Human dentin characteristics of patients with osteogenesis imperfecta: insights into collagen-based biomaterials.

Osteogenesis imperfecta (OI), also known as "brittle bone disease", is a rare genetic disorder of the skeleton, whose most benign form I corresponds to autosomal dominant mutations in the genes encoding type I collagen (COLA1, COLA2). Several associated skeletal manifestations are often observed but, surprisingly, while dentin defects often reflect genetic bone disorders, about half of OI patients have no obvious oral manifestations. Here, we investigated the collagen, mineral and mechanical properties of dentin from deciduous teeth collected from patients with mild form of OI and displaying no obvious clinical signs of dentinogenesis imperfecta. For the first time, an increase in the hardness of OI dentin associated with an increase in mineral content compared to healthy patients was reported. In addition, OI altered the tissue characteristics of the dentin-enamel junction but the interfacial gradient was preserved. The impact of changes in molecular structure due to mutations in OI was assessed by Raman microspectroscopy. Our results highlighted a change in the hydroxyproline-proline ratio in direct association with collagen mineralization. Our findings suggest that the evaluation of teeth could be an important aid for mild types of OI that are often difficult to diagnose clinically and provide experimental evidence that hydroxyproline content should be considered in future studies on collagen-based biomaterials.

Differences between inflammatory and catabolic mediators of peri-implantitis and periodontitis lesions following initial mechanical therapy: An exploratory study.

BACKGROUND AND OBJECTIVE: The aim of this study was to analyze the differences in inflammatory and catabolic mediators expressed in peri-implantitis compared to periodontitis lesions after non-surgical therapy. Peri-implantitis is associated with a faster rate of bone loss when compared with periodontitis, and peri-implant non-surgical therapy is ineffective to cure peri-implantitis. This may be due to persistent inflammation in peri-implantitis tissues after initial mechanical treatment. MATERIAL AND METHODS: Eleven patients with peri-implantitis and 10 with severe chronic periodontitis received non-surgical therapy. They were included at re-evaluation (8 weeks) if they presented pocket depth ≥6 mm with bleeding on probing, and the indication for open flap debridement surgery. Connective tissues were harvested during surgery from diseased sites. Healthy gingiva were harvested during third molar extraction in a third group of healthy patients (n=10). Explants were incubated for 24 hours in media culture and the release of cytokines, chemokines, growth factors, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand (RANKL), matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase (TIMP) in the conditioned media was analyzed by an exploratory multiplex immunoassay. When difference was found in the conditioned media, an immunohistochemistry was performed to compare expression in the tissues. RESULTS: Connective tissues from non-stabilized peri-implantitis exhibited a distinct cytokine profile compared to periodontitis lesions that did not respond to initial therapy. Indeed, TIMP-2 was significantly increased in media from peri-implantitis (P≤.05). In addition, the in situ expression of TIMP-2, interleukin-10 and RANKL was also significantly increased in peri-implantitis tissues (P≤.05). However, the ratio of RANKL/osteoprotegerin-positive cells did not vary (P≥.05). CONCLUSION: This study suggests that peri-implantitis and periodontitis connective tissues exhibit differences in response to non-surgical treatment, which may contribute to a different pattern of disease evolution.

Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

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.

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.

Phosphate and Vitamin D Prevent Periodontitis in X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH) is a rare genetic skeletal disease where increased phosphate wasting in the kidney leads to hypophosphatemia and prevents normal mineralization of bone and dentin. Here, we examined the periodontal status of 34 adults with XLH and separated them according to the treatment they received for hypophosphatemia. We observed that periodontitis frequency and severity were increased in adults with XLH and that the severity varied according to the hypophosphatemia treatment. Patients who benefited from an early and continuous vitamin D and phosphate supplementation during their childhood presented less periodontal attachment loss than patients with late or incomplete supplementation. Continued hypophosphatemia treatment during adulthood further improved the periodontal health. Extracted teeth from patients with late or incomplete supplementation showed a strong acellular cementum hypoplasia when compared with age-matched healthy controls. These results show that XLH disturbs not only bone and dentin formation but also cementum and that the constitutional defect of the attachment apparatus is associated with attachment loss.

[Endocrine control of serum phosphate: from the discoveries of phosphatonins to novel therapies]. / Régulation endocrine de la phosphatémie : de l'identification des facteurs aux nouvelles thérapies.

Phosphate is absorbed through the gut, stored in the bone and reabsorbed through the proximal renal tubule. More importantly, PTH and FGF23 have been identified as the main phosphaturic factors that control the expression of the phosphate co-transporters NaPi-IIa et IIc. By allowing the adjustment of the urinary phosphate reabsorption, these two phosphatonins play a major role in bone and tooth mineralization and growth. Recently, novel therapies have successfully targeted the FGF23 signaling pathway to treat the hypophosphatemia in patients affected with molecular defects of this pathway (mutations in the PHEX gene).

Abnormal osteopontin and matrix extracellular phosphoglycoprotein localization, and odontoblast differentiation, in X-linked hypophosphatemic teeth.

Mutations in phosphate-regulating gene (PHEX) lead to X-linked hypophosphatemic rickets (XLH), a genetic disease characterized by impaired mineralization in bones and teeth. In human XLH tooth dentin, calcospherites that would normally merge as part of the mineralization process are separated by unmineralized interglobular spaces where fragments of matrix proteins accumulate. Here, we immunolocalized osteopontin (OPN) in human XLH teeth, in a three-dimensional XLH human dental pulp stem cell-collagen scaffold culture model and in a rat tooth injury repair model treated with acidic serine- and aspartate-rich motif peptides (ASARM). In parallel, matrix extracellular phosphoglycoprotein (MEPE) immunolocalization and alkaline phosphatase (ALP) activity were assessed in XLH teeth. OPN was expressed by odontoblasts in the XLH models, and localized to the abnormal calcospherites of XLH tooth dentin. In addition, ALP activity and MEPE localization were abnormal in human XLH teeth, with MEPE showing an accumulation in the unmineralized interglobular spaces in dentin. Furthermore, XLH odontoblasts failed to form a well-polarized odontoblast layer. These data suggest that both MEPE and OPN are involved in impaired tooth mineralization associated with XLH, possibly through different effects on the mineralization process.

Minimal intervention dentistry: part 8. Biotherapies for the dental pulp.

The development of new therapies, the biotherapies, can now be envisioned in dentistry thanks to the important development of research in the stem cell and biomaterial fields. Indeed, the recent discovery of stem cells in different tissues of the body, and in particular in the dental pulp, should allow the development of new therapeutic approaches for pulpo-dentinal lesions. In case of moderate pulp injury, the stimulation by biomolecules of pulp stem cells located in the remaining pulp is the approach to be considered. In case of more severe lesions requiring a total removal of pulp tissues, a cell therapy could be considered. The objective of this article is to present these innovative therapeutic approaches for the future of dentistry.

Dental caries and enamelin haplotype.

In the literature, the enamelin gene ENAM has been repeatedly designated as a possible candidate for caries susceptibility. Here, we checked whether ENAM variants could increase caries susceptibility. To this aim, we sequenced coding exons and exon-intron boundaries of ENAM in 250 children with a severe caries phenotype and in 149 caries-free patients from 9 French hospital groups. In total, 23 single-nucleotide polymorphisms (SNPs) were found, but none appeared to be responsible for a direct change of ENAM function. Six SNPs had a high minor allele frequency (MAF) and 6 others were identified for the first time. Statistical and evolutionary analyses showed that none of these SNPs was associated with caries susceptibility or caries protection when studied separately and challenged with environmental factors. However, haplotype interaction analysis showed that the presence, in a same variant, of 2 exonic SNPs (rs7671281 and rs3796704; MAF 0.12 and 0.10, respectively), both changing an amino acid in the protein region encoded by exon 10 (p.I648T and p.R763Q, respectively), increased caries susceptibility 2.66-fold independent of the environmental risk factors. These findings support ENAM as a gene candidate for caries susceptibility in the studied population.

Mineralization of dense collagen hydrogel scaffolds by human pulp cells.

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Common SNPs of AmelogeninX (AMELX) and dental caries susceptibility.

Genetic approaches have shown that several genes could modify caries susceptibility; AmelogeninX (AMELX) has been repeatedly designated. Here, we hypothesized that AMELX mutations resulting in discrete changes of enamel microstructure may be found in children with a severe caries phenotype. In parallel, possible AMELX mutations that could explain resistance to caries may be found in caries-free patients. In this study, coding exons of AMELX and exon-intron boundaries were sequenced in 399 individuals with extensive caries (250) or caries-free (149) individuals from nine French hospital groups. No mutation responsible for a direct change of amelogenin function was identified. Seven single-nucleotide polymorphisms (SNPs) were found, 3 presenting a high allele frequency, and 1 being detected for the first time. Three SNPs were located in coding regions, 2 of them being non-synonymous. Both evolutionary and statistical analyses showed that none of these SNPs was associated with caries susceptibility, suggesting that AMELX is not a gene candidate in our studied population.

ES cells-derived ectomesenchymal cells for tooth engineering.

Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells.

Effect of a calcium-silicate-based restorative cement on pulp repair.

In cases of pulp injury, capping materials are used to enhance tertiary dentin formation; Ca(OH)(2) and MTA are the current gold standards. The aim of this study was to evaluate the capacity of a new calcium-silicate-based restorative cement to induce pulp healing in a rat pulp injury model. For that purpose, cavities with mechanical pulp exposure were prepared on maxillary first molars of 27 six-week-old male rats, and damaged pulps were capped with either the new calcium-silicate-based restorative cement (Biodentine), MTA, or Ca(OH)(2). Cavities were sealed with glass-ionomer cement, and the repair process was assessed at several time-points. At day 7, our results showed that both the evaluated cement and MTA induced cell proliferation and formation of mineralization foci, which were strongly positive for osteopontin. At longer time-points, we observed the formation of a homogeneous dentin bridge at the injury site, secreted by cells displaying an odontoblastic phenotype. In contrast, the reparative tissue induced by Ca(OH)(2) showed porous organization, suggesting a reparative process different from those induced by calcium silicate cements. Analysis of these data suggests that the evaluated cement can be used for direct pulp-capping.

Tooth dentin defects reflect genetic disorders affecting bone mineralization.

Several genetic disorders affecting bone mineralization may manifest during dentin mineralization. Dentin and bone are similar in several aspects, especially pertaining to the composition of the extracellular matrix (ECM) which is secreted by well-differentiated odontoblasts and osteoblasts, respectively. However, unlike bone, dentin is not remodelled and is not involved in the regulation of calcium and phosphate metabolism. In contrast to bone, teeth are accessible tissues with the shedding of deciduous teeth and the extractions of premolars and third molars for orthodontic treatment. The feasibility of obtaining dentin makes this a good model to study biomineralization in physiological and pathological conditions. In this review, we focus on two genetic diseases that disrupt both bone and dentin mineralization. Hypophosphatemic rickets is related to abnormal secretory proteins involved in the ECM organization of both bone and dentin, as well as in the calcium and phosphate metabolism. Osteogenesis imperfecta affects proteins involved in the local organization of the ECM. In addition, dentin examination permits evaluation of the effects of the systemic treatment prescribed to hypophosphatemic patients during growth. In conclusion, dentin constitutes a valuable tool for better understanding of the pathological processes affecting biomineralization.

Interest in a new test for caries risk in adolescents undergoing orthodontic treatment.

It has been reported that patients undergoing orthodontic treatment present a high risk of caries. Recently, an immediate chair-side test was proposed, displaying the intra-oral lactic acid production of cariogenic bacteria. The aim of this 12-month follow-up prospective cohort study was to evaluate the association between having a high score on this test and caries occurrence in 110 young patients scheduled for orthodontic treatment. Caries occurrence was studied by Kaplan-Meier curves and Multivariate Cox models allowed the examination of its association with covariates. Fifty four patients developed at least one carious lesion during the follow-up period. At baseline, approximately 70% of the patients presented a high risk of caries according to the test and this number came close to 80% by the study's completion. According to the Kaplan-Meier estimator, 51% (CI(95%) 0.40, 0.60) of the sample would have developed at least one carious lesion during the follow-up. The test score was then associated with age, DMFT, and caries occurrence. This study showed that a high test score at baseline associated with a high DMFT predicted a high risk of caries (RR = 2.6). Taking the patient's age into consideration, an increase of 1 year resulted in a 10% decrease of the risk of caries occurrence (RR = 0.89). Within the limits of this longitudinal study, it may be concluded that this test is useful to evaluate the risk for dental caries in adolescents with orthodontic treatment. Furthermore, the distribution of the lesions in our sample suggests specific clinical approaches for this group of patients.