We report the case of a patient who underwent endovascular retrieval of a conical inferior vena cava (IVC) filter with a ruptured retraction hook that was attached to the IVC wall. A 21-year-old woman with a Celect (Cook) filter, implanted 1,522 days prior, requested retrieval. Preoperative ultrasound and CT examinations showed that the filter was inclined, the retraction hook was attached to the IVC wall, and one of the filter’s pedicles was broken. The inferior vena cava was patent, with no thrombus. Old superficial femoral vein thrombosis could be seen in the right lower extremity. The filter retrieval equipment  failed to capture the retraction hook. By means of a pigtail catheter (with a partly removed catheter tip) and loach guidewire, we applied a modified loop-snare technique to successfully cut the proliferative tissue near the tip of the retraction hook, by which the hook re-entered the inferior vena cava. Although the snare successfully captured the retraction hook and retrieved the filter, the broken pedicle was retained in the inferior vena cava. We used forceps to capture and pull it to the distal end. In the end, the inferior vena cava became patent, with no contrast agent spillage or residual, and no symptomatic pulmonary embolization. A simultaneous occurrence of oblique adherence and fracture is rarely found in the same filter; however, by using the modified loop-snare technique and biopsy forceps technique, we successfully retrieved the filter and broken pedicle. Our case provides a practical auxiliary technique for regular clinical practice. The American College of Chest Physicians’ (ACCP) 2016 guidelines for the management of venous thromboembolism state that an inferior vena cava (IVC) filter should be removed immediately once the risk of pulmonary embolism has decreased . EndovasHere, we report the case of a 21-year-old woman who requested retrieval of a Celect IVC filter, which had been implanted 1,522 days ago. The patient had been in a traffic accident more than 4 years ago, which had caused pelvic fracture, multiple fractures of the lower extremity, rib fracture with pleural effusion, and hemorrhage in the subarachnoid space. She had been treated with multiple surgeries. Due to deep vein thrombosis in the right lower extremity, the Celect filter was implanted  to prevent fatal pulmonary embolization. She also underwent open internal fixation surgery for unstable pelvic fractures and closed intramedullary nailing for femoral shaft fractures. The patient had been discharged without postoperative anticoagulation therapy due to factors such as a low hemoglobin count (7.2 g/L), subarachnoid hemorrhage, and pleural effusion. She returned to the local high school to continue her education, and no follow-up had been done in the last 4 years.The patient complained of dull pain in the right lower abdomen 3 months before admission. Upon admission, venous ultrasound showed an old superficial femoral vein thrombosis in the right lower extremity, but no thrombosis was found in the inferior vena cava. An enhanced abdominal CT also showed that the inferior vena cava was patent, with no hematoma visible. However, the filter was severely inclined, the retraction hook was adhered to the vena cava wall, the main pedicle of the filter was broken, and the broken pedicle was embedded in the wall of the inferior vena cava. Due to the adherence of the retraction hook to the wall and the presence of the broken pedicle, the preferred treatment options were to retrieve the filter by the endovascular method with minimal invasiveness or retrieve the filter laparoscopically . Open suAngiography via the right common femoral venous access showed that no thrombosis was found in the inferior vena cava, and that it was patent; it also showed that the main pedicle of the Celect filter was broken, the filter was obliquely attached to the retraction hook, and proliferative tissue surrounded the hook . A filteThe filter retrieval set and biopsy forceps were introduced via the right common femoral venous access, and the broken pedicle was successfully captured . HoweverThe broken pedicle was captured again, and the biopsy forceps and the retrieval sheath were used to pull the broken pedicle to the distal end. Then, the angle and coaxial distance were gradually reduced. The broken pedicle was entered into the retrieval sheath  and was No symptomatic pulmonary embolization occurred during the perioperative period, and the patient received oral rivaroxaban 20 mg QD as an anticoagulation therapy  postoperThe possible complications of long-term IVC filter implantation include obstruction and perforation of the vena cava and approaching or entering of the filter pedicle into the surrounding organs, which can manifest with associated symptoms –15. WhenDue to abnormalities in the inferior vena cava, the presence of thrombus blocked by the filter, the consequences of the IVC filter placement, or other unknown reasons, the conical filter is more prone to adhere obliquely. Furthermore, the attachment of the retraction hook to proliferative tissues can easily result in failure to capture the hook. The following methods could increase the chances of successfully retrieving the filter: (1) Use of the loop-snare technique  to form The fracture rate of the Celect filter is only 1.1%, which is the lowest rate among all kinds of IVC filters . NeverthThe patient in our study experienced two complications: oblique adherence and fracture of the filter. The modified loop-snare technique and biopsy forceps technique were used to resolve these complications, with an initial failure to capture the retraction hook and retrieve the broken pedicle and filter. There are few similar reports in the recent literature. In contrast to the use of a reverse-curve catheter, as reported by Desai et al. , the looOur surgical method has the following limitations: (1) The head of the pigtail catheter is not robust, and it is therefore easily bent or deformed. The guidewire can be introduced into the pigtail catheter end to increase the supporting force ; (2) The presence of a gap between the filter and the inferior vena cava wall is critical to the success of the modified loop-snare technique. When the filter is severely tilted or deformed, there may be no gap to allow the guidewire and catheter to pass through, making the procedure less successful. (3) A preoperative enhanced CT of the abdomen is a good way to evaluate and initially select the loop-snare technique.It should be noted that the technique of grasping a broken pedicle using biopsy forceps cannot be performed routinely, and there is a risk of the pedicle falling off. A balloon can be used to block the inferior vena cava at the proximal end, which may reduce the risk of the broken pedicle falling off into the pulmonary artery.Longer retention times can spell more serious damage for the inferior vena cava when the filter is retrieved, especially if any complications such as a broken pedicle are present. Regular postoperative anticoagulation therapy and follow-up are therefore indispensable. In accordance with the ACCP guidelines , 2, 11, In summary, oblique adherence and fracture of conical IVC filters are common complications, but the simultaneous occurrence of both in the same filter is rare. The filter and broken pedicle were removed successfully using a modified loop-snare technique and biopsy forceps technique, avoiding the major trauma of open surgery. Our method can therefore serve as an auxiliary technique for regular clinical practice.


Antibody therapies targeting FcRn, testing those anti-FcRn inhibitors that effectively catabolize the IgG4 antibody subclass, may be especially promising.The main IgG4 antibody–mediated neurological disorders (IgG4-ND) include MuSK myasthenia; CIDP with nodal/paranodal antibodies to Neurofascin-155, contactin-1/caspr-1, or pan-neurofascins; anti-LGI1 and CASPR2-associated limbic encephalitis, Morvan syndrome, or neuromyotonia; and several cases of the anti-IgLON5 and anti-DPPX-spectrum CNS diseases. The paper is centered on the clinical spectrum of IgG4-ND and their immunopathogenesis highlighting the unique functional effects of the IgG4 subclass compared to IgG1-3 antibody subclasses. The IgG4 antibodies exert pathogenic effects on their targeted antigens by blocking enzymatic activity or disrupting protein–protein interactions affecting signal transduction pathways, but not by activating complement, binding to inhibitory FcγRIIb receptor or engaging in cross-linking of the targeted antigen with immune complex formation as the IgG1-IgG3 antibody subclasses do. IgG4 can even inhibit the classical complement pathway by affecting the affinity of IgG1-2 subclasses to C1q binding. Because the IgG4 antibodies do not trigger inflammatory processes or complement-mediated immune responses, the conventional anti-inflammatory therapies, especially with IVIg, immunosuppressants, and plasmapheresis, are ineffective or not sufficiently effective in inducing long-term remissions. In contrast, aiming at the activated plasmablasts connected with IgG4 antibody production is a meaningful therapeutic target in IgG4-ND. Indeed, data from large series of patients with MuSK myasthenia, CIDP with nodal/paranodal antibodies, and anti-LGI1 and CASPR2-associated syndromes indicate that B cell depletion therapy with rituximab exerts long-lasting clinical remissions by targeting memory B cells and IgG4-producing CD20-positive short-lived plasma cells. Because IgG4 antibody titers seem reduced in remissions and increased in exacerbation, they may serve as potential biomarkers of treatment response supporting further the pathogenic role of self-reacting B cells. Controlled trials are needed in IgG4-ND not only with rituximab but also with the other anti-B cell agents that target CD19/20, especially those like The online version contains supplementary material available at 10.1007/s13311-022-01210-1. The IgG4 subclass of autoantibodies has been associated with a broad spectrum of more than 12, multisystemic or fibroinflammatory autoimmune disorders, referred to as IgG4-related diseases (IgG4-RD). These disorders, insidiously and sometimes subclinically, affect lacrimal and salivary glands, thyroid, lungs, bile ducts, kidneys, pancreas, aorta, retroperitoneum, and orbits in a form of orbital myositis . They arIn contrast to their IgG1-3-associated counterparts, the IgG4-ND exhibit most of the times poor long-term response to IVIg and inadequate long-term response to steroids or plasmapheresis, but excellent response to anti-B cell therapies, like rituximab , 3.The paper is a detailed extension of the recently published review on the same topic which was focused on why the patients with IgG4 subclass of antibodies do not respond to IVIg . The preAnti-MuSK-MGThe IgG4 antibodies arise after chronic antigenic exposure late in the immune response and, after undergoing several rounds of affinity maturation and somatic hypermutation, they exhibit very high affinity for their target antigen , 11. In About 7% of all AChR-negative MG patients have IgG4 antibodies against muscle-specific kinase (MuSK), a post-synaptic transmembrane polypeptide expressed at the neuromuscular junction. MuSK, via interactions with agrin and rapsyn, plays a fundamental role in AChR clustering which is essential for efficient neuromuscular transmission , 6, 12. 2.CIDP with paranodal antibodiesAlthough patients with MuSK-MG may have a phenotype similar to AChR-MG, they most often have unique presentation with selective weakness and atrophy of the neck, tongue, shoulder, and bulbar muscles –14. In MA breakthrough in CIDP antibody autoimmunity has been the remarkable observation that a subset of patients who do not respond to IVIg or plasmapheresis have IgG4 antibodies to nodal/paranodal antigens directed against neurofascin-155 (Nfasc155), neurofascin-140/186 (Nfasc140/186), contactin-1 (CNTN1), and contactin-associated protein 1 (Caspr1) , 15–19. NF155 is a Schwann cell adhesion protein that, at the paranodal terminal myelin loops, interacts with CNTN1/Caspr1 complex anchoring myelin to axon, forming a protein complex critical for maintaining the integrity of the nodal structures ensuring rapid impulse propagation –21. The 3.LGI-1 and CASPR2 autoimmune syndromesThe autoimmune nodopathy patients may have the typical clinical phenotype of CIDP but most often have distinct characteristics highlighted by subacute onset of severe neuropathy, tremor, and sensory ataxia. Most importantly, and in contrast to their IgG1-3 counterparts, they respond poorly to IVIg and plasmapheresis but excellently to rituximab that induces long-lasting remissions, necessitating increased awareness to identify them from the outset , 15–21. Leucine-rich glioma-inactivated-1 (LGI1) and contactin-associated protein 2 (CASPR2) function to stabilize the voltage-gated potassium channel complex into the membrane , 9; LGI1LGI1 and CASPR2 are expressed in both the CNS and PNS, including peripheral nerves and dorsal root ganglia. Sera from patients with CASPR2—but not LGI1—antibodies bind in vitro to unmyelinated human sensory neurons and rodent dorsal root ganglia , suggest4.Anti-IgLON5 disorderAlthough patients with anti-LGI-1 and CASPR2 antibodies have clinical heterogeneity, they also exhibit significant overlapping clinical symptomatology; the antibodies to LGI1 are most commonly associated with limbic encephalitis and epilepsy while the antibodies to CASPR2 with Morvan syndrome, neuromyotonia, and neuropathic pain , 9, 25. IgLON5 is a neuronal cell adhesion molecule attached to the cell membrane by a glycosyl-phosphatidylinositol anchor protein, known to play crucial roles in cell adhesion and signaling by interacting with other cytoskeletal proteins . Anti-Ig5.Anti-DPPX disorderAnti-IgLON5 antibodies define a complex syndrome of chronic progressive brainstem symptomatology, gait instability, distinct non-rapid eye movement (REM) and REM parasomnias, obstructive sleep apnea, sleep-disordered breathing, cognitive decline, and movement disorders as recently identified, most commonly craniofacial dyskinesias, dystonia, chorea, and abnormal eye movements , 28. Mul–like protein (DPPX), a regulatory subunit of neuronal Kv4.2, rapidly inactivating potassium channel complex, responsible for transient inhibitory currents that regulate repetitive firing rates into neuronal dendrites. The DPPX antibodies are both of the IgG4 and IgG1 subclass and reduce cell membrane protein levels of DPPX and Kv4.2 potassium channels in cultured neurons increasing neuronal excitability in myenteric neurons [These patients have antibodies to dipeptidyl peptidase neurons . Because neurons –31. The  neurons , 30; intIn healthy adults, the IgG4 is the least common IgG subclass, comprising less than 5% of the total IgG with concentration ranging from 0.08 to 1.4 g/l –4. The Imonospecific and bivalent  identical for all antibodies of the same subclass by which bind to cell surface allowing phagocytosis, and two antigen-binding fragments (Fab) that bind to a specific antigen Neutralization of pathogenic autoantibodies by idiotypic antibodies of the IgG1subclass. The IVIg preparations contain minimal IgG4 (from 0.7 to 2.6%) because more than 95% of IgG is of the IgG1 subclass. IVIg, derived from thousand donors, contains anti-idiotypic IgG1 antibodies forming dimers connected between their F(αβ′)2 domains Inhibition of complement binding, preventing the formation of membranolytic attack complex (MAC). IVIg inhibits complement uptake by forming covalent or non-covalent bonds with C3b, intercepting the formation and deposition of MAC by the complement-fixing IgG1 antibodies on the targeted tissues preventing antibody-mediated cytotoxicity [toxicity , 46, 47.iii)Upregulation of the inhibitory FcγRIIB receptors. IgG molecules bind through their Fc region to various FcγR on monocytes, macrophages, dendritic cells, and B cells and exert either activating signaling via the FcγRIA, FcγRIIA, and FcγRIIIA or inhibitory signals via the FcγRIIB, mediating inflammatory and immune effector functions including cellular activation and cytokine production [oduction , 48–51. oduction –42. CIDPoduction , 48–51. oduction .iv)uppression of pathogenic cytokines and immunoinflammatory molecules. IVIg effectively suppresses pro-inflammatory cytokines [Sytokines –40 contrytokines –3.v)Saturation of the FcRn receptors, enhancing catabolism of IgG1-3 antibody subclass. IgG antibodies bind to FcRn to return intact back into the circulation, being protected from degradation by the lysosomes [ysosomes . The supysosomes , 53, redysosomes . IVIg isA series of observations, especially in CIDP patients with paranodal antibodies, have consistently shown that these patients did not respond from the outset to conventional therapies with IVIG, which is the treatment of choice for CIDP based on controlled studies, generating dilemmas even about the diagnosis , 20, 22.Because in IgG4-RD, the B cells are activated and expanded and the IgG4 antibodies correlate with disease activity, B cell depletion therapy with rituximab is a reasonable therapeutic target –4. This flCD20_CD27hiCD38hi plasmablasts are increased and oligoclonally proliferate [A number of non-randomized trials, summarized from 105 articles, have shown that rituximab is effective in patients with IgG4-RD who do not respond or have become refractory to corticosteroids , 4. In tliferate  but afteMusk-MG. In a multicenter, blinded, prospective review, 14/24 (58%) of patients treated with rituximab reached the primary outcome after a median follow-up of 3.5 years, compared to 5/31 (16%) of controls (p = 0.002) [p = 0.005) [= 0.002) . In addiw, 14/24 % of pati= 0.005) ; in one = 0.005) , 61. SimNeurofascin-155 and CASPR1/CNTN1 CIDP. The evidence that these patients are unresponsive to IVIg has been overwhelming and almost a hallmark of autoimmune nodopathies, as more than 90% of these patients have been originally diagnosed as CIDP-unresponsive to IVIG based on many reports [ reports , 15–20.  reports ; in cont reports .LGI1/CASPR2- and DPPX-associated autoimmunities. In several retrospective but small series, the first-line immunotherapy for those patient subsets presented with encephalitis and epilepsy has been steroids and IVIG with either encouraging or inconsistent responses [LGI1/CASPR2-associated spectrum of CNS and PNS autoimmunities is not only clinically heterogeneous but the antibodies in some of these patients may be of IgG1-3 subclass and a partial or even a very good response to IVIg or steroids may not be surprising, as observed in a very well done controlled study [esponses , 64. Altesponses , 64, greesponses . In one esponses . Howeveresponses  justifieed study , patiented study . Case reed study . NeverthThe effect of anti-B cell therapies in IgG4-ND is very similar to IgG4-RD but even more compelling based on larger but also uncontrolled series, as summarized below for each disorder:In several series of IgG4-RD, the risk of relapse has been lower while on rituximab maintenance, compared to only one rituximab induction therapy , 4, 67; In IgG4-ND, the IgG4 levels are irrelevant because, based on a series of CIDP patents with IgG4-nodal antibodies that we have examined, the IgG4 concentration was normal . In contrast, the IgG4-specific antibody titers can serve as key biomarkers because they correlate with disease activity, as shown for MuSK-MG , CIDP-asThe role of follow-up rituximab infusions to achieve long-term remissions in IgG4-ND, although still empirical, is very reassuring. In contrast to IgG1-3-ND where patients have more labile disease and may require 2 g every 6 months or 1 g every 3 months to ensure stability , 56, 69,Anti-B cell agents. They target CD20 by antibody-dependent cell cytotoxicity (ADCC) involving the Fcγ receptors (FcγRs), by complement-dependent cytotoxicity (CDC) or by directly inducing cellular apoptosis [Inebilizumab, a monoclonal anti-CD19 antibody approved in neuromyelitis optica [s optica , 74. Of s optica .Ocrelizumab, a recombinant anti-CD 20 monoclonal antibody that represents the humanized version of rituximab, approved for relapsing and progressive multiple sclerosis (MS).Ofatumumab, a fully humanized anti-CD-20 monoclonal already, approved for relapsing–remitting MS, given monthly subcutaneously. Ofatumumab, compared to rituximab, binds not only the large loop of CD20 but also the small loop closer to the B cell membrane, leading to more effective B cell lysis [ll lysis .Obexelimab, that targets CD19/FcγRIIB. This is especially attractive in IgG4-ND because it binds simultaneously to both CD19 and FcγRIIb, promoting internalization of CD19 in the lipid rafts [id rafts , 72. BecObinutuzumab, approved for chronic lymphocytic leukemia. Being type II anti-CD20 has increased efficiency in B cell depletion compared to type I. Because it exerts a prolonged action via the FcγRIIb, it may be attractive for the IgG-4-ND, by affecting also antigen presentation. In two rituximab-resistant patients with anti-MAG IgM antibodies and demyelinating neuropathy we have treated, no beneficial effect was however noted [er noted .poptosis . They arpoptosis .InebilizBruton’s tyrosine kinase inhibitors . These oral agents approved for CLL are now in phase II trials in pemphigus vulgaris [Tolebrutinib is now showing very promising results in a phase II trial multiple sclerosis [vulgaris . They arclerosis .Elotuzumab. This is a monoclonal anti-SLAMF7 antibody ready to be tested in IGg4-RD [ IGg4-RD . Elotuzu IGg4-RD .Anti-FcRn. The FcRn inhibitors bind to FcRn with high affinity leading to catabolism of all IgG subclasses and selective reduction of pathogenic and nonpathogenic serum IgG [Efgartigimod, a humanized IgG1 Fc fragment with increased binding to FcRn at neutral and acidic pH, now approved in myasthenia gravis [a gravis . It was a gravis , 61,Rozanolixizumab, a high affinity humanized anti-FcRn IgG4 monoclonal antibody, andNipocalimab, a humanized deglycosylated IgG1 monoclonal antibody that binds with high affinity to FcRn throughout the recycling pathway.erum IgG . They caerum IgG , 78. Alterum IgG , 61; in The extensive somatic hypermutation shown by immunoglobulin sequencing of expanded plasmablast clones in IgG4-RD, in conjunction with the effect of rituximab in B cell depletion, provides by analogy a strong rationale for more promising effective therapies not only in IgG4-RD but also in IgG4-ND. This is especially relevant to neurology where several anti-B cell monoclonals have already been approved in autoimmune neurological diseases. Collectively, future agents, ready to explore in all aforementioned IgG4-ND, include:The IgG4, due to its unique structural features in the hinge region, has non-inflammatory properties being functionally monovalent, unable to engage in cross-linking and internalization of targeted antigen exerting pathogenicity by blocking protein–protein interactions and signal transduction. Because IgG4 does not activate complement and cannot bind to inhibitory FcγRIIb receptor to activate cellular and complement-mediated immune responses, all the key targets inhibited by IVIg and conventional immunotherapies, IgG4-ND do not respond to IVIg like their IgG1 counterparts, but robustly respond to anti-B cell therapies. Because rituximab effectively targets the production of pathogenic IgG-4 antibodies, it should be the preferred treatment in IgG4-ND. Controlled studies with other anti-CD19/20 monoclonals, including those that also activate FcγRIIb, may be even more promising in treating IgG4-ND. Other anti-B cell agents, including Bruton’s tyrosine kinase inhibitors, and agents targeting FcRn that increase the catabolism of pathogenic antibodies are also of interest. At the molecular level, examining somatic hypermutation by immunoglobulin sequencing of expanded plasmablast clones after anti-B cell treatments my shed light in understanding IgG4-secretring B cell clones. At the clinical level, IgG4 antibody titers may become a monitoring tool in assessing disease activity and treatment response because they are reduced in remissions, increased in exacerbations, and seem to correlate with the patients’ clinical status.Supplementary file1 (PDF 584 KB)Below is the link to the electronic supplementary material.


Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150–300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells. Targeted nanoparticles (NPs) have been designed to enhance therapeutic efficacy and reduce the systemic toxicity of different pharmaceuticals and, in particular, anti-cancer agents ,2,3. TheThe realm of targeting ligands is vast and comprises peptides, antibodies (and their fragments), aptamers, and small molecules . Monoclonal antibodies (mAbs) are one of the most studied classes of ligands, especially their variable domains, and several antibody-targeting NPs have entered clinical trials . HoweverWhen designing efficient ligand-targeted NPs, a targeting moiety with high binding affinity and selectivity should be employed along with a suitable conjugation strategy. The latter has been shown to detrimentally affect ligand orientation and conformation ,22,23. FIn terms of optimal ligand density, some lessons can be learned from viruses. Viruses, like nanomedicines, aim to avoid immune recognition, and the density and distribution of spike proteins on the virion envelope is one of the mechanisms utilized. The HIV virus has a low spike density (on average 14 spikes per virion), whereas lassa and influenza viruses have 270 and 300–400 spikes per virion, respectively, while having the same particle size of around 100 nm ,29,30. TTargeted NPs face another obstacle once they reach the tumor site. The biomolecules and particularly plasma proteins that might have been adsorbed onto their surface in the circulation (the antifouling PEG chains fail to completely prevent protein adsorption ) can hidThis study therefore aimed to elucidate the relationship between Nb density and targeting efficacy of PEGylated liposomes towards cancer cells as well as liposome association with immune cells. PEGylated liposomes were conjugated with a varying number of Nbs raised against the hepatocyte growth factor receptor (MET)  via To investigate the impact of nanobody density on targeting efficiency, MET-TLs were prepared by the functionalization of the liposome surface with Nbs (15.6 kDa) targeting the MET receptor (MET-Nbs). These Nbs contain an engineered C-terminal cysteine that reacts with MAL-PEG-DSPE via maleimide-thiol click chemistry A. The coDifferent amounts of MET-Nbs were conjugated to the surface of liposomes by varying the amount of MET-Nbs added to the reaction mix. The degree of conjugation was determined by SDS-PAGE, where the MET-Nb-MAL-PEG-DSPE complex should yield a band at 18.5 kDa. Liposomes were first prepared with 8% of MAL-PEG-DSPE, which yielded two bands of a higher molecular weight than 15.6 kDa C. HoweveNTLs and six MET-TL formulations with a different MET-Nb density were successfully prepared by varying the molar ratio between MET-Nbs and MAL-PEG-DSPE from 1:500 to 1:12. As shown in 2, respectively . However, the number of MET-Nbs that can be conjugated to the liposomes is limited by the amount of MAL-PEG-DSPE and the steric hindrance imparted by the first MET-Nbs that react with maleimide. Transmission electron microscopy images  and MET-negative (TOV112D) tumor cell lines. The MET protein expression levels were confirmed by flow cytometry A.MET-TLs bound more efficiently to TOV+MET cells than to TOV112D cells and also bound more efficiently to TFK1 cells than TOV+MET cells in a lipid concentration- and conjugation density-dependent fashion after 1 h of incubation at 4 °C. These data indicate a correlation between MET-TL binding and MET expression, as TFK1 cells showed the highest expression of MET receptors. In contrast, NTLs interacted very weakly with all cell lines. Altogether, these results confirm that the interaction of MET-TLs was mediated by the MET receptor B. FurtheWhen net binding to TOV+MET cells was plotted by subtracting the TOV112D binding traces B, the 1:Association studies were performed by incubating the cells with NTLs and MET-TLs for 5, 15, and 30 min at 37 °C. The association with the MET-negative (TOV112D) cells was very limited for both NTLs and the different MET-TLs A. For thThe specificity of the association of MET-TLs with MET-expressing cells was validated with a competition assay, where cells were pre-incubated with a saturating concentration of free MET-Nbs prior to their exposure to liposomes . Under tThe uptake of fluorescently labeled liposomes on MET-negative and MET-positive cells was assessed with confocal microscopy C,D. As s2 , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000 (PEG-DSPE), maleimide PEG-DSPE, and Cy5.5-PE were obtained from Avanti Polar Lipids . Cholesterol, L-cysteine hydrochloride, and magnesium chloride were acquired from Sigma-Aldrich . Bolt 4–12%, Bis-Tris, 1.0 mm, mini protein gels, and Bolt MES SDS running buffer (20×) were obtained from Thermo Scientific. 1,4-Dithiothreitol (DTT) was acquired from Sigma Aldrich. Coomassie Blue PageBlue protein staining solution and PageRuler pre-stained protein ladder were obtained from Thermo Scientific. Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Dulbecco’s modified Eagle medium (DMEM) high glucose, fetal bovine serum (FBS) (sterile filtered), trypsin EDTA solution (1×), antibiotic/antimycotic solution (100×), Dulbecco’s phosphate-buffered saline (PBS), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Thermo Scientific. Vivaspin 2 100,000 MWCO (PES membrane) was obtained from Sartorius Stedim Lab . All organic solvents were obtained from Biosolve . Human plasma was obtained from Sigma-Aldrich and from healthy volunteers through the mini donor service of the University Medical Center Utrecht, the Netherlands. Donors provided informed consent. Institutional review board approval was waived for this route of whole blood curation.Phosphate buffered saline 10× solution  was purchased from Fisher Bioreagents . DNAse I was obtained from Roche Diagnostics . One-mL CaptureSelect C-tagXL pre-packed columns were purchased from Thermo Scientific  and HiTrap desalting columns were obtained from GE Healthcare . Dipalmitoyl-E. coli transformed with a pET28 vector containing a C-terminal cysteine and an EPEA affinity purification tag. Single colonies were grown overnight in LB medium supplemented with 2% glucose and 100 μg/mL kanamycin. Overnight pre-culture (1:10 dilution) was expanded in a 5-L BioFlo/CelliGen 115 benchtop fermenter  in a Terrific Broth medium containing 0.1% glucose and 100 μg/mL kanamycin. Once the log growth phase was reached, isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added to induce Nb expression. Bacteria were kept overnight at 25 °C. The next day, bacteria were centrifuged at 4800× g for 20 min at 4 °C, and the resulting pellet was resuspended in PBS and stored at −20 °C.MET-Nbs were produced and purified as described for other Nbs  with sevE. coli was isolated with two cycles of freeze–thawing and two rounds of centrifugation at 4 °C . Periplasm was treated with DNAse and MgCl2 at a final concentration of 5 µg/mL and 25 mM, respectively, to reduce nucleic acid contamination and sample viscosity, and filtered through a 0.2-µm aPES membrane.The periplasm fraction of 2 was applied at 0.5 mL/min. The Nb fractions were collected and buffer-exchanged to PBS (pH = 7.4) using 5-mL HiTrap desalting columns . Nb concentration was determined using absorbance at 280 nm  and a molar extinction coefficient of 37,025 M−1·cm−1 .Nbs were purified by affinity chromatography using an ÄKTA Pure system  with a 1-mL CaptureSelect C-tagXL pre-packed column (Thermo Scientific). The column was equilibrated with PBS (pH = 7.4) and the periplasm fraction was applied to the column at a flow rate of 0.8 mL/min. The column was washed with 10 mL PBS, and subsequently a linear gradient elution with 10 mL of 20 mM Tris (pH = 7.0) and 2 M MgCl2 (standard culture conditions).The human extrahepatic bile duct carcinoma TFK-1 cell line ACC 344) was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The human ovarian carcinoma cell line TOV112D (CRL-11731) was obtained from the American Tissue Culture Collection . TOV112D cells that stably express MET (here described as TOV+MET) were previously described [4 was purTFK1, TOV-112D, and TOV+MET cells  were fixed for 15 min with 4% paraformaldehyde (PFA), blocked with PBS containing 1% bovine serum albumin, and incubated with 50 µL of the primary antibody diluted 1:30 in blocking buffer  for 45 min at room temperature (RT). The cells were washed twice and incubated with 50 µL of the secondary antibody diluted 1:200 in the blocking buffer  for 30 min at RT. Next, the cells were washed, resuspended in 100 µL of blocking buffer, and transferred to a 96-well plate. Fluorescence intensity was measured with a flow cytometer  using the 488 nm laser line and a 530/30 nm filter. At least 10,000 events were collected per sample. Mean fluorescence intensity was calculated using FlowLogic Software .sn-glycero-3-phosphocholine (DPPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000 (PEG-DSPE), and maleimide PEG2000-DSPE (MAL-PEG-DSPE) at a molar ratio of 77:15:4:4. During the optimization process to find the most suitable amount of MAL-PEG-DSPE, the fraction of PEG-DSPE and MAL-PEG-DSPE was kept at 8 mol%. Fluorescently labeled liposomes were prepared by adding 0.2 mol% of 18:0 Cy5.5-PE at the expense of DPPC. The liposomes were prepared by the lipid film hydration method as previously described [w/v) NaCl, 10 mM HEPES (pH = 7.4) to a final lipid concentration of 10 mM. Liposomes were extruded 2× through 400-nm polycarbonate membranes and 4× through 200- and 100-nm polycarbonate membranes using a 10-mL thermobarrel Lipex extruder .The MET-TLs and NTLs were composed of 1,2-dipalmitoyl-escribed . In briew/v) NaCl, 10 mM HEPES (pH = 7.4) with Zeba spin desalting columns . MET-Nbs were mixed with liposomes at different ratios of Nb:MAL-PEG-DSPE and incubated in an orbital shaker for 2 h at RT and overnight at 4 °C. To quench the unreacted maleimide groups, a cysteine solution at 100× the molar concentration of MAL-PEG-DSPE prepared in 0.8% (w/v) NaCl, 10 mM HEPES (pH = 7.4) was added to the liposome dispersion (1:10 dilution) and left for 2 h at RT while rotating. NTLs were prepared by blocking the maleimide groups with cysteine as described for the MET-TLs. To remove non-conjugated and reacted MET-Nbs, the liposomes were dialyzed  against 0.8% (w/v) NaCl, 10 mM HEPES (pH = 7.4) for 16 h with 3× buffer exchange, followed by centrifugation at 3000× g with Vivaspin 100 kDa (Sartorius) to concentrate the liposome dispersion to the initial 10-mM lipid concentration.For the conjugation of the MET-Nbs onto the liposome surface, MET-Nbs in PBS were reduced with TCEP at an Nb:TCEP molar ratio of 1:200 and incubated at RT for 5 min. Next, TCEP was removed by buffer exchange to 0.8% . The zeta potential was measured with a ZetaSizer Nano Z . For both measurements, the liposomes were diluted 100× in 10 mM HEPES buffer (pH = 7.4).The number of liposomes was determined by NTA. NTA measurements were performed using a NanoSight LM-10SH device  equipped with a 532-nm laser. The liposomes were diluted 10,000-fold so that less than 100 particles were identified per frame. The liposomes were video-tracked with an EMCCD camera at RT. The average size was calculated with NanoSight 3.4 software from five measurements, each based on a 60-s video segment.The size and morphology of the NTLs and MET-TLs were assessed by transmission electron microscopy (TEM) using a TFS Tecnai 20 microscope  operated at 200 kV. For this analysis, a drop of the suspensions under study was placed on a carbon-coated copper grid for 2 min and stained with uranyl for 1 min.v/v, 30 µL total sample volume) and PBS and incubated at 90 °C for 10 min. After cooling down, the samples were loaded onto an SDS-PAGE gel  and run at 100 V for 50 min, employing 1 × 2-(N-morpholino)-ethanesulfonic acid (MES) buffer as running solution. Afterwards, the gel was stained with Coomassie blue (PageBlue protein staining solution) and scanned with a ChemiDoc Imaging System . Conjugation efficiency was determined by comparing the band intensity of free and conjugated Nbs using the Plot Lanes tool in ImageJ software .The total lipid concentration was determined by an inorganic phosphate assay modified from . The rec2 was estimated by dividing the average number of MET-Nbs per liposome by the surface area of a liposome with an average radius of 122.8 nm . The number of MET-Nbs per mL was determined by multiplying the number of moles by Avogadro’s constant and correcting for the conjugation efficiency . The num4 cells per sample) were added to a 96 round-bottom well plate and kept on ice for 15 min. Next, the cells were exposed to fluorescently labeled NTLs and MET-TLs serially diluted (from 500 µM starting lipid concentration and a dilution factor of 3) in RPMI containing 1% (w/v) BSA and 25 mM HEPES (pH = 7.4), and incubated for 1 h at 4 °C. After 3× washing to remove unbound liposomes and fixation with 4% PFA for 15 min, liposome binding was quantified by flow cytometry (Canto II) using the 633 nm laser line and a 780/60 filter. At least 10,000 events were collected in the gated region per sample. The mean fluorescence intensity was determined for each sample and corrected for the background signal intensity of non-treated cells using FlowLogic 8.3 Software.TFK1, TOV-112D, or TOV+MET cells  for 5, 15, and 30 min at 37 °C in a humidified atmosphere with 5% CO2. A competition group with free Nbs was included in the assay. In this case, the cells were first incubated for 15 min with 1 µM of MET-Nbs, followed by either NTL or MET-TL incubation for 30 min under standard culture conditions. After washing thrice to remove unbound liposomes, the cells were detached with 100 µL of Accutase , transferred into a round-bottom well plate, and fixed with 4% PFA for 15 min. The liposome association with cells was measured by flow cytometry as described in TFK-1, TOV-112D, and TOV+MET cells were seeded at a density of 3.5 × 104, 1.2 × 104, and 1.2 × 104 cells per well in a black 96-well plate with a glass transparent bottom. The next day, NTLs and MET-TLs (500 µM lipid concentration was used to facilitate detection) were diluted in RPMI containing 1% BSA and 25 mM HEPES (pH = 7.4), which were added to cells and incubated for 1 h or 24 h at 37 °C under standard culture conditions. After 3× washing to remove unbound liposomes and fixation with 4% PFA for 20 min, the cells were stained with Hoechst 33342 (Thermo Scientific) for 20 min at 37 °C (1:750 dilution from 1 mg/mL stock in RPMI medium supplemented with 10% FBS). The cells were imaged with a Yokogawa High Content Imaging Platform  using a 20× objective. Quantification of the fluorescence signal was performed using ImageJ and corrected for the background signal.TFK-1, TOV-112D, and TOV+MET cells were seeded at a density of 2.0 × 10NTLs and MET-TLs were diluted in RPMI with 50% FBS or 50% human plasma  or obtained from fresh whole blood ) to dispn = 6) into citrate-anticoagulated Vacutainer tubes . Complete whole blood counts were obtained using a CELL-DYN Emerald hematology analyzer . To obtain the plasma only, whole blood was centrifuged at 900× g for 15 min at RT. The plasma collected from the first centrifugation was again centrifuged at 950× g for 10 min at RT to remove any residual cells and stored at −20 °C.Fresh human blood was collected from healthy subjects (11 particles/mL) in a 2-mL Eppendorf tube for 1 h at 37 °C using a thermoshaker (400 rpm). Afterwards, red blood cells lysis buffer was added and the blood samples were incubated for 30 min on ice. Samples were washed twice with PBS  and fixed with 1% PFA. Liposome association with immune cells was measured by flow cytometry as described in Whole blood (180 µL) was incubated with NTLs and MET-TLs  overcome the protein corona. The studies with ex vivo human blood showed that the MET-Nb density on liposomes affects immune cell interactions. Moreover, we confirmed that a panel of in vitro assays can be used to optimize formulations and, in part, steer clinical translation of nanomedicines. Future in vivo experiments will be conducted to verify the trends observed here and assess the drug delivery capacity of MET-TLs.


Streptococcus mutans biofilms. The present study represents an effort to characterize the shear-bond strength (SBS) and color stability (CS) of these novel biomaterials. Experimental adhesives were obtained by dispersing nitrogen and fluorine co-doped titanium dioxide nanoparticles  into OptiBond Solo Plus (OPTB). Dentin surfaces were wet-polished (600-Grit). Specimens (n = 5/group) of Tetric EvoCeram were fabricated and bonded using either OPTB or experimental (OPTB + NF_TiO2) adhesives. Specimens were stored in water (37 °C) for twenty-four hours (T1), three months (T2), and six months (T3). At T1, T2, or T3, specimens were removed from water storage and were tested for SBS. Disc-shaped specimens  of adhesives investigated were fabricated and subjected to thermocycling . Specimens’ colors were determined with a VITA Easyshade® V spectrophotometer (after every 1000 cycles). SBS data was analyzed using two-way ANOVA and post-hoc Tukey tests, while CS data was analyzed using one-way ANOVA and post-hoc Tukey tests (α = 0.05). Mean values of SBS ranged from 16.39 ± 4.20 MPa (OPTB + 30%NF_TiO2) to 19.11 ± 1.11 MPa (OPTB), from 12.99 ± 2.53 MPa (OPTB + 30% NF_TiO2) to 14.87 ± 2.02 (OPTB) and from 11.37 ± 1.89 (OPTB + 20% NF_TiO2) to 14.19 ± 2.24 (OPTB) after twenty-four hours, three months, and six months of water storage, respectively. Experimental materials had SBS values that were comparable (p > 0.05) to those from OPTB independently of nanoparticle concentration or time-point considered. Experimental materials with higher NF_TiO2 concentrations had less intense color variations and were more color stable than OPTB even after 10,000 thermocycles. In combination, the results reported have demonstrated that experimental adhesives can establish strong and durable bonds to human dentin while displaying colors that are more stable, thereby suggesting that the antibacterial nanotechnology investigated can withstand the harsh conditions within the oral cavity without compromising the esthetic component of dental restorations.Experimental adhesives containing co-doped metaloxide nanoparticles were demonstrated to display strong and long-term antibacterial properties against  Composite resins and dental adhesive resins are typically used to repair and augment the function and esthetics of mineralized dental tissues. These mercury-free restorative materials display outstanding handling and esthetic properties, have good mechanical and physical properties, and are associated with minimally invasive  and ultr2) [2 produced via sol-gel (N-TiO2) was demonstrated to enhance the strength and in vitro antibacterial properties of composite resins against Escherichia coli when used as a filler [2 prevented the growth of Streptococcus mutans over a period of three months, which was considered to be an effective strategy in preventing enamel demineralization during orthodontic therapy [2 in orthodontic resins, found that experimental materials containing TiO2  promoted significant in vitro microbial reductions against S. mutans, Streptococcus sanguinis, and Lactobacillus acidophilus [2 (Ag_TiO2) has also been evaluated [2 was capable of eradicating S. mutans planktonic cultures (strain NG8) when exposed to visible light irradiation (1500 lux) [Several approaches have been investigated to improve the clinical performance and longevity of dental adhesive resins. These include the incorporation of fluoride, quaternary ammonium dimethacrylates (QADM), silver (Ag) , zinc ox2) ,17 into 2) ,20,21,22a filler . Salehi  therapy . Sodagardophilus . The antvaluated . Results500 lux) . Despite2, 6–15 nm) [2 were spherical, had smooth surfaces, and were capable of absorbing two times more visible light when compared to undoped TiO2  [2  were demonstrated to have strong antibacterial and biomimetic properties when irradiated with visible light  [2  [p > 0.05) to those of three commercially available and FDA-approved materials , Clearfil SE Protect , and Adper Scotchbond ) [Nitrogen-doped titanium dioxide nanoparticles obtained using two-step solvothermal reactions (N_TiO6–15 nm)  have beeGermany) . Experim7 J/cm2) . These p0%, v/v) . MateriaN, USA)) .2-containing materials could withstand the harsh conditions in the oral cavity and could express strong antibacterial properties at the adhesive interface without adversely impacting the viability of human pulp cells. Esteban Florez et al. [2) or nitrogen and silver (NAg_TiO2)) into OPTB, have demonstrated that strategies used resulted in the covalent functionalization of nanoparticles, the establishment of smooth interfaces between nanoparticles and polymer chains, and experimental materials displaying polymeric networks with preserved morphology, structure and three-dimensional lamellar distribution, which indicates that experimental materials tested could potentially display good mechanical properties [In combination, previous results cited indicate that N_TiOz et al. , while ioperties . Despiteoperties ,31 indicv/v%) of antibacterial NF_TiO2. Null hypotheses tested were that experimental materials would display values of shear bond strength and color stability (in terms of ΔEab and ΔE00) that were similar (p > 0.05) to those attained with the commercially available dental adhesive resin.These results are critical because micromechanical interlocking is the primary mechanism of adhesion to enamel and dentin , and the2 was synthesized in the laboratories of the Division of Dental Biomaterials at the University of Oklahoma Health Sciences Center College of Dentistry using protocols recently published [2 nanoparticles were then stored in ethanol (concentration ≅ 40 mg/mL).NF_TiOublished ,43,44,45v/v, suspended in ethyl alcohol) of NF_TiO2 into OptiBond Solo Plus  using a Q700 sonicator . The rationale for selecting these concentrations was based on previous publications demonstrating that single-doped or co-doped nanoparticles displayed strong initial antibacterial and biomimetic properties with or without visible light irradiation at similar concentrations [Experimental dental adhesive resins were formulated by ultrasonically dispersing 10%, 20%, and 30%  were longitudinally sectioned  under copious water irrigation to expose dentin surfaces. Each half tooth section was embedded in an acrylic block and wet-polished through 600-grit SiC paper  before being randomly distributed to groups investigated. Specimens  of Tetric EvoCeram  were fabricated using a mounting jig  and were bonded  using the proper adhesive in each group. n = 15/group/time-point) were fabricated using the same procedures previously described but were bonded (following manufacturer instructions) using unaltered and commercially-available OptiBond Solo Plus , and served as the control group in the present study. At specific time-points , specimens pertaining to each group investigated were removed from water storage and were lightly dried in preparation for SBS testing procedures.De-identified and caries-free extracted human molars  were manually fabricated in a custom-made metallic mold using the adhesives investigated  or experimental (OPTB+ NF_TiO2)). All specimens were photopolymerized  against glass.Disc-shaped specimens  specimens from each group were individually tested for SBS. Individual specimens were secured using a metal clamp coupled to an Ultradent shear bond strength testing jig  coupled TM 600 Dualbeam FIB/SEM  at 2 kV accelerating voltage and 0.34 nA beam current. The detector used was a solid-state annular backscatter (ABS) detector. FEI MAPS 2.0 software was used to acquire and stitch together individual SEM images, given that the ROI was not completely within the field of view at the lowest magnification of the SEM.Samples were allowed to dry for approximately 18 hrs at room temperature and then mounted to aluminum SEM stubs using a conductive carbon adhesive tape. A grounding strip of copper tape was applied to connect the top surface of the sample to the Al stub. Samples were coated with Au/Pd in a Denton Desk V sputter coater system to ensure conductivity. Imaging was performed in a Helios Nanolab® V, VITA, Bad Säckingen, Germany). Specimens’ colors were determined immediately after fabrication and after every increment of 1000 thermocycles  to a total of 10,000 thermocycles. Raw experimental data was then tabulated, and specimens’ color stability was calculated (in terms of ΔEab and ΔE00) using the CIELAB and CIEDE2000 [Specimens fabricated as described in IEDE2000  equationIEDE2000 . The ratIEDE2000  that indwww.graphpad.com accessed on 15 November 2022) and SPSS 23 (IBM).Mean and standard deviation values of SBS were calculated and were used to determine statistically significant differences among groups investigated using two-way ANOVA and post-hoc Tukey tests (α = 0.05). Color stability data were assessed for normality and homoscedasticity. Mean values that met statistical assumptions were analyzed using one-way ANOVA and post hoc Tukey tests. Statistical analyses in the present study were performed (α = 0.05) using GraphPad Prism version 9.0.0 for Windows .2 after six months of water storage .2) to 19.11 ± 1.11 MPa (OPTB), from 12.99 ± 2.53 MPa (OPTB + 30% NF_TiO2) to 14.87 ± 2.02 (OPTB), and from 11.37 ± 1.89 (OPTB + 20% NF_TiO2) to 14.19 ± 2.24 (OPTB) after twenty-four hours, three months, and six months of water storage, respectively. Even though parameters “time” (p < 0.0001) and “adhesive” (p = 0.0005) were observed to be significant predictors of response when analyzed individually, the interaction between parameters of interest (time*adhesive) was shown to not be significant (p = 0.8536), which indicates that the functionalization of NF_TiO2 into OPTB did not adversely impacted OPTB’s ability to establish adhesive interfaces that were strong and durable.Mean and standard deviation values of SBS from each group investigated were calculated as described in p < 0.001) decreased overtime and independently of the adhesive  and nanoparticles’ concentrations  considered.2 displayed variations in SBS values that were either positive (4.5%) or negative (−2.4%), respectively. After three months of water storage, and except for the group containing 10% of NF_TiO2 that had a positive variation in SBS values (+3.3%), all other groups had SBS variations that were negative in nature and ranged from −5.5% (OPTB + 30% NF_TiO2) to −1.4% (OPTB + 20% NF_TiO2). This trend could not be observed after six months of water storage, and all experimental groups were associated with positive variations in SBS values that ranged from 0.4% (OPTB + 30% NF_TiO2) to 5.5% (OPTB + 10% NF_TiO2).2 into OPTB resulted in experimental materials that can establish strong and durable bonds to human dentin. In addition, the statistical analysis performed indicated that materials investigated  had similar  SBS values and would be appropriate for clinical use, thereby supporting the utilization of the antibacterial nanotechnology investigated in the present study. E00 and ΔEab, respectively), where it is possible to observe that after 1000 thermocycles, experimental adhesives investigated displayed color variations that were smaller than that of the parental polymer and were below the perceptibility threshold, which indicates that experimental materials were more color stable than OPTB independently of the concentration of nanoparticles considered. From 3000 to 10,000 thermocycles, all materials investigated  were observed to display color variations that were higher than the thresholds of perceptibility (PT) and acceptability (AT). The analysis of ΔE00 obtained from 5000 to 10,000 thermocycles indicated that experimental adhesives containing 30% of NF_TiO2 displayed color variation values that were consistently lower when compared to the control group (OPTB). These results have clearly demonstrated that the incorporation of NF_TiO2 into OPTB did not alter the optical behavior of OPTB and seemed to downregulate the color variations observed, which further supports the clinical utilization of the antibacterial nanotechnology investigated here.In combination, these results suggest that the functionalization of NF_TiO2, ZnO, Ag_TiO2, N_TiO2 and NF_TiO2) have gained the attention of researchers due to their ability to generate reactive oxygen species (ROS) and their proven strong antimicrobial and biomimetic effects [Recent advancements in the field of nanotechnology allowed the utilization of nanoparticles and nanostructured materials in many health-related fields, including biosensing, cancer treatment, and drug delivery . Metalox effects . Nanopar effects . Previou effects ,54,55. H effects ,31.2. Results reported in 2) into dental adhesive resins results in materials that are less soluble, absorb less water, and have higher specific gravity, which in theory could decrease the detrimental effects of water on bond strength. Al-Saleh et al. [2 or ZrO2 displayed non-Newtonian rheological behaviors, pseudo-plasticity properties, and improved flow, which suggests that nanofilled adhesive resins could establish strong interfaces with the tooth structure. Results reported [2 nanoparticles were shown to produce enhanced hybrid layers when compared to those attained with ZrO2-containing adhesives, as denoted by the formation of resin tags that were longer [The present study represents an effort to characterize the SBS and the CS of experimental adhesive resins containing varying concentrations of NF_TiOh et al. , while ireported  have alse longer . In comb2  in adhesive resins demonstrated that experimental materials displayed SBS values that were comparable (p > 0.05) to the parental polymer and indicated the presence of concentration thresholds (minimum and maximum) in which TiO2 would yield a positive effect on bond strength. In contrast, the results of the present study revealed that all materials investigated displayed comparable (p > 0.05) mean values of SBS at twenty-four hours, three months, and six months independently of concentration of nanoparticles, thereby further corroborating that the functionalization of nanoparticles did not adversely impact the SBS of OPTB. Prior research comparing several self-etch adhesives found that OPTB displayed SBS values that were higher and statistically different (p < 0.05) when compared to those from AdheSE, Adper Prompt Self-Etch Adhesive, Clearfil SE Bond, and One-Up Bond F [p = 0.8131).Sun et al. , while ip Bond F . Even th2. This was an unexpected behavior, because previous studies [The results of the present study indicated that no differences in SBS were detected between experimental adhesives containing NF_TiO studies ,31 have  studies , while c studies , which f2. From 4000 to 10,000 thermocycles, all materials investigated displayed variations in color that consistently increased above PT and AT. When comparing  that were consistently lower than that of OPTB, thereby suggesting that NF_TiO2 functionalization reduced the impact of thermocycling on the color of OPTB. 2) were numerically smaller and statistically significant  compared with those from OPTB after 10,000 thermocycles. Šimunović et al. [Color stability results are shown in omparing  the coloć et al. , while iE00) of commercially available composite resins, have demonstrated that all materials investigated  and Filtek Z350 XT ) displayed color variations that were statistically significant (p < 0.001) and considered unacceptable (above PT and AT) after 10,000 cycles. The results published by Štruncová et al. [Previous studies ,61 have 2 would display SBS values that were comparable (p > 0.05) to those of commercially available adhesive resins. The second part of the null hypothesis was rejected, because experimental materials displayed color variations that were numerically smaller and statistically different  than those from OPTB after 10,000 thermocycles. Once fully developed and characterized, materials reported in the present study may hold the promise to increase the service lives of polymer-based restorations and decrease the incidence of secondary caries and the costs of oral health care. Future studies in the field should investigate the long-term  shear bond strength and color stability of experimental materials in challenging conditions [Results reported in the present study have allowed us to fully accept the first part of the null hypothesis tested, that experimental materials with varying concentrations  of NF_TiOnditions  that res2 were observed to be the most color stable material after 10,000 thermocycles. Experimental materials displayed positive percent changes in SBS values after six months of water storage, which indicates that adhesive interfaces established with experimental materials may become mechanically more stable when compared to the parental polymer. In combination, results reported support the continued development and characterization of the antibacterial nanotechnology investigated here.The present study has demonstrated that co-doped nanoparticles synthesized via one-step solvothermal reactions can be successfully functionalized into a self-etch and commercially available dental adhesive resin. Experimental materials investigated were observed to display shear bond strength values that were comparable to the parental polymer after twenty-four hours, three months, or six months of water storage. Adhesives containing 30% of NF_TiO


The compressive strength of concrete is not the same in high temperature humid environments and normal temperature dry environments. In this study, quasi-static uniaxial compression experiments of concrete with different temperatures and water contents were carried out to investigate the variation pattern of the compressive strength of concrete under combined heat and moisture conditions. The results showed that the temperature softening effect and water softening effect of the compressive strength of concrete were coupled with each other. The compressive strength exhibited a variation trend from increase to decrease with the increase in both temperature and water content, and the relations among the heat–moisture coupling factor, temperature, and relative saturation ratio were obtained. The water absorption of concrete after immersion had a more significant effect on the compressive strength than the free water content stored inside the specimen before immersion. The “pseudo-temperature strengthening effect” distinguished the thermodynamic response of immersed concrete from that of dry concrete, and the functional relationships among the heat–moisture coupling factor, temperature, and relative water absorption ratio were established. The evolutionary mechanism of the competition between the microcrack expansion and healing of concrete under combined heat and moisture conditions was revealed. Concrete structures, with their advantages of better corrosion resistance, impact resistance, and high temperature resistance, have a wide range of applications in national defense constructions and civil engineering. Studies ,2,3,4,5 (1)The effects of temperature and water on the strength of wet concrete are independent. The compressive strength of concrete is the superimposed result of the independent effects of the temperature softening effect and water softening effect. Due to the secondary hydration during water immersion, a large number of cracks occur within the concrete, which does not heal or shrink with the phase change and loss of water at high temperature. This means that the damage formed in the process of hydration is irreversible at high temperature. The compressive strength of concrete after immersion monotonically decreases with the increase in temperature.(2)When wet concrete is exposed to high temperatures, the water is evaporated and the water content decreases. With the loss of free water, the splitting effect of water at the crack tip disappears, and the hydration cracks formed in the immersion process are completely healed, and the damage is reversible. The phase transition of water does not affect the healing of cracks caused by the loss of liquid water. The compressive strength of wet concrete gradually increases with the increase in temperature until the water absorbed during the immersion process is completely lost and the compressive strength reaches the peak, which is equal to the compressive strength of dry concrete at normal temperature. Then, it shows the same decay pattern as that of the compressive strength of dry concrete at high temperature. The absorbed water delays but does not affect the temperature softening effect on the compressive strength. The effect of water on the high temperature strength of concrete materials is fully reversible such as that of the compressive strength at normal temperature, that is, the phase transition of water has no effect on the damage of concrete materials and the water content has no effect on the peak strength and the attenuation pattern of post-peak strength.(3)The effects of temperature and water on the strength of wet concrete are coupled. Under the action of high temperature, wet concrete gradually becomes dry concrete due to the evaporation of internal moisture. The compressive strength of concrete first increases to a certain peak strength and then decreases with the rise of temperature. Both the peak strength and the attenuation pattern of post-peak strength are related to the water content. The peak strength and attenuation pattern of post-peak strength are not the same as those of dry concrete under the same temperature conditions. The phase transition of water at high temperature has an irreversible effect on the evolution of cracks in concrete materials, at least on part of the crack evolution. The temperature and water effects on the compressive strength are interactive. Under the above three possibilities, the general relationship between the compressive strength and temperature is sketched as shown below :Compared to the research on the water softening effect of wet concrete at normal temperature and the temperature softening effect of dry concrete at high temperature, scholars are less unified in their views on the pattern of the effect of temperature and water on the compressive strength of concrete materials under combined heat–moisture conditions. Rather, there are uncertainties and controversies on some key issues. For example, some scholars believe that the main reason for the damage of concrete materials under high temperature is that the pore pressure inside the material increases due to the obstruction of water vapor diffusion under high temperature, thus leading to the phenomenon of high temperature bursting of concrete materials ,32. HoweIn response to the above controversies and uncertainties and to accurately grasp the joint effect of temperature and moisture on the compressive strength of concrete materials under combined heat and moisture conditions, thus determining the variation pattern of compressive strength, this study carried out quasi-static uniaxial compression experiments of concrete under different temperatures  and different water contents  and obtained the effects of temperature and water on the compressive strength of the concrete material. The different effects on the compressive strength under high temperature between the free water stored in the first hydration process of concrete and the water absorbed in the second hydration process of concrete were observed. The special phenomenon of “pseudo-temperature strengthening” was discovered in the concrete specimens after immersion. The heat–moisture coupling pattern of the compressive strength of concrete was determined, and the meso-evolution mechanism of microcracks inside the material under combined heat and moisture conditions was revealed.The raw material for the specimens consisted of coarse aggregate (crushed stone with a maximum particle size of 8 mm), cement with a strength class of 42.5 MPa, medium sand with a fineness modulus of 2.3, and a small amount of water reducing agent. The mix proportions of the specimens are shown in 0. The air-drying specimens were heated in the oven at 105 °C and were weighed and recorded several times during the heating process until the mass no longer changed. Then, they were considered completely stoved specimens, the mass of which was measured as Md. The mass of free water and the water content in the air-drying specimen were measured as δM (δM = M0 − Md) and δM/M0, respectively. The experiments showed that the water content stored in the air-drying specimen placed in normal temperature and dry environment for three months was approximately 1.87%.In this experiment, the curing of concrete method adopted was in accordance with clause 5.2.4 of the “Standard of Test Methods for Mechanical Properties of Ordinary Concrete, GB/T50081” currently practiced in China. The standard curing environment of concrete specimens is: within 28 days, the temperature is maintained at 20 ± 2 °C, the relative ambient humidity is >95%, the specimens should not be stacked, the interval between each other is required to be 10–20 mm, and the surface should be kept wet, but not directly wet with water, but by using atomization humidification treatment. After the specimens were fabricated and cured in the standard way for 28 days, they were placed in a dry indoor environment at normal temperature for at least three months. The specimens under this condition were considered as air drying concrete specimens, whose mass was recorded as MThe production process of the immersed concrete specimen included two hydration actions. The first hydration action occurred during the process of production and curing of the initial concrete. The initial concrete specimen was made in accordance with the predetermined mix ratio and cured for 28 days in the standard way. The initial specimens placed in a normal temperature dry environment for more than three months were referred to as air drying specimen. Then, the air-drying specimens were immersed in water to make immersed concrete specimens with different water contents. The second hydration occurred in the process of immersing the air-drying concrete specimen. The water content in the concrete specimen maintained in a dry room at normal temperature before immersion was called the initial water content, and the water content of the specimen after immersion increased with the increase in immersion time until saturation, when the mass of the specimen no longer increased with the increase in immersion time.0 as the reference. The water absorption mass ΔM of the specimen was obtained . The water absorption mass, ΔM, does not include the free water mass, δM, stored inside the air-drying specimen, but the water absorption mass of the specimen during the immersion process for a predetermined time. The water absorption ratio, S, is the water absorption mass ΔM/the air-drying specimen mass M0, i.e., S = ΔM/M0. The water content of the immersed concrete is: ΔM + δM, and the water content ratio, W%, is the mass of water in the concrete specimen/the mass of the concrete completely stoved in the oven, i.e., W% = (ΔM + δM)/Md.The air-drying specimen was immersed in water . Different water contents inside the specimen were achieved by controlling the immersion time. The change in the mass of the specimen under different periods of immersion during the immersion process was measured, taking the mass of the air-drying specimen MThe real time water absorption of the specimen is not only related to the immersion time, but is also affected by the time that the specimen is placed in the dry environment before immersing and the size, shape, and mix proportions of the specimen. In this experiment, a cylindrical specimen (specimen size: ϕ50 mm × 100 mm) placed in a dry environment for 3 months after standard curing and two sets of cylindrical specimens of different sizes (ϕ70 mm × 35 mm and ϕ50 mm × 100 mm) placed for 9 months, all of which were obtained by coring from the same concrete cube embryo, were tested for the variation law of water content with time. The test results are listed in The variation pattern of mass and water absorption ratio with immersion time for three groups of specimens is shown in From Therefore, the following conclusions can be drawn from the three sets of specimen-immersion experiments: the size and shape of the specimen affect the water absorption mass, rate of change of water absorption mass, water content ratio, and time to reach water saturation, but there is no effect on the water absorption and water content ratio at saturation. Although the time that the specimen was placed in a dry environment after the fabrication and maintenance affects the initial water content mass of the specimen, the water absorption mass after immersion, the water absorption ratio, the time to reach water saturation, and the rate of the change of the water absorption mass; it has no effect on the final water absorption mass, water content mass, and water content ratio of the specimen at saturation.d is the mass of the concrete specimen after being stoved in an oven, and Ms is the mass of the immersed concrete specimen. At the same time, the relative water absorption ratio W was used to describe the water absorption of concrete from the surrounding environment, which was defined as the real water absorption mass of the specimen/total water absorption mass of the specimen at saturation, i.e.,0 is the mass of the air-drying concrete specimen before immersion.In addition, the differences in the aggregate type, mix ratio, and water reducing agent of concrete materials may affect its water absorption ratio and water content ratio. Therefore, to reduce the influence of the differences of the materials themselves on the research results, this study used the relative saturation ratio and relative water absorption ratio to describe the moisture level of concrete in a humid or water environment, and defined the relative saturation ratio S as the water content mass of the immersed specimen/the water content mass of the specimen at saturation. That is,−5/s. The test system’s installed heating chamber is shown in After production and curing, six groups of specimens (size ϕ50 mm × 100 mm) were placed in a dry environment for three months, which were then stoved in an oven, immersed for 2 h, 6 h, 60 h, immersed to full saturation, and without any treatment as the test subjects. The water content and water absorption of the immersed specimen used in the test are shown in The size of the selected specimens was ϕ50 mm × 100 mm and the test temperatures were normal temperature (20 °C), 200 °C, 400 °C, and 520 °C, respectively. Before the test, we drilled holes in the core of concrete specimen column, and the depth of holes was about half of the length of the specimen. The thermocouple was inserted into the holes in the center of the specimens. At the set target temperatures , 3–5 heating tests were carried out to determine the constant temperature time required to ensure the uniform distribution of the temperature inside the concrete after the heating chamber was heated to the target temperature. The results showed that the constant temperature time required to reach the target temperature inside the specimen was as follows: the constant temperature time is 60 min at 200 °C, 120 min at 400 °C, and 150 min at 520 °C. In addition, before the experiment, it was necessary to check the insulation of the heating chamber, and insulation cotton was used to wrap the heating chamber, so as to ensure the heating efficiency and the accuracy of the test results.The quasi-static uniaxial compressive strengths of the concrete specimens with different water contents at different temperatures were obtained, and the specimens had typical damage morphology after the tests, as shown in During the heating process of the specimen, the color of the specimen changed with the rise in temperature. The color of the specimen did not change much before the temperature reached 200 °C, but when the temperature reached 400 °C and 520 °C, the specimen presented a light yellow and gray-white color, respectively. After the specimen reached the predetermined temperature, there were no obvious cracks before loading. However, in some reports  of high-T, with temperature. The temperature softening factor, KT, of compressive strength is often defined as the ratio of the high temperature quasi-static uniaxial compressive strength, σT, of air-drying concrete to the normal temperature quasi-static uniaxial compressive strength, σ0, i.e., KT = σT/σ0.The compressive strength of concrete materials shows a significant softening effect at high temperatures, and the effect of temperature on the strength of concrete materials is generally characterized by the variation pattern of softening factor, KT, with temperature for air-drying concrete specimens were obtained from the above experiments, as shown in T–T curves obtained by Ping Li [In this study, the experimental data of the temperature softening factor, K Ping Li  by summaT, and the temperature, T, obtained is given by Ping Li [c is the reference temperature, Tc = 20 °C, a = 1.1, m = 0.1, and n = 27.The relationship between the temperature softening factor, K Ping Li :(3)KT=a1w (Kw = wσ is the compressive strength of the immersed specimens and σd is the compressive strength of the specimens stoved in an oven) with the relative saturation ratio S. The comparison between the experimental results in this paper and those in existing literature [w, and the relative saturation ratio, S, is:c = 116.8, and k = −0.04.The quasi-static uniaxial compressive strengths of concrete specimens with different water contents (expressed by relative saturation ratio in this paper) at normal temperature were obtained, along with the variation curves of the water softening factor, Kterature ,36,37,38Ts/σd, where σTs is the compressive strength of concrete materials with different water contents  at temperature, T, and σd is the compressive strength of concrete at normal temperature after being stoved in an oven. The variation relationship between the heat–moisture combined effect factor DIF and the relative saturation ratio is obtained and shown in The above high temperature compression experiments on dry concrete specimens and normal temperature compression experiments on immersed concrete specimens verify the temperature softening effect and water softening effect on the compressive strength of concrete, respectively. However, under combined heat and moisture conditions, the water inside the concrete will undergo phase transformation and disappear, and the variation pattern of the compressive strength of the concrete material is yet to be determined. In this study, on the basis of the high temperature uniaxial quasi-static compression experiments of dry and wet concrete, the variation factor of the compressive strength of the concrete material under combined heat and moisture conditions is defined as DIF = σWe found two interesting phenomena from Despite the fact that the free water content stored inside the concrete differs when it is placed in the dry normal temperature environment for different times after production and curing, and the free water content decreases with the increase in placing time, the effect of the free water retained in the concrete during the first hydration process on the compressive strength at high temperature is not significant compared to that of the water absorbed during the second hydration process. Therefore, this study mainly addressed the variation pattern of the compressive strength of the immersed concrete specimens at high temperature and found the coupling relationship among water absorption ratio, temperature, and compressive strength of concrete specimens undergoing the second hydration. This has a more practical significance not only for the prediction of compressive strength of concrete when working in a hot and humid environment but also for the design of structural protection.ab and the heat–moisture coupling effect factor DIF of the compressive strength of concrete specimens at different temperatures are obtained by fitting the above experimental results, as shown in The relationship curves between the relative water absorption ratio WThe following patterns can be observed from (1)The variation patterns of the compressive strength of immersed concrete at high temperature and normal temperature are remarkably different. At high temperature, the quasi-static compressive strength of immersed concrete shows a trend from rise to decline with the increase in relative water absorption ratio of the specimen, which is from approximately 45% to 65%. The high temperature compressive strength of concrete reaches a peak, and after that the strength decreases rapidly. Further, as the water absorption increases, the higher the temperature of concrete is, the faster the rate of strength decrease is after the peak. The relationship among the heat–moisture coupling effect factor DIF, the corresponding relative water absorption ratio, and temperature at the peak can be obtained by the fitted curve. As can be observed in (2)Compared with the compressive strength of air-drying concrete at normal temperature, the high-temperature compressive strength of immersed concrete increases in a certain range with the increase in water absorption mass (or relative water absorption), but the increase in the value of the compressive strength and the range of the relative water absorption ratio corresponding to the increase in compressive strength are temperature dependent. When T = 200 °C, the high-temperature compressive strength of the air-drying concrete specimens is slightly lower than their normal-temperature compressive strength, and the high-temperature compressive strength of the immersed concrete specimens is higher than the normal-temperature compressive strength of the air-drying concrete specimens. Even in a larger range of relative water absorption ratio (approximately 10% to 90%), their compressive strength is higher than that of the concrete stoved in an oven at normal temperature. When T = 400 °C, the compressive strength of the immersed concrete is lower than that of the concrete stoved in an oven at normal temperature, and it is only slightly higher than the compressive strength of the air-drying concrete specimens at normal temperature near the peak. Further, the corresponding range of the relative water absorption ratio is significantly narrower than that at T = 200 °C. When T = 520 °C, the compressive strength of the immersed concrete is lower than that of the air-drying concrete specimens at normal temperature.(3)ab is less than 2%, approximately). At T = 400 °C, when the relative water absorption ratio, Wab, is less than 22%, the compressive strength is higher than that at normal temperature and with the same relative water absorption ratio. At T = 520 °C, when the relative water absorption ratio, Wab, is greater than 33% and less than 90%, the compressive strength is higher than that at normal temperature and with the same relative water absorption ratio. However, when Wab is greater than 90%, the compressive strength is lower than that at normal temperature with the same water absorption ratio.Under the same relative water absorption ratio, the compressive strength of immersed concrete at T = 200 °C is higher than that at normal temperature −Wab curve shifts back with the increase in temperature. The DIF (20 °C)−Wab curve reflects the water softening effect of the compressive strength of concrete under water immersion conditions at normal temperature. At this intersection, the heat–moisture coupling effect at temperature T is equivalent to the water absorption softening effect at normal temperature. Beyond this intersection point, within a certain range of relative water absorption ratio, the temperature plays a role in reducing the water softening effect, which is macroscopically manifested as the temperature strengthening phenomenon of the immersed concrete. This occurs because the free water absorbed during the material immersion process undergoes phase change and is lost at high temperature, thus weakening the softening effect of water on the compressive strength and leading to the result that the high-temperature compressive strength of immersed concrete is higher than that at normal temperature. This “pseudo-temperature strengthening effect” is a distinctive feature that distinguishes the thermodynamic response of immersed concrete from that of dry concrete (including air drying concrete and concrete stoved in an oven).Before the quasi-static high-temperature compressive strength of concrete reaches its peak, the intersection point of the DIF −W(4)ab, is:The fitted equation of the heat–moisture coupling effect factor DIF and relative water absorption ratio, W0 = 20 °C.The variation relationship of A(T), B(T), and C(T) with respect to temperature is obtained from the experiment, as follows:The equation reflects the change pattern of compressive strength of the concrete material under the influence of the heat–moisture coupling effect. When DIF(T) = 1, it means that, under the action of the heat–moisture coupling effect, the compressive strength of this concrete specimen and that of the concrete specimen stoved in an oven at normal temperature are equal. In this case, the heat–moisture coupling effect has the same effect on the crack growth and healing inside the material. Macroscopically, the heat–moisture coupling effect has no influence on the compressive strength of concrete at this temperature and moisture condition. When DIF(T) > 1, it indicates that the high-temperature compressive strength of immersed concrete under the action of the heat–moisture coupling effect is greater than that of the concrete stoved in an oven at normal temperature. In this case, the role of the heat–moisture coupling effect in promoting the healing of cracks within the material is greater than the role in promoting the growth of cracks, which is macroscopically manifested as the increase in the compressive strength of concrete at this temperature and moisture condition. When DIF(T) < 1, the compressive strength of concrete under the action of the heat–moisture coupling effect is less than that of the concrete stoved in an oven at normal temperature. In this case, the role of the heat–moisture coupling effect in promoting the growth of cracks within the material is greater than the role in promoting the healing of cracks, which is macroscopically manifested as the attenuation of the compressive strength of concrete at this temperature and moisture condition.The variation pattern of the heat–moisture coupling effect factor DIF with temperature for the concrete specimens with different relative water absorption ratio and concrete specimens stoved in an oven are shown in (1)The heat–moisture coupling effect factor DIF of quasi-static compressive strength of concrete stoved in an oven decreases linearly with increasing temperature, T. The fitted equation for the DIF–T relationship is0 and T0 is the reference temperature (T0 = 20 °C). DIF (stoved) = σT/σd, where σd is the quasi-static normal temperature compressive strength of the concrete specimen stoved in an oven and σT is the quasi-static high temperature compressive strength of the concrete specimen stoved in an oven. Equation (9) reflects the temperature softening effect of concrete specimens that are not affected by free water.From the variation curve of DIF−T in (2)ab = 0%)−T change curve of the heat–moisture coupling effect factor DIF (Wab = 0%) of the quasi-static compressive strength of the air-drying concrete (Wab = 0%) with temperature, T, it can be observed that, compared to the concrete specimens stoved in an oven, the DIF (Wab = 0%)−T curve shows an obvious non-linear decline characteristic, and its decline rate is more gentle until 350 °C. Some researchers [From the DIF ab = 60% and Wab = 90% are close to each other, whereas the peak of the saturated concrete specimen (Wab = 100%) is significantly lower than that of the specimens with the above two water absorption ratios and the temperature corresponding to the peak is smaller.The heat–moisture coupling effect factor DIF of the compressive strength of immersed concrete shows a trend from rise to decline with the increase in temperature. The DIF reaches its maximum value between 200 and 300 °C, and then decreases rapidly, and the rate of decrease is faster than that of concrete air drying or that stoved in an oven. The peak of DIF declines with the increase in water absorption, which also indicates that the moisture effect on material strength is not fully reversible. Based on the fitting curves, it is found that when the DIF reaches its peak, the corresponding peak temperatures of the concrete specimens with the relative water absorption ratio of WIn addition, after the heat–moisture coupling effect factor DIF of the immersed concrete reaches its peak, the decline rate of DIF accelerates with the increase in water absorption of the specimens Therefore, the specimens with greater water absorption are more likely to burst at high-temperature.(4)From the fitted experimental curves, it can be observed that the strength of concrete with the relative water absorption ratios of 60% and 90% is greater than that of stoved concrete at normal temperature within a certain temperature interval corresponding to the vicinity of the DIF peak. For concrete specimens with 60% relative water absorption ratio, when the temperatures range from approximately 150 to 400 °C, their compressive strengths are greater than that of stoved concrete specimens at normal temperature, that is, DIF > 1; when the temperature is between 100 and 450 °C, their compressive strengths are greater than that of air-drying concrete specimens at normal temperature; when the temperature is between 80 and 520 °C, their compressive strengths are greater than that of air-drying concrete specimens at the same high temperature. For concrete specimens with 90% relative water absorption ratio, when the temperatures range from approximately 200 to 350 °C, their compressive strengths are greater than that of stoved concrete specimens at normal temperature, that is, DIF > 1; when the temperature is between 140 and 420 °C, their compressive strengths are greater than that of air drying concrete specimens at normal temperature; when the temperature is between 135 and 520 °C, their compressive strengths are greater than that of air-drying concrete specimens at the same high temperature. The high temperature compressive strength of fully saturated concrete specimens is lower than the normal temperature compression strength of both air-drying specimens and specimens stoved in an oven. The compressive strength of fully saturated concrete specimens is greater than that of air-drying concrete at the same temperature ranging from 200 to 400 °C.After water absorption in concrete, the water inside the specimen undergoes a series of physical and chemical processes during heating: water absorbed during immersion is evaporated, then free water stored in the specimen before immersion is evaporated → physically adsorbed water adsorbed on the surface of the solid phase is lost due to drying → water between the layers of the C-S-H structure is lost due to intense drying → chemically bound water is lost along with hydride decomposition. According to the Feldman–Sereda  model, tAt normal temperature, approximately 50% of the hydrogen bonds of water are broken, and in this state, the surface charge of the material is unsaturated. Thus, the surface energy increases, which leads to surface tension and further causes a large number of molecules that tend to agglomerate together. The water that accumulates at the crack tip has a wedge-like splitting effect on crack extension. After curing, the concrete will shrink and form cracks, G0 (including reversible cracks G1 and irreversible cracks G2), due to water loss in its interior under normal temperature and air-drying conditions, especially in the interface transition zone between the aggregate and cement mortar, where more microcracks and microporosity exist. Because of this, the interface transition zone is the weakest link in the strength chain and is usually considered as the strength limiting phase in concrete, which plays a key role in the compressive strength of concrete. Assuming that the evolution process of reversible and irreversible cracks within the material is independent. After dry concrete is immersed in water, a small number of reversible cracks, G1, are closed, and a large number of irreversible cracks, G2, are continuously derived, grown, and connected on the basis of the original damage nucleus caused by the wedge splitting effect of water, Finally, a new crack group, G, with a larger scale and number will be formed. Therefore, the compressive strength of immersed concrete specimens is less than that of dry concrete at normal temperature.The new cracks group, G, after water immersion also contain reversible crack part G3 and irreversible crack part G4. As the temperature rises, the water inside the material will undergo a series of physical and chemical reactions. The volume expansion of water from liquid phase to gas phase at high temperature may lead to the increase in pore tension in the cracks. However, because of the existence of a large number of microcracks, G, inside the material during the hydration process after water immersion, the gas generated by the water phase transition is rapidly transferred to the surrounding medium through these microcracks. Moreover, this process of mass and heat transfer makes the temperature distribution inside the immersed specimen more uniform than that of the dry specimen, and the thermal stress caused by the temperature gradient is smaller. As indicated by the results of the present experiments in the temperature range of 20 to 520 °C, the pore tension inside the immersed concrete specimen is not large, which has also been proved by the test results of pore pressure of concrete materials under the combination of heat and moisture conducted by Mindeguia . In addi(1)Air-drying stage at normal temperature after production and curingAt this stage, the dry shrinkage phenomenon occurs in the specimen due to air drying and water loss, forming an initial crack group, G0, which includes reversible dry shrinkage cracks, G1, and irreversible dry shrinkage cracks, G2.(2)Immersion stageDuring water immersion of the specimen, the reversible dry shrinkage cracks, G1, heal while the irreversible dry shrinkage cracks, G2, expand and connect under the wedge splitting action of water, forming a larger size crack cluster, G, which includes reversible hydration cracks, G3, and irreversible hydration cracks, G4.(3)Heating phaseAt high temperature, the free water inside the material is gradually lost, the reversible hydration cracks, G3, close, while the irreversible hydration cracks, G4, further expand with a series of physicochemical reactions inside the material to form a new crack group, G5.At normal temperature, the amount of cracks formed by dry shrinkage of the specimen is smaller than the amount of hydration cracks formed by water absorption of the specimen, i.e., G0 < G, and thus the compressive strength of immersed concrete is smaller than that of dry concrete. However, at high temperature, the crack evolution process is influenced by the coupling effect of temperature and moisture. With the increase in temperature, the reversible hydration cracks, G3, in concrete specimens with different water contents began to heal, and the compressive strength of wet concrete gradually rises. In the process of reversible hydration crack healing, the amount of cracks (G3 + G4) gradually decreases. The compressive strength of wet concrete with the same water content at high temperature is greater than that at normal temperature. When the amount of cracks (G3 + G4) < G0, the high temperature compression strength of wet concrete is greater than the compression strength of dry concrete at normal temperature. When G3 is completely healed, the high temperature compression strength of wet concrete reaches its peak, and the irreversible hydration crack, G4, began to develop rapidly, forming a new crack group, G5. When G5 < G0, the wet concrete high temperature compression strength is greater than the compression strength of dry concrete at normal temperature. When G5 = G0, the wet concrete high temperature compression strength is equal to the compression strength of dry concrete at normal temperature. When G5 > G0, the wet concrete high temperature compression strength is lower than the compression strength of dry concrete at normal temperature. From the DIF−T change curve, it can be found that after the compressive strength of wet concrete reaches its peak, DIF decreases with the increase in temperature, The higher the water content is, the faster the rate of DIF decline, indicating that the expansion rate of crack group G5 is significantly greater than the crack expansion rate of the dry concrete material at the same temperature. Further, the higher the relative saturation rate of concrete after water absorption, the higher the propagation rate of the G5 crack group after the compression strength reaches the peak, and the more likely the high temperature bursting phenomenon in specimens is to occur.In this study, by carrying out experimental studies on quasi-static uniaxial compression of concrete materials at different temperatures and different water contents, the coupling relationship among compression strength of concrete, temperature, and water absorption ratio is determined, and the mechanism of the heat–moisture coupling effect on the compression strength of concrete is analyzed based on the experimental results. The following conclusions are obtained.(1)Under combined heat and moisture conditions, the change in concrete compressive strength no longer shows a monotonic decay law, but a parabolic variation characterized by an increase followed by a decrease. When the relative water absorption ratio of the specimen is approximately 45% to 65% and the temperature is approximately 200 to 300 °C, compressive strength of the immersed concrete reaches its peak.(2)There is a significant difference in the effect of water on the compressive strength of concrete before and after immersing. That is, the free water stored during the process of production, curing, and air drying  and the water absorbed during the process of immersing  have different effects on the crack evolution of concrete materials, and the effect of water absorption of concrete during the second hydration is more significant on its compression strength. However, the microscopic mechanism of the effect of the two hydration processes on the evolution of microcracks needs to be further investigated in depth.(3)The expansion and closure of cracks within the concrete material are the result of the heat and moisture coupling effect. The compression strength of the concrete rises when the crack closure rate dominates, and conversely, the compression strength of the concrete material decreases when the crack expansion rate dominates. On the basis of previous studies and combined with our experimental results, we have given a reasonable explanation for the evolution process of microcracks under the combined heat and moisture conditions. However, more micro-scale experimental evidence is needed. This is also the work we are currently carrying out, and some results have been obtained, which we believe will be published in the near future. In addition, the specimen size used in the heat and moisture coupling experiment and theoretical research of concrete materials in this paper is slightly small. In the heat and mass transfer process, the internal crack evolution law caused by the temperature and moisture gradients of large-size concrete specimens under combined heat and moisture conditions may differ from that of small specimens. These are more prone to high-temperature cracking due to the more significant temperature and moisture gradients under the heat and moisture coupling conditions. The change pattern of the heat–moisture coupling effect factor of the large-size concrete specimens have to be further verified.(4)ab, is:The equation for the heat–moisture coupling effect factor DIF of the compressive strength, temperature, T, and relative water absorption ratio, W