牙本质发育异常和牙本质发育不全分子遗传学的研究进展

最近,牙本质疾病的进展非常迅速。这些疾病主要分为2类,并带有不同的亚型。一类为牙本质发育异常（dentin dysplasia,DD）Ⅰ型和Ⅱ型,另一类为牙本质发育不全（dentinogenesis imperfecta,DGI）Ⅰ型、Ⅱ型和Ⅲ型。遗传连锁分析证明了DD-Ⅱ、DGI-Ⅱ和DGI-Ⅲ的关键位点位于4号染色体长臂上,这些位点包括了分泌型焦磷酸蛋白（SPP1）、骨唾液酸蛋白（BSP）、细胞外基质磷酸化糖蛋白（MEPE）、牙本质基质蛋白1（DMP1）和牙本质唾液酸焦磷酸蛋白（DSPP）基因。目前,只有DSPP的突变被证实。现将最新进展做一综述。     

Analysis of DSPP gene mutation in a Chinese pedigree affected with hereditary dentinogenesis imperfectaCSCD

Objective: To analyze the clinical phenotype of a Chinese pedigree affected with hereditary dentinogenesis imperfecta and mutation of dentin sialophosphoprotein (DSPP) gene. Methods: Affected members underwent intraoral photography, dental film and panoramic radiography. Genomic DNA was extracted from peripheral venous blood samples. Coding regions of the DSPP gene were subjected to PCR amplification and Sanger sequencing. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. The tertiary structure of wild type and mutant proteins were predicted by Swiss-Port. Results: A heterozygous c. 50C>T (p. P17L) mutation was identified in exon 2 of the DSPP gene in the proband and her father. The same mutation was not found among 200 unrelated healthy controls. The Pro-17 residues and its surrounding positions in DSPP are highly conserved across various species. The mutation was predicted to be damaging to the structure of DSPP protein. Conclusion: The c. 50C>T (p. P17L) mutation of the DSPP gene probably underlies the disease in this pedigree. Above finding has expanded the spectrum of DSPP gene mutations and provided a basis for genetic counseling and prenatal diagnosis for this family. © 2018 MeDitorial Ltd. All rights reserved.     

Morphological analysis and chemical content of natural dentin carious lesion zones

Dentin is one of the earliest bio-mineralization products to appear in the evolution of vertebrates. Dentin reactions to infection mimic earlier phylogenetic patterns, and carious lesions are divided into different zones which reflect the natural patho-morphological reaction of dentin to the carious attack. It was the aim of this study to investigate deep dentin carious lesions of human molars with combined polarization light microscopy, scanning electron microscopy and energy dispersive X-ray element analysis (EDX) for the determination of different zones of the carious lesions, their extent and the chemical content. Sixteen extracted teeth with deep dentin carious lesions were embedded in Technovit 9100 (Kulzer) and serial sections of 80 microm thickness were made. These sections were then examined with polarized light microscopy to identify the different zones of the lesions. The outlines of the zones were traced consecutively and 3D-reconstructions were made for the determination of the extent and calculation of the volumes of the different zones. From the volumes of the demineralizing dentin and the translucent zone a Dentin Demineralization Index (DDI) was calculated. Three sections of each lesion were then coated with carbon and studied with a scanning electron microscope. 3D-reconstruction of the teeth showed the rather stable translucent zone, interrupted by remnants of dead tracts, and very different volumes of demineralizing dentin. Therefore, with increasing size of the demineralizing dentin, the DDI increased. The chemical content was measured using energy dispersive X-ray analysis (EDX) in areas of intertubular dentin. The content of Ca, P, and C was significantly different in all zones. The Ca/P ratio was significantly different between sound dentin and demineralizing dentin. From the results we conclude that the mineral content of intertubular dentin of the translucent zone and demineralizing dentin is different from that of sound dentin, and the unique mineralization pattern of the translucent zone is a biological reaction to the carious attack. Because active dentin lesions exhibit many non-occluded open dentin tubules, further bacterial invasion or, in case of dentin treatment, the penetration of bonding agents towards the pulp is morphologically not prevented and therefore of clinical importance.     

Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root

Tissue engineering strategies to reconstruct tooth roots are an effective therapy for the treatment of tooth loss. However, strategies to successfully regenerate tooth roots have not been developed and optimized. In the present study, rat dental follicle stem cells (DFCs) were characterized, followed by a thorough investigation of tooth roots regeneration for a combination of DFCs seeding cells, treated dentin matrix (TDM) scaffolds, and an inductive alveolar fossa microenvironment. Eighteen clones derived from single DFCs were harvested; however, only three clones were amplified successfully more than five passages and 90-95 days in culture. Following 270 days or 30 passages, the heterogeneous DFCs showed suitable characteristics for seeding cells to regenerate tooth roots. However, various features, such as variable proliferation rates, differentiation characteristics, apoptosis rates, and total lifespan were observed in DFCs and the three clones. Importantly, upon transplantation of DFCs combined with TDM for four weeks, root-like tissues stained positive for markers of dental pulp and periodontal tissues were regenerated in the alveolar fossa, but not in the skull and omental pockets. These results indicate that tooth roots were successfully regenerated and suggest that the combination of DFCs with TDM in the alveolar fossa is a feasible strategy for tooth roots regeneration. This strategy could be a promising approach for the treatment of clinical tooth loss and provides a perspective with potential applications to regeneration of other tissues and organs.     

Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix - based scaffold

Stem cell mediated tissue engineering has been acknowledged as a prospective strategy for repairing and replacing damaged and lost tissues. However, the low survival rate of implanted stem cells proves to be a major challenge in the management of transplantation failures. While previous studies have indicated the effectiveness of tissue engineered cell sheets in improving the survival rate of implanted cells, we have recently demonstrated the use of treated dentin matrix (TDM) as a biological scaffold and dental follicle cells (DFCs) as the seeding cells for dentinogenesis and tooth root construction. This study proposes a strategy utilizing TDM with human dental follicle cell sheets (DFCSs) for root regeneration. The biological characteristics and changes of human DFCSs under the effect of TDM were studied with scanning electron microscopy, transmission electron microscopy, immunofluorescence microscopy, immunohistochemistry and quantitative real-time PCR. DFCSs combined with TDM were implanted subcutaneously into the dorsum of mice. Histological examination of the harvested grafts revealed a whirlpool-like alignment of the DFCs in multiple layers that were positive for COLI, integrinβ1, fibronectin and alkaline phosphatase (ALP), suggestive of the formation of a rich extracellular matrix. DFCSs, under the effect of TDM, highly expressed DMP-1 and bone sialoprotein (BSP), indicating their potential for odontogenesis and osteogenesis. Importantly, in vivo, TDM could induce and support DFCSs to develop new dentin-pulp like tissues and cementum-periodontal complexes that were positive for markers such as DSP, nestin and VIII factors, COLI and cementum attachment protein (CAP), implying successful root regeneration. Therefore, DFCSs combined with TDM may prove to be a better strategy for the construction of tooth root, and is a prospective approach that could be utilized for the treatment of root or tooth defect or loss in future.     

Odontogenic differentiation and dentin formation of dental pulp cells under nanobioactive glass induction

Bioactive glass (BG) has been widely used in bone regeneration; however, reports on the biological effects of BG on dental pulp cells are rare. This study aims to investigate the effects of nanoscale BG (n-BG) on odontogenic differentiation and dentin formation of dental pulp cells and to compare these effects with those of microscale BG (m-BG). Human dental pulp cells (hDPCs) from third molars were cultured directly with m-BG and n-BG in vitro. The cell proliferation increased at 0.1 mg ml⁻¹ BG, which also had a chemotactic effect on hDPCs. The mineralization capacity and expression of odontogenic-related proteins and genes (dentin sialophosphoprotein, dentin matrix protein 1 and collagen type I) of hDPCs were significantly up-regulated under BG induction, and were particularly higher in the n-BG group than in the control group. m-BG and n-BG combined with pulp tissues were transplanted into the dorsum of immunodeficient mice to observe their biological effects on dental pulp cells in vivo. A continuous layer of dentin-like tissue with uniform thickness, a well-organized dentinal tubule structure and polarizing odontoblast-like cells aligned along it was generated upon the n-BG layer, whereas some irregular sporadic osteodentin-like mineralized tissues were observed in the control group. This study reveals that BG, especially n-BG, induces the odontogenic differentiation and dentin formation of dental pulp cells and may serve as a potential material for pulp repair and dentin regeneration.     

Gluma脱敏剂对牙本质黏结强度影响的系统评价

BACKGROUND: Desensitization agent is widely used to relieve tooth sensitivity after tooth preparation. However, some components in the desensitization agent for certain affect the dentin bond strength between prosthesis and dentin. Given this, choosing a proper desensitization agent is under discussion. OBJECTIVE: To investigate the influence of Gluma desensitizing agents on bond strength to dentin by Meta-analysis. METHODS: A computer-based retrieval of PubMed, EMbase, EBSCO EDLINE, The chrane Library, ScienceDirect, Web Of Science, OVID, CBMbase, CJFD, CqVip and WanFang databases combined with manual searching was performed for randomized controlled trials or controlled clinical trials about effect of desensitizing agents on dentin bond strength. The retrieval time ranged from the creation date to June 18, 2016, without region and language limits. In accordance with inclusion/exclusion criteria, related data were extracted, and the qualitative and quantitative analysis of the included studies was performed by two researchers independently, and then the Meta-analysis was conducted using RevMan 5.2 software. RESULTS AND CONCLUSION: There was no significant difference in the shear bond strength of self-etching or total-etching adhesive to dentin between two groups, and the same results were found when comparing the shear bond strength of self-etching and total-etching adhesive to dentin. The Gluma had a beneficial effect on the shear bond strength of self-adhesive. These results indicate that when Gluma used as the desensitizing agent, self-adhesive materials are the first choice, and secondly the self-etching or total-etching adhesive materials containing acid or able to dissolve protein crystals are preferred.     

The structure and development of the collar enameloid in two teleost fishes, Halichoeres poecilopterus and Pagrus major

Summary Histologically the outer layer of the collar enameloid obviously differs from the inner layer, and it has a degree of mineralization nearly as high as the cap enameloid which has the highest. In the stage of matrix formation, the organic matrix of the collar enameloid contains a number of collagen fibers, and odontoblasts display features suggesting that these cells actively synthesized and secreted collagen. A number of cell processes, matrix vesicles and some cell debris which were probably derived from the odontoblasts were observed in the organic matrix of the collar enameloid. We consider that the majority of the organic matrix in collar enameloid originates from the odontoblasts. In the stage of maturation, collagen fibers were not observed in the outer layer of the collar enameloid in demineralized specimens. In the IDE cells during this stage, the complex infoldings of cell membranes developed in the distal portion, and several lysosomal granules and irregular-shaped granules containing many tubular structures, were observed in the distal cytoplasm. In the ODE cells, abundant labyrinthine canals appeared in the cytoplasm, and capillary vessels were found close to the outer surface of the ODE cells. We assume that the higher mineralized outer layer of the collar enameloid is made possible by the absorptive and transport functions of the epithelial cells during the stage of maturation. It is considered that the collar enameloid in this study was initially produced by the odontoblasts and then reconstructed by the epithelial cells, so that the collar enameloid differs from true enamel.     

A novel mutation in the FOXC1 gene in a family with Axenfeld-Rieger syndrome and Peters' anomaly

Peters anomaly and Axenfeld-Rieger syndrome (ARS) belong to the overlapping spectrum of disorders summarized as anterior segment dysgenesis (ASD). Five patients from a family with Peters' anomaly and ARS were screened for mutations in the PITX2, CYP1B1 and FOXC1 genes by direct sequencing. All affected family members examined were heterozygous for a single nucleotide substitution, resulting in a nonsense mutation (Q120X) at a highly conserved residue of the FOXC1 gene that is essential for DNA binding. In this pedigree, all affected family members were diagnosed with ARS except for one who shows bilateral Peters' anomaly. Our findings support the role of FOXC1 mutations in the spectrum of ASD.     

The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli

In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.     

Dual reporter system to dissect cis- and trans-effects influencing the mutation rate in a hypermutating cell line

Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18–81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18–81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18–81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.     

Functional consequences of an LMNA mutation associated with a new cardiac and non-cardiac phenotype

Heritable dilated cardiomyopathy is a genetically highly heterogeneous disease. To date 17 different chromosomal loci have been described for autosomal dominant forms of dilated cardiomyopathy with or without additional clinical manifestations. Among the 10 mutated genes associated with dilated cardiomyopathy, the lamin A/C (LMNA) gene has been reported in forms associated with conduction-system disease with or without skeletal muscle myopathy. For the first time, we report here a French family affected with a new phenotype composed of an autosomal dominant severe dilated cardiomyopathy with conduction defects or atrial/ventricular arrhythmias, and a specific quadriceps muscle myopathy. In all previously reported cases with both cardiac and neuromuscular involvement, neuromuscular disorders preceded cardiac abnormalities. The screening of the coding sequence of the LMNA gene on all family members was performed and we identified a missense mutation (R377H) in the lamin A/C gene that cosegregated with the disease in the family. Cell transfection experiments showed that the R377H mutation leads to mislocalization of both lamin and emerin. These results were obtained in both muscular (C2C12) and non-muscular cells (COS-7). This new phenotype points out the wide spectrum of neuromuscular and cardiac manifestations associated with lamin A/C mutations, with the functional consequence of this mutation seemingly associated with a disorganization of the lamina.     

Developmental abnormalities in the Nuc1 rat retina: a spontaneous mutation that affects neuronal and vascular remodeling and retinal function

The retina serves as an excellent model in which to study vertebrate CNS development. We have discovered a spontaneous mutation in the Sprague-Dawley rat that results in a novel and unusual ocular phenotype, including retinal abnormalities, that we have named Nuc1. We have previously shown that the Nuc1 mutation appears to suppress programmed cell death in the developing retina. Here we report that maturation of both the retinal neurons and the retinal vessels is abnormal in Nuc1 homozygous rats. The developmental changes in the retinal neurons and vasculature are correlated with regard to degree of abnormality. As Nuc1 homozygotes mature, focal retinal detachment begins at approximately 3 months after birth, and near total traction retinal detachment, associated with pre-retinal fibrosis and neovascularization, is evident by 18 months. Electroretinographic studies at 2.5 months of age indicate that functional retinal degeneration precedes retinal detachment. The functional abnormality is most evident in rods and the inner retina, and is present in homozygous but not heterozygous mutants. Immunocytochemical studies of rod and cone photoreceptors indicate abnormalities in rod, but not cone, photoreceptors in Nuc1 homozygotes, consistent with the electroretinographic findings. In Nuc1 animals, the Muller cells are activated. Although such activation may result from inflammation, Muller cells in Nuc1 may be reacting to a neuronal influence. It appears that the Nuc1 mutation plays a regulatory role in both developing and maturing ocular tissues. The Nuc1 mutation may also serve as an important genetic tool to explore the relationships that may exist among gliosis, normal neuronal development, and normal vascular development and how abnormalities in these associations lead to common retinal diseases.     

High-affinity uptake of L-[3H]glutamate and D-[3H]aspartate during postnatal development of the hippocampal formation: a quantitative autoradiographic study

Summary Quantitative autoradiography was used to determine the topographical and time patterns of L-[³H]glutamate and D-[³H]aspartate high-affinity uptake system in the hippocampal formation of the rat during postnatal development. Extended control experiments were performed to verify the specificity of labelling. For short incubation periods of 3–10 min, the data demonstrated a conspicuously low rate of glutamate accumulation in the hippocampal formation of newborn animals and a marked increase in labelling of hippocampal neuropil areas during the first weeks of postnatal life. Our autoradiographic data on developmental increase in glutamate high-affinity uptake levels are consistent, in terms of time and topography, in many ways with other parameters of maturation of glutamatergic and/or aspartatergic structures in the hippocampal formation.