Vasorin as an actor of bone turnover?

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/ß-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.

Vasorin plays a critical role in vascular smooth muscle cells and arterial functions.

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

NAMPT expression in osteoblasts controls osteoclast recruitment in alveolar bone remodeling.

In bone remodeling, osteoclasts are recruited via increased production of RANKL (receptor activator of nuclear factor-κB ligand) and migrate to the bone surface, aided by matrix metalloproteinases (MMPs). NAMPT (nicotinamide phosphoribosyl transferase), which catalyzes the rate-limiting step in the NAD+ salvage pathway, increases during in vitro osteogenic differentiation and inhibits RANKL-induced osteoclast differentiation. Alveolar bone loss, due to disturbance of the remodeling process, is a major feature of periodontitis. Thus, we investigated the role of NAMPT in a synchronized alveolar bone remodeling rat model. NAMPT expression increased in osteogenic cells during the remodeling activation phase, in parallel with RANKL and MMP-2 expression. Inhibition of NAMPT activity, by systemic delivery of its selective inhibitor FK866, decreased the recruitment of osteoclasts, but not their activity. In vitro, NAMPT mRNA, and protein expression also increased during osteoblast differentiation in primary calvarial osteoblast cultures. Recombinant NAMPT and NMN, its direct metabolite, dose-dependently increased bone marker expression, including that of sialoprotein (BSP) and osteocalcin (OC), whereas their expression was inhibited by FK866 treatment. Recombinant NAMPT did not regulate MMP-2, -9, MMP-13, or RANKL/OPG mRNA expression in osteoblasts. Our data suggest that de novo NAMPT synthesis in osteoblasts controls cell differentiation through osteoclast recruitment during the activation of bone remodeling.

Osteogenic Effect of Fisetin Doping in Bioactive Glass/Poly(caprolactone) Hybrid Scaffolds.

Treating large bone defects or fragile patients may require enhancing the bone regeneration rate to overcome a weak contribution from the body. This work investigates the osteogenic potential of nutrient fisetin, a flavonoid found in fruits and vegetables, as a doping agent inside the structure of a SiO2-CaO bioactive glass-poly(caprolactone) (BG-PCL) hybrid scaffold. Embedded in the full mass of the BG-PCL hybrid during one-pot synthesis, we demonstrate fisetin to be delivered sustainably; the release follows a first-order kinetics with active fisetin concentration being delivered for more than 1 month (36 days). The biological effect of BG-PCL-fisetin-doped scaffolds (BG-PCL-Fis) has been highlighted by in vitro and in vivo studies. A positive impact is demonstrated on the adhesion and the differentiation of rat primary osteoblasts, without an adverse cytotoxic effect. Implantation in critical-size mouse calvaria defects shows bone remodeling characteristics and remarkable enhancement of bone regeneration for fisetin-doped scaffolds, with the regenerated bone volume being twofold that of nondoped scaffolds and fourfold that of a commercial trabecular bovine bone substitute. Such highly bioactive materials could stand as competitive alternative strategies involving biomaterials loaded with growth factors, the use of the latter being the subject of growing concerns.

Dental pulp stem cells as a promising model to study imprinting diseases.

Parental imprinting is an epigenetic process leading to monoallelic expression of certain genes depending on their parental origin. Imprinting diseases are characterized by growth and metabolic issues starting from birth to adulthood. They are mainly due to methylation defects in imprinting control region that drive the abnormal expression of imprinted genes. We currently lack relevant animal or cellular models to unravel the pathophysiology of growth failure in these diseases. We aimed to characterize the methylation of imprinting regions in dental pulp stem cells and during their differentiation in osteogenic cells (involved in growth regulation) to assess the interest of this cells in modeling imprinting diseases. We collected dental pulp stem cells from five controls and four patients (three with Silver-Russell syndrome and one with Beckwith-Wiedemann syndrome). Methylation analysis of imprinting control regions involved in these syndromes showed a normal profile in controls and the imprinting defect in patients. These results were maintained in dental pulp stem cells cultured under osteogenic conditions. Furthermore, we confirmed the same pattern in six other loci involved in imprinting diseases in humans. We also confirmed monoallelic expression of H19 (an imprinted gene) in controls and its biallelic expression in one patient. Extensive imprinting control regions methylation analysis shows the strong potential of dental pulp stem cells in modeling imprinting diseases, in which imprinting regions are preserved in culture and during osteogenic differentiation. This will allow to perform in vitro functional and therapeutic tests in cells derived from dental pulp stem cells and generate other cell-types.

Combining sclerostin neutralization with tissue engineering: An improved strategy for craniofacial bone repair.

Scaffolds associated with different types of mesenchymal stromal stem cells (MSC) are extensively studied for the development of novel therapies for large bone defects. Moreover, monoclonal antibodies have been recently introduced for the treatment of cancer-associated bone loss and other skeletal pathologies. In particular, antibodies against sclerostin, a key player in bone remodeling regulation, have demonstrated a real benefit for treating osteoporosis but their contribution to bone tissue-engineering remains uncharted. Here, we show that combining implantation of dense collagen hydrogels hosting wild-type (WT) murine dental pulp stem cells (mDPSC) with weekly systemic injections of a sclerostin antibody (Scl-Ab) leads to increased bone regeneration within critical size calvarial defects performed in WT mice. Furthermore, we show that bone formation is equivalent in calvarial defects in WT mice implanted with Sost knock-out (KO) mDPSC and in Sost KO mice, suggesting that the implantation of sclerostin-deficient MSC similarly promotes new bone formation than complete sclerostin deficiency. Altogether, our data demonstrate that an antibody-based therapy can potentialize tissue-engineering strategies for large craniofacial bone defects and urges the need to conduct research for antibody-enabled local inhibition of sclerostin. STATEMENT OF SIGNIFICANCE: The use of monoclonal antibodies is nowadays broadly spread for the treatment of several conditions including skeletal bone diseases. However, their use to potentialize tissue engineering constructs for bone repair remains unmet. Here, we demonstrate that the neutralization of sclerostin, through either a systemic inhibition by a monoclonal antibody or the implantation of sclerostin-deficient mesenchymal stromal stem cells (MSC) directly within the defect, improves the outcome of a tissue engineering approach, combining dense collagen hydrogels and MSC derived from the dental pulp, for the treatment of large craniofacial bone defects.

Microvascular maturation by mesenchymal stem cells in vitro improves blood perfusion in implanted tissue constructs.

Blood perfusion of grafted tissue constructs is a hindrance to the success of stem cell-based therapies by limiting cell survival and tissue regeneration. Implantation of a pre-vascularized network engineered in vitro has thus emerged as a promising strategy for promoting blood supply deep into the construct, relying on inosculation with the host vasculature. We aimed to fabricate in vitro tissue constructs with mature microvascular networks, displaying perivascular recruitment and basement membrane, taking advantage of the angiogenic properties of dental pulp stem cells and self-assembly of endothelial cells into capillaries. Using digital scanned light-sheet microscopy, we characterized the generation of dense microvascular networks in collagen hydrogels and established parameters for quantification of perivascular recruitment. We also performed original time-lapse analysis of stem cell recruitment. These experiments demonstrated that perivascular recruitment of dental pulp stem cells is driven by PDGF-BB. Recruited stem cells participated in deposition of vascular basement membrane and vessel maturation. Mature microvascular networks thus generated were then compared to those lacking perivascular coverage generated using stem cell conditioned medium. Implantation in athymic nude mice demonstrated that in vitro maturation of microvascular networks improved blood perfusion and cell survival within the construct. Taken together, these data demonstrate the strong potential of in vitro production of mature microvasculature for improving cell-based therapies.

Bioactive Glass/Polycaprolactone Hybrid with a Dual Cortical/Trabecular Structure for Bone Regeneration.

Organic-inorganic hybrid biomaterials stand as a promise for combining bone bonding and bone mineral-forming ability, stimulation of osteogenic cells, and adequate mechanical properties. Bioactive glass (BG)-polycaprolactone (PCL) hybrids are of special interest as they gather the ability of BG to enhance osteoblast-mediated bone formation with the slow degradation rate and the toughness of PCL. In this study, BG-PCL hybrids were synthesized in the form of scaffold, owing to a dual cortical/trabecular structure mimicking the bone architecture. Their biological potential was evaluated both in vitro using rat primary osteoblasts (RPO) and in vivo in a mice model of critical-size calvarial defects. BG-PCL scaffolds were compared to Lubboc (BTB), a commercial purified bovine xenograft widely used in orthopedics and periodontal procedures and known for its efficiency. BG-PCL hybrids were found to facilitate RPO adhesion at their surface and to enhance RPO differentiation when compared to BTB. An in vivo micro-CT study demonstrates a higher bone ingrowth with BG-PCL scaffolds and a complete chemical conversion of the remaining BG-PCL after 3 months of implantation, while histological data show the vascularization of BG-PCL scaffolds and confirm the well-advanced bone regeneration with ongoing remodeling. Finally, we evidence the complete chemical conversion of the remaining BG-PCL into a bone-like mineral.

Priming Dental Pulp Stem Cells from Human Exfoliated Deciduous Teeth with Fibroblast Growth Factor-2 Enhances Mineralization Within Tissue-Engineered Constructs Implanted in Craniofacial Bone Defects.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue-engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor-2 (FGF-2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF-2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF-2 priming. After implantation in immunodeficient mice, the tissue-engineered constructs seeded with FGF-2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF-2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844&857.

Phosphorylated and Non-phosphorylated Leucine Rich Amelogenin Peptide Differentially Affect Ameloblast Mineralization.

The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the amelogenin gene. As full length amelogenin, LRAP has been shown, in precipitation experiments, to regulate hydroxyapatite (HAP) crystal formation depending on its phosphorylation status. However, very few studies have questioned the impact of its phosphorylation status on enamel mineralization in biological models. Therefore, we have analyzed the effect of phosphorylated (+P) or non-phosphorylated (-P) LRAP on enamel formation in ameloblast-like cell lines and ex vivo cultures of murine postnatal day 1 molar germs. To this end, the mineral formed was analyzed by micro-computed tomography, Field Emission Scanning Electron Microscopy, Transmission Electron Microscopy, Selected Area Electon Diffraction imaging. Amelogenin gene transcription was evaluated by qPCR analysis. Our data show that, in both cells and germ cultures, LRAP is able to induce an up-regulation of amelogenin transcription independently of its phosphorylation status. Mineral formation is promoted by LRAP(+P) in all models, while LRAP(-P) essentially affects HAP crystal formation through an increase in crystal length and organization in ameloblast-like cells. Altogether, these data suggest a differential effect of LRAP depending on its phosphorylation status and on the ameloblast stage at the time of treatment. Therefore, LRAP isoforms can be envisioned as potential candidates for treatment of enamel lesions or defects and their action should be further evaluated in pathological models.

Early angiogenesis detected by PET imaging with 64Cu-NODAGA-RGD is predictive of bone critical defect repair.

Therapies using stem cells may be applicable to all fields of regenerative medicine, including craniomaxillofacial surgery. Dental pulp stem cells (DPSCs) have demonstrated in vitro and in vivo osteogenic and proangiogenic properties. The aim of the study was to evaluate whether early angiogenesis investigated by nuclear imaging can predict bone formation within a mouse critical bone defect. Two symmetrical calvarial critical-sized defects were created. Defects were left empty or filled with i) DPSC-containing dense collagen scaffold, ii) 5% hypoxia-primed DPSC-containing dense collagen scaffold, iii) acellular dense collagen scaffold, or iv) left empty. Early angiogenesis assessed by PET using 64Cu-NODAGA-RGD as a tracer was found to be correlated with bone formation determined by micro-CT within the defects from day 30, and to be correlated to the late calcium apposition observed at day 90 using 18F-Na PET. These results suggest that nuclear imaging of angiogenesis, a technique applicable in clinical practice, is a promising approach for early prediction of bone grafting outcome, thus potentially allowing to anticipate alternative regenerative strategies. STATEMENT OF SIGNIFICANCE: Bone defects are a major concern in medicine. As life expectancy increases, the number of bone lesions grows, and occurring complications lead to a delay or even lack of consolidation. Therefore, to be able to predict healing or the absence of scarring at early times would be very interesting. This would not "waste time" for the patient. We report here that early nuclear imaging of angiogenesis, using 64Cu-NODAGA-RGD as a tracer, associated with nuclear imaging of mineralization, using 18F-Na as a tracer, is correlated to late bone healing objectivized by classical histology and microtomography. This nuclear imaging represents a promising approach for early prediction of bone grafting outcome in clinical practice, thus potentially allowing to anticipate alternative regenerative strategies.

Strategies Developed to Induce, Direct, and Potentiate Bone Healing.

Bone exhibits a great ability for endogenous self-healing. Nevertheless, impaired bone regeneration and healing is on the rise due to population aging, increasing incidence of bone trauma and the clinical need for the development of alternative options to autologous bone grafts. Current strategies, including several biomolecules, cellular therapies, biomaterials, and different permutations of these, are now developed to facilitate the vascularization and the engraftment of the constructs, to recreate ultimately a bone tissue with the same properties and characteristics of the native bone. In this review, we browse the existing strategies that are currently developed, using biomolecules, cells and biomaterials, to induce, direct and potentiate bone healing after injury and further discuss the biological processes associated with this repair.

Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis.

The transition from osteoblast to osteocyte is described to occur through passive entrapment mechanism (self-buried, or embedded by neighboring cells). Here, we provide evidence of a new pathway where osteoblasts are "more" active than generally assumed. We demonstrate that osteoblasts possess the ability to migrate and differentiate into early osteocytes inside dense collagen matrices. This step involves MMP-13 simultaneously with IBSP and DMP1 expression. We also show that osteoblast migration is enhanced by the presence of apatite bone mineral. To reach this conclusion, we used an in vitro hybrid model based on both the structural characteristics of the osteoid tissue (including its density, texture and three-dimensional order), and the use of bone-like apatite. This finding highlights the mutual dynamic influence of osteoblast cell and bone extra cellular matrix. Such interactivity extends the role of physicochemical effects in bone morphogenesis complementing the widely studied molecular signals. This result represents a conceptual advancement in the fundamental understanding of bone formation.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells.

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

Periosteum Metabolism and Nerve Fiber Positioning Depend on Interactions between Osteoblasts and Peripheral Innervation in Rat Mandible.

The sympathetic nervous system controls bone remodeling by regulating bone formation and resorption. How nerves and bone cells influence each other remains elusive. Here we modulated the content or activity of the neuropeptide Vasoactive Intestinal Peptide to investigate nerve-bone cell interplays in the mandible periosteum by assessing factors involved in nerve and bone behaviors. Young adult rats were chemically sympathectomized or treated with Vasoactive Intestinal Peptide or Vasoactive Intestinal Peptide10-28, a receptor antagonist. Sympathectomy depleted the osteogenic layer of the periosteum in neurotrophic proNerve Growth Factor and neurorepulsive semaphorin3a; sensory Calcitonin-Gene Related Peptide-positive fibers invaded this layer physiologically devoid of sensory fibers. In the periosteum non-osteogenic layer, sympathectomy activated mast cells to release mature Nerve Growth Factor while Calcitonin-Gene Related Peptide-positive fibers increased. Vasoactive Intestinal Peptide treatment reversed sympathectomy effects. Treating intact animals with Vasoactive Intestinal Peptide increased proNerve Growth Factor expression and stabilized mast cells. Vasoactive Intestinal Peptide10-28 treatment mimicked sympathectomy effects. Our data suggest that sympathetic Vasoactive Intestinal Peptide modulate the interactions between nervous fibers and bone cells by tuning expressions by osteogenic cells of factors responsible for mandible periosteum maintenance while osteogenic cells keep nervous fibers at a distance from the bone surface.

Pulp cell tracking by radionuclide imaging for dental tissue engineering.

Pulp engineering with dental mesenchymal stem cells is a promising therapy for injured teeth. An important point is to determine the fate of implanted cells in the pulp over time and particularly during the early phase following implantation. Indeed, the potential engraftment of the implanted cells in other organs has to be assessed, in particular, to evaluate the risk of inducing ectopic mineralization. In this study, our aim was to follow by nuclear imaging the radiolabeled pulp cells after implantation in the rat emptied pulp chamber. For that purpose, indium-111-oxine (¹¹¹In-oxine)-labeled rat pulp cells were added to polymerizing type I collagen hydrogel to obtain a pulp equivalent. This scaffold was implanted in the emptied pulp chamber space in the upper first rat molar. Labeled cells were then tracked during 3 weeks by helical single-photon emission computed tomography (SPECT)/computed tomography performed on a dual modality dedicated small animal camera. Negative controls were performed using lysed radiolabeled cells obtained in a hypotonic solution. In vitro data indicated that ¹¹¹In-oxine labeling did not affect cell viability and proliferation. In vivo experiments allowed a noninvasive longitudinal follow-up of implanted living cells for at least 3 weeks and indicated that SPECT signal intensity was related to implanted cell integrity. Notably, there was no detectable systemic release of implanted cells from the tooth. In addition, histological analysis of the samples showed mitotically active fibroblastic cells as well as neoangiogenesis and nervous fibers in pulp equivalents seeded with entire cells, whereas pulp equivalents prepared from lysed cells were devoid of cell colonization. In conclusion, our study demonstrates that efficient labeling of pulp cells can be achieved and, for the first time, that these cells can be followed up after implantation in the tooth by nuclear imaging. Furthermore, it appears that grafted cells retained the label and are viable to follow the repair process. This technique is expected to be of major interest for monitoring implanted cells in innovative therapies for injured teeth.

MEPE-derived ASARM peptide inhibits odontogenic differentiation of dental pulp stem cells and impairs mineralization in tooth models of X-linked hypophosphatemia.

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide - a substrate for PHEX and a strong inhibitor of mineralization - derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.