The effect of prolonged holding time on the mechanical property and microstructural property of lithium disilicate glass-ceramic.

Repeat firing produces uncertainty about stabilizing lithium disilicate glass-ceramic (LDGC) material properties, even though prolonged holding time can enhance the mechanical property of LDGC during a single firing cycle. However, the effect of prolonged holding time and repeat firing on the mechanical property and microstructure of LDGC is not fully understood. In the present study, three groups of LDGC material were created: (i) extension of holding time (7 vs. 14 vs. 28 min) at 780-800 °C; (ii) holding time extension (7 vs. 14 min) and dual sintering at 800-820 °C, respectively; (iii) dual sintering with prolonged holding time (7 vs. 14 min) at 820-840 °C. The nano-indenter test revealed that prolonged holding time (14 and 28 min) promoted the enhancement of LDGC hardness and Young's modulus. X-ray photoelectron spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy confirmed that prolonged holding time increased and stabilized LD phase in LDGC, as well as induced residual compressive stress. Scanning electron microscopy showed that prolonged holding time increased LD crystal grains homogeneously and facilitated LDGC to form dense interlocking structure without enlarging crystal size grains significantly. In contrast, LDGC that dual sintered alone at 820-840 °C possessed inferior mechanical properties, coupled with heterogeneous crystal phases, residual tensile stress, and melted crystals grains in the porous microstructure. Interestingly, these deteriorated properties of LDGC caused by dual sintering alone could be counteracted by prolonging the holding time. Nevertheless, the LDGC materials displayed an excellent biocompatibility throughout the study. This study identified that prolonged holding time during repeated firing cycles stabilized LD phase and crystal grain size of LDGC, thus enhanced the mechanical properties, which provided a new insight to extend the repeat fired restoration longevity of LDGC. Graphical abstract.

Single-cell RNA-seq of in vitro expanded cells from cranial neural crest reveals a rare odontogenic sub-population.

Ecto-mesenchymal cells of mammalian tooth germ develops from cranial neural crest cells. These cells are recognised as a promising source for tooth development and regeneration. Despite the high heterogeneity of the neural crest, the cellular landscape of in vitro cultured cranial neural crest cells (CNCCs) for odontogenesis remains unclear. In this study, we used large-scale single-cell RNA sequencing to analyse the cellular landscape of in vitro cultured mouse CNCCs for odontogenesis. We revealed distinct cell trajectories from primary cells to passage 5 and identified a rare Alx3+/Barx1+ sub-population in primary CNCCs that differentiated into two odontogenic clusters characterised by the up-regulation of Pax9/Bmp3 and Lhx6/Dmp1. We successfully induced whole tooth-like structures containing enamel, dentin, and pulp under the mouse renal capsule using in vitro cultured cells from both cranial and trunk neural crests with induction rates of 26.7% and 22.1%, respectively. Importantly, we confirmed only cells sorted from odontogenic path can induce tooth-like structures. Cell cycle and DNA replication genes were concomitantly upregulated in the cultured NCCs of the tooth induction groups. Our data provide valuable insights into the cell heterogeneity of in vitro cultured CNCCs and their potential as a source for tooth regeneration.

Branched hydrophobic tails in lipid nanoparticles enhance mRNA delivery for cancer immunotherapy.

Efficient and safe delivery of vulnerable mRNA is a long-standing challenge for the broad application of the emerging mRNA-based therapeutics. Herein, a combinatorial library containing 119 novel lipids was constructed via sequential aza-Michael addition reactions of arylates and varying amines to tackle the ongoing challenge in mRNA delivery. Through in vitro screening of the lipid library on IGROV 1 cells, we identified several synthetic lipids with superior mRNA delivery efficacy. The delivery capability of these lipids was verified by the potent expression of luciferase in BALB/c mice upon intravenous administration of luciferase-encoding mRNA lipid nanoparticles (LNPs). Further investigations on the structure-activity relationship revealed that lipids with branched hydrophobic tails were better at delivering mRNA than those containing linear tails at the similar total number of carbons. In comparison to linear tails, the branched tails endowed LNPs with less inner hydrophobicity, fewer surface charges, and proper stability, which benefits the cellular uptake of LNPs and the intracellular trafficking of mRNA, thus improves the delivery efficacy of mRNA. The therapeutical potential of the lead LNPs was evaluated by delivering ovalbumin (OVA)-encoding mRNA to mice bearing B16-OVA melanoma tumors. The results demonstrated that the administration of OVA mRNA LNPs significantly activated CD8+ T cells in tumor microenvironment and substantially prohibited the growth of the aggressive B16-OVA tumors. The robust antitumor efficacy highlights the great potential of these LNPs in cancer immunotherapy.

Single cell atlas of developing mouse dental germs reveals populations of CD24+ and Plac8+ odontogenic cells.

The spatiotemporal relationships in high-resolution during odontogenesis remain poorly understood. We report a cell lineage and atlas of developing mouse teeth. We performed a large-scale (92,688 cells) single cell RNA sequencing, tracing the cell trajectories during odontogenesis from embryonic days 10.5 to 16.5. Combined with an assay for transposase-accessible chromatin with high-throughput sequencing, our results suggest that mesenchymal cells show the specific transcriptome profiles to distinguish the tooth types. Subsequently, we identified key gene regulatory networks in teeth and bone formation and uncovered spatiotemporal patterns of odontogenic mesenchymal cells. CD24+ and Plac8+ cells from the mesenchyme at the bell stage were distributed in the upper half and preodontoblast layer of the dental papilla, respectively, which could individually induce nonodontogenic epithelia to form tooth-like structures. Specifically, the Plac8+ tissue we discovered is the smallest piece with the most homogenous cells that could induce tooth regeneration to date. Our work reveals previously unknown heterogeneity and spatiotemporal patterns of tooth germs that may lead to tooth regeneration for regenerative dentistry.

Synthesis and characterization of PEGylated toll like receptor 7 ligands.

Toll-like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid, or poly(ethylene glycol) (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1ß, and type 1 interferon, as well as for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.

Study on the anti-infection ability of vancomycin cationic liposome combined with polylactide fracture internal fixator.

A polylactide composite fracture fixator loaded with vancomycin cationic liposome (PLA@VL) was prepared by reverse evaporation method. The method of cationic liposome encapsulating vancomycin could effectively improve antibacterial property and achieve drug sustained release effect, so as to reduce toxicity of antibiotics in vivo. Scanning electron microscope (SEM) was used to observe morphology and Fourier transform infrared spectroscopy (FTIR) was used to detect the composition of the internal fixator. In vitro drug release model, in vitro degradation model and body fluid osteogenesis model were designed in this study. On the other hand, the experiments of inhibition zone and MC3T3-E1 osteoblasts in mice were conducted to explore antibacterial property, cell activity and adhesion of the PLA@VL composite internal fixator. Alkaline phosphatase (ALP) staining method and alizarin red assay were used to detect the osteogenic induction ability of the composite internal fixator. Finally, mice fracture models were established to verify osteogenic and anti-infection abilities of the composite internal fixator in vivo. The results showed that MC3T3-E1 cells had better adhesion and proliferation abilities on the PLA@VL composite internal fixator than on the PLA fixator, which indicated that the PLA@VL composite internal fixator possessed excellent osteogenic and anti-infection abilities both in vivo and in vitro. Therefore, the above experiments showed that the fracture internal fixator combined with vancomycin cationic liposome had better biocompatibility, antibacterial ability and osteogenic ability, which provides a promising anti-infection material for the clinical field of fracture.

Layer-by-layer immobilizing of polydopamine-assisted ε-polylysine and gum Arabic on titanium: Tailoring of antibacterial and osteogenic properties.

Bacterial infection has become a crucial reason that give rise to failure of medical implants in clinical applications. In this regard, various antibacterial surface modifications of implants have been developed in recent years. However, it remains a challenge to enable the implant surfaces with both suitable antibacterial and osteogenic properties. In this work, ε-polylysine and gum Arabic multilayer composite films were immobilized layer by layer (LBL) on anodized titanium with the assistance of polydopamine for the first time. In vitro antibacterial results showed that the bacteria numbers decreased with an increase in the loading amount of ε-polylysine. Furthermore, long-term antibacterial property up to 3 weeks against both gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) was obtained combined with the merits of covalent binding and LBL methods. Meanwhile, the cell proliferation and osteogenic differentiation of BMSCs on titanium dioxide nanotubes (TNTs) modified with composite films was significantly improved. Remarkably, a facile method to optimize anti-infective and osteogenic properties of medical titanium has been developed, and it was demonstrated that the ε-polylysine and gum Arabic multilayer composite films with assistance of polydopamine were able to endow the orthopedic implant materials both improved antibacterial property and excellent biocompatibility, which is of profound significance for practical application of titanium-based implants.

Rapid mussel-inspired synthesis of PDA-Zn-Ag nanofilms on TiO2 nanotubes for optimizing the antibacterial activity and biocompatibility by doping polydopamine with zinc at a higher temperature.

Mussel-inspired deposition of polydopamine (PDA) is a green chemical method that has been used to load silver nanoparticles on titanium oxide nanotubes (TiO2 NTs) to kill bacteria. However, a long reaction time is required for both the polymerization of dopamine and the reaction between PDA and silver nitrate. In addition, the deposition of silver nanoparticles is difficult to control, which may increase the risk of cytotoxicity. In this study, a rapid polymerization of dopamine was achieved by performing the reaction in a water bath at 90 °C (PDA-H). Furthermore, the reduction of Ag+ ions was markedly accelerated by the PDA-Zn film that was formed on the surface of TiO2 NTs from a solution of dopamine and zinc nitrate under the same heating conditions. The reaction between the PDA-Zn film and silver nitrate was dramatically reduced to 10 min, and the silver nanoparticles deposited on the PDA-Zn film were more uniform than those by PDA-H film. This PDA-Zn-Ag-TiO2 NTs material exhibited good antibacterial activity, as evidenced by the inhibition zone. The WST-1 assay indicated that the PDA-Zn-Ag film possessed a lower cell cytotoxicity and better biocompatibility than other Ag containing PDA films.

[Lipopolysaccharide of Porphyromonas gingivalis promotes the autophagy of human gingival fibroblasts].

Objective To investigate the impact of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) on the autophagy of human gingival fibroblasts (HGFs). Methods Firstly, HGFs was stimulated with 10 µg/mL Pg-LPS for 12 hours or 24 hours. Rapamycin was used as a positive control. The expression of LC3B was detected by Western blotting and the distribution of autophagosomes was observed by indirect immunofluorescence staining. At the same time, mitochondrial ROS (mtROS) was labeled by MitoSOX Red. The levels of mtROS and mitochondrial autophagy were measured in HGFs after treated with Pg-LPS. Then, T-butyl-4 (BHA), N-acetylcysteine (NAC) and coenzyme Q10 (CoQ10) were used separately to block the ROS and the expression of LC3B in Pg-LPS-treated HGFs was tested by Western blotting. Results After treatment with Pg-LPS, the ratio of LC3BII/LC3BI and the number of autophagic cells significantly increased, and the increase in the 24-hour treatment group was the more obvious than that in the 12-hour treatment group. The mtROS production and mitochondrial autophagy were significantly promoted after Pg-LPS treatment. In addition, CoQ10 effectively reduced Pg-LPS-induced autophagy of HGFs. Conclusion Pg-LPS can induce the autophagy of HGFs by raising mtROS production, and autophagy is involved in the degradation of damaged mitochondria to maintain cellular homeostasis.

Poly(ε-benzyloxycarbonyl-L-lysine)-grafted branched polyethylenimine as efficient nanocarriers for indomethacin with enhanced oral bioavailability and anti-inflammatory efficacy.

Star-block copolymers PEI-g-PZLL with a branched polyethylenimine (PEI) core and multiple grafted poly(ε-benzyloxycarbonyl-L-lysine) (PZLL) peripheral chains were designed, synthesized, and evaluated as nanocarriers for indomethacin (IND). In an aqueous solution, PEI-g-PZLL self-assembled into spherical nanoparticles capable of encapsulating IND at high loading capacity and loading efficiency. Differential scanning calorimetry and X-ray diffraction measurements indicated that IND was molecularly or amorphously dispersed in the nanoparticles. Fourier transform infrared spectra revealed the presence of multiple intermolecular interactions, including hydrogen bonding, electrostatic forces, π-π stacking and hydrophobic interactions, between the block copolymer and the IND molecules. IND-loaded nanoparticles exhibited fast release under intestinal pH. Compared with raw IND, the utilization of PEI-g-PZLL as a carrier significantly enhanced the oral bioavailability of IND and improved its protective effect on renal ischemia-reperfusion injury, as evidenced by in vivo pharmacokinetic and pharmacodynamic studies. Cytotoxicity assay, histological observation and cellular uptake study suggested that PEI-g-PZLL was fairly biocompatible. All these results indicated that star-block copolymers PEI-g-PZLL could be used as efficient nanocarriers for IND and other poorly water-soluble drugs. STATEMENT OF SIGNIFICANCE: The use of polyethylenimine (PEI) as an oral drug delivery carrier is limited because it is not biodegradable and the use of higher molecular weight PEI leads to improved efficiency but also increased cytotoxicity. The design of functionalized PEIs with low cytotoxicity and high efficiency is crucial for developing a successful oral drug delivery system. In our study, poly(ε-benzyloxycarbonyl-L-lysine) (PZLL)-grafted branched PEI (PEI-g-PZLL) was reported as an oral nanocarrier for indomethacin (IND). The low cytotoxicity and biodegradability, well-defined self-assembled nano-sized polymeric micelles, high loading capacity and loading efficiency, amorphous state of the encapsulated IND, as well as the enhanced oral bioavailability of IND, makes the copolymer PEI-g-PZLL a promising nanocarrier for the oral administration of IND and possibly other poorly water-soluble drugs.

Antimicrobial activity and cytocompatibility of silver nanoparticles coated catheters via a biomimetic surface functionalization strategy.

Catheter-related bloodstream infections are a significant problem in the clinic and may result in a serious infection. Here, we developed a facile and green procedure for buildup of silver nanoparticles (AgNPs) on the central venous catheters (CVCs) surface. Inspired by mussel adhesive proteins, dopamine was used to form a thin polydopamine layer and induce AgNPs formation without additional reductants or stabilizers. The chemical and physicochemical properties of AgNPs coated CVCs were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and water contact angle. The Staphylococcus aureus culture experiment was used to study the antibacterial properties. The cytocompatibility was assessed by water soluble tetrazolium salts (WST-1) assay, fluorescence staining, and scanning electron microscopy analysis. The results indicated that the CVCs surface was successfully coated with compact AgNPs. AgNPs were significantly well separated and spherical with a size of 30-50 nm. The density of AgNPs could be modulated by the concentration of silver nitrate solution. The antibacterial activity was dependent on the AgNPs dose. The high dose of AgNPs showed excellent antibacterial activity while associated with increased cytotoxicity. The appropriate density of AgNPs coated CVCs could exhibit improved biocompatibility and maintained evident sterilization effect. It is promising to design mussel-inspired silver releasing CVCs with both significant antimicrobial efficacy and appropriate biological safety.

Effect of Octacalcium-Phosphate-Modified Micro/Nanostructured Titania Surfaces on Osteoblast Response.

Surface structures and properties of titanium implants play a vital role in successful bone replacement. To mimic the natural bone structure, some strategies have recently focused on the preparation of multiscaled morphology on medical titanium and shown some promising results; however, relatively few efforts have been made for further enhancing the biocompatibility of such a hierarchical hybrid structure without compromising the superior bioactivity of the starting micro/nano roughness. In this study, a thin ribbonlike octacalcium phosphate (OCP) coating was electrodeposited on a hierarchically structured titania surface, maintaining its micro/nanospongelike morphology. It is indicated that the micro/nanostructured surface with deposited OCP showed an improved biomineralization ability, in comparison to that without OCP modification, when immersed in simulated body fluid (SBF). Further evaluations of cellular activities demonstrated that the introduction of OCP to the micro/nano spongelike-structured surface remarkably enhanced MC3T3-E1 cell proliferation, alkaline phosphatase activity, and extracellular matrix mineralization compared to that of cells on the micro/nanospongelike titania surface during 14 days of culturing. Meanwhile, the OCP-deposited micro/nanostructured surface displayed much a smaller passive current density and lower current response to the applied potential, resulting in the improvement of corrosion resistance. All of the evaluations suggested that the modification of the OCP coating on the prepared micro/nanospongelike titania is of superior chemical stability, biomineralization, and osteoblast activities, which indicates a favorable implant microenvironment for osseointegration in vivo.

Application of iPS cells in dental bioengineering and beyond.

The stem-cell-based tissue-engineering approaches are widely applied in establishing functional organs and tissues for regenerative medicine. Successful generation of induced pluripotent stem cells (iPS cells) and rapid progress of related technical platform provide great promise in the development of regenerative medicine, including organ regeneration. We have previously reported that iPS cells could be an appealing stem cells source contributing to tooth regeneration. In the present paper, we mainly review the application of iPS technology in dental bioengineering and discuss the challenges for iPS cells in the whole tooth regeneration.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells.

BACKGROUND: Tooth is vital not only for a good smile, but also good health. Yet, we lose tooth regularly due to accidents or diseases. An ideal solution to this problem is to regenerate tooth with patients' own cells. Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells (ifhU-iPSCs). RESULTS: We first differentiated ifhU-iPSCs to epithelial sheets, which were then recombined with E14.5 mouse dental mesenchymes. Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC. We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth. CONCLUSION: Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Ras GTPases modulate morphogenesis, sporulation and cellulase gene expression in the cellulolytic fungus Trichoderma reesei.

BACKGROUND: The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2(G16V) allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. CONCLUSIONS/SIGNIFICANCE: Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.