Structure and composition of dissolved organic matters in sludge by ultrasonic treatment.

The leaching of dissolved organic matter (DOM) from the sludge into the liquid phase is induced by ultrasound. However, there is limited investigation into the structure and molecular composition of sludge DOM in this process. The molecular structure and composition of sludge DOM in ultrasonic treatment were comprehensively elucidated in this study. The sludge dissolved organic carbon (DOC) and three-dimensional fluorescence spectroscopy (3D-EEM) image had most significant change at 15-min ultrasonic time and 1.2 W/mL ultrasonic density, respectively. Gas Chromatography-Mass Spectrometry (GC-MS) analysis indicated that ultrasonic treatment of sludge reduced the macromolecules to small molecules in DOM. Then, electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI FT-ICR-MS) analysis revealed that lignin, tannins, and carbohydrates were the main components of sludge DOMs after ultrasound treatment. analysis revealed that lignin, tannins, and carbohydrates were the main components of sludge DOMs after ultrasound treatment. Furthermore, through the Van Krevelen analysis, the major components were CHO (48.50%) and CHOS (23.20%) in the DOM of ultrasonicated sludge. This research provides the basis for the practical application of ultrasonic treatment of sludge and provides basic information for DOM components.

Evaluation of enhancing effect of soybean oil on polymalic acid production by Aureobasidium pullulans HA-4D.

Polymalic acid (PMA) is a water-soluble polyester produced by Aureobasidium pullulans. In this study, the physiological response of A. pullulans after the addition of vegetable oils was investigated. Soybean oil (SBO) is pivotal for shortening fermentation time and achieving high PMA titer. With the addition of 1% (w/v) SBO, the titer and productivity of PMA was, respectively, increased by 34.2% and 80%. SBO acted as a chemical stimulatory agent rather than a carbon source, the enhancement on PMA production was attributed to the component of fatty acid. SBO induced the dimorphism (yeast-like cells and mycelia) of A. pullulans, in vitro enzyme activities indicated that the TCA oxidative branch for malic acid synthesis might be strengthened, which could generate more ATP for PMA synthesis, and the assay of intracellular energy supply validated this deduction. This study provided a new sight for recognizing the regulatory behavior of SBO in A. pullulans.

Probing Membrane Fouling via Infrared Attenuated Total Reflection Mapping Coupled with Multivariate Curve Resolution.

Understanding membrane fouling induced by dissolved organic matter (DOM) is of primary importance for developing effective fouling control and prevention strategies. In this work, we combine multivariate curve resolution-alternating least squares analysis with infrared attenuated total reflection mapping to explore the fouling process of microfiltration and ultrafiltration membranes caused by two typical DOMs, humic acid (HA) and bovine serum albumin (BSA). The spectral contributions of different foulants and the membrane substrate were successfully discriminated, thereby enabling the diagnosis of fouling origins. Membrane fouling caused by HA is more severe than that by BSA. Three periods, the initial adsorption stage, the equilibrium stage, and the accumulation stage, were observed for the HA-induced fouling process. The integrated approach presented herein elegantly demonstrates the spatial and temporal characterization of membrane fouling processes, along with relative concentrations of the involved species, and suggests a promising perspective for understanding the interaction mechanisms between foulant species and membranes at the molecular level.

Regulation of extracellular polymers based on quorum sensing in wastewater biological treatment from mechanisms to applications: A critical review.

Extracellular polymeric substances (EPS) regulated by quorum sensing (QS) could directly mediate adhesion between microorganisms and form tight microbial aggregates. Besides, EPS have redox properties, which can facilitate electron transfer for promoting electroactive bacteria. Currently, the applications research on improving wastewater biological treatment performance based on QS regulated EPS have been widely reported, but reviews on the level of QS regulated EPS to enhance EPS function in microbial systems are still lacking. This work proposes the potential mechanisms of EPS synthesis by QS regulation from the viewpoint of material metabolism and energy metabolism, and summarizes the effects of QS on EPS synthesis. By synthesizing the role of QS in EPS regulation, we further point out the applications of QS-regulated EPS in wastewater biological treatment, which involve a series of aspects such as strengthening microbial colonization, mitigating membrane biofouling, improving the shock resistance of microbial metabolic systems, and strengthening the electron transfer capacity of microbial metabolic systems. According to this comprehensive review, future research on QS-regulated EPS should focus on the exploration of the micro-mechanisms, and economic regulation strategies for QS-regulated EPS should be developed, while the stability of QS-regulated EPS in long-term production experimental research should be further demonstrated.

A new strategy for accelerating recovery of anaerobic granular sludge after low-temperature shock: In situ regulation of quorum sensing microorganisms embedded in polyvinyl alcohol sodium alginate.

Low-temperature could inhibit the performance of anaerobic granular sludge (AnGS). Quorum sensing (QS), as a communication mode between microorganisms, can effectively regulate AnGS. In this study, a kind of embedded particles (PVA/SA@Serratia) based on signal molecule secreting bacteria was prepared by microbial immobilization technology based on polyvinyl alcohol and sodium alginate to accelerate the recovery of AnGS system after low temperature. Low-temperature shock experiment verified the positive effect of PVA/SA@Serratia on restoring the COD removal rate and methanogenesis capacity of AnGS. Further analysis by metagenomics analysis showed that PVA/SA@Serratia stimulated higher QS activity and promoted the secretion of extracellular polymeric substance (EPS) in AnGS. The rapid construction of EPS protective layer effectively accelerated the establishment of a robust microbial community structure. PVA/SA@Serratia also enhanced multiple methanogenic pathways, including direct interspecies electron transfer. In conclusion, this study demonstrated that PVA/SA@Serratia could effectively strengthen AnGS after low-temperature shock.

Adsorption-desorption behaviors of ciprofloxacin onto aged polystyrene fragments in aquatic environments.

As two emerging pollutants of great concern, microplastics (MPs) and antibiotics inevitably cooccur in various aquatic environments and interact with each other, impacting the fate and ecological risks. Aging obviously complicates their interaction and deserves further study. Therefore, the adsorption-desorption behaviors of ciprofloxacin (CIP) onto polystyrene (PS) fragments with various aging extent were investigated, and the key physiochemical properties influencing the interaction and the interaction mechanisms were clarified by redundancy analysis, FTIR and XPS spectra. The physicochemical properties of PS MPs were significantly changed with aging time, and the morphological and chemical changes seemed to occur asynchronously. The adsorption of CIP onto the pristine PS MPs relied on physisorption, especially the ion-involving electrostatic and cation-π interaction. Due to the hydrogen bonding formed by the C-OH, CO, and O-CO groups of PS and CIP, the adsorption capacities of the aged PS MPs were greatly increased. The desorption efficiency of CIP from MPs in the gastric fluid was closely related to the solution ionic strengths, C-OH and CO groups of MPs, while that in the intestinal fluid was associated with O-CO groups of MPs. The different impact factors could be well described by the differences in the chemical components and pHs of the simulated gastric and intestinal fluids. This study gives a comprehensive understanding of the adsorption-desorption behaviors of antibiotics onto MPs at a molecular level and indicates that MPs could act as Trojan horses to transport antibiotics into aquatic organisms.

Collagen-based scaffolds with high wet-state cyclic compressibility for potential oral application.

Soft tissue substitutes have been developed to treat gingival recessions to avoid a second surgical site. However, products of pure collagen for clinical application lack their original mechanical strengths and tend to degrade fast in vivo. In this study, a collagen-based scaffold crosslinked with oxidized sodium alginate (OSA-Col) was developed to promote mechanical properties. Compared with commercial products collagen matrix (CM) and collagen sponge (CS), OSA-Col scaffolds presented higher wet-state cyclic compressibility, early anti-degradation ability, similar hemocompatibility and cytocompatibility. Furthermore, in the subcutaneous implantation experiment, OSA2-Col3 scaffolds showed better anti-degradation performance than CS scaffolds and superior neovascularization than CM scaffolds. These results demonstrated that OSA2-Col3 scaffolds had potential as a new soft tissue substitute for the treatment of gingival recessions.

Variables Impacting Silicone Oil Migration and Biologics in Prefilled Syringes.

Prefilled syringes (PFS) as a primary container for parenteral drug products offer significant advantages, such as fast delivery time, ease of self-administration and fewer dosing errors. Despite the benefits that PFS can provide to patients, the silicone oil pre-coated on the glass barrels has shown migration into the drug product, which can impact particle formation and syringe functionality. Health authorities have urged product developers to better understand the susceptibility of drug products to particle formation in PFS due to silicone oil. In the market, there are multiple syringe sources provided by various PFS suppliers. Due to current supply chain shortages and procurement preferences for commercial products, the PFS source may change in the middle of development. Additionally, health authorities require establishing source duality. Therefore, it is crucial to understand how different syringe sources and formulation compositions impact the drug product quality. Here, several design of experiments (DOE) are executed that focus on the risk of silicone oil migration induced by syringe sources, surfactants, protein types, stress, etc. We utilized Resonant Mass Measurement (RMM) and Micro Flow Imaging (MFI) to characterize silicone oil and proteinaceous particle distribution in both micron and submicron size ranges, as well as ICP-MS to quantify silicon content. The protein aggregation and PFS functionality were also monitored in the stability study. The results show that silicone oil migration is impacted more by syringe source, siliconization process and surfactant (type & concentration). The break loose force and extrusion force across all syringe sources increase significantly as protein concentration and storage temperature increase. Protein stability is found to be impacted by its molecular properties and is less impacted by the presence of silicone oil, which is the same inference drawn in other literatures. A detailed evaluation described in this paper enables a thorough and optimal selection of primary container closure and de-risks the impact of silicone oil on drug product stability.

Ethanol-wet bonding technique may enhance the bonding performance of contemporary etch-and-rinse dental adhesives.

PURPOSE: To determine whether bonds of contemporary etch-and-rinse adhesives made with ethanol-wet bonding are stronger and more durable than those made with water-wet bonding, and to explore the possible reasons for the bonding results. MATERIALS AND METHODS: Flat surfaces of midcoronal dentin were made in extracted human third molars. The dentin surfaces were randomized into 6 groups according to bonding techniques (water- vs ethanol-wet bonding) and dental adhesives [Single Bond 2 (SB), Prime Bond NT (PB), and Gluma Comfort Bond (GB)]. After etching and rinsing, dentin surfaces were either left water-moist or immersed in ethanol. Following adhesive application and composite buildups, the bonded teeth were sectioned into beams for microtensile bond strength evaluation with or without NaOCl challenge. The morphology of the hybrid layer was analyzed with SEM. The wettability of water- vs. ethanol-saturated dentin was evaluated. The concentrations of non-volatile ingredients in the adhesives were compared. RESULTS: Compared to water-wet bonding, ethanol-wet bonding yielded similar (p > 0.05 for PB and GB) or higher (p < 0.05 for SB) 24-h bond strength, displayed significantly higher bond strength after chemical challenge (p < 0.05, for all three adhesives), and produced more even hybrid layers. Moreover, ethanol-saturated dentin exhibited a lower contact angle than water-saturated specimens, and the concentrations of non-volatile ingredients of the adhesives decreased in the order of SB > GB > PB. CONCLUSION: Ethanol-wet bonding could improve the bonding efficacy of contemporary etch-and-rinse adhesives, probably due to the good wettability of ethanol-saturated dentin and the structure of the hybrid layer. Moreover, this positive effect of ethanol-wet bonding might be influenced by the composition of adhesives.

Bonding efficiency of contemporary adhesives to the dentinoenamel junction zone.

The aim of this study was to investigate the bonding of dental adhesives to the dentinoenamel junction (DEJ) zone. Bonding of four adhesives [two etch-and-rinse adhesives (ERAs) and two self-etching adhesives (SEAs)] to enamel, dentin, and the DEJ zone was evaluated using a micro-shear test. Based on the measured bond strengths of dentin/enamel and on the area percentages of dentin in the DEJ zone, predicted bond strengths for the DEJ zone were calculated and compared with those measured. The DEJ zone was analyzed, using scanning electron microscopy, after conditioning, resin infiltration, and debonding. Regardless of the adhesive, bond strengths were significantly influenced by substrates, exhibiting the following order of bond strength (strongest to weakest): dentin >DEJ zone > enamel. The predicted values of the DEJ zone for the ERA groups were significantly higher than the measured values. Analysis of the ERA specimens using scanning electron microscopy showed distinct etching textures of enamel and dentin, which outlined the DEJ and increased the adhesive thickness at the enamel side of the DEJ. Those characteristics could not be detected in the SEA groups. The DEJ zone displayed bond properties that were stronger than enamel but weaker than dentin and therefore may be considered as transitional bond properties. Enamel/dentin within the DEJ zone might bond more weakly to ERAs than its counterpart of the bulk tissue. The presence of the DEJ in the bond area might compromise the bonding efficiency of ERAs.

Peptide-based supramolecular hydrogels for bioimaging applications.

Owing to their superior loading capacity and biocompatibility, imaging agent-conjugated (or encapsulated) peptide-based supramolecular hydrogels are capable of imaging in vivo biological events with enhanced signals. Notably, by rational design of the hydrogelators, the hydrogelation process can "smartly" occur on the pathological site (or region of interest), rendering precise and sensitive bioimaging of the disease (or event) in vivo. Considering their importance in disease diagnosis, herein, we provide a review on the recent advances in peptide-based supramolecular hydrogels for bioimaging applications. Besides, we provide an outlook on the challenges (or chances) for these types of biomaterials in the field of bioimaging.

Fabrication and applications of bioactive chitosan-based organic-inorganic hybrid materials: A review.

Organic-inorganic hybrid materials like bone, shells, and teeth can be found in nature, which are usually composed of biomacromolecules and nanoscale inorganic ingredients. Synergy of organic-inorganic components in hybrid materials render them outstanding and versatile performance. Chitosan is commonly used organic materials in bionic hybrid materials since its bioactive properties and could be controllable tailored by various means to meet complex conditions in different applications. Among these fabrication means, hybridization was favored for its convenience and efficiency. This review discusses three kinds of chitosan-based hybrid materials: hybridized with hydroxyapatite, calcium carbonate, and clay respectively, which are the representative of phosphate, carbonate, and hydrous aluminosilicates. Here, we reported the latest developments of the preparation methods, composition, structure and applications of these bioactive hybrid materials, especially in the biomedical field. Despite the great progress was made in bioactive organic-inorganic hybrid materials based on chitosan, some challenges and specific directions are still proposed for future development in this review.

Probing protein-induced membrane fouling with in-situ attenuated total reflectance fourier transform infrared spectroscopy and multivariate curve resolution-alternating least squares.

Proteins are one of the major contributors to membrane fouling. The interaction between proteins and the polymer membrane at the molecular level is essential for the alleviation/prevention of membrane fouling, but remains unclear. In this work, time-dependent in-situ attenuated total reflectance Fourier transform infrared spectroscopy is applied to investigate the interaction process between two model proteins, bovine serum albumin and lysozyme, and the poly(vinylidene fluoride) (PVDF) membrane. Multivariate curve resolution-alternating least squares is integrated with two-dimensional correlation spectroscopy analysis to resolve the membrane-induced conformational changes of proteins. The multivariate curve resolution-alternating least squares analysis reveals a two-step process in the protein-membrane interaction and provides the kinetics of the conformational transition, which aids the segmentation of the spectral dataset. By applying two-dimensional correlation spectroscopy analysis to different groups of the time-dependent spectra, the sequential order of the secondary structural changes of proteins is determined. The proteins initially undergo unfolding transition to a more open, less structured state, which appears to be triggered by the hydrophobic membrane surface. Afterwards, the proteins become aggregated with the high anti-parallel ß-sheet content, aggravating the membrane fouling. The conformational transition process of proteins was also confirmed by the atomic force microscopic images and quartz crystal microbalance measurement. Overall, this work provides an in-depth understanding of the interaction between proteins and the membrane surface, which is helpful for the development of membrane anti-fouling strategies.

Self-assembled lignin-silica hybrid material derived from rice husks as the sustainable reinforcing fillers for natural rubber.

Biomass derived fillers have been developed as sustainable substitution of carbon black (CB) used in rubber industry to reduce the dependence on fossil fuels. Lignin is the abundant component of biomass, but has poor reinforcing performance due to its huge particle size. In this work, we prepared a lignin/silica (LS) hybrid material from rice husks via a facile self-assembly method. The formation of LS cut lignin macromolecules into fragments and resulted in a small average particle size of 320 nm. When LS was filled into the natural rubber to substitute 10 phr CB, both filler-filler interaction and filler-rubber interaction were enhanced compared with the single use of CB. In consequence, the obtained 10LS/40CB/NR vulcanizates showed overall improvement in tensile strength, tear strength and abrasion resistance compared with the 50CB/NR vulcanizates. For the sustainable development of rubber industry, the natural LS hybrid material with low production cost is a promising reinforcing filler for the partial replacement of CB.

Photosensitizer-Doped and Plasma Membrane-Responsive Liposomes for Nuclear Drug Delivery and Multidrug Resistance Reversal.

Clinically approved doxorubicin (Dox)-loaded liposomes (e.g., Doxil) guarantee good biosafety, but their insufficient nuclear delivery of Dox (<0.4%) after cellular uptake significantly hampers their final anticancer efficacy. Here, we report that simply doping protoporphyrin IX (PpIX, a commonly used hydrophobic photosensitizer) into the lipid bilayers of Dox-loaded liposomes (the resultant product is termed PpIX/Dox liposomes) is a feasible way to promote the nuclear delivery of Dox. This facile strategy relies on a unique property of PpIX-it presents considerably higher affinity for the real plasma membrane over its liposomal carrier, which drives the doped PpIX molecules to detach from the liposomes when encountering cancer cells. We demonstrate that this process can trigger the efficient release of the loaded Dox molecules and allow them to enter the nuclei of MCF-7 breast cancer cells without being trapped by lysosomes. Regarding the drug-resistant MCF-7/ADR cells, the aberrant activation of the efflux pumps in the plasma membranes expels the internalized Dox. However, we strikingly find that the robust drug resistance can be reversed upon mild laser irradiation because the photodynamic effect of PpIX disrupts the drug efflux system (e.g., P-glycoprotein) and facilitates the nuclear entry of Dox. As a proof-of-concept, this PpIX doping strategy is also applicable for enhancing the effectiveness of cisplatin-loaded liposomes against both A549 and A549/DDP lung cancer cells. In vivo experimental results prove that a single injection of PpIX/Dox liposomes completely impedes the growth of MCF-7 tumors in nude mice within 2 weeks and, in combination with laser irradiation, can synergistically ablate MCF-7/ADR tumors. Biosafety assessments reveal no significant systemic toxicity caused by PpIX/Dox liposomes. This work exemplifies a facile method to modulate the subcellular fate of liposomal drugs and may inspire the optimization of nanopharmaceuticals in the near future.

Construction of Dually Responsive Nanotransformers with Nanosphere-Nanofiber-Nanosphere Transition for Overcoming the Size Paradox of Anticancer Nanodrugs.

Tumor microenvironment (TME)-responsive nanosystems represent a category of intelligent nanomaterials for precise anticancer drug delivery. Herein, we report a smart size-/morphology-switchable nanodrug that can respond to the acidic TME and near-infrared (NIR) laser irradiation for effective tumor ablation and tumor metastasis inhibition. The nanoagent is physically assembled by a cytolytic peptide, melittin (MEL), an NIR-absorbing molecule, cypate, and a tumor-targeting polymer, hyaluronic acid (HA). At pH 7.4, the as-formed MEL/Cypate@HA complexes are negatively charged nanospheres (∼50 nm), which are suitable for long-term systemic circulation. When these nanospheres actively target tumors, the weakly acidic TME triggers an in situ transformation of the nanospheres to net-like nanofibers. Compared with the nanospheres, the nanofibers not only exhibit an inhibitory effect on tumor cell mobility but also significantly prolong the retention time of MEL/Cypate@HA in tumor tissues for MEL-based chemotherapy. Moreover, the nanofibers can be photodegraded into small nanospheres (∼25 nm) by NIR laser irradiation during cypate-mediated photothermal therapy, which enables deep tumor penetration of the loaded MEL and thus achieves effective tumor eradication. This work provides a facile strategy for converting naturally occurring therapeutic peptides into a TME-responsive drug delivery system and may inspire the development of nanomaterials with changeable structures for therapeutic purposes.

Plasma membrane-anchorable photosensitizing nanomicelles for lipid raft-responsive and light-controllable intracellular drug delivery.

The past decade has witnessed a growing number of nanoparticulate drug delivery systems for cancer treatment. However, insufficient cellular uptake by cancer cells and the undesirable endo/lysosomal entrapment of internalized therapeutic drugs remain the "Achilles heel" of many developed nanoagents. Here, we develop a novel lipid raft-responsive and light-controllable polymeric drug for efficient cytosolic delivery of photosensitizers. Conjugating a photosensitizer protoporphyrin IX (PpIX) to a polyethylene glycol-cholesterol polymer affords the amphiphilic drug (denoted as Chol-PEG-PpIX) that forms micelles in aqueous solutions. The Chol-PEG-PpIX with two hydrophobic units (cholesterol and PpIX) showed robust binding to plasma membranes and enabled significant cellular uptake via two pathways: (1) cholesterol moiety triggered the lipid raft-mediated endocytosis of Chol-PEG-PpIX with minimized endo/lysosomal trafficking after internalization; (2) the membrane-bound PpIX acted as a light-controlled trigger and can augment the permeability of plasma membranes upon laser irradiation, allowing the rapid influx of extracellular Chol-PEG-PpIX within 5 min. For systemic drug delivery, Chol-PEG-PpIX was anchored on the surface of liposomes via in situ membrane modification, which substantially avoided nonspecific binding of Chol-PEG-PpIX to red blood cells during circulation. Besides, the Chol-PEG-PpIX-anchored liposomes exhibited enhanced in vivo fluorescence, reduced liver uptake, prolonged tumor retention, and effective tumor ablation by photodynamic therapy. This work illustrates a new strategy for direct and efficient cytosolic delivery of photosensitizers, which may hold great promise in cancer therapy.

Failed percutaneous kyphoplasty in treatment of stage 3 Kummell disease: A case report and literature review.

RATIONALE: Albeit it is rare, the authors report a stage 3 Kummell disease case. It is diagnosed by dynamic thoracic magnetic resonance imaging (MRI). Because there is no established strategy on stage 3 Kummell disease, we performed percutaneous kyphoplasty at first, but unfortunately made a revision surgery to remove the polymethylmethacrylate (PMMA) cement after the failure of percutaneous kyphoplasty. PATIENT CONCERNS: A 73-year-old lady with severe back pain due to osteoporosis vertebral fracture was admitted to our hospital on June 23, 2016. She underwent percutaneous kyphoplasty with the back pain improved shortly. Unfortunately, she went back to our hospital due to aggravated back pain and partial paralysis on July 25, 2016. DIAGNOSES: Kummell disease, lumbar stenosis, lacunar infarction. OUTCOMES: Specially, the compression of spinal cord was obvious in the hyperflexion position on dynamic MRI. Even though there was no improvement in muscle strength, the patient was satisfied with the back pain relief after percutaneous kyphoplasty. But, the same back pain reappeared after about 1 month. Then, we took out the PMMA cement and performed posterior vertebral column resection. At last, the back pain was relieved again but the muscle strength was improved not obviously after operation. At the same time, the kyphosis was corrected and the intravertebral stability was achieved. LESSONS: Dynamic MRI is helpful in Kummell disease with neurologic symptoms, even if there is no obvious compression in the neutral position. Furthermore, the intravertebral instability is probably the main reason of the neurologic symptoms in this case. Percutaneous kyphoplasty could not reconstruct the intravertebral stability. Stage 3 Kummell disease with obvious intravertebral instability should be treated by open surgery.

Local structure of channel ions in carbonate apatite.

Refinement of the single-crystal X-ray diffraction structure of a type A carbonate apatite [CAp; Ca10(PO4)6-y(CO3)x+(3/2)y(OH)2-2x, x=0.75, y=0.0; space group P3 ] has been continued with independent positional and isotropic displacement parameters for the carbonate oxygen atoms, reducing the residual indices significantly (R=0.024, Rw=0.020) and confirming the earlier structure assignment. The carbonate ion is located in the apatite channel at z approximately 0.5, and oriented with two oxygen atoms close to the c-axis. Rigid body refinement, giving a preferred structure, used a novel procedure for defining the ideal equilateral triangular geometry of the channel carbonate ion. Resolution of the channel carbonate ions in type A-B CAp (x=0.69, y=0.57; P63/m) is also improved. Channel carbonate ions in CAp are canted, rotated and displaced to optimize Ca2-O bond distances. The rotation of the A1 carbonate in type A-B CAp is opposite to that of the channel carbonate in type A CAp, due mainly to the accommodation of a second channel carbonate ion (A2). These structures simulate the local structure of type A carbonate in hydroxyapatite of bone and dental enamel.