Safe and Durable Treatment of Dentin Hypersensitivity via Nourishing and Remineralizing Dentin Based on ß-Chitooligosaccharide Graft Derivative.

Dentin hypersensitivity (DH) is a common symptom of various dental diseases that usually produces abnormal pain with external stimuli. Various desensitizers are developed to treat DH by occluding dentine tubules (DTs) or blocking intersynaptic connections of dental sensory nerve cells. However, the main limitations of currently available techniques are the chronic toxic effects of chemically active ingredients and their insufficiently durable efficacy. Herein, a novel DH therapy with remarkable biosafety and durable therapeutic value based on ß-chitooligosaccharide graft derivative (CAD) is presented. Particularly, CAD indicates the most energetic results, restoring the amino polysaccharide protective membrane in DTs, significantly promoting calcium and phosphorus ion deposition and bone anabolism, and regulating the levels of immunoglobulin in saliva and cellular inflammatory factors in plasma. Exposed DTs are occluded by remineralized hydroxyapatite with a depth of over 70 µm, as shown in in vitro tests. The bone mineral density of Sprague-Dawley rats' molar dentin increases by 10.96%, and the trabecular thickness of bone improves to about 0.03 µm in 2 weeks in the CAD group compared to the blank group. Overall, the ingenious concept that modified marine biomaterial can be a safe and durable therapy for DH is demonstrated by nourishing and remineralizing dentin.

A basic protein, N25, from a mollusk modifies calcium carbonate morphology and shell biomineralization.

Biomineralization is a widespread biological process in the formation of shells, teeth, or bones. Matrix proteins in biominerals have been widely investigated for their roles in directing biomineralization processes such as crystal morphologies, polymorphs, and orientations. Here, we characterized a basic matrix protein, named mantle protein N25 (N25), identified previously in the Akoya pearl oyster (Pinctada fucata). Unlike some known acidic matrix proteins containing Asp or Glu as possible Ca2+-binding residues, we found that N25 is rich in Pro (12.4%), Ser (12.8%), and Lys (8.8%), suggesting it may perform a different function. We used the recombinant protein purified by refolding from inclusion bodies in a Ca(HCO3)2 supersaturation system and found that it specifically affects calcite morphologies. An X-ray powder diffraction (XRD) assay revealed that N25 could help delay the transformation of vaterites (a metastable calcium carbonate polymorph) to calcite. We also used fluorescence super-resolution imaging to map the distribution of N25 in CaCO3 crystals and transfected a recombinant N25-EGFP vector into HEK-293T cells to mimic the native process in which N25 is secreted by mantle epithelial cells and integrated into mineral structures. Our observations suggest N25 specifically affects crystal morphologies and provide evidence that basic proteins lacking acidic groups can also direct biomineralization. We propose that the attachment of N25 to specific sites on CaCO3 crystals may inhibit some crystal polymorphs or morphological transformation.

The Microstructure, Proteomics and Crystallization of the Limpet Teeth.

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs, and clathrin, are identified by proteomics. A goethite-binding experiment coupled with proteomics and RNA-seq highlights six chitin-binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.

Pf-Sp8/9, a novel member of the specificity protein family in Pinctada fucata, potentially participates in biomineralization.

Specificity protein (Sp) belong to a transcription factor family that contains nine subgroups with essential functions in development, including skeletogenesis, tooth development, neural tube closure, and limb formation. In molluscs, functions of the Sp protein family members have not been reported in detail. In this study, we report the first Sp protein-encoding gene in Pinctada fucata. We named the translated protein Pf-Sp8/9, based on the phylogenetic development tree constructed using Sp protein sequences from six model organisms, which showed that it was a Sp8/9 homolog. Alignment of the Pf-Sp8/9 sequence with the amino acid sequences of related proteins showed that Pf-Sp8/9 had conserved domains, including three DNA-binding motifs. The tissue distribution showed that while Pf-Sp8/9 mRNA expression was detected in all tested tissues, it was particularly high in the mantle. The luciferase reporter assay results showed that Pf-Sp8/9 had the ability to activate the transcription of a number of matrix proteins. The expression pattern of Pf-Sp8/9 during P. fucata pearl sac development was similar to that of some genes that encode matrix proteins, suggesting Pf-Sp8/9 may be involved in mantle-related physiological activities and biomineralization.

Microplastics impact shell and pearl biomineralization of the pearl oyster Pinctada fucata.

Microplastics are extremely widespread aquatic pollutants that severely detriment marine life. In this study, the influence of microplastics on biomineralization was investigated. For the first time, multiple forms and types of microplastics were detected and isolated from the shells and pearls of Pinctada fucata. According to the present study, the abundance of microplastics in shells and pearls was estimated at 1.95 ± 1.43 items/g and 0.53 ± 0.37 items/g respectively. Interestingly, microplastics were less abundant in high-quality round pearls. Microplastics may hinder the growth of calcite and aragonite crystals, which are crucial components required for shell formation. During the process of biomineralization microplastics became embedded in shells, suggesting the existence of a novel pathway by which microplastics accumulate in bivalves. After a 96-h exposure to microplastics, the expression level of typical biomineralization-related genes increased, including amorphous calcium carbonate binding protein (ACCBP) gene which experienced a significant increase. ACCBP promotes the formation of amorphous calcium carbonate (ACC), which is the pivotal precursor of shell formation-related biominerals. ACCBP is highly expressed during the developmental stage of juvenile oysters and the shell-damage repair process. The increased expression of ACCBP suggests biomineralization is enhanced as a result of microplastics exposure. These results provide important evidence that microplastics exposure may impact the appearance of biominerals and the expression of biomineralization-related genes, posing a new potential threat to aquatic organisms.

Biomineral proteomics: A tool for multiple disciplinary studies.

The hard tissues of animals, such as skeletons and teeth, are constructed by a biologically controlled process called biomineralization. In invertebrate animals, biominerals are considered important for their evolutionary success. These biominerals are hieratical biocomposites with excellent mechanical properties, and their formation has intrigued researchers for decades. Although proteins account for ~5 wt% of biominerals, they are critical players in biomineralization. With the development of high-throughput analysis methods, such as proteomics, biomineral protein data are rapidly accumulating, thus necessitating a refined model for biomineralization. This review focuses on biomineral proteomics in invertebrate animals to highlight the diversity of biomineral proteins (generally 40-80 proteins), and the results indicate that biomineralization includes thermodynamic crystal growth as well as intense extracellular matrix activity and/or vesicle transport. Biominerals have multiple functions linked to biological immunity and antipathogen activity. A comparison of proteomes across species and biomineral types showed that von Willebrand factor type A and epidermal growth factor, which frequently couple with other extracellular domains, are the most common domains. Combined with species-specific repetitive low complexity domains, shell matrix proteins can be employed to predict biomineral types. Furthermore, this review discusses the applications of biomineral proteomics in diverse fields, such as tissue regeneration, developmental biology, archeology, environmental science, and material science.

PU14, a Novel Matrix Protein, Participates in Pearl Oyster, Pinctada Fucata, Shell Formation.

Biomineralization is a widespread biological process, involved in the formation of shells, teeth, and bones. Shell matrix proteins have been widely studied for their importance during shell formation. In 2015, our group identified 72 unique shell matrix proteins in Pinctada fucata, among which PU14 is a matrix protein detected in the soluble fraction that solely exists in the prismatic layer. However, the function of PU14 is still unclear. In this study, the full-length cDNA sequence of PU14 was obtained and functional analyses of PU14 protein during shell formation were performed. The deduced protein has a molecular mass of 77.8 kDa and an isoelectric point of 11.34. The primary protein structure contains Gln-rich and random repeat units, which are typical characteristics of matrix protein and indicate its potential function during shell formation. In vivo and in vitro experiments indicated PU14 has prismatic layer functions during shell formation. The tissue expression patterns showed that PU14 was mainly expressed in the mantle tissue, which is consistent with prismatic layer formation. Notching experiments suggested that PU14 responded to repair and regenerate the injured shell. After inhibiting gene expression by injecting PU14-specific double-stranded RNA, the inner surface of the prismatic layer changed significantly and became rougher. Further, in vitro experiments showed that recombinant protein rPU14 impacted calcite crystal morphology. Taken together, characterization and functional analyses of a novel matrix protein, PU14, provide new insights about basic matrix proteins and their functions during shell formation.

[Effect of nanoparticle with vascular endothelial growth factor gene transferred into ischemic myocardium: experiment with rabbits].

OBJECTIVE: To evaluate the possibility and efficiency of nanoparticle as a new vector in vascular endothelial growth factor (VEGF) gene transference, and investigate the efficacy of direct gene transfer of nanoparticle with VEGF(165) gene into ischemic myocardium. METHODS: Nanoparticle-VEGF (Np/VEGF) complex was prepared with poly (D, L-lactide-co-glycolide) (PLGA) loading VEGF(165) gene and the envelopment efficiency and size of the complex were determined. The Np/VEGF was transfected into the cultured myocardial cells, RT-PCR and ELISA were used to evaluate the transfection of VEGF. Suspension of Np/VEGF was injected into the myocardial tissue of 4 rabbits. 96 hours after operation myocardial tissue was obtained, made into sections, and observed with electron microscope. New Zealand White rabbits underwent thoracotomy followed by ligation of left anterior descending coronary artery to establish ischemic models. The New Zealand White rabbits were divided into 3 groups: Np/VEGF group (n = 12, nanoparticle with VEGF(165) were injected into the cordial myocardium), blank plasmid group (n = 12, injected with blank VEGF(165) plasmid), and control group (n = 8, injected with normal saline). Ultrasonography and immunohistochemistry with factor VIII related antigen were conducted to evaluate the cardiac function and the collateral circulation of the occluded artery. One month later the rabbits were killed to observe the vascularization of capillaries in the ischemic myocardium. RESULTS: The envelopment efficiency of the Np/VEGF complex thus prepared, 50 - 300 nm in size, were 1.87% y. RT-PCR and ELISA showed that VEGF gene had been successfully transfected into myocardial cells by the nanoparticles. A great number of nanoparticles were observed in the myocardial cytoplasm and nuclei. One month after operation, the ventricular wall motor amplitude of the Np/VEGF group was 1.87 mm +/- 0.32 mm, significantly larger than those of the blank plasmid group (1.59 mm +/- 0.24 mm, P < 0.05) and control group (0.93 mm +/- 0.40 mm, P < 0.05); and the left ventricular ejection fraction of the Np/VEGF group was 60% +/- 10%, significantly higher than those of the blank plasmid group (50% +/- 6%, P < 0.05) and control group (40% +/- 8%, P < 0.05). The capillary density at low power field (x 100) of the Np/VEGF group was 57 +/- 12, significantly higher than those of the VEGF group (41 +/- 14) and control group (24 +/- 8). CONCLUSION: Nanoparticle can act as a vector to transfect specific gene in vitro and in vivo. Direct gene transfer of nanoparticle with DNA encoding VEGF into the ischemic rabbit myocardium can increase capillary number; therefore it may be a novel therapeutic approach for myocardial ischemia.

Extensible byssus of Pinctada fucata: Ca(2+)-stabilized nanocavities and a thrombospondin-1 protein.

The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca(2+) by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the ß-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater.