Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice.

BACKGROUND: Extracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA. MATERIALS AND METHODS: To determine whether Dspp+/- mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/-, and Dspp homozygous (Dspp-/-) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/- mice over time, condyles of Dspp+/- and WT mice were analyzed radiologically, histologically, and immunohistochemically. RESULTS: Micro-CT and histomorphometric analyses revealed that Dspp+/- and Dspp-/- mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp-/- mice which exhibited tooth loss, Dspp+/- mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/- mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/- mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage. CONCLUSIONS: Dspp+/- mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.

Identification of DSPP novel variants and phenotype analysis in dentinogenesis dysplasia Shields type II patients.

OBJECTIVES: To investigate the genetic causes and teeth characteristics of dentin dysplasia Shields type II(DD-II) in three Chinese families. MATERIALS AND METHODS: Data from three Chinese families affected with DD-II were collected. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) were conducted to screen for variations, and Sanger sequencing was used to verify mutation sites. The physical and chemical characteristics of the affected teeth including tooth structure, hardness, mineral content, and ultrastructure were investigated. RESULTS: A novel frameshift deletion mutation c.1871_1874del(p.Ser624fs) in DSPP was found in families A and B, while no pathogenic mutation was found in family C. The affected teeth's pulp cavities were obliterated, and the root canals were smaller than normal teeth and irregularly distributed comprising a network. The patients' teeth also had reduced dentin hardness and highly irregular dentinal tubules. The Mg content of the teeth was significantly lower than that of the controls, but the Na content was obviously higher than that of the controls. CONCLUSIONS: A novel frameshift deletion mutation, c.1871_1874del (p.Ser624fs), in the DPP region of the DSPP gene causes DD-II. The DD-II teeth demonstrated compromised mechanical properties and changed ultrastructure, suggesting an impaired function of DPP. Our findings expand the mutational spectrum of the DSPP gene and strengthen the understanding of clinical phenotypes related to the frameshift deletion in the DPP region of the DSPP gene. CLINICAL RELEVANCE: A DSPP mutation can alter the characteristics of the affected teeth, including tooth structure, hardness, mineral content, and ultrastructure.

Novel dentin sialophosphoprotein gene frameshift mutations affect dentin mineralization.

OBJECTIVE: This study aimed to identify candidate genes for inheritable dentin defects in three Chinese pedigrees and characterize the property of affected teeth. DESIGN: Clinical and radiological features were recorded for the affected individuals. Genomic DNA obtained from peripheral venous blood or saliva were analyzed by whole-exome sequencing. The density and microhardness of affected dentin was measured. Scanning electron microscopy (SEM) was also performed to obtain the microstructure phenotype. RESULTS: 1) General appearance: the affected dentitions shared yellowish-brown or milky color. Radiographs showed that the pulp cavity and root canals were obliterated in varying degrees or exhibited a pulp aspect in the 'thistle tube'. Some patients exhibited periapical infections without pulpal exposure, and some affected individuals showed shortened, abnormally thin roots accompanied by severe alveolar bone loss. 2) Genomic analysis: three new frameshift mutations (NM_014208.3: c.2833delA, c.2852delGand c.3239delA) were identified in exon 5 of dentin sialophosphoprotein (DSPP) gene, altering dentin phosphoprotein (DPP) as result. In vitro studies showed that the density and microhardness of affected dentin were decreased, the dentinal tubules were sparse and arranged disorderly, and the dentinal-enamel-junction (DEJ) was abnormal. CONCLUSIONS: In this study, we identified three novel frameshift mutations of dentin sialophosphoprotein gene related to inherited dentin defects. These mutations are speculated to cause abnormal coding of dentin phosphoprotein C-terminus, which affect dentin mineralization. These results expand the spectrum of dentin sialophosphoprotein gene mutations causing inheritable dentin defects and broaden our understanding of the biological mechanisms by which dentin forms.

A novel DSPP frameshift mutation causing dentin dysplasia type 2 and disease management strategies.

The present study aims to investigate the mutation in a Chinese family with dentin dysplasia type II (DD-II) and to summarize mutation hotspots, clinical manifestations, and disease management strategies. Phenotype analysis, clinical intervention, mutation screening, and cosegregation analysis within the enrolled family were performed. A summary of the reported mutations in the dentin phosphoprotein (DPP) region of dentin sialophosphoprotein (DSPP) was analyzed. Pathogenicity prediction analysis of the physical properties and function of DSPP variants was performed by bioinformatic processing. Clinical management strategies are discussed. A novel pathogenic mutation (c.2035delA) in the DPP region of DSPP was identified, which was cosegregated in the family. The immature permanent teeth of patients with DD-II presented with X-shaped root canal phenotypes. Most of the identified mutations for DD-II were clustered in the DPP region between nucleotides 1686-2134. Points of differential diagnosis, clinical interventions, and management strategies are proposed. This study revealed a novel DSPP frameshift mutation and presented new clinical features of DD-II. The locus involving nucleotides 1686-2134 of DSPP may represent a mutational hotspot for the disease. Appropriate management of DD-II at different stages is important to avoid the development of secondary dental lesions.

Human Dental Pulp Stem Cells Differentiate into Cementoid-Like-Secreting Cells on Decellularized Teeth Scaffolds.

Periodontitis is a common inflammatory disease that in some cases can cause tooth loss. Cementum is a mineralized tissue that forms part of the insertion periodontium and serves to fix the teeth to the alveolar bone. In addition, it acts as a reservoir of different growth and differentiation factors, which regulate the biology of the teeth. Cementogenesis is a complex process that is still under investigation and involves different factors, including dentin sialophosphoprotein (DSPP). In this work we studied the role of surface microtopography in the differentiation of human dental pulp stem cells (hDPSCs) into cementoid-like secreting cells. We cultured hDPSCs on decellularized dental scaffolds on either dentin or cementum surfaces. Cell morphology was evaluated by light and electron microscopy. We also evaluated the DSPP expression by immunohistochemistry. The hDPSCs that was cultured on surfaces with accessible dentinal tubules acquired an odontoblastic phenotype and emitted characteristic processes within the dentinal tubules. These cells synthesized the matrix components of a characteristic reticular connective tissue, with fine collagen fibers and DSPP deposits. The hDPSCs that was cultured on cementum surfaces generated a well-organized tissue consisting of layers of secretory cells and dense fibrous connective tissue with thick bundles of collagen fibers perpendicular to the scaffold surface. Intra- and intercellular deposits of DSPP were also observed. The results presented here reinforce the potential for hDPSCs to differentiate in vitro into cells that secrete a cementoid-like matrix in response to the physical stimuli related to the microtopography of contact surfaces. We also highlight the role of DSPP as a component of the newly formed matrix.

The role of Dentin Sialophosphoprotein (DSPP) in craniofacial development.

DSPP is known to be important in the formation of dentin. In DSPP's absence, a severely hypomineralized dentin is formed, in a condition known as dentinogenesis imperfecta (DGI). DSPP has recently been found in several different tissues, including the mandibular condylar cartilage and craniofacial skeleton. However, there is limited literature on the role of DSPP in these tissues. Therefore, the objective of the present study was to investigate the role of DSPP in craniofacial development. Two mice strains, DSPP knockout and C57BL/6J wild type, were compared at 1, 3, and 6-months of age. Skulls and condyles were investigated through morphological and histological analyses. Cell culture was also conducted to investigate the potential effects of DSPP absence in osteoblasts from the calvaria. Mineralization defects were noticed in the structures of skulls and MCC, with the most significant impact at 1 month of age. Therefore, DSPP is an essential protein for the normal mineralization of craniofacial tissues.

Characterization of SIBLING Proteins in the Mineralized Tissues.

The SIBLING proteins are a family of non-collagenous proteins (NCPs) previously thought to be expressed only in dentin but have been demonstrated in other mineralized and non-mineralized tissues. They are believed to play vital roles in both osteogenesis and dentinogenesis. Since they are tightly regulated lifelong processes and involve a peak of mineralization, three different age groups were investigated. Fifteen wild-type (WT) mice were euthanized at ages 1, 3, and 6 months. Hematoxylin and eosin staining (H&E) was performed to localize various microscopic structures in the mice mandibles and tibias. The immunostaining pattern was compared using antibodies for dentin sialoprotein (DSP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). Immunostaining of DSP in tibia showed its most noticeable staining in the 3-month age group. DSP was expressed in alveolar bone, cellular cementum, and PDL. A similar expression of DMP1 was seen in the tibia and dentin. BSP was most noticeably detected in the tibia and acellular cementum. OPN was mainly expressed in the bone. A lower level of OPN was observed at all age groups in the teeth. The immunostaining intensity was the least detected for all proteins in the 6-month tibia sample. The expression patterns of the four SIBLING proteins showed variations in their staining intensity and temporospatial patterning concordant with skeletal and dental maturity. These findings suggest some role in this tightly regulated mineralization process.

Translated Mutant DSPP mRNA Expression Level Impacts the Severity of Dentin Defects.

Hereditary dentin defects are conventionally classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). Mutations in the dentin sialophosphoprotein (DSPP) gene have been identified to cause DGI type II and III and DD type II; therefore, these are not three different conditions, but rather allelic disorders. In this study, we recruited three families with varying clinical phenotypes from DGI-III to DD-II and performed mutational analysis by candidate gene analysis or whole-exome sequencing. Three novel mutations including a silent mutation (NM_014208.3: c.52-2del, c.135+1G>C, and c.135G>A; p.(Gln45=)) were identified, all of which affected pre-mRNA splicing. Comparison of the splicing assay results revealed that the expression level of the DSPP exon 3 deletion transcript correlated with the severity of the dentin defects. This study did not only expand the mutational spectrum of DSPP gene, but also advanced our understanding of the molecular pathogenesis impacting the severity of hereditary dentin defects.

Enamel Defects Associated With Dentin Sialophosphoprotein Mutation in Mice.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is highly expressed in odontoblasts, but only transiently expressed in presecretory ameloblasts during tooth development. We previously generated a knockin mouse model expressing a mouse equivalent (DSPP, p.P19L) of human mutant DSPP (p.P17L; referred to as "DsppP19L/+ "), and reported that DsppP19L/+ and DsppP19L/P19L mice manifested a dentin phenotype resembling human dentinogenesis imperfecta (DGI). In this study, we analyzed pathogenic effects of mutant P19L-DSPP on enamel development in DsppP19L/+ and DsppP19L/P19L mice. Micro-Computed Tomography (µCT) analyses of 7-week-old mouse mandibular incisors showed that DsppP19L/P19L mice had significantly decreased enamel volume and/or enamel density at different stages of amelogenesis examined. Acid-etched scanning electron microscopy (SEM) analyses of mouse incisors demonstrated that, at the mid-late maturation stage of amelogenesis, the enamel of wild-type mice already had apparent decussating pattern of enamel rods, whereas only minute particulates were found in DsppP19L/+ mice, and no discernible structures in DsppP19L/P19L mouse enamel. However, by the time that incisor enamel was about to erupt into oral cavity, distinct decussating enamel rods were evident in DsppP19L/+ mice, but only poorly-defined enamel rods were revealed in DsppP19L/P19L mice. Moreover, µCT analyses of the mandibular first molars showed that DsppP19L/+ and DsppP19L/P19L mice had a significant reduction in enamel volume and enamel density at the ages of 2, 3, and 24weeks after birth. Backscattered and acid-etched SEM analyses revealed that while 3-week-old DsppP19L/+ mice had similar pattern of enamel rods in the mandibular first molars as age-matched wild-type mice, no distinct enamel rods were observed in DsppP19L/P19L mice. Yet neither DsppP19L/+ nor DsppP19L/P19L mice showed well-defined enamel rods in the mandibular first molars by the age of 24weeks, as judged by backscattered and acid-etched SEM. In situ hybridization showed that DSPP mRNA level was markedly reduced in the presecretory ameloblasts, but immunohistochemistry revealed that DSP/DSPP immunostaining signals were much stronger within the presecretory ameloblasts in Dspp mutant mice than in wild-type mice. These results suggest that mutant P19L-DSPP protein caused developmental enamel defects in mice, which may be associated with intracellular retention of mutant DSPP in the presecretory ameloblasts.

Effects of DSPP Gene Mutations on Periodontal Tissues.

Dentin sialophosphoprotein ( DSPP ) gene mutations cause autosomal dominantly inherited diseases. DSPP gene mutations lead to abnormal expression of DSPP, resulting in a series of histological, morphological, and clinical abnormalities. A large number of previous studies demonstrated that DSPP is a dentinal-specific protein, and DSPP gene mutations lead to dentin dysplasia and dentinogenesis imperfecta. Recent studies have found that DSPP is also expressed in bone, periodontal tissues, and salivary glands. DSPP is involved in the formation of the periodontium as well as tooth structures. DSPP deficient mice present furcation involvement, cementum, and alveolar bone defect. We speculate that similar periodontal damage may occur in patients with DSPP mutations. This article reviewed the effects of DSPP gene mutations on periodontal status. However, almost all of the research is about animal study, there is no evidence that DSPP mutations cause periodontium defects in patients yet. We need to conduct systematic clinical studies on DSPP mutation families in the future to elucidate the effect of DSPP gene on human periodontium.

The role of NMT induction on odontogenic proliferation and differentiation of dental pulp stem cells.

This study was conducted to investigate the odontogenic proliferation and differentiation of dental pulp stem cells (DPSCs) after induction by nanoparticle mineral trioxide (NMT). DPSCs were isolated from permanent teeth and placed in tubes containing Dulbecco's modified Eagle's medium, followed by immunocytochemistry analysis. The viability of DPSCs exposed to NMT was measured using MTT assay with trypan blue dye exclusion. Alkaline phosphatase (ALP) activity was evaluated using ALP colorimetric reactions by reacting NMT supernatants with fluorescent-specific ALP substrates. The concentration of osteocalcin was determined using an instant human osteocalcin enzyme-linked immunosorbent assay (ELISA) kit. A human dentin sialophosphoprotein (DSPP) ELISA kit coated with anti-human DSPP antibody was employed to measure DSPP levels. There was a significant difference between ALP activity after exposing the cells to NMT and trioxide mineral aggregate on days 3, 7, and 21. Osteocalcin activity showed a significant difference on days 3, 7, 14, and 21. There was a significant difference in DSPP levels on days 7 and 21. DPSCs exposed to NMT and to trioxide mineral aggregate showed extracellular matrix formation on day 7 and 14, respectively. Furthermore, NMT may effectively increase the proliferation and differentiation of DPSCs as well as their maturation toward odontoblasts.

A Bioinformatics Systems Biology Analysis of the Current Oral Proteomic Biomarkers and Implications for Diagnosis and Treatment of External Root Resorption.

External root resorption (ERR) is a silent destructive phenomenon detrimental to dental health. ERR may have multiple etiologies such as infection, inflammation, traumatic injuries, pressure, mechanical stimulations, neoplastic conditions, systemic disorders, or idiopathic causes. Often, if undiagnosed and untreated, ERR can lead to the loss of the tooth or multiple teeth. Traditionally, clinicians have relied on radiographs and cone beam computed tomography (CBCT) images for the diagnosis of ERR; however, these techniques are not often precise or definitive and may require exposure of patients to more ionizing radiation than necessary. To overcome these shortcomings, there is an immense need to develop non-invasive approaches such as biomarker screening methods for rapid and precise diagnosis for ERR. In this review, we performed a literature survey for potential salivary or gingival crevicular fluid (GCF) proteomic biomarkers associated with ERR and analyzed the potential pathways leading to ERR. To the best of our knowledge, this is the first proteomics biomarker survey that connects ERR to body biofluids which represents a novel approach to diagnose and even monitor treatment progress for ERR.

Primary observation of the role of posttranslational modification of dentin sialophosphoprotein (DSPP) on postnatal development of mandibular condyle in mice.

OBJECTIVES: We aimed to observe the posttranslational role of dentin sialophosphoprotein (DSPP) on postnatal development of mandibular condyle in mice. METHODS: To explore the function of full-length DSPP, four groups of mice were employed: (1) wild type (WT) mice; (2)Dspp knockout (Dspp KO) mice; (3) mice expressing the normal DSPP transgene in the Dspp KO background (Dspp KO/normal Tg); (4) mice expressing the uncleavable full-length DSPP in the Dspp KO background (Dspp KO/D452A Tg). Firstly, Plain X-ray Radiography and Micro-computed Tomography were used to observe the condylar morphology changes of Dspp KO/D452A Tg mice in comparison with the other three groups. Then, Hematoxylin & eosin and toluidine blue staining were applied to uncover the histological changes of mandibular condylar cartilage (MCC) of Dspp KO/D452A Tg mice. To explore the function of the NH2-terminal fragments (i.e. DSP/DSP-PG), three groups of mice were employed: (1) WT mice; (2) Dspp KO mice; (3) mice expressing the NH2-terminal fragments of DSPP in the Dspp-null background (Dspp KO/DSP Tg). The former strategies were utilized to examine the differences of condylar morphology and histological structures changes within three groups of mice. RESULTS: Transgenic full-length DSPP partially maintained mandibular condylar morphology and MCC thickness of Dspp KO mice. Transgenic DSP failed to do so, but led to smaller mandibular condyle and disordered cartilage structure. CONCLUSIONS: Our observations provide insight into the role of posttranslational modification of DSPP in the postnatal development of healthy MCC and maintenance of condylar morphology.

Expression of biological markers in gingival crevicular fluid of teeth with orthodontically induced root resorption.

AIM: The goal was to investigate interleukin­1 receptor antagonist (IL-1ra) and dentin sialophosphoprotein (DSPP) levels in gingival crevicular fluid (GCF) as potential biomarkers for orthodontically induced root resorption (OIRR) using enzyme-linked immunosorbent immunoassay (ELISA). MATERIALS AND METHODS: In all, 74 subjects were assigned to one of three groups: (1) orthodontic group included orthodontic patients who showed radiographic evidence of 1-3 mm root resorption of a maxillary central incisor, (2) pediatric group included pediatric patients who showed radiographic evidence of physiologic root resorption of a lower second primary molar, and (3) control group included subjects who had no orthodontic treatment and showed no radiographic evidence of root resorption. Samples from the GCF were collected with endodontic absorbent paper points inserted 1 mm below the gingival margin of the tooth. The IL-1ra and DSPP levels were evaluated using ELISA. RESULTS: The IL-1ra levels were 657.5 ± 51.5, 319.9 ± 181.3, and 129.4 ± 54.9 pg/ml for the control, orthodontic, and pediatric groups, respectively. The DSPP levels were 1.6 ± 1.0, 30.1 ± 9.6, and 39.2 ± 3.3 pg/ml for the control, orthodontic, and pediatric groups, respectively. Post hoc analyses revealed significant differences for IL-1ra and DSPP between any two groups. Sensitivity and specificity of IL-1ra for the diagnosis of OIRR showed 80% reliability and a cutoff value of ≤432.6 pg/ml, while the analysis of DSPP showed 100% reliability and a cutoff value of ≥7.33 pg/ml. CONCLUSIONS: The levels of IL-1ra and DSPP detected in the orthodontic and pediatric groups indicate a possible association with OIRR. Efforts to develop tests for screening, diagnosis, and monitoring OIRR based on biological markers should continue.

Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT-mTOR signaling in the presence of CD44.

Purpose: In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation.Methods: We searched for a compound other than hyaluronan that induces odontoblastic differentiation of dental pulp stem cells and used western blotting to determine whether CD44 is involved in the induction of odontoblastic differentiation by the compound. We further validated the cell signaling details of the compound-induced expression of dentin sialophosphoprotein (DSPP), which is known as a marker of odontoblastic differentiation.Results: We investigated shikonin, which is one of the derivatives of naphthoquinone, the skeleton of vitamin K. Shikonin-induced expression of DSPP was inhibited by PI3K, AKT, and mTOR inhibitors. Additionally, shikonin-induced expression of DSPP was inhibited in dental pulp stem cells transfected with siRNA against CD44.Conclusions: Shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT-mTOR signaling pathway and CD44. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT-mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggests that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of the dental pulp.

Bobby sox homolog regulates tooth root formation through modulation of dentin sialophosphoprotein.

Tooth root development occurs through the interaction of multiple growth factors and transcription factors expressed in Hertwig's epithelial root sheath (HERS) and dental mesenchyme. Previously, we demonstrated that bobby sox homolog (Bbx) regulates odontoblast differentiation of human dental pulp stem cells. Here, we generated Bbx knockout (Bbx-/- ) mice to address the functional role of Bbx in tooth formation. During tooth development, Bbx was expressed in both dental epithelium and mesenchyme. However, molar and incisor morphology in Bbx-/- mice at postnatal Day 0 (P0) exhibited no prominent abnormalities compared with their wild-type (Bbx+/+ ) littermates. Until P28, the crown morphology in Bbx-/- mice was not distinctively different from Bbx+/+ littermates. Meanwhile, the length of the mandibular base in Bbx-/- mice was notably less at P28. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first molar were reduced by 21.33% and 16.28% at P14 and 16.28% and 16.24% at P28, respectively, in Bbx-/- mice. The second molar of Bbx-/- mice also showed 10.16% and 6.4% reductions at P28 in the mesial and distal lengths, compared with Bbx+/+ mice, respectively. The gene expression analysis during early tooth root formation (P13) showed that the expression of dentin sialophosphoprotein (Dspp) was significantly decreased in Bbx-/- mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression.

Phenotype and molecular characterizations of a family with dentinogenesis imperfecta shields type II with a novel DSPP mutation.

BACKGROUND: Dentinogenesis imperfecta (DGI), Shields type-II is an autosomal dominant genetic disease which severely affects the function of the patients' teeth. The dentin sialophosphoprotein (DSPP) gene is considered to be the pathogenic gene of DGI-II. In this study, a DGI-II family with a novel DSPP mutation were collected, functional characteristics of DGI cells and clinical features were analyzed to better understand the genotype-phenotype relationship of this disease. METHODS: Clinical data were collected, whole exome sequencing (WES) was conducted, and Sanger sequencing was used to verify the mutation sites. Physical characteristics of the patient's teeth were examined using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The localization of green fluorescent protein (GFP)-fused wild-type (WT) dentin sialoprotein (DSP) and its variant were evaluated via an immunocytochemistry (ICC) assay. The behaviors of human dental pulp stem cells (hDPSCs) were investigated by flow cytometry, osteogenic differentiation, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: A novel heterozygous mutation c.53T > G (p. Val18Gly) in DSPP was found in this family. The SEM results showed that the participants' teeth had reduced and irregular dentinal tubes. The EDS results showed that the Ca/P ratio of the patients' teeth was significantly higher than that of the control group. The ICC assay showed that the mutant DSP was entrapped in the endoplasmic reticulum (ER), while the WT DSP located mainly in the Golgi apparatus. In comparison with normal cells, the patient's cells exhibited significantly decreased mineralization ability and lower expression levels of DSPP and RUNX2. CONCLUSIONS: The c.53T > G (p. Val18Gly) DSPP variant was shown to present with rare hypoplastic enamel defects. Functional analysis revealed that this novel variant disturbs dentinal characteristics and pulp cell behavior.

Effect of aspartic acid on the crystallization kinetics of ACP and dentin remineralization.

Type I collagen and non-collagen proteins are the main organic components of dentin. This study aimed to investigate the biomimetic remineralization of demineralized dentin by aspartic acid (Asp), which is abundant in non-collagenous proteins (NCPs). Asp was added to a mineralizing solution containing polyacrylic acid (PAA) to explore the mechanism of Asp regulating the pure amorphous calcium phosphate (ACP) phase transition process. The remineralization process and superstructure of the remineralized layer of demineralized dentin were evaluated and analyzed by transmission electron microscope (TEM) and scanning electron microscope (SEM), and the biological stability of the remineralized layer was investigated by collagenase degradation experiment. It demonstrated that Asp promoted the crystallization kinetics of PAA-stabilized amorphous calcium phosphate to hydroxyapatite (HAP), and shortened the remineralization time of demineralized dentin from 7 days to 2 days. The newly formed remineralized dentin had similar morphology and biological stability to the natural dentin layer. The presence of a large number of Asp residues in NCPs promoted the phase transformation of ACP, and further revealed the mechanism of action of NCPs in dentin biomineralization. This experiment also showed that Asp promoted the biomimetic remineralization of dentin; the morphology and hierarchical structure of remineralized layer was similar to that of natural teeth, and had good biological properties.

Odontogenic differentiation of inflamed dental pulp stem cells (IDPSCs) on polycaprolactone (PCL) nanofiber blended with hydroxyapatite.

The objective of this study was to develop electrospun polycaprolactone (PCL) membranes blended with hydroxyapatite (HA) and evaluate its potential in differentiating inflamed dental pulp stem/progenitor cells (IDPSCs) into odontoblasts. Electrospun nanofibrous membrane consisting of PCL blended with 10 wt% and 15 wt% of HA were fabricated and the characterization was done by Scanning electron microscopy (SEM), Fourier- transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and contact angle analysis. Cytocompatibility, cell adhesion and odontogenic differentiation ability of the membranes were assessed by MTT, Live/Dead, SEM/DAPI and qPCR studies. The mineral deposition ability of the membranes with IDPSCs was estimated by SEM-EDS. The SEM analysis revealed a nanofibrous texture with an average fiber diameter of 140 nm for PCL, 220 nm for PCL10%HA and 250 nm for PCL15%HA. Among the membranes tested, PCL10%HA favored positive cell attachments, upregulated expression of DSPP and ALP gene and higher Ca/P ratio compared to PCL and PCL15%HA.

Dentin Sialophosphoprotein Deletion Leads to Femoral Head Cartilage Attenuation and Subchondral Bone Ill-mineralization.

Dentin sialophosphoprotein (DSPP), which expresses and synthesizes in odontoblasts of dental pulp, is a critical protein for normal teeth mineralization. Originally, DSPP was identified as a dentin-specific protein. In 2010, DSPP was also found in femoral head cartilage, and it is still unclear what roles DSPP play in femoral head cartilage formation, growth, and maintenance. To reveal biological functions of DSPP in the femoral head cartilage, we examined Dspp null mice compared with wild-type (WT) mice to observe DSPP expression as well as localization in WT mice and to uncover differences of femoral head cartilage, bone morphology, and structure between these two kinds of mice. Expression data demonstrated that DSPP had heterogeneous fragments, expressed in each layer of femoral head cartilage and subchondral bone of WT mice. Dspp null mice exhibited a significant reduction in the thickness of femoral head cartilage, with decreases in the amount of proliferating cartilage cells and increases in apoptotic cells. In addition, the subchondral bone mineralization decreased, and the expressions of vessel markers (vascular endothelial growth factor [VEGF] and CD31), osteoblast markers (Osterix and dentin matrix protein 1 [DMP1]), osteocyte marker (sclerostin [SOST]), and osteoclast marker (tartrate-resistant acid phosphatase [TRAP]) were remarkably altered. These indicate that DSPP deletion can affect the proliferation of cartilage cells in the femoral head cartilage and endochondral ossification in subchondral bone. Our data clearly demonstrate that DSPP plays essential roles in the femoral head cartilage growth and maintenance and subchondral biomineralization.