Julie Lesieur

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.

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.

Sclerostin Deficiency Promotes Reparative Dentinogenesis

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice (Sost–/–). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost–/– and wild-type (WT) mice (n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro–computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost–/– mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost–/– pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein–positive cells, had expanded in both groups but remained significantly larger in Sost–/– pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost–/– dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies.

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson’s trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge—the microarchitecture of which significantly differed from the native dentin—was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.

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.

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.

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.

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.