Caveolae-Mediated Sensing of Nanoporous Cues in Fibroblasts Reprogramming During Wound Healing

Caveolae-Mediated Sensing of Nanoporous Cues in Fibroblasts Reprogramming During Wound Healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Caveolae-Mediated Sensing of Nanoporous Cues in Fibroblasts Reprogramming During Wound Healing Keqing Shi, Feixia Guo, Wei Xie, Wante Lin, Meilin Yi, Haobing Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5955293/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Scaffolds with pores can influence cellular fate and tissue microenvironments by altering intracellular and intercellular signaling pathways, which are crucial for guiding tissue regeneration. Despite their significance, the cellular mechanisms behind the response to pores, especially at the nanoscale, are not well understood. Our study shows that scaffolds with different nanopore characteristics can enhance skin regeneration in various ways, with fibroblasts being the primary cellular responders. We have demonstrated that caveolae formation is a critical mechanism by which fibroblasts interact with nanopores. The phosphorylation of caveolin-1 (Cav1) is a key event in this process, enabling caveolae-mediated endocytosis and the subsequent internalization of cellular substances. This initiates a cascade of events involving the internalization of cell surface receptors such as PDGFRβ, activation of tyrosine kinase pathways including PI3K, AKT, and ERK1/2, and the phosphorylation of the transcription factor RUNX1. The nuclear translocation of RUNX1 upregulates the expression of fibroblast growth factor binding protein 3 (FGFBP3), which is a key factor in tissue repair. Additionally, cytoplasmic Cav1 can competitively bind to Filamin-A, releasing CBFβ, which then translocates to the nucleus and enhances RUNX1's DNA-binding affinity, synergistically activating FGFBP3 transcription and promoting tissue healing. Collectively, our findings underscore the importance of considering nanopore characteristics in scaffold design for tissue regeneration and highlight caveolae formation as a central mediator of cellular responses to nanoporous environments, initiating a multitude of biological processes essential for tissue repair and regeneration. Biological sciences/Biophysics/Endocytosis Health sciences/Diseases/Trauma fibroblast caveolae wound healing scaffold RUNX1 FGFBP3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Severe skin injuries, including full-thickness defects and chronic ulcers, pose significant health challenges and can be life-threatening 1 , 2 . Timely and sophisticated medical interventions are crucial for addressing the loss or damage to skin tissue 3 . Recent advancements in biomaterial science have led to the development of synthetic scaffolds, such as artificial dermis, which capitalize on the body's innate healing capabilities 4 , 5 . These scaffolds harness the potential of endogenous cells to repair or replace damaged tissues, providing a less invasive and more sustainable solution compared to traditional methods 6 . By directing cell reprogramming, these scaffolds can facilitate cell attachment, migration, and differentiation, thereby guiding the tissue regeneration process. While the biophysical and biochemical cues of scaffolds have been extensively studied for their role in controlling cell state transitions 7 , the impact of scaffold porosity on cell fate is often underappreciated. It is well-established that the biophysical properties of biomaterials significantly influence the local tissue microenvironment and cellular behavior 8 . The stiffness of the extracellular matrix (ECM) is crucial for cell adhesion, spreading, and mechanotransduction 9 . Additionally, various pore morphologies found in biological materials and natural ECM direct cellular responses 10 . Studies have shown that nanoscale pores of appropriate dimensions can initiate cell anchorage, promote proliferation, influence differentiation, protect cells from apoptosis, and activate cytoskeletal reorganization—all of which are critical for tissue repair and homeostasis 11 – 14 . Despite the recognized importance of nanoscale pores in scaffolds for tissue repair 15 , the specific mechanisms by which repair cells respond to these nanoscale features remain poorly understood. In this study, we have developed scaffolds with distinct nanopore characteristics that maintain structural integrity and resist deformation. This allows us to explore the mechanisms underlying cell responses to various nanopores. We focus on fibroblasts, which are the primary responders to nanopores in scaffolds and exhibit diverse behaviors in terms of migration and proliferation. Our findings reveal that caveolae formation is a critical response of fibroblasts to nanopores, induced by the phosphorylation of Cav1. This process initiates the biological processes necessary for regeneration. We propose that caveolae induced by nanopores could serve as a key parameter in the design of scaffolds for tissue engineering applications. Materials and Methods Preparation and characterization of nanopore scaffolds To evaluate the impact of scaffold nanopores on tissue repair and regeneration, we fabricated scaffolds with distinct nanopores using polystyrene (a material commonly used for cell culture dishes), which include round (Named as Nanopore #1), 3×-stretched oval (Nanopore #2), and 6×-stretched oval (Nanopore #3) configurations (Extended Data Fig. 1), exhibiting resistance to deformation and degradation, as described in our previous study. Briefly, colloidal crystal templates made from uniform silica nanoparticles were created on glass slides using the vertical deposition technique. These templates were then soaked in a polystyrene solution and left to dry at room temperature (20°C) for four days before they were allowed to detach naturally, yielding an unetched anti-opal film. Following this, the anti-opal film and silica composite were removed from the slides after a 30-minute immersion in a 4% hydrofluoric acid solution. The films were then subjected to etching with a 1% hydrofluoric acid solution for 12 hours at 4°C to eliminate all silica nanoparticles, resulting in an anti-opal nano-scaffold with a consistent nanopores. To achieve this, the two sections of the film were affixed to a vernier caliper, which was placed in an 80°C water bath. The film was stretched by pulling the caliper's slider, and the stretch ratio was determined using the caliper's reading, thus obtaining virous nanopores in scaffolds with 3× and 6× stretch ratios. Finally, the acquired scaffolds were analyzed using scanning electron microscopy (SEM) to examine their nanopores morphology. Cell culture Primary cells (fibroblasts, macrophages, keratinocytes) were extracted from the skin of C57BL/6J mice. Mouse embryonic cells, NIH-3T3 and human umbilical vein endothelial cells (HUVECs) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were maintained in DMEM culture medium (Biological Industries, Israel) supplemented with 10% fetal bovine serum (Gemini, USA) and 1% streptomycin and penicillin (Invitrogen Corp., Carlsbad, CA, USA) in a humidified atmosphere with 5% CO 2 at 37°C. Regular identification of cells was conducted to ensure the elimination of mycoplasma contamination. Wound healing Approximately 10,000 cells of a 70 μL cell suspension were added to each well of the cell migration insert. The insert was then placed in a CO2 incubator at 37°C for 12 hours. After 8 hours, the insert was carefully removed, and serum-free medium was added to the well plate. The culture was maintained for 12 and 24 hours from the time the medium was introduced. Following the removal of the culture medium, 4% paraformaldehyde was used for fixation. The well was then washed three times with PBS, and images were captured and observed using an inverted phase contrast microscope (Olympus, Japan). The cell migration rate was calculated by comparing it to the state at 0 hours. In vitro tube formation The ability of HUVECs to form tube-like structures was assessed using a modified tube formation assay. Twenty-four well plates were layered with a cold Matrigel solution (BD Biosciences, New Jersey, USA) and incubated at 37°C for 30 minutes to permit the Matrigel to gel. The HUVECs were collected, stained with calcein-AM, and resuspended in a medium reduced in fetal bovine serum (FBS) to 1%. These cells were then plated at a concentration of 20,000 cells/mL per well onto the Matrigel-coated wells and pre-incubated at 37°C for 2 hours to facilitate cell adhesion. Subsequently, 50 μL of concentrated supernatant, derived from fibroblasts that had been cultured on plates or scaffolds, was added to the cell-laden plates. Photographs of the tube-like structures were captured using a Nikon confocal microscope (Nikon C2, Japan) over a period of 8 to 16 hours. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from primary fibroblasts or NIH-3T3 cells using TRIzol Reagent (catalog #15596026, Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the Prime Script RT Kit (catalog #KR107, Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's guidelines. Quantitative real-time PCR (qRT-PCR) was performed on a Light Cycler Real-time PCR system (Roche, Shanghai, China) with SYBR Green master Mix (catalog #FP205, Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer's protocols. The Ct values of the target genes were analyzed using the 2^-ΔΔCT method, with β-actin expression serving as the endogenous control for normalization. Specific primer sequences utilized in this study are provided in Supplementary Table 1 (Sangon Biotech Co., Ltd., Shanghai, China). DNA pull down and liquid chromatography-tandem mass spectrometry NIH-3T3 cells were grown in a 150 mm culture dish until they reached 80% confluence. After rinsing the cells three times with cold PBS, they were treated with trypsin and collected via centrifugation. The nuclear lysate was extracted using a nuclear and cytoplasmic protein separation kit (catalog #P0028, Beyotime Biological, Shanghai, China) in accordance with the manufacturer's protocol. Dynabeads® C1 Streptavidin Magnetic Beads were rinsed three times with B&W buffer (5 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM EDTA), and then 500 μL of the magnetic beads were incubated with 50 μg of biotinylated target DNA for 30 minutes at room temperature. DNA pull-down was conducted at 4°C with nuclear supernatant extracts and magnetic bead-DNA complexes. The complexes were subsequently washed six times with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA) and finally resuspended in 100 μL of elution buffer. Shotgun Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then carried out by Genechem, Shanghai, China. Lastly, the LC-MS/MS raw data were searched and qualitatively analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and MASCOT 2.6 (Matrix Science). The protein database utilized was UNIPROT_Mus musculus_2023_03. Co-immunoprecipitation and liquid chromatography-tandem mass spectrometry NIH-3T3 cells were cultured to 80% confluence in a 10 cm dish. The cells were washed three times with cold PBS, and then cell lysates were collected. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (catalog #P008, Beyotime Biological, Shanghai, China) following the manufacturer's protocol. The cell lysates were incubated with specific primary antibodies or control IgG overnight at 4°C with rotation. Protein A/G Agarose (catalog #37478, Cell Signaling Technology, Inc., CST, USA) was added to the lysates and incubated with rotation at 4°C for 3 hours to precipitate protein-protein complexes. Subsequently, the bead-protein complex precipitate was washed seven times with PBS, and 50 μL of protein loading buffer was added to elute the proteins at 100°C for 10 minutes. Immunoblotting was conducted to verify the elution of the target protein. Shotgun Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then carried out by Genechem, Shanghai, China. Finally, the LC-MS/MS raw data were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and MASCOT 2.6 (Matrix Science). The protein database used for the analysis was UNIPROT_Mus musculus_2023_03. Chromatin immunoprecipitation (ChIP) The enrichment of RUNX1 in the promoter region of FGFBP3 was quantified using an EZ-Magna ChIP™ A/G ChIP Kit (catalog #9003, Cell Signaling Technology, Inc., CST, USA) following the manufacturer's guidelines. In brief, cells were lysed and sonicated to shear the cross-linked chromatin and protein into fragments of appropriate size (200-1000 bp). The sheared DNA was sonicated for 28 cycles (15 seconds on and 10 seconds off) using a Diagenode Biorupter Pico (Diagenode, Belgium). After centrifugation at 10,000 × g for 10 minutes at 4 °C, the supernatant was collected and prepared for immunoprecipitation. Three aliquots of sheared DNA were added to 20 μL of fully resuspended protein A/G magnetic beads along with positive control (Anti-RNA Polymerase II), negative control (normal rabbit IgG), and the antibody against RUNX1 (catalog ab229482, abcam, USA), respectively. Following overnight incubation at 4 °C with rotation, protein/DNA complexes were eluted, and free DNA was reverse crosslinked from these complexes. The DNA was then purified using spin columns and analyzed by qRT-PCR with specific primers targeting the FGFBP3 gene promoter region (Supplementary Table 2). Dual-luciferase reporter assay The binding site of RUNX1 to the FGFBP3 gene promoter was predicted using the online prediction tool, JASPAR 3.0, which can be accessed at http://jaspar.genereg.net. Fibroblasts in the logarithmic growth phase were evenly distributed into a 6-well plate beforehand. When the cell density reached an appropriate level, wild type or mutant plasmids were transfected into the fibroblast cells using Lipofectamine 3000 (catalog #L3000015, Invitrogen, USA). Specific promoter sequences of FGFBP3 (sites #1, #2, #3, and #4, detailed in Supplementary Table 3) were all cloned into pGL3 reporter plasmids by Repobio (Hangzhou, China). Eight hours post-transfection, the supernatant was aspirated, and the cells were cultured in standard medium for an additional 24 hours. Luciferase activity was quantified as chemiluminescence using the Dual-Luciferase® Reporter (DLR™) Assay System (catalog #E1910, Promega, USA) following the manufacturer's instructions. The relative luciferase activity, as measured by the Multiskan SkyHigh (Thermo Scientific, USA), was determined based on the readings from both firefly and Renilla luciferase. Each transfection was carried out in triplicate to ensure reproducibility. Plasmids, virus and cell transfection Lipofectamine 3000 Transfection Reagent (catalog #L3000015, Invitrogen, USA) was employed for cell transfection following the manufacturer's recommendations. Plasmids and RNA interference lentiviruses were packaged and procured from Repobio, Hangzhou, China. NIH-3T3 cells were infected with RNA interference lentiviruses LV-shNC and LV-shCav1, and subsequently isolated using puromycin (3 µg/mL, Life Technologies, Gibco, USA) selection to achieve stable knockdown of Cav1. Total protein extracts were obtained from the transfected cells, and the knockdown efficiency of the target protein was verified by immunoblotting. The sequences of shCav1 and several siRNAs are detailed in Supplementary Table 4. In this study, AAV vectors for the knockdown of Cav1 and RUNX1 were constructed and produced by Cyagen Technology (Shanghai) Corp., Ltd. We utilized an Adeno-Associated Virus 9 (AAV9) vector to express short hairpin RNA (shRNA) that specifically targets Cav1 or RUNX1: AAV9-U6-shRNA (Cav1)-CMV-WPRE, AAV9-U6-shRNA (RUNX1)-CMV-WPRE, and AAV9-U6-shRNA (NC)-CMV-WPRE. The shRNA sequences targeting Cav1 and RUNX1 were inserted into the shRNA AAV vector, and the recombinant plasmids were co-transfected into HEK293T cells with PEI. Cells were collected by centrifugation at 4°C, 800 g for 10 minutes, 72 hours post-transfection. After resuspension in lysis buffer (150 mM NaCl and 20 mM Tris, pH 8.0) by vortexing, the cell pellet was incubated with a concentration reagent overnight at 4°C. Following quantification of the AAV vector concentration, the viral stock was mixed in HN buffer (containing 50 mM HEPES, pH 7.4, and 0.15 M NaCl) for injection. The shRNA sequences used in this study are as follows: shRNA (Cav1): 5'-GCTTCCTGATTGAGATTCAGT-3', shRNA ( RUNX1 ): 5'-CACCTACCATAGAGCCATCAA-3'. To knock down Cav1 or RUNX1 in mouse skin, AAV9 adenovirus carrying shCav1 or shRUNX1 was injected intradermally two weeks prior to establishing the wound model. Control groups received an equal amount of AAV9 vehicle containing a negative control sequence (AAV9-shNC). Scanning Electron Microscopy (SEM) Electron microscope samples were processed and examined using an electron microscope at the Laboratory of our institute. Cells cultured on scaffolds and glass slides were fixed with 2.5% glutaraldehyde, followed by ethanol gradient dehydration and air drying. The cell samples were then sputter-coated with platinum and observed under a scanning electron microscope (SEM, HITACHI SU8010, Japan). Transmission Electron Microscopy (TEM) Fibroblasts and NIH-3T3 cells were collected and resuspended in a 2.5% glutaraldehyde fixative solution at 4°C overnight, followed by incubation with 1% osmic acid for 2 hours at room temperature. After gradient dehydration with ethanol and air drying, the cell samples were sputter-coated with platinum and examined under a scanning electron microscope (SEM, HITACHI SU8010, Japan). The number of caveolae was analyzed using NIH ImageJ software (National Institutes of Health, Bethesda, MD) Western blotting The skin tissues or cultured cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (P0013C, Beyotime, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, China) to extract total proteins. Protein concentrations were determined using a BCA protein assay kit (P008, Beyotime Biological, Shanghai, China) following the manufacturer's instructions. Equal amounts of proteins were loaded and separated by 10% or 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp, USA). The membrane was blocked with 5% non-fat dry milk (BD Biosciences) for 1 hour at room temperature. Following incubation with specific primary antibodies overnight at 4°C, the antibodies included: FGFBP3 (24725-1-AP, Proteintech, China), CAV1 (3267S, CST, USA), p-CAV1 (3251S, CST, USA), RUNX1 (ab229482, Abcam, USA), p-RUNX (ab182659, Abcam, USA), PDGFRβ (ab69506, Abcam, USA), CBFβ (ab133600, Abcam, USA), FLNA (ab76289, Abcam, USA), p-ERK1/2 (ab201015, Abcam, USA), ERK1/2 (ab17942, Abcam, USA), p-PI3K (17366S, CST, USA), PI3K (4292S, CST, USA), p-AKT (4056S, CST, USA), AKT (9272S, CST, USA), and GAPDH (ab245355, Abcam, USA), respectively. The dilution ratio for all antibodies was 1:1000. Immunodetection was performed with an EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Haemek, Israel). Protein expression was quantified and analyzed using NIH ImageJ software (National Institutes of Health, Bethesda, MD). GAPDH served as an internal reference. Immunofluorescence (IF) staining For cell samples, cover-slip cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) for 30 minutes, and treated with 0.25% Triton X-100 for 15 minutes at room temperature. Then, the cells were blocked with 5% bovine serum albumin (BSA) for 1 hour. The cells were incubated with primary antibodies: FGFBP3 (24725-1-AP, Proteintech, China), Cav1 (3267S, CST, USA), p- Cav1 (3251S, CST, USA), RUNX1 (ab229482, Abcam, USA), p-RUNX (ab182659, Abcam, USA), PDGFRβ (ab69506, Abcam, USA), CBFβ (ab133600, Abcam, USA), and FLNA (ab76289, Abcam, USA) at 4°C overnight. The dilution ratio for all antibodies was 1:200. After staining, they were cultured with corresponding secondary antibodies coupled with Alexa Fluor 488 or Alexa Fluor 596 and incubated at 37°C for 1 hour. The nuclei were stained with DAPI for 5 minutes. Finally, the analysis was performed using a confocal microscope (Nikon C2, Japan). Image analysis was conducted using NIH ImageJ software (National Institutes of Health, Bethesda, MD). For skin tissue samples, immunofluorescent staining was performed on frozen sections of skin tissue. Briefly, the sections were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature. They were then incubated with primary antibodies: FGFBP3 (24725-1-AP, Proteintech, China), Cav1 (3267S, CST, USA), RUNX1 (ab229482, Abcam, USA), PDGFRa (3174S, CST, USA), α-SMA (19245S, CST, USA), CD31 (ab222783, Abcam, USA) overnight at 4°C. Concurrently, IgG isotype control antibodies were used as negative controls to confirm the specificity of the primary antibodies. Subsequently, the slides were washed three times with PBS and incubated with the appropriate fluorescently conjugated secondary antibodies coupled with 488 or Alexa Fluor 596 for 30 minutes at 37°C. The nuclei of tissue sections were stained with DAPI containing an anti-fluorescence quencher, and images were captured using a Nikon confocal laser scanning microscope (Nikon C2, Japan). Image analysis was conducted using NIH ImageJ software (National Institutes of Health, Bethesda, MD). Imaging of total endocytosis. Primary fibroblasts and NIH-3T3 cells were cultured on plates or scaffolds for a duration of 24 hours. After aspirating the culture medium, the cells were rinsed with PBS. Thereafter, 1 mL of 2 μM FM 1-43 dye (T35356, ThermoFisher, USA) was added to each well of a 24-well plate, and the cells were incubated in a CO2 incubator at 37°C for 20 minutes. Post-incubation, the dye solution was aspirated, and the cells were further incubated with 1 mL of PBS for an additional 20 minutes. The samples were subsequently fixed and counterstained with Hoechst. Images were acquired using a Nikon confocal laser scanning microscope (Nikon C2, Japan). Image analysis was executed using NIH ImageJ software (National Institutes of Health, Bethesda, MD). The analysis entailed quantifying FM 1-43 dye puncta in each focal plane and determining the total intensity of the puncta per cell. RNA sequencing To assess differences in gene expression, total RNA was extracted from NIH-3T3 cells cultured on plates or scaffolds using TRIzol reagent (catalog #15596026, Invitrogen, Carlsbad, CA, USA). RNA purity and quantification were determined using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Thereafter, cDNA libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit following the manufacturer's protocol. Transcriptome sequencing was conducted on the Illumina platform (Illumina NovaSeq 6000, China), yielding 150 bp paired-end reads. Raw sequencing reads for each sample were cleaned to remove low-quality reads and adapters using fastp (version 0.22.0). The clean reads were aligned to the reference genome using HISAT2. The FPKM values for each gene were calculated, and the read counts for each gene were obtained using HTSeq-count. Differential expression analysis was performed using DESeq2. A Q value 2 or fold change < 0.5 were set as the criteria for significantly differentially expressed genes (DEGs). The Volcano Plot of differential expression genes was generated to visualize the expression of up-regulated or down-regulated DEGs using the R package ggradar. The transcriptome sequencing and analysis were carried out by OE Biotech Co., Ltd. (Shanghai, China). Sample preparation for proteomics and phosphoproteomics Protein extraction The samples were subjected to three rounds of sonication while on ice using a high-intensity ultrasonic processor (Scientz) in lysis buffer containing 8M urea, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail. Following centrifugation at 12,000 × g for 10 minutes at 4°C, the remaining debris was pelleted and removed. The supernatant was then collected, and protein concentration was measured using a BCA protein assay kit according to the manufacturer's instructions. Trypsin digestion To facilitate digestion, the protein solution was initially treated with 5 mM dithiothreitol (DTT, Sigma-Aldrich) for 30 minutes at 56°C to reduce disulfide bonds, followed by alkylation with 11 mM iodoacetamide (Sigma-Aldrich) for 15 minutes at room temperature in the dark. The protein sample was then diluted by adding 100 mM TEAB (Sigma-Aldrich) to reduce the urea concentration to less than 2 M. Subsequently, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the primary overnight digestion, followed by a secondary 4-hour digestion at a 1:100 trypsin-to-protein mass ratio. Finally, the peptides were purified using a C18 SPE column (Phenomenex). Phosphopeptides enrichment The peptide mixtures were first incubated with IMAC microspheres in a loading buffer consisting of 50% acetonitrile/0.5% acetic acid, with gentle agitation. Non-specifically adsorbed peptides were then removed by washing the IMAC microspheres with 50% acetonitrile/0.5% acetic acid followed by 30% acetonitrile/0.1% trifluoroacetic acid. Afterward, an elution buffer containing 10% NH4OH was added to elute the enriched phosphopeptides, which were collected with further agitation. The supernatant containing the phosphopeptides was then collected and subjected to lyophilization for subsequent LC-MS/MS analysis. Liquid chromatography-mass spectrometry analysis Peptides were resuspended in liquid chromatography mobile phase A and separated using the Nano-Elute Ultra Performance Liquid Chromatography (UPLC) system. Mobile phase A consists of 0.1% formic acid and 2% acetonitrile in water; mobile phase B consists of 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: from 0 to 70 minutes, 6% to 24% B; from 70 to 144 minutes, 24% to 35% B; from 144 to 147 minutes, 35% to 80% B; and from 147 to 150 minutes, holding at 80% B, with a flow rate of 450 nL/min. The peptides were resolved by the UPLC system, introduced into a capillary ion source, ionized, and analyzed by TOF Pro mass spectrometry. The ion source voltage was set to 1.75 kV, and both the peptide precursor ions and their subsequent fragments were detected and analyzed using a high-resolution time-of-flight (TOF) mass spectrometer. The secondary mass spectrum scan range was set from 100 to 1700 m/z. Data acquisition was performed in Parallel Cumulative Serial Fragmentation (PASEF) mode. After 10 cycles of PASEF, a primary mass spectrum was acquired to collect secondary spectra with precursor ion charge states ranging from 0 to 5. The dynamic exclusion time for tandem mass spectrometry was set to 30 seconds to prevent the repetitive scanning of precursor ions. Phosphoproteomic analysis The MS/MS data acquired were processed using the MaxQuant search engine (version 1.6.15.0). Tandem mass spectra were aligned against the Mus_musculus_10090_SP_20230103.fasta database (containing 17,132 entries), as well as a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, permitting up to two missed cleavages. The minimum amino acid length was set to seven. The precursor ion mass tolerance was set to 20 ppm for the initial search and 4.5 ppm for the main search, while the fragment ion mass tolerance was maintained at 20 ppm. Carbamidomethylation on Cys was designated as a fixed modification, and acetylation at the protein N-terminus and oxidation on Met were designated as variable modifications. The false discovery rate (FDR) was controlled to be less than 1%. Protein quantities were calculated and normalized using the median-centering method. Genes with missing values in at least 50% of the samples were excluded, and the remaining missing values were imputed using the K-Nearest Neighbor (KNN) method with the R package impute88 (version 1.70.0). Differentially expressed proteins were identified using the limma package (version 3.54.2) with a threshold of log2FoldChange >0.3 (or <−0.3) and a P value < 0.05. Gene set enrichment analysis (GSEA) was performed using the R package cluster Profiler. Further details regarding the differential analysis of quantitative proteomics data are provided in Supplementary Data 2. Animal Studies Male wild-type C57BL/6J mice, aged 6 to 8 weeks, were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All mice were maintained under specific-pathogen-free conditions, with ad libitum access to chow and water, and were housed on a 12-hour light-dark cycle. Prior to the experiments, the mice were acclimated for at least one week in the animal facility. All animal procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Animal Care and Use Committee of our institute. Following general inhalation anesthesia using halothane, the dorsal hair was shaved from the mice. Skin wounds were created with a 6-mm skin punch. The mice were then randomly assigned to one of four groups, with each group receiving different experimental interventions: the control group received a PBS solution, while the experimental groups were treated with scaffolds of varying pore structures. At four, seven and ten days post-wounding, the healing tissues were harvested from the mice for further analysis. After a two-week period, the mice were humanely euthanized by cervical dislocation under general inhalation anesthesia with halothane. Histology Portions of the skin samples were fixed with 4% paraformaldehyde for 24 hours and then embedded in paraffin for histomorphological analysis. The remaining skin tissue was stored at -80°C for subsequent molecular studies. Tissue sections, 4 micrometers thick, were prepared and stained with hematoxylin and eosin (H&E) and Masson's trichrome. The tissue sections underwent dehydration, rehydration, and staining following the manufacturer's instructions for the H&E staining kit (Solarbio, G1120) and Masson's trichrome staining kit (Solarbio, G1340). All images of the stained sections were acquired using digital scanners (3DHISTECH Pannoramic 250 FLASH, Hungary). For Oil Red O staining, 10-micrometer-thick frozen tissue sections were prepared. These sections were fixed with 4% paraformaldehyde for 30 minutes. Post-fixation, the samples were washed three times with PBS at room temperature. The Oil Red O working solution was prepared by mixing the Oil Red O stock solution (Sigma, O1391) with double-distilled H2O in a 3:2 ratio. Following a 20-minute staining period, the samples were washed twice with 60% isopropanol, counterstained with hematoxylin, mounted with glycerol gelatin (Solarbio, S2150), and imaged using digital scanners (3DHISTECH Pannoramic 250 FLASH, Hungary). Immunohistochemistry (IHC) analysis IHC was employed to qualitatively evaluate the expression of phenotypic markers. Paraffin-embedded renal tissue sections (4 μm) were mounted on adhesive microscope slides for analysis. The sections were deparaffinized and rehydrated using xylene and a series of graded alcohol concentrations. They were then treated with an enhanced endogenous peroxidase blocking buffer (P0100B, Beyotime, China) for 30 minutes to quench endogenous peroxidase activity. Antigen retrieval was achieved by microwaving the sections in citrate-EDTA solution (P0086, Beyotime, China) for 20 minutes. After cooling to room temperature, the sections were blocked with 5% bovine serum albumin (A1933, Sigma, Missouri, USA) for 30 minutes. The sections were incubated overnight at 4°C with primary antibodies targeting FGFBP3 (Proteintech, 24725-1-AP, China) and p-RUNX (ab182659, Abcam, USA). After rewarming and washing, the sections were incubated with appropriate secondary antibodies for 1 hour at 37°C. The signals were then visualized using 3,3'-diaminobenzidine (DAB, LI-9018, ZSGB-Bio) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, USA). Statistical analysis All experiments were conducted three times, and the data are presented as the mean ± standard deviation (SD). The experimental data were analyzed using GraphPad Prism software (version 8.02, USA). Statistical comparisons between two groups were made using a two-tailed Student's t-test, while one-way or two-way ANOVA with Tukey's post-hoc test was employed for multiple group comparisons (> 2 groups). Differences between groups were considered significant at P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***), and were considered highly statistically significant at P < 0.0001 (****). Results Nanopores affect tissue repair and regeneration The nanoporous scaffolds, constructed from polystyrene a material known for its resistance to rapid degradation or deformation effectively leverage the advantages of nanopores for tissue regeneration in this study. The nanopores in these scaffolds are comparable to those found in artificial dermis used clinically for deep skin wounds (Extended Data Fig. 2). We employed three types of nanoporous scaffolds to evaluate their impact on 6 mm full-thickness dorsal skin wounds in mice. All wounds were covered with the respective scaffolds, while control wounds were treated with saline (Fig. 1a). Macroscopic observation of wound closure in mice showed a significant reduction in wound size at 7, 10, and 14 days after treatment. Nanopore #3 group exhibited the most rapid decrease in wound size, followed by Nanopore #2 group, while Nanopore #1 and control groups showed the slowest reduction (Fig. 1b, c). In line with this, H&E staining revealed substantial increases in wound closure rates, ranging from 82-90% on day 14 in Nanopore #3 group, compared to the control group, which had closure rates of 53-69%. Furthermore, re-epithelialization in the Nanopore #3 group at day 14 covered 82-91% of the wound area, while Nanopore #2, Nanopore #1, and control groups covered 75-83%, 66-73%, and 53-69%, respectively (Fig.1d). Notably, the Nanopore #3 group showed a greater deposition of well-organized collagen fibers and a significant increase in type I collagen levels, as indicated by Masson's trichrome staining (Fig. 1e, f). Immunofluorescence staining for CD31 and α-SMA, markers for vascular endothelial cells and smooth muscle cells, respectively, demonstrated a substantial enhancement in blood vessel formation within the Nanopore #3 group, while the other groups showed only a limited number of positively stained cells (Fig.1g, h). Oil Red O staining also revealed a significant increase in the formation of new sebaceous glands and dermal appendages in the Nanopore #3 group (Extended Data Fig. 3). Collectively, these findings suggest that scaffolds with varying nanopores differentially promote skin wound repair throughout the entire healing process. Fibroblasts sense the scaffolds through caveolae formation We extracted fibroblasts, macrophages, and keratinocytes from mouse skin, which are critical skin tissue cells essential for wound healing, and cultured them on Nanopore #3 scaffold, which showed superior cytocompatibility and facilitated swift wound repair. Notably, primary fibroblasts grew directionally along the scaffold, aligning neatly - a pattern not observed in other cell types (Extended Data Fig. 4a). Furthermore, both staining and SEM analyses indicate that Nanopore #3 promotes the reorganization of fibroblast cytoskeletons into linear configurations (Extended Data Fig. 4b), an effect also observed in NIH-3T3 cells (Extended Data Fig. 5). In line with this, cell counting kit-8 and wound healing assays demonstrate that scaffolds with varying nanostructures differentially impact the proliferation and migration of fibroblasts, with Nanopore #3 exhibiting the most pronounced enhancement (Extended Data Fig. 6). These findings collectively suggest that fibroblasts are the principal cells that sense to nanopore cue, potentially initiating a series of pro-repair effects and playing a pivotal role in managing full-thickness skin injuries. To elucidate the response of fibroblasts to nanopore environments, we undertook an extensive proteomic and phosphoproteomic analysis to identify phosphorylated proteins that exhibit differential expression between fibroblasts cultured on conventional plates and those cultured on Nanopore #3. Principal component analysis revealed that fibroblasts cultured on identical substrates exhibited a high degree of similarity, as evidenced by their tight clustering (Fig. 2a). Our analysis of the top 8 enriched KEGG pathways has uncovered a marked upregulation of caveolar endocytosis signaling pathways (Fig. 2b). Cav1 can be phosphorylated at a conserved tyrosine residue, specifically at position 14 ( Y14 p-Cav1) which is a differentially expressed phosphorylated protein sites between fibroblasts cultured on plates and Nanopore #3 (Fig. 2c), while total Cav1 protein levels were not significantly increased as shown in proteomic analysis. Previous research has established that Y14 p-Cav1 plays a critical role in modulating caveolar endocytosis. 16,17 Similarly, the levels of Y14 p-Cav1were found to be increased in primary fibroblasts and NIH-3T3 cells when cultured on Nanopore #3 (Fig. 2d, e). Additionally, a gradient increase in Y14 p-Cav1 content was observed across different substrates, including plates, Nanopore #1, Nanopore #2, and Nanopore #3 (Extended Data Fig. 7). This gradient increase in Y14 p-Cav1 correlates with the progressive changes in cell proliferation and migration, suggesting a link between substrate topography, Cav1 phosphorylation, and cellular behavior. Consistently, the expression of Y14 p-Cav1 was significantly enhanced in cells cultured on artificial dermis (Extended Data Fig. 8). Moreover, immunofluorescence analysis was employed to visualize both the localization and expression of Y14 p- Cav1 in fibroblasts. Our findings indicated that Y14 p- Cav1 was notably increased in the cytoplasm and membrane of cells cultured on Nanopore #3, whereas in cells cultured on traditional plates, Y14 p- Cav1was primarily localized to the cell membrane (Fig. 2f-h and Extended Data Fig. 9a-c). These results confirm that Nanopore #3 scaffold significantly enhance the phosphorylation level of Cav1 in fibroblasts and alter the distribution of Y14 p- Cav1. To investigate whether the effect of Nanopore #3 on fibroblast caveolae formation and endocytosis is mediated by Cav1. Utilizing transmission electron microscopy, we observed a marked increase in the number of caveolae in both primary fibroblasts and NIH-3T3 cells when cultured on Nanopore #3. Furthermore, the decrease in caveolae numbers following Cav1 knockdown implies that Cav1 is crucial for the formation of caveolae, underscoring its central role in the biogenesis of these membrane invaginations (Fig. 2i and Extended Data Fig. 9d). To ascertain the role of Cav1 in caveolae-mediated endocytosis, primary fibroblasts and NIH-3T3 cells were treated with the membrane-impermeable FM 1-43 dye for a duration of 15 minutes, after which the dye was washed away. Subsequent quantitative analysis of confocal fluorescence microscopy images revealed that cells cultured on Nanopore #3 exhibited a significantly higher number of FM 1-43-positive endocytic vesicles compared to those on traditional plate, while fibroblasts with Cav1 knockdown displayed a reduction in the number of endocytosed vesicles. Furthermore, fibroblasts treated with 2 μM PP2, a Src family kinase inhibitor known to inhibit Cav1 phosphorylation, showed a reduction of endocytosed vesicles (Fig. 2j and Extended Data Fig. 9e). This observation suggests that Y14 p- Cav1 is specifically required for caveolar endocytosis in fibroblasts cultured on Nanopore #3. In light of our observations that fibroblasts respond to Nanopore #3 by phosphorylating Cav1 and engaging in caveolar endocytosis, we next aimed to investigate whether the knockdown of Cav1 in fibroblasts would impact the efficacy of the scaffold with Nanopore #3 in facilitating wound healing and tissue regeneration. Initially, we evaluated the effect of Cav1 knockdown on fibroblast proliferation in vitro . We found that the migration of fibroblasts with Cav1 knockdown was significantly compromised compared to those treated with shNC (Extended Data Fig. 10). In vivo , following the intradermal injection of adeno-associated virus (AAV)-shCav1 for two weeks (Fig. 3a), skin wound models were established and subsequently treated with Nanopore #3 scaffolds for a further two-week period. The administration of AAV-shCav1 significantly impeded the pro-wound healing effects induced by Nanopore #3 scaffolds. This was evidenced by a larger skin wound size at days 1, 3, 7, 10, and 14 following wounding, as well as reduced rates of skin wound closure and re-epithelialization in AAV-shCav1-treated mice compared to those treated with AAV-shNC (Fig. 3b-e). Furthermore, the efficiency of Cav1 knockdown was confirmed by the significantly lower levels of Cav1 protein in the skin samples of mice treated with AAV-shCav1 and Nanopore #3 scaffolds, as compared to those treated with AAV-shNC (Fig. 3f and g). Additionally, the Nanopore #3 scaffold-induced increase in neovascular markers, specifically α-SMA and CD31-positive cells, was significantly attenuated in mice treated with AAV-shCav1 (Fig. 3h). To further substantiate the role of Cav1 protein in the pro-wound healing effects mediated by Nanopore #3, we examined the development of new sebaceous glands and dermal appendages at the wound sites. Oil Red O staining demonstrated a marked reduction in the formation of new sebaceous glands and dermal appendages in mice treated with AAV-shCav1 (Fig. 3i). Together, our data indicate that the suppression of Cav1 in skin fibroblasts significantly impairs the wound healing process. Caveolae-Mediated Endocytic Internalization of PDGFRβ Caveolae interact with a variety of signaling molecules to modulate intracellular signaling cascades, notably those involving growth factor receptors 18,19 , the interleukin-1 beta receptor 20 , and the vasoactive intestinal peptide binding VPAC2 receptor 21 . To elucidate the proteins that interact with Cav1, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Fig. 4a). Notably, platelet-derived growth factor receptor β (PDGFRβ), a receptor tyrosine kinase, was identified as an interacting protein through LC-MS/MS analysis (Fig. 4b and Supplementary Table 5). Immunoprecipitation assays revealed a robust interaction between Cav1 and PDGFRβ in primary fibroblasts (Fig. 4c). Similarly, Y14 p- Cav1 was found to interact with PDGFRβ in these cells (Extended Data Fig. 11). Immunofluorescence assays revealed a significant increase in PDGFRβ levels within the cytoplasm of fibroblasts cultured on the Nanopore #3 scaffold, accompanied by a notable enhancement in the co-localization of Cav1 and PDGFRβ. Knockdown of Cav1 led to a reduction in the internalization of PDGFRβ into the cytoplasm of fibroblasts cultured on the Nanopore #3 scaffold (Fig. 4d, e and Extended Data Fig.12a). Immunofluorescence assays were utilized to visualize the localization and expression patterns of PDGFRβ. Our results indicated a significant increase in the intensity of internalized PDGFRβ within the cytoplasm. In primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffold, activated PDGFRβ was predominantly localized to the cell membrane surface in those with Cav1 knockdown, whereas in control cells, it was distributed throughout the cytoplasm (Fig. 4f-h and Extended Data Fig. 12b-d). These findings suggest that nanopore structures can enhance the internalization of PDGFRβ into the cytoplasm and that a portion of PDGFRβ internalization occurs through Cav1-mediated endocytosis. As a member of the receptor tyrosine kinase family, the internalization of PDGFRβ signifies its activation, which subsequently modulates downstream signaling pathways to regulate a range of cellular activities 22,23 . Phosphoproteomic analyses utilizing KEGG and Wikipathways databases reveal that proteins involved in the PI3K-AKT and ERK signaling pathways are significantly upregulated (Fig. 4i). Immunoblotting analysis demonstrated a significant increase in the phosphorylation levels of PI3K, AKT, and ERK1/2 proteins, which are key components of the PI3K-AKT and ERK signaling pathways, in primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffold (Fig. 4j and Extended Data Fig. 13). Importantly, Cav1 knockdown cells cultured on the Nanopore #3 scaffold exhibited a significant reduction in the phosphorylation levels of PI3K, AKT, and ERK1/2 proteins, which are integral to the PI3K-AKT and ERK pathways (Extended Data Fig. 14). This finding suggests that caveolae-mediated endocytosis is essential for Nanopore #3 scaffold's role in accelerating wound healing by facilitating PDGFRβ internalization and the subsequent activation of the PI3K-AKT and ERK signaling pathways. Caveolae-Mediated Endocytosis Drives Wound Healing through RUNX/FGFBP3 To further elucidate the roles of fibroblasts subsequent to caveolae formation in Nanopore #3 scaffold, RNA sequencing was performed on NIH-3T3 cells cultured on both standard plates and Nanopore #3 scaffold. The RNA sequencing analysis revealed a total of 581 differentially expressed genes, encompassing several genes known to promote regeneration, such as FGFBP3, FGF2, CEMIP, PTX3, and additional related genes (Fig. 5a, b and Supplementary Table 6). Recognizing that FGFBP3 is a secreted protein that binds FGF2, preventing its heparin interaction and facilitating FGFR signaling in wound healing 24 . We initially assessed the expression of FGFBP3 in cells cultured on the Nanopore #3 scaffold. Our findings revealed that both mRNA and protein levels of FGFBP3 were significantly elevated in primary fibroblasts and NIH-3T3 cells cultured on this scaffold, as evidenced by ELISA, immunoblotting, and immunofluorescence assays (Fig. 5c-f). In line with these results, we also noted a significant enhancement in FGFBP3 expression levels in artificial dermis (Extended Data Fig. 15). Furthermore, increased FGFBP3 expression was observed in the concentrated cell supernatants obtained from primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffold, as determined by ELISA analysis (Extended Data Fig. 16a). In addition, tubule formation in HUVECs treated with concentrated supernatant from primary fibroblasts cultured on various nanopore structure scaffolds was differentially enhanced (Extended Data Fig. 16b). However, Cav1 knockdown significantly impaired the angiogenesis ability of HUVECs in vitro (Extended Data Fig. 16c). This suggests that the scaffold may promote wound healing and regeneration through Cav1 regulating the secreted protein FGFBP3. Furthermore, in vivo studies demonstrated that the levels of skin FGFBP3 at the wound sites of mice treated with the Nanopore #3 scaffold were significantly higher on days 7, 10, and 14 post-wounding, as determined by immunoblotting analysis (Fig. 5g, h). Immunohistochemical analysis of skin wound tissue on day 14 indicated that the Nanopore #3 scaffold markedly increased the expression of FGFBP3 protein in the wound tissue (Fig. 5i). Furthermore, the formation of tubules in HUVECs treated with concentrated supernatant from primary fibroblasts cultured on Nanopore #3 scaffold was significantly reduced upon FGFBP3 knockdown (Fig. 5j and Extended Data Fig. 17), which affects angiogenesis. The data collectively suggest that the increased secretion of FGFBP3 could be a potential mechanism through which the nanoporous scaffold enhances wound regeneration and repair. We further explored the mechanism through which caveolae-mediated endocytosis enhances FGFBP3 expression. Using DNA pull-down assays in conjunction with LC-MS/MS analysis, we identified 2216 proteins capable of binding to the DNA sequence of FGFBP3 in mouse primary fibroblasts (Supplementary Table 7). Much research highlights the critical role of transcription factors (TFs) as intermediaries between cellular signaling pathways and gene regulation 25 . With this understanding, we utilized the JASPAR online TF prediction tool for bioinformatics analysis to identify potential TFs that could bind to the FGFBP3 promoter. The analysis identified three candidate TFs: RUNX1, MECOM, and CREB1 (Fig. 6a). To confirm these results, we inserted the FGFBP3 promoter sequence into the pGL3 reporter vector and co-transfected it with expression plasmids for RUNX1, MECOM, and CREB1 into 293T cells. Dual-luciferase reporter assays revealed that co-transfection with the RUNX1 overexpression plasmid significantly enhanced luciferase activity driven by the pGL3-FGFBP3 promoter (Extended Data Fig. 18a). Furthermore, the RUNX1 protein was identified in the protein complex precipitated by the FGFBP3 promoter, as detected by immunoblotting (Extended Data Fig. 18b). In primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffolds, both RUNX1 mRNA and protein levels were confirmed to increase, with a significant elevation in phosphorylated RUNX1, as assessed by immunoblotting (Fig. 6b, c). Moreover, p-RUNX1 immunofluorescence staining revealed that the levels of activated RUNX1 that translocated to the nucleus were elevated in cells treated with Nanopore #3 scaffold (Fig. 6d and Extended Data Fig. 19). To determine whether RUNX1 binds directly to FGFBP3, JASPAR predicted four potential RUNX1 binding sites within the FGFBP3 promoter region (Fig. 6e, Supplementary Table 8). Following this, we cloned partial promoter sequences of FGFBP3, containing sites #1 through #4, into pGL3 reporter plasmids (Fig. 6f). These plasmids were then transfected into 293T cells, both with and without the RUNX1 overexpression plasmid. Dual-luciferase reporter assays indicated that co-transfection with the RUNX1 overexpression plasmid and the pGL3-FGFBP3 site #2 promoter significantly increased luciferase activity, whereas co-transfection with sites #1, #3, and #4 along with the RUNX1 overexpression plasmid had no significant effect on luciferase activity in 293T cells (Fig. 6g). Concentrating on RUNX1 binding site #2, ChIP assays confirmed substantial RUNX1-binding activity at this site within the FGFBP3 promoter (Fig. 6h). We then evaluated the impact of RUNX1 silencing on wound repair and regeneration in mice treated with Nanopore #3 scaffolds. FGFBP3 mRNA and protein levels were found to be downregulated in RUNX1 knockdown primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffolds (Fig 6i, j and Extended Data Fig. 20). Furthermore, RUNX1 knockdown significantly impaired the in vitro angiogenic capacity, as indicated by reduced tubule formation in HUVECs treated with concentrated supernatant from primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffolds (Extended Data Fig. 21). In vivo , the levels of phosphorylated RUNX1 and its downstream target protein FGFBP3 in the skin of AAV-shCav1-treated mice with the scaffold were significantly decreased compared to those in AAV-shNC-treated mice, as determined by immunoblot and immunohistochemical analysis (Extended Data Fig. 22). In vitro , the scaffold-induced elevation of phosphorylated RUNX1 and FGFBP3 in fibroblasts with Cav1 knockdown was significantly attenuated, as confirmed by both immunoblot and immunofluorescence analyses (Extended Data Fig. 23). Moreover, after two weeks of intradermal injection with adeno-associated virus (AAV)-shRUNX1 (Fig. 6k), skin wound models were created. In the mice treated with AAV-shRUNX1 and equipped with the Nanopore #3 scaffold, the levels of RUNX1 and FGFBP3 at the wound site were markedly reduced compared to those in mice treated with AAV-shNC (Fig. 6l and Extended Data Fig. 24a-d). Furthermore, the delivery of AAV-shRUNX1 notably suppressed the pathological progression associated with scaffold-induced pro-wound healing. This was evidenced by a larger skin wound size observed on days 1, 3, 7, 10, and 14 following injuries, as well as a decrease in skin wound closure and re-epithelialization rates in comparison to mice treated with AAV-shNC (Fig. 6m-p). In addition, the Nanopore #3 scaffold-induced increase in neovascular marker proteins and collagen fiber content, were substantially diminished in mice treated with AAV-shRUNX1 (Extended Data Fig. 24e, f). Collectively, these findings suggest that the fibroblast caveolae in the skin mediate tissue repair through the RUNX1-FGFBP3 pathway. Building on our previous observations that caveolae formation may enhance Nanopore #3-induced wound healing by internalizing PDGFRβ, thereby activating the PI3K-AKT and ERK signaling pathways, we further explored the regulatory impact of caveolae-mediated PDGFRβ internalization on RUNX1, which in turn increases the secretion of FGFBP3. This could represent a potential mechanism for promoting wound regeneration and repair. We then assessed the effect of PDGFRβ silencing on scaffold-induced FGFBP3 increases in fibroblasts. As anticipated, immunoblot, qPCR, and immunofluorescence analyses showed that scaffold-induced increases in FGFBP3 protein and mRNA levels were significantly downregulated in PDGFRβ knockdown fibroblasts (Extended Data Fig. 25a-c). Consistently, PDGFRβ knockdown also notably reduced FGFBP3 levels in the cell supernatant (Extended Data Fig. 25d). Therefore, we hypothesize that PDGFRβ, internalized into the cytoplasm with Cav1, activates downstream PI3K-AKT and ERK signaling pathways, which in turn promote RUNX1 phosphorylation and FGFBP3 transcription. To further confirm the hypothesis, we treated fibroblasts cultured on Nanopore #3 scaffold with the PI3K inhibitor Thioridazine (SIGMA, USA, #T905, 2 μM, 24 h) and the ERK inhibitor FR180204 (SIGMA, USA, #328010, 2 μM, 24 h). Immunoblot analysis revealed that the expression of p-RUNX1 and FGFBP3 proteins upregulated by the scaffold was markedly reduced in fibroblasts treated with ERK or PI3K inhibitors (Extended Data Fig. 26). Conversely, RUNX1 knockdown did not significantly affect the PI3K-AKT and ERK pathways in fibroblasts cultured on the scaffold (Extended Data Fig. 27). Collectively, these data indicate that PDGFRβ internalization with caveolae formation activates PI3K-AKT and ERK signaling, thereby promoting RUNX1 phosphorylation and FGFBP3 transcription. CAV1 Mediated Release of CBFβ from FLNA Enhances RUNX1 Activity to Promote FGFBP3 Transcription Post-Caveolae Endocytosis Interestingly, caveolae interact with the dynamic cytoskeleton, which is crucial for sensing and converting mechanical forces into cellular signals 26,27 . Previous studies have shown that FLNA engages with Cav1 via the hinge region between the 23rd and 24th homologous repeats of 96 amino acids each repeats 28,29 . Results from double immunofluorescence staining demonstrated substantial co-localization of Cav1 and FLNA in fibroblasts cultured on scaffolds (Fig. 7a and Extended Data Fig. 29a). CBFβ, a co-transcription factor that binds to RUNX1, is composed of two domains with distinct structural and functional characteristics. The first domain is a loosely structured regulatory region that is capable of interacting with a variety of molecules 30 . For example, when the 23-24 repeat hinge region of FLNA binds to this regulatory domain, it sequesters CBFβ in the cytoplasm, thereby preventing its involvement in the transcription factor complex. The second domain is an execution domain with a rigid structure that specifically interacts with the RUNX1 protein. Once CBFβ forms a complex with RUNX1, it acts as a transcription factor within the cell nucleus 31,32 . CBFβ and CAV1 compete for the same binding region on FLNA. Our double immunofluorescence data show that the nuclear localization of CBFβ bound to RUNX1 increased in fibroblasts cultured on the scaffold, while the cytoplasmic retention of CBFβ bound to FLNA was markedly decreased, in contrast to fibroblasts cultured on standard plates (Fig. 7b, c and Extended Data Fig. 28b, c). Moreover, immunoprecipitation assays corroborated the findings from the immunofluorescence double staining (Fig. 7d, e). Notably, these assays also indicated a stronger interaction between Y14 p-Cav1 and FLNA in fibroblasts cultured on scaffolds compared to control cells (Extended Data Fig. 29). This suggests that both Cav1 and Y14 p-Cav1 can bind to FLNA, thereby preventing it from binding to CBFβ. As a result, CBFβ is released to enter the nucleus and function as a transcriptional cofactor, binding to RUNX1. Understanding that CBFβ is a co-transcription factor that interacts with RUNX1, we explored its role in regulating FGFBP3, a target gene of the RUNX1 transcription factor. Immunoblot analysis indicated a significant increase in the nuclear localization of both RUNX1 and CBFβ proteins in fibroblasts cultured on Nanopore #3 scaffold, with a corresponding decrease in their cytoplasmic localization (Fig. 7f, g). Furthermore, immunoblot, qRT-PCR, and immunofluorescence analysis revealed that the scaffold-induced elevation of FGFBP3 mRNA and protein levels was reduced in CBFβ knockdown fibroblasts, although still slightly higher compared to siNC cells (Fig. 7h-j and Extended Data Fig. 28d-f). Together, these results suggest that the scaffold enhances the interaction between Cav1 or Y14 p-Cav1 and FLNA, thereby preventing FLNA from binding to CBFβ. Consequently, CBFβ is released to translocate into the nucleus, where it acts as a transcriptional cofactor with RUNX1 to facilitate the production of the FGFBP3 protein (Fig. 7k). Discussion A comprehensive understanding of the biophysical and biochemical properties of scaffolds is crucial for optimizing the use of endogenous cells and promoting cellular reprogramming in tissue engineering. The precise integration of these cues, customized to meet the specific needs of various target tissues, could pave the way for new advances in biomaterial-based therapies. In our study, we examined the effects of scaffolds with varying nanopores on wound healing and cell fate. Our findings show that scaffolds with distinct nanopores differentially enhance skin wound healing. Fibroblasts, which are the primary cells that sense and respond to scaffolds, play a pivotal role in addressing full-thickness skin defects, potentially initiating the repair process and leading to the formation of new tissue structures. The formation of caveolae and the initiation of endocytosis on the cell surface during fibroblast deformation on the scaffold are among the cellular responses to the nanopore features. During this biological process, caveolae can bud off from the plasma membrane to form endocytic vesicles. These vesicles, in concert with Cav1 or other lipid-interacting proteins, are internalized into the cytoplasm, thereby triggering a series of signaling pathways 33 . Additionally, Cav1 within the endocytic vesicles can interact with cytoplasmic proteins, thereby modulating key cellular processes 34 . Our findings indicate that the formation of caveolae in fibroblasts represents an initial step in the wound healing response to the nanopores n scaffolds. Tissue-resident fibroblasts, a subset of mesenchymal cells, display significant plasticity, enabling them to adjust their phenotype in response to environmental signals. For instance, during the wound healing process, myofibroblasts are capable of transdifferentiating into adipocytes, demonstrating their adaptability within the microenvironment 35 . These cells are crucial for maintaining tissue homeostasis and are involved in physiological processes such as wound healing, tissue repair, and fibrosis. The biophysical cues of biomaterials can significantly impact fibroblast behavior, encompassing epigenetic and metabolic reprogramming 36 , 37 . Our research demonstrates that the nanopores of scaffolds regulate fibroblast biology through transcriptomic, proteomic, and phosphoproteomic analyses, clarifying how fibroblasts detect and react to environmental stimuli. The interaction between fibroblasts and nanopores may trigger the reparative process, with scaffolds guiding cellular functions that are vital for tissue regeneration, which is essential for harnessing the body's inherent regenerative capacity 38 , 39 . Previous research has demonstrated that the nanopores within scaffolds significantly influence tissue regeneration, with pore size and architecture being critical factors in cellular adhesion, proliferation, and matrix deposition 40 . Advancements in this field may yield interventions that can modulate fibroblast activity to promote tissue regeneration without aggravating fibrosis, offering novel therapeutic strategies for diseases marked by abnormal tissue repair. To elucidate the fibroblast sensing nanopores, we conducted comprehensive proteomic and phosphoproteomic analyses. These omics data, in conjunction with the established role of caveolae in sensing external microenvironments and stimuli, prompted us to hypothesize that caveolae formation in fibroblasts is pivotal for nanopore detection. Our study demonstrates that fibroblasts detect nanopores through the phosphorylation of Cav1 and the endocytosis of caveolae. Beyond their role in sensing and responding to mechanical forces, caveolae formation also appears to be a response to nanopores. Caveolae, which are enriched with specific lipids and proteins, form distinct nanodomains that play a crucial role in cellular signaling pathways. These membrane invaginations interact with various signaling molecules, and their dynamics are vital for intracellular signaling processes. Receptor tyrosine kinases, such as PDGFRβ, are a class of cell-surface receptors that sense and transmit environmental signals, activating their inherent enzymatic activity and functioning as enzymes. Upon binding with Cav1, PDGFRβ is internalized into the cytoplasm during caveolae endocytosis, initiating its tyrosine kinase activity, which subsequently activates the phospho-PI3K, phospho-AKT, and phospho-ERK1/2 pathways. PDGFRβ can also interact with Src family kinases, including Lyn and Fyn, further activating its tyrosine kinase activity and leading to the phosphorylation of tyrosine residues on proteins 41 . In the context of nanopore interactions, we have found that the transcription factor RUNX1 is phosphorylated by tyrosine kinase pathways during caveolae endocytosis. The phosphorylated RUNX1 then translocates to the nucleus, where it promotes the transcription of FGFBP3. FGFBP3 can bind to FGF2, preventing its interaction with heparin and likely inhibiting the immobilization of FGF2 on extracellular matrix glycosaminoglycans. This allows for the release and subsequent activation of FGFR signaling. By modulating the activity and bioavailability of FGF2, FGFBP3 influences wound healing processes. This discovery is consistent with studies showing that RUNX1 significantly modulates the regenerative capacity of wound-activated fibroblasts, thereby affecting healing outcomes 42 , 43 . Additionally, in this study, we observed that the expression of Y 14 p-Cav1 is associated with fibroblast proliferation, suggesting that reparative fibroblasts possess an inherent yet adaptable regenerative potential. Another key focus of this research is the examination of the role of Cav1 within the cytoplasm. Cav1, a key component of caveolae, is internalized into the cytoplasm where it interacts with a variety of molecules, thereby participating in the regulation of numerous cellular processes. FLNA, a member of the actin-binding protein family, engages with a broad spectrum of molecules, including transcription factors and co-activators 44 . Studies have demonstrated that Cav1 can interact with Filamin-A in the cytoplasm 45 . Significantly, CBFβ and Cav1 compete for the same binding site on FLNA. Upon binding to FLNA, Cav1 forms a complex that affects several critical cellular processes. One of these processes is the release of CBFβ from FLNA, which then promotes its nuclear translocation. Within the nucleus, CBFβ forms heterodimers with Runx family members (RUNX), thereby enhancing their DNA-binding affinity and transcriptional activity 32 . While CBFβ does not directly interact with DNA, it significantly enhances RUNX1's ability to activate the transcription of FGFBP3. These results emphasize that caveolae, upon sensing the nanostructures of a scaffold, initiate a complex series of cellular events that are crucial for tissue regeneration. In conclusion, fibroblasts have an innate yet adaptable regenerative capacity that is triggered by their recognition of scaffold nanostructures. The formation of caveolae indicates the cell's response to these nanostructures and marks the beginning of reparative processes. This response initiates a series of complex biological events that ultimately lead to tissue regeneration and repair. Consequently, the cellular caveolae response can be harnessed as a key parameter in the design of scaffold nanostructures, potentially improving therapeutic outcomes for patients. Abbreviations BCA, bicinchoninic acid; Cav1, caveolin-1; ChIP, chromatin immunoprecipitation; ECM, extracellular matrix; FBS, fetal bovine serum; FGFBP3, fibroblast growth factor binding protein 3; FLNA, filamin-A; HUVECs, human umbilical vein endothelial cells; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TFs, transcription factors Declarations Ethical approval and consent to participate: The study protocol was granted ethical approval by the Ethics Committee of our institute (Reference Number: 2023-R15, WYYY-AEC-YS-2023-0606). Conflicts of Interest: The authors declare no conflict of interest. Acknowledgements: The present study was financially supported by the National Natural Science Foundation of China (No. 82241031), Science and Technology Bureau (No. Y20240095) and Natural Science Foundation of Science and Technology Bureau (No. 2023J254). Data Availability Statement: The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Authors’ Contributions: Conceptualization: K.Q.S. Methodology and investigation: F.X.G, W.X, W.T.L, M.L.Y, H.B.L, S.F.W, X.Y.N, E.A, H.W.W, and J.W. Data analysis: F.X.G, W.X, W.T.L and K.Q.S. Writing (original draft): F.X.G and K.Q.S. Writing (review and editing): J.X, Z.M.C, and K.Q.S. Funding and supervision: F.X.G and K.Q.S. References Falanga, V. et al. Chronic wounds. Nat Rev Dis Primers 8 , 50, doi:10.1038/s41572-022-00377-3 (2022). Martin, P., Pardo-Pastor, C., Jenkins, R. G. & Rosenblatt, J. Imperfect wound healing sets the stage for chronic diseases. Science 386 , eadp2974, doi:10.1126/science.adp2974 (2024). Freedman, B. R. et al. Breakthrough treatments for accelerated wound healing. Sci Adv 9 , eade7007, doi:10.1126/sciadv.ade7007 (2023). Wang., C. et al. 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Nat Rev Mol Cell Biol 2 , 138-145, doi:10.1038/35052082 (2001). Dhanda, A. S., Vogl, A. W., Ness, F., Innocenti, M. & Guttman, J. A. mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading. mBio 12 , e0293921, doi:10.1128/mBio.02939-21 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary information SupplementaryTable5.listforTyrosinerelatedmembranereceptorLCMS.xlsx Supplementary Table 5 SupplementaryTable6.listforRNAseqDDEPs.xls Supplementary Table 6 SupplementaryTable7.listforDNApulldownJASPAR.xlsx Supplementary Table 7 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5955293","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":416049717,"identity":"0b63ca7e-0344-437a-8282-576ac7e353cc","order_by":0,"name":"Keqing 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06:00:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5955293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5955293/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76634612,"identity":"9039de47-6824-432b-a72a-25d2d15c837d","added_by":"auto","created_at":"2025-02-19 07:16:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4410566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScaffolds with various nanopores affect skin wound repair.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e) Schematic of wound healing studies. \u003cstrong\u003eb\u003c/strong\u003e) Wound healing dynamics of wild-type C57BL/6J mice treated with diverse scaffolds on day 1 to day 14. \u003cstrong\u003ec\u003c/strong\u003e) Representative images of the healing process in wounds treated with PBS, Nanopore #1, Nanopore #2 and Nanopore #3 scaffolds. \u003cstrong\u003ed\u003c/strong\u003e) H\u0026amp;E-stained histologic longitudinal sections of various groups after 14 days, and the wound closure and re-epithelialization rates of control and experimental groups. Scale bars, 0.5 mm; ep, epithelialization; gt, granulation tissue. \u003cstrong\u003ee\u003c/strong\u003e) Masson's trichrome staining of various groups after 14 days. Scale bars, 0.5 mm. \u003cstrong\u003ef\u003c/strong\u003e) Quantitative analysis of collagen volume fraction. \u003cstrong\u003eg\u003c/strong\u003e) Immunofluorescence staining of α-SMA and CD31 for neovascularization, with the vessels indicated by white arrows. Scale bars, 2 mm. \u003cem\u003en \u003c/em\u003e= 6. Data are presented as mean ± SD, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. The scaffolds with distinct nanopores include round (Named as Nanopore #1), 3×-stretched oval (Nanopore #2), and 6×-stretched oval (Nanopore #3).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/66b786176e3be0e0918f7139.png"},{"id":76636090,"identity":"210287b4-a36f-4ed4-9dbe-5a9e576ca58b","added_by":"auto","created_at":"2025-02-19 07:32:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1451911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibroblasts sense nanopores through phosphorylation of Cav1 and formation of caveolae. a\u003c/strong\u003e) Principal component analysis (PCA) in protein phosphorytomics sequencing. \u003cstrong\u003eb\u003c/strong\u003e) The bubble chart displayed different expressive\u003cstrong\u003e \u003c/strong\u003etop 8 enriched KEGG pathways of NIH-3T3 cells. \u003cstrong\u003ec\u003c/strong\u003e) The distinct, highly-expressed phosphorylated genes of NIH-3T3 cells cultured on plate or Nanopore #3 scaffold are shown in the volcano plots. \u003cstrong\u003ed, e\u003c/strong\u003e) \u003csup\u003eY14\u003c/sup\u003ep-Cav1 protein levels in primary fibroblasts and NIH-3T3 cells. \u003cstrong\u003ef\u003c/strong\u003e) Immunofluorescence staining of \u003csup\u003eY14\u003c/sup\u003ep-Cav1 in fibroblasts. Scale bars, 50 μm. \u003cstrong\u003eg\u003c/strong\u003e) Quantitative analysis of the fluorescence intensity across the white line in (f). \u003cstrong\u003eh\u003c/strong\u003e) Quantitative analysis of the fluorescence of \u003csup\u003eY14\u003c/sup\u003ep-Cav1 protein. \u003cstrong\u003ei\u003c/strong\u003e) TEM of Cav1knockdown and control cells. Red arrows highlight caveolar rosettes and caveolae. Scale bars, 0.5 μm. \u003cstrong\u003ej\u003c/strong\u003e) Fluorescence images of endocytosed FM 1- 43 dye in fibroblasts. Scale bars, 50 μm. \u003cem\u003en \u003c/em\u003e= 3. Data are presented as mean ± SD, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/364337e085db1fa2dbd6a350.png"},{"id":76634613,"identity":"e9b39cb9-5ac4-4b1e-beb6-70311e095dae","added_by":"auto","created_at":"2025-02-19 07:16:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3895028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWound healing is disturbed by AAVshCav1 in skin fibroblasts.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e) Flow chart of Cav1 knockdown impact on healing in a skin wound model. \u003cstrong\u003eb\u003c/strong\u003e) Wound healing dynamics of wild-type C57BL/6J mice treated with PBS (control) and Nanopore #3 scaffold in shNC or shCav1 groups on day 1 to day 10. \u003cstrong\u003ec\u003c/strong\u003e) Representative images of the healing process in wounds treated with control and Nanopore #3 scaffold in shNC or shCav1 groups. \u003cstrong\u003ed\u003c/strong\u003e) H\u0026amp;E-stained histologic longitudinal sections of various groups after 14 days. Scale bars, 0.5 mm; ep, epithelialization; gt, granulation tissue. \u003cstrong\u003ee\u003c/strong\u003e) Wound closure and re-epithelialization rates of various groups. \u003cstrong\u003ef\u003c/strong\u003e) Skin FGFBP3 contents in wound tissue determined by immunoblot analysis. \u003cstrong\u003eg\u003c/strong\u003e) Immunofluorescence staining of PDGFRa (green) and Cav1 (red) in various groups. Scale bars, 100 mm. \u003cstrong\u003eh\u003c/strong\u003e) Immunofluorescence staining of α-SMA and CD31 for neovascularization, with the vessels indicated by white arrows. Scale bars, 2 mm. \u003cstrong\u003ei\u003c/strong\u003e) Oil Red O staining of wounds treated with PBS or Nanopore #3 scaffold after 14 days, scale bars, 100 mm, with corresponding wound images, scale bars, 0.5 mm. \u003cstrong\u003ej\u003c/strong\u003e) Quantitative analysis of dermal appendages and Oil Red O positive area. n = 5. Data are presented as mean ± SD, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/1249c1429981472e31d2e462.png"},{"id":76634614,"identity":"90372443-a40f-4d04-a406-7b95e5b210dc","added_by":"auto","created_at":"2025-02-19 07:16:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1813179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNanopore-mediated PDGFRβ internalization and tyrosine kinase pathway activation via Cav1 binding in caveolae endocytosis. a\u003c/strong\u003e) Schematic diagram of the qualitative protein analysis workflow using LC-MS/MS. \u003cstrong\u003eb\u003c/strong\u003e) LC-MS/MS analysis of PDGFRβ interactions with Cav1 peptide segments. \u003cstrong\u003ec\u003c/strong\u003e) Co-immunoprecipitation of endogenous PDGFRβ and Cav1 or endogenous Cav1 and PDGFRβ in primary fibroblasts. \u003cstrong\u003ed\u003c/strong\u003e) Co-localization of endogenous PDGFRβ and Cav1 examined by double immunofluorescence staining. Scale bars, 50 μm. \u003cstrong\u003ee\u003c/strong\u003e) Cav1 and PDGFRβ protein levels in the cell membrane and cytoplasm of fibroblasts. \u003cstrong\u003ef) \u003c/strong\u003eRepresentative immunofluorescence images of \u003csup\u003eY14\u003c/sup\u003ep-Cav1 in fibroblasts. Scale bars, 50 μm. \u003cstrong\u003eg\u003c/strong\u003e) Quantitative analysis of the fluorescence intensity across the white line in (f). \u003cstrong\u003eh\u003c/strong\u003e) Quantitative analysis of the internalized PDGFRβ intensity. \u003cstrong\u003ei\u003c/strong\u003e) Bubble chart displaying the top 8 enriched Wikipathways in fibroblasts. \u003cstrong\u003ej\u003c/strong\u003e) Expression of ERK and PI3K-AKT signaling pathway-related proteins determined by immunoblot analysis. \u003cem\u003en \u003c/em\u003e=3. Data are presented as mean ± SD, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/862c7d05501c4503369e155d.png"},{"id":76634609,"identity":"ad8f5484-f38d-4b88-b51c-adbed62324fb","added_by":"auto","created_at":"2025-02-19 07:16:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1943843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe nanopores facilitate expression and secretion of FGFBP3 promoting improved wound healing. a\u003c/strong\u003e) Schematic diagram of the RNA transcriptome analysis workflow.\u003cstrong\u003e b\u003c/strong\u003e) The volcano plots of distinct expressive genes in primary fibroblasts planted on plate or Nanopore #3 scaffolds. \u003cstrong\u003ec and d\u003c/strong\u003e) \u003cem\u003eFGFBP3\u003c/em\u003emRNA and protein levels in primary fibroblasts and NIH-3T3 cells. \u003cstrong\u003ee and f\u003c/strong\u003e) Immunofluorescence staining of FGFBP3 in primary fibroblast and NIH-3T3 cells. Scale bars, 50 μm. \u003cstrong\u003eg\u003c/strong\u003e) Images of wounds treated with PBS and scaffolds on day 7 to day 14. \u0026nbsp;\u003cstrong\u003eh\u003c/strong\u003e) Skin tissue FGFBP3 contents in wound tissue determined by immunoblot analysis. \u003cstrong\u003ei\u003c/strong\u003e) Skin tissue FGFBP3 contents examined by immunohistochemical staining. Scale bars, 100 mm.\u003cstrong\u003e j\u003c/strong\u003e) Fluorescence images and analysis of HUVECs forming tubule-like structures with the supernatant of FGFBP3 knockdown primary fibroblasts cultured on plates or Nanopore #3 scaffolds. Scale bars, 200 μm. \u003cem\u003en \u003c/em\u003e=3. Data are presented as mean ± SD, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/dad668b77a7be6600a0fb9d7.png"},{"id":76635756,"identity":"fa19b5f4-33d4-43ad-b6d9-7cf595d1ccbe","added_by":"auto","created_at":"2025-02-19 07:24:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2136570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRUNX1 is a transcription factor of FGFBP3 affecting skin wound healing. a\u003c/strong\u003e) DNA pull down assay and JASPAR database prediction of FGFBP3 transcription factor. \u003cstrong\u003eb and c\u003c/strong\u003e) \u003cem\u003eRUNX1\u003c/em\u003e mRNA and protein levels in primary fibroblasts and NIH-3T3 cells planted on plates or Nanopore #3 scaffold, respectively. \u003cstrong\u003ed\u003c/strong\u003e) Immunofluorescence staining of p-RUNX1 in primary fibroblasts. Scale bars, 50 μm.\u003cstrong\u003e e\u003c/strong\u003e) The predicted sites of the putative RUNX1 binding motif in \u003cem\u003eFGFBP3 \u003c/em\u003epromoter. \u003cstrong\u003ef\u003c/strong\u003e) A schematic diagram of constructed luciferase reporter plasmids. \u003cstrong\u003eg\u003c/strong\u003e) Dual luciferase reporter assays were applied to detect the RUNX1 binding site within the \u003cem\u003eFGFBP3\u003c/em\u003epromoter region. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e. empty plasmid group. \u003cstrong\u003eh\u003c/strong\u003e) Ch-IP analysis of RUNX1 occupancy in site#2 of the\u003cem\u003e FGFBP3\u003c/em\u003e promoter in fibroblasts. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e. anti-IgG group. \u003cstrong\u003ei and j\u003c/strong\u003e) \u003cem\u003eFGFBP3 \u003c/em\u003emRNA and protein levels in fibroblasts with RUNX1 knockdown. \u003cstrong\u003ek\u003c/strong\u003e) Flow chart for RUNX1 knockdown in skin wound model. \u003cstrong\u003el\u003c/strong\u003e) Skin FGFBP3 contents in wound tissue determined by immunoblot analysis. \u003cstrong\u003em\u003c/strong\u003e) Representative images of the healing process in wounds treated with PBS or Nanopore #3 scaffold in AAV-shRUNX1-mice or AAV-shNC-mice. \u003cstrong\u003en\u003c/strong\u003e) H\u0026amp;E-stained histologic longitudinal sections after 14 days. Scale bars, 0.5 mm. \u003cstrong\u003eo\u003c/strong\u003e) Wound healing dynamics of WT mice treated with Nanopore #3 scaffold on day 1 to day 14. \u003cstrong\u003ep\u003c/strong\u003e) Wound closure and re-epithelialization rates. \u003cem\u003en \u003c/em\u003e=3. Data are presented as mean ± SD, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/19b570e0119dbf6c8db6e563.png"},{"id":76635759,"identity":"f440e47d-ba22-4c0b-b51a-63993dbfc209","added_by":"auto","created_at":"2025-02-19 07:24:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1946035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCav1-mediated CBFβ release from FLNA synergistically activating RUNX1 to promote FGFBP3 transcription following caveolae endocytosis. a\u003c/strong\u003e) Confocal microscopy analysis of Cav1 (red) and FLNA (green) colocalization in primary fibroblasts. Scale bars, 20 μm. \u003cstrong\u003eb\u003c/strong\u003e) Confocal microscopy analysis of CBFβ (red) and FLNA (green) localization in the primary fibroblasts. Scale bars, 20 μm. \u003cstrong\u003ec\u003c/strong\u003e) Confocal microscopy analysis of exogenous eGFP-RUNX1 (green) and mCherry-CBFβ (red) in fibroblasts. Scale bars, 20 μm. \u003cstrong\u003ed\u003c/strong\u003e) Co-immunoprecipitation of endogenous FLNA with Cav1, FLNA with CBFβ in primary fibroblasts. \u003cstrong\u003ee\u003c/strong\u003e) Co-immunoprecipitation of endogenous CBFβ with FLNA, CBFβ with RUNX1 in fibroblasts. \u003cstrong\u003ef\u003c/strong\u003e) RUNX1 and CBFβ protein levels in total, nucleus and cytoplasm of primary fibroblasts planted on plates and Nanopore 3# scaffold. \u003cstrong\u003eg)\u003c/strong\u003e Localization and interaction analysis of RUNX1 and CBFβ in primary fibroblasts. \u003cstrong\u003eh and i\u003c/strong\u003e) \u003cem\u003eFGFBP3\u003c/em\u003e mRNA and protein levels in fibroblasts with CBFβ knockdown. \u003cstrong\u003ej\u003c/strong\u003e) Immunofluorescence staining of FGFBP3 in fibroblasts with CBFβ knockdown. Scale bars, 50 μm. \u003cstrong\u003ek\u003c/strong\u003e) Schematic representation of Cav1-mediated CBFβ release from FLNA and nuclear translocation to synergize with RUNX1 in activating FGFBP3 transcription post-caveolae endocytosis. \u003cem\u003en \u003c/em\u003e=3. Data are presented as mean ± SD, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/e631d4a79a59a7067ffb39d3.png"},{"id":92984238,"identity":"43a165f6-e949-4e2d-a6eb-b0dbefe98776","added_by":"auto","created_at":"2025-10-07 21:03:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20168793,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/102c2045-60f2-40fc-891e-71125330fc70.pdf"},{"id":76634622,"identity":"17c898e7-a5a7-4d1f-8ac7-c613ca8e41c2","added_by":"auto","created_at":"2025-02-19 07:16:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14344816,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/c8f64acc93581c6fe50e0235.docx"},{"id":76636091,"identity":"6c8638c3-afee-441f-80fc-b01c29ef8377","added_by":"auto","created_at":"2025-02-19 07:32:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":903034,"visible":true,"origin":"","legend":"Supplementary Table 5","description":"","filename":"SupplementaryTable5.listforTyrosinerelatedmembranereceptorLCMS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/c453e66da2cb370228afb1e0.xlsx"},{"id":76637227,"identity":"d368a968-fedb-47b4-9899-fecbd46cd034","added_by":"auto","created_at":"2025-02-19 07:40:40","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":180224,"visible":true,"origin":"","legend":"Supplementary Table 6","description":"","filename":"SupplementaryTable6.listforRNAseqDDEPs.xls","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/a6d54416c0c4f593dbe35d21.xls"},{"id":76635758,"identity":"9cc36ef8-aa51-4f03-a920-7e029a642df0","added_by":"auto","created_at":"2025-02-19 07:24:40","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":247522,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 7\u003c/p\u003e","description":"","filename":"SupplementaryTable7.listforDNApulldownJASPAR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5955293/v1/bc61772cda06908295fbd589.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Caveolae-Mediated Sensing of Nanoporous Cues in Fibroblasts Reprogramming During Wound Healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere skin injuries, including full-thickness defects and chronic ulcers, pose significant health challenges and can be life-threatening\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Timely and sophisticated medical interventions are crucial for addressing the loss or damage to skin tissue\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recent advancements in biomaterial science have led to the development of synthetic scaffolds, such as artificial dermis, which capitalize on the body's innate healing capabilities\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These scaffolds harness the potential of endogenous cells to repair or replace damaged tissues, providing a less invasive and more sustainable solution compared to traditional methods\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. By directing cell reprogramming, these scaffolds can facilitate cell attachment, migration, and differentiation, thereby guiding the tissue regeneration process. While the biophysical and biochemical cues of scaffolds have been extensively studied for their role in controlling cell state transitions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, the impact of scaffold porosity on cell fate is often underappreciated.\u003c/p\u003e \u003cp\u003eIt is well-established that the biophysical properties of biomaterials significantly influence the local tissue microenvironment and cellular behavior\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The stiffness of the extracellular matrix (ECM) is crucial for cell adhesion, spreading, and mechanotransduction\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, various pore morphologies found in biological materials and natural ECM direct cellular responses\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies have shown that nanoscale pores of appropriate dimensions can initiate cell anchorage, promote proliferation, influence differentiation, protect cells from apoptosis, and activate cytoskeletal reorganization\u0026mdash;all of which are critical for tissue repair and homeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Despite the recognized importance of nanoscale pores in scaffolds for tissue repair\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, the specific mechanisms by which repair cells respond to these nanoscale features remain poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we have developed scaffolds with distinct nanopore characteristics that maintain structural integrity and resist deformation. This allows us to explore the mechanisms underlying cell responses to various nanopores. We focus on fibroblasts, which are the primary responders to nanopores in scaffolds and exhibit diverse behaviors in terms of migration and proliferation. Our findings reveal that caveolae formation is a critical response of fibroblasts to nanopores, induced by the phosphorylation of Cav1. This process initiates the biological processes necessary for regeneration. We propose that caveolae induced by nanopores could serve as a key parameter in the design of scaffolds for tissue engineering applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of nanopore scaffolds \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of scaffold nanopores on tissue repair and regeneration, we fabricated scaffolds with distinct nanopores using polystyrene (a material commonly used for cell culture dishes), which include round (Named as Nanopore #1), 3\u0026times;-stretched oval (Nanopore #2), and 6\u0026times;-stretched oval (Nanopore #3) configurations (Extended Data Fig. 1), exhibiting resistance to deformation and degradation, as described in our previous study. Briefly, colloidal crystal templates made from uniform silica nanoparticles were created on glass slides using the vertical deposition technique. These templates were then soaked in a polystyrene solution and left to dry at room temperature (20\u0026deg;C) for four days before they were allowed to detach naturally, yielding an unetched anti-opal film. Following this, the anti-opal film and silica composite were removed from the slides after a 30-minute immersion in a 4% hydrofluoric acid solution. The films were then subjected to etching with a 1% hydrofluoric acid solution for 12 hours at 4\u0026deg;C to eliminate all silica nanoparticles, resulting in an anti-opal nano-scaffold with a consistent nanopores. To achieve this, the two sections of the film were affixed to a vernier caliper, which was placed in an 80\u0026deg;C water bath. The film was stretched by pulling the caliper\u0026apos;s slider, and the stretch ratio was determined using the caliper\u0026apos;s reading, thus obtaining virous nanopores in scaffolds with 3\u0026times; and 6\u0026times; stretch ratios. Finally, the acquired scaffolds were analyzed using scanning electron microscopy (SEM) to examine their nanopores morphology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cells (fibroblasts, macrophages, keratinocytes) were extracted from the skin of C57BL/6J mice. Mouse embryonic cells, NIH-3T3 and human umbilical vein endothelial cells (HUVECs) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were maintained in DMEM culture medium (Biological Industries, Israel) supplemented with 10% fetal bovine serum (Gemini, USA) and 1% streptomycin and penicillin (Invitrogen Corp., Carlsbad, CA, USA) in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Regular identification of cells was conducted to ensure the elimination of mycoplasma contamination. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 10,000 cells of a 70 \u0026mu;L cell suspension were added to each well of the cell migration insert. The insert was then placed in a CO2 incubator at 37\u0026deg;C for 12 hours. After 8 hours, the insert was carefully removed, and serum-free medium was added to the well plate. The culture was maintained for 12 and 24 hours from the time the medium was introduced. Following the removal of the culture medium, 4% paraformaldehyde was used for fixation. The well was then washed three times with PBS, and images were captured and observed using an inverted phase contrast microscope (Olympus, Japan). The cell migration rate was calculated by comparing it to the state at 0 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e tube formation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ability of HUVECs to form tube-like structures was assessed using a modified tube formation assay. Twenty-four well plates were layered with a cold Matrigel solution (BD Biosciences, New Jersey, USA) and incubated at 37\u0026deg;C for 30 minutes to permit the Matrigel to gel. The HUVECs were collected, stained with calcein-AM, and resuspended in a medium reduced in fetal bovine serum (FBS) to 1%. These cells were then plated at a concentration of 20,000 cells/mL per well onto the Matrigel-coated wells and pre-incubated at 37\u0026deg;C for 2 hours to facilitate cell adhesion. Subsequently, 50 \u0026mu;L of concentrated supernatant, derived from fibroblasts that had been cultured on plates or scaffolds, was added to the cell-laden plates. Photographs of the tube-like structures were captured using a Nikon confocal microscope (Nikon C2, Japan) over a period of 8 to 16 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from primary fibroblasts or NIH-3T3 cells using TRIzol Reagent (catalog #15596026, Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 \u0026mu;g of total RNA using the Prime Script RT Kit (catalog #KR107, Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer\u0026apos;s guidelines. Quantitative real-time PCR (qRT-PCR) was performed on a Light Cycler Real-time PCR system (Roche, Shanghai, China) with SYBR Green master Mix (catalog #FP205, Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer\u0026apos;s protocols. The Ct values of the target genes were analyzed using the 2^-\u0026Delta;\u0026Delta;CT method, with \u0026beta;-actin expression serving as the endogenous control for normalization. Specific primer sequences utilized in this study are provided in Supplementary Table 1 (Sangon Biotech Co., Ltd., Shanghai, China). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA pull down and liquid chromatography-tandem mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNIH-3T3 cells were grown in a 150 mm culture dish until they reached 80% confluence. After rinsing the cells three times with cold PBS, they were treated with trypsin and collected via centrifugation. The nuclear lysate was extracted using a nuclear and cytoplasmic protein separation kit (catalog #P0028, Beyotime Biological, Shanghai, China) in accordance with the manufacturer\u0026apos;s protocol. Dynabeads\u0026reg; C1 Streptavidin Magnetic Beads were rinsed three times with B\u0026amp;W buffer (5 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM EDTA), and then 500 \u0026mu;L of the magnetic beads were incubated with 50 \u0026mu;g of biotinylated target DNA for 30 minutes at room temperature. DNA pull-down was conducted at 4\u0026deg;C with nuclear supernatant extracts and magnetic bead-DNA complexes. The complexes were subsequently washed six times with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA) and finally resuspended in 100 \u0026mu;L of elution buffer. Shotgun Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then carried out by Genechem, Shanghai, China. Lastly, the LC-MS/MS raw data were searched and qualitatively analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and MASCOT 2.6 (Matrix Science). The protein database utilized was UNIPROT_Mus musculus_2023_03.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation and liquid chromatography-tandem mass spectrometry\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eNIH-3T3 cells were cultured to 80% confluence in a 10 cm dish. The cells were washed three times with cold PBS, and then cell lysates were collected. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (catalog #P008, Beyotime Biological, Shanghai, China) following the manufacturer\u0026apos;s protocol. The cell lysates were incubated with specific primary antibodies or control IgG overnight at 4\u0026deg;C with rotation. Protein A/G Agarose (catalog #37478, Cell Signaling Technology, Inc., CST, USA) was added to the lysates and incubated with rotation at 4\u0026deg;C for 3 hours to precipitate protein-protein complexes. Subsequently, the bead-protein complex precipitate was washed seven times with PBS, and 50 \u0026mu;L of protein loading buffer was added to elute the proteins at 100\u0026deg;C for 10 minutes. Immunoblotting was conducted to verify the elution of the target protein. Shotgun Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then carried out by Genechem, Shanghai, China. Finally, the LC-MS/MS raw data were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and MASCOT 2.6 (Matrix Science). The protein database used for the analysis was UNIPROT_Mus musculus_2023_03.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enrichment of RUNX1 in the promoter region of FGFBP3 was quantified using an EZ-Magna ChIP\u0026trade; A/G ChIP Kit (catalog #9003, Cell Signaling Technology, Inc., CST, USA) following the manufacturer\u0026apos;s guidelines. In brief, cells were lysed and sonicated to shear the cross-linked chromatin and protein into fragments of appropriate size (200-1000 bp). The sheared DNA was sonicated for 28 cycles (15 seconds on and 10 seconds off) using a Diagenode Biorupter Pico (Diagenode, Belgium). After centrifugation at 10,000 \u0026times; g for 10 minutes at 4 \u0026deg;C, the supernatant was collected and prepared for immunoprecipitation. Three aliquots of sheared DNA were added to 20 \u0026mu;L of fully resuspended protein A/G magnetic beads along with positive control (Anti-RNA Polymerase II), negative control (normal rabbit IgG), and the antibody against RUNX1 (catalog ab229482, abcam, USA), respectively. Following overnight incubation at 4 \u0026deg;C with rotation, protein/DNA complexes were eluted, and free DNA was reverse crosslinked from these complexes. The DNA was then purified using spin columns and analyzed by qRT-PCR with specific primers targeting the FGFBP3 gene promoter region (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding site of RUNX1 to the FGFBP3 gene promoter was predicted using the online prediction tool, JASPAR 3.0, which can be accessed at http://jaspar.genereg.net. Fibroblasts in the logarithmic growth phase were evenly distributed into a 6-well plate beforehand. When the cell density reached an appropriate level, wild type or mutant plasmids were transfected into the fibroblast cells using Lipofectamine 3000 (catalog #L3000015, Invitrogen, USA). Specific promoter sequences of FGFBP3 (sites #1, #2, #3, and #4, detailed in Supplementary Table 3) were all cloned into pGL3 reporter plasmids by Repobio (Hangzhou, China). Eight hours post-transfection, the supernatant was aspirated, and the cells were cultured in standard medium for an additional 24 hours. Luciferase activity was quantified as chemiluminescence using the Dual-Luciferase\u0026reg; Reporter (DLR\u0026trade;) Assay System (catalog #E1910, Promega, USA) following the manufacturer\u0026apos;s instructions. The relative luciferase activity, as measured by the Multiskan SkyHigh (Thermo Scientific, USA), was determined based on the readings from both firefly and Renilla luciferase. Each transfection was carried out in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids, virus and cell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipofectamine 3000 Transfection Reagent (catalog #L3000015, Invitrogen, USA) was employed for cell transfection following the manufacturer\u0026apos;s recommendations. Plasmids and RNA interference lentiviruses were packaged and procured from Repobio, Hangzhou, China. NIH-3T3 cells were infected with RNA interference lentiviruses LV-shNC and LV-shCav1, and subsequently isolated using puromycin (3 \u0026micro;g/mL, Life Technologies, Gibco, USA) selection to achieve stable knockdown of Cav1. Total protein extracts were obtained from the transfected cells, and the knockdown efficiency of the target protein was verified by immunoblotting. The sequences of shCav1 and several siRNAs are detailed in Supplementary Table 4.\u003c/p\u003e\n\u003cp\u003eIn this study, AAV vectors for the knockdown of Cav1 and RUNX1 were constructed and produced by Cyagen Technology (Shanghai) Corp., Ltd. We utilized an Adeno-Associated Virus 9 (AAV9) vector to express short hairpin RNA (shRNA) that specifically targets Cav1 or RUNX1: AAV9-U6-shRNA (Cav1)-CMV-WPRE, AAV9-U6-shRNA (RUNX1)-CMV-WPRE, and AAV9-U6-shRNA (NC)-CMV-WPRE. The shRNA sequences targeting Cav1 and RUNX1 were inserted into the shRNA AAV vector, and the recombinant plasmids were co-transfected into HEK293T cells with PEI. Cells were collected by centrifugation at 4\u0026deg;C, 800 g for 10 minutes, 72 hours post-transfection. After resuspension in lysis buffer (150 mM NaCl and 20 mM Tris, pH 8.0) by vortexing, the cell pellet was incubated with a concentration reagent overnight at 4\u0026deg;C. Following quantification of the AAV vector concentration, the viral stock was mixed in HN buffer (containing 50 mM HEPES, pH 7.4, and 0.15 M NaCl) for injection. The shRNA sequences used in this study are as follows: shRNA (Cav1): 5\u0026apos;-GCTTCCTGATTGAGATTCAGT-3\u0026apos;, shRNA (\u003cem\u003eRUNX1\u003c/em\u003e): 5\u0026apos;-CACCTACCATAGAGCCATCAA-3\u0026apos;. To knock down Cav1 or RUNX1 in mouse skin, AAV9 adenovirus carrying shCav1 or shRUNX1 was injected intradermally two weeks prior to establishing the wound model. Control groups received an equal amount of AAV9 vehicle containing a negative control sequence (AAV9-shNC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectron microscope samples were processed and examined using an electron microscope at the Laboratory of our institute. Cells cultured on scaffolds and glass slides were fixed with 2.5% glutaraldehyde, followed by ethanol gradient dehydration and air drying. The cell samples were then sputter-coated with platinum and observed under a scanning electron microscope (SEM, HITACHI SU8010, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFibroblasts and NIH-3T3 cells were collected and resuspended in a 2.5% glutaraldehyde fixative solution at 4\u0026deg;C overnight, followed by incubation with 1% osmic acid for 2 hours at room temperature. After gradient dehydration with ethanol and air drying, the cell samples were sputter-coated with platinum and examined under a scanning electron microscope (SEM, HITACHI SU8010, Japan). The number of caveolae was analyzed using NIH ImageJ software (National Institutes of Health, Bethesda, MD)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe skin tissues or cultured cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (P0013C, Beyotime, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, China) to extract total proteins. Protein concentrations were determined using a BCA protein assay kit (P008, Beyotime Biological, Shanghai, China) following the manufacturer\u0026apos;s instructions. Equal amounts of proteins were loaded and separated by 10% or 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp, USA). The membrane was blocked with 5% non-fat dry milk (BD Biosciences) for 1 hour at room temperature. Following incubation with specific primary antibodies overnight at 4\u0026deg;C, the antibodies included: FGFBP3 (24725-1-AP, Proteintech, China), CAV1 (3267S, CST, USA), p-CAV1 (3251S, CST, USA), RUNX1 (ab229482, Abcam, USA), p-RUNX (ab182659, Abcam, USA), PDGFR\u0026beta; (ab69506, Abcam, USA), CBF\u0026beta; (ab133600, Abcam, USA), FLNA (ab76289, Abcam, USA), p-ERK1/2 (ab201015, Abcam, USA), ERK1/2 (ab17942, Abcam, USA), p-PI3K (17366S, CST, USA), PI3K (4292S, CST, USA), p-AKT (4056S, CST, USA), AKT (9272S, CST, USA), and GAPDH (ab245355, Abcam, USA), respectively. The dilution ratio for all antibodies was 1:1000. Immunodetection was performed with an EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Haemek, Israel). Protein expression was quantified and analyzed using NIH ImageJ software (National Institutes of Health, Bethesda, MD). GAPDH served as an internal reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence (IF) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell samples, cover-slip cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) for 30 minutes, and treated with 0.25% Triton X-100 for 15 minutes at room temperature. Then, the cells were blocked with 5% bovine serum albumin (BSA) for 1 hour. The cells were incubated with primary antibodies: FGFBP3 (24725-1-AP, Proteintech, China), Cav1 (3267S, CST, USA), p- Cav1 (3251S, CST, USA), RUNX1 (ab229482, Abcam, USA), p-RUNX (ab182659, Abcam, USA), PDGFR\u0026beta; (ab69506, Abcam, USA), CBF\u0026beta; (ab133600, Abcam, USA), and FLNA (ab76289, Abcam, USA) at 4\u0026deg;C overnight. The dilution ratio for all antibodies was 1:200. After staining, they were cultured with corresponding secondary antibodies coupled with Alexa Fluor 488 or Alexa Fluor 596 and incubated at 37\u0026deg;C for 1 hour. The nuclei were stained with DAPI for 5 minutes. Finally, the analysis was performed using a confocal microscope (Nikon C2, Japan). Image analysis was conducted using NIH ImageJ software (National Institutes of Health, Bethesda, MD).\u003c/p\u003e\n\u003cp\u003eFor skin tissue samples, immunofluorescent staining was performed on frozen sections of skin tissue. Briefly, the sections were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature. They were then incubated with primary antibodies: FGFBP3 (24725-1-AP, Proteintech, China), Cav1 (3267S, CST, USA), RUNX1 (ab229482, Abcam, USA), PDGFRa (3174S, CST, USA), \u0026alpha;-SMA (19245S, CST, USA), CD31 (ab222783, Abcam, USA) overnight at 4\u0026deg;C. Concurrently, IgG isotype control antibodies were used as negative controls to confirm the specificity of the primary antibodies. Subsequently, the slides were washed three times with PBS and incubated with the appropriate fluorescently conjugated secondary antibodies coupled with 488 or Alexa Fluor 596 for 30 minutes at 37\u0026deg;C. The nuclei of tissue sections were stained with DAPI containing an anti-fluorescence quencher, and images were captured using a Nikon confocal laser scanning microscope (Nikon C2, Japan). Image analysis was conducted using NIH ImageJ software (National Institutes of Health, Bethesda, MD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging of total endocytosis. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary fibroblasts and NIH-3T3 cells were cultured on plates or scaffolds for a duration of 24 hours. After aspirating the culture medium, the cells were rinsed with PBS. Thereafter, 1 mL of 2 \u0026mu;M FM 1-43 dye (T35356, ThermoFisher, USA) was added to each well of a 24-well plate, and the cells were incubated in a CO2 incubator at 37\u0026deg;C for 20 minutes. Post-incubation, the dye solution was aspirated, and the cells were further incubated with 1 mL of PBS for an additional 20 minutes. The samples were subsequently fixed and counterstained with Hoechst. Images were acquired using a Nikon confocal laser scanning microscope (Nikon C2, Japan). Image analysis was executed using NIH ImageJ software (National Institutes of Health, Bethesda, MD). The analysis entailed quantifying FM 1-43 dye puncta in each focal plane and determining the total intensity of the puncta per cell.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess differences in gene expression, total RNA was extracted from NIH-3T3 cells cultured on plates or scaffolds using TRIzol reagent (catalog #15596026, Invitrogen, Carlsbad, CA, USA). RNA purity and quantification were determined using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Thereafter, cDNA libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit following the manufacturer\u0026apos;s protocol. Transcriptome sequencing was conducted on the Illumina platform (Illumina NovaSeq 6000, China), yielding 150 bp paired-end reads. Raw sequencing reads for each sample were cleaned to remove low-quality reads and adapters using fastp (version 0.22.0). The clean reads were aligned to the reference genome using HISAT2. The FPKM values for each gene were calculated, and the read counts for each gene were obtained using HTSeq-count. Differential expression analysis was performed using DESeq2. A Q value \u0026lt; 0.05 and fold change \u0026gt; 2 or fold change \u0026lt; 0.5 were set as the criteria for significantly differentially expressed genes (DEGs). The Volcano Plot of differential expression genes was generated to visualize the expression of up-regulated or down-regulated DEGs using the R package ggradar. The transcriptome sequencing and analysis were carried out by OE Biotech Co., Ltd. (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample preparation for proteomics and phosphoproteomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e Protein extraction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were subjected to three rounds of sonication while on ice using a high-intensity ultrasonic processor (Scientz) in lysis buffer containing 8M urea, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail. Following centrifugation at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C, the remaining debris was pelleted and removed. The supernatant was then collected, and protein concentration was measured using a BCA protein assay kit according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e Trypsin digestion\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo facilitate digestion, the protein solution was initially treated with 5 mM dithiothreitol (DTT, Sigma-Aldrich) for 30 minutes at 56\u0026deg;C to reduce disulfide bonds, followed by alkylation with 11 mM iodoacetamide (Sigma-Aldrich) for 15 minutes at room temperature in the dark. The protein sample was then diluted by adding 100 mM TEAB (Sigma-Aldrich) to reduce the urea concentration to less than 2 M. Subsequently, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the primary overnight digestion, followed by a secondary 4-hour digestion at a 1:100 trypsin-to-protein mass ratio. Finally, the peptides were purified using a C18 SPE column (Phenomenex).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e Phosphopeptides enrichment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peptide mixtures were first incubated with IMAC microspheres in a loading buffer consisting of 50% acetonitrile/0.5% acetic acid, with gentle agitation. Non-specifically adsorbed peptides were then removed by washing the IMAC microspheres with 50% acetonitrile/0.5% acetic acid followed by 30% acetonitrile/0.1% trifluoroacetic acid. Afterward, an elution buffer containing 10% NH4OH was added to elute the enriched phosphopeptides, which were collected with further agitation. The supernatant containing the phosphopeptides was then collected and subjected to lyophilization for subsequent LC-MS/MS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e Liquid chromatography-mass spectrometry analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeptides were resuspended in liquid chromatography mobile phase A and separated using the Nano-Elute Ultra Performance Liquid Chromatography (UPLC) system. Mobile phase A consists of 0.1% formic acid and 2% acetonitrile in water; mobile phase B consists of 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: from 0 to 70 minutes, 6% to 24% B; from 70 to 144 minutes, 24% to 35% B; from 144 to 147 minutes, 35% to 80% B; and from 147 to 150 minutes, holding at 80% B, with a flow rate of 450 nL/min. The peptides were resolved by the UPLC system, introduced into a capillary ion source, ionized, and analyzed by TOF Pro mass spectrometry. The ion source voltage was set to 1.75 kV, and both the peptide precursor ions and their subsequent fragments were detected and analyzed using a high-resolution time-of-flight (TOF) mass spectrometer. The secondary mass spectrum scan range was set from 100 to 1700 m/z. Data acquisition was performed in Parallel Cumulative Serial Fragmentation (PASEF) mode. After 10 cycles of PASEF, a primary mass spectrum was acquired to collect secondary spectra with precursor ion charge states ranging from 0 to 5. The dynamic exclusion time for tandem mass spectrometry was set to 30 seconds to prevent the repetitive scanning of precursor ions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e Phosphoproteomic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MS/MS data acquired were processed using the MaxQuant search engine (version 1.6.15.0). Tandem mass spectra were aligned against the Mus_musculus_10090_SP_20230103.fasta database (containing 17,132 entries), as well as a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, permitting up to two missed cleavages. The minimum amino acid length was set to seven. The precursor ion mass tolerance was set to 20 ppm for the initial search and 4.5 ppm for the main search, while the fragment ion mass tolerance was maintained at 20 ppm. Carbamidomethylation on Cys was designated as a fixed modification, and acetylation at the protein N-terminus and oxidation on Met were designated as variable modifications. The false discovery rate (FDR) was controlled to be less than 1%. Protein quantities were calculated and normalized using the median-centering method. Genes with missing values in at least 50% of the samples were excluded, and the remaining missing values were imputed using the K-Nearest Neighbor (KNN) method with the R package impute88 (version 1.70.0). Differentially expressed proteins were identified using the limma package (version 3.54.2) with a threshold of log2FoldChange \u0026gt;0.3 (or \u0026lt;\u0026minus;0.3) and a P value \u0026lt; 0.05. Gene set enrichment analysis (GSEA) was performed using the R package cluster\u003cins cite=\"mailto:飞霞%20郭\" datetime=\"2025-01-10T09:53\"\u003e \u003c/ins\u003eProfiler. Further details regarding the differential analysis of quantitative proteomics data are provided in Supplementary Data 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale wild-type C57BL/6J mice, aged 6 to 8 weeks, were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All mice were maintained under specific-pathogen-free conditions, with ad libitum access to chow and water, and were housed on a 12-hour light-dark cycle. Prior to the experiments, the mice were acclimated for at least one week in the animal facility. All animal procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Animal Care and Use Committee of our institute.\u003c/p\u003e\n\u003cp\u003eFollowing general inhalation anesthesia using halothane, the dorsal hair was shaved from the mice. Skin wounds were created with a 6-mm skin punch. The mice were then randomly assigned to one of four groups, with each group receiving different experimental interventions: the control group received a PBS solution, while the experimental groups were treated with scaffolds of varying pore structures. At four, seven and ten days post-wounding, the healing tissues were harvested from the mice for further analysis. After a two-week period, the mice were humanely euthanized by cervical dislocation under general inhalation anesthesia with halothane.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePortions of the skin samples were fixed with 4% paraformaldehyde for 24 hours and then embedded in paraffin for histomorphological analysis. The remaining skin tissue was stored at -80\u0026deg;C for subsequent molecular studies. Tissue sections, 4 micrometers thick, were prepared and stained with hematoxylin and eosin (H\u0026amp;E) and Masson\u0026apos;s trichrome. The tissue sections underwent dehydration, rehydration, and staining following the manufacturer\u0026apos;s instructions for the H\u0026amp;E staining kit (Solarbio, G1120) and Masson\u0026apos;s trichrome staining kit (Solarbio, G1340). All images of the stained sections were acquired using digital scanners (3DHISTECH Pannoramic 250 FLASH, Hungary).\u003c/p\u003e\n\u003cp\u003eFor Oil Red O staining, 10-micrometer-thick frozen tissue sections were prepared. These sections were fixed with 4% paraformaldehyde for 30 minutes. Post-fixation, the samples were washed three times with PBS at room temperature. The Oil Red O working solution was prepared by mixing the Oil Red O stock solution (Sigma, O1391) with double-distilled H2O in a 3:2 ratio. Following a 20-minute staining period, the samples were washed twice with 60% isopropanol, counterstained with hematoxylin, mounted with glycerol gelatin (Solarbio, S2150), and imaged using digital scanners (3DHISTECH Pannoramic 250 FLASH, Hungary).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC was employed to qualitatively evaluate the expression of phenotypic markers. Paraffin-embedded renal tissue sections (4 \u0026mu;m) were mounted on adhesive microscope slides for analysis. The sections were deparaffinized and rehydrated using xylene and a series of graded alcohol concentrations. They were then treated with an enhanced endogenous peroxidase blocking buffer (P0100B, Beyotime, China) for 30 minutes to quench endogenous peroxidase activity. Antigen retrieval was achieved by microwaving the sections in citrate-EDTA solution (P0086, Beyotime, China) for 20 minutes. After cooling to room temperature, the sections were blocked with 5% bovine serum albumin (A1933, Sigma, Missouri, USA) for 30 minutes. The sections were incubated overnight at 4\u0026deg;C with primary antibodies targeting FGFBP3 (Proteintech, 24725-1-AP, China) and p-RUNX (ab182659, Abcam, USA). After rewarming and washing, the sections were incubated with appropriate secondary antibodies for 1 hour at 37\u0026deg;C. The signals were then visualized using 3,3\u0026apos;-diaminobenzidine (DAB, LI-9018, ZSGB-Bio) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted three times, and the data are presented as the mean \u0026plusmn; standard deviation (SD). The experimental data were analyzed using GraphPad Prism software (version 8.02, USA). Statistical comparisons between two groups were made using a two-tailed Student\u0026apos;s t-test, while one-way or two-way ANOVA with Tukey\u0026apos;s post-hoc test was employed for multiple group comparisons (\u0026gt; 2 groups). Differences between groups were considered significant at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*),\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01 (**), or \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (***), and were considered highly statistically significant at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (****).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eNanopores affect tissue repair and regeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nanoporous scaffolds, constructed from polystyrene a material known for its resistance to rapid degradation or deformation effectively leverage the advantages of nanopores for tissue regeneration in this study. The nanopores in these scaffolds are comparable to those found in artificial dermis used clinically for deep skin wounds (Extended Data Fig. 2). We employed three types of nanoporous scaffolds to evaluate their impact on 6 mm full-thickness dorsal skin wounds in mice. All wounds were covered with the respective scaffolds, while control wounds were treated with saline (Fig. 1a). Macroscopic observation of wound closure in mice showed a significant reduction in wound size at 7, 10, and 14 days after treatment. Nanopore #3 group exhibited the most rapid decrease in wound size, followed by Nanopore #2 group, while Nanopore #1 and control groups showed the slowest reduction (Fig. 1b, c). In line with this, H\u0026amp;E staining revealed substantial increases in wound closure rates, ranging from 82-90% on day 14 in Nanopore #3 group, compared to the control group, which had closure rates of 53-69%. Furthermore, re-epithelialization in the Nanopore #3 group at day 14 covered 82-91% of the wound area, while Nanopore #2, Nanopore #1, and control groups covered 75-83%, 66-73%, and 53-69%, respectively (Fig.1d). Notably, the Nanopore #3 group showed a greater deposition of well-organized collagen fibers and a significant increase in type I collagen levels, as indicated by Masson\u0026apos;s trichrome staining (Fig. 1e, f). Immunofluorescence staining for CD31 and \u0026alpha;-SMA, markers for vascular endothelial cells and smooth muscle cells, respectively, demonstrated a substantial enhancement in blood vessel formation within the Nanopore #3 group, while the other groups showed only a limited number of positively stained cells (Fig.1g, h). Oil Red O staining also revealed a significant increase in the formation of new sebaceous glands and dermal appendages in the Nanopore #3 group (Extended Data Fig. 3). Collectively, these findings suggest that scaffolds with varying nanopores differentially promote skin wound repair throughout the entire healing process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFibroblasts sense the scaffolds through caveolae formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extracted fibroblasts, macrophages, and keratinocytes from mouse skin, which are critical skin tissue cells essential for wound healing, and cultured them on Nanopore #3 scaffold, which showed superior cytocompatibility and facilitated swift wound repair. Notably, primary fibroblasts grew directionally along the scaffold, aligning neatly - a pattern not observed in other cell types (Extended Data Fig. 4a). Furthermore, both staining and SEM analyses indicate that Nanopore #3 promotes the reorganization of fibroblast cytoskeletons into linear configurations (Extended Data Fig. 4b), an effect also observed in NIH-3T3 cells (Extended Data Fig. 5). In line with this, cell counting kit-8\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand wound healing assays demonstrate that scaffolds with varying nanostructures differentially impact the proliferation and migration of fibroblasts, with Nanopore #3 exhibiting the most pronounced enhancement (Extended Data Fig. 6). These findings collectively suggest that fibroblasts are the principal cells that sense to nanopore cue, potentially initiating a series of pro-repair effects and playing a pivotal role in managing full-thickness skin injuries.\u003c/p\u003e\n\u003cp\u003eTo elucidate the response of fibroblasts to nanopore environments, we undertook an extensive proteomic and phosphoproteomic analysis to identify phosphorylated proteins that exhibit differential expression between fibroblasts cultured on conventional plates and those cultured on Nanopore #3. Principal component analysis revealed that fibroblasts cultured on identical substrates exhibited a high degree of similarity, as evidenced by their tight clustering (Fig. 2a). Our analysis of the top 8 enriched KEGG pathways has uncovered a marked upregulation of caveolar endocytosis signaling pathways (Fig. 2b). Cav1 can be phosphorylated at a conserved tyrosine residue, specifically at position 14 (\u003csup\u003eY14\u003c/sup\u003ep-Cav1) which is a differentially expressed phosphorylated protein sites between fibroblasts cultured on plates and Nanopore #3 (Fig. 2c), while total Cav1 protein levels were not significantly increased as shown in proteomic analysis. Previous research has established that \u003csup\u003eY14\u003c/sup\u003ep-Cav1\u0026nbsp;plays a critical role in modulating caveolar endocytosis.\u003csup\u003e16,17\u003c/sup\u003e Similarly, the levels of \u003csup\u003eY14\u003c/sup\u003ep-Cav1were found to be increased in primary fibroblasts and NIH-3T3 cells when cultured on Nanopore #3 (Fig. 2d, e). Additionally, a gradient increase in \u003csup\u003eY14\u003c/sup\u003ep-Cav1\u0026nbsp;content was observed across different substrates, including plates, Nanopore #1, Nanopore #2, and Nanopore #3 (Extended Data Fig. 7). This gradient increase in \u003csup\u003eY14\u003c/sup\u003ep-Cav1\u0026nbsp;correlates with the progressive changes in cell proliferation and migration, suggesting a link between substrate topography,\u0026nbsp;Cav1\u0026nbsp;phosphorylation, and cellular behavior. Consistently, the expression of \u003csup\u003eY14\u003c/sup\u003ep-Cav1 was significantly enhanced in cells cultured on artificial dermis (Extended Data Fig. 8). Moreover, immunofluorescence analysis was employed to visualize both the localization and expression of \u003csup\u003eY14\u003c/sup\u003ep- Cav1 in fibroblasts. Our findings indicated that \u003csup\u003eY14\u003c/sup\u003ep- Cav1\u0026nbsp;was notably increased in the cytoplasm and membrane of cells cultured on Nanopore #3, whereas in cells cultured on traditional plates, \u003csup\u003eY14\u003c/sup\u003ep- Cav1was primarily localized to the cell membrane (Fig. 2f-h and Extended Data Fig. 9a-c). These results confirm that Nanopore #3 scaffold significantly enhance the phosphorylation level of Cav1 in fibroblasts and alter the distribution of \u003csup\u003eY14\u003c/sup\u003ep- Cav1.\u003c/p\u003e\n\u003cp\u003eTo investigate whether the effect of Nanopore #3 on fibroblast caveolae formation and endocytosis is mediated by Cav1. Utilizing transmission electron microscopy, we observed a marked increase in the number of caveolae in both primary fibroblasts and NIH-3T3 cells when cultured on Nanopore #3. Furthermore, the decrease in caveolae numbers following Cav1 knockdown implies that Cav1 is crucial for the formation of caveolae, underscoring its central role in the biogenesis of these membrane invaginations (Fig. 2i and Extended Data Fig. 9d). To ascertain the role of Cav1 in caveolae-mediated endocytosis, primary fibroblasts and NIH-3T3 cells were treated with the membrane-impermeable FM 1-43 dye for a duration of 15 minutes, after which the dye was washed away. Subsequent quantitative analysis of confocal fluorescence microscopy images revealed that cells cultured on Nanopore #3 exhibited a significantly higher number of FM 1-43-positive endocytic vesicles compared to those on traditional plate, while fibroblasts with Cav1\u0026nbsp;knockdown\u0026nbsp;displayed a reduction in the number of endocytosed vesicles. Furthermore, fibroblasts treated with 2 \u0026mu;M PP2, a Src family kinase inhibitor known to inhibit\u0026nbsp;Cav1\u0026nbsp;phosphorylation, showed a reduction of endocytosed vesicles (Fig. 2j and Extended Data Fig. 9e). This observation suggests that \u003csup\u003eY14\u003c/sup\u003ep-\u0026nbsp;Cav1\u0026nbsp;is specifically required for caveolar endocytosis in fibroblasts cultured on Nanopore #3.\u003c/p\u003e\n\u003cp\u003eIn light of our observations that fibroblasts respond to Nanopore #3 by phosphorylating\u0026nbsp;Cav1\u0026nbsp;and engaging in caveolar endocytosis, we next aimed to investigate whether the knockdown of Cav1 in fibroblasts would impact the efficacy of the scaffold with Nanopore #3 in facilitating wound healing and tissue regeneration. Initially, we evaluated the effect of\u0026nbsp;Cav1 knockdown on fibroblast proliferation \u003cem\u003ein vitro\u003c/em\u003e. We found that the migration of fibroblasts with Cav1 knockdown was significantly compromised compared to those treated with shNC (Extended Data Fig. 10). \u003cem\u003eIn vivo\u003c/em\u003e, following the intradermal injection of adeno-associated virus (AAV)-shCav1 for two weeks (Fig. 3a), skin wound models were established and subsequently treated with Nanopore #3 scaffolds for a further two-week period. The administration of AAV-shCav1 significantly impeded the pro-wound healing effects induced by Nanopore #3 scaffolds. This was evidenced by a larger skin wound size at days 1, 3, 7, 10, and 14 following wounding, as well as reduced rates of skin wound closure and re-epithelialization in AAV-shCav1-treated mice compared to those treated with AAV-shNC (Fig. 3b-e). Furthermore, the efficiency of Cav1 knockdown was confirmed by the significantly lower levels of Cav1\u0026nbsp;protein in the skin samples of mice treated with AAV-shCav1 and Nanopore #3 scaffolds, as compared to those treated with AAV-shNC (Fig. 3f and g). Additionally, the Nanopore #3 scaffold-induced increase in neovascular markers, specifically \u0026alpha;-SMA and CD31-positive cells, was significantly attenuated in mice treated with AAV-shCav1\u0026nbsp;(Fig. 3h). To further substantiate the role of\u0026nbsp;Cav1\u0026nbsp;protein in the pro-wound healing effects mediated by Nanopore #3, we examined the development of new sebaceous glands and dermal appendages at the wound sites. Oil Red O staining demonstrated a marked reduction in the formation of new sebaceous glands and dermal appendages in mice treated with AAV-shCav1 (Fig. 3i). Together, our data indicate that the suppression of\u0026nbsp;Cav1\u0026nbsp;in skin fibroblasts significantly impairs the wound healing process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaveolae-Mediated Endocytic Internalization of PDGFR\u0026beta;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaveolae interact with a variety of signaling molecules to modulate intracellular signaling cascades, notably those involving growth factor receptors\u003csup\u003e18,19\u003c/sup\u003e, the interleukin-1 beta receptor\u003csup\u003e20\u003c/sup\u003e, and the vasoactive intestinal peptide binding VPAC2 receptor\u003csup\u003e21\u003c/sup\u003e. \u0026nbsp;To elucidate the proteins that interact with\u0026nbsp;Cav1, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Fig. 4a). Notably, platelet-derived growth factor receptor \u0026beta; (PDGFR\u0026beta;), a receptor tyrosine kinase, was identified as an interacting protein through LC-MS/MS analysis (Fig. 4b and\u0026nbsp;Supplementary Table 5). Immunoprecipitation assays revealed a robust interaction between\u0026nbsp;Cav1\u0026nbsp;and PDGFR\u0026beta; in primary fibroblasts\u0026nbsp;(Fig. 4c). Similarly, \u003csup\u003eY14\u003c/sup\u003ep- Cav1 was found to interact with PDGFR\u0026beta; in these cells (Extended Data Fig. 11). Immunofluorescence assays revealed a significant increase in PDGFR\u0026beta; levels within the cytoplasm of fibroblasts cultured on the Nanopore #3 scaffold, accompanied by a notable enhancement in the co-localization of Cav1\u0026nbsp;and PDGFR\u0026beta;.\u0026nbsp;Knockdown of\u0026nbsp;Cav1\u0026nbsp;led to a reduction in the internalization of PDGFR\u0026beta; into the cytoplasm of fibroblasts cultured on the Nanopore #3 scaffold (Fig. 4d, e and\u0026nbsp;Extended Data Fig.12a). Immunofluorescence assays were utilized to visualize the localization and expression patterns of PDGFR\u0026beta;. Our results indicated a significant increase in the intensity of internalized PDGFR\u0026beta; within the cytoplasm. In primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffold, activated PDGFR\u0026beta; was predominantly localized to the cell membrane surface in those with Cav1\u0026nbsp;knockdown, whereas in control cells, it was distributed throughout the cytoplasm (Fig. 4f-h and\u0026nbsp;Extended Data Fig. 12b-d). These findings suggest that nanopore structures can enhance the internalization of PDGFR\u0026beta; into the cytoplasm and that a portion of PDGFR\u0026beta; internalization occurs through Cav1-mediated endocytosis.\u003c/p\u003e\n\u003cp\u003eAs a member of the receptor tyrosine kinase family, the internalization of PDGFR\u0026beta; signifies its activation, which subsequently modulates downstream signaling pathways to regulate a range of cellular activities\u003csup\u003e22,23\u003c/sup\u003e. Phosphoproteomic analyses utilizing KEGG and Wikipathways databases reveal that proteins involved in the PI3K-AKT and ERK signaling pathways are significantly upregulated (Fig. 4i). Immunoblotting analysis demonstrated a significant increase in the phosphorylation levels of PI3K, AKT, and ERK1/2 proteins, which are key components of the PI3K-AKT and ERK signaling pathways, in primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffold (Fig. 4j and Extended Data Fig. 13). Importantly,\u0026nbsp;Cav1\u0026nbsp;knockdown cells cultured on the Nanopore #3 scaffold exhibited a significant reduction in the phosphorylation levels of PI3K, AKT, and ERK1/2 proteins, which are integral to the PI3K-AKT and ERK pathways (Extended Data Fig. 14). This finding suggests that caveolae-mediated endocytosis is essential for Nanopore #3 scaffold\u0026apos;s role in accelerating wound healing by facilitating PDGFR\u0026beta; internalization and the subsequent activation of the PI3K-AKT and ERK signaling pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaveolae-Mediated Endocytosis Drives Wound Healing through\u003c/strong\u003e \u003cstrong\u003eRUNX/FGFBP3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the roles of fibroblasts subsequent to caveolae formation in Nanopore #3 scaffold, RNA sequencing was performed on NIH-3T3 cells cultured on both standard plates and Nanopore #3 scaffold. The RNA sequencing analysis revealed a total of 581 differentially expressed genes, encompassing several genes known to promote regeneration, such as FGFBP3, FGF2, CEMIP, PTX3, and additional related genes (Fig. 5a, b and Supplementary Table 6). Recognizing that FGFBP3 is a secreted protein that binds FGF2, preventing its heparin interaction and facilitating FGFR signaling in wound healing\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e. We initially assessed the expression of FGFBP3 in cells cultured on the Nanopore #3 scaffold. Our findings revealed that both mRNA and protein levels of FGFBP3 were significantly elevated in primary fibroblasts and NIH-3T3 cells cultured on this scaffold, as evidenced by ELISA, immunoblotting, and immunofluorescence assays (Fig. 5c-f). In line with these results, we also noted a significant enhancement in FGFBP3 expression levels in artificial dermis (Extended Data Fig. 15). \u0026nbsp;Furthermore, increased FGFBP3 expression was observed in the concentrated cell supernatants obtained from primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffold, as determined by ELISA analysis (Extended Data Fig. 16a). In addition, tubule formation in HUVECs treated with concentrated supernatant from primary fibroblasts cultured on various nanopore structure scaffolds was differentially enhanced (Extended Data Fig. 16b). However, Cav1\u0026nbsp;knockdown significantly impaired the angiogenesis ability of HUVECs \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(Extended Data Fig. 16c). This suggests that the scaffold may promote wound healing and regeneration through Cav1 regulating the secreted protein FGFBP3. Furthermore, \u003cem\u003ein vivo\u003c/em\u003e studies demonstrated that the levels of skin FGFBP3 at the wound sites of mice treated with the Nanopore #3 scaffold were significantly higher on days 7, 10, and 14 post-wounding, as determined by immunoblotting analysis (Fig. 5g, h). Immunohistochemical analysis of skin wound tissue on day 14 indicated that the Nanopore #3 scaffold markedly increased the expression of FGFBP3 protein in the wound tissue (Fig. 5i). Furthermore, the formation of tubules in HUVECs treated with concentrated supernatant from primary fibroblasts cultured on Nanopore #3 scaffold was significantly reduced upon FGFBP3 knockdown (Fig. 5j and Extended Data Fig. 17), which affects angiogenesis. The data collectively suggest that the increased secretion of FGFBP3 could be a potential mechanism through which the nanoporous scaffold enhances wound regeneration and repair.\u003c/p\u003e\n\u003cp\u003eWe further explored the mechanism through which caveolae-mediated endocytosis enhances FGFBP3 expression. Using DNA pull-down assays in conjunction with LC-MS/MS analysis, we identified 2216 proteins capable of binding to the DNA sequence of FGFBP3 in mouse primary fibroblasts (Supplementary Table 7). Much research highlights the critical role of transcription factors (TFs) as intermediaries between cellular signaling pathways and gene regulation\u003csup\u003e25\u003c/sup\u003e. With this understanding, we utilized the JASPAR online TF prediction tool for bioinformatics analysis to identify potential TFs that could bind to the FGFBP3 promoter. The analysis identified three candidate TFs: RUNX1, MECOM, and CREB1 (Fig. 6a). To confirm these results, we inserted the FGFBP3 promoter sequence into the pGL3 reporter vector and co-transfected it with expression plasmids for RUNX1, MECOM, and CREB1 into 293T cells. Dual-luciferase reporter assays revealed that co-transfection with the RUNX1 overexpression plasmid significantly enhanced luciferase activity driven by the pGL3-FGFBP3 promoter (Extended Data Fig. 18a). Furthermore, the RUNX1 protein was identified in the protein complex precipitated by the FGFBP3 promoter, as detected by immunoblotting (Extended Data Fig. 18b). In primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffolds, both RUNX1 mRNA and protein levels were confirmed to increase, with a significant elevation in phosphorylated RUNX1, as assessed by immunoblotting (Fig. 6b, c). Moreover, p-RUNX1 immunofluorescence staining revealed that the levels of activated RUNX1 that translocated to the nucleus were elevated in cells treated with Nanopore #3 scaffold (Fig. 6d and Extended Data Fig. 19). To determine whether RUNX1 binds directly to FGFBP3, JASPAR predicted four potential RUNX1 binding sites within the FGFBP3 promoter region (Fig. 6e, Supplementary Table 8). Following this, we cloned partial promoter sequences of FGFBP3, containing sites #1 through #4, into pGL3 reporter plasmids (Fig. 6f). These plasmids were then transfected into 293T cells, both with and without the RUNX1 overexpression plasmid. Dual-luciferase reporter assays indicated that co-transfection with the RUNX1 overexpression plasmid and the pGL3-FGFBP3 site #2 promoter significantly increased luciferase activity, whereas co-transfection with sites #1, #3, and #4 along with the RUNX1 overexpression plasmid had no significant effect on luciferase activity in 293T cells (Fig. 6g). Concentrating on RUNX1 binding site #2, ChIP assays confirmed substantial RUNX1-binding activity at this site within the FGFBP3 promoter (Fig. 6h). We then evaluated the impact of RUNX1 silencing on wound repair and regeneration in mice treated with Nanopore #3 scaffolds. FGFBP3 mRNA and protein levels were found to be downregulated in RUNX1 knockdown primary fibroblasts and NIH-3T3 cells cultured on the Nanopore #3 scaffolds (Fig 6i, j and Extended Data Fig. 20). Furthermore, RUNX1 knockdown significantly impaired the \u003cem\u003ein vitro\u003c/em\u003e angiogenic capacity, as indicated by reduced tubule formation in HUVECs treated with concentrated supernatant from primary fibroblasts and NIH-3T3 cells cultured on Nanopore #3 scaffolds (Extended Data Fig. 21).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e, the levels of phosphorylated RUNX1 and its downstream target protein FGFBP3 in the skin of AAV-shCav1-treated mice with the scaffold were significantly decreased compared to those in AAV-shNC-treated mice, as determined by immunoblot and immunohistochemical analysis (Extended Data Fig. 22).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eIn vitro\u003c/em\u003e,\u0026nbsp;the scaffold-induced elevation of phosphorylated RUNX1 and FGFBP3 in fibroblasts with Cav1 knockdown was significantly attenuated, as confirmed by both immunoblot and immunofluorescence analyses (Extended Data Fig. 23). Moreover, after two weeks of intradermal injection with adeno-associated virus (AAV)-shRUNX1 (Fig. 6k), skin wound models were created. In the mice treated with AAV-shRUNX1 and equipped with the Nanopore #3 scaffold, the levels of RUNX1 and FGFBP3 at the wound site were markedly reduced compared to those in mice treated with AAV-shNC (Fig. 6l and Extended Data Fig. 24a-d). Furthermore, the delivery of AAV-shRUNX1 notably suppressed the pathological progression associated with scaffold-induced pro-wound healing. This was evidenced by a larger skin wound size observed on days 1, 3, 7, 10, and 14 following injuries, as well as a decrease in skin wound closure and re-epithelialization rates in comparison to mice treated with AAV-shNC (Fig. 6m-p). In addition, the Nanopore #3 scaffold-induced increase in neovascular marker proteins and collagen fiber content, were substantially diminished in mice treated with AAV-shRUNX1 (Extended Data Fig. 24e, f). Collectively, these findings suggest that the fibroblast caveolae in the skin mediate tissue repair through the RUNX1-FGFBP3 pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBuilding on our previous observations that caveolae formation may enhance Nanopore #3-induced wound healing by internalizing PDGFR\u0026beta;, thereby activating the PI3K-AKT and ERK signaling pathways, we further explored the regulatory impact of caveolae-mediated PDGFR\u0026beta; internalization on RUNX1, which in turn increases the secretion of FGFBP3. This could represent a potential mechanism for promoting wound regeneration and repair. We then assessed the effect of PDGFR\u0026beta; silencing on scaffold-induced FGFBP3 increases in fibroblasts. As anticipated, immunoblot, qPCR, and immunofluorescence analyses showed that scaffold-induced increases in FGFBP3 protein and mRNA levels were significantly downregulated in PDGFR\u0026beta; knockdown fibroblasts (Extended Data Fig. 25a-c). Consistently, PDGFR\u0026beta; knockdown also notably reduced FGFBP3 levels in the cell supernatant (Extended Data Fig. 25d). Therefore, we hypothesize that PDGFR\u0026beta;, internalized into the cytoplasm with Cav1, activates downstream PI3K-AKT and ERK signaling pathways, which in turn promote RUNX1 phosphorylation and FGFBP3 transcription. To further confirm the hypothesis, we treated fibroblasts cultured on Nanopore #3 scaffold with the PI3K inhibitor Thioridazine (SIGMA, USA, #T905, 2 \u0026mu;M, 24 h) and the ERK inhibitor FR180204 (SIGMA, USA, #328010, 2 \u0026mu;M, 24 h). Immunoblot analysis revealed that the expression of p-RUNX1 and FGFBP3 proteins upregulated by the scaffold was markedly reduced in fibroblasts treated with ERK or PI3K inhibitors (Extended Data Fig. 26). Conversely, RUNX1 knockdown did not significantly affect the PI3K-AKT and ERK pathways in fibroblasts cultured on the scaffold (Extended Data Fig. 27). Collectively, these data indicate that PDGFR\u0026beta; internalization with caveolae formation activates PI3K-AKT and ERK signaling, thereby promoting RUNX1 phosphorylation and FGFBP3 transcription.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAV1 Mediated Release of CBF\u0026beta; from FLNA Enhances RUNX1 Activity to Promote FGFBP3 Transcription Post-Caveolae Endocytosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInterestingly, caveolae interact with the dynamic cytoskeleton, which is crucial for sensing and converting mechanical forces into cellular signals\u003csup\u003e26,27\u003c/sup\u003e. Previous studies have shown that FLNA engages with Cav1 via the hinge region between the 23rd and 24th homologous repeats of 96 amino acids each repeats\u003csup\u003e28,29\u003c/sup\u003e. Results from double immunofluorescence staining demonstrated substantial co-localization of Cav1 and FLNA in fibroblasts cultured on scaffolds (Fig. 7a and Extended Data Fig. 29a). CBF\u0026beta;, a co-transcription factor that binds to RUNX1, is composed of two domains with distinct structural and functional characteristics. The first domain is a loosely structured regulatory region that is capable of interacting with a variety of molecules\u003csup\u003e30\u003c/sup\u003e. For example, when the 23-24 repeat hinge region of FLNA binds to this regulatory domain, it sequesters CBF\u0026beta; in the cytoplasm, thereby preventing its involvement in the transcription factor complex. The second domain is an execution domain with a rigid structure that specifically interacts with the RUNX1 protein. Once CBF\u0026beta; forms a complex with RUNX1, it acts as a transcription factor within the cell nucleus\u003csup\u003e31,32\u003c/sup\u003e. CBF\u0026beta; and CAV1 compete for the same binding region on FLNA. Our double immunofluorescence data show that the nuclear localization of CBF\u0026beta; bound to RUNX1 increased in fibroblasts cultured on the scaffold, while the cytoplasmic retention of CBF\u0026beta; bound to FLNA was markedly decreased, in contrast to fibroblasts cultured on standard plates (Fig. 7b, c and Extended Data Fig. 28b, c). Moreover, immunoprecipitation assays corroborated the findings from the immunofluorescence double staining (Fig. 7d, e). Notably, these assays also indicated a stronger interaction between \u003csup\u003eY14\u003c/sup\u003ep-Cav1 and FLNA in fibroblasts cultured on scaffolds compared to control cells (Extended Data Fig. 29). This suggests that both Cav1 and \u003csup\u003eY14\u003c/sup\u003ep-Cav1 can bind to FLNA, thereby preventing it from binding to CBF\u0026beta;. As a result, CBF\u0026beta; is released to enter the nucleus and function as a transcriptional cofactor, binding to RUNX1.\u003c/p\u003e\n\u003cp\u003eUnderstanding that CBF\u0026beta; is a co-transcription factor that interacts with RUNX1, we explored its role in regulating FGFBP3, a target gene of the RUNX1 transcription factor. Immunoblot analysis indicated a significant increase in the nuclear localization of both RUNX1 and CBF\u0026beta; proteins in fibroblasts cultured on Nanopore #3 scaffold, with a corresponding decrease in their cytoplasmic localization (Fig. 7f, g). Furthermore, immunoblot, qRT-PCR, and immunofluorescence analysis revealed that the scaffold-induced elevation of FGFBP3 mRNA and protein levels was reduced in CBF\u0026beta; knockdown fibroblasts, although still slightly higher compared to siNC cells (Fig. 7h-j and Extended Data Fig. 28d-f). Together, these results suggest that the scaffold enhances the interaction between Cav1 or \u003csup\u003eY14\u003c/sup\u003ep-Cav1 and FLNA, thereby preventing FLNA from binding to CBF\u0026beta;. Consequently, CBF\u0026beta; is released to translocate into the nucleus, where it acts as a transcriptional cofactor with RUNX1 to facilitate the production of the FGFBP3 protein (Fig. 7k).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA comprehensive understanding of the biophysical and biochemical properties of scaffolds is crucial for optimizing the use of endogenous cells and promoting cellular reprogramming in tissue engineering. The precise integration of these cues, customized to meet the specific needs of various target tissues, could pave the way for new advances in biomaterial-based therapies. In our study, we examined the effects of scaffolds with varying nanopores on wound healing and cell fate. Our findings show that scaffolds with distinct nanopores differentially enhance skin wound healing. Fibroblasts, which are the primary cells that sense and respond to scaffolds, play a pivotal role in addressing full-thickness skin defects, potentially initiating the repair process and leading to the formation of new tissue structures. The formation of caveolae and the initiation of endocytosis on the cell surface during fibroblast deformation on the scaffold are among the cellular responses to the nanopore features. During this biological process, caveolae can bud off from the plasma membrane to form endocytic vesicles. These vesicles, in concert with Cav1 or other lipid-interacting proteins, are internalized into the cytoplasm, thereby triggering a series of signaling pathways\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Additionally, Cav1 within the endocytic vesicles can interact with cytoplasmic proteins, thereby modulating key cellular processes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that the formation of caveolae in fibroblasts represents an initial step in the wound healing response to the nanopores n scaffolds. Tissue-resident fibroblasts, a subset of mesenchymal cells, display significant plasticity, enabling them to adjust their phenotype in response to environmental signals. For instance, during the wound healing process, myofibroblasts are capable of transdifferentiating into adipocytes, demonstrating their adaptability within the microenvironment\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These cells are crucial for maintaining tissue homeostasis and are involved in physiological processes such as wound healing, tissue repair, and fibrosis. The biophysical cues of biomaterials can significantly impact fibroblast behavior, encompassing epigenetic and metabolic reprogramming\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our research demonstrates that the nanopores of scaffolds regulate fibroblast biology through transcriptomic, proteomic, and phosphoproteomic analyses, clarifying how fibroblasts detect and react to environmental stimuli. The interaction between fibroblasts and nanopores may trigger the reparative process, with scaffolds guiding cellular functions that are vital for tissue regeneration, which is essential for harnessing the body's inherent regenerative capacity\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Previous research has demonstrated that the nanopores within scaffolds significantly influence tissue regeneration, with pore size and architecture being critical factors in cellular adhesion, proliferation, and matrix deposition\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Advancements in this field may yield interventions that can modulate fibroblast activity to promote tissue regeneration without aggravating fibrosis, offering novel therapeutic strategies for diseases marked by abnormal tissue repair.\u003c/p\u003e \u003cp\u003eTo elucidate the fibroblast sensing nanopores, we conducted comprehensive proteomic and phosphoproteomic analyses. These omics data, in conjunction with the established role of caveolae in sensing external microenvironments and stimuli, prompted us to hypothesize that caveolae formation in fibroblasts is pivotal for nanopore detection. Our study demonstrates that fibroblasts detect nanopores through the phosphorylation of Cav1 and the endocytosis of caveolae. Beyond their role in sensing and responding to mechanical forces, caveolae formation also appears to be a response to nanopores. Caveolae, which are enriched with specific lipids and proteins, form distinct nanodomains that play a crucial role in cellular signaling pathways. These membrane invaginations interact with various signaling molecules, and their dynamics are vital for intracellular signaling processes. Receptor tyrosine kinases, such as PDGFRβ, are a class of cell-surface receptors that sense and transmit environmental signals, activating their inherent enzymatic activity and functioning as enzymes. Upon binding with Cav1, PDGFRβ is internalized into the cytoplasm during caveolae endocytosis, initiating its tyrosine kinase activity, which subsequently activates the phospho-PI3K, phospho-AKT, and phospho-ERK1/2 pathways. PDGFRβ can also interact with Src family kinases, including Lyn and Fyn, further activating its tyrosine kinase activity and leading to the phosphorylation of tyrosine residues on proteins\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In the context of nanopore interactions, we have found that the transcription factor RUNX1 is phosphorylated by tyrosine kinase pathways during caveolae endocytosis. The phosphorylated RUNX1 then translocates to the nucleus, where it promotes the transcription of FGFBP3. FGFBP3 can bind to FGF2, preventing its interaction with heparin and likely inhibiting the immobilization of FGF2 on extracellular matrix glycosaminoglycans. This allows for the release and subsequent activation of FGFR signaling. By modulating the activity and bioavailability of FGF2, FGFBP3 influences wound healing processes. This discovery is consistent with studies showing that RUNX1 significantly modulates the regenerative capacity of wound-activated fibroblasts, thereby affecting healing outcomes\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Additionally, in this study, we observed that the expression of \u003csup\u003eY\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003ep-Cav1 is associated with fibroblast proliferation, suggesting that reparative fibroblasts possess an inherent yet adaptable regenerative potential.\u003c/p\u003e \u003cp\u003eAnother key focus of this research is the examination of the role of Cav1 within the cytoplasm. Cav1, a key component of caveolae, is internalized into the cytoplasm where it interacts with a variety of molecules, thereby participating in the regulation of numerous cellular processes. FLNA, a member of the actin-binding protein family, engages with a broad spectrum of molecules, including transcription factors and co-activators\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that Cav1 can interact with Filamin-A in the cytoplasm\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Significantly, CBFβ and Cav1 compete for the same binding site on FLNA. Upon binding to FLNA, Cav1 forms a complex that affects several critical cellular processes. One of these processes is the release of CBFβ from FLNA, which then promotes its nuclear translocation. Within the nucleus, CBFβ forms heterodimers with Runx family members (RUNX), thereby enhancing their DNA-binding affinity and transcriptional activity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. While CBFβ does not directly interact with DNA, it significantly enhances RUNX1's ability to activate the transcription of FGFBP3. These results emphasize that caveolae, upon sensing the nanostructures of a scaffold, initiate a complex series of cellular events that are crucial for tissue regeneration.\u003c/p\u003e \u003cp\u003eIn conclusion, fibroblasts have an innate yet adaptable regenerative capacity that is triggered by their recognition of scaffold nanostructures. The formation of caveolae indicates the cell's response to these nanostructures and marks the beginning of reparative processes. This response initiates a series of complex biological events that ultimately lead to tissue regeneration and repair. Consequently, the cellular caveolae response can be harnessed as a key parameter in the design of scaffold nanostructures, potentially improving therapeutic outcomes for patients.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eBCA, bicinchoninic acid; Cav1, caveolin-1; ChIP, chromatin immunoprecipitation; ECM, extracellular matrix; FBS, fetal bovine serum; FGFBP3, fibroblast growth factor binding protein 3; FLNA, filamin-A; HUVECs, human umbilical vein endothelial cells; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TFs, transcription factors\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical approval and consent to participate:\u003c/h2\u003e\n\u003cp\u003eThe study protocol was granted ethical approval by the Ethics Committee of our institute (Reference Number: 2023-R15, WYYY-AEC-YS-2023-0606).\u003c/p\u003e\n\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\n\u003cp\u003eThe present study was financially supported by the National Natural Science Foundation of China (No. 82241031), Science and Technology Bureau (No. Y20240095) and Natural Science Foundation of Science and Technology Bureau (No. 2023J254).\u003c/p\u003e\n\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\n \u003ch2\u003eAuthors\u0026rsquo; Contributions:\u003c/h2\u003e\n \u003cp\u003eConceptualization: K.Q.S. Methodology and investigation: F.X.G, W.X, W.T.L, M.L.Y, H.B.L, S.F.W, X.Y.N, E.A, H.W.W, and J.W. Data analysis: F.X.G, W.X, W.T.L and K.Q.S. Writing (original draft): F.X.G and K.Q.S. Writing (review and editing): J.X, Z.M.C, and K.Q.S. Funding and supervision: F.X.G and K.Q.S.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFalanga, V.\u003cem\u003e et al.\u003c/em\u003e Chronic wounds. \u003cem\u003eNat Rev Dis Primers\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 50, doi:10.1038/s41572-022-00377-3 (2022).\u003c/li\u003e\n\u003cli\u003eMartin, P., Pardo-Pastor, C., Jenkins, R. G. \u0026amp; Rosenblatt, J. Imperfect wound healing sets the stage for chronic diseases. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e386\u003c/strong\u003e, eadp2974, doi:10.1126/science.adp2974 (2024).\u003c/li\u003e\n\u003cli\u003eFreedman, B. 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A. mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0293921, doi:10.1128/mBio.02939-21 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fibroblast, caveolae, wound healing, scaffold, RUNX1, FGFBP3","lastPublishedDoi":"10.21203/rs.3.rs-5955293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5955293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eScaffolds with pores can influence cellular fate and tissue microenvironments by altering intracellular and intercellular signaling pathways, which are crucial for guiding tissue regeneration. Despite their significance, the cellular mechanisms behind the response to pores, especially at the nanoscale, are not well understood. Our study shows that scaffolds with different nanopore characteristics can enhance skin regeneration in various ways, with fibroblasts being the primary cellular responders. We have demonstrated that caveolae formation is a critical mechanism by which fibroblasts interact with nanopores. The phosphorylation of caveolin-1 (Cav1) is a key event in this process, enabling caveolae-mediated endocytosis and the subsequent internalization of cellular substances. This initiates a cascade of events involving the internalization of cell surface receptors such as PDGFRβ, activation of tyrosine kinase pathways including PI3K, AKT, and ERK1/2, and the phosphorylation of the transcription factor RUNX1. The nuclear translocation of RUNX1 upregulates the expression of fibroblast growth factor binding protein 3 (FGFBP3), which is a key factor in tissue repair. Additionally, cytoplasmic Cav1 can competitively bind to Filamin-A, releasing CBFβ, which then translocates to the nucleus and enhances RUNX1's DNA-binding affinity, synergistically activating FGFBP3 transcription and promoting tissue healing. Collectively, our findings underscore the importance of considering nanopore characteristics in scaffold design for tissue regeneration and highlight caveolae formation as a central mediator of cellular responses to nanoporous environments, initiating a multitude of biological processes essential for tissue repair and regeneration.\u003c/p\u003e","manuscriptTitle":"Caveolae-Mediated Sensing of Nanoporous Cues in Fibroblasts Reprogramming During Wound Healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-19 07:16:35","doi":"10.21203/rs.3.rs-5955293/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5a05844-d7c9-4108-ae67-8597541cd152","owner":[],"postedDate":"February 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44369615,"name":"Biological sciences/Biophysics/Endocytosis"},{"id":44369616,"name":"Health sciences/Diseases/Trauma"}],"tags":[],"updatedAt":"2025-10-07T20:55:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-19 07:16:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5955293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5955293","identity":"rs-5955293","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}