Pyruvate metabolism dictates fibroblast sensitivity to GLS1 inhibition during fibrogenesis.

Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-ß1 (TGF-ß1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-ß1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-ß1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-ß1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-ß1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-ß1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Thrombospondins: Conserved mediators and modulators of metazoan extracellular matrix.

This review provides a personal overview of significant scientific developments in the thrombospondin field during the course of my career. Thrombospondins are multidomain, multimeric, calcium-binding extracellular glycoproteins with context-specific roles in tissue organisation. They act at cell surfaces and within ECM to regulate cell phenotype and signalling, differentiation and assembly of collagenous ECM, along with tissue-specific roles in cartilage, angiogenesis and synaptic function. More recently, intracellular, homeostatic roles have also been identified. Resolution of structures for the major domains of mammalian thrombospondins has facilitated major advances in understanding thrombospondin biology from molecule to tissue; for example, in illuminating molecular consequences of disease-causing coding mutations in human pseudoachrondroplasia. Although principally studied in vertebrates, thrombospondins are amongst the most ancient of animal ECM proteins, with many invertebrates encoding a single thrombospondin and the thrombospondin gene family of vertebrates originating through gene duplications. Moreover, thrombospondins form one branch of a thrombospondin superfamily that debuted at the origin of metazoans. The super-family includes additional sub-groups, present only in invertebrates, that differ in N-terminal domain organisation, share the distinctive TSP C-terminal region domain architecture and, to the limited extent studied to date, apparently contribute to tissue development and organisation. Finally, major lines of translational research are discussed, related to fibrosis; TSP1, TSP2 and inhibition of angiogenesis; and the alleviation of chronic cartilage tissue pathologies in pseudoachrondroplasia.

The Role of Collagens in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting both children and adults. The clinical picture of AD manifests in typical skin lesions, such as localized eczema and dry skin, with dominant, persistent itching that leads to sleep disturbances. The pathophysiology of AD has been extensively investigated with respect to epigenetic and genetic factors, skin barrier defects, as well as immunological and microbial disorders. However, to date, the involvement of extracellular matrix (ECM) elements has received limited attention. Collagen, a major component of the ECM, may serve as a therapeutic target for the future treatment of AD. This paper summarizes the role of collagens, which are the most abundant components of the extracellular matrix in AD.

Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size.

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-ß1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-ß1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.

Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-ß1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-ß1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-ß1 signaling axis.

Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV-driven sinusoidal remodeling.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB-dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

Role of the mitochondrial protein cyclophilin D in skin wound healing and collagen secretion.

Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif-KO mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.

Investigating the Role of SNAI1 and ZEB1 Expression in Prostate Cancer Progression and Immune Modulation of the Tumor Microenvironment.

Prostate cancer (PCa) is an immunologically cold tumor and the molecular processes that underlie this behavior are poorly understood. In this study, we investigated a primary cohort of intermediate-risk PCa (n = 51) using two NanoString profiling panels designed to study cancer progression and immune response. We identified differentially expressed genes (DEGs) and pathways associated with biochemical recurrence (BCR) and clinical risk. Confirmatory analysis was performed using the TCGA-PRAD cohort. Noteworthy DEGs included collagens such as COL1A1, COL1A2, and COL3A1. Changes in the distribution of collagens may influence the immune activity in the tumor microenvironment (TME). In addition, immune-related DEGs such as THY1, IRF5, and HLA-DRA were also identified. Enrichment analysis highlighted pathways such as those associated with angiogenesis, TGF-beta, UV response, and EMT. Among the 39 significant DEGs, 11 (28%) were identified as EMT target genes for ZEB1 using the Harmonizome database. Elevated ZEB1 expression correlated with reduced BCR risk. Immune landscape analysis revealed that ZEB1 was associated with increased immunosuppressive cell types in the TME, such as naïve B cells and M2 macrophages. Increased expression of both ZEB1 and SNAI1 was associated with elevated immune checkpoint expression. In the future, modulation of EMT could be beneficial for overcoming immunotherapy resistance in a cold tumor, such as PCa.

Composite Zonal Scaffolds of Collagen I/II for Meniscus Regeneration.

The meniscus is divided into three zones according to its vascularity: an external vascularized red-red zone mainly comprising collagen I, a red-white interphase zone mainly comprising collagens I and II, and an internal white-white zone rich in collagen II. Known scaffolds used to treat meniscal injuries do not reflect the chemical composition of the vascular areas of the meniscus. Therefore, in this study, four composite zonal scaffolds (named A, B, C, and D) were developed and characterized; the developed scaffolds exhibited the main chemical components of the external (collagen I), interphase (collagens I/II), and internal (collagen II) zones of the meniscus. Noncomposite scaffolds were also produced (named E), which had the same shape as the composite scaffolds but were entirely made of collagen I. The composite zonal scaffolds were prepared using different concentrations of collagen I and the same concentration of collagen II and were either cross-linked with genipin or not cross-linked. Porous, biodegradable, and hydrophilic scaffolds with an expected chemical composition were obtained. Their pore size was smaller than the size reported for the meniscus substitutes; however, all scaffolds allowed the adhesion and proliferation of human adipose-derived stem cells (hADSCs) and were not cytotoxic. Data from enzymatic degradation and hADSC proliferation assays were considered for choosing the cross-linked composite scaffolds along with the collagen I scaffold and to test if composite zonal scaffolds seeded with hADSC and cultured with differentiation medium produced fibrocartilage-like tissue different from that formed in noncomposite scaffolds. After 21 days of culture, hADSCs seeded on composite scaffolds afforded an extracellular matrix with aggrecan, whereas hADSCs seeded on noncomposite collagen I scaffolds formed a matrix-like fibrocartilage without aggrecan.

Myocardial fibrosis in right heart dysfunction.

Cardiac fibrosis, associated with right heart dysfunction, results in significant morbidity and mortality. Stimulated by various cellular and humoral stimuli, cardiac fibroblasts, macrophages, CD4+ and CD8+ T cells, mast and endothelial cells promote fibrogenesis directly and indirectly by synthesizing numerous profibrotic factors. Several systems, including the transforming growth factor-beta and the renin-angiotensin system, produce type I and III collagen, fibronectin and α-smooth muscle actin, thus modifying the extracellular matrix. Although magnetic resonance imaging with gadolinium enhancement remains the gold standard, the use of circulating biomarkers represents an inexpensive and attractive means to facilitate detection and monitor cardiovascular fibrosis. This review explores the use of protein and nucleic acid (miRNAs) markers to better understand underlying pathophysiology as well as their role in the development of therapeutics to inhibit and potentially reverse cardiac fibrosis.

Lupus dermal fibroblasts are proinflammatory and exhibit a profibrotic phenotype in scarring skin disease.

Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in patients with cutaneous lupus erythematosus (CLE) remain poorly understood. Dermal fibroblasts in patients with systemic lupus erythematosus (SLE) experience increased cytokine signaling in vivo, but the effect of inflammatory mediators on fibroblast responses in nonscarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from nonlesional skin of 22 patients with SLE and CLE and 34 individuals acting as healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared with those from individuals acting as healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single-cell RNA-Seq of SFRP2+ and inflammatory fibroblast subsets, and TGF-ß was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of nonlesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-ß, whereas inflammatory pathways were more prominent in nonscarring patients. Our study revealed that SLE fibroblasts are poised to hyperrespond to inflammation, with differential responses among patients with scarring versus nonscarring disease, providing a potential skin-specific target for mitigating damage.

Dysfunctional latent transforming growth factor ß activation after corneal injury in a classical Ehlers-Danlos model.

Patients with classical Ehlers Danlos syndrome (cEDS) suffer impaired wound healing and from scars formed after injuries that are atrophic and difficult to close surgically. Haploinsufficiency in COL5A1 creates systemic morphological and functional alterations in the entire body. We investigated mechanisms that impair wound healing from corneal lacerations (full thickness injuries) in a mouse model of cEDS (Col5a1+/-). We found that collagen V reexpression in this model is upregulated during corneal tissue repair and that wound healing is delayed, impaired, and results in large atrophic corneal scars. We noted that in a matrix with a 50 % content of collagen V, activation of latent Transforming Growth Factor (TGF) ß is dysregulated. Corneal myofibroblasts with a haploinsufficiency of collagen V failed to mechanically activate latent TGF ß. Second harmonic imaging microscopy showed a disorganized, undulated, and denser collagen matrix in our Col5a1+/- model that suggested alterations in the extracellular matrix structure and function. We hypothesize that a regenerated collagen matrix with only 50 % content of collagen V is not resistant enough mechanically to allow adequate activation of latent TGF ß by fibroblasts and myofibroblasts.

Giant peritoneal loose body and its protein composition: a case report.

Peritoneal loose body (PLB) is a kind of lesions located in the abdominal cavity or pelvic cavity, which is rare and difficult to diagnose. The diameter of PLB is mostly 0.5-2.5 cm. Most PLBS are asymptomatic. Here we reported a case of giant PLB in the pelvis and analyzed its structure and protein composition. Surgical exploration revealed a white oval mass (4.5*4*3 cm) in the pelvic cavity. After the mass was removed, the symptoms of hematuria disappeared and the patient was discharged on the second postoperative day. Histochemical staining showed that PLB was mainly composed of collagen and scattered calcification. The protein components of PLB were detected by proteome analysis, and a variety of proteins related to collagen deposition and calcification were identified in PLB.

Restraining of glycoprotein VI- and integrin α2ß1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2ß1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2ß1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2ß1 engagement.

'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

Collagen Mimicry with a Short Collagen Model Peptide.

Mimicking triple helix and fibrillar network of collagen through collagen model peptide(CMP) with short GPO tripeptide repeats is a great challenge. Herein, a minimalistic CMP comprising only five GPO repeats [(GPO)5 ] is presented. This novel approach involves the fusion of ultrashort peptide with the synergetic power of π-system and ß-sheet formation to short CMP (GPO)5 . Accordingly, a hydrogel-forming, fluorenylmethoxycarbonyl (Fmoc)-functionalized ultrashort peptide (NFGAIL) is fused at the N-terminus and phenylalanine at the C-terminus of (GPO)5 (Fmoc-NFGAIL-(GPO)5 -F-COOH, FmP-5GPO). At room temperature, it forms a robust triple helix in aqueous buffer solution and has a relatively high melting point of 35 °C. The fluorenyl motif stabilizes the triple helix by aromatic π-π interactions as in its absence, triple helix is not formed. NFGAIL, which forms a ß-sheet, also aids in triple helix stabilization via intermolecular hydrogen bonding and hydrophobic interactions. FmP-5GPO forms highly entangled nanofibrils with a micrometer length, which have excellent cell viability. The achievement of stable triple helix and fibrils in such a short CMP(FmP-5GPO) sequence is a challenging feat, and its significance in CMP-based biomaterials is undeniable. The present strategy highlights the potential for developing new CMP sequences through intelligent tuning of fusion peptides and GPO repeats.

Rapid Intrafibrillar Mineralization Strategy Enhances Adhesive-Dentin Interface.

Biomimetic mineralization of dentin collagen appears to be a promising strategy to optimize dentin bonding durability. However, traditional postbonding mineralization strategies based on Ca/P ion release still have some drawbacks, such as being time-consuming, having a spatiotemporal mismatch, and having limited intrafibrillar minerals. To tackle these problems, a prebonding rapid intrafibrillar mineralization strategy was developed in the present study. Specifically, polyacrylic acid-stabilized amorphous calcium fluoride (PAA-ACF) was found to induce rapid intrafibrillar mineralization of the single-layer collagen model and dentin collagen at just 1 min and 10 min, as identified by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. This strategy has also been identified to strengthen the mechanical properties of demineralized dentin within a clinically acceptable timeframe. Significantly, the bonding strength of the PAA-ACF-treated groups outperformed the control group irrespective of aging modes. In addition, the endogenous matrix metalloproteinases as well as exogenous bacterial erosion were inhibited, thus reducing the degradation of dentin collagen. High-quality integration of the hybrid layer and the underlying dentin was also demonstrated. On the basis of the present results, the concept of "prebonding rapid intrafibrillar mineralization" was proposed. This user-friendly scheme introduced PAA-ACF-based intrafibrillar mineralization into dentin bonding for the first time. As multifunctional primers, PAA-ACF precursors have the potential to shed new light on prolonging the service life of adhesive restorations, with promising significance.