Piezoelectric Heterojunctions as Bacteria-Killing Bone-Regenerative Implants.

Heterojunctions are widely used in energy conversion, environmental remediation, and photodetection, but have not been fully explored in regenerative medicine. In particular, piezoelectric heterojunctions have never been examined in tissue regeneration. Here the development of piezoelectric heterojunctions is shown to promote bone regeneration while eradicating pathogenic bacteria through light-cellular force-electric coupling. Specifically, an array of heterojunctions (TiO2/Bi2WO6), made of piezoelectric nanocrystals (Bi2WO6) decorating TiO2 nanowires, is fabricated as a biocompatible implant. Upon exposure to near-infrared light, the piezoelectric heterojunctions generate reactive oxygen species and heat to kill bacteria through photodynamic and photothermal therapy, respectively. Meanwhile, the mechanical forces of the stem cells grown on the implant trigger the heterojunctions to produce electric fields that further promote osteogenesis to achieve osteointegration. The heterojunctions effectively suppress postoperative recurrent infections while promoting osseointegration through the local electric fields induced by cells. Therefore, the piezoelectric heterojunctions represent a promising antibacterial tissue-regenerative implant.

LSPR-enhanced photoresponsive antibacterial efficiency of Bi/MoS2-loaded fibrin gel for management of diabetic wounds.

Chronic diabetic wounds present formidable challenges, marked by uncontrolled bacterial infections, prolonged inflammation, and impaired angiogenesis. The evolving landscape of photo-responsive antibacterial therapy holds great promise in addressing these multifaceted issues, with a particular focus on leveraging the distinctive properties of 2D heterojunction materials. In this investigation, we engineered composite sprayed hydrogels, seamlessly integrating Bi/MoS2 nano-heterojunctions. Capitalizing on the synergistic interplay between photocatalytic antibacterial and photothermal antibacterial mechanisms, the Bi/MoS2 heterojunction, guided by its localized surface plasmon resonance, demonstrated outstanding antibacterial efficacy within a mere 10-minute exposure to 808 nm near-infrared light. This accelerated sterilization both in vitro and in vivo, consequently expediting wound healing. The sprayed composite gel not only furnishes protective shielding for skin tissues but also fosters endothelial cell proliferation, vascularization, and angiogenesis. This safe and ultrafast sterilizing hydrogel presents immense potential for application in antimicrobial dressings, thereby offering a promising avenue for diabetic wound healing.

Hydrogel Coatings of Implants for Pathological Bone Repair.

Hydrogels are well-suited for biomedical applications due to their numerous advantages, such as excellent bioactivity, versatile physical and chemical properties, and effective drug delivery capabilities. Recently, hydrogel coatings have developed to functionalize bone implants which are biologically inert and cannot withstand the complex bone tissue repair microenvironment. These coatings have shown promise in addressing unique and pressing medical needs. This review begins with the major functionalized performance and interfacial bonding strategy of hydrogel coatings, with a focus on the novel external field response properties of the hydrogel. Recent advances in the fabrication strategies of hydrogel coatings and their use in the treatment of pathologic bone regeneration are highlighted. Finally, challenges and emerging trends in the evolution and application of physiological environment-responsive and external electric field-responsive hydrogel coatings for bone implants are discussed.

Sea Anemone-Inspired Conducting Polymer Sensing Platform for Integrated Detection of Tumor Protein Marker and Circulating Tumor Cell.

Combining the detection of tumor protein markers with the capture of circulating tumor cells (CTCs) represents an ultra-promising approach for early tumor detection. However, current methodologies have not yet achieved the necessary low detection limits and efficient capture. Here, a novel polypyrrole nanotentacles sensing platform featuring anemone-like structures capable of simultaneously detecting protein biomarkers and capturing CTCs is introduced. The incorporation of nanotentacles significantly enhances the electrode surface area, providing abundant active sites for antibody binding. This enhancement allows detecting nucleus matrix protein22 and bladder tumor antigen with 2.39 and 3.12 pg mL-1 detection limit, respectively. Furthermore, the developed sensing platform effectively captures MCF-7 cells in blood samples with a detection limit of fewer than 10 cells mL-1, attributed to the synergistic multivalent binding facilitated by the specific recognition antibodies and the positive charge on the nanotentacles surface. This sensing platform demonstrates excellent detection capabilities and outstanding capture efficiency, offering a simple, accurate, and efficient strategy for early tumor detection.

Tetramethylpyrazine-loaded electroconductive hydrogels promote tissue repair after spinal cord injury by protecting the blood-spinal cord barrier and neurons.

Spinal cord injury (SCI) usually induces profound microvascular dysfunction. It disrupts the integrity of the blood-spinal cord barrier (BSCB), which could trigger a cascade of secondary pathological events that manifest as neuronal apoptosis and axonal demyelination. These events can further lead to irreversible neurological impairments. Thus, reducing the permeability of the BSCB and maintaining its substructural integrity are essential to promote neuronal survival following SCI. Tetramethylpyrazine (TMP) has emerged as a potential protective agent for treating the BSCB after SCI. However, its therapeutic potential is hindered by challenges in the administration route and suboptimal bioavailability, leading to attenuated clinical outcomes. To address this challenge, traditional Chinese medicine, TMP, was used in this study to construct a drug-loaded electroconductive hydrogel for synergistic treatment of SCI. A conductive hydrogel combined with TMP demonstrates good electrical and mechanical properties as well as superior biocompatibility. Furthermore, it also facilitates sustained local release of TMP at the implantation site. Furthermore, the TMP-loaded electroconductive hydrogel could suppress oxidative stress responses, thereby diminishing endothelial cell apoptosis and the breakdown of tight junction proteins. This concerted action repairs BSCB integrity. Concurrently, myelin-associated axons and neurons are protected against death, which meaningfully restore neurological functions post spinal cord injury. Hence, these findings indicate that combining the electroconductive hydrogel with TMP presents a promising avenue for potentiating drug efficacy and synergistic repair following SCI.

Localized Surface Plasmon Resonance-Enhanced Photocatalytic Antibacterial of In Situ Sprayed 0D/2D Heterojunction Composite Hydrogel for Treating Diabetic Wound.

Chronic diabetic wounds pose significant challenges due to uncontrolled bacterial infections, prolonged inflammation, and impaired angiogenesis. The rapid advancement of photo-responsive antibacterial therapy shows promise in addressing these complex issues, particularly utilizing 2D heterojunction materials, which offer unique properties. Herein, an in situ sprayed Bi/BiOCl 0D/2D heterojunction composite fibrin gel with the characteristics of rapid formation and effective near-infrared activation is designed for the treatment of non-healing diabetes-infected wounds. The sprayed composite gel can provide protective shielding for skin tissues and promote endothelial cell proliferation, vascularization, and angiogenesis. The Bi/BiOCl 0D/2D heterojunction, with its localized surface plasmon resonance (LSPR), can overcome the wide bandgap limitation of BiOCl, enhancing the generation of local heat and reactive oxygen species under near-infrared irradiation. This facilitates bacterial elimination and reduced inflammation, supporting the accelerated healing of diabetes-infected wounds. This study underscores the potential of LSPR-enhanced heterojunctions as advanced wound therapies for chronic diabetic wounds.

Injectable ultrasound-powered bone-adhesive nanocomposite hydrogel for electrically accelerated irregular bone defect healing.

The treatment of critical-size bone defects with irregular shapes remains a major challenge in the field of orthopedics. Bone implants with adaptability to complex morphological bone defects, bone-adhesive properties, and potent osteogenic capacity are necessary. Here, a shape-adaptive, highly bone-adhesive, and ultrasound-powered injectable nanocomposite hydrogel is developed via dynamic covalent crosslinking of amine-modified piezoelectric nanoparticles and biopolymer hydrogel networks for electrically accelerated bone healing. Depending on the inorganic-organic interaction between the amino-modified piezoelectric nanoparticles and the bio-adhesive hydrogel network, the bone adhesive strength of the prepared hydrogel exhibited an approximately 3-fold increase. In response to ultrasound radiation, the nanocomposite hydrogel could generate a controllable electrical output (-41.16 to 61.82 mV) to enhance the osteogenic effect in vitro and in vivo significantly. Rat critical-size calvarial defect repair validates accelerated bone healing. In addition, bioinformatics analysis reveals that the ultrasound-responsive nanocomposite hydrogel enhanced the osteogenic differentiation of bone mesenchymal stem cells by increasing calcium ion influx and up-regulating the PI3K/AKT and MEK/ERK signaling pathways. Overall, the present work reveals a novel wireless ultrasound-powered bone-adhesive nanocomposite hydrogel that broadens the therapeutic horizons for irregular bone defects.

NIR-responsive micropatterned nanocomposite functionalized implant for sequential antibacterial and osteogenesis.

The long-term durability of the implant is influenced by two significant clinical challenges, namely bacterial infection and fixation loosening. Conventional implant materials have failed to meet the demands of the dynamic process of infectious bone repair, which necessitates early-stage bacterial sterilization and a conducive environment for late-stage osteogenesis. Consequently, there is an urgent requirement for an implant material that can sequentially regulate antibacterial properties and promote osteogenesis. The study aimed to develop a micropatterned graphene oxide nanocomposite on titanium implant (M-NTO/GO) for the sequential management of bacterial infection and osteogenic promotion. M-NTO/GO exhibited a micropattern nanostructure surface and demonstrated responsiveness to near-infrared (NIR) light. Upon NIR light irradiation, M-NTO/GO exhibited effective antibacterial properties, achieving antibacterial rates of 96.9% and 98.6% against E. coli and S. aureus, respectively. Under no-light condition, the micropatterned topography of M-NTO/GO exhibited the ability to induce directed cell growth, enhance cell adhesion and spreading, and facilitate osteogenic differentiation. These findings suggest the successful development of a functionalized micropatterned nanocomposite implant capable of sequentially regulating antibacterial and osteogenesis activity. Consequently, this highly effective strategy holds promise for expanding the potential applications of orthopedic implants.

[Bionic design, preparation and clinical translation of oral hard tissue restorative materials].

Oral diseases concern almost every individual and are a serious health risk to the population. The restorative treatment of tooth and jaw defects is an important means to achieve oral function and support the appearance of the contour. Based on the principle of "learning from the nature", Deng Xuliang's group of Peking University School and Hospital of Stomatology has proposed a new concept of "microstructural biomimetic design and tissue adaptation of tooth/jaw materials" to address the worldwide problems of difficulty in treating dentine hypersensitivity, poor prognosis of restoration of tooth defects, and vertical bone augmentation of alveolar bone after tooth loss. The group has broken through the bottleneck of multi-stage biomimetic technology from the design of microscopic features to the enhancement of macroscopic effects, and invented key technologies such as crystalline/amorphous multi-level assembly, ion-transportation blocking, and multi-physical properties of the micro-environment reconstruction, etc. The group also pioneered the cationic-hydrogel desensitizer, digital stump and core integrated restorations, and developed new crown and bridge restorative materials, gradient functionalisation guided tissue regeneration membrane, and electrically responsive alveolar bone augmentation restorative membranes, etc. These products have established new clinical strategies for tooth/jaw defect repair and achieved innovative results. In conclusion, the research results of our group have strongly supported the theoretical improvement of stomatology, developed the technical system of oral hard tissue restoration, innovated the clinical treatment strategy, and led the progress of the stomatology industry.

Piezo-enhanced near infrared photocatalytic nanoheterojunction integrated injectable biopolymer hydrogel for anti-osteosarcoma and osteogenesis combination therapy.

Preventing local tumor recurrence while promoting bone tissue regeneration is an urgent need for osteosarcoma treatment. However, the therapeutic efficacy of traditional photosensitizers is limited, and they lack the ability to regenerate bone. Here, a piezo-photo nanoheterostructure is developed based on ultrasmall bismuth/strontium titanate nanocubes (denoted as Bi/SrTiO3), which achieve piezoelectric field-driven fast charge separation coupling with surface plasmon resonance to efficiently generate reactive oxygen species. These hybrid nanotherapeutics are integrated into injectable biopolymer hydrogels, which exhibit outstanding anticancer effects under the combined irradiation of NIR and ultrasound. In vivo studies using patient-derived xenograft models and tibial osteosarcoma models demonstrate that the hydrogels achieve tumor suppression with efficacy rates of 98.6 % and 67.6 % in the respective models. Furthermore, the hydrogel had good filling and retention capabilities in the bone defect region, which exerted bone repair therapeutic efficacy by polarizing and conveying electrical stimuli to the cells under mild ultrasound radiation. This study provides a comprehensive and clinically feasible strategy for the overall treatment and tissue regeneration of osteosarcoma.

Tough, Antifreezing, and Piezoelectric Organohydrogel as a Flexible Wearable Sensor for Human-Machine Interaction.

Piezoelectric hydrogel sensors are becoming increasingly popular for wearable sensing applications due to their high sensitivity, self-powered performance, and simple preparation process. However, conventional piezoelectric hydrogels lack antifreezing properties and are thus confronted with the liability of rupture in low temperatures owing to the use of water as the dispersion medium. Herein, a kind of piezoelectric organohydrogel that integrates piezoelectricity, low-temperature tolerance, mechanical robustness, and stable electrical performance is reported by using poly(vinylidene fluoride) (PVDF), acrylonitrile (AN), acrylamide (AAm), p-styrenesulfonate (NaSS), glycerol, and zinc chloride. In detail, the dipolar interaction of the PVDF chain with the PAN chain facilitates the crystal phase transition of PVDF from the α to ß phase, which endows the organohydrogels with a high piezoelectric constant d33 of 35 pC/N. In addition, the organohydrogels are highly ductile and can withstand significant tensile and compressive forces through the synergy of the dipolar interaction and amide hydrogen bonding. Besides, by incorporating glycerol and zinc chloride, the growth of ice crystals is inhibited, allowing the organohydrogels to maintain stable flexibility and sensitivity even at -20 °C. The real-time monitoring of the pulse signal for up to 2 min indicates that the gel sensor has stable sensitivity. It is believed that our organohydrogels will have good prospects in future wearable electronics.

Zwitterion modified cochlear implants resist postoperative infection and inflammation.

The cochlear implant (CI), an advanced electronic device replacing the entire cochlear function, is the ultimate treatment for over 466 million people with disabling hearing loss. Infection after cochlear implantation is a common and worrisome complication despite the routine administration of the antibiotic. The bacterial biofilms formed on the surface of CI are the main cause of antibiotic failure. To solve this problem, we developed a copper-containing zwitterionic coating consisting of anti-adherent poly sulfobetaine methacrylate (PSB) and steadfast polydopamine (PDA). CuSO4/H2O2. was added to accelerate this co-deposition reaction and enhance the anti-bacterial property. The preparation method was simple, rapid, and suitable for clinical use. In our in vitro and in vivo studies, the PSB/PDA(Cu) coating showed high biocompatibility, and conferred CI implants excellent anti-inflammatory, strong anti-bacterial effects, and great anti-biofilm properties to representative Gram-positive and Gram-negative bacteria. These findings implied that the PSB/PDA(Cu) coating was a unique anti-bacterial strategy for enhancing CI performance.

Electro-mechanical coupling directs endothelial activities through intracellular calcium ion deployment.

Conversion between mechanical and electrical cues is usually considered unidirectional in cells with cardiomyocytes being an exception. Here, we discover a material-induced external electric field (Eex) triggers an electro-mechanical coupling feedback loop in cells other than cardiomyocytes, human umbilical vein endothelial cells (HUVECs), by opening their mechanosensitive Piezo1 channels. When HUVECs are cultured on patterned piezoelectric materials, the materials generate Eex (confined at the cellular scale) to polarize intracellular calcium ions ([Ca2+]i), forming a built-in electric field (Ein) opposing Eex. Furthermore, the [Ca2+]i polarization stimulates HUVECs to shrink their cytoskeletons, activating Piezo1 channels to induce influx of extracellular Ca2+ that gradually increases Ein to balance Eex. Such an electro-mechanical coupling feedback loop directs pre-angiogenic activities such as alignment, elongation, and migration of HUVECs. Activated calcium dynamics during the coupling further modulate the downstream angiogenesis-inducing eNOS/NO pathway. These findings lay a foundation for developing new ways of electrical stimulation-based disease treatment.

All-Polymer Piezoelectric Elastomer with High Stretchability, Low Hysteresis, Self-Adhesion, and UV-Blocking as Flexible Sensor.

All-polymer piezoelectric elastomers that integrate self-powered, soft, and elastic performance are attractive in the fields of flexible wearable electronics and human-machine interfaces. However, a lack of adhesion and UV-blocking performances greatly hinders the potential applications of elastomers in these emerging fields. Here, a high-performance piezoelectric elastomer with piezoelectricity, mechanical robustness, self-adhesion, and UV-resistance was developed by using poly(vinylidene fluoride) (PVDF), acrylonitrile (AN), acrylamide (AAm), and oxidized tannic acid (OTA) (named PPO). In this design, the dipole-dipole interactions between the PVDF and PAN chains promoted the content of ß-PVDF, endowing high piezoelectric coefficient (d33, 58 pC/N). Besides, high stretchability (∼500%), supercompressibility (∼98%), low Young's modulus (∼0.02 MPa), and remarkable elasticity (∼13.8% hysteresis ratio) were achieved simultaneously for the elastomers. Inspired by the mussel adhesion chemistry, the OTA containing abundant catechol and quinone groups provided high adhesion (93.26 kPa to wood) and an exceptional UV-blocking property (∼99.9%). In addition, the elastomers can produce a reliable electric signal output (Vocmax = 237 mV) and show a fast response (24 ms) when subjected to external force. Furthermore, the elastomer can be easily assembled as a wearable sensor for human physiological (body pulse and speech identification) monitoring and communication.

Flexible, Breathable, and Self-Powered Patch Assembled of Electrospun Polymer Triboelectric Layers and Polypyrrole-Coated Electrode for Infected Chronic Wound Healing.

Chronic wound healing is often impaired by bacterial infection and weak trans-epithelial potential. Patches with electrical stimulation and bactericidal activity may solve this problem. However, inconvenient power and resistant antibiotics limit their application. Here, we proposed a self-powered and intrinsic bactericidal patch based on a triboelectric nanogenerator (TENG). Electrospun polymer tribo-layers and a chemical vapor-deposited polypyrrole electrode are assembled as the TENG, offering the patch excellent flexibility, breathability, and wettability. Electrical stimulations by harvesting mechanical motions and positive charges on the polypyrrole surface kill over 96% of bacteria due to their synergistic effects on cell membrane disruption. Moreover, the TENG patch promotes infected diabetic rat skin wounds to heal within 2 weeks. Cell culture and animal tests suggest that electrical stimulation enhances gene expression of growth factors for accelerated wound healing. This work provides new insights into the design of wearable and multifunctional electrotherapy devices for chronic wound treatment.

Piezoelectric Bioactive Glasses Composite Promotes Angiogenesis by the Synergistic Effect of Wireless Electrical Stimulation and Active Ions.

Insufficient angiogenesis frequently occurs after the implantation of orthopedic materials, which greatly increases the risk of bone defect reconstruction failure. Therefore, the development of bone implant with improved angiogenic properties is of great importance. Mimicking the extracellular matrix clues provides a more direct and effective strategy to modulate angiogenesis. Herein, inspired by the bioelectrical characteristics of the bone microenvironment, a piezoelectric bioactive glasses composite (P-KNN/BG) based on the incorporation of polarized potassium sodium niobate is constructed, which could effectively promote angiogenesis. It is found that P-KNN/BG has exceptional wireless electrical stimulation performance and sustained active ions release. In vitro cell experiments reveal that P-KNN/BG enhances endothelial cell adhesion, migration, and differentiation via activating the eNOS/NO signaling pathway, which might be contributed to cell membrane hyperpolarization induced by wireless electrical stimulation increase the influx of active ions into the cells. In vivo chick chorioallantoic membrane experiment demonstrates that P-KNN/BG shows excellent pro-angiogenic capacity and biocompatibility. This work broadens the current understanding of bioactive materials with bionic electrical properties, which brings new insights into the clinical treatment of bone defect repair.

Injectable Intrinsic Photothermal Hydrogel Bioadhesive with On-Demand Removability for Wound Closure and MRSA-Infected Wound Healing.

Photothermal hydrogel adhesives have yielded promising results for wound closure and infected wound treatment in recent years. However, photothermal hydrogel bioadhesives with on-demand removability without additional nanomaterials-based photothermal agents have rarely been reported in the literature. In this work, an injectable intrinsic photothermal hydrogel bioadhesive with an on-demand removal trait is developed through dynamic cross-linking of gelatin (Gel), tannic acid (TA) quinone, and borax for closing skin incisions and accelerating methicillin-resistant Staphylococcus aureus (MRSA) infected wound healing. The TA quinone containing polyphenol and quinone groups with multifunctional adhesiveness and intrinsic photothermal performance confer the hydrogel adhesive with near-infrared (NIR) responsive antibacterial activity. The cross-linking of pH-sensitive boronic ester (polyphenol-B) and Schiff base bonds endow the hydrogel with great self-healing capacity and on-demand removability. Moreover, the hydrogel possesses good biocompatibility, injectability, and hemostasis. The in vivo experiment in a rat cutaneous incision model and full-thickness MRSA-infected wound model indicate that the smart hydrogel can close wounds efficiently and treat infected ones, demonstrating its superiority in noninvasive treatment of cutaneous incisions and enhancing infected full-thickness wound healing.

Soft, Super-Elastic, All-Polymer Piezoelectric Elastomer for Artificial Electronic Skin.

Piezoelectric sensors are widely used in wearable devices to mimic the functions of human skin. However, it is considerably challenging to develop soft piezoelectric materials that can exhibit high sensitivity, stretchability, super elasticity, and suitable modulus. In this study, a soft skin-like piezoelectric polymer elastomer composed of poly(vinylidene fluoride) (PVDF) and a novel elastic substrate polyacrylonitrile is prepared by combining the radical polymerization and freeze-drying processes. Dipole-dipole interaction results in the phase transition of PVDF (α phase to ß phase), which enhances the electrical and mechanical performances. Thus, we achieve a high piezoelectric coefficient (d33max = 63 pC/N), good stretchability (211.3-259.3%), super compressibility (subjected to 99% compression strain without cracking), and super elasticity (100% recovery after extreme compression) simultaneously for the elastomer. The soft composite elastomer produces excellent electrical signal output (Vocmax = 253 mV) and responds rapidly (15 ms) to stress-induced polarization effects. In addition, the elastomer-based sensor accurately detects various physiological signals such as gestures, throat vibrations, and pulse waves. The developed elastomers exhibit excellent mechanical properties and high sensitivity, which helps facilitate their application as artificial electronic skin to sense subtle external pressure in real time.

Light-Programmable Nanocomposite Hydrogel for State-Switchable Wound Healing Promotion and Bacterial Infection Elimination.

Developing an ideal wound dressing that not only accelerates wound healing but also eliminates potential bacterial infections remains a difficult balancing act. This work reports the design of a light-programmable sodium alginate nanocomposite hydrogel loaded with BiOCl/polypyrrole (BOC/PPy) nanosheets for state-switchable wound healing promotion and bacterial infection elimination remotely. The nanocomposite hydrogel possesses programmable photoelectric or photothermal conversion due to the expanded light absorption range, optimized electron transmission interface, promoted photo-generated charge separation, and transfer of the BOC/PPy nanosheets. Under white light irradiation state, the nanocomposite hydrogel induces human umbilical vein endothelial cells migration and angiogenesis, and accelerates the healing efficiency of mouse skin in vivo. Under near-infrared light irradiation state, the nanocomposite hydrogel presents superior antibacterial capability in vitro, and reaches an antibacterial rate of 99.1% for Staphylococcus aureus infected skin wound in vivo. This light-programmable nanocomposite hydrogel provides an on-demand resolution of biological state-switching to balance wound healing and elimination of bacterial infection.

Strong Biopolymer-Based Nanocomposite Hydrogel Adhesives with Removability and Reusability for Damaged Tissue Closure and Healing.

Bioadhesives are widely used in a variety of medical settings due to their ease of use and efficient wound closure and repair. However, achieving both strong adhesion and removability/reusability is highly needed but challenging. Here, we reported an injectable mesoporous bioactive glass nanoparticle (MBGN)-incorporated biopolymer hydrogel bioadhesive that demonstrates a strong adhesion strength (up to 107.55 kPa) at physiological temperatures that is also removable and reusable. The incorporation of MBGNs in the biopolymer hydrogel significantly enhances the tissue adhesive strength due to an increased cohesive and adhesive property compared to the hydrogel adhesive alone. The detachment of bioadhesive results from temperature-induced weakening of interfacial adhesive strength. Moreover, the bioadhesive displays injectability, self-healing, and excellent biocompatibility. We demonstrate potential applications of the bioadhesive in vitro, ex vivo, and in vivo for hemostasis and intestinal leakage closure and accelerated skin wound healing compared to surgical wound closures. This work provides a novel design of strong and removable bioadhesives.