Mapping Connective Tissue Molecular Blueprints to Illuminate Human Keratinized and Non-Keratinized Oral Mucosa

Mapping Connective Tissue Molecular Blueprints to Illuminate Human Keratinized and Non-Keratinized Oral Mucosa | 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 Mapping Connective Tissue Molecular Blueprints to Illuminate Human Keratinized and Non-Keratinized Oral Mucosa Shoucheng Chen, Ruoxuan Huang, Leyao Xu, Chunxin Xu, Yuanxiang Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5368489/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 A fundamental question in oral science is elucidating the factors that underpin the distinct tissue characteristics of human keratinized and non-keratinized mucosa. Clinical autotransplantation and large animal studies have observed that intrinsic regulation within connective tissue defines mucosal phenotypes, emphasizing the need for in-depth molecular characterization, which remains largely unexplored. This study aimed to map the molecular blueprints of in situ connective tissues and isolated fibroblasts of human keratinized oral mucosa (gingiva, GIN) and non-keratinized oral mucosa (alveolar mucosa, ALV). Distinct variations were observed in extracellular matrix composition, retinoic acid metabolism (closely associated with keratinization), and immune function between GIN and ALV. GIN displayed higher expression of collagen-related genes (notably COL1 and COL3) and lower expression of elastin-related genes. In GIN, the retinol metabolism pathway was enriched, with downregulation of retinoic acid synthesis and upregulation of its catabolism. In contrast, the complement and coagulation cascade were notably upregulated in ALV, with significantly elevated expression of C3. This study is the first to systematically dissect and compare the molecular profiles of connective tissue in GIN and ALV providing foundational insights that could drive future advancements in mucosal phenotype modulation and regenerative therapies. Health sciences/Health care/Dentistry/Periodontics Health sciences/Health care/Dentistry/Dental treatments/Dental implants Keratinized Oral Mucosa Non-Keratinized Oral Mucosa Connective Tissue Fibroblast Molocular Profile Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In the oral cavity, keratinized and non-keratinized mucosa are anatomically adjacent yet exhibit marked differences in tissue characteristics. Keratinized mucosa has a denser extracellular matrix, increased abrasion resistance, and enhanced barrier functions, which together protect periodontal and peri-implant tissues from mechanical and microbial challenges. In contrast, non-keratinized mucosa lacks a keratin layer, making it softer and more elastic to accommodate oral movements; however, it tends to elicit a stronger inflammatory response under stressors [ 1 – 5 ] (Fig. 1 A). Understanding the factors responsible for these distinct characteristics is a shared focus of both basic researchers and clinicians, as it is essential for advancing mucosal phenotype modulation and regenerative therapies. Despite this interest, the underlying determinants of these phenotypic distinctions remain elusive. Current perspectives on keratinized and non-keratinized mucosal phenotypes emphasize the role of local environmental factors. For instance, mechanical stimuli have been shown to promote keratinization in the oral mucosa [ 6 ]. Additionally, the presence of natural teeth appears to foster keratinization, with spontaneous keratinized mucosa formation reported following removal of the keratinized epithelium—even in the absence of connective tissue grafting (CTG) [ 7 ]. Transplanting non-keratinized tissue adjacent to natural teeth similarly results in keratinization of the transplanted tissue [ 8 , 9 ].However, given the complexity and variability of these external stimuli, achieving consistent and controllable soft tissue regeneration remains challenging, underscoring the need to explore endogenous mechanisms that may provide more stable and predictable regulation of mucosal phenotype. Recently, increasing evidence points to the importance of connective tissue in determining mucosal phenotype. Both clinical outcomes and animal experiments suggest that connective tissue intrinsically determines human keratinized and non-keratinized oral mucosal phenotypes. In clinical practice, free gingival grafting, regarded as the "gold standard" for increasing the width of keratinized mucosa, demonstrates that transplanted keratinized mucosa retains its phenotype when integrated into a non-keratinized area [ 10 ]. This indicates that mucosal keratinization may be largely determined by intrinsic genetic factors rather than external factors such as mechanical stimuli. In basic research, animal studies have further confirmed that when connective tissue from keratinized mucosa is transplanted into a non-keratinized region and the original epithelium is removed, the transplanted connective tissue retains its keratinized properties, and the newly formed epithelium also exhibits keratinized features [ 11 , 12 ](Fig. 1 B). These growing evidences underscore a pressing need for in-depth molecular characterization of connective tissue in these mucosal types, which could unlock new, targeted approaches for modulating mucosal phenotypes and advancing regenerative therapies. To address this gap, the present study aims to dissect the molecular profiles of human keratinized (gingiva, GIN) and non-keratinized (alveolar mucosa, ALV) oral mucosa by examining in situ connective tissues and isolated fibroblasts (Fig. 1 C). Based on bioinformatics analyses, we investigated and validated differences in three key domains: extracellular matrix composition, metabolic pathways, and immune responses. Key gene expression was validated via qPCR, while protein expression and localization were confirmed using immunohistochemistry, western blotting, and immunofluorescence. Our objective is to provide a molecular blueprint that lays the groundwork for future advances in phenotype modulation and mucosal regeneration. Results Transcriptomic analysis revealed extensive molecular differences between the GIN and ALV groups The boxplot of FPKM values reveals a generally consistent distribution of gene expression levels across samples, with the ALV group demonstrating greater consistency (Supplementary Fig. 1a). Principal Component Analysis (PCA) illustrates distinct spatial separation between GIN and ALV samples, indicating significant differences between the two groups. Specifically, ALV samples cluster more closely together, whereas GIN samples exhibit greater dispersion, reflecting increased heterogeneity within the GIN group (Supplementary Fig. 1b). Differential expression analysis identified 1,309 genes with significant expression changes (p-value 1), including 480 genes that were upregulated and 829 genes that were downregulated in GIN compared to ALV group (Supplementary Fig. 1c). Heatmaps visually represent the extensive differences in gene expression profiles between GIN and ALV samples, while volcano plots further illustrate the distribution of differentially expressed genes between the two groups. Notably, many genes upregulated in GIN compared to ALV group are associated with extracellular matrix and collagen ( e.g. , COL1A1, COL3A1, COL5A1, COL7A1). Conversely, the most significantly downregulated genes in GIN are linked to the complement system ( e.g. , C3) and vitamin A metabolism ( e.g. , AOX1, ADH4, ADH1A) (Fig. 2 a). The extracellular matrix (ECM) composition emerged as the primary distinction between the molecular profiles of the GIN and ALV groups GO enrichment analysis of differentially expressed genes initially revealed a predominant prevalence of terms associated with the extracellular matrix across Biological Process, Cellular Component, and Molecular Function categories, highlighting 726 enriched terms. Notably, most of the enriched terms within the Cellular Component (CC) classification were related to the extracellular matrix (Fig. 2 b, Supplementary Fig. 2). The heatmap of differentially expressed extracellular matrix-related genes indicates a considerable number of upregulated genes in both the GIN and ALV groups; however, these genes are clustered within distinct functional modules. Specifically, genes upregulated in GIN are concentrated in collagen synthesis-related functions, while those upregulated in ALV are associated with elastin and collagen regulation (Fig. 2 c). Protein-protein interaction (PPI) analysis, followed by MCODE clustering, identified core genes upregulated in GIN primarily related to collagen synthesis and remodeling, such as fibril-forming collagens ( e.g. , COL1A1, COL1A2, COL3A1, COL5A1, COL11A1), basement membrane collagen (COL7A1), short-chain collagens ( e.g. , COL10A1, COL6A1, COL8A1), and genes associated with collagen fiber synthesis and remodeling ( e.g. , MMP1, MMP11, ADAMTS2, FMOD, LOX, PCOLCE, and SERPINH1). Conversely, genes related to elastin fibers and their synthesis and remodeling, including ELN1, FBLN1, EMILIN2, MFAP4, and MFAP5, were upregulated in ALV (Fig. 2 d,e). Additionally, GSEA results revealed upregulation of terms related to extracellular matrix scaffolding and barrier function in GIN (Supplementary Fig. 3). RT-qPCR validation corroborated the transcriptome sequencing results, showing significantly higher expression of COL1A1, COL5A1, COL5A2, COL6A2, and the collagen synthesis-related gene SERPINH1 in GIN compared to ALV. Conversely, MFAP4, involved in elastin fiber assembly, was significantly less expressed in GIN (Fig. 3 a,b). Immunohistochemistry (IHC) staining revealed widespread expression of Type I collagen (COL1) in the extracellular matrix of GIN, with semi-quantitative analysis indicating significantly higher COL1 expression in GIN compared to ALV ( p = 0.009). Type III collagen (COL3) was expressed sparsely in both GIN and ALV but at significantly higher levels in GIN ( p = 0.049) (Fig. 3 c, Supplementary Fig. 4). Verification at both tissue and cellular levels showed that collagen-related genes—COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, COL6A2, COL12A1, and COL16A1—were more highly expressed in gingival fibroblasts (HGF) compared to alveolar mucosal fibroblasts (HAMF), whereas elastin (ELN) expression was lower in HGF. Statistical analysis confirmed that COL5A2 ( p = 0.015), COL6A2 ( p = 0.016), and COL12A1 ( p = 0.025) were significantly more expressed in HGF than in HAMF(Fig. 3 d). Western blot analysis revealed significantly higher COL1 expression in HGF compared to ALV ( p = 0.033), while COL3 expression did not differ significantly (Fig. 3 e). Immunofluorescence staining further demonstrated that COL1 expression is notably higher in HGF compared to HAMF (Fig. 3 f). The retinol metabolism pathway, which is closely associated with keratinization, exhibited significant differences between the GIN and ALV groups KEGG enrichment analysis of differentially expressed genes revealed significant involvement of the vitamin A metabolic pathway, which plays a critical role in epithelial keratinization (Fig. 4 a). GSEA results indicated that upstream genes of this pathway were significantly upregulated in GIN, whereas downstream genes were downregulated (Fig. 4 b). Further examination showed that genes involved in retinoic acid synthesis, including alcohol dehydrogenases (ADH1A, ADH1B, ADH1C, ADH4), aldehyde dehydrogenases (ALDH1A2, ALDH1A3), and aldehyde oxidase (AOX1), exhibited lower expression in GIN compared to ALV. Conversely, genes related to retinoic acid metabolism, such as cytochrome P450 enzymes (CYP26B1, CYP2C18, CYP3A5), were upregulated in GIN (Fig. 4 c, d). RT-qPCR validation confirmed that the expression trends were consistent with transcriptome sequencing results. Specifically, ADH1 ( p = 0.037), ALDH1A3 ( p = 0.043), and AOX1 ( p < 0.001) were significantly upregulated in GIN (Fig. 5 a). Immunohistochemistry (IHC) staining showed that alcohol dehydrogenase (ADH) was sparsely expressed in ALV, predominantly in fibroblasts, with minimal ADH staining in GIN. Semi-quantitative analysis further revealed significantly higher ADH expression in ALV compared to GIN ( p = 0.004). Similarly, aldehyde oxidase 1 (AOX1) was observed in endothelial cells and fibroblasts in both GIN and ALV, with a higher number of AOX1-positive fibroblasts in ALV. Semi-quantitative analysis confirmed that AOX1 expression was significantly higher in ALV than in GIN ( p = 0.025). Cytochrome P450 26B1 (CYP26B1) was diffusely expressed in both GIN and ALV, with no significant difference in expression levels between the two groups (Fig. 5 b, Supplementary Fig. 4). Further RT-qPCR validation in fibroblasts (HGF and HAMF) showed that ADH1, ALDH1A2, and AOX1 were more highly expressed in HGF compared to HAMF (Fig. 5 c). Western blot analysis indicated a significantly lower expression of AOX1 in HGF compared to ALV (p = 0.014), while the expression levels of ADH and CYP26B1 did not exhibit statistically significant differences (Fig. 5 d). Consistently, immunofluorescence staining revealed markedly higher AOX1 expression in HAMF relative to HGF, with no significant differences observed for ADH and CYP26B1 expression (Fig. 5 e, Supplementary Fig. 5). The complement system was more active in the ALV group KEGG analysis indicated that nearly 20% of the differentially expressed genes were enriched in immune system-related pathways (Supplementary Fig. 2). GO enrichment analysis of differentially expressed genes revealed significant enrichment in immune-related categories, particularly those associated with complement pathways (Fig. 6 a). The differential expression heatmap showed that immune-related genes are predominantly downregulated in GIN relative to ALV (Fig. 6 b). Enrichment analysis using ClueGO highlighted that these immune-related genes are notably involved in complement activation and T lymphocyte chemotaxis functions (Fig. 6 c). The GO enrichment circle plot confirmed that complement pathway genes are uniformly downregulated in GIN, with most genes related to "immune response" and "inflammatory response" also exhibiting lower expression levels (Fig. 6 d). Furthermore, GSEA analysis revealed significant downregulation of gene sets associated with the complement and coagulation cascade pathways in GIN (Fig. 6 e). Analysis of the complement and coagulation cascade pathway genes demonstrated that complement activation-related genes are substantially downregulated in GIN. Core components of the recognition phase, including C1QA, C1QB, and C1QC, were expressed at 2.74, 2.95, and 2.61 times higher levels in ALV compared to GIN, respectively. In the activation phase, C3 was expressed 26 times higher in ALV than in GIN. For the membrane attack phase, core components C6 and C7 were expressed 56.37 and 11.09 times higher in ALV, respectively (Fig. 6 f). RT-qPCR validation of key genes in the complement system confirmed that expression trends align with transcriptome sequencing results. Specifically, complement components C1QA ( p = 0.006), C1QB ( p = 0.012), C1QC ( p = 0.009), C3 ( p = 0.038), C6 ( p = 0.034), and C7 ( p = 0.012) were all significantly upregulated in GIN (Fig. 6 g). IHC staining results further demonstrated that complement component C3 is predominantly expressed in endothelial cells of the superficial connective tissue. Semi-quantitative analysis indicated that C3 expression levels are significantly higher in ALV compared to GIN (p = 0.046) (Fig. 6 h. Supplementary Fig. 5). Discussion Studies have shown that connective tissue is a key determinant of oral mucosal phenotype, but the specific characteristics contributing to this role are not yet fully understood. A comprehensive understanding of the differences between keratinized and non-keratinized mucosa is crucial for exploring the regulatory mechanisms of mucosal phenotypes [ 12 ]. In this study, we performed transcriptomic sequencing on connective tissues from healthy human gingiva and alveolar mucosa, analyzing gene expression differences in three key areas: extracellular matrix composition, metabolic characteristics, and immune responses. Our aim was to provide direction and evaluation metrics for understanding the molecular mechanisms underlying mucosal keratinization and phenotype regulation. Literatures have shown that keratinization can be modulated by certain elements of ECM. Specific type of collagen, including type VI and type XVIII collagen, has been found to enhance the expression of keratinization-associated genes, while elastin has been found to inhibit keratinization[ 13 – 15 ]. The findings of this study demonstrate that extracellular matrix-related genes are the primary contributors to the differences between keratinized and non-keratinized mucosal connective tissues, consistent with histological observations shown in Supplementary Fig. 4 and supported by recent studies [ 16 ]. Further analysis of differentially expressed genes revealed that type I and type III collagen, the major structural proteins of connective tissue, exhibited gene expression levels more than five times higher in keratinized mucosa compared to non-keratinized mucosa. This significant upregulation suggests a prominent role for these collagens in maintaining the structural integrity and function of keratinized mucosa. Furthermore, genes related to type V collagen [ 17 ], type VI collagen [ 18 – 20 ], and FACIT collagens [ 21 ], which play important roles in collagen fiber assembly and functional regulation of cells, were more highly expressed in gingiva. Other genes directly involved in collagen synthesis and remodeling, such as SERPINH1, which encodes heat shock protein 47—a collagen-specific molecular chaperone essential for correct procollagen folding—were significantly upregulated in gingiva [ 22 ]. Genes such as PCOLCE (procollagen C-endopeptidase enhancer protein), ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs), and LOX (lysyl oxidase), which positively regulate collagen synthesis, assembly, and degradation, were also upregulated in gingiva [ 21 – 29 ]. Conversely, TIMP3, a tissue inhibitor of metalloproteinases, was downregulated in gingiva. These results suggest that collagen fibers in gingiva may have a more active synthesis and metabolic process. qPCR validation revealed no statistically significant differences for most genes, indicating that further studies with larger sample sizes are needed to confirm these results. Vitamin A (retinol) and its metabolic derivatives, retinoids, play important regulatory roles in epithelial cell proliferation, differentiation, and oral mucosal phenotype transformation, with all-trans retinoic acid (ATRA) being the most biologically active metabolite [ 30 , 31 ]. Studies have shown that retinoids inhibit epithelial keratinization and influence mucosal phenotype transformation by downregulating keratinized epithelial markers K1 and K10 and upregulating non-keratinized epithelial markers K4 and K13 [ 31 – 33 ]. In vitro studies by Ozaki et al. demonstrated that fetal bovine serum inhibited keratinization of mouse epithelial cells, but the addition of a retinoic acid receptor inhibitor reversed this effect [ 34 ]. Additionally, Miyazono et al. confirmed that ATRA inhibits keratinization of epithelial cells in mice, pigs, and humans [ 35 ].In the vitamin A metabolic pathway, retinol is oxidized by alcohol dehydrogenase to retinal, which is further oxidized by aldehyde dehydrogenase and aldehyde oxidase to retinoic acid [ 36 , 37 ], and then metabolized by cytochrome P450 enzymes (particularly CYP26) [ 38 , 39 ]. In our study, genes related to the retinoic acid metabolic pathway were significantly enriched in alveolar mucosa (ALV). Genes such as ADH1A/B/C, ADH4, ALDH1A2/3, and AOX1, which promote retinoic acid synthesis, were highly expressed in ALV, whereas CYP26B1, CYP2C18, and CYP3A5 were expressed at lower levels in ALV. This suggests that retinoic acid synthesis may be more active in ALV, thereby inhibiting epithelial keratinization and influencing mucosal phenotype. Further qPCR and IHC analyses confirmed differences in the expression of ADH and AOX1, with IHC showing significant ADH expression in ALV fibroblasts but minimal expression in gingival tissue (GIN). There are seven isoenzymes of ADH in humans, with ADH1A-C (class I) and ADH4 (class II) playing important roles in vitamin A metabolism [ 36 ]. Studies have shown that ADH1 and ADH4 knockout mice have significantly reduced levels of retinoic acid [ 40 ], suggesting that retinoic acid synthesis may be inhibited in gingival tissue. Fibroblast validation results showed higher AOX1 expression in gingival fibroblasts compared to alveolar mucosa, but no significant difference in ADH expression between the two, possibly due to regulation by substances such as retinoic acid and glucocorticoids, which cannot be replicated in vitro [ 41 , 42 ]. These findings suggest that differences in ADH expression between gingiva and alveolar mucosa may be influenced by environmental factors rather than intrinsic genetic characteristics of fibroblasts. In summary, the high expression of genes and proteins related to retinoic acid synthesis in non-keratinized alveolar mucosal connective tissue suggests that retinoic acid synthesis is more active in ALV, which may be one of the pathways through which connective tissue regulates mucosal phenotype transformation. In terms of immune response, the oral mucosa, as the first line of defense against external stimuli, is exposed to abundant microorganisms and mechanical stress [ 1 ]. Our study found differences in immune activity between different mucosal phenotypes, with genes related to "immune response" and "inflammatory response" highly expressed in alveolar mucosa, indicating higher immune activity. The differences were mainly observed in the activation of the complement system, with related genes significantly upregulated in ALV. The lack of keratinization in ALV results in weaker barrier function, making it more susceptible to microbial and environmental influences [ 1 ], which may lead to more active immune responses compared to GIN. The complement system is a key component of innate immunity and can be activated through the classical, lectin, and alternative pathways. The classical pathway is initiated by C1q recognition of antigen-antibody complexes, while the alternative pathway is rapidly activated upon pathogen stimulation [ 43 ]. Complement activation can induce immune cell activation, leading to tissue inflammation and specific immune responses [ 43 , 44 ]. Studies have shown that tissue destruction in periodontitis is mediated not only by microorganisms but also by the host immune response, with excessive complement activation closely related to the onset of periodontitis and tissue damage [ 1 , 44 – 47 ]. Moreover, differences in complement activation may explain the higher prevalence of peri-implantitis and more severe marginal bone loss in the absence of keratinized mucosa around implants [ 48 , 49 ]. To our knowledge, this is the first study to perform transcriptomic sequencing of gingival and alveolar mucosal connective tissues in healthy individuals, systematically characterizing their gene expression differences under physiological conditions. The discovered differences in extracellular matrix components, vitamin A metabolism, and complement system activation may provide new directions and evaluation metrics for studying mucosal phenotype modulation and regeneration (Fig. 7 ). Based on these findings, future strategies for mucosal phenotype regulation include altering extracellular matrix composition, structure, or mechanical properties; applying bioactive molecules ( e.g. , retinoic acid) to regulate epithelial cell behavior; and utilizing specific bioactive materials to optimize the cellular microenvironment, precisely regulating keratinized or non-keratinized oral mucosal phenotypes to achieve long-term stability and functional maintenance of soft tissues. Despite the valuable insights gained, this study has some limitations. Firstly, the limited sample size may affect the generalizability of the findings, and larger sample sizes are needed to confirm the observed gene expression differences. Additionally, transcriptomic sequencing was based on whole tissue RNA, limiting the resolution of cell-type-specific gene expression. Single-cell RNA sequencing (scRNA-seq) or spatial transcriptomics could provide more detailed insights into the role of connective tissue in regulating epithelial phenotypes. In conclusion , the transcriptomic analysis of gingival (GIN) and alveolar (ALV) mucosal tissues revealed significant differences, particularly in the extracellular matrix composition. Genes associated with collagen synthesis and remodeling were notably upregulated in GIN. In terms of metabolic pathways, the retinol metabolism pathway displayed marked differences, with genes involved in retinoic acid synthesis downregulated in GIN. Immunologically, the complement system was more active in ALV, as evidenced by the upregulation of related genes. This study is the first to systematically map the molecular profiles of connective tissue in human keratinized and non-keratinized oral mucosa, providing foundational insights that could drive future advancements in mucosal phenotype modulation and regenerative therapies. Materials and Methods Tissue Sample Collection Gingival and alveolar mucosal samples were obtained from healthy individuals undergoing dental implant surgery or tooth extraction at Sun Yat-sen University Affiliated Stomatological Hospital in 2022. The study was approved by the Medical Ethics Committee of Sun Yat-sen University Affiliated Stomatological Hospital (KQEC-2021-73-02), and all participants provided informed consent. The inclusion and exclusion criteria were as follows: Inclusion Criteria: 1. Individuals aged 18-40 years; 2. Need for dental implant restoration or tooth extraction; 3. Healthy gingiva with no signs of acute or chronic inflammation; 4. Keratinized mucosa width ≥8 mm at the implant or extraction site; 5. Adequate bone volume at the implant site, without the need for bone augmentation. Exclusion Criteria: 1. Poor oral hygiene; 2. Uncontrolled oral or systemic diseases; 3. Metabolic disorders such as diabetes; 4. Smoking index >200; 5. Individuals unwilling to participate. Tissue Sample Processing The surgical site was disinfected with iodine tincture. Gingival tissue (GIN) was collected from the alveolar ridge crest, at least 2 mm from adjacent teeth, while alveolar mucosa (ALV) was collected as close to the mucosal junction as possible(Supplementary File 1: Fig. S7). The collected samples were washed in sterile 4°C physiological saline, transferred to DMEM, and processed within 30 minutes. The tissues were placed in culture dishes and treated with 2.4 mg/mL Dispase II solution, incubated at 37°C for 2.5 hours. Following treatment, the tissues were washed with sterile PBS three times, and the epithelium was gently separated from the connective tissue using micro-forceps. The connective tissue was immersed in RNAlater and stored at 4°C overnight, then at -20°C. RNA Extraction and Sequencing RNA was extracted using an RNA extraction kit according to the manufacturer’s protocol. RNA purity and quantity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific), with quality standards set at RIN ≥ 7 and 28S/18S ratio ≥ 0.7. mRNA was enriched using magnetic beads (Vazyme, N401-2), and cDNA libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit (NEB, E7765). Library concentration and fragment size were evaluated with Qubit and Agilent 2100 systems, respectively. Sequencing was performed on an Illumina Novaseq 6000 platform. Bioinformatics Analysis Raw sequencing data were processed using fastp software to filter low-quality reads, resulting in Clean reads for analysis. HISAT2 was used for genome alignment. Gene expression levels were calculated using the FPKM method, and read counts for each gene were obtained using HTSeq-count. Differentially expressed genes (DEGs) were identified with DESeq2, applying a p-value threshold of 2 or < 0.5. Volcano plots and heat maps were generated using the Ouyi Biological Cloud Platform ( https://cloud.oebiotech.com/task/ ). Principal Component Analysis (PCA), Gene Ontology (GO) enrichment analysis, KEGG pathway analysis, and Gene Set Enrichment Analysis (GSEA) were conducted. Protein-Protein Interaction (PPI) networks were analyzed using the STRING database, with ClueGO enrichment analysis and Hub gene identification performed using Cytoscape and the MCODE plugin. Data visualization was done using the OmicStudio Cloud Platform ( https://www.omicstudio.cn/tool ) and TBtools software. RT-qPCR of Tissue Samples Tissue samples stored in RNAlater were transferred to RNAse-free EP tubes, homogenized with RNAzol and RNAse-free zirconia grinding beads, and vortexed. The mixture was centrifuged at 12,000 g for 15 minutes. The supernatant was collected, and RNA was precipitated with 75% ethanol. The pellet was washed twice with 75% ethanol, air-dried for 5 minutes, and resuspended in DEPC water. RNA concentration was measured with a micro-spectrophotometer and stored at -80°C. RNA was reverse transcribed using the Hifair® III 1st Strand cDNA Synthesis SuperMix Kit. RNA, RNase-Free H2O, and gDNA digester Mix were incubated at 42°C for 2 minutes to remove genomic DNA. Hifair® III SuperMix plus was added, and the mixture was incubated at 25°C for 5 minutes, 55°C for 15 minutes, and 85°C for 5 minutes. The cDNA was stored at -20°C. Primers were designed based on cDNA sequences from NCBI and validated in the BLAST database. GAPDH was used as a reference gene. Gene expression was assessed using SYBR real-time quantitative PCR premix on a 384-well plate. The two-step reaction protocol was employed, and data were analyzed using the 2-ΔΔCt method. Primer sequences are provided in Supplementary File 1: Table S1 . Histological Processing Tissue samples were fixed in 4% paraformaldehyde for 24 hours, followed by dehydration, embedding, and sectioning. Paraffin sections (5 µm) were stained with hematoxylin/eosin and Masson’s trichrome, and imaged using an Axio Imager M2 microscope (Carl Zeiss). Collagen content was quantified using the Fiji distribution of ImageJ. Primary Fibroblast Culturing Gingival and alveolar mucosal tissues were collected and processed within 30 minutes. The tissues were treated with 2.4 mg/mL Dispase II solution, incubated at 37°C for 2.5 hours, and then separated into epithelial and connective tissue. Connective tissue was digested with 0.25% trypsin and 0.5% collagenase I, filtered, and cultured in DMEM containing 15% fetal bovine serum and 3% penicillin/streptomycin. Cells were passaged at a 1:3 ratio once they reached 80%-90% confluence.Cells were seeded into a 96-well plate at a density of 1×10^4 cells/wel for proliferation measurement. Proliferation was assessed using the CCK-8 method. Absorbance at 450 nm was measured using a microplate reader. Cell Differentiation Assays 1) Collagen Synthesis: Cells were seeded in a 12-well plate at 1×10^5 cells/well and cultured in differentiation medium for 48 hours. Cells were fixed with 4% paraformaldehyde and collagen synthesis was detected by immunofluorescence. 2) Mineralization: Cells were seeded in a 24-well plate at 5×10^4 cells/well and cultured in osteogenic medium for 14 days. Cells were fixed with 4% paraformaldehyde and stained with alizarin red S to assess mineralization. Statistical Analysis Data are expressed as means ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (version 8.0). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. A p-value < 0.05 was considered statistically significant. Declarations Acknowledgements This work was supported by National Natural Science Foundation of China (82201119, 81970975, 82071167), Guangdong Basic and Applied Basic Research Foundation (2023A0505050138, 2021A1515110380), Guangdong Financial Fund for High-Caliber Hospital Construction, Academy of Osseointegration Research Grant and Osteology Research Academy Grant. Conflict of Interest The authors declare no conflict of interest. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Author Contributions Conceptualization, Shoucheng Chen; Methodology and Analyses, Ruoxuan Huang, Leyao Xu, Chunxin Xu, Yuanxiang Liu, Runheng Liu, Shudan Deng, and Zhipeng Li; Writing—Original Draft Preparation, Ruoxuan Huang, Leyao Xu and Chunxin Xu; Writing—Review and Editing, Ruoxuan Huang, Shoucheng Chen, Zhuofan Chen and Zetao Chen; Supervision and funding acquisition, Shoucheng Chen, Zhuofan Chen and Zetao Chen. All authors have read and agreed to the version of this manuscript. References Moutsopoulos, N. M. & Konkel, J. E. Tissue-specific immunity at the oral mucosal barrier. Trends Immunol. 39, 276–287 (2018). Williams, D. W. et al. Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity. Cell 184, 4090–4104.e15 (2021). Larjava, H., Wiebe, C., Gallant-Behm, C., Hart, D. A. & Heino, J. Exploring scarless healing of oral soft tissues. J. Can. Dent. Assoc. 77, b18 (2011). Rojas, M. A. et al. Gene expression profiles of oral soft tissue-derived fibroblasts from healing wounds: correlation with clinical outcome, autophagy activation, and fibrotic markers expression. J. Clin. Periodontol. 48, 705–720 (2021). Kabakov, L., Nemcovsky, C. E., Plasmanik-Chor, M. & Baruch, Y. Fibroblasts from the oral masticatory and lining mucosa have different gene expression profiles and proliferation rates. J. Clin. Periodontol. 48, 1393–1401 (2021). Caffesse, R. G., Nasjleti, C. J., Kowalski, C. J. & Castelli, W. A. The effect of mechanical stimulation on the keratinization of sulcular epithelium. J. Periodontol. 53, 89–92 (1982). Liñares, A., Rubinos, A., Puñal, A., Muñoz, F. & Blanco, J. Regeneration of keratinized tissue around teeth and implants following coronal repositioning of alveolar mucosa with and without a connective tissue graft: An experimental study in dogs. J. Clin. Periodontol. 49, 1133–1144 (2022). European Federation of Periodontology. What happens after healing when keratinised and non-keratinised tissue are transpositioned? EFP website. https://www.efp.org/publications/perio-insight/what-happens-after-healing-when-keratinised-and-non-keratinised-tissue-are-transpositioned/ (2024). Nguyen, H. T. T. et al. Type XVIII collagen modulates keratohyalin granule formation and keratinization in oral mucosa. Int. J. Mol. Sci. 20, 4739 (2019). Tavelli, L. et al. Peri-implant soft tissue phenotype modification and its impact on peri-implant health: A systematic review and network meta-analysis. J. Periodontol. 92, 21–44 (2021). Karring, T., Ostergaard, E. & Löe, H. Conservation of tissue specificity after heterotopic transplantation of gingiva and alveolar mucosa. J. Periodontal Res. 6, 282–293 (1971). Karring, T., Lang, N. P. & Löe, H. The role of gingival connective tissue in determining epithelial differentiation. J. Periodontal Res. 10, 1–11 (1975). Nguyen, H. T. T. et al. Type XVIII collagen modulates keratohyalin granule formation and keratinization in oral mucosa. Int. J. Mol. Sci. 20, 4739 (2019). Komori, T. et al. Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium. Sci. Rep. 8, 2612 (2018). Hsieh, P. C., Jin, Y. T., Chang, C. W., Huang, C. C., Liao, S. C. & Yuan, K. Elastin in oral connective tissue modulates the keratinization of overlying epithelium. J. Clin. Periodontol. 37, 705–711 (2010). Mu, X. et al. Exploring the regulators of keratinization: Role of BMP-2 in oral mucosa. Cells 13, 807 (2024). Wenstrup, R. J., Florer, J. B., Brunskill, E. W., Bell, S. M., Chervoneva, I. & Birk, D. E. Type V collagen controls the initiation of collagen fibril assembly. J. Biol. Chem. 279, 53331–53337 (2004). Fitzgerald, J., Holden, P. & Hansen, U. The expanded collagen VI family: new chains and new questions. Connect. Tissue Res. 54, 345–350 (2013). Lettmann, S. et al. Col6a1 null mice as a model to study skin phenotypes in patients with collagen VI-related myopathies: Expression of classical and novel collagen VI variants during wound healing. PLoS One 9, e105686 (2014). Theocharidis, G. et al. Type VI collagen regulates dermal matrix assembly and fibroblast motility. J. Invest. Dermatol. 136, 74–83 (2016). Izu, Y. & Birk, D. E. Collagen XII-mediated cellular and extracellular mechanisms in development, regeneration, and disease. Front. Cell Dev. Biol. 11, 1129000 (2023). Ito, S. & Nagata, K. Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone. Semin. Cell Dev. Biol. 62, 142–151 (2017). Wong, V. W., You, F., Januszyk, M., Gurtner, G. C. & Kuang, A. A. Transcriptional profiling of rapamycin-treated fibroblasts from hypertrophic and keloid scars. Ann. Plast. Surg. 72, 711–719 (2014). Kelwick, R., Desanlis, I., Wheeler, G. N. & Edwards, D. R. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family. Genome Biol. 16, 113 (2015). Hubmacher, D. & Apte, S. S. ADAMTS proteins as modulators of microfibril formation and function. Matrix Biol. 47, 34–43 (2015). Vallet, S. D. & Ricard-Blum, S. Lysyl oxidases: from enzyme activity to extracellular matrix cross-links. Essays Biochem. 63, 349–364 (2019). Zheng, Z., Granado, H. S. & Li, C. Fibromodulin, a multifunctional matricellular modulator. J. Dent. Res. 102, 125–134 (2023). Van Doren, S. R. Matrix metalloproteinase interactions with collagen and elastin. Matrix Biol. 44–46, 224–231 (2015). Nagase, H., Visse, R. & Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562–573 (2006). Szymański, Ł. et al. Retinoic acid and its derivatives in skin. Cells 9, 2660 (2020). Törmä, H. Regulation of keratin expression by retinoids. Dermatoendocrinol. 3, 136–140 (2011). Virtanen, M., Törmä, H. & Vahlquist, A. Keratin 4 upregulation by retinoic acid in vivo : a sensitive marker for retinoid bioactivity in human epidermis. J. Invest. Dermatol. 114, 487–493 (2000). Li, J., Li, Q. & Geng, S. All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and – 4 and epidermal barrier function-associated genes in the epidermis. Int. J. Mol. Med. 43, 1789–1805 (2019). Ozaki, A. et al. Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling. Histochem. Cell Biol. 151, 315–326 (2019). Miyazono, S. et al. The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium. Histochem. Cell Biol. 153, 225–237 (2020). Duester, G. Genetic dissection of retinoid dehydrogenases. Chem. Biol. Interact. 130–132, 469–480 (2001). Zhong, G. et al. Aldehyde oxidase contributes to all-trans-retinoic acid biosynthesis in human liver. Drug Metab. Dispos. 49, 202–211 (2021). Veit, J. G. S. et al. Characterization of CYP26B1-selective inhibitor, DX314, as a potential therapeutic for keratinization disorders. J. Invest. Dermatol. 141, 72–83.e6 (2021). Gudas, L. J. Retinoid metabolism: new insights. J. Mol. Endocrinol. 69, T37–T49 (2022). Molotkov, A., Deltour, L., Foglio, M. H., Cuenca, A. E. & Duester, G. Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice. J. Biol. Chem. 277, 13804–13811 (2002). Harding, P. P. & Duester, G. Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3. J. Biol. Chem. 267, 14145–14150 (1992). Dong, Y. et al. Regulation of gene expression of class I alcohol dehydrogenase by glucocorticoids. Proc. Natl Acad. Sci. USA 85, 767–771 (1988). Pouw, R. B. & Ricklin, D. Tipping the balance: intricate roles of the complement system in disease and therapy. Semin. Immunopathol. 43, 757–771 (2021). Damgaard, C., Holmstrup, P., Van Dyke, T. E. & Nielsen, C. H. The complement system and its role in the pathogenesis of periodontitis: current concepts. J. Periodontal Res. 50, 283–293 (2015). Huang, R. Y. et al. Complement components C3b and C4b as potential reliable site-specific diagnostic biomarkers for periodontitis. J. Periodontal Res. 58, 1020–1030 (2023). Attström, R., Laurel, A. B., Lahsson, U. & Sjöholm, A. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. J. Periodontal Res. 10, 19–27 (1975). Hajishengallis, G. Complement and periodontitis. Biochem. Pharmacol. 80, 1992–2001 (2010). Ramanauskaite, A., Schwarz, F. & Sader, R. Influence of width of keratinized tissue on the prevalence of peri-implant diseases: a systematic review and meta-analysis. Clin Oral Implants Res. 33, 8–31 (2022). Ravidà, A., Arena, C., Tattan, M., Caponio, V. C. A., Saleh, M. H. A., Wang, H. L. & Troiano, G. The role of keratinized mucosa width as a risk factor for peri-implant disease: A systematic review, meta-analysis, and trial sequential analysis. Clin. Implant Dent. Relat. Res. 24, 287–300 (2022). Additional Declarations There is no conflict of interest Supplementary Files TableS1.docx Table S1 SupplementaryFile.pdf SupplementaryFigsLegends.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5368489","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376927095,"identity":"2d09775d-c472-48f6-a741-4ebe32aee5f1","order_by":0,"name":"Shoucheng Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDACCSBmbGBI4GdvYGBIKIAJsxGhRbLnAFCLASlaDG4kAFnEaOGf3Xzs4c8ddnmSM18nfnhgwGBvcP6MAcOHssNAqQbsltw5lm7Meya5mF86d7ME0GGJG27kGDDOOHcYKHUAqxYDiRwzaca2A4kzZ+duAGkBupDHgJm37TBQKgGHlvxvkj+BWjbcPLv5RwLUYcx/8WrJYZPgBWm5wbsNZAvjhgM5BsyMeLRI3Egzk+ZtS06c2ZO7zSLBQCJx5o20goM959J5JG5g18I/I/kZ0GF2if3sZzff/FFhY893/vDGBz/KrOX4Z2DXgmErg8IBBgYgYuAhSj0YyDcQr3YUjIJRMApGBgAAqH9h/DRV2ygAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0154-220X","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":true,"prefix":"","firstName":"Shoucheng","middleName":"","lastName":"Chen","suffix":""},{"id":376927096,"identity":"5ef35f16-44ac-4fae-9194-3ba66aa9c22a","order_by":1,"name":"Ruoxuan Huang","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Ruoxuan","middleName":"","lastName":"Huang","suffix":""},{"id":376927097,"identity":"ea2d1bc2-ed5d-494c-addd-57c69080d772","order_by":2,"name":"Leyao Xu","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Leyao","middleName":"","lastName":"Xu","suffix":""},{"id":376927098,"identity":"3deccb96-1a50-4c2b-87c2-b2269850e0e0","order_by":3,"name":"Chunxin Xu","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Chunxin","middleName":"","lastName":"Xu","suffix":""},{"id":376927099,"identity":"cb949cc2-e4e8-482a-9fac-a5f46a6736df","order_by":4,"name":"Yuanxiang Liu","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Yuanxiang","middleName":"","lastName":"Liu","suffix":""},{"id":376927100,"identity":"fdc80368-1b58-46b2-8982-417d6f399cd4","order_by":5,"name":"Runheng Liu","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Runheng","middleName":"","lastName":"Liu","suffix":""},{"id":376927101,"identity":"29c08378-ef0e-4036-b315-5a80b08bba95","order_by":6,"name":"Shudan Deng","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Shudan","middleName":"","lastName":"Deng","suffix":""},{"id":376927102,"identity":"fd3ce503-72a4-4417-b36b-bb53cc64d61c","order_by":7,"name":"Zhipeng Li","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Zhipeng","middleName":"","lastName":"Li","suffix":""},{"id":376927103,"identity":"86318eb1-49be-4719-8fcf-bab9c59d4427","order_by":8,"name":"Zetao Chen","email":"","orcid":"https://orcid.org/0000-0003-1982-8847","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Zetao","middleName":"","lastName":"Chen","suffix":""},{"id":376927104,"identity":"efc692c6-ecf0-4d11-a10e-38e5a7afcdb3","order_by":9,"name":"Zhuofan Chen","email":"","orcid":"","institution":"Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Zhuofan","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-10-31 17:25:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5368489/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5368489/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68959315,"identity":"76efcf7a-9e33-45c7-b231-87f2d3d88328","added_by":"auto","created_at":"2024-11-14 02:30:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":623363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResearch Background and Experimental Design.\u003c/strong\u003e (A) Keratinized and non-keratinized mucosa are anatomically adjacent in the oral cavity yet exhibit significant differences in tissue characteristics. (B) Recent clinical and preclinical studies suggest that connective tissue intrinsically determines these mucosal phenotypes; however, its underlying molecular profile remains unclear. (C) This study aims to dissect the molecular blueprints of human keratinized oral mucosa (gingiva, GIN) and non-keratinized oral mucosa (alveolar mucosa, ALV) by investigating \u003cem\u003ein situ\u003c/em\u003e connective tissues and isolated fibroblasts. Based on bioinformatics analysis, we systematically analyzed and validated key differences in three domains -- extracellular matrix composition, metabolism, and immune responses.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/13677df95e65224711cda9da.png"},{"id":68959312,"identity":"bb706ff4-7697-42c3-9742-75f0aaf4cb2f","added_by":"auto","created_at":"2024-11-14 02:30:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":606504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression analysis of extracellular matrix-related genes between gingival (GIN) and alveolar (ALV) mucosa. (a) \u003c/strong\u003eVolcano plot illustrated the distribution of differentially expressed genes between the two groups.\u003cstrong\u003e (b\u003c/strong\u003e) GO enrichment analysis showing the top 10 categories with the lowest p-values. \u003cstrong\u003e(c)\u003c/strong\u003e Heatmap of differential expression of extracellular matrix-related genes. \u003cstrong\u003e(d)\u003c/strong\u003e PPI network analysis of differential expression genes related to extracellular matrix (pink indicates higher expression in GIN, blue indicates lower expression in GIN). \u003cstrong\u003e(e)\u003c/strong\u003eDifferential expression and classification of key extracellular matrix-related genes.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/c7a9e52e89387540e8c558b8.png"},{"id":68959310,"identity":"111b1e7e-2b3d-4412-827b-2a49ce1ea828","added_by":"auto","created_at":"2024-11-14 02:30:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1083367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of extracellular matrix-related gene expression between gingival (GIN) and alveolar (ALV) mucosa. (a)\u003c/strong\u003eHeatmap and \u003cstrong\u003e(b) \u003c/strong\u003eBar charts of RT-qPCR validated expression levels of key extracellular matrix-related genes in gingival tissues, demonstrating the consistency across different samples. \u003cstrong\u003e(c)\u003c/strong\u003e Immunohistochemical staining images of collagen types COL1 and COL3 in sections of GIN and ALV tissues. Representative images show the localization and intensity of staining. Scale bar, 50 µm. \u003cstrong\u003e(d) \u003c/strong\u003eExpression profiles of collagen and elastin-related genes in fibroblasts derived from GIN and ALV, assessed via quantitative RT-qPCR. Data are presented as mean ± SD. \u003cstrong\u003e(e)\u003c/strong\u003e Protein expression of COL1 and COL3 in fibroblasts derived from GIN and ALV: A) Representative Western blot bands demonstrating specific protein bands for COL1 and COL3. B) Semi-quantitative analysis of COL1 protein levels, normalized to a housekeeping protein. C) Semi-quantitative analysis of COL3 protein levels. \u003cstrong\u003e(f)\u003c/strong\u003eImmunofluorescence staining for COL1 in fibroblasts originating from GIN and ALV, with representative images displaying the cellular distribution of COL1. Scale bar, 50 µm. Data represent means ± SD from three independent experiments performed with three different cell donors, in duplicates. Significant differences to the control are indicated as ***\u003cem\u003ep \u003c/em\u003e\u0026lt;0.001, **\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/37e9bcd1d3718af7c2b7ddc9.png"},{"id":68959642,"identity":"0f41e297-547d-4f74-bf76-7d9d8334765c","added_by":"auto","created_at":"2024-11-14 02:38:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":921159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential Expression of Vitamin A Metabolism-Related Genes Between GIN and ALV. (a) \u003c/strong\u003eTop 10 pathways from KEGG enrichment analysis ranked by p-value, highlighting significant metabolic pathways influenced by differential gene expression. \u003cstrong\u003e(b)\u003c/strong\u003e GSEA plot for retinol metabolism pathways, with heatmap displaying expression of pathway-specific genes. \u003cstrong\u003e(c)\u003c/strong\u003e Heatmap showing upregulated and downregulated genes within the retinol metabolism pathway. \u003cstrong\u003e(d) \u003c/strong\u003eThe schematic illustrates gene functions within the pathway, showing upregulated genes in red and downregulated in blue, with the bar chart depicting the fold changes and p-values of key genes in the vitamin A metabolism pathway. ATRA: all-trans retinoic acid.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/9808561b5dcde4d66140516f.png"},{"id":68959313,"identity":"592fab90-4c8a-43d6-b0da-76a5115c77bb","added_by":"auto","created_at":"2024-11-14 02:30:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1154665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of Vitamin A Metabolism-Related Gene Expression Between GIN and ALV. (a)\u003c/strong\u003e Heatmap and bar chart showing RT-qPCR validated differential expression of key vitamin A metabolism genes in GIN and ALV. \u003cstrong\u003e(b)\u003c/strong\u003e Immunohistochemical staining for ADH, AOX1, and CYP26B1 in GIN and ALV tissues illustrates protein localization (Scale bar, 100 µm). \u003cstrong\u003e(c)\u003c/strong\u003e RT-qPCR profiles of vitamin A metabolism-related genes in fibroblasts from GIN and ALV. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot analysis for ADH, AOX1, and CYP26B1 in fibroblasts from GIN and ALV: \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative bands. \u003cstrong\u003e(B-D)\u003c/strong\u003e Protein level quantification normalized to a housekeeping protein. \u003cstrong\u003e(e)\u003c/strong\u003e Immunofluorescence staining of AOX1 in fibroblasts from GIN and ALV, showing protein distribution (Scale bar, 50 µm). Data represent means ± SD from three independent experiments for RT-qPCR and Western blot, and from five samples per tissue type for immunofluorescence. Significant differences between GIN and ALV are indicated: ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/0cf71f12bbd4f387050c1c89.png"},{"id":68959317,"identity":"ce4b2666-0600-494d-aa7a-93fe9f546fc5","added_by":"auto","created_at":"2024-11-14 02:30:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":778630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential Expression of Immune-Related Genes Between GIN and ALV. (a) \u003c/strong\u003eBar chart of the top 10 immune-related GO categories with the lowest p-values, illustrating significant pathway enrichments. \u003cstrong\u003e(b)\u003c/strong\u003e Heatmap of immune-related gene expression differences between GIN and ALV, indicating upregulated and downregulated genes. \u003cstrong\u003e(c)\u003c/strong\u003e ClueGO enrichment analysis plot showing key immune-related pathways influenced by differential gene expression. \u003cstrong\u003e(d)\u003c/strong\u003e GO enrichment circle plot for immune-related terms (First circle: Categories and terms. Second circle: Number of genes and p-values. Third circle: Ratio of upregulated to downregulated genes. Fourth circle: RichFactor values, reflecting the degree of enrichment.). \u003cstrong\u003e(e\u003c/strong\u003e) GSEA plot for the complement and coagulation cascade pathways, detailing enrichment scores and statistical significance. \u003cstrong\u003e(f)\u003c/strong\u003eFlowchart illustrating the complement activation pathways, with significantly differentially expressed genes highlighted in red. The bar chart shows fold changes and p-values for these complement-related genes. Bar chart displaying fold changes and p-values for differentially expressed complement-related genes. \u003cstrong\u003e(g)\u003c/strong\u003eHeatmap and bar chart showing RT-qPCR results for key complement system genes, highlighting expression differences between GIN and ALV. \u003cstrong\u003e(h)\u003c/strong\u003eImmunohistochemical staining for C3 in GIN and ALV, depicting differential localization and intensity. The bar chart shows fold changes and p-values for these complement-related genes. Bar chart displaying fold changes and p-values for differentially expressed complement-related genes.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/00d16584c96cf9f8880f40f7.png"},{"id":68959321,"identity":"52e87715-0641-4555-b146-410b6e15f6c0","added_by":"auto","created_at":"2024-11-14 02:30:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":745175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating distinct connective tissue molecular blueprints in human keratinized and non-keratinized oral mucosa. \u003c/strong\u003eIn keratinized mucosal connective tissue, upregulated genes related to collagen synthesis and remodeling strengthen the extracellular matrix, while downregulation of genes involved in retinoic acid synthesis favors keratinization. In non-keratinized mucosal connective tissue, increased complement system activity, indicated by upregulated immune-related genes, highlights an enhanced immune response. These gene expression differences underscore the unique roles of keratinized and non-keratinized mucosal connective tissues in extracellular matrix composition, metabolic processes, and immune function, reflecting a connective tissue-based foundation for their phenotypic distinctions.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/84c320d946bb2bac98cffca0.png"},{"id":77815465,"identity":"5c8fae67-84fb-474b-9f5c-a38906de6262","added_by":"auto","created_at":"2025-03-05 18:58:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7088208,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/dedba61f-a2bd-44e3-ad96-76fd90462f6a.pdf"},{"id":68959314,"identity":"b6638f51-417f-4abc-98a1-1dd61e4a590f","added_by":"auto","created_at":"2024-11-14 02:30:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18426,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/41a661ed269215cb7768a2b5.docx"},{"id":68959311,"identity":"2f5d709f-6bcd-4ab9-9f7f-a403f3aa4ced","added_by":"auto","created_at":"2024-11-14 02:30:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16511630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/a6d84a402e17caaf1f5c64ca.pdf"},{"id":68959644,"identity":"446a70bf-905d-4d47-809e-d4ef44600416","added_by":"auto","created_at":"2024-11-14 02:38:26","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14980,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigsLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5368489/v1/4f222e3815ea26af72243d6e.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Mapping Connective Tissue Molecular Blueprints to Illuminate Human Keratinized and Non-Keratinized Oral Mucosa","fulltext":[{"header":"Introduction","content":"\u003cp\u003e In the oral cavity, keratinized and non-keratinized mucosa are anatomically adjacent yet exhibit marked differences in tissue characteristics. Keratinized mucosa has a denser extracellular matrix, increased abrasion resistance, and enhanced barrier functions, which together protect periodontal and peri-implant tissues from mechanical and microbial challenges. In contrast, non-keratinized mucosa lacks a keratin layer, making it softer and more elastic to accommodate oral movements; however, it tends to elicit a stronger inflammatory response under stressors [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Understanding the factors responsible for these distinct characteristics is a shared focus of both basic researchers and clinicians, as it is essential for advancing mucosal phenotype modulation and regenerative therapies. Despite this interest, the underlying determinants of these phenotypic distinctions remain elusive.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCurrent perspectives on keratinized and non-keratinized mucosal phenotypes emphasize the role of local environmental factors. For instance, mechanical stimuli have been shown to promote keratinization in the oral mucosa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, the presence of natural teeth appears to foster keratinization, with spontaneous keratinized mucosa formation reported following removal of the keratinized epithelium\u0026mdash;even in the absence of connective tissue grafting (CTG) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Transplanting non-keratinized tissue adjacent to natural teeth similarly results in keratinization of the transplanted tissue [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].However, given the complexity and variability of these external stimuli, achieving consistent and controllable soft tissue regeneration remains challenging, underscoring the need to explore endogenous mechanisms that may provide more stable and predictable regulation of mucosal phenotype.\u003c/p\u003e \u003cp\u003eRecently, increasing evidence points to the importance of connective tissue in determining mucosal phenotype. Both clinical outcomes and animal experiments suggest that connective tissue intrinsically determines human keratinized and non-keratinized oral mucosal phenotypes. In clinical practice, free gingival grafting, regarded as the \"gold standard\" for increasing the width of keratinized mucosa, demonstrates that transplanted keratinized mucosa retains its phenotype when integrated into a non-keratinized area [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This indicates that mucosal keratinization may be largely determined by intrinsic genetic factors rather than external factors such as mechanical stimuli. In basic research, animal studies have further confirmed that when connective tissue from keratinized mucosa is transplanted into a non-keratinized region and the original epithelium is removed, the transplanted connective tissue retains its keratinized properties, and the newly formed epithelium also exhibits keratinized features [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These growing evidences underscore a pressing need for in-depth molecular characterization of connective tissue in these mucosal types, which could unlock new, targeted approaches for modulating mucosal phenotypes and advancing regenerative therapies.\u003c/p\u003e \u003cp\u003eTo address this gap, the present study aims to dissect the molecular profiles of human keratinized (gingiva, GIN) and non-keratinized (alveolar mucosa, ALV) oral mucosa by examining \u003cem\u003ein situ\u003c/em\u003e connective tissues and isolated fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Based on bioinformatics analyses, we investigated and validated differences in three key domains: extracellular matrix composition, metabolic pathways, and immune responses. Key gene expression was validated via qPCR, while protein expression and localization were confirmed using immunohistochemistry, western blotting, and immunofluorescence. Our objective is to provide a molecular blueprint that lays the groundwork for future advances in phenotype modulation and mucosal regeneration.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic analysis revealed extensive molecular differences between the GIN and ALV groups\u003c/h2\u003e \u003cp\u003eThe boxplot of FPKM values reveals a generally consistent distribution of gene expression levels across samples, with the ALV group demonstrating greater consistency (Supplementary Fig.\u0026nbsp;1a). Principal Component Analysis (PCA) illustrates distinct spatial separation between GIN and ALV samples, indicating significant differences between the two groups. Specifically, ALV samples cluster more closely together, whereas GIN samples exhibit greater dispersion, reflecting increased heterogeneity within the GIN group (Supplementary Fig.\u0026nbsp;1b). Differential expression analysis identified 1,309 genes with significant expression changes (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026amp; |log2FC|\u0026gt;1), including 480 genes that were upregulated and 829 genes that were downregulated in GIN compared to ALV group (Supplementary Fig.\u0026nbsp;1c). Heatmaps visually represent the extensive differences in gene expression profiles between GIN and ALV samples, while volcano plots further illustrate the distribution of differentially expressed genes between the two groups. Notably, many genes upregulated in GIN compared to ALV group are associated with extracellular matrix and collagen (\u003cem\u003ee.g.\u003c/em\u003e, COL1A1, COL3A1, COL5A1, COL7A1). Conversely, the most significantly downregulated genes in GIN are linked to the complement system (\u003cem\u003ee.g.\u003c/em\u003e, C3) and vitamin A metabolism (\u003cem\u003ee.g.\u003c/em\u003e, AOX1, ADH4, ADH1A) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe extracellular matrix (ECM) composition emerged as the primary distinction between the molecular profiles of the GIN and ALV groups\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGO enrichment analysis of differentially expressed genes initially revealed a predominant prevalence of terms associated with the extracellular matrix across Biological Process, Cellular Component, and Molecular Function categories, highlighting 726 enriched terms. Notably, most of the enriched terms within the Cellular Component (CC) classification were related to the extracellular matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;2). The heatmap of differentially expressed extracellular matrix-related genes indicates a considerable number of upregulated genes in both the GIN and ALV groups; however, these genes are clustered within distinct functional modules. Specifically, genes upregulated in GIN are concentrated in collagen synthesis-related functions, while those upregulated in ALV are associated with elastin and collagen regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eProtein-protein interaction (PPI) analysis, followed by MCODE clustering, identified core genes upregulated in GIN primarily related to collagen synthesis and remodeling, such as fibril-forming collagens (\u003cem\u003ee.g.\u003c/em\u003e, COL1A1, COL1A2, COL3A1, COL5A1, COL11A1), basement membrane collagen (COL7A1), short-chain collagens (\u003cem\u003ee.g.\u003c/em\u003e, COL10A1, COL6A1, COL8A1), and genes associated with collagen fiber synthesis and remodeling (\u003cem\u003ee.g.\u003c/em\u003e, MMP1, MMP11, ADAMTS2, FMOD, LOX, PCOLCE, and SERPINH1). Conversely, genes related to elastin fibers and their synthesis and remodeling, including ELN1, FBLN1, EMILIN2, MFAP4, and MFAP5, were upregulated in ALV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e). Additionally, GSEA results revealed upregulation of terms related to extracellular matrix scaffolding and barrier function in GIN (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eRT-qPCR validation corroborated the transcriptome sequencing results, showing significantly higher expression of COL1A1, COL5A1, COL5A2, COL6A2, and the collagen synthesis-related gene SERPINH1 in GIN compared to ALV. Conversely, MFAP4, involved in elastin fiber assembly, was significantly less expressed in GIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Immunohistochemistry (IHC) staining revealed widespread expression of Type I collagen (COL1) in the extracellular matrix of GIN, with semi-quantitative analysis indicating significantly higher COL1 expression in GIN compared to ALV (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009). Type III collagen (COL3) was expressed sparsely in both GIN and ALV but at significantly higher levels in GIN (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVerification at both tissue and cellular levels showed that collagen-related genes\u0026mdash;COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, COL6A2, COL12A1, and COL16A1\u0026mdash;were more highly expressed in gingival fibroblasts (HGF) compared to alveolar mucosal fibroblasts (HAMF), whereas elastin (ELN) expression was lower in HGF. Statistical analysis confirmed that COL5A2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015), COL6A2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016), and COL12A1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025) were significantly more expressed in HGF than in HAMF(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Western blot analysis revealed significantly higher COL1 expression in HGF compared to ALV (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033), while COL3 expression did not differ significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Immunofluorescence staining further demonstrated that COL1 expression is notably higher in HGF compared to HAMF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe retinol metabolism pathway, which is closely associated with keratinization, exhibited significant differences between the GIN and ALV groups\u003c/b\u003e \u003c/p\u003e \u003cp\u003eKEGG enrichment analysis of differentially expressed genes revealed significant involvement of the vitamin A metabolic pathway, which plays a critical role in epithelial keratinization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). GSEA results indicated that upstream genes of this pathway were significantly upregulated in GIN, whereas downstream genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Further examination showed that genes involved in retinoic acid synthesis, including alcohol dehydrogenases (ADH1A, ADH1B, ADH1C, ADH4), aldehyde dehydrogenases (ALDH1A2, ALDH1A3), and aldehyde oxidase (AOX1), exhibited lower expression in GIN compared to ALV. Conversely, genes related to retinoic acid metabolism, such as cytochrome P450 enzymes (CYP26B1, CYP2C18, CYP3A5), were upregulated in GIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRT-qPCR validation confirmed that the expression trends were consistent with transcriptome sequencing results. Specifically, ADH1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), ALDH1A3 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and AOX1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were significantly upregulated in GIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Immunohistochemistry (IHC) staining showed that alcohol dehydrogenase (ADH) was sparsely expressed in ALV, predominantly in fibroblasts, with minimal ADH staining in GIN. Semi-quantitative analysis further revealed significantly higher ADH expression in ALV compared to GIN (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). Similarly, aldehyde oxidase 1 (AOX1) was observed in endothelial cells and fibroblasts in both GIN and ALV, with a higher number of AOX1-positive fibroblasts in ALV. Semi-quantitative analysis confirmed that AOX1 expression was significantly higher in ALV than in GIN (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025). Cytochrome P450 26B1 (CYP26B1) was diffusely expressed in both GIN and ALV, with no significant difference in expression levels between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther RT-qPCR validation in fibroblasts (HGF and HAMF) showed that ADH1, ALDH1A2, and AOX1 were more highly expressed in HGF compared to HAMF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Western blot analysis indicated a significantly lower expression of AOX1 in HGF compared to ALV (p\u0026thinsp;=\u0026thinsp;0.014), while the expression levels of ADH and CYP26B1 did not exhibit statistically significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Consistently, immunofluorescence staining revealed markedly higher AOX1 expression in HAMF relative to HGF, with no significant differences observed for ADH and CYP26B1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe complement system was more active in the ALV group\u003c/h3\u003e\n\u003cp\u003eKEGG analysis indicated that nearly 20% of the differentially expressed genes were enriched in immune system-related pathways (Supplementary Fig.\u0026nbsp;2). GO enrichment analysis of differentially expressed genes revealed significant enrichment in immune-related categories, particularly those associated with complement pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The differential expression heatmap showed that immune-related genes are predominantly downregulated in GIN relative to ALV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Enrichment analysis using ClueGO highlighted that these immune-related genes are notably involved in complement activation and T lymphocyte chemotaxis functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The GO enrichment circle plot confirmed that complement pathway genes are uniformly downregulated in GIN, with most genes related to \"immune response\" and \"inflammatory response\" also exhibiting lower expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Furthermore, GSEA analysis revealed significant downregulation of gene sets associated with the complement and coagulation cascade pathways in GIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the complement and coagulation cascade pathway genes demonstrated that complement activation-related genes are substantially downregulated in GIN. Core components of the recognition phase, including C1QA, C1QB, and C1QC, were expressed at 2.74, 2.95, and 2.61 times higher levels in ALV compared to GIN, respectively. In the activation phase, C3 was expressed 26 times higher in ALV than in GIN. For the membrane attack phase, core components C6 and C7 were expressed 56.37 and 11.09 times higher in ALV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eRT-qPCR validation of key genes in the complement system confirmed that expression trends align with transcriptome sequencing results. Specifically, complement components C1QA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006), C1QB (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), C1QC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009), C3 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038), C6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034), and C7 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) were all significantly upregulated in GIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). IHC staining results further demonstrated that complement component C3 is predominantly expressed in endothelial cells of the superficial connective tissue. Semi-quantitative analysis indicated that C3 expression levels are significantly higher in ALV compared to GIN (p\u0026thinsp;=\u0026thinsp;0.046) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh. Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have shown that connective tissue is a key determinant of oral mucosal phenotype, but the specific characteristics contributing to this role are not yet fully understood. A comprehensive understanding of the differences between keratinized and non-keratinized mucosa is crucial for exploring the regulatory mechanisms of mucosal phenotypes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, we performed transcriptomic sequencing on connective tissues from healthy human gingiva and alveolar mucosa, analyzing gene expression differences in three key areas: extracellular matrix composition, metabolic characteristics, and immune responses. Our aim was to provide direction and evaluation metrics for understanding the molecular mechanisms underlying mucosal keratinization and phenotype regulation.\u003c/p\u003e \u003cp\u003eLiteratures have shown that keratinization can be modulated by certain elements of ECM. Specific type of collagen, including type VI and type XVIII collagen, has been found to enhance the expression of keratinization-associated genes, while elastin has been found to inhibit keratinization[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The findings of this study demonstrate that extracellular matrix-related genes are the primary contributors to the differences between keratinized and non-keratinized mucosal connective tissues, consistent with histological observations shown in Supplementary Fig.\u0026nbsp;4 and supported by recent studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Further analysis of differentially expressed genes revealed that type I and type III collagen, the major structural proteins of connective tissue, exhibited gene expression levels more than five times higher in keratinized mucosa compared to non-keratinized mucosa. This significant upregulation suggests a prominent role for these collagens in maintaining the structural integrity and function of keratinized mucosa. Furthermore, genes related to type V collagen [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], type VI collagen [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and FACIT collagens [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which play important roles in collagen fiber assembly and functional regulation of cells, were more highly expressed in gingiva. Other genes directly involved in collagen synthesis and remodeling, such as SERPINH1, which encodes heat shock protein 47\u0026mdash;a collagen-specific molecular chaperone essential for correct procollagen folding\u0026mdash;were significantly upregulated in gingiva [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Genes such as PCOLCE (procollagen C-endopeptidase enhancer protein), ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs), and LOX (lysyl oxidase), which positively regulate collagen synthesis, assembly, and degradation, were also upregulated in gingiva [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Conversely, TIMP3, a tissue inhibitor of metalloproteinases, was downregulated in gingiva. These results suggest that collagen fibers in gingiva may have a more active synthesis and metabolic process. qPCR validation revealed no statistically significant differences for most genes, indicating that further studies with larger sample sizes are needed to confirm these results.\u003c/p\u003e \u003cp\u003eVitamin A (retinol) and its metabolic derivatives, retinoids, play important regulatory roles in epithelial cell proliferation, differentiation, and oral mucosal phenotype transformation, with all-trans retinoic acid (ATRA) being the most biologically active metabolite [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Studies have shown that retinoids inhibit epithelial keratinization and influence mucosal phenotype transformation by downregulating keratinized epithelial markers K1 and K10 and upregulating non-keratinized epithelial markers K4 and K13 [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e studies by Ozaki et al. demonstrated that fetal bovine serum inhibited keratinization of mouse epithelial cells, but the addition of a retinoic acid receptor inhibitor reversed this effect [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, Miyazono et al. confirmed that ATRA inhibits keratinization of epithelial cells in mice, pigs, and humans [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].In the vitamin A metabolic pathway, retinol is oxidized by alcohol dehydrogenase to retinal, which is further oxidized by aldehyde dehydrogenase and aldehyde oxidase to retinoic acid [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and then metabolized by cytochrome P450 enzymes (particularly CYP26) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In our study, genes related to the retinoic acid metabolic pathway were significantly enriched in alveolar mucosa (ALV). Genes such as ADH1A/B/C, ADH4, ALDH1A2/3, and AOX1, which promote retinoic acid synthesis, were highly expressed in ALV, whereas CYP26B1, CYP2C18, and CYP3A5 were expressed at lower levels in ALV. This suggests that retinoic acid synthesis may be more active in ALV, thereby inhibiting epithelial keratinization and influencing mucosal phenotype. Further qPCR and IHC analyses confirmed differences in the expression of ADH and AOX1, with IHC showing significant ADH expression in ALV fibroblasts but minimal expression in gingival tissue (GIN). There are seven isoenzymes of ADH in humans, with ADH1A-C (class I) and ADH4 (class II) playing important roles in vitamin A metabolism [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Studies have shown that ADH1 and ADH4 knockout mice have significantly reduced levels of retinoic acid [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], suggesting that retinoic acid synthesis may be inhibited in gingival tissue. Fibroblast validation results showed higher AOX1 expression in gingival fibroblasts compared to alveolar mucosa, but no significant difference in ADH expression between the two, possibly due to regulation by substances such as retinoic acid and glucocorticoids, which cannot be replicated \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These findings suggest that differences in ADH expression between gingiva and alveolar mucosa may be influenced by environmental factors rather than intrinsic genetic characteristics of fibroblasts. In summary, the high expression of genes and proteins related to retinoic acid synthesis in non-keratinized alveolar mucosal connective tissue suggests that retinoic acid synthesis is more active in ALV, which may be one of the pathways through which connective tissue regulates mucosal phenotype transformation.\u003c/p\u003e \u003cp\u003eIn terms of immune response, the oral mucosa, as the first line of defense against external stimuli, is exposed to abundant microorganisms and mechanical stress [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Our study found differences in immune activity between different mucosal phenotypes, with genes related to \"immune response\" and \"inflammatory response\" highly expressed in alveolar mucosa, indicating higher immune activity. The differences were mainly observed in the activation of the complement system, with related genes significantly upregulated in ALV. The lack of keratinization in ALV results in weaker barrier function, making it more susceptible to microbial and environmental influences [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which may lead to more active immune responses compared to GIN. The complement system is a key component of innate immunity and can be activated through the classical, lectin, and alternative pathways. The classical pathway is initiated by C1q recognition of antigen-antibody complexes, while the alternative pathway is rapidly activated upon pathogen stimulation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Complement activation can induce immune cell activation, leading to tissue inflammation and specific immune responses [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Studies have shown that tissue destruction in periodontitis is mediated not only by microorganisms but also by the host immune response, with excessive complement activation closely related to the onset of periodontitis and tissue damage [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, differences in complement activation may explain the higher prevalence of peri-implantitis and more severe marginal bone loss in the absence of keratinized mucosa around implants [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first study to perform transcriptomic sequencing of gingival and alveolar mucosal connective tissues in healthy individuals, systematically characterizing their gene expression differences under physiological conditions. The discovered differences in extracellular matrix components, vitamin A metabolism, and complement system activation may provide new directions and evaluation metrics for studying mucosal phenotype modulation and regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Based on these findings, future strategies for mucosal phenotype regulation include altering extracellular matrix composition, structure, or mechanical properties; applying bioactive molecules (\u003cem\u003ee.g.\u003c/em\u003e, retinoic acid) to regulate epithelial cell behavior; and utilizing specific bioactive materials to optimize the cellular microenvironment, precisely regulating keratinized or non-keratinized oral mucosal phenotypes to achieve long-term stability and functional maintenance of soft tissues. Despite the valuable insights gained, this study has some limitations. Firstly, the limited sample size may affect the generalizability of the findings, and larger sample sizes are needed to confirm the observed gene expression differences. Additionally, transcriptomic sequencing was based on whole tissue RNA, limiting the resolution of cell-type-specific gene expression. Single-cell RNA sequencing (scRNA-seq) or spatial transcriptomics could provide more detailed insights into the role of connective tissue in regulating epithelial phenotypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn conclusion\u003c/b\u003e, the transcriptomic analysis of gingival (GIN) and alveolar (ALV) mucosal tissues revealed significant differences, particularly in the extracellular matrix composition. Genes associated with collagen synthesis and remodeling were notably upregulated in GIN. In terms of metabolic pathways, the retinol metabolism pathway displayed marked differences, with genes involved in retinoic acid synthesis downregulated in GIN. Immunologically, the complement system was more active in ALV, as evidenced by the upregulation of related genes. This study is the first to systematically map the molecular profiles of connective tissue in human keratinized and non-keratinized oral mucosa, providing foundational insights that could drive future advancements in mucosal phenotype modulation and regenerative therapies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTissue Sample Collection\u003c/h2\u003e \u003cp\u003e Gingival and alveolar mucosal samples were obtained from healthy individuals undergoing dental implant surgery or tooth extraction at Sun Yat-sen University Affiliated Stomatological Hospital in 2022. The study was approved by the Medical Ethics Committee of Sun Yat-sen University Affiliated Stomatological Hospital (KQEC-2021-73-02), and all participants provided informed consent. The inclusion and exclusion criteria were as follows:\u003c/p\u003e \n\u003cp\u003e\u003cstrong\u003eInclusion Criteria:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Individuals aged 18-40 years;\u003c/p\u003e\n\u003cp\u003e2. Need for dental implant restoration or tooth extraction;\u003c/p\u003e\n\u003cp\u003e3. Healthy gingiva with no signs of acute or chronic inflammation;\u003c/p\u003e\n\u003cp\u003e4. Keratinized mucosa width \u0026ge;8 mm at the implant or extraction site;\u003c/p\u003e\n\u003cp\u003e5. Adequate bone volume at the implant site, without the need for bone augmentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion Criteria:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Poor oral hygiene;\u003c/p\u003e\n\u003cp\u003e2. Uncontrolled oral or systemic diseases;\u003c/p\u003e\n\u003cp\u003e3. Metabolic disorders such as diabetes;\u003c/p\u003e\n\u003cp\u003e4. Smoking index \u0026gt;200;\u003c/p\u003e\n\u003cp\u003e5. Individuals unwilling to participate.\u003c/p\u003e\n \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTissue Sample Processing\u003c/h2\u003e \u003cp\u003eThe surgical site was disinfected with iodine tincture. Gingival tissue (GIN) was collected from the alveolar ridge crest, at least 2 mm from adjacent teeth, while alveolar mucosa (ALV) was collected as close to the mucosal junction as possible(Supplementary File 1: Fig. S7). The collected samples were washed in sterile 4\u0026deg;C physiological saline, transferred to DMEM, and processed within 30 minutes. The tissues were placed in culture dishes and treated with 2.4 mg/mL Dispase II solution, incubated at 37\u0026deg;C for 2.5 hours. Following treatment, the tissues were washed with sterile PBS three times, and the epithelium was gently separated from the connective tissue using micro-forceps. The connective tissue was immersed in RNAlater and stored at 4\u0026deg;C overnight, then at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA Extraction and Sequencing\u003c/h3\u003e\n\u003cp\u003eRNA was extracted using an RNA extraction kit according to the manufacturer\u0026rsquo;s protocol. RNA purity and quantity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific), with quality standards set at RIN\u0026thinsp;\u0026ge;\u0026thinsp;7 and 28S/18S ratio\u0026thinsp;\u0026ge;\u0026thinsp;0.7. mRNA was enriched using magnetic beads (Vazyme, N401-2), and cDNA libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit (NEB, E7765). Library concentration and fragment size were evaluated with Qubit and Agilent 2100 systems, respectively. Sequencing was performed on an Illumina Novaseq 6000 platform.\u003c/p\u003e\n\u003ch3\u003eBioinformatics Analysis\u003c/h3\u003e\n\u003cp\u003eRaw sequencing data were processed using fastp software to filter low-quality reads, resulting in Clean reads for analysis. HISAT2 was used for genome alignment. Gene expression levels were calculated using the FPKM method, and read counts for each gene were obtained using HTSeq-count. Differentially expressed genes (DEGs) were identified with DESeq2, applying a p-value threshold of \u0026lt;\u0026thinsp;0.05 and a Foldchange\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.5.\u003c/p\u003e \u003cp\u003eVolcano plots and heat maps were generated using the Ouyi Biological Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com/task/\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com/task/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Principal Component Analysis (PCA), Gene Ontology (GO) enrichment analysis, KEGG pathway analysis, and Gene Set Enrichment Analysis (GSEA) were conducted. Protein-Protein Interaction (PPI) networks were analyzed using the STRING database, with ClueGO enrichment analysis and Hub gene identification performed using Cytoscape and the MCODE plugin. Data visualization was done using the OmicStudio Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omicstudio.cn/tool\u003c/span\u003e\u003cspan address=\"https://www.omicstudio.cn/tool\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TBtools software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR of Tissue Samples\u003c/h2\u003e \u003cp\u003eTissue samples stored in RNAlater were transferred to RNAse-free EP tubes, homogenized with RNAzol and RNAse-free zirconia grinding beads, and vortexed. The mixture was centrifuged at 12,000 g for 15 minutes. The supernatant was collected, and RNA was precipitated with 75% ethanol. The pellet was washed twice with 75% ethanol, air-dried for 5 minutes, and resuspended in DEPC water. RNA concentration was measured with a micro-spectrophotometer and stored at -80\u0026deg;C. RNA was reverse transcribed using the Hifair\u0026reg; III 1st Strand cDNA Synthesis SuperMix Kit. RNA, RNase-Free H2O, and gDNA digester Mix were incubated at 42\u0026deg;C for 2 minutes to remove genomic DNA. Hifair\u0026reg; III SuperMix plus was added, and the mixture was incubated at 25\u0026deg;C for 5 minutes, 55\u0026deg;C for 15 minutes, and 85\u0026deg;C for 5 minutes. The cDNA was stored at -20\u0026deg;C. Primers were designed based on cDNA sequences from NCBI and validated in the BLAST database. GAPDH was used as a reference gene. Gene expression was assessed using SYBR real-time quantitative PCR premix on a 384-well plate. The two-step reaction protocol was employed, and data were analyzed using the 2-ΔΔCt method. Primer sequences are provided in Supplementary File 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistological Processing\u003c/h2\u003e \u003cp\u003eTissue samples were fixed in 4% paraformaldehyde for 24 hours, followed by dehydration, embedding, and sectioning. Paraffin sections (5 \u0026micro;m) were stained with hematoxylin/eosin and Masson\u0026rsquo;s trichrome, and imaged using an Axio Imager M2 microscope (Carl Zeiss). Collagen content was quantified using the Fiji distribution of ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrimary Fibroblast Culturing\u003c/h2\u003e \u003cp\u003eGingival and alveolar mucosal tissues were collected and processed within 30 minutes. The tissues were treated with 2.4 mg/mL Dispase II solution, incubated at 37\u0026deg;C for 2.5 hours, and then separated into epithelial and connective tissue. Connective tissue was digested with 0.25% trypsin and 0.5% collagenase I, filtered, and cultured in DMEM containing 15% fetal bovine serum and 3% penicillin/streptomycin. Cells were passaged at a 1:3 ratio once they reached 80%-90% confluence.Cells were seeded into a 96-well plate at a density of 1\u0026times;10^4 cells/wel for proliferation measurement. Proliferation was assessed using the CCK-8 method. Absorbance at 450 nm was measured using a microplate reader.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Differentiation Assays\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1) Collagen Synthesis: Cells were seeded in a 12-well plate at 1\u0026times;10^5 cells/well and cultured in differentiation medium for 48 hours. Cells were fixed with 4% paraformaldehyde and collagen synthesis was detected by immunofluorescence.\u003c/p\u003e \u003cp\u003e2) Mineralization: Cells were seeded in a 24-well plate at 5\u0026times;10^4 cells/well and cultured in osteogenic medium for 14 days. Cells were fixed with 4% paraformaldehyde and stained with alizarin red S to assess mineralization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (version 8.0). One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used for comparisons between groups. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82201119, 81970975, 82071167), Guangdong Basic and Applied Basic Research Foundation (2023A0505050138, 2021A1515110380), Guangdong Financial Fund for High-Caliber Hospital Construction, Academy of Osseointegration Research Grant and Osteology Research Academy Grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Shoucheng Chen; Methodology and Analyses, Ruoxuan Huang, Leyao Xu, Chunxin Xu, Yuanxiang Liu, Runheng Liu, Shudan Deng, and Zhipeng Li; Writing\u0026mdash;Original Draft Preparation, Ruoxuan Huang, Leyao Xu and Chunxin Xu; Writing\u0026mdash;Review and Editing, Ruoxuan Huang, Shoucheng Chen, Zhuofan Chen and Zetao Chen; Supervision and funding acquisition, Shoucheng Chen, Zhuofan Chen and Zetao Chen. All authors have read and agreed to the version of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoutsopoulos, N. M. \u0026amp; Konkel, J. E. Tissue-specific immunity at the oral mucosal barrier. Trends Immunol. 39, 276\u0026ndash;287 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, D. W. et al. Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity. Cell 184, 4090\u0026ndash;4104.e15 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarjava, H., Wiebe, C., Gallant-Behm, C., Hart, D. A. \u0026amp; Heino, J. Exploring scarless healing of oral soft tissues. J. Can. Dent. Assoc. 77, b18 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRojas, M. A. et al. Gene expression profiles of oral soft tissue-derived fibroblasts from healing wounds: correlation with clinical outcome, autophagy activation, and fibrotic markers expression. J. Clin. Periodontol. 48, 705\u0026ndash;720 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKabakov, L., Nemcovsky, C. E., Plasmanik-Chor, M. \u0026amp; Baruch, Y. Fibroblasts from the oral masticatory and lining mucosa have different gene expression profiles and proliferation rates. J. Clin. Periodontol. 48, 1393\u0026ndash;1401 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaffesse, R. G., Nasjleti, C. J., Kowalski, C. J. \u0026amp; Castelli, W. A. The effect of mechanical stimulation on the keratinization of sulcular epithelium. J. Periodontol. 53, 89\u0026ndash;92 (1982).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi\u0026ntilde;ares, A., Rubinos, A., Pu\u0026ntilde;al, A., Mu\u0026ntilde;oz, F. \u0026amp; Blanco, J. Regeneration of keratinized tissue around teeth and implants following coronal repositioning of alveolar mucosa with and without a connective tissue graft: An experimental study in dogs. J. Clin. Periodontol. 49, 1133\u0026ndash;1144 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Federation of Periodontology. What happens after healing when keratinised and non-keratinised tissue are transpositioned? EFP website. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.efp.org/publications/perio-insight/what-happens-after-healing-when-keratinised-and-non-keratinised-tissue-are-transpositioned/\u003c/span\u003e\u003cspan address=\"https://www.efp.org/publications/perio-insight/what-happens-after-healing-when-keratinised-and-non-keratinised-tissue-are-transpositioned/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, H. T. T. et al. Type XVIII collagen modulates keratohyalin granule formation and keratinization in oral mucosa. Int. J. Mol. Sci. 20, 4739 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavelli, L. et al. Peri-implant soft tissue phenotype modification and its impact on peri-implant health: A systematic review and network meta-analysis. J. Periodontol. 92, 21\u0026ndash;44 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarring, T., Ostergaard, E. \u0026amp; L\u0026ouml;e, H. Conservation of tissue specificity after heterotopic transplantation of gingiva and alveolar mucosa. J. Periodontal Res. 6, 282\u0026ndash;293 (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarring, T., Lang, N. P. \u0026amp; L\u0026ouml;e, H. The role of gingival connective tissue in determining epithelial differentiation. J. Periodontal Res. 10, 1\u0026ndash;11 (1975).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, H. T. T. et al. Type XVIII collagen modulates keratohyalin granule formation and keratinization in oral mucosa. Int. J. Mol. Sci. 20, 4739 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomori, T. et al. Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium. Sci. Rep. 8, 2612 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh, P. C., Jin, Y. T., Chang, C. W., Huang, C. C., Liao, S. C. \u0026amp; Yuan, K. Elastin in oral connective tissue modulates the keratinization of overlying epithelium. J. Clin. Periodontol. 37, 705\u0026ndash;711 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu, X. et al. Exploring the regulators of keratinization: Role of BMP-2 in oral mucosa. Cells 13, 807 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWenstrup, R. J., Florer, J. B., Brunskill, E. W., Bell, S. M., Chervoneva, I. \u0026amp; Birk, D. E. Type V collagen controls the initiation of collagen fibril assembly. J. Biol. Chem. 279, 53331\u0026ndash;53337 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzgerald, J., Holden, P. \u0026amp; Hansen, U. The expanded collagen VI family: new chains and new questions. Connect. Tissue Res. 54, 345\u0026ndash;350 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLettmann, S. et al. Col6a1 null mice as a model to study skin phenotypes in patients with collagen VI-related myopathies: Expression of classical and novel collagen VI variants during wound healing. PLoS One 9, e105686 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheocharidis, G. et al. Type VI collagen regulates dermal matrix assembly and fibroblast motility. J. Invest. Dermatol. 136, 74\u0026ndash;83 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzu, Y. \u0026amp; Birk, D. E. Collagen XII-mediated cellular and extracellular mechanisms in development, regeneration, and disease. Front. Cell Dev. Biol. 11, 1129000 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIto, S. \u0026amp; Nagata, K. Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone. Semin. Cell Dev. Biol. 62, 142\u0026ndash;151 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong, V. W., You, F., Januszyk, M., Gurtner, G. C. \u0026amp; Kuang, A. A. Transcriptional profiling of rapamycin-treated fibroblasts from hypertrophic and keloid scars. Ann. Plast. Surg. 72, 711\u0026ndash;719 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelwick, R., Desanlis, I., Wheeler, G. N. \u0026amp; Edwards, D. R. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family. Genome Biol. 16, 113 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHubmacher, D. \u0026amp; Apte, S. S. ADAMTS proteins as modulators of microfibril formation and function. Matrix Biol. 47, 34\u0026ndash;43 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVallet, S. D. \u0026amp; Ricard-Blum, S. Lysyl oxidases: from enzyme activity to extracellular matrix cross-links. Essays Biochem. 63, 349\u0026ndash;364 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Z., Granado, H. S. \u0026amp; Li, C. Fibromodulin, a multifunctional matricellular modulator. J. Dent. Res. 102, 125\u0026ndash;134 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Doren, S. R. Matrix metalloproteinase interactions with collagen and elastin. Matrix Biol. 44\u0026ndash;46, 224\u0026ndash;231 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagase, H., Visse, R. \u0026amp; Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562\u0026ndash;573 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzymański, Ł. et al. Retinoic acid and its derivatives in skin. Cells 9, 2660 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026ouml;rm\u0026auml;, H. Regulation of keratin expression by retinoids. Dermatoendocrinol. 3, 136\u0026ndash;140 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirtanen, M., T\u0026ouml;rm\u0026auml;, H. \u0026amp; Vahlquist, A. Keratin 4 upregulation by retinoic acid \u003cem\u003ein vivo\u003c/em\u003e: a sensitive marker for retinoid bioactivity in human epidermis. J. Invest. Dermatol. 114, 487\u0026ndash;493 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, J., Li, Q. \u0026amp; Geng, S. All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and \u0026ndash;\u0026thinsp;4 and epidermal barrier function-associated genes in the epidermis. Int. J. Mol. Med. 43, 1789\u0026ndash;1805 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzaki, A. et al. Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling. Histochem. Cell Biol. 151, 315\u0026ndash;326 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazono, S. et al. The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium. Histochem. Cell Biol. 153, 225\u0026ndash;237 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuester, G. Genetic dissection of retinoid dehydrogenases. Chem. Biol. Interact. 130\u0026ndash;132, 469\u0026ndash;480 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong, G. et al. Aldehyde oxidase contributes to all-trans-retinoic acid biosynthesis in human liver. Drug Metab. Dispos. 49, 202\u0026ndash;211 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeit, J. G. S. et al. Characterization of CYP26B1-selective inhibitor, DX314, as a potential therapeutic for keratinization disorders. J. Invest. Dermatol. 141, 72\u0026ndash;83.e6 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGudas, L. J. Retinoid metabolism: new insights. J. Mol. Endocrinol. 69, T37\u0026ndash;T49 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolotkov, A., Deltour, L., Foglio, M. H., Cuenca, A. E. \u0026amp; Duester, G. Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice. J. Biol. Chem. 277, 13804\u0026ndash;13811 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarding, P. P. \u0026amp; Duester, G. Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3. J. Biol. Chem. 267, 14145\u0026ndash;14150 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, Y. et al. Regulation of gene expression of class I alcohol dehydrogenase by glucocorticoids. Proc. Natl Acad. Sci. USA 85, 767\u0026ndash;771 (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePouw, R. B. \u0026amp; Ricklin, D. Tipping the balance: intricate roles of the complement system in disease and therapy. Semin. Immunopathol. 43, 757\u0026ndash;771 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamgaard, C., Holmstrup, P., Van Dyke, T. E. \u0026amp; Nielsen, C. H. The complement system and its role in the pathogenesis of periodontitis: current concepts. J. Periodontal Res. 50, 283\u0026ndash;293 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, R. Y. et al. Complement components C3b and C4b as potential reliable site-specific diagnostic biomarkers for periodontitis. J. Periodontal Res. 58, 1020\u0026ndash;1030 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAttstr\u0026ouml;m, R., Laurel, A. B., Lahsson, U. \u0026amp; Sj\u0026ouml;holm, A. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. J. Periodontal Res. 10, 19\u0026ndash;27 (1975).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHajishengallis, G. Complement and periodontitis. Biochem. Pharmacol. 80, 1992\u0026ndash;2001 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamanauskaite, A., Schwarz, F. \u0026amp; Sader, R. Influence of width of keratinized tissue on the prevalence of peri-implant diseases: a systematic review and meta-analysis. Clin Oral Implants Res. 33, 8\u0026ndash;31 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRavid\u0026agrave;, A., Arena, C., Tattan, M., Caponio, V. C. A., Saleh, M. H. A., Wang, H. L. \u0026amp; Troiano, G. The role of keratinized mucosa width as a risk factor for peri-implant disease: A systematic review, meta-analysis, and trial sequential analysis. Clin. Implant Dent. Relat. Res. 24, 287\u0026ndash;300 (2022).\u003c/span\u003e\u003c/li\u003e\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":"Keratinized Oral Mucosa, Non-Keratinized Oral Mucosa, Connective Tissue, Fibroblast, Molocular Profile","lastPublishedDoi":"10.21203/rs.3.rs-5368489/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5368489/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e A fundamental question in oral science is elucidating the factors that underpin the distinct tissue characteristics of human keratinized and non-keratinized mucosa. Clinical autotransplantation and large animal studies have observed that intrinsic regulation within connective tissue defines mucosal phenotypes, emphasizing the need for in-depth molecular characterization, which remains largely unexplored. This study aimed to map the molecular blueprints of \u003cem\u003ein situ\u003c/em\u003e connective tissues and isolated fibroblasts of human keratinized oral mucosa (gingiva, GIN) and non-keratinized oral mucosa (alveolar mucosa, ALV). Distinct variations were observed in extracellular matrix composition, retinoic acid metabolism (closely associated with keratinization), and immune function between GIN and ALV. GIN displayed higher expression of collagen-related genes (notably COL1 and COL3) and lower expression of elastin-related genes. In GIN, the retinol metabolism pathway was enriched, with downregulation of retinoic acid synthesis and upregulation of its catabolism. In contrast, the complement and coagulation cascade were notably upregulated in ALV, with significantly elevated expression of C3. This study is the first to systematically dissect and compare the molecular profiles of connective tissue in GIN and ALV providing foundational insights that could drive future advancements in mucosal phenotype modulation and regenerative therapies.\u003c/p\u003e","manuscriptTitle":"Mapping Connective Tissue Molecular Blueprints to Illuminate Human Keratinized and Non-Keratinized Oral Mucosa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 02:30:14","doi":"10.21203/rs.3.rs-5368489/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":"d2e742f2-8936-4d67-8add-4aef4610d094","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40123969,"name":"Health sciences/Health care/Dentistry/Periodontics"},{"id":40123970,"name":"Health sciences/Health care/Dentistry/Dental treatments/Dental implants"}],"tags":[],"updatedAt":"2025-03-05T18:50:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-14 02:30:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5368489","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5368489","identity":"rs-5368489","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}