Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma.

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)-spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

The Role of DSPP in Dentine Formation and Hereditary Dentine Defects.

The dentine sialophosphoprotein (DSPP) gene is the only identified causative gene for dentinogenesis imperfecta type 2 (DGI-II), dentinogenesis imperfecta type 3 (DGI-III) and dentine dysplasia type 2 (DD-II). These three disorders may have similar molecular mechanisms involved in bridging the DSPP mutations and the resulting abnormal dentine mineralisation. The DSPP encoding proteins DSP (dentine sialoprotein) and DPP (dentine phosphoprotein) are positive regulators of dentine formation and perform a function during dentinogenesis. The present review focused on the recent findings and viewpoints regarding the relationship between DSPP and dentinogenesis as well as mineralisation from multiple perspectives, involving studies relating to spatial structure and tissue localisation of DSPP, DSP and DPP, the biochemical characteristics and biological function of these molecules, and the causative role of the proteins in phenotypes of the knockout mouse model and in hereditary dentine defects.

A newly developed kit for dental apical root resorption detection: efficacy and acceptability.

OBJECTIVES: To determine the efficacy of a newly developed kit in dentine sialophosphoprotein (DSPP) detection and compare it with enzyme-linked immunosorbent assay (ELISA). User acceptance was also determined. MATERIALS AND METHODS: This cross-sectional study consisted of 45 subjects who were divided into 3 groups based on the severity of root resorption using radiographs: normal (RO), mild (RM), and severe (RS). DSPP in GCF samples was analyzed using both methods. Questionnaires were distributed to 30 orthodontists to evaluate future user acceptance. RESULTS: The sensitivity and specificity of the kit were 0.98 and 0.8 respectively. The DSPP concentrations measured using ELISA were the highest in the RS group (6.33 ± 0.85 ng/mL) followed by RM group (3.77 ± 0.36 ng/mL) and the RO group had the lowest concentration (2.23 ± 0.55 ng/mL). The new kit portrayed similar results as the ELISA, the optical density (OD) values were the highest in the RS group (0.62 ± 0.10) followed by RM group (0.33 ± 0.03) and the RO group (0.19 ± 0.06). The differences among all the groups were statistically significant (p < 0.05) for both methods. The Pearson correlation coefficient showed a statistically significant (p < 0.001) strong and positive correlation between DSPP concentrations and OD values. CONCLUSIONS: The new kit was validated to detect the colour intensities of different severity of root resorptions. Most of the responses to the survey were positive towards the new kit for being a safer and simpler method to detect apical root resorption.

Simple Binding and Dissociation of a Sialoglycoprotein Using Boronic Acid-Modified Functional Interfaces on Microparticles.

An overexpression of sialic acid is an indicator of metastatic cancer, and selective detection of sialic acid shows potential for cancer diagnosis. Boronic acid is a promising candidate for this purpose because of its ability to specifically bind to sialic acid under acidic conditions. Notably, the binding strength can be easily modulated by adjusting the pH, which allows for a simple dissociation of the bound sialic acid. In this study, we developed 5-boronopicolinic acid (5-BPA)-modified magnetic particles (BMPs) to selectively capture sialic acid biomolecules. We successfully captured fetuin, a well-known sialoglycoprotein, on BMPs at >104 molecules/particle using an acetate buffer (pH 5.0). Facile dissociation then occurred when the system was changed to a pH 7.6 phosphate buffer. This capture-and-release process could be repeated at least five times. Moreover, this system could enrich fetuin by more than 20 times. In summary, BMPs are functional particles for facile purification and concentration through the selective capture of sialic acid proteins and can improve detection sensitivity compared with conventional methods. This technology shows potential for the detection of sialic acid overexpression by biological particles.

Isomeric Separation of α2,3/α2,6-Linked 2-Aminobenzamide (2AB)-Labeled Sialoglycopeptides by C18-LC-MS/MS.

Determination of the relative expression levels of the α2,3/α2,6-sialic acid linkage isomers on glycoproteins is critical to the analysis of various human diseases such as cancer, inflammation, and viral infection. However, it remains a challenge to separate and differentiate site-specific linkage isomers at the glycopeptide level. Some derivatization methods on the carboxyl group of sialic acid have been developed to generate mass differences between linkage isomers. In this study, we utilized chemical derivatization that occurred on the vicinal diol of sialic acid to separate linkage isomers on a reverse-phase column using a relatively short time. 2-Aminobenzamide (2AB) labeling derivatization, including periodate oxidation and reductive amination, took only ∼3 h and achieved high labeling efficiency (>90%). Within a 66 min gradient, the sialic acid linkage isomers of 2AB-labeled glycopeptides from model glycoproteins can be efficiently resolved compared to native glycopeptides. Two different methods, neuraminidase digestion and higher-energy collision dissociation tandem mass spectrometry (HCD-MS2) fragmentation, were utilized to differentiate those isomeric peaks. By calculating the diagnostic oxonium ion ratio of Gal2ABNeuAc and 2ABNeuAc fragments, significant differences in chromatographic retention times and in mass spectral peak abundances were observed between linkage isomers. Their corresponding MS2 PCA plots also helped to elucidate the linkage information. This method was successfully applied to human blood serum. A total of 514 2AB-labeled glycopeptide structures, including 152 sets of isomers, were identified, proving the applicability of this method in linkage-specific structural characterization and relative quantification of sialic acid isomers.

Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs.

Several sialoglycopeptides were isolated from several fish eggs and exerted anti-osteoporosis effects. However, few papers have explored sialoglycopeptide from tuna eggs (T-ES). Here, a novel T-ES was prepared through extraction with KCl solution and subsequent enzymolysis. Pure T-ES was obtained through DEAE-Sepharose ion exchange chromatography and sephacryl S-300 gel filtration chromatography. The T-ES was composed of 14.07% protein, 73.54% hexose, and 8.28% Neu5Ac, with a molecular weight of 9481 Da. The backbone carbohydrate in the T-ES was →4)-ß-D-GlcN-(1→3)-α-D-GalN-(1→3)-ß-D-Glc-(1→2)-α-D-Gal-(1→2)-α-D-Gal-(1→3)-α-D-Man-(1→, with two branches of ß-D-GlcN-(1→ and α-D-GalN-(1→ linking at o-4 in →2,4)-α-D-Gal-(1→. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1→. A peptide chain, Ala-Asp-Asn-Lys-Ser*-Met-Ile that was connected to the carbohydrate chain through O-glycosylation at the -OH of serine. Furthermore, in vitro data revealed that T-ES could remarkably enhance bone density, bone biomechanical properties, and bone microstructure in SAMP mice. The T-ES elevated serum osteogenesis-related markers and reduced bone resorption-related markers in serum and urine. The present study's results demonstrated that T-ES, a novel sialoglycopeptide, showed significant anti-osteoporosis effects, which will accelerate the utilization of T-ES as an alternative marine drug or functional food for anti-osteoporosis.

[Recognition on dentin dysplasia type â ¡].

Dentin dysplasia type Ⅱ (DD-Ⅱ) is a subtype of hereditary dentin disorders. The dentin sialophosphoprotein (DSPP) gene has been revealed to be the causative gene, whose mutations could affect the normal tooth development process. The lesions involve both deciduous and permanent dentition, mainly manifested as tooth discoloration, attrition and even the subsequent malocclusion. If not treated in time, it will significantly affect the physical and psychological health of patients. The disease is difficult to be diagnosed in clinic accurately as its low incidence and hidden manifestations. The present article aims to discuss the clinical and radiographic characteristics, diagnosis, treatment of DD-Ⅱ, in order to improve the overall understanding on DD-Ⅱ for clinicians.

A novel strategy for quantification of α2,3- and α2,6-linked sialic acids in sialylated glycoproteins.

Sialic acid, a monosaccharide containing nine carbon atoms, is widely distributed in eukaryotic cells. The bound sialic acids are mainly present at the glycan ends of glycoconjugates via α2-3 or α2-6 glycosidic bonds, and alterations in their expression levels and linkage types are associated with the progress of many diseases and tumors. The present study provides a new strategy for quantification of α2,3- and α2,6-linked sialic acids in sialylated glycoproteins. In fact, quantification of α2,3-linked sialic acids were based on the difference of the bound sialic acids in the sample before and after treatment with α2-3 neuraminidase, whereas the α2,6-linked sialic acids were equal to the bound sialic acids in the α2-3 neuraminidase-treated sample. Subsequently, α2,3/6-linked sialic acids in salivary glycoproteins from healthy volunteers and diabetic patients were quantified in accordance with this method. This work provides an accurate method for the quantification of α2,3- and α2,6-linked sialic acids in the sialoglycoproteins, which is more instructive for understanding the biological roles of α2,3/6-linked sialic acid in sialoglycoproteins.

Thermodynamic Evolution of a Metamorphic Protein: A Theoretical-Computational Study of Human Lymphotactin.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins.

Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.

Bone sialoprotein promotes lung cancer osteolytic bone metastasis via MMP14-dependent mechanisms.

Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.

AAV6-Mediated Gene Therapy Prevents Developmental Dentin Defects in a Dentinogenesis Imperfecta Type â ¢ Mouse Model.

Dentin is a major type of hard tissue of teeth and plays essential roles for normal tooth function. Odontoblasts are responsible for dentin formation. Mutations or deficiency in various genes affect the differentiation of odontoblasts, leading to irreversible dentin developmental defects in animals and humans. Whether such dentin defects can be reversed by gene therapy for odontoblasts remains unknown. In this study, we compare the infection efficiencies of six commonly used adeno-associated virus (AAV) serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVDJ) in cultured mouse odontoblast-like cells (OLCs). We show that AAV6 serotype infects OLCs with the highest efficiency among the six AAVs. Two cellular receptors, which are able to recognize AAV6, AAV receptor (AAVR), and epidermal growth factor receptor (EGFR), are strongly expressed in the odontoblast layer of mouse teeth. After local administration to mouse molars, AAV6 infects the odontoblast layer with high efficiency. Furthermore, AAV6-Mdm2 was successfully delivered to teeth and prevents the defects in odontoblast differentiation and dentin formation in Mdm2 conditional knockout mice (a mouse model of dentinogenesis imperfecta type Ⅲ). These results suggest that AAV6 can serve as a reliable and efficient vehicle for gene delivery to odontoblasts through local injection. In addition, human OLCs were also successfully infected by AAV6 with high efficiency, and both AAVR and EGFR are strongly expressed in the odontoblast layer of extracted human developing teeth. These findings suggest that AAV6-mediated gene therapy through local injection may be a promising treatment approach for hereditary dentin disorders in humans.

Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.

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.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

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.

[Mutation of dentin sialophosphoprotein and hereditary malformations of dentin].

The classification as well as the clinical manifestations of hereditary malformations of dentin are of great concern and have been deeply elucidated. The understanding of its genetic basis also increases progressively. Dentin sialophosphoprotein (DSPP) is the pathogenic gene of dentinogenesis imperfecta type Ⅱ, dentinogenesis imperfecta type Ⅲ and dentin dysplasia type Ⅱ. In this article, the classification of DSPP mutations as well as the resultant dysfunction of the mutant DSPP are summarized respectively and the corresponding clinical manifestations are analyzed. This work will provide a reference for the diagnosis and treatment of hereditary malformations of dentin.

Tubular dentin formation by TGF-ß/BMP signaling in dental epithelial cells.

OBJECTIVES: This study aimed to identify formation of tubular dentin induced by transforming growth factor-ß (TGF-ß) and bone morphogenic protein (BMP) signaling pathway in dental epithelial cells. METHODS: We collected conditioned medium (CM) of rTGF-ß1/rBMP-2-treated HAT-7 and treated to MDPC-23 cells. The expression levels of odontoblast differentiation markers, KLF4, DMP1, and DSP were evaluated by real-time PCR and Western blot analysis. To evaluate whether CM of rTGF-ß1/rBMP-2 induces tubular dentin formation, we made a beagle dog tooth defect model. RESULTS: Here, we show that Cpne7 is regulated by Smad4-dependent TGF-ß1/BMP2 signaling pathway in dental epithelial cells. CM of rTGF-ß1/rBMP-2 treated HAT-7 or rCPNE7 raises the expression levels of KLF4, DMP1, and DSP in MDPC-23 cells. When rTGF-ß1 or rBMP-2 is directly treated to MDPC-23 cells, however, expression levels of Cpne7-regulated genes remain unchanged. In a beagle dog defect model, application of rTGF-ß1/BMP2-treated CM resulted in tubular tertiary dentin mixed with osteodentin at cavity-prepared sites, while rTGF-ß1 group exhibited homogenous osteodentin. CONCLUSIONS: Taken together, Smad4-dependent TGF-ß1/BMP2 signaling regulates Cpne7 in dental epithelial cells, and CPNE7 protein secreted from pre-ameloblasts mediates odontoblast differentiation via epithelial-mesenchymal interaction.

The BMP and FGF pathways reciprocally regulate odontoblast differentiation.

PURPOSE: Previous studies demonstrated that the exposure of primary dental pulp (DP) cultures to fibroblast growth factor 2 (FGF2) between days 3-7 exerted significant and long-lasting stimulatory effects on odontoblast differentiation and Dspp expression. These effects involved the increased expression of components of bone morphogenetic protein (BMP) signaling and were reverted by a BMP inhibitor noggin. FGF2 also transiently stimulated osteoblast differentiation and the expression of Ibsp and Dmp1. The present study aimed to further explore interactions between BMP and FGF signaling during odontoblast and osteoblast differentiation in DP cultures. MATERIALS AND METHODS: Cultures were established using DP tissue isolated from non-transgenic and fluorescent reporter (DSPP-Cerulean, BSP-GFP, and DMP1-mCherry) transgenic mice and exposed to BMP2, FGF2, SU5402 (an FGF receptor inhibitor), and noggin between days 3-7. Mineralization, gene expression, fluorescent protein expression, and odontoblast formation were examined using xylenol orange, quantitative PCR, fluorometric analysis, and immunocytochemistry, respectively. RESULTS: BMP2 activated SMAD1/5/8 but not ERK1/2 signaling, whereas FGF2 exerted opposite effects. BMP2 did not affect mineralization, the expression of Ibsp and Dmp1, and the percentage of DSPP-Cerulean+ odontoblasts but significantly increased Dspp and DSPP-Cerulean. In cultures exposed to BMP2 and FGF2, respectively, both SU5402 and noggin led to long-lasting decreases in Dspp and DSPP-Cerulean and transient decreases in Dmp1 and DMP1-mCherry without affecting Ibsp and BSP-GFP. CONCLUSION: BMP2 and FGF2 exerted reciprocal stimulatory effects on odontoblast differentiation, whereas their effects on osteoblast differentiation were mediated independently. These data will further elucidate the perspectives of using BMP2 and FGF2 for dentin regeneration/repair.

Biodentine but not MTA induce DSPP expression of dental pulp cells with different severity of LPS-induced inflammation.

OBJECTIVES: To explore the inflammatory and differentiation response in inflamed dental pulp cells (DPCs) induced by lipopolysaccharide (LPS) under different conditions with Biodentine and mineral trioxide aggregate (MTA) treatment. MATERIALS AND METHODS: DPCs were treated with 0.001-1 µg/mL LPS for different periods to induce inflammation. Normal and inflamed DPCs were further treated with 0.14 mg/mL Biodentine or 0.13 mg/mL MTA for different periods. mRNA expression level of IL-6, IL-8 and ALP were analysed by qPCR. DSPP protein expression was detected by western blot. The data were analysed by the Mann-Whitney test, unpaired t test or two-way ANOVA. RESULTS: After treatment for different times and with different concentrations of LPS, different severity of pulp inflammation was revealed by the expressions of IL-6 and IL-8. Higher concentrations of LPS induced higher IL-6 and IL-8 expressions, and these expressions first increased and then decreased (p < 0.0001). At 96 and 192 h, Biodentine significantly suppressed IL-6 expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine suppressed ALP expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine induced DSPP expressions in both normal and inflamed DPCs (p < 0.05). CONCLUSION: Biodentine enhanced more DSPP differentiation of both normal and inflamed DPCs under different treatment durations than MTA. CLINICAL RELEVANCE: The prognosis of vital pulp therapy may depend on the severity of pulp inflammation which is difficult to be determined in clinical settings. Therefore, Biodentine may enhance odontogenic differentiation in different severity of pulp inflammation imply its clinical indications.