Self-assembled branched polypeptides as amelogenin mimics for enamel repair.

The regeneration of demineralized enamel holds great significance in the treatment of dental caries. Amelogenin (Ame), an essential protein for mediating natural enamel growth, is no longer secreted after enamel has fully matured in childhood. Although biomimetic mineralization based on peptides or proteins has made significant progress, easily accessible, low-cost, biocompatible and highly effective Ame mimics are still lacking. Herein, we construct a series of amphiphilic branched polypeptides (CAMPs) by facile coupling of the Ame's C-terminal segment and poly(γ-benzyl-L-glutamate), which serves to simulate the Ame's hydrophobic N-terminal segment. Among them, CAMP15 is the best biomimetic mineralization template with great self-assembly performance to guide the oriented crystallization of hydroxyapatite and is capable of inhibiting the adhesion of Streptococcus mutans and Staphylococcus aureus on the enamel surfaces. This work highlights the potential application of amphiphilic branched polypeptide as Ame mimics in repairing defected enamel, providing a promising strategy for prevention and treatment of dental caries.

Self-assembly nanoplatform of platinum (â £) prodrug for enhanced ovarian cancer therapy.

Cisplatin is a metal platinum complex commonly used in the field of anti-tumor and one of the most commonly used drugs in combination chemotherapy. However, chemotherapy with Cisplatin induced overexpression of cyclooxygenase-2 (COX-2) protein in tumor cells, which could impair the therapeutic effect of chemotherapy on tumor progression. Here, we presented a novel method for the treatment of ovarian cancer with a self-assembly based nano-system. Cisplatin and tolfenamic acid were each linked to linoleic acid to give them the ability to self-assemble into nanoparticles in water. TPNPs had flexible drug ratio adjustability, homogeneous stability, and high drug loading capacity. Compared with Cisplatin, TPNPs could promote cellular uptake and tumor aggregation, co-induce enhanced apoptosis and tumor growth inhibition by inhibiting COX-2 in the mice xenograft model of human ovarian cancer, and reduce systemic toxicity. Therefore, TPNPs is a promising antitumor drug as a kind of self-assembly nano-prodrug with high drug load.

Direct control of shell regeneration by the mantle tissue in the pearl oyster Pinctada fucata.

Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells.

Proteomics of Shell Matrix Proteins from the Cuttlefish Bone Reveals Unique Evolution for Cephalopod Biomineralization.

In contrast to the external shells in bivalves and gastropods, most cephalopods are missing this external protection. The cuttlefish, belonging to class cephalopod, has an internal biomineralized structure made of mainly calcium carbonate for controlling buoyancy. However, the macromolecules, especially proteins that control cuttlebone mineral formation, are not sufficiently understood, limiting our understanding of the evolution of this internal shell. In this study, we extracted proteins from the cuttlebone of pharaoh cuttlefish Sepia pharaonis and performed liquid chromatography-tandem mass spectrometry to identify the shell matrix proteins (SMPs). In total, 41 SMPs were identified. Among them, hemocyanin, an oxygen-carrying protein, was the most abundant SMP. By comparison with SMPs of other marine biominerals, hemocyanin, apolipophorin, soul domain proteins, transferrin, FL-rich, and enolase were found to be unique to the cuttlebone. In contrast, typical SMPs of external shells such as carbonic anhydrase complement control protein, fibronectin type III, and G/A-rich proteins were lacking from the cuttlebone. Furthermore, the cluster analysis of biomineral SMPs suggests that the SMP repertoire of the cuttlebone does not resemble that of other species with external shells. Taken together, this study implies a potential relationship of the cuttlefish internal shell with other internal biominerals, which highlights a unique shell evolutionary pathway in invertebrates.

Phosphorylated and Phosphonated Low-Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel.

Biomimetic mineralization based on self-assembly has made great progress, providing bottom-up strategies for the construction of new organic-inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self-assembling low-complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

Heterogeneous distribution of shell matrix proteins in the pearl oyster prismatic layer.

Shell formation in molluscan bivalves is regulated by organic matrices composed of biological macromolecules, but how these macromolecules assemble in vitro remains elusive. Prismatic layer in the pearl oyster Pinctada fucata consists of polygonal prisms enveloped by thick organic matrices. In this study, we found that the organic matrices were heterogeneously distributed, with highly acidic fractions (EDTA-soluble and EDTA-insoluble) embedded inside the prism columns, while basic EDTA-insoluble faction as inter-column framework enveloping the prisms. The intra-column matrix was enriched in aspartic acid whereas the inter-column matrix was enriched in glycine, tyrosine and phenylalanine. Moreover, the intra-column matrix contained sulfo group further contributing to its acidic property. Proteomics data showed that the intra-column proteins mainly consisted of acidic proteins, while some typical matrix proteins were absent. The absent matrix proteins such as shematrin family and KRMP family were highly basic and contained aromatic amino acids, suggesting that electric charge and hydrophobic effect might play a role in the matrix heterogeneity. Interestingly, chitin metabolism related proteins were abundant in the inter-column matrix, which may be involved in reconstructing the prism organic matrix. Overall, our study suggests that each single prism grew in an enclosed organic envelope and the organic matrix undergoes rearrangement, thus leading to the peculiar growth of the prismatic layer.

Transition from horizontal expansion to vertical growth in the oyster prismatic layer.

Biomimetic materials inspired by biominerals have substantial applications in various fields. The prismatic layer of bivalve molluscs has extraordinary flexibility compared to inorganic CaCO3. Previous studies showed that in the early stage, minerals expanded horizontally and formed prism domains as a Voronoi division, while the evolution of the mature prisms were thermodynamically driven, which was similar to grain growth. However, it was unclear how the two processes were correlated during shell formation. In this study, we used scanning electronic microscopy and laser confocal scanning microscopy to look into the microstructure of the columnar prismatic layer in the pearl oyster Pinctada fucata. The Dirichlet centers of the growing domains in mature prisms were calculated, and the corresponding Voronoi division was reconstructed. It was found that the domain pattern did not fit the Voronoi division, indicating the driving forces of the mature prisms evolution and the initiation stage were different. During the transition from horizontal expansion to vertical growth, the minerals broke through the inner periostracum and squeezed out the organic materials to the inter-prism space. Re-arrangement of the organic framework pattern was driven by elastic relaxation at the vertices, indicating the transition process was thermodynamically driven. Our study provided insights into shell growth in bivalves and pave the way to synthesize three-dimensional material biomimetically.

Functional identification of a rare vascular endothelial growth factor a (VEGFA) variant associating with the nonsyndromic cleft lip with/without cleft palate.

Vascular endothelial growth factor A (VEGFA) is a crucial growth factor, which participates in multiple processes of human growth and development, such as angiogenesis and osteogenesis and is also necessary for development of palate. The purpose of this study was to investigate the effect of a rare VEGFA mutation (NM_001025366.2 773 T > C p.Val258Ala) on the cell functions and osteogenesis. Here, we found that the VEGFA mutation has adverse effects on the function of human embryonic palatal plate mesenchymal (HEPM) cells, and may affect the development of palate. The VEGFA mutation has adverse effects on promoting cell proliferation and migration and inhibiting apoptosis in HEPM and HEK-293 cells. In addition, the mutant VEGFA allele has a negative influence on osteogenesis. Taken together, the rare variant of the VEGFA gene had an adverse effect on cell functions and osteogenesis, which may impact the development of the palate. And these findings suggested that VEGFA mutation (c.773 T > C) may lead to nonsyndromic cleft lip with or without cleft palate and also provide a new insight into the mechanism of VEGFA gene in osteogenesis and palatogenesis.

Loss of BMP signaling mediated by BMPR1A in osteoblasts leads to differential bone phenotypes in mice depending on anatomical location of the bones.

Bone morphogenetic protein (BMP) signaling in osteoblasts plays critical roles in skeletal development and bone homeostasis. Our previous studies showed loss of function of BMPR1A, one of the type 1 receptors for BMPs, in osteoblasts results in increased trabecular bone mass in long bones due to an imbalance between bone formation and bone resorption. Decreased bone resorption was associated with an increased mature-to-immature collagen cross-link ratio and mineral-matrix ratios in the trabecular compartments, and increased tissue-level biomechanical properties. Here, we investigated the bone mass, bone composition and biomechanical properties of ribs and spines in the same genetically altered mouse line to compare outcomes by loss of BMPR1A functions in bones from different anatomic sites and developmental origins. Bone mass was significantly increased in both cortical and trabecular compartments of ribs with minimal to modest changes in compositions. While tissue-levels of biomechanical properties were not changed between control and mutant animals, whole bone levels of biomechanical properties were significantly increased in association with increased bone mass in the mutant ribs. For spines, mutant bones showed increased bone mass in both cortical and trabecular compartments with an increase of mineral content. These results emphasize the differential role of BMP signaling in osteoblasts in bones depending on their anatomical locations, functional loading requirements and developmental origin.

Cloning, characterization and functional analysis of an Alveoline-like protein in the shell of Pinctada fucata.

Shell matrix proteins (SMPs) have important functions in biomineralization. In the past decades, the roles of SMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata, among which Alveoline-like (Alv) protein was reported to have homologous genes in Pinctada maxima and Pinctada margaritifera. In this study, the full-length cDNA sequence of Alv and the functional analysis of Alv protein during shell formation were explored. The deduced protein (Alv), which has a molecular mass of 24.9 kDa and an isoelectric point of 11.34, was characterized, and the functional analyses was explored in vivo and in vitro. The Alv gene has high expression in mantle and could response to notching damage. The functional inhibition of Alv protein in vivo by injecting recombinant Alv (rAlv) antibodies destroyed prism structure but accelerated nacre growth. Western blot and immunofluorescence staining showed that native Alv exists in the EDTA-insoluble matrix of both prismatic and nacreous layers and has different distribution patterns in the inner or outer prismatic layer. Taken together, the characterization and functional analyses of matrix protein Alv could expand our understanding of basic matrix proteins and their functions during shell formation.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization.

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

The receptor genes PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in the pearl oyster Pinctada fucata.

Mounting evidence suggests that TGFß/BMP signaling pathway is most likely involved in shell biomineralization in molluscs, but the function of pathway receptors is poorly studied. Here, we cloned and identified two homologous BMP receptor genes, PfBMPR1B and PfBAMBI, from the pearl oyster Pinctada fucata. Real-time quantitative PCR and in situ hybridization revealed that these genes were expressed in mantle edge and pallial, specifically located at the outer epithelia. Knockdown of PfBMPR1B by RNA interference (RNAi) significantly decreased the expression levels of matrix protein (MP) genes and induced the abnormal ultrastructure of prismatic and nacreous layers. Conversely, knockdown of PfBAMBI significantly increased the expression levels of a portion of MP genes and induced the overgrowth of nacreous layer crystals. In the RNAi and shell notching experiments, MP gene expressions were competitively regulated by PfBMPR1B and PfBAMBI. In addition, the receptor inhibitor LDN193189 reduced the expression levels of MP genes in mantle primary cells and larvae, and induced abnormal D-shaped shell formation during larval development. Collectively, these results clearly show that PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in P. fucata. Our study therefore provides the direct evidence that BMP receptors participate in mollusc biomineralization.

Hemocytes participate in calcium carbonate crystal formation, transportation and shell regeneration in the pearl oyster Pinctada fucata.

In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, hemocyte release and transportation, and hemocyte distribution at the shell regeneration area and to analyse the proteome of hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of hemocytes in shell regeneration. Overall, this study provided evidence that hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of hemocyte-mediated biomineralization in shelled molluscs.

Transcriptome and biomineralization responses of the pearl oyster Pinctada fucata to elevated CO2 and temperature.

Ocean acidification and global warming have been shown to significantly affect the physiological performances of marine calcifiers; however, the underlying mechanisms remain poorly understood. In this study, the transcriptome and biomineralization responses of Pinctada fucata to elevated CO2 (pH 7.8 and pH 7.5) and temperature (25 °C and 31 °C) are investigated. Increases in CO2 and temperature induced significant changes in gene expression, alkaline phosphatase activity, net calcification rates and relative calcium content, whereas no changes are observed in the shell ultrastructure. "Ion and acid-base regulation" related genes and "amino acid metabolism" pathway respond to the elevated CO2 (pH 7.8), suggesting that P. fucata implements a compensatory acid-base mechanism to mitigate the effects of low pH. Additionally, "anti-oxidation"-related genes and "Toll-like receptor signaling", "arachidonic acid metabolism", "lysosome" and "other glycan degradation" pathways exhibited responses to elevated temperature (25 °C and 31 °C), suggesting that P. fucata utilizes anti-oxidative and lysosome strategies to alleviate the effects of temperature stress. These responses are energy-consuming processes, which can lead to a decrease in biomineralization capacity. This study therefore is important for understanding the mechanisms by which pearl oysters respond to changing environments and predicting the effects of global climate change on pearl aquaculture.

Interactive Effects of Seawater Acidification and Elevated Temperature on the Transcriptome and Biomineralization in the Pearl Oyster Pinctada fucata.

Interactive effects of ocean acidification and ocean warming on marine calcifiers vary among species, but little is known about the underlying mechanisms. The present study investigated the combined effects of seawater acidification and elevated temperature (ambient condition: pH 8.1 × 23 °C, stress conditions: pH 7.8 × 23 °C, pH 8.1 × 28 °C, and pH 7.8 × 28 °C, exposure time: two months) on the transcriptome and biomineralization of the pearl oyster Pinctada fucata, which is an important marine calcifier. Transcriptome analyses indicated that P. fucata implemented a compensatory acid-base mechanism, metabolic depression and positive physiological responses to mitigate the effects of seawater acidification alone. These responses were energy-expensive processes, leading to decreases in the net calcification rate, shell surface calcium and carbon content, and changes in the shell ultrastructure. Elevated temperature (28 °C) within the thermal window of P. fucata did not induce significant enrichment of the sequenced genes and conversely facilitated calcification, which was detected to alleviate the negative effects of seawater acidification on biomineralization and the shell ultrastructure. Overall, this study will help elucidate the mechanisms by which pearl oysters respond to changing seawater conditions and predict the effects of global climate change on pearl aquaculture.

In-depth proteomic analysis of shell matrix proteins of Pinctada fucata.

The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

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.

Interactive effects of seawater acidification and elevated temperature on biomineralization and amino acid metabolism in the mussel Mytilus edulis.

Seawater acidification and warming resulting from anthropogenic production of carbon dioxide are increasing threats to marine ecosystems. Previous studies have documented the effects of either seawater acidification or warming on marine calcifiers; however, the combined effects of these stressors are poorly understood. In our study, we examined the interactive effects of elevated carbon dioxide partial pressure (P(CO2)) and temperature on biomineralization and amino acid content in an ecologically and economically important mussel, Mytilus edulis. Adult M. edulis were reared at different combinations of P(CO2) (pH 8.1 and 7.8) and temperature (19, 22 and 25°C) for 2 months. The results indicated that elevated P(CO2) significantly decreased the net calcification rate, the calcium content and the Ca/Mg ratio of the shells, induced the differential expression of biomineralization-related genes, modified shell ultrastructure and altered amino acid content, implying significant effects of seawater acidification on biomineralization and amino acid metabolism. Notably, elevated temperature enhanced the effects of seawater acidification on these parameters. The shell breaking force significantly decreased under elevated P(CO2), but the effect was not exacerbated by elevated temperature. The results suggest that the interactive effects of seawater acidification and elevated temperature on mussels are likely to have ecological and functional implications. This study is therefore helpful for better understanding the underlying effects of changing marine environments on mussels and other marine calcifiers.

Morphology and classification of hemocytes in Pinctada fucata and their responses to ocean acidification and warming.

Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of hemocytes in the pearl oyster, Pinctada fucata. Three types of hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major hemocyte population. Morphological analyses indicated that these hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of hemocyte in the pearl oyster, P. fucata, and also reveals the immune responses of hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters.