR. Well characterized PDECs: (a) could not be expanded adequately with the usual mature ECs culture media; (b) were able to colonize and grow on fibrin glue; (c) exhibited higher resistance to oxidative stress than HSVECs and HUVECs; (d) withstood physiological shear stress when lining both substrates in flow chambers, and their gene expression was regulated; (e) colonized a collagen-impregnated vascular prosthesis and were able to sense mechanical forces. Our results provide an improved qualification of PDECs for vascular tissue engineering.

Effect of xylitol on an in vitro model of oral biofilm.

PURPOSE: The aim of the present study was to examine whether xylitol, at different concentrations, inhibits the formation of an experimental model of oral biofilm. MATERIALS AND METHODS: Biofilms of six bacterial species (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, Actinomyces viscosus, Porphyromonas gingivalis and Fusobacterium nucleatum) were prepared on hydroxyapatite (HA) discs according to the Zürich Biofilm Model. Xylitol was tested at two concentrations, 1% and 3%. At the end of their designated incubation times, some HA discs were destined for confocal laser scanning microscopy (CLSM) and the others were harvested using a sterile surgical instrument. Aliquots of harvested biofilms were diluted and plated onto specific media. After a 48-h anaerobic incubation at 37 degrees C, the colony-forming units (CFUs) were counted. RESULTS: CLSM images showed that only a small amount of isolated bacteria was observed on the surface of HA discs. Culture of harvested biofilms showed an inhibition in the growth of different species included in the biofilms. CONCLUSIONS: Xylitol has a clear inhibitory effect on the formation of the experimental biofilms. This study shows that xylitol is not only efficient in inhibiting the acid production of cariogenic bacteria, but also in preventing the formation of a multispecies biofilm; it confirms the relevance of the use of this polyol for the prevention of oral diseases caused by dental plaque.

Human endothelial progenitor cell attachment to polysaccharide-based hydrogels: a pre-requisite for vascular tissue engineering.

A hydrogel was prepared from polysaccharides (pullulan/dextran/fucoidan) and evaluated as a novel biomaterial for Endothelial Progenitor Cell (EPC) culture. Using a cross-linking process with sodium trimetaphosphate in aqueous solution, homogeneous, transparent and easy to handle gels were obtained with a water content higher than 90%. Circular scaffolds (6 mm diameter and 2 mm thickness discs) were used for cell culture. Different types of EPCs were used: CD34+ derived ECs from cord blood and two sorts of CD133+ derived ECs from human bone marrow, old (30 days) and young (4 days) cells. EPCs were characterised as endothelial cells by immunofluorescent stainings for CD31 and Dil-Ac-LDL. CD133+ derived ECs from bone marrow were characterized by RT-PCR for CD31, VE-cadherin and KDR. HSVECs (Human Saphenous Vein Endothelial Cells) were used as control cells. We evaluated whether different kinds of EPCs could adhere on this novel hydrogel 4 h and 24 h after seeding, by a colorimetric quantitative test. EPCs adhered to hydrogels in serum- free conditions with values being over than 80% for young CD133+ cells at 4 h and 24 h. This pullulan-based hydrogel could constitute a suitable support for vascular cell adhesion as a pre-requisite for vascular tissue engineering.