Wet Adhesion Research Articles

Wet-Adaptive Electronic Skin.

Skin electronics provides remarkable opportunities for non-invasive and long-term monitoring of a wide variety of biophysical and physiological signals that are closely related to health, medicine, and human-machine interactions. Nevertheless, conventional skin electronics fabricated on elastic thin films are difficult to adapt to the wet microenvironments of the skin: Elastic thin films are non-permeable, which block the skin perspiration; Elastic thin films are difficult to adhere to wet skin; Most skin electronics are difficult to work underwater. Here, a Wet-Adaptive Electronic Skin (WADE-skin) is reported, which consists of a next-to-skin wet-adhesive fibrous layer, a next-to-air waterproof fibrous layer, and a stretchable and permeable liquid metal electrode layer. While the electronic functionality is determined by the electrode design, this WADE-skin simultaneously offers superb stretchability, wet adhesion, permeability, biocompatibility, and waterproof property. The WADE-skin can rapidly adhere to human skin after contact for a few seconds and stably maintain the adhesion over weeks even under wet conditions, without showing any negative effect to the skin health. The use of WADE-skin is demonstrated for the stable recording of electrocardiogram during intensive sweating as well as underwater activities, and as the strain sensor for the underwater operation of virtual reality-mediated human-machine interactions.

A tough bio-adhesive inspired by pearl layer and arthropod cuticle structure with desirable water resistance, flame-retardancy, and antibacterial property

Petroleum-derived formaldehyde resin adhesives are serious hazards to human health and depend on limited resources. Abundant, cheap and renewable biomass materials are expected to replace them. However, the contradictory mechanisms of high mechanical strength and fracture toughness affect the use of bioadhesives. Herein, a biomimetic soybean meal (SM) adhesive inspired by the structure of insect cuticles and shell pearl layer was proposed. Specifically, chitosan (CS@DA) modified 3,4-dihydroxybenzoic acid (DA, rich in catechol moiety) was anchored on molybdenum disulfide nanosheets (MoS2) to construct a biomimetic structure with copper hydroxide and SM substrate (SM-MoS2/CS@DA-Cu). Schiff base, ionic, and hydrogen bonding strengthened the cohesion of the adhesive. The ordered alternating stacking “brick-mortar” structure stimulated the lamellar sliding and crack deflection of MoS2, synergistically reinforcing the toughness. Compared to SM adhesive (0.57 MPa and 0.148 J), the wet shear strength and adhesion work of the SM-MoS2/CS@DA-Cu were 1.68 MPa and 0.867 J, with 194.7 % and 485.8 % increases, respectively. The multiple antimicrobial effects of CS@DA, Schiff base, and Cu2+ increased the applicability period of the adhesive to 40 days. The adhesive also displayed favorable water resistance and flame retardancy. Therefore, this peculiar and efficient biomimetic structural design inspired the development of multi-functional composites.

Injectable Antiswelling and High-Strength Bioactive Hydrogels with a Wet Adhesion and Rapid Gelling Process to Promote Sutureless Wound Closure and Scar-free Repair of Infectious Wounds.

Developing injectable antiswelling and high-strength bioactive hydrogels with wet tissue adhesiveness and a rapid gelling process to meet the requirements for rapid hemostasis, sutureless wound closure, and scar-free repair of infected skin wounds continues to have ongoing challenges. Herein, injectable, antibacterial, and antioxidant hydrogel adhesives based on poly(citric acid-co-polyethylene glycol)-g-dopamine and amino-terminated Pluronic F127 (APF) micelles loaded with astragaloside IV (AS) are prepared. The H2O2/horseradish peroxidase (HRP) system is used to cause cross-linking of the hydrogel network through oxidative coupling between catechol groups and chemical cross-linking between the catechol group and the amino group. The hydrogels exhibit a rapid gelling process, high mechanical strength, an antiswelling effect, good antioxidant property, H2O2 release behavior, and degradability. In addition, the hydrogels present good wet tissue adhesiveness, high bursting pressure, excellent antibacterial activity, long-term sustained release of AS, and good biocompatibility. The hydrogels perform good hemostasis on mouse liver, rat liver, and rabbit femoral vein bleeding models and achieve much better closure and healing of skin incisions than biomedical glue and surgical sutures. Furthermore, the hydrogel dressing significantly improved the scar-free repair of MRSA-infected full thickness skin defect wounds by modulating inflammation, regulating the ratio of collagen I/III, and improving the vascularization and granulation tissue formation. Thus, AS-loaded hydrogels show huge potential as multifunctional dressings for in vivo hemostasis, sutureless wound closure, and scar-free repair of infected skin wounds.

Mussel‐Inspired Adhesive Hydrogels: Chemistry and Biomedical Applications†

Comprehensive SummaryAdhesive hydrogels are an emerging class of hydrogels that combine three‐dimensional hydrated networks with adhesive properties. These properties facilitate intimate tissue‐material contact in diverse biomedical applications, enhancing tissue joining, drug transport, and signal transmission. Inspired by the universal adhesiveness of mussel foot proteins, 3,4‐dihydroxyphenyl‐L‐alanine (DOPA) and its analogs have been extensively exploited for the fabrication of adhesive hydrogels, within which the DOPA moieties can not only serve as cross‐linking mediators but also participate in various intermolecular and surface interactions to mediate wet adhesion. This mini‐review highlights recent achievements in the development of mussel‐inspired adhesive hydrogels, focusing on: (1) elucidating DOPA‐mediated adhesion mechanisms through nanomechanical characterizations, (2) designing injectable adhesive hydrogels toward applications in drug delivery, hemostasis, and wound closure, which includes in situ gelling liquids and shear‐thinning preformed hydrogels, and (3) fabricating tough adhesive hydrogels with enhanced mechanical properties for use in tissue regeneration, biosensing, and bioimaging, with typical examples of nanocomposite and double‐network hydrogels. The challenges and prospects in this rapidly developing field are also discussed.

Chemical Structure Evaluations of Amine Hardeners to Ensure and Predict the Performance of Wet Adhesion of Epoxies

Abstract The relationship between the chemical structure and performance of a water-sorbed epoxy adhesive (wet adhesion) provides fundamental data for epoxy adhesives for application in wet and underwater environments. However, data on the effect of the chemical structure on wet adhesion remains insufficient. This study systematically examined the wet adhesion strengths of epoxies comprising bisphenol A diglycidyl ether and various amines. The use of numerical parameters quantifying the features of the chemical structure and physicochemical properties via theoretical calculations to analyze the correlation between wet adhesion and the chemical structure of amine yielded clear linear relationships. This enabled the extraction of the amine molecular structural features that were superior in wet adhesion, in addition to quantification of the certainties of the features contributing to the physical properties. Furthermore, a prediction model for wet adhesive strength was prepared using machine-learning least absolute shrinkage and selection operator regression analysis. The model exhibited a reasonable accuracy, even using only 14 experimental values, and its effectiveness was verified experimentally. This process facilitates the rational design and selection of amine hardeners for preparing epoxies with excellent performance in wet conditions and underwater environments.

Facilely Prepared Thirsty Granules Arouse Tough Wet Adhesion on Overmoist Wounds for Hemostasis and Tissue Repair.

Bioadhesives have been widely used in hemostasis and tissue repair, but the overmoist and wet nature of wound surface (due to the presence of blood and/or wound exudate) has led to poor wet adhesion of bioadhesives, which interrupts the continuous care of wounds. Here, a thirsty polyphenolic silk granule (Tan@SF-pwd-hydro), which absorbs blood and exudate to self-convert to robust bioadhesives (Tan@SF-gel-hydro) in situ, was facilely developed in this study for enhanced wet adhesion toward hemostasis and tissue repair. Tan@SF-pwd-hydro could shield wounds' wetness and immediately convert itself to Tan@SF-gel-hydro to seal wounds for hemorrhage control and wound healing. The maximum adhesiveness of Tan@SF-gel-hydro over wet pigskin was as high as 59.8 ± 2.1 kPa. Tan@SF-pwd-hydro is a promising transformative dressing for hemostasis and tissue repair since its hemostatic time was approximately half of that of the commercial hemostatic product, CeloxTM, and its healing period was much shorter than that of the commercial bioadhesive product, TegadermTM. This pioneering study utilized adverse wetness over wounds to arouse robust adhesiveness by converting thirsty granules to bioadhesives in situ, creatively turning adversity into opportunities. The facile fabrication approach also offers new perspectives for manufacturing sustainability of biomaterials.

Cellulose‐Based pH‐Responsive Janus Dressing with Unidirectional Moisture Drainage for Exudate Management and Diabetic Wounds Healing

AbstractDiabetic wounds with hyperglycemic tissue exudates and bacterial infections have become a great challenge that seriously delays the healing of diabetic wounds. Therefore, multifunctional dressings for exudate management of diabetic wound are urgently needed. Herein, a smart dressing with rapid moisture‐drained and non‐adhesive, pH‐responsive, and antibacterial capabilities is proposed for exudate management and wound monitoring. The Janus dressing is assembled with an antioxidant, pH‐sensitive, and hydrophilic cellulose cover and an antibacterial hydrophobic polycaprolactone bottom layer that directly contacts with the wounds, which can unidirectionally and irreversibly drain the wound exudates and weaken the wet adhesion to the wound. In response to the humoral pH variations (5‐9) during the healing process ofdiabetic wounds, the healing process can be in situ monitored according to the distinct pH‐responsive colors of the Janus dressing at different healing stages. In vivo assays and histopathological studies suggest that the Janus dressing has a superior pro‐healing rate, collagen deposition, and angiogenesis than commercial gauze. Notably, such a mulitfunctional dressing provides real‐time wound monitoring simply through a smartphone integrated with Python‐RGB programs, which can significantly alleviate the hyperglycemic exudate‐flooded environment as an alternative strategy for diabetic wound treatment.

Bioadhesive Microcarriers Encapsulated with IL-27 High Expressive MSC Extracellular Vesicles for Inflammatory Bowel Disease Treatment.

Mesenchymal stem cell (MSC) therapy is a promising candidate for inflammatory bowel disease (IBD) treatment, while overcoming the limitations of naive seeding cells function and realizing efficient intestinal targeting remains a challenge. Here, a bioadhesive microparticle carrying interleukin-27 (IL-27) MSC-derived extracellular vesicles (MSCIL-27 EVs) is developed to treat IBD. The MSCIL-27 EVs prepared through lentivirus-mediated gene transfection technology show ideal anti-inflammatory and damage repair function. By encapsulating MSCIL-27 EVs into dopamine methacrylamide-modified hydrogel, a bioadhesive EVs microcarrier via microfluidic technology is fabricated. The resultant microcarriers exhibit ideal MSCIL-27 EVs sustained release effect and effective wet adhesion property. Furthermore, the therapeutic potential of MSCIL-27 EVs-loaded microcarriers in treating IBD is demonstrated. Through giving IBD rats a rectal administration, it is found that the microcarriers can firmly anchor to the surface of colon, reduce the inflammatory response, and repair the damaged barrier. Therefore, the bioadhesive MSCIL-27 EVs-loaded microcarriers provide a promising strategy for the biomedical application of MSC-derived EVs, and broaden the clinical potential of MSC therapy.

Stretchable Multi‐Channel Ionotronic Electrodes for In Situ Dual‐Modal Monitoring of Muscle–Vascular Activity

AbstractIn situ dual‐modal detection of electrophysiology (EP) and blood oxygen (BO) during muscle–vascular activity is crucial for healthcare and human–machine interaction. It requires an integrated monitoring system: a transparent electrode for EP detection directly adhered to skin and a near‐infrared spectroscopy (NIRs) layer for BO detection. In particular, the multi‐channel, highly transparent, stretchable electrodes should be well developed. In this study, 16‐channel transparent ionotronic electrodes are fabricated based on a stretchable and printable ionogel, demonstrating excellent properties such as high transparency, large stretchability, wet adhesion, biocompatibility, softness, and long‐term stability. The electrode exhibits stable contact impedance for 24 h and a 2‐week periodic EP monitoring. The 16‐channel transparent ionotronic electrode outperforms stretchable gold electrodes by providing stable EP monitoring with minimal interference in NIRs for blood oxygen detection. An in situ negative correlation is found between EP intensity and blood oxygen level over time. This study paves the way for designing transparent ionotronics for synchronous optical‐related monitoring and highlights the potential use of an in situ dual‐modal monitoring system for the study of muscle–vascular coupling in neurocience and rehabilitation.

3D-exosomes laden multifunctional hydrogel enhances diabetic wound healing via accelerated angiogenesis

Impaired vascular networks, local insufficiency of neovascularization, and tissue inflammation caused by the accumulation of reactive oxygen species (ROS) and bacterial infections contribute to the delayed healing of wounds in patients with diabetes. Thus, an urgent need is to develop effective diabetic wound treatments that promote angiogenesis, inhibit bacterial infections, and reduce oxidative damage and tissue inflammation. Exosome therapy promotes angiogenesis and wound healing. However, traditional exosomes have limitations such as low yield and rapid release. Compared with two-dimensional (2D) cell culture, three-dimensional (3D) cell culture results in a higher yield of exosomes and better healing effects. In this study, we designed a multifunctional hydrogel with 3D-exosome-sustained release features to improve diabetic wound healing. Chitosan-grafted-dihydrocaffeic acid (CS-DA) and benzaldehyde-terminated Pluronic®F127 (PF127-CHO) were combined using a dynamic Schiff base bond to form a dynamic hydrogel network and simultaneously fused tannic acid (TA) and 3D adipose-derived mesenchymal stem cells-derived exosomes (3D ADSCs-Exos, referred to in this study as 3D-Exo). The phosphoric acid groups of 3D-Exo combine with the polyphenol groups of DA/TA through reversible interactions, enabling the sustained release of 3D-Exo. The CS-DA/PF/TA/3D-Exo hydrogel exhibited tissue-adhesive, self-healing, antibacterial, anti-inflammatory, and antioxidant properties. The CS-DA/PF/TA/3D-Exo hydrogel significantly accelerated the recovery of diabetic wounds by promoting angiogenesis and collagen deposition in vivo. Furthermore, rapid hemostasis was achieved owing to the wet tissue adhesion and platelet activation of the hydrogel. In general, this multifunctional hydrogel could be used as a wound dressing for diabetic wound management.

Robust but On-Demand Detachable Wet Tissue Adhesive Hydrogel Enhanced with Modified Tannic Acid.

Adhesives with robust but readily detachable wet tissue adhesion are of great significance for wound closure. Polyelectrolyte complex adhesive (PECA) is an important wet tissue adhesive. However, its relatively weak cohesive and adhesive strength cannot satisfy clinical applications. Herein, modified tannic acid (mTA) with a catechol group, a long alkyl hydrophobic chain, and a phenyl group was prepared first, and then, it was mixed with acrylic acid (AA) and polyethylenimine (PEI), followed by UV photopolymerization to make a wet tissue adhesive hydrogel with tough cohesion and adhesion strength. The hydrogel has a strong wet tissue interfacial toughness of ∼1552 J/m2, good mechanical properties (∼7220 kPa cohesive strength, ∼873% strain, and ∼33,370 kJ/m3 toughness), and a bursting pressure of ∼1575 mmHg on wet porcine skin. The hydrogel can realize quick and effective adhesion to various wet biological tissues including porcine skin, liver, kidney, and heart and can be changed easily with triggering urea solution to avoid tissue damage or uncomfortable pain to the patient. This biosafe adhesive hydrogel is very promising for wound closure and may provide new ideas for the design of robust wet tissue adhesives.

Characterization of Morphologically Distinct Components in the Tarsal Secretion of Medauroidea extradentata (Phasmatodea) Using Cryo-Scanning Electron Microscopy.

Attachment to the substrate is an important phenomenon that determines the survival of many organisms. Most insects utilize wet adhesion to support attachment, which is characterized by fluids that are secreted into the interface between the tarsus and the substrates. Previous research has investigated the composition and function of tarsal secretions of different insect groups, showing that the secretions are likely viscous emulsions that contribute to attachment by generating capillary and viscous adhesion, leveling surface roughness and providing self-cleaning of the adhesive systems. Details of the structural organization of these secretions are, however, largely unknown. Here, we analyzed footprints originating from the arolium and euplantulae of the stick insect Medauroidea extradentata using cryo-scanning electron microscopy (cryo-SEM) and white light interferometry (WLI). The secretion was investigated with cryo-SEM, revealing four morphologically distinguishable components. The 3D WLI measurements of the droplet shapes and volumes over time revealed distinctly different evaporation rates for different types of droplets. Our results indicate that the subfunctionalization of the tarsal secretion is facilitated by morphologically distinct components, which are likely a result of different proportions of components within the emulsion. Understanding these components and their functions may aid in gaining insights for developing adaptive and multifunctional biomimetic adhesive systems.

Adaptive Adhesions of Barnacle-Inspired Adhesive Peptides.

The strategy of robust adhesion employed by barnacles renders them fascinating biomimetic candidates for developing novel wet adhesives. Particularly, barnacle cement protein 19k (cp19k) has been speculated to be the key adhesive protein establishing the priming layer in the initial barnacle cement construction. In this work, we systematically studied the sequence design rationale of cp19k by designing adhesive peptides inspired by the low-complexity STGA-rich and the charged segments of cp19k. Combining structure analysis and the adhesion performance test, we found that cp19k-inspired adhesive peptides possess excellent disparate adhesion strategies for both hydrophilic mica and hydrophobic self-assembled monolayer surfaces. Specifically, the low-complexity STGA-rich segment offers great structure flexibility for surface adhesion, while the hydrophobic and charged residues can contribute to the adhesion of the peptides on hydrophobic and charged surfaces. The adaptive adhesion strategy identified in this work broadens our understanding of barnacle adhesion mechanisms and offers valuable insights for designing advanced wet adhesives with exceptional performance on various types of surfaces.

Construction of Poly(hydrophobic deep eutectic solvent)/Ethylcellulose Composite Films for Green Recyclable Tapes.

Constructing green recyclable cellulose-based tapes with high transparency, mechanical robustness, and strong wet adhesion using natural components is highly desirable but challenging. Herein, novel cellulose-based self-adhesive tapes were reported by coating a polymerizable hydrophobic deep eutectic solvent (DES) on ethylcellulose followed by photopolymerization. The prepared ethylcellulose-based self-adhesive tape (ECSAT) exhibited an optical transmittance of up to ∼88% and could provide strong adhesion by interfacial intermolecular interactions without obstructing information. Due to the hydrophobic nature of the overall structure, ECSAT does not exhibit significant adhesive strength and mechanical degradation under water, acid, and alkali environments. Notably, ECSAT can be completely dissolved in the resultant DES and furthermore reused as a self-adhesive coating. The recycled ECSAT still maintained good optical transparency, mechanical strength, and wet adhesion. We believe that ECSATs with all-around performances have a wide range of applications in packaging and other engineering fields.

All natural mussel-inspired bioadhesives from soy proteins and plant derived polyphenols with marked water-resistance and favourable antibacterial profile for wound treatment applications

HypothesisImplementation of tissue adhesives from natural sources endowed with good mechanical properties and underwater resistance still represents a challenging research goal. Inspired by the extraordinary wet adhesion properties of mussel byssus proteins resulting from interaction of catechol and amino residues, hydrogels from soy protein isolate (SPI) and selected polyphenols i.e. caffeic acid (CA), chlorogenic acid (CGA) and gallic acid (GA) under mild aerial oxidative conditions were prepared. ExperimentsThe hydrogels were subjected to chemical assays, ATR FT-IR and EPR spectroscopy, rheological and morphological SEM analysis. Mechanical tests were carried out on hydrogels prepared by inclusion of agarose. Biological tests included evaluation of the antibacterial and wound healing activity, and hemocompatibility. FindingsThe decrease of free NH2 and SH groups of SPI, the EPR features, the good cohesive strength and excellent underwater resistance (15 days for SPI/GA) under conditions relevant to their use as surgical glues indicated an efficient interaction of the polyphenols with the protein in the hydrogels. The polyphenols greatly also improved the mechanical properties of the SPI/ agarose/polyphenols hydrogels. These latter proved biocompatible, hemocompatible, not harmful to skin, displayed durable adhesiveness and good water-vapour permeability. Excellent antibacterial properties and in some cases (SPI/CGA) a favourable wound healing activity on dermal fibroblasts was obtained.

Wet adhesion enhancement through citric-acid-regulated supramolecular network

Adhesion on wet surface is of significance but remains challenging. Despite large steps forward in wet adhesives, they suffer from complicated preparation procedures and unbalanced properties. Herein, we develop a simple one-pot approach to fabricate a supramolecular adhesive with simultaneous enhancement in wet adhesion and mechanical toughness, which is achieved via citric acid (CA)-regulated supramolecular network of N-hydroxy succinimide (NHS) grafted polyacrylic acid chain and gelatin. CA not only reinforces supramolecular network by forming abundant hydrogen bonds, but also repels the NHS groups exposed outward to increase the opportunity for bonding with the target surface. The resultant adhesive shows fast and robust wet adhesion, high mechanical strength and elongation, as well as good biocompatibility. Such features enable the resultant adhesive with high potential in a variety of biomedical settings, such as tissue adhesion, sealant, and skin-adhesive strain sensor. This study opens a new avenue for the development of advanced bioadhesives, holding broad clinical application prospects.

Rapid hemostatic antibacterial self-gelling powder based on methacryloylsulfonyl betaine and quaternized carboxymethyl chitosan

Hemostatic powders can be used for deep wounds and wounds with irregular shapes that are frequently inaccessible to traditional hemostatic dressings like hemostatic gauze, sponges, and foams. In this study, sulfobetaine methacrylate (SBMA) and quaternized carboxymethyl chitosan (QCCS) were combined to create an antibacterial hemostatic hydrogel through photopolymerization under green LED irradiation, which was then changed into PSBMA/QCCS powder. PSBMA/QCCS powder could quickly form hydrogel with strong wet adhesion. The internal structure, water absorption capacity, and adhesion properties of the powder were evaluated. The coagulation ability, antimicrobial properties, and biocompatibility of the powder were also characterized. The PSBMA/QCCS powder could aggregate blood cells and platelets to enhance hemostasis. Meanwhile, PSBMA/QCCS powder also showed effective antibacterial ability against both gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). In summary, PSBMA/QCCS powder is a promising hemostatic agent with the characteristics of quick hemostasis, tough wet adhesion, satisfactory biocompatibility, considerable antibacterial effect, and adaptability to any irregularly shaped wounds.

Strengthening the mechanical characteristics and cathodic delamination resistance of fiber-reinforced polymer through chemical surface modification of glass fibers.

This work aims to scrutinize the effect of the silanization of glass fibers (GF) on the mechanical properties and cathodic disbonding resistance of an epoxy composite coating. Successful silanization was approved based on different characterization techniques, including Fourier transform infrared spectra, field emission-scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy, and thermogravimetric analysis. Tensile strength measurement exhibited a significant effect of silanization on the mechanical performance of the fiber-reinforced polymer (FRP). FE-SEM cross-sectional images illustrated improved interfacial bonding between the epoxy matrix and GF upon silanization. Pull-off measurements revealed improved wet adhesion strength of the FRP to the mild steel surface after exposure to the salt spray chamber when the GF were silanized. In addition, silanization revealed enhanced resistance to cathodic delamination (CD). Electrochemical impedance spectroscopy and electrochemical noise assessments proved silanization's significant influence on the FRP's CD resistance.

Water-repellent and self-attachable flexible conductive patch

Achieving exceptional water-repellency and reliable reversible adhesion is crucial for the development of wearable flexible electronics. However, simultaneously achieving these properties presents a significant challenge, as water-repellency requires maximizing the presence of air while robust adhesion necessitates enhancing the solid fraction. In this study, we present a flexible and transparent conductive patch that addresses this challenge by offering simultaneous robust superhydrophobicity and strong adhesion in both dry and wet conditions. The device incorporates a unique combination of overhang micropillars, microgrids and a percolating network of carbon nanotubes. The proposed patch demonstrates outstanding water repellency with a contact angle exceeding 150°, while delivering impressive dry adhesion (>200 kPa) and wet adhesion (>150 kPa) performance. Furthermore, the device exhibits tunable electrical conductivity and optical transmittance.

Er:YAG Laser Physical Etching and Ultra-High-Molecular-Weight Cross-Linked Sodium Polyacrylate Chemical Etching for a Reliable Dentin Dry Bonding.

Dentin bond interface stability is the key issue of dental adhesion in present clinical dentistry. The concept of selective extrafibrillar demineralization has opened a new way to maintain intrafibrillar minerals to prevent interface degradation. Here, using ultra-high-molecular-weight sodium polyacrylate [Carbopol (Carbo) > 40 kDa] as a calcium chelator, we challenge this concept and propose a protocol for reliable dentin dry bonding. The results of high-resolution transmission electron microscopy revealed periodic bands of 67 nm dentin collagen fibrils after Carbo etching, and the hydroxyproline concentration increasing with prolonged chelating time denied the concept of extrafibrillar demineralization. The results that wet and dry bonding with Carbo-based demineralization produced a weaker bond strength than the traditional phosphoric acid wet adhesion suggested that the Carbo-based demineralization is an unreliable adhesion strategy. A novel protocol of Er:YAG laser physical etching followed by Carbo chemical etching for dentin adhesion revealed that a micro-/nano-level rough, rigid, and non-collagen exposed dentin surface was produced, the micro-tensile bond strength was maintained after aging under dry and wet bonding modes, and in situ zymography and nanoleakage within the hybrid layers presented lower signals after aging. Cell culture in vitro and a rabbit deep dentin adhesion model in vivo proved that this protocol is safe and biocompatible. Taken together, the concept of extrafibrillar demineralization is limited and insufficient to use in the clinic. The strategy of Er:YAG laser physical etching followed by Carbo chemical etching for dentin adhesion produces a bonding effect with reliability, durability, and safety.