Origins of Alterations to Rankl Null Mutant Mouse Dental Root Development.

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

Paradoxical side effects of bisphosphonates on the skeleton: What do we know and what can we do?

Bisphosphonates are considered the most effective drugs for controlling adult and pediatric osteolytic diseases. Although they have been used successfully for many years, several side effects, such as osteonecrosis of the jaw, delayed dental eruption, atypical femoral fracture, and alterations to the bone growth system, have been described. After an overview of nitrogenous bisphosphonate, the purpose of this article is to describe their mechanisms of action and current applications, review the preclinical and clinical evidence of their side effects in the skeleton ("what we know"), and describe current recommendations for preventing and managing these effects ("what we can do"). Finally, promising future directions on how to limit the occurrence of these side effects will be presented.

Distorted Patterns of Dentinogenesis and Eruption in Msx2 Null Mutants: Involvement of Sost/Sclerostin.

The muscle segment homeogenes Msx1 and Msx2 play a major role in tooth and bone formation. Periodontal osteoclast impairment also occurs in Msx2 null mutant mice, which is restored by overexpression of the receptor activator of NF-κB targeted in osteoclast lineage. Here, we investigated the role of Msx2 in dentinogenesis. Experiments were performed on Msx2(-/-) mice and the MDPC-23 odontoblastic cell line. After Msx2 gene silencing, real-time quantitative RT-PCR data showed significant overexpression of Runx2, Bglap, Dspp, and Alpl. Of three inhibitors of Wnt/ß-catenin signaling (Dkk1, SostDc1, and Sost/Sclerostin), only Sost was expressed in postnatal teeth and overexpressed in Msx2(-/-) tooth samples. Initial crown dentin formation-primary dentinogenesis-occurred fairly normally in Msx2(-/-) teeth, albeit with distorted cusp patterns. Later stages of tooth development were characterized by a deviation from secondary toward tertiary dentinogenesis with osteodentin formation and impaired dentin deposition leading to limited root elongation. In Msx2(-/-)/receptor activator of NF-κB-transgenic double mutants, the dentin phenotype, notably in the roots, was rescued and sclerostin levels were normalized. These data suggest that Msx2 may act indirectly on dentinogenesis by controlling osteoclast activity and the signaling network related to eruption, supporting and further extending the concept that Msx2 controls formation of mineralized tissues by inhibition of the Wnt/ß-catenin pathway; Sost in dentin and Dkk1 in bone, as previously demonstrated.

Choroidal mast cells in retinal pathology: a potential target for intervention.

Mast cells are important in the initiation of ocular inflammation, but the consequences of mast cell degranulation on ocular pathology remain uncharacterized. We induced mast cell degranulation by local subconjunctival injection of compound 48/80. Initial degranulation of mast cells was observed in the choroid 15 minutes after the injection and increased up to 3 hours after injection. Clinical signs of anterior segment inflammation paralleled mast cell degranulation. With the use of optical coherence tomography, dilation of choroidal vessels and serous retinal detachments (SRDs) were observed and confirmed by histology. Subconjunctival injection of disodium cromoglycate significantly reduced the rate of SRDs, demonstrating the involvement of mast cell degranulation in posterior segment disorders. The infiltration of polymorphonuclear and macrophage cells was associated with increased ocular media concentrations of tumor necrosis factor-α, CXCL1, IL-6, IL-5, chemokine ligand 2, and IL-1ß. Analysis of the amounts of vascular endothelial growth factor and IL-18 showed an opposite evolution of vascular endothelial growth factor compared with IL-18 concentrations, suggesting that they regulate each other's production. These findings suggest that the local degranulation of ocular mast cells provoked acute ocular inflammation, dilation, increased vascular permeability of choroidal vessels, and SRDs. The involvement of mast cells in retinal diseases should be further investigated. The pharmacologic inhibition of mast cell degranulation may be a potential target for intervention.

Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit.

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK-RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities.

Pharmacological options in the treatment of osteogenesis imperfecta: A comprehensive review of clinical and potential alternatives.

Osteogenesis imperfecta (OI) is a genetically heterogeneous connective tissue disorder characterized by bone fragility and different extra-skeletal manifestations. The severity of these manifestations makes it possible to classify OI into different subtypes based on the main clinical features. This review aims to outline and describe the current pharmacological alternatives for treating OI, grounded on clinical and preclinical reports, such as antiresorptive agents, anabolic agents, growth hormone, and anti-TGFß antibody, among other less used agents. The different options and their pharmacokinetic and pharmacodynamic properties will be reviewed and discussed, focusing on the variability of their response and the molecular mechanisms involved to attain the main clinical goals, which include decreasing fracture incidence, improving pain, and promoting growth, mobility, and functional independence.

Bone resorption control of tooth eruption and root morphogenesis: Involvement of the receptor activator of NF-κB (RANK).

Activation of the receptor activator of NF-κB (RANK) is a crucial step in osteoclastogenesis. Loss- and gain-of-function mutations in the Rank gene cause, respectively, osteopetrosis and several forms of extensive osteolysis. Tooth and alveolar bone alterations are associated with these pathologies but remain to be better characterized. The aim of the present study was to establish the tooth and alveolar bone phenotype of a transgenic mouse model of RANK over-expression in osteoclast precursors. Early tooth eruption and accelerated tooth root elongation were observed subsequent to an increase in osteoclast numbers surrounding the tooth. The final root length appeared not to be affected by RANK over-expression, but a significant reduction in root diameter occurred in both control and root-morphogenesis-defective Msx2 null mutant mice. These results indicate that root length is independent of the surrounding bone resorption activity. In contrast, root diameter is sensitive to the activity of alveolar bone osteoclasts. These data suggest that early eruption and thin root are phenotypic features that could be associated with extensive osteolytic pathologies.

Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

Primary Retention of Molars and RANKL Signaling Alteration during Craniofacial Growth.

The primary retention of molars observed in clinic corresponds to a still-unexplained absence of molar eruption despite the presence of an eruption pathway, resembling the experimental transient inhibition of RANKL signaling in mice. The aim of the present study was to confront the hypothesis according to which the primary retention of molars is associated with transitory perturbations to RANKL signaling during growth as part of a wider craniofacial skeleton pattern. The experimental strategy was based on combining a clinical study and an animal study corresponding to the characterization of the craniofacial phenotypes of patients with primary retention of molars and analyses in mice of the consequences of transient inhibition of RANKL signaling on molar eruption and craniofacial growth. The clinical study validated the existence of a particular craniofacial phenotype in patients with primary retention of molars: a retromandibular skeletal class II typology with reduced mandibular dimensions which manifests itself at the dental level by a class II/2 with palatoversion of the upper incisors and anterior overbite. The animal study demonstrated that transient invalidation of RANKL signaling had an impact on the molar eruption process, the severity of which was dependent on the period of inhibition and was associated with a reduction in two craniofacial morphometric parameters: total skull length and craniofacial vault length. In conclusion, primary retention of molars may be proposed as part of the craniofacial skeleton phenotype associated with a transitory alteration in RANKL signaling during growth.

Genetically-achieved disturbances to the expression levels of TNFSF11 receptors modulate the effects of zoledronic acid on growing mouse skeletons.

Zoledronic acid (ZOL), a nitrogen bisphosphonate (N-BP), is currently used to treat and control pediatric osteolytic diseases. Variations in the intensity of the effects and side effects of N-BPs have been reported with no clear explanations regarding their origins. We wonder if such variations could be associated with different levels of RANKL signaling activity in growing bone during and after the treatment with N-BPs. To answer this question, ZOL was injected into neonate C57BL/6J mice with different genetically-determined RANKL signaling activity levels (Opg+/+\RankTg-, Opg+/+\RankTg+, Opg+/-\RankTg-, Opg+/-\RankTg+, Opg-/-\RankTg- and Opg-/-\RankTg+ mice) following a protocol (4 injections from post-natal day 1 to 7 at the dose of 50 µg/kg) that mimics those used in onco-pediatric patients. At the end of pediatric growth (1 and half months) and at an adult age (10 months), the bone morphometric and mineral parameters were measured using µCT in the tibia and skull for the different mice. A histologic analysis of the dental and periodontal tissues was also performed. At the end of pediatric growth, a delay in long bone and skull bone growth, a blockage of tooth eruption, some molar root alterations and a neoplasia-like structure associated with incisor development were found. Interestingly, the magnitude of these side effects was reduced by Opg deficiency (Opg-/-) but increased by Rank overexpression (RankTg). Analysis of the skeletal phenotype at ten months confirmed respectively the beneficial and harmful effects of Opg deficiency and Rank overexpression. These results validated the hypothesis that the RANKL signaling activity level in the bone microenvironment is implicated in the modulation of the response to ZOL. Further studies will be necessary to understand the underlying molecular mechanisms, which will help decipher the variability in the effects of N-BPs reported in the human population. SIGNIFICANT STATEMENTS: The present study establishes that in mice the RANKL signaling activity level is a major modulator of the effects and side-effects of bisphosphonates on the individual skeleton during growth. However, the modulatory actions are dependent on the ways in which this level of activity is increased. A decrease in OPG expression is beneficial to the skeletal phenotype observed at the end of growth, while RANK overexpression deteriorates it. Far removed from pediatric treatment, in adults, the skeletal phenotypes initially observed at the end of growth for the different levels of RANKL signaling activity were maintained, although significant improvement was associated only with reductions in OPG expression.

[Effects of post-natal inhibition of RANKL on molar eruption and root formation in C57BL/6 mice]. / Effets de l'inhibition post-natale de RANKL sur l'éruption et la formation radiculaire des molaires de souris C57BL/6.

INTRODUCTION: Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG. MATERIALS AND METHODS: C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination. RESULTS: The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted. DISCUSSION: This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases.

Physiological implications of DLX homeoproteins in enamel formation.

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression.

Maternal RANKL Reduces the Osteopetrotic Phenotype of Null Mutant Mouse Pups.

RANKL signalization is implicated in the morphogenesis of various organs, including the skeleton. Mice invalidated for Rankl present an osteopetrotic phenotype that was less severe than anticipated, depending on RANKL's implication in morphogenesis. The hypothesis of an attenuated phenotype, as a result of compensation during gestation by RANKL of maternal origin, was thus brought into question. In order to answer this question, Rankl null mutant pups from null mutant parents were generated, and the phenotype analyzed. The results validated the presence of a more severe osteopetrotic phenotype in the second-generation null mutant with perinatal lethality. The experiments also confirmed that RANKL signalization plays a part in the morphogenesis of skeletal elements through its involvement in cell-to-cell communication, such as in control of osteoclast differentiation. To conclude, we have demonstrated that the phenotype associated with Rankl invalidation is attenuated through compensation by RANKL of maternal origin.

RANK/RANKL/OPG Signalization Implication in Periodontitis: New Evidence from a RANK Transgenic Mouse Model.

Periodontitis is based on a complex inflammatory over-response combined with possible genetic predisposition factors. The RANKL/RANK/OPG signaling pathway is implicated in bone resorption through its key function in osteoclast differentiation and activation, as well as in the inflammatory response. This central element of osteo-immunology has been suggested to be perturbed in several diseases, including periodontitis, as it is a predisposing factor for this disease. The aim of the present study was to validate this hypothesis using a transgenic mouse line, which over-expresses RANK (RTg) and develops a periodontitis-like phenotype at 5 months of age. RTg mice exhibited severe alveolar bone loss, an increased number of TRAP positive cells, and disorganization of periodontal ligaments. This phenotype was more pronounced in females. We also observed dental root resorption lacunas. Hyperplasia of the gingival epithelium, including Malassez epithelial rests, was visible as early as 25 days, preceding any other symptoms. These results demonstrate that perturbations of the RANKL/RANK/OPG system constitute a core element of periodontitis, and more globally, osteo-immune diseases.

Nano alterations of membrane structure on both γ-irradiated and stored human erythrocytes.

PURPOSE: Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality. MATERIALS AND METHODS: Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes. RESULTS: RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered. CONCLUSIONS: Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.

Hepatitis C virus infection and non-Hodgkin's lymphoma: a review and case report of nine patient.

The role of hepatitis C virus (HCV) is well established in the development of chronic hepatitis, cirrhosis and hepatic carcinoma, as well as in mixed type II cryoglobulinemia, membranoproliferative glomerulonephritis(MPGN) and porphyria cutanea tarda (PCT). Increasing evidence has been reported of a close association of HCV infection with autoimmune and hematological processes, mainly cytopenias and lymphoproliferative disorders such as B cell non-Hodgkin's lymphoma. We describe the demographic, clinical and histopathological findings of nine patients from the Mexican population with non-Hodgkin's lymphoma and HCV infection.

Bone resorption: an actor of dental and periodontal development?

Dental and periodontal tissue development is a complex process involving various cell-types. A finely orchestrated network of communications between these cells is implicated. During early development, communications between cells from the oral epithelium and the underlying mesenchyme govern the dental morphogenesis with successive bud, cap and bell stages. Later, interactions between epithelial and mesenchymal cells occur during dental root elongation. Root elongation and tooth eruption require resorption of surrounding alveolar bone to occur. For years, it was postulated that signaling molecules secreted by dental and periodontal cells control bone resorbing osteoclast precursor recruitment and differentiation. Reverse signaling originating from bone cells (osteoclasts and osteoblasts) toward dental cells was not suspected. Dental defects reported in osteopetrosis were associated with mechanical stress secondary to defective bone resorption. In the last decade, consequences of bone resorption over-activation on dental and periodontal tissue formation have been analyzed with transgenic animals (RANK (Tg) and Opg (-∕-) mice). Results suggest the existence of signals originating from osteoclasts toward dental and periodontal cells. Meanwhile, experiments consisting in transitory inhibition of bone resorption during root elongation, achieved with bone resorption inhibitors having different mechanisms of action (bisphosphonates and RANKL blocking antibodies), have evidenced dental and periodontal defects that support the presence of signals originating bone cells toward dental cells. The aim of the present manuscript is to present the data we have collected in the last years that support the hypothesis of a role of bone resorption in dental and periodontal development.

Skeletal consequences of RANKL-blocking antibody (IK22-5) injections during growth: mouse strain disparities and synergic effect with zoledronic acid.

High doses of bone resorption inhibitors are currently under evaluation in pediatric oncology. Previous works have evidenced transient arrest in long bone and skull bone growth and tooth eruption blockage when mice were treated with zoledronic acid (ZOL). The question of potential similar effects with a RANKL-blocking antibody (IK22.5) was raised. Sensitivity disparities in these inhibitors between mouse strains and synergic effects of zoledronic acid and a RANKL-blocking antibody were subsidiary questions. In order to answer these questions, newborn C57BL/6J and CD1 mice were injected every two or three days (4 injections in total so 7 or 10 days of treatment length) with high doses of a RANKL-blocking antibody. The consequences on the tibia, craniofacial bones and teeth were analyzed by µCT and histology at the end of the treatment and one, two and three months later. The results obtained showed that RANKL-blocking antibody injections induced a transient arrest of tibia and skull bone growth and an irreversible blockage of tooth eruption in C57BL/6J mice. In CD1 mice, tooth eruption defects were also present but only at much higher doses. Similar mouse strain differences were obtained with zoledronic acid. Finally, a synergic effect of the two inhibitors was evidenced. In conclusion as previously observed for bisphosphonates (ZOL), a RANKL-blocking antibody induced a transient arrest in long bone and skull bone growth and a blockage of tooth eruption with however disparities between mouse strains with regard to this last effect. A synergic effect of both bone resorption inhibitors was also demonstrated.

Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies.

High doses of zoledronic acid (ZOL), one of the most potent inhibitors of bone resorption, are currently evaluated in phase III clinical trials in Europe for the treatment of malignant pediatric primary bone tumors. The impact of such an intensive treatment on the craniofacial skeleton growth is a critical question in the context of patients with actively growing skeleton; in particular, in light of our previous studies evidencing that endochondral bone formation was transiently disturbed by high doses of ZOL. Two protocols adapted from pediatric treatments were developed for newborn mice (a total of 5 or 10 injections of ZOL 50µg/kg every two days). Their impact on skull bones and teeth growth was analyzed by X-rays, microCT and histology up to 3months after the last injection. ZOL administrations induced a transient delay of skull bone growth and an irreversible delay in incisor, first molar eruption and root elongation. Other teeth were affected, but most were erupted by 3months. Root histogenesis was severely impacted for all molars and massive odontogenic tumor-like structures were observed in all mandibular incisors. High doses of ZOL irreversibly disturbed teeth eruption and elongation, and delayed skull bone formation. These preclinical observations are essential for the follow-up of onco-pediatric patients treated with ZOL.