Transcriptomic and Proteomic Integrated Analysis Reveals Molecular Mechanisms of 3D Bioprinted Vaginal Scaffolds in Vaginal Regeneration

Transcriptomic and Proteomic Integrated Analysis Reveals Molecular Mechanisms of 3D Bioprinted Vaginal Scaffolds in Vaginal Regeneration | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transcriptomic and Proteomic Integrated Analysis Reveals Molecular Mechanisms of 3D Bioprinted Vaginal Scaffolds in Vaginal Regeneration Xuemei Zhang, Jiahua Zheng, Liye Zhang, Jingkun Zhang, Lin Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5146586/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract 3D bioprinting technology has been applied to vaginal reconstruction with satisfactory results. Understanding the transcriptome and proteome of regenerated vaginas is essential for knowing how biomaterials and seed cells contribute to vaginal regeneration. There are no reports on the systemic analysis of vaginal regeneration transcriptomes or proteomes. This study aims to explore the transcriptomic and proteomic features of vaginal tissue reconstructed with 3D bioprinted scaffolds. The scaffolds were made with biomaterials and bone marrow-derived mesenchymal stem cells (BMSCs) and then transplanted into a rabbit model.RNA sequencing was used to analyze the transcriptomes of reconstructed and normal vaginal tissues, identifying 11,956 differentially expressed genes (DEGs). Proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and data-independent acquisition (DIA) identified 7,363 differentially expressed proteins (DEPs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on DEGs and DEPs. Results showed that DEGs and DEPs were involved in extracellular matrix remodeling, angiogenesis, inflammatory response, epithelialization, and muscle formation. This study shows that 3D bioprinted scaffolds are feasible for vaginal reconstruction and offers new insights into the molecular mechanisms involved. Biological sciences/Biological techniques Biological sciences/Biotechnology Vaginal reconstruction Biological scaffold Bone marrow-derived mesenchymal stem cells Proteomics Transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The vagina is a key part of the female reproductive system and is important for sexual health and reproduction. Congenital defects, trauma, or diseases can cause vaginal deformities, requiring either non-surgical or surgical treatments for reconstruction[ 1 ]. Traditional reconstruction methods can cause complications and discomfort, affecting patients' quality of life[ 2 ]. Recent advances in tissue engineering and regenerative medicine offer promising methods for vaginal tissue repair and reconstruction. Utilizing bioengineered implants mimicking the extracellular matrix (ECM) of tissues is key to overcoming existing limitations. ECM scaffolds from specific organs are preferred because they preserve organ structure and guide cell migration, anchoring, and organization[ 3 ].Combining ECM with gelatin methacryloyl (GelMA) and silk fibroin (SF) improves photocrosslinking and mechanical strength, resulting in excellent tissue repair performance[ 4 , 5 ]. Vaginal reconstruction is challenging because of its multi-layered structure, complex blood vessels, and nerve distribution[ 6 ]. Regenerating all functional levels requires precise cellular loading and transplantation[ 7 ]. Bone marrow-derived mesenchymal stem cells (BMSCs) are promising because they can renew themselves, differentiate into various cell types, modulate immune responses, promote tissue remodeling, and act as seeding cells in vaginal reconstruction[ 8 , 9 ]. Regeneration is a tissue renewal process that depends on wound healing principles and involves multiple tissues and cells. It includes cell migration, proliferation, extracellular matrix deposition, remodeling, and coordinated inflammation and angiogenesis[ 10 ]. While small-scale defects heal quickly, but larger defects often cause fibrotic scarring, narrowing, or tissue dysfunction[ 11 ]. The tissue matrix, made of hydrated macromolecules, proteins, and polysaccharides, provides a vital microenvironment for tissue formation and function. These proteins create complex extracellular networks that control cell adhesion, proliferation, and tissue regeneration[ 12 ]. ECM from different organs helps understand tissue-specific niches, aiding in integrating native tissues with implants for better tissue regeneration and functional recovery[ 13 ]. Cell therapy currently holds a leading position in tissue engineering and regenerative medicine. MSCs secrete bioactive factors like soluble proteins, lipids, nucleic acids, and extracellular vesicles, which have anti-inflammatory, anti-scar, immune-regulatory, and anti-apoptotic properties[ 14 ]. Combining BMSCs with biomaterials in preclinical studies has shown promising results in regenerating bone, cartilage, and tendons. Choosing the right stem cells and creating biocompatible scaffolds are essential for repairing damaged tissues. Our recent study suggests that personalized 3D bioprinted biomimetic scaffolds could be a viable option for restoring damaged vaginal structure and function[ 15 ]. This technology creates bio-inks from vaginal tissue of animal donors, recellularizes them with allogeneic BMSCs, and uses 3D bioprinting to make personalized tissue-engineered vaginal implants. Our research shows that 3D bioprinted vaginal scaffolds achieve cellularization in vivo, whether or not they are seeded with cells. Cell seeding can improve the structural and functional regeneration of the reconstructed vagina. The mechanisms of bioprinted biomimetic scaffolds in tissue reconstruction involve complex interactions and regulation at multiple levels. This study is the first to comprehensively explore differentially expressed genes and proteins, and their functional pathways, in vaginal tissue reconstruction using integrated transcriptomic and proteomic analysis, revealing the molecular mechanisms of biological scaffolds in this process. Results Construction and in situ transplantation of 3D bioengineered vaginal scaffolds. P3-BMSCs observed under an inverted phase contrast microscope showed a uniform elongated morphology. (Fig. 1 A). These cells were encapsulated in ECM-GelMA-SF bioink (Fig. 1 B) and loaded into a 3D bioprinter syringe. A 3D bioengineered scaffold was printed according to preset parameters (Fig. 1 C). After 7 days of in vitro culture, live-dead cell staining showed many green live cells, indicating strong cell growth within the scaffold (Fig. 1 D). A New Zealand rabbit vaginal defect model was constructed, and the scaffold was successfully transplanted in situ (Fig. 1 E). Histological staining of reconstructed vaginal tissues. Samples were collected 12 weeks after in situ transplantation of scaffolds. Gross examination of reconstructed vaginal tissues revealed a rich vascular network, smooth mucosa, and prominent folds, with a resilient and elastic texture similar to normal vaginal tissue, especially in the 3D cell bioengineered scaffold group (Fig. 2 ). Histologically, HE staining showed a well-organized epithelial structure with distinct folds in normal vaginal tissue. After 12 weeks post-transplantation, both reconstructed vaginal tissue groups had epithelial cells. In the 3D cell bioengineered scaffold group, epithelial cells were plump, regularly arranged, and featured prominent mucosal folds. Beneath the epithelium was loose submucosal tissue rich in blood vessels and collagen fibers, similar to normal tissue, with no significant differences observed. In contrast, the 3D cell-free bioengineered scaffold group exhibited thin and sparse epithelial layers with irregular cell arrangement. Periodic acid-Schiff (PAS) staining confirmed glycogen synthesis in newly formed epithelial cells, showing purple-red positive cells in both normal and reconstructed vaginal epithelium, with more pronounced positivity in the 3D cell bioengineered scaffold group compared to the 3D cell-free bioengineered scaffold group. Masson's trichrome and Van Gieson (VG) staining revealed a smooth muscle layer beneath the normal vaginal mucosa, with an inner circular and outer longitudinal arrangement of blood vessels and collagen fibers. After 12 weeks post-transplantation, both reconstructed vaginal tissue groups showed many blood vessels in the submucosal fibers, with muscle fibers (red in Masson, yellow in VG) surrounding the vessel walls. In the 3D cell bioengineered scaffold group, muscle fibers were regularly arranged and dense, closely resembling smooth muscle fibers in normal vaginal tissue (Fig. 2 ). Analysis of differentially expressed genes (DEGs). To study how different bioengineered scaffolds affect vaginal regeneration, we analyzed the gene activity in normal vaginal (N), the 3D cell-free biomimetic scaffold (Z), and the 3D cell-loaded biomimetic scaffold (C). We statistically analyzed genes from each sample and created UpSet plots to display unique and common gene expressions among them (Fig. 3 A). We used FPKM to normalize gene expression levels for comparison across different genes and samples (Fig. 3 B). Checking gene expression level correlations between samples helps verify experimental reliability. Results showed strong correlations (above 0.8) within each group, which indicates high experimental reliability (Fig. 3 C). Principal component analysis (PCA) grouped similar samples together, with closer distances showing higher similarity (Fig. 3 D). This analysis confirmed that the sample grouping based on gene expression profiles was consistent and robust. Differential expression gene (DEG) analysis. To explore how different bioengineered scaffolds might regulate vaginal regeneration, we conducted DEG analysis comparing group N, group C, and group Z. Among the 11,956 annotated genes, the C group showed 7,249 DEGs compared to N, with 4,023 genes upregulated and 3,226 genes downregulated. The Z group had 6,684 DEGs, including 3,374 upregulated and 3,310 downregulated genes. Comparing C to Z, we identified 5,960 DEGs, with 3,607 genes upregulated and 2,353 genes downregulated (Fig. 4 A). Volcano plots (Fig. 4 B-D) illustrate the distribution of DEGs. A Venn diagram (Fig. 4 E) shows the overlap of DEGs among the comparison groups. There are 1,467 DEGs common to all three comparison groups, and 2,595 DEGs unique to the C-N and Z-N comparisons. These DEGs may be important for understanding how different treatments regulate vaginal tissue regeneration. Cluster heatmaps (Fig. 4 F) show the gene expression patterns and clustering relationships among the genes and samples. The results show clear separation and color differences among the N, C, and Z groups, indicating significant differences in gene expression patterns. However, samples within each group are closely clustered, showing similar gene expression patterns within each treatment group. Analysis of differentially expressed proteins (DEPs). To understand how different bioengineered scaffolds affect vaginal regeneration, we performed proteomic analysis. PCA clustered similar samples together, with closer distances showing higher similarity between samples (Fig. 5 A), which confirmed the reliability of protein levels and the appropriateness of sample selection (Fig. 5 B). DEPs identified a total of 7,363 proteins across all groups. Compared to N, the Z group had 460 DEPs, including 270 upregulated and 190 downregulated proteins. The C group had 557 DEPs, with 454 upregulated and 103 downregulated proteins. Comparing C to Z, we found 496 DEPs, with 367 upregulated and 129 downregulated proteins. The volcano plot (Fig. 5 C) shows the distribution of DEPs among the groups. A heatmap (Fig. 5 D) shows the hierarchical clustering analysis of DEPs from the three comparison groups. The heatmap demonstrates that DEPs meeting the fold change criteria (> 2) and statistical significance (p < 0.05) effectively separate the comparison groups, confirming the validity of DEP selection. Protein-protein interaction (PPI) network analysis. Proteins do not act alone; their activities depend on interactions with other proteins and regulation. Combining PPI network analysis with pathway annotation offers a detailed model of cellular activities, aiding in the exploration of molecular mechanisms. Figure 6 A shows the PPI network constructed from DEPs of the three comparison groups. Further analysis identified the top 30 DEPs with the highest connectivity, which are highlighted in the PPI network graph (Fig. 6 B). This approach identifies key proteins and reveals their interactions within biological systems, providing insights into their roles and potential impact on vaginal tissue regeneration. Gene Ontology (GO) enrichment analysis. GO enrichment analysis was conducted on the top 30 key proteins from each comparison group to clarify their functional roles. In the Z-N comparison group, key proteins were significantly enriched in processes like cell migration, regulation of cell shape, cell adhesion, positive regulation of translation, peptide cross-linking, acute-phase response, and tissue homeostasis. Identified proteins were notably enriched in cellular components such as the extracellular region, extracellular space, stress fibers, intermediate filaments, and the lateral plasma membrane. These proteins are involved in functions like heparin binding, calcium dependent protein binding, integrin binding, and fibronectin binding. In the C-N comparison group, key proteins were primarily associated with biological processes including cell adhesion, acute-phase response, integrin-mediated signaling pathway, protein polymerization, response to calcium ion, adaptive immune response, and platelet activation. The enriched cellular components included extracellular region, collagen trimer, phagocytic vesicle, basement membrane, focal adhesion, and endoplasmic reticulum exit site. These proteins participate in molecular functions such as heparin binding, fibronectin binding, collagen binding, integrin binding, extracellular matrix structural constituent, and signaling receptor binding. In the C-Z comparison group, key proteins were significantly enriched in biological processes such as complement activation, acute-phase response, platelet activation, positive regulation of phagocytosis, and blood coagulation. They predominantly localized to extracellular space and cell surface. These proteins are involved in molecular functions including heparin binding, fibronectin binding, collagen binding, antioxidant activity, laminin binding, and calcium ion binding (Fig. 7 A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. KEGG pathway enrichment analysis was performed on the top 30 key proteins from each comparison group to find enriched pathways. In the Z-N comparison group, key proteins were enriched in pathways such as Vascular smooth muscle contraction, Oxytocin signaling pathway, MicroRNAs in cancer, cGMP-PKG signaling pathway, Focal adhesion, Rap1 signaling pathway, and cAMP signaling pathway. The altered DEPs included FN1, VIM, THBS1, FMOD, TIMP1, CCN2, MYH11, MYL9, and ANXA1, involved in processes like angiogenesis, wound healing, cell adhesion, inflammation response, muscle contraction, and epithelial cell formation and differentiation. In the C-N comparison group, key proteins were enriched in pathways including Complement and coagulation cascades, C-type lectin receptor signaling pathway, Platelet activation, Lysosome, Phagosome, Rap1 signaling pathway, and Proteoglycans in cancer. The altered DEPs such as FN1, VIM, THBS1, FMOD, TIMP1, ITGB3, FGA, CTSS, CRP, HP, and COL1A2, participate in processes related to extracellular matrix remodeling, angiogenesis, cell adhesion, and inflammation response. Proteases are involved in collagen degradation in the extracellular matrix, fibronectin aids in protein polymerization, and LUM mitigates scar formation during regeneration, collectively regulating extracellular matrix remodeling. In the C-Z comparison group, key proteins were enriched in pathways such as Complement and coagulation cascades, Cholesterol metabolism, Vitamin digestion and absorption, Fat digestion and absorption, PPAR signaling pathway, ECM-receptor interaction, Platelet activation, and Lysosome. The altered DEPs including PLG, FGA, HRG, HP, C3, C9, CRP, C5, C1Q1, ORM1, and SERPIND1, play roles in regulating complement activation, cell lysis, vascular endothelial growth factor production, lipid transport and metabolism, inflammation response, coagulation reaction, wound healing, vascular development, and extracellular matrix assembly (Fig. 7 B). These analyses provide comprehensive insights into the functional roles and pathways influenced by different bioengineered scaffolds during vaginal tissue regeneration. Integration of mRNA and protein analysis. Transcriptomics and proteomics were integrated in the analysis, as shown in the Venn diagrams (Fig. 8 A-B). The correlation analysis showed significant relationships between differentially expressed genes and proteins in the N, Z, and C comparison groups. Compared to the N group, the C group had 49 upregulated and 11 downregulated genes and proteins, while the Z group had 11 upregulated and 19 downregulated genes and proteins. Compared to the Z group, the C group had 48 upregulated and 19 downregulated genes and proteins. Correlation coefficient analysis (Fig. 8 C) assessed the relationship between differentially expressed proteins and genes. The heatmap showed significant correlations between the selected differentially expressed proteins and genes. This approach helps us understand how gene expression is regulated at both the mRNA and protein levels, shedding light on the molecular mechanisms of vaginal tissue regeneration. GO enrichment analysis. GO enrichment analysis was performed on the differentially co-expressed genes and proteins from the comparisons between the N, Z, and C groups. The results show that compared to the N group, the Z group's differentially co-expressed genes and proteins were mainly enriched in GO terms like antioxidant activity, protein homodimerization, fibronectin binding, laminin binding, and hypotaurine dehydrogenase activity. They participate in biological processes including immune system process, positive regulation of transforming growth factor beta receptor signaling pathway, cell adhesion, negative regulation of cell-matrix adhesion, negative regulation of fibroblast growth factor receptor signaling pathway, and positive regulation of smooth muscle cell proliferation. These proteins are primarily localized in cellular components such as the actin cytoskeleton, bicellular tight junction, axon, endoplasmic reticulum membrane, brush border, cornified envelope, stress fiber, and ubiquitin ligase complex, potentially influencing immune responses, cell-cell and cell-matrix interactions, and smooth muscle proliferation. The differentially co-expressed genes and proteins in the C group, compared to the N group, were enriched in GO terms such as fibronectin binding, laminin binding, actin binding, calcium ion binding, hormone activity, misfolded protein binding, and antioxidant activity. They participate in biological processes such as immune system process, camera-type eye development, positive regulation of intrinsic apoptotic signaling pathway, positive regulation of protein kinase B signaling, regulation of cell population proliferation, and positive regulation of angiogenesis. These proteins are primarily localized in extracellular space, cornified envelope, cell-cell junction, skeletal muscle myofibril, cortical cytoskeleton, and myelin sheath, potentially regulating cell proliferation and apoptosis, angiogenesis, organ development, and inflammatory responses. Compared to the Z group, the differentially co-expressed genes and proteins in the C group were enriched in GO terms like fibronectin binding, collagen binding, calmodulin binding, structural molecule activity, hormone activity, AMP-activated protein kinase activity, and antioxidant activity. They participate in biological processes including cellular acute-phase response, inflammatory response, collagen catabolic process, vasoconstriction, cellular response to xenobiotic stimulus, glycogen catabolic process, positive regulation of epidermal growth factor receptor signaling pathway, and ventricular cardiac muscle tissue morphogenesis. These proteins are significantly enriched in extracellular space, phagocytic cup, cell-cell junction, membrane raft, skeletal muscle myofibril, and protein folding chaperone complex, potentially influencing inflammation responses, extracellular matrix remodeling, vascular regeneration, and organ morphogenesis (Fig. 9 A). This GO enrichment analysis reveals the functional roles and cellular processes affected by differentially expressed genes and proteins during vaginal tissue regeneration across the comparison groups. KEGG pathway enrichment analysis. KEGG pathway enrichment analysis was conducted on the differentially co-expressed genes and proteins identified from the correlation analysis across the Z-N, C-N, and C-Z groups. In the Z-N group, these genes and proteins were significantly enriched in pathways such as leukocyte transendothelial migration, tight junctions, TGF-beta signaling, FoxO signaling, cell adhesion molecules, the renin-angiotensin system, taurine and hypotaurine metabolism, motor proteins, and focal adhesion. Key protein products involved include MYL9, THBS1, and HP, which are associated with inflammation and immunity, vascular formation, cell-matrix interactions, tissue injury, and remodeling. In the C-N group, the differentially co-expressed genes and proteins were enriched in pathways including Phagosome, Glycolysis / Gluconeogenesis, p53 signaling pathway, Cytoskeleton in muscle cells, Fc gamma R-mediated phagocytosis, Natural killer cell mediated cytotoxicity, Protein digestion and absorption, and Renin-angiotensin system. Protein products such as CTSS, THBS1, COL8A1, and PYGM were identified, which are involved in inflammation and immunity, protein synthesis metabolism, cell proliferation, epithelial cell differentiation, endothelial formation, and embryonic organ morphogenesis. In the C-Z group, the differentially co-expressed genes or proteins were enriched in pathways such as Hypertrophic cardiomyopathy, Complement and coagulation cascades, Tight junction, Starch and sucrose metabolism, Cytoskeleton in muscle cells, PPAR signaling pathway, and Vascular smooth muscle contraction. Key protein products including MMP1, ORM1, and PYGM were highlighted, predominantly associated with cellular metabolism, immune response, extracellular matrix remodeling, and muscle organ morphogenesis (Fig. 9 B). These KEGG pathway enrichment findings reveal the biological processes and molecular pathways affected by differentially expressed genes and proteins during vaginal tissue regeneration across the comparison groups. PPI Network Analysis. The differentially co-expressed genes and proteins from correlation analysis were visualized in the PPI network (Fig. 10 ), helping to analyze protein-protein interactions and identify key proteins. In the Z-N group, the protein corresponding to the HP gene occupies a central position in the network, primarily associated with oxidative stress and inflammatory responses. In the C-N group, proteins corresponding to the HP, CTSS, THBS1, CRP, and MMP7 genes are centrally located in the network. These proteins are predominantly involved in cellular metabolism, inflammation, and extracellular matrix remodeling. In the C-Z group, proteins corresponding to the CTSS, CRP, HP, MMP1, and ORM1 genes are central to the network. They are mainly associated with collagen degradation metabolism, vascular processes in the circulatory system, inflammation, and are implicated in cell proliferation, extracellular matrix remodeling, and immune responses. These key genes and proteins, located in the dense core regions of the network, interact with each other and include many associated proteins. They collectively regulate organ development in animals and may be important targets for vaginal tissue regeneration. Integrated Analysis of Results. In the Z group compared to N, we identified 10 key upregulated proteins: FN1, FMOD, VIM, THBS1, TIMP1, CCN2, MYH11, MYL9, and ANXA1. HP was downregulated in the Z group. In the C group compared to N, we identified 14 key proteins upregulated: FN1, VIM, THBS1, FMOD, TIMP1, ITGB3, FGA, CTSS, CRP, HP, COL1A2, MMP7, COL8A1. PYGM was downregulated in the C group. In the C group compared to Z, we identified 14 key proteins upregulated: PLG, FGA, C9, CRP, C5, C3, C1Q1, ORM1, HRG, HP, MMP1, CTSS, SERPIND1. PYGM was downregulated in the C group. TIMP1 is involved in cell proliferation through the HIF-1, p53, and PI3K-Akt signaling pathways. THBS1 regulates cells via the Rap1 signaling pathway. MYL9 is linked to cell proliferation and vascular formation through the cAMP and oxytocin signaling pathways, which collectively aid in vaginal tissue regeneration. These findings highlight how interactions among these proteins regulate vaginal tissue regeneration by affecting cellular proliferation, vascular formation, and inflammatory responses. Discussion The application of 3D bioprinting technology in repairing tissue defects and reconstructing organs is very promising. This technology offers personalized treatment options for patients by printing biomaterials, marking an innovative breakthrough in vaginal reconstruction[ 15 ]. This personalized printing approach ensures that reconstructed vaginas closely mimic natural morphology and function. Scaffold materials have excellent biocompatibility and bioactivity, allowing them to integrate with patient tissues and reducing the risks of rejection and infection. This makes vaginal reconstruction surgeries safer and more reliable[ 16 ]. Biomaterials support cells physically, mimic the extracellular matrix structure and function, and promote cell adhesion, proliferation, and differentiation[ 17 ]. Seed cells mixed with bioinks can differentiate into target cells like epithelial and smooth muscle cells under specific conditions. This process promotes tissue repair and regeneration and improves the local environment to enhance the survival and function of transplanted tissues[ 18 , 19 ]. We printed a new ECM-GelMA-SF bioink using ECM from porcine vaginal tissue and BMSCs, and implanted it in rabbits for vaginal reconstruction. Macroscopic analysis showed that all New Zealand white rabbits with the biomimetic scaffold had good vaginal structure and function. Biomimetic scaffolds loaded with cells showed better regenerative effects, indicating that these scaffolds are effective in promoting vaginal regeneration. However, the mechanism of biomimetic scaffolds made with 3D bioprinting in tissue reconstruction is complex, involving many levels of interaction and regulation[ 20 ]. Mechanistic studies of tissue engineering technologies have been reported for oral mucosa, glands, muscles, blood vessels, and neural tissue reconstruction[ 21 – 24 ]. Currently, no study has explained the molecular mechanisms of 3D bioprinted biomimetic scaffolds in vaginal reconstruction. Building on our successful construction of vaginal tissue, we used combined transcriptomic and proteomic analyses. Transcriptomic analysis found 3,374 upregulated and 3,310 downregulated genes in the 3D scaffold group compared to the normal group, and 4,023 upregulated and 3,226 downregulated genes in the 3D cell scaffold group. This difference may reflect changes in gene expression regulation when scaffolds are loaded with BMSCs. Proteomic analysis further corroborated the transcriptomic findings. We identified key proteins involved in vaginal regeneration, including those related to inflammation, vascular formation, extracellular matrix remodeling, epithelial and muscle regeneration, neural regeneration, and glycogen synthesis. Scaffolds with stem cells also promote immune activation and coagulation during the regeneration process. The tissue compatibility of biomaterials is closely related to inflammatory responses during tissue regeneration. When inflammation is well-regulated, it can improve interactions between cells and materials and aid in vascular formation. Vascularization is essential for tissue regeneration because it supplies nutrients and oxygen to new tissues. Neovascularization also affects the migration of inflammatory cells, as new vessels can attract these cells, speeding up wound healing. Our study found changes in inflammation-related proteins during vaginal reconstruction, such as CRP, THBS1, ANXA1, HRG, HP, ORM1, MYL9, and CTSS, and their associated pathways. Notably, CRP, THBS1, ANXA1, HRG, and CTSS are involved in vascular formation. CRP, an acute-phase protein, is produced and released during infection or tissue injury. It directly regulates angiogenesis through the Wnt pathway and promotes liver cell regeneration in vitro and in vivo[ 25 ]. Local inflammation at the wound site is crucial for healing because it clears debris and protects against bacterial invasion. Fibroplasia and granulation tissue formation help restore the protective skin layer along with epithelialization[ 26 ]. However, excessive inflammation can cause further tissue damage and excessive scarring, so it must be strictly regulated and limited at the injury site. THBS1 is a multifunctional glycoprotein released by platelets, epithelial cells, and stromal cells during development, wound healing, angiogenesis, platelet aggregation, and cell adhesion. Without THBS1, inflammation can persist, wound healing may be delayed, and scab detachment can be slow[ 27 ]. Studies have shown that THBS1 mRNA is undetectable in normal skin but present in early wounds, primarily sourced from macrophage-like cells in inflammatory infiltrates. Inhibiting THBS1 significantly delays wound repair by reducing re-epithelialization rates and slowing dermal remodeling, showing that THBS1 produced by macrophages aids the repair process[ 28 ]. THBS1 works with angiogenic factors to control capillary angiogenesis and maintain vascular balance during regeneration[ 29 ]. These biological processes may involve pathways like the Rap1, p53, and TGF-beta signaling pathways. ANXA1, an anti-inflammatory protein mediated by glucocorticoids, reduces inflammation by inhibiting leukocyte activation and migration. ANXA1 regulates cell migration, promotes tissue repair, and plays a crucial role in liver proliferation and regeneration[ 30 ]. ANXA1 helps resolve inflammation by promoting wound closure and restoring epithelial barriers in intestinal cells in vivo[ 31 ]. ANXA1 inhibits fibrosis gene expression, shows anti-fibrotic activity, promotes corneal epithelial wound healing, and reduces corneal inflammation[ 32 ]. The expression of HRG and CTSS was higher in the scaffold with stem cells compared to the scaffold alone. HRG is a liver-produced glycoprotein that regulates angiogenesis in both pro- and anti-angiogenic ways[ 33 ]. HRG reduces inflammation by inhibiting excessive neutrophil activation in circulation and protects the vascular endothelial barrier by regulating clotting pathways[ 34 ]. CTSS, a key cysteine protease, degrades anti-angiogenic peptides and adhesion proteins, promoting neovascularization, tumor cell invasion, and metastasis. CTSS degrades the endothelial basement membrane and promotes VSMC migration and proliferation through the p38MAPK/Akt signaling pathway, aiding injury-related vascular repair[ 35 , 36 ]. We hypothesize that stem cells may positively regulate inflammation and vascular formation. In summary, regulating both pro-inflammatory and anti-inflammatory responses, as well as vascular formation, is crucial throughout vaginal regeneration. The ECM is a flexible structure found in all tissues that supports cells and tissues and changes constantly in a controlled way. Our results indicate upregulation of ECM remodeling-associated components such as MMP1, MMP7, TIMP1, COLIA2, COL8A1, FMOD, FN1, VIM, ITGB3 in the scaffold group. The breakdown of ECM components is a main part of ECM remodeling, and matrix metalloproteinases (MMPs) are crucial for this process. MMP activity is usually low but rises during repair, remodeling, and in diseased or inflamed tissues[ 37 ]. Both bioengineered scaffolds for vaginal reconstruction showed a significant increase in MMPs (MMP-1, MMP-7) and higher levels of tissue inhibitors of metalloproteinases (TIMPs). MMPs are involved in tissue remodeling, growth, angiogenesis, tissue defense, and immune responses[ 38 ]. TIMPs usually regulate MMPs, and the balance between them determines how much ECM protein is broken down and how tissue is remodeled[ 39 ]. MMP1 helps assemble and repair the basement membrane, supporting wound re-epithelialization under oxidative stress and anti-fibrotic conditions[ 40 , 41 ]. This makes MMP1 a potential anti-fibrotic agent for preventing or treating hypertrophic scars[ 42 ]. Angiogenesis needs the breakdown of the vascular basement membrane and ECM remodeling for endothelial cell migration into nearby tissues. MMPs facilitate the actions of various angiogenic factors through their proteolytic activity, promoting blood vessel formation[ 43 ]. Additionally, MMPs may exert anti-angiogenic effects by degrading specific collagen chains and plasminogen activators[ 44 ]. Thus, MMPs are important regulators of angiogenesis, usually promoting it. Collagen is the most abundant part of the ECM and is essential for tissue structure and function. Collagen fibers provide the foundation for tissue shape, strength, and wound healing[ 45 , 46 ]. Type I collagen is the most abundant type and is found in blood vessels, cornea, sclera, tendons, ligaments, and skin, making up over 90% of ECM levels[ 47 ]. TIMP-1 and COL1A1 work together in the proliferation and remodeling stages of periodontal wound healing, crucially preventing ECM and collagen breakdown during remodeling[ 48 ]. Collagen I interacts with different pro-fibrotic tissue remodeling proteins to form the ECM. Other ECM molecules, like FN1 and TIMP-1, integrate into the network to balance the construction and breakdown of damaged tissues[ 49 ]. COL8A1 is a secreted protein found in many rapidly growing cells and promotes smooth muscle cell migration and proliferation during vascular remodeling[ 50 ]. Col8a1 genes may be biomarkers for heart remodeling[ 51 ]. Endothelial-derived Col8a1 is a key factor in endothelial cell proliferation and may positively influence endothelial repair and the integrity of new intimal layers[ 52 ]. Fibronectin-1 (FN1) is a glycoprotein found on cell surfaces, in extracellular fluids, and in connective tissues, interacting with collagens, fibronectins, and integrins. FN1 primarily supports cell adhesion and is involved in cytoskeletal organization, cell migration, and important physiological processes like wound healing, thrombosis, and aging[ 53 ]. High levels of FN1 interact with integrin ITGB3, regulating gene activity that influences various signaling pathways and is involved in ovarian follicle development, maturation, ovulation, and corpus luteum formation[ 54 ]. During tissue injury, mRNA levels of ITGB3 and FN1 increase under low oxygen conditions, allowing them to stay in local areas, stimulate cell proliferation, speed up blood vessel formation, and enhance wound healing[ 55 ]. Adding stem cells increases ITGB3 protein expression. Integrins are transmembrane receptors that help cells stick to the ECM, bind to various growth factors, generate different intracellular signals, and regulate cell proliferation, differentiation, and fusion[ 56 ]. Phosphorylation of ITGB3 can stimulate angiogenesis and VEGF expression via the MAPK/ERK pathway, regulating vascular formation and stability[ 57 ]. Vimentin is a type III intermediate filament protein that is important for cell functions like migration, proliferation, and division. It is also linked to cell adhesion and migration during epithelial-mesenchymal transition, ECM remodeling, and inflammatory responses, which are essential for normal wound healing[ 58 ]. Vimentin promotes nerve sprout growth and sensory recovery, providing neuroprotective functions[ 59 ]. Vimentin is essential for regulating vascular contraction, tension, endothelial integrity, and barrier function. It participates in vascular remodeling and smooth muscle cell differentiation, which are important for tissue homeostasis and repair[ 60 ]. Fibromodulin (FMOD) binds to ECM structural components, regulating collagen cross-linking, assembly, and fiber structure. It also interacts with signaling molecules, influencing cell adhesion, proliferation, migration, invasion, differentiation, and metastasis. Therefore, FMOD promotes migration, angiogenesis, anti-inflammation, and anti-fibrosis, playing important roles in determining cell fate and tissue regeneration[ 61 ]. FMOD is involved in wound healing and the assembly of ECM components, such as collagen, and is closely related to muscle regeneration and myoblast proliferation and differentiation[ 62 ]. FMOD can induce a more "fetal-like" migratory and contractile phenotype in adult dermal fibroblasts, which reduces scar formation without weakening tensile strength in adult wound models[ 63 ]. During vaginal reconstruction, increased expression of collagen, fibronectin, and vimentin offers support and stability for new tissues, while MMPs, TIMPs, and FMOD aid in regeneration and repair by degrading and remodeling ECM components. The balance between these factors is essential for successful vaginal tissue reconstruction. We observed increased expression of CCN family members in the scaffold group. CCNs can promote tissue regeneration through various mediators like growth factors, matrix proteins, and integrins[ 64 ]. Studies show that CCN2 speeds up the healing rate of burns and diabetic wounds[ 65 , 66 ]. CCN2 regulates keratinocyte migration, promoting re-epithelialization during skin wound healing[ 67 ]. Dynamic expression of CCNs during tissue regeneration drives epithelial regeneration and mediates fibrogenesis and anti-fibrotic activity during repair. FMOD coordinates the timing and location of TGF-β ligands and receptors in vivo, regulating collagen fiber generation and reducing scar formation in the extracellular matrix. In oral mucosal wound healing, FMOD's location and abundance in the wound epithelium are regulated over time and space, promoting wound re-epithelialization and connective tissue regeneration[ 68 ]. As a hollow organ, the vagina shows different contractile behaviors, such as peristalsis, tension maintenance, and spasms, due to its smooth muscle components. Beta-3 integrin mediates myogenesis by regulating myogenic gene expression and satellite cell migration[ 69 ]. Myosin heavy chain subtype I (MYH1) is part of the MYH gene family and encodes the MyHC-IIX protein, which is expressed in intermediate muscle fibers and helps form skeletal muscle fibers[ 70 , 71 ]. MYH1 may be linked to SMSC differentiation and muscle fiber hypertrophy, and it is highly expressed in the late stages of C2C12 cell differentiation[ 70 ]. THBS1 promotes axon growth in neurons in vitro and aids axon regeneration through intrinsic mechanisms in retinal ganglion cells[ 72 ]. In addition, VIM, ITGB1 and CTSS can also regulate axon regeneration[ 73 – 75 ]. Vaginal epithelial cells produce glycogen, which converts to lactate when shed, creating a unique microenvironment that prevents pathogenic bacteria from invading[ 76 ]. We found that PYGM, a protein involved in glycogen breakdown metabolism, is downregulated in the reconstructed vaginal tissue with added cells. PYGM mainly provides energy for muscle contraction but is also expressed in other tissues and plays a key role in the early steps of glycogen breakdown through phosphorylation[ 77 ]. Inhibiting PYGM reduces glycogen breakdown, promoting glycogen storage within epithelial cells, beneficial for maintaining the vaginal microenvironment. The upregulation of CCN2, FMOD, MYH1, THBS1, VIM, and CTSS, along with the downregulation of PYGM, suggests that bioengineered scaffolds promote vaginal epithelialization, muscle regeneration, vaginal smooth muscle contraction, axon regeneration, and increased glycogen storage, aiding in the formation and recovery of vaginal tissue structure. Compared to the scaffold-only group, the cell-added group showed increased levels of proteins related to the coagulation cascade and anticoagulant systems, such as FGA, PLG, and SERPIND1, as well as proteins associated with the complement cascade and immune responses, including C9, C5, C3, C1Q1, HP, and ORM1. The coagulation, fibrinolysis, and complement systems are essential for maintaining tissue homeostasis, with many interactions between these cascades that regulate fibrinolysis and complement activation during injury and acute inflammation[ 78 ]. Tissue injury, trauma, or systemic inflammation trigger complement activation and the coagulation and fibrinolysis pathways, where coordinated enzymatic cascades repair damaged vessels and regulate the inflammatory process, despite their opposing functions[ 79 ]. The final step of the coagulation cascade involves converting fibrinogen alpha chain (FGA) to fibrin, which then polymerizes to form a blood clot[ 80 ]. Elevated FGA in inflammation-driven acute responses promotes hemostasis by activating platelets, which help distribute red blood cells, macrophages, and fibroblasts around wounds. Controlling these processes is essential for hemostasis, wound healing, and tissue regeneration[ 81 ].In addition to its role in hemostasis, FGA regulates leukocyte function during inflammation, with soluble fibrinogen influencing neutrophil adhesion and affecting leukocyte recruitment in vivo, thereby promoting inflammatory responses[ 82 ]. FGA and its degradation products regulate vascular generation potential[ 83 ]. PLG is one of the most abundant proteins involved in hemostasis and is activated by t-PA and u-PA to produce plasmin, which starts fibrinolysis[ 84 ]. Plasminogen has functions beyond the fibrinolysis system, such as removing misfolded or aggregated proteins, triggering other enzyme cascades like complement activation, regulating cell behavior, and affecting immune and inflammatory processes, enhancing thrombolysis and speeding up wound repair[ 85 ]. FGA and PLG work together to maintain balance between the coagulation cascade and the fibrinolysis system, promoting tissue structure and functional reconstruction, especially in epithelial injury and reshaping[ 86 ]. The complement system is a key part of the innate immune system, serving as the first line of defense against invading pathogens. It also removes apoptotic and necrotic cells and regulates the adaptive immune system and inflammatory processes[ 87 ]. Complement has been shown to enhance coagulation by inhibiting fibrinolysis, while proteases from the coagulation and fibrinolysis systems cleave and activate C3 and generate C5a[ 88 , 89 ]. During infection, local complement-mediated coagulation activation boosts clotting, creating a barrier against bacterial spread and aiding in the production of antimicrobial peptides while supporting the inflammatory response[ 90 ]. In neovaginal formation, the coagulation system, fibrinolysis system, and complement system are essential for maintaining tissue homeostasis. They ensure vascular integrity, prevent thrombus formation, resist foreign pathogens, and maintain tissue stability through cooperation and precise regulation. Conclusion This study revealed the critical roles of genes and proteins related to extracellular matrix remodeling, vascular regeneration, inflammatory response, epithelialization, and muscle formation in vaginal reconstruction through combined transcriptomic and proteomic analysis. This research offers a new perspective on vaginal tissue regeneration and highlights the potential of personalized bio-scaffolds for promoting tissue repair and functional recovery. In the future, further exploration of these molecular mechanisms and improvements in 3D printing and cell therapy technologies aim to achieve better clinical outcomes. Materials and methods Experimental Animals and Grouping. Female New Zealand White rabbits aged 1–2 months, weighing 1.0-1.5 kg, were used to harvest BMSCs. Mature female New Zealand White rabbits aged 7–8 months, weighing 3.5-4.0 kg, were used to create a vaginal defect model for testing vaginal bioengineered scaffolds. All animals were bought from Tonghui Breeding Co., Ltd. in Wangdu County, Hebei Province, China. All procedures involving these animals strictly adhered to the Guidelines for Care and Use of Laboratory Animals of Hebei Medical University and received approval from the Animal Ethics Committee of the Second Hospital of Hebei Medical University(Approval Letter No.2024-AE011). All methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org).Eac h animal was housed separately in the Animal Experimental Center of the Second Hospital of Hebei Medical University. There were three experimental groups: 1) Normal group (n = 4): sham-operated group; 2) 3D cell-free biomimetic scaffold group (n = 4): each rabbit received a bioengineered scaffold; 3) 3D cell-seeded biomimetic scaffold group (n = 4): each rabbit received a scaffold seeded with rabbit BMSCs. Following the completion of the experiments, to ensure the comfort of the rabbits and alleviate their suffering, the researchers adhered to the guidance of the Animal Ethics Committee and employed euthanasia through overdose anesthesia. This method is considered a humane and effective means of euthanasia for experimental animals. Preparation of BMSCs. Rabbit BMSCs were prepared by density gradient centrifugation and adherent culture. P3 cells were used for the experiments. When the cell fusion rate reached 80%-90%, the cells were detached with trypsin and resuspended as a single-cell suspension for further use. Preparation of vECM-GelMA-SF Bioink. ECM was prepared as described previously[ 18 ]. The lyophilized ECM was ground into a powder and mixed with 3 ml of 0.01 mol/L HCl solution containing 45 mg of pepsin. Digestion was carried out at 37°C, 80 rpm for 12 hours to prepare vECM hydrogel. The pH was adjusted from acidic to neutral using 0.1 mol/L NaOH. GelMA (8% concentration) and photoinitiator (5%) were dissolved in 3 ml of sterile distilled water at 60°C for 40 minutes, filtered through a 0.22 µm sterile filter, and sterilized by autoclaving. This solution was mixed with the prepared 3 ml vECM solution and 5% SF (50 mg/ml) to obtain vECM-GelMA-SF bioink. P3-BMSCs were added to the bioink at a concentration of 1x10^7 cells/ml, loaded into sterile syringes, and kept at 4°C until use. The 3D biomimetic vaginal scaffold was printed using a JNOV Life 3D bioprinting system. An adapter needle (diameter 0.34 mm) was installed and fixed on the bioprinter's low-temperature nozzle. The scaffold parameters were set to X = 10 mm, Y = 10 mm, Z = 15 mm, with a layer thickness of 0.34 mm and 30 layers. Biocompatibility Testing of vECM-GelMA-SF Bioink. P3-BMSCs loaded scaffolds were cultured in vitro for 7 days. Cell viability was assessed with a live/dead cell staining kit, and fluorescence confocal microscopy was used to observe cell survival. Scaffold In Situ Transplantation. Experimental animals were anesthetized with 1% pentobarbital sodium (0.4 ml/100 g) via ear marginal vein injection. After anesthesia, the abdominal area was prepared and disinfected with iodine, and a midline incision was made in the lower abdomen to expose the uterus and vagina. Partial vaginal excision was performed by cutting the upper end of the vagina 5 mm below the cervix and the lower end 1 cm above the urethral meatus. The prepared bioengineered scaffold was fixed at the upper end to the external uterine orifice and aligned with the remaining vaginal end, then the abdominal cavity was closed layer by layer. All surgeries were performed by the same surgical team. To prevent infection, 1 million units of sodium penicillin were injected into the abdominal cavity once a day for 3 days. Sample Collection and Evaluation. Three months post-surgery, newly formed vaginal tissues were collected for histological examination. The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 mm thick sections. Sections were stained with HE, Masson, VG, and PAS stains to observe epithelialization, vascularization, smooth muscle formation, and glycogen synthesis in the regenerated vaginal epithelium. RNA Sequencing. Vaginal tissues from the three groups, with three samples per group, were collected. Total RNA was extracted using TRIzol Reagent. RNA concentration and purity were assessed using an Agilent 2100 Bioanalyzer and RNA-specific agarose gel electrophoresis. mRNA enriched with polyA structure was selected using Oligo(dT) magnetic beads. RNA was then fragmented to approximately 300 bp using ion disruption, and 300 bp fragments were synthesized into cDNA using RNA as a template. After library construction, PCR amplification was performed to enrich the library fragments. Library size selection was set to 450 bp. The quality and concentration of the libraries were evaluated using the Agilent 2100 Bioanalyzer. The libraries were then subjected to next-generation sequencing (NGS) using the Illumina sequencing platform for paired-end (PE) sequencing. DEGs Analysis. Initially, the expression level correlation among samples was examined using Pearson correlation coefficien. PCA based on expression levels was conducted using the DESeq package in R. Differential gene expression analysis was performed using DESeq package in R. volcano plots were generated using ggplot2 package in R to display the distribution of genes, fold change differences, and significance results. DEGs specific to and shared among the comparison groups were statistically analyzed. Heatmaps were constructed using the pheatmap package in R to perform bidirectional clustering analysis of DEGs across all comparison groups, based on Euclidean distance and complete linkage method. DEGs were annotated using GO database ( http://www.geneontology.org/ ) and GO annotation analysis was conducted using Goatools software. DEGs were also mapped to KEGG database ( http://www.genome.jp/kegg/ ) for pathway enrichment analysis using KOBAS software. Proteomic Analysis DIA Analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptide sequences (n = 3) from vaginal tissues of each group, which were then used for proteomic analysis. Adequate protein samples were used to construct a spectral library. Protein samples from each group were subjected to SDS-PAGE to assess sample consistency. Trypsin digestion was performed using FASP method. Peptide concentration was determined by OD280, and peptides were fractionated using HPRP method. Each fraction was analyzed by data-dependent acquisition (DDA) mass spectrometry after addition of iRT standard peptides. Nano-flow HPLC system Nanoelute was used for chromatographic separation. Peptides were analyzed by timsTOF Pro mass spectrometer after liquid chromatography separation. Maxquant software was used for data processing, and Mus musculus_uniprot database was employed for database searching with the following parameters: trypsin enzyme, maximum missed cleavage sites set to 2, fixed modification of Carbamidomethyl (C), and dynamic modifications of Oxidation (M) and Acetyl (Protein N-term). Identified proteins were filtered based on a false discovery rate (FDR) < 1%. Spectral libraries were constructed by importing raw files and search results into Maxquant software. DIA mass spectrometry was performed on all samples, with chromatographic separation using Nanoelute and the same liquid chromatography gradient and DDA testing conditions. Peptides separated by nano-flow high-performance liquid chromatography were analyzed by timsTOF Pro mass spectrometer using DIA data acquisition mode. Data were processed using Maxquant software with the same database used for library construction. DEPs Analysis. Annotation information corresponding to species was downloaded from uniprot. Direct and indirect interactions among DEPs were investigated based on STRING database ( http://string-db.org/ ) information for PPI network analysis. Results from PPI network analysis were imported into Cytoscape (v3.7.1) to analyze and select proteins with top 30 Degree values. These proteins were further subjected to GO functional enrichment analysis and KEGG pathway enrichment analysis. Integration Analysis of Transcriptomic and Proteomic Data. Transcriptomic and proteomic data were integrated to identify genes and proteins that were differentially expressed and regulated in the same way between the two sequencing methods. Venn diagrams were used to display the intersection of upregulated/downregulated genes and proteins. Correlation analysis was performed to examine the relationship between differentially expressed proteins and genes. GO functional enrichment analysis and KEGG pathway enrichment analysis were conducted for differentially expressed genes and proteins. Direct and indirect interactions between differentially expressed genes and proteins were explored using STRING database, followed by PPI analysis to identify proteins with high connectivity. Statistical Analysis. GraphPad Prism 8.0 software was used for data processing. Continuous variables with a normal distribution were presented as mean ± standard deviation (x ± s). Student's t-test was used to analyze the significance of differential gene and protein expression, with fold change (FC) criteria typically set at p 2 or < 0.5. GO and KEGG database analyses were conducted using Fisher's exact test for functional and pathway enrichment, with p < 0.05 indicating significant enrichment, where smaller values indicated more significant enrichment. Declarations Author Contribution Xuemei Zhang and Jiahua Zheng, contributed to conception, design, acquisition, analysis, and interpretation of data, drafed manuscript, revised manuscript. Xianghua Huang and Lin Zhang, contributed to acquisition, analysis, and interpretation of data, critically revised manuscript, gave fnal approval. Liye Zhang and Jingkun Zhang, acquisited and analyzed the datas. All authors edited and approved the final manuscript. Acknowledgement We extend our gratitude to all members of the Hebei Key Laboratory of Regenerative Medicine of Obstetrics and Gynecology and Hebei Medical University's Core Facilities and Centers for their valuable discussions regarding this work and the manuscript. The authors extend their gratitude to technicist from Shiyanjia Lab (www.shiyanjia.com) for the sequencing services and bioinformatic analysis. Data Availability Sequence data that support the findings of this study have been deposited in the NCBI with the primary accession code PRJNA1175434. Web Links：ID 1175434 - BioProject - NCBI. References McQuillan, S.K. & Grover, S.R. Dilation and surgical management in vaginal agenesis: a systematic review. Int Urogynecol J 25 , 299-311 (2014). Cheikhelard, A. et al. Surgery is not superior to dilation for the management of vaginal agenesis in Mayer-Rokitansky-Küster-Hauser syndrome: a multicenter comparative observational study in 131 patients. 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The contact system--a novel branch of innate immunity generating antibacterial peptides. Embo j 25 , 5569-5578 (2006). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Feb, 2025 Reviews received at journal 19 Dec, 2024 Reviewers agreed at journal 08 Dec, 2024 Reviews received at journal 26 Nov, 2024 Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 07 Nov, 2024 Editor invited by journal 07 Nov, 2024 Submission checks completed at journal 04 Nov, 2024 First submitted to journal 24 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-5146586","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":375570684,"identity":"9e83c527-6975-4531-9db0-c0c4430944c1","order_by":0,"name":"Xuemei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACNv6GhAMJPBL1/PKPDxCnhU/iwMMHH2RsEiQb0hKI0yLHkPjYcIZNWoLBgRwDIh3GcDhNmifncJ7BgTMfb7xhsJPTbSCkhbkNqOXM4WLJg72bLecwJBubHSBoy5k0ad6ew4x9h3m3SfMwHEjcRlhL/jdp3n+HGRuO8TwjVktCsuEMnrTECWd42IjUInEg8cEHHhtjyRlsxpZzDIjwi3w/JCrl+CWYH954U2EnR1ALCpDgITJqkLWQqmMUjIJRMApGBAAAlFFEW5+hAXAAAAAASUVORK5CYII=","orcid":"","institution":"Department of Pelvic floor clinic, Cangzhou Central Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Zhang","suffix":""},{"id":375570685,"identity":"ee95c31c-f01e-465b-9abe-3284647b7c02","order_by":1,"name":"Jiahua Zheng","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, the Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiahua","middleName":"","lastName":"Zheng","suffix":""},{"id":375570686,"identity":"d3af8dae-19f0-4f86-a9a8-ece410139734","order_by":2,"name":"Liye Zhang","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liye","middleName":"","lastName":"Zhang","suffix":""},{"id":375570687,"identity":"09e21c8b-fe7f-4098-9ce2-94cc1f40853c","order_by":3,"name":"Jingkun Zhang","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, the Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingkun","middleName":"","lastName":"Zhang","suffix":""},{"id":375570688,"identity":"05ab4fb0-c691-4fc1-b6c6-9e33a7281819","order_by":4,"name":"Lin Zhang","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, the Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":375570689,"identity":"32d2bf36-a6a0-4123-a22f-5ce0c0734898","order_by":5,"name":"Xianghua Huang","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, the Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianghua","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-09-24 16:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5146586/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5146586/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-00507-3","type":"published","date":"2025-05-28T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69100526,"identity":"bd4d0e24-6094-452c-bff8-c2d4ff4546c8","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1812246,"visible":true,"origin":"","legend":"\u003cp\u003eThe process of 3D printing and orthotopic transplantation of vaginal biomimetic scaffold. A The morphology of BMSCs at the P3. Scale bar 200μm. B Porcine vaginal ECM powder and vECM-GelMA-SF hydrogel. C The process of bioink loading and 3D bioprinted vaginal biomimetic scaffold. D Staining of live/dead cells from biomimetic scaffolds encasing BMSCs after 7 days in vitro culture. Scale bar 200μm. E Morphological observation of RPE cells. Scale bar 25 μm. D The orthotopic transplantation process of rabbit vagina.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/7e5dbbb070019a99a0c677d6.png"},{"id":69100536,"identity":"17db4ac9-adf9-44b4-b443-e397160b5972","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3437436,"visible":true,"origin":"","legend":"\u003cp\u003eAfter 12 weeks, gross view after specimen collection, HE staining, PAS staining, Masson's trichrome staining and VG staining of newly formed vaginas each group. Scale bar 200μm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/b73d44c0756bd73ca34868ec.png"},{"id":69101393,"identity":"daf217cf-3206-434c-91ba-09c05a14cfa9","added_by":"auto","created_at":"2024-11-15 15:56:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":209406,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of gene expression in each group includes: A The gene UpSet map for each sample identification, B The FPKM density distribution map, C The sample correlation test, with colored squares showing correlation levels between samples, D PCA analysis, where different shapes represent samples and different colors indicate groupings.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/8040a14f12c52c042649bddf.png"},{"id":69100528,"identity":"eb2376ff-aa84-4187-9215-e447f6f1772b","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256321,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of gene differential expression in the three sample groups includes: A A bar graph showing the results of differential expression analysis, B-D Volcano plots for differentially expressed genes in the CvsN, ZvsN, and CvsZ comparisons, with the x-axis representing log2FoldChange and the y-axis representing -log10(p-value); up-regulated genes are shown in red and down-regulated genes in blue, E Venn diagrams illustrating shared and unique differential genes between the comparison groups, F A heatmap of clustered differentially expressed genes, with highly expressed genes shown in red and low-expressed genes in green.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/a96e3bb3422f45e2e1f40446.png"},{"id":69100529,"identity":"cd9fcf4d-3870-41d2-9b8e-3fc46078ffc1","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341119,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic results for reconstructed and normal vaginal tissues include: A PCA analysis, with different shapes representing samples and colors indicating groupings, B Sample correlation test, where colored squares show high or low correlations between samples, C Volcano plot of DEPs for the three comparison groups, D Heatmap of hierarchical clustering analysis for DEPs across the three comparison groups.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/b4b3985de1e7f97e81cc49bd.png"},{"id":69100527,"identity":"79fca12f-5fcd-484a-8d8f-1939fedbb3d0","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2569948,"visible":true,"origin":"","legend":"\u003cp\u003ePPI network plots of DEPs between the three comparison groups. A all DEPs for the three comparison groups: C vs. N, C vs. Z, and Z vs. N. B The top 30 DEPs for C vs. N, C vs. Z, and Z vs. N.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/982b578213880d97a1223977.png"},{"id":69100761,"identity":"1cf80f5e-17a5-46a0-afbc-30d71a51d962","added_by":"auto","created_at":"2024-11-15 15:48:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":234630,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analysis of the top 30 DEPs. A GO terms enriched in GO enrichment analysis for the three comparison groups: C vs. N, C vs. Z, and Z vs. N. B Pathways enriched in KEGG enrichment analysis for the three comparison groups: C vs. N, C vs. Z, and Z vs. N.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/4c15adb73f8491b21bb415cf.png"},{"id":69101394,"identity":"2d0435ac-4eb5-47ce-bf6e-24a79c9328bd","added_by":"auto","created_at":"2024-11-15 15:56:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":633866,"visible":true,"origin":"","legend":"\u003cp\u003eA Venn diagram of upregulated genes and proteins in each comparison group, B Venn diagram of downregulated genes and proteins in each comparison group, C Heatmap showing the correlation between differentially expressed genes and proteins in each comparison group.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/87be5976e527264da76ec6a3.png"},{"id":69100530,"identity":"8870e051-8da8-4af7-8f0c-a1feadaf4c13","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":265678,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analysis for each comparison group includes: A Enriched GO terms for the three comparisons: C vs. N, C vs. Z, and Z vs. N, B Enriched pathways from KEGG analysis for the same comparisons: C vs. N, C vs. Z, and Z vs. N.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/2096dbfc016d0eaac3763b81.png"},{"id":69100534,"identity":"31ac0a2b-6c9f-48a9-9ec7-cc2569443fb7","added_by":"auto","created_at":"2024-11-15 15:40:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":616211,"visible":true,"origin":"","legend":"\u003cp\u003ePPI networks of differentially co-expressed genes and proteins for each comparison group: C vs. N, C vs. Z, and Z vs. N.\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/4683ceef8dbad2438a9d3e19.png"},{"id":83782986,"identity":"c37ab5e3-393a-4e02-916f-fc85b212be38","added_by":"auto","created_at":"2025-06-02 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12611019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5146586/v1/96e291b1-ea82-449f-bbd0-8eed9fe8a6ad.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTranscriptomic and Proteomic Integrated Analysis Reveals Molecular Mechanisms of 3D Bioprinted Vaginal Scaffolds in Vaginal Regeneration\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe vagina is a key part of the female reproductive system and is important for sexual health and reproduction. Congenital defects, trauma, or diseases can cause vaginal deformities, requiring either non-surgical or surgical treatments for reconstruction[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Traditional reconstruction methods can cause complications and discomfort, affecting patients' quality of life[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recent advances in tissue engineering and regenerative medicine offer promising methods for vaginal tissue repair and reconstruction. Utilizing bioengineered implants mimicking the extracellular matrix (ECM) of tissues is key to overcoming existing limitations. ECM scaffolds from specific organs are preferred because they preserve organ structure and guide cell migration, anchoring, and organization[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Combining ECM with gelatin methacryloyl (GelMA) and silk fibroin (SF) improves photocrosslinking and mechanical strength, resulting in excellent tissue repair performance[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vaginal reconstruction is challenging because of its multi-layered structure, complex blood vessels, and nerve distribution[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Regenerating all functional levels requires precise cellular loading and transplantation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bone marrow-derived mesenchymal stem cells (BMSCs) are promising because they can renew themselves, differentiate into various cell types, modulate immune responses, promote tissue remodeling, and act as seeding cells in vaginal reconstruction[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegeneration is a tissue renewal process that depends on wound healing principles and involves multiple tissues and cells. It includes cell migration, proliferation, extracellular matrix deposition, remodeling, and coordinated inflammation and angiogenesis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While small-scale defects heal quickly, but larger defects often cause fibrotic scarring, narrowing, or tissue dysfunction[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The tissue matrix, made of hydrated macromolecules, proteins, and polysaccharides, provides a vital microenvironment for tissue formation and function. These proteins create complex extracellular networks that control cell adhesion, proliferation, and tissue regeneration[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. ECM from different organs helps understand tissue-specific niches, aiding in integrating native tissues with implants for better tissue regeneration and functional recovery[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cell therapy currently holds a leading position in tissue engineering and regenerative medicine. MSCs secrete bioactive factors like soluble proteins, lipids, nucleic acids, and extracellular vesicles, which have anti-inflammatory, anti-scar, immune-regulatory, and anti-apoptotic properties[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Combining BMSCs with biomaterials in preclinical studies has shown promising results in regenerating bone, cartilage, and tendons. Choosing the right stem cells and creating biocompatible scaffolds are essential for repairing damaged tissues.\u003c/p\u003e \u003cp\u003eOur recent study suggests that personalized 3D bioprinted biomimetic scaffolds could be a viable option for restoring damaged vaginal structure and function[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This technology creates bio-inks from vaginal tissue of animal donors, recellularizes them with allogeneic BMSCs, and uses 3D bioprinting to make personalized tissue-engineered vaginal implants. Our research shows that 3D bioprinted vaginal scaffolds achieve cellularization in vivo, whether or not they are seeded with cells. Cell seeding can improve the structural and functional regeneration of the reconstructed vagina. The mechanisms of bioprinted biomimetic scaffolds in tissue reconstruction involve complex interactions and regulation at multiple levels. This study is the first to comprehensively explore differentially expressed genes and proteins, and their functional pathways, in vaginal tissue reconstruction using integrated transcriptomic and proteomic analysis, revealing the molecular mechanisms of biological scaffolds in this process.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eConstruction and in situ transplantation of 3D bioengineered vaginal scaffolds.\u003c/b\u003e P3-BMSCs observed under an inverted phase contrast microscope showed a uniform elongated morphology. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These cells were encapsulated in ECM-GelMA-SF bioink (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and loaded into a 3D bioprinter syringe. A 3D bioengineered scaffold was printed according to preset parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). After 7 days of in vitro culture, live-dead cell staining showed many green live cells, indicating strong cell growth within the scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). A New Zealand rabbit vaginal defect model was constructed, and the scaffold was successfully transplanted in situ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological staining of reconstructed vaginal tissues.\u003c/b\u003e Samples were collected 12 weeks after in situ transplantation of scaffolds. Gross examination of reconstructed vaginal tissues revealed a rich vascular network, smooth mucosa, and prominent folds, with a resilient and elastic texture similar to normal vaginal tissue, especially in the 3D cell bioengineered scaffold group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Histologically, HE staining showed a well-organized epithelial structure with distinct folds in normal vaginal tissue. After 12 weeks post-transplantation, both reconstructed vaginal tissue groups had epithelial cells. In the 3D cell bioengineered scaffold group, epithelial cells were plump, regularly arranged, and featured prominent mucosal folds. Beneath the epithelium was loose submucosal tissue rich in blood vessels and collagen fibers, similar to normal tissue, with no significant differences observed. In contrast, the 3D cell-free bioengineered scaffold group exhibited thin and sparse epithelial layers with irregular cell arrangement. Periodic acid-Schiff (PAS) staining confirmed glycogen synthesis in newly formed epithelial cells, showing purple-red positive cells in both normal and reconstructed vaginal epithelium, with more pronounced positivity in the 3D cell bioengineered scaffold group compared to the 3D cell-free bioengineered scaffold group. Masson's trichrome and Van Gieson (VG) staining revealed a smooth muscle layer beneath the normal vaginal mucosa, with an inner circular and outer longitudinal arrangement of blood vessels and collagen fibers. After 12 weeks post-transplantation, both reconstructed vaginal tissue groups showed many blood vessels in the submucosal fibers, with muscle fibers (red in Masson, yellow in VG) surrounding the vessel walls. In the 3D cell bioengineered scaffold group, muscle fibers were regularly arranged and dense, closely resembling smooth muscle fibers in normal vaginal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of differentially expressed genes (DEGs).\u003c/b\u003e To study how different bioengineered scaffolds affect vaginal regeneration, we analyzed the gene activity in normal vaginal (N), the 3D cell-free biomimetic scaffold (Z), and the 3D cell-loaded biomimetic scaffold (C). We statistically analyzed genes from each sample and created UpSet plots to display unique and common gene expressions among them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We used FPKM to normalize gene expression levels for comparison across different genes and samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Checking gene expression level correlations between samples helps verify experimental reliability. Results showed strong correlations (above 0.8) within each group, which indicates high experimental reliability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Principal component analysis (PCA) grouped similar samples together, with closer distances showing higher similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This analysis confirmed that the sample grouping based on gene expression profiles was consistent and robust.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential expression gene (DEG) analysis.\u003c/b\u003e To explore how different bioengineered scaffolds might regulate vaginal regeneration, we conducted DEG analysis comparing group N, group C, and group Z. Among the 11,956 annotated genes, the C group showed 7,249 DEGs compared to N, with 4,023 genes upregulated and 3,226 genes downregulated. The Z group had 6,684 DEGs, including 3,374 upregulated and 3,310 downregulated genes. Comparing C to Z, we identified 5,960 DEGs, with 3,607 genes upregulated and 2,353 genes downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Volcano plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D) illustrate the distribution of DEGs. A Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) shows the overlap of DEGs among the comparison groups. There are 1,467 DEGs common to all three comparison groups, and 2,595 DEGs unique to the C-N and Z-N comparisons. These DEGs may be important for understanding how different treatments regulate vaginal tissue regeneration. Cluster heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) show the gene expression patterns and clustering relationships among the genes and samples. The results show clear separation and color differences among the N, C, and Z groups, indicating significant differences in gene expression patterns. However, samples within each group are closely clustered, showing similar gene expression patterns within each treatment group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of differentially expressed proteins (DEPs).\u003c/b\u003e To understand how different bioengineered scaffolds affect vaginal regeneration, we performed proteomic analysis. PCA clustered similar samples together, with closer distances showing higher similarity between samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which confirmed the reliability of protein levels and the appropriateness of sample selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). DEPs identified a total of 7,363 proteins across all groups. Compared to N, the Z group had 460 DEPs, including 270 upregulated and 190 downregulated proteins. The C group had 557 DEPs, with 454 upregulated and 103 downregulated proteins. Comparing C to Z, we found 496 DEPs, with 367 upregulated and 129 downregulated proteins. The volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) shows the distribution of DEPs among the groups. A heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) shows the hierarchical clustering analysis of DEPs from the three comparison groups. The heatmap demonstrates that DEPs meeting the fold change criteria (\u0026gt;\u0026thinsp;2) and statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) effectively separate the comparison groups, confirming the validity of DEP selection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein-protein interaction (PPI) network analysis.\u003c/b\u003e Proteins do not act alone; their activities depend on interactions with other proteins and regulation. Combining PPI network analysis with pathway annotation offers a detailed model of cellular activities, aiding in the exploration of molecular mechanisms. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows the PPI network constructed from DEPs of the three comparison groups. Further analysis identified the top 30 DEPs with the highest connectivity, which are highlighted in the PPI network graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This approach identifies key proteins and reveals their interactions within biological systems, providing insights into their roles and potential impact on vaginal tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene Ontology (GO) enrichment analysis.\u003c/b\u003e GO enrichment analysis was conducted on the top 30 key proteins from each comparison group to clarify their functional roles. In the Z-N comparison group, key proteins were significantly enriched in processes like cell migration, regulation of cell shape, cell adhesion, positive regulation of translation, peptide cross-linking, acute-phase response, and tissue homeostasis. Identified proteins were notably enriched in cellular components such as the extracellular region, extracellular space, stress fibers, intermediate filaments, and the lateral plasma membrane. These proteins are involved in functions like heparin binding, calcium dependent protein binding, integrin binding, and fibronectin binding. In the C-N comparison group, key proteins were primarily associated with biological processes including cell adhesion, acute-phase response, integrin-mediated signaling pathway, protein polymerization, response to calcium ion, adaptive immune response, and platelet activation. The enriched cellular components included extracellular region, collagen trimer, phagocytic vesicle, basement membrane, focal adhesion, and endoplasmic reticulum exit site. These proteins participate in molecular functions such as heparin binding, fibronectin binding, collagen binding, integrin binding, extracellular matrix structural constituent, and signaling receptor binding. In the C-Z comparison group, key proteins were significantly enriched in biological processes such as complement activation, acute-phase response, platelet activation, positive regulation of phagocytosis, and blood coagulation. They predominantly localized to extracellular space and cell surface. These proteins are involved in molecular functions including heparin binding, fibronectin binding, collagen binding, antioxidant activity, laminin binding, and calcium ion binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.\u003c/b\u003e KEGG pathway enrichment analysis was performed on the top 30 key proteins from each comparison group to find enriched pathways. In the Z-N comparison group, key proteins were enriched in pathways such as Vascular smooth muscle contraction, Oxytocin signaling pathway, MicroRNAs in cancer, cGMP-PKG signaling pathway, Focal adhesion, Rap1 signaling pathway, and cAMP signaling pathway. The altered DEPs included FN1, VIM, THBS1, FMOD, TIMP1, CCN2, MYH11, MYL9, and ANXA1, involved in processes like angiogenesis, wound healing, cell adhesion, inflammation response, muscle contraction, and epithelial cell formation and differentiation. In the C-N comparison group, key proteins were enriched in pathways including Complement and coagulation cascades, C-type lectin receptor signaling pathway, Platelet activation, Lysosome, Phagosome, Rap1 signaling pathway, and Proteoglycans in cancer. The altered DEPs such as FN1, VIM, THBS1, FMOD, TIMP1, ITGB3, FGA, CTSS, CRP, HP, and COL1A2, participate in processes related to extracellular matrix remodeling, angiogenesis, cell adhesion, and inflammation response. Proteases are involved in collagen degradation in the extracellular matrix, fibronectin aids in protein polymerization, and LUM mitigates scar formation during regeneration, collectively regulating extracellular matrix remodeling. In the C-Z comparison group, key proteins were enriched in pathways such as Complement and coagulation cascades, Cholesterol metabolism, Vitamin digestion and absorption, Fat digestion and absorption, PPAR signaling pathway, ECM-receptor interaction, Platelet activation, and Lysosome. The altered DEPs including PLG, FGA, HRG, HP, C3, C9, CRP, C5, C1Q1, ORM1, and SERPIND1, play roles in regulating complement activation, cell lysis, vascular endothelial growth factor production, lipid transport and metabolism, inflammation response, coagulation reaction, wound healing, vascular development, and extracellular matrix assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These analyses provide comprehensive insights into the functional roles and pathways influenced by different bioengineered scaffolds during vaginal tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIntegration of mRNA and protein analysis.\u003c/b\u003e Transcriptomics and proteomics were integrated in the analysis, as shown in the Venn diagrams (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). The correlation analysis showed significant relationships between differentially expressed genes and proteins in the N, Z, and C comparison groups. Compared to the N group, the C group had 49 upregulated and 11 downregulated genes and proteins, while the Z group had 11 upregulated and 19 downregulated genes and proteins. Compared to the Z group, the C group had 48 upregulated and 19 downregulated genes and proteins. Correlation coefficient analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) assessed the relationship between differentially expressed proteins and genes. The heatmap showed significant correlations between the selected differentially expressed proteins and genes. This approach helps us understand how gene expression is regulated at both the mRNA and protein levels, shedding light on the molecular mechanisms of vaginal tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGO enrichment analysis.\u003c/b\u003e GO enrichment analysis was performed on the differentially co-expressed genes and proteins from the comparisons between the N, Z, and C groups. The results show that compared to the N group, the Z group's differentially co-expressed genes and proteins were mainly enriched in GO terms like antioxidant activity, protein homodimerization, fibronectin binding, laminin binding, and hypotaurine dehydrogenase activity. They participate in biological processes including immune system process, positive regulation of transforming growth factor beta receptor signaling pathway, cell adhesion, negative regulation of cell-matrix adhesion, negative regulation of fibroblast growth factor receptor signaling pathway, and positive regulation of smooth muscle cell proliferation. These proteins are primarily localized in cellular components such as the actin cytoskeleton, bicellular tight junction, axon, endoplasmic reticulum membrane, brush border, cornified envelope, stress fiber, and ubiquitin ligase complex, potentially influencing immune responses, cell-cell and cell-matrix interactions, and smooth muscle proliferation. The differentially co-expressed genes and proteins in the C group, compared to the N group, were enriched in GO terms such as fibronectin binding, laminin binding, actin binding, calcium ion binding, hormone activity, misfolded protein binding, and antioxidant activity. They participate in biological processes such as immune system process, camera-type eye development, positive regulation of intrinsic apoptotic signaling pathway, positive regulation of protein kinase B signaling, regulation of cell population proliferation, and positive regulation of angiogenesis. These proteins are primarily localized in extracellular space, cornified envelope, cell-cell junction, skeletal muscle myofibril, cortical cytoskeleton, and myelin sheath, potentially regulating cell proliferation and apoptosis, angiogenesis, organ development, and inflammatory responses. Compared to the Z group, the differentially co-expressed genes and proteins in the C group were enriched in GO terms like fibronectin binding, collagen binding, calmodulin binding, structural molecule activity, hormone activity, AMP-activated protein kinase activity, and antioxidant activity. They participate in biological processes including cellular acute-phase response, inflammatory response, collagen catabolic process, vasoconstriction, cellular response to xenobiotic stimulus, glycogen catabolic process, positive regulation of epidermal growth factor receptor signaling pathway, and ventricular cardiac muscle tissue morphogenesis. These proteins are significantly enriched in extracellular space, phagocytic cup, cell-cell junction, membrane raft, skeletal muscle myofibril, and protein folding chaperone complex, potentially influencing inflammation responses, extracellular matrix remodeling, vascular regeneration, and organ morphogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). This GO enrichment analysis reveals the functional roles and cellular processes affected by differentially expressed genes and proteins during vaginal tissue regeneration across the comparison groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKEGG pathway enrichment analysis.\u003c/b\u003e KEGG pathway enrichment analysis was conducted on the differentially co-expressed genes and proteins identified from the correlation analysis across the Z-N, C-N, and C-Z groups. In the Z-N group, these genes and proteins were significantly enriched in pathways such as leukocyte transendothelial migration, tight junctions, TGF-beta signaling, FoxO signaling, cell adhesion molecules, the renin-angiotensin system, taurine and hypotaurine metabolism, motor proteins, and focal adhesion. Key protein products involved include MYL9, THBS1, and HP, which are associated with inflammation and immunity, vascular formation, cell-matrix interactions, tissue injury, and remodeling. In the C-N group, the differentially co-expressed genes and proteins were enriched in pathways including Phagosome, Glycolysis / Gluconeogenesis, p53 signaling pathway, Cytoskeleton in muscle cells, Fc gamma R-mediated phagocytosis, Natural killer cell mediated cytotoxicity, Protein digestion and absorption, and Renin-angiotensin system. Protein products such as CTSS, THBS1, COL8A1, and PYGM were identified, which are involved in inflammation and immunity, protein synthesis metabolism, cell proliferation, epithelial cell differentiation, endothelial formation, and embryonic organ morphogenesis. In the C-Z group, the differentially co-expressed genes or proteins were enriched in pathways such as Hypertrophic cardiomyopathy, Complement and coagulation cascades, Tight junction, Starch and sucrose metabolism, Cytoskeleton in muscle cells, PPAR signaling pathway, and Vascular smooth muscle contraction. Key protein products including MMP1, ORM1, and PYGM were highlighted, predominantly associated with cellular metabolism, immune response, extracellular matrix remodeling, and muscle organ morphogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). These KEGG pathway enrichment findings reveal the biological processes and molecular pathways affected by differentially expressed genes and proteins during vaginal tissue regeneration across the comparison groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePPI Network Analysis.\u003c/b\u003e The differentially co-expressed genes and proteins from correlation analysis were visualized in the PPI network (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), helping to analyze protein-protein interactions and identify key proteins. In the Z-N group, the protein corresponding to the HP gene occupies a central position in the network, primarily associated with oxidative stress and inflammatory responses. In the C-N group, proteins corresponding to the HP, CTSS, THBS1, CRP, and MMP7 genes are centrally located in the network. These proteins are predominantly involved in cellular metabolism, inflammation, and extracellular matrix remodeling. In the C-Z group, proteins corresponding to the CTSS, CRP, HP, MMP1, and ORM1 genes are central to the network. They are mainly associated with collagen degradation metabolism, vascular processes in the circulatory system, inflammation, and are implicated in cell proliferation, extracellular matrix remodeling, and immune responses. These key genes and proteins, located in the dense core regions of the network, interact with each other and include many associated proteins. They collectively regulate organ development in animals and may be important targets for vaginal tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIntegrated Analysis of Results.\u003c/b\u003e In the Z group compared to N, we identified 10 key upregulated proteins: FN1, FMOD, VIM, THBS1, TIMP1, CCN2, MYH11, MYL9, and ANXA1. HP was downregulated in the Z group. In the C group compared to N, we identified 14 key proteins upregulated: FN1, VIM, THBS1, FMOD, TIMP1, ITGB3, FGA, CTSS, CRP, HP, COL1A2, MMP7, COL8A1. PYGM was downregulated in the C group. In the C group compared to Z, we identified 14 key proteins upregulated: PLG, FGA, C9, CRP, C5, C3, C1Q1, ORM1, HRG, HP, MMP1, CTSS, SERPIND1. PYGM was downregulated in the C group. TIMP1 is involved in cell proliferation through the HIF-1, p53, and PI3K-Akt signaling pathways. THBS1 regulates cells via the Rap1 signaling pathway. MYL9 is linked to cell proliferation and vascular formation through the cAMP and oxytocin signaling pathways, which collectively aid in vaginal tissue regeneration. These findings highlight how interactions among these proteins regulate vaginal tissue regeneration by affecting cellular proliferation, vascular formation, and inflammatory responses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe application of 3D bioprinting technology in repairing tissue defects and reconstructing organs is very promising. This technology offers personalized treatment options for patients by printing biomaterials, marking an innovative breakthrough in vaginal reconstruction[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This personalized printing approach ensures that reconstructed vaginas closely mimic natural morphology and function. Scaffold materials have excellent biocompatibility and bioactivity, allowing them to integrate with patient tissues and reducing the risks of rejection and infection. This makes vaginal reconstruction surgeries safer and more reliable[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Biomaterials support cells physically, mimic the extracellular matrix structure and function, and promote cell adhesion, proliferation, and differentiation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Seed cells mixed with bioinks can differentiate into target cells like epithelial and smooth muscle cells under specific conditions. This process promotes tissue repair and regeneration and improves the local environment to enhance the survival and function of transplanted tissues[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We printed a new ECM-GelMA-SF bioink using ECM from porcine vaginal tissue and BMSCs, and implanted it in rabbits for vaginal reconstruction. Macroscopic analysis showed that all New Zealand white rabbits with the biomimetic scaffold had good vaginal structure and function. Biomimetic scaffolds loaded with cells showed better regenerative effects, indicating that these scaffolds are effective in promoting vaginal regeneration. However, the mechanism of biomimetic scaffolds made with 3D bioprinting in tissue reconstruction is complex, involving many levels of interaction and regulation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Mechanistic studies of tissue engineering technologies have been reported for oral mucosa, glands, muscles, blood vessels, and neural tissue reconstruction[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Currently, no study has explained the molecular mechanisms of 3D bioprinted biomimetic scaffolds in vaginal reconstruction.\u003c/p\u003e \u003cp\u003eBuilding on our successful construction of vaginal tissue, we used combined transcriptomic and proteomic analyses. Transcriptomic analysis found 3,374 upregulated and 3,310 downregulated genes in the 3D scaffold group compared to the normal group, and 4,023 upregulated and 3,226 downregulated genes in the 3D cell scaffold group. This difference may reflect changes in gene expression regulation when scaffolds are loaded with BMSCs. Proteomic analysis further corroborated the transcriptomic findings. We identified key proteins involved in vaginal regeneration, including those related to inflammation, vascular formation, extracellular matrix remodeling, epithelial and muscle regeneration, neural regeneration, and glycogen synthesis. Scaffolds with stem cells also promote immune activation and coagulation during the regeneration process.\u003c/p\u003e \u003cp\u003eThe tissue compatibility of biomaterials is closely related to inflammatory responses during tissue regeneration. When inflammation is well-regulated, it can improve interactions between cells and materials and aid in vascular formation. Vascularization is essential for tissue regeneration because it supplies nutrients and oxygen to new tissues. Neovascularization also affects the migration of inflammatory cells, as new vessels can attract these cells, speeding up wound healing. Our study found changes in inflammation-related proteins during vaginal reconstruction, such as CRP, THBS1, ANXA1, HRG, HP, ORM1, MYL9, and CTSS, and their associated pathways. Notably, CRP, THBS1, ANXA1, HRG, and CTSS are involved in vascular formation. CRP, an acute-phase protein, is produced and released during infection or tissue injury. It directly regulates angiogenesis through the Wnt pathway and promotes liver cell regeneration in vitro and in vivo[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Local inflammation at the wound site is crucial for healing because it clears debris and protects against bacterial invasion. Fibroplasia and granulation tissue formation help restore the protective skin layer along with epithelialization[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, excessive inflammation can cause further tissue damage and excessive scarring, so it must be strictly regulated and limited at the injury site. THBS1 is a multifunctional glycoprotein released by platelets, epithelial cells, and stromal cells during development, wound healing, angiogenesis, platelet aggregation, and cell adhesion. Without THBS1, inflammation can persist, wound healing may be delayed, and scab detachment can be slow[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies have shown that THBS1 mRNA is undetectable in normal skin but present in early wounds, primarily sourced from macrophage-like cells in inflammatory infiltrates. Inhibiting THBS1 significantly delays wound repair by reducing re-epithelialization rates and slowing dermal remodeling, showing that THBS1 produced by macrophages aids the repair process[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. THBS1 works with angiogenic factors to control capillary angiogenesis and maintain vascular balance during regeneration[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These biological processes may involve pathways like the Rap1, p53, and TGF-beta signaling pathways. ANXA1, an anti-inflammatory protein mediated by glucocorticoids, reduces inflammation by inhibiting leukocyte activation and migration. ANXA1 regulates cell migration, promotes tissue repair, and plays a crucial role in liver proliferation and regeneration[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. ANXA1 helps resolve inflammation by promoting wound closure and restoring epithelial barriers in intestinal cells in vivo[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. ANXA1 inhibits fibrosis gene expression, shows anti-fibrotic activity, promotes corneal epithelial wound healing, and reduces corneal inflammation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The expression of HRG and CTSS was higher in the scaffold with stem cells compared to the scaffold alone. HRG is a liver-produced glycoprotein that regulates angiogenesis in both pro- and anti-angiogenic ways[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. HRG reduces inflammation by inhibiting excessive neutrophil activation in circulation and protects the vascular endothelial barrier by regulating clotting pathways[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CTSS, a key cysteine protease, degrades anti-angiogenic peptides and adhesion proteins, promoting neovascularization, tumor cell invasion, and metastasis. CTSS degrades the endothelial basement membrane and promotes VSMC migration and proliferation through the p38MAPK/Akt signaling pathway, aiding injury-related vascular repair[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We hypothesize that stem cells may positively regulate inflammation and vascular formation. In summary, regulating both pro-inflammatory and anti-inflammatory responses, as well as vascular formation, is crucial throughout vaginal regeneration.\u003c/p\u003e \u003cp\u003eThe ECM is a flexible structure found in all tissues that supports cells and tissues and changes constantly in a controlled way. Our results indicate upregulation of ECM remodeling-associated components such as MMP1, MMP7, TIMP1, COLIA2, COL8A1, FMOD, FN1, VIM, ITGB3 in the scaffold group. The breakdown of ECM components is a main part of ECM remodeling, and matrix metalloproteinases (MMPs) are crucial for this process. MMP activity is usually low but rises during repair, remodeling, and in diseased or inflamed tissues[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Both bioengineered scaffolds for vaginal reconstruction showed a significant increase in MMPs (MMP-1, MMP-7) and higher levels of tissue inhibitors of metalloproteinases (TIMPs). MMPs are involved in tissue remodeling, growth, angiogenesis, tissue defense, and immune responses[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. TIMPs usually regulate MMPs, and the balance between them determines how much ECM protein is broken down and how tissue is remodeled[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. MMP1 helps assemble and repair the basement membrane, supporting wound re-epithelialization under oxidative stress and anti-fibrotic conditions[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This makes MMP1 a potential anti-fibrotic agent for preventing or treating hypertrophic scars[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Angiogenesis needs the breakdown of the vascular basement membrane and ECM remodeling for endothelial cell migration into nearby tissues. MMPs facilitate the actions of various angiogenic factors through their proteolytic activity, promoting blood vessel formation[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, MMPs may exert anti-angiogenic effects by degrading specific collagen chains and plasminogen activators[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, MMPs are important regulators of angiogenesis, usually promoting it. Collagen is the most abundant part of the ECM and is essential for tissue structure and function. Collagen fibers provide the foundation for tissue shape, strength, and wound healing[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Type I collagen is the most abundant type and is found in blood vessels, cornea, sclera, tendons, ligaments, and skin, making up over 90% of ECM levels[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. TIMP-1 and COL1A1 work together in the proliferation and remodeling stages of periodontal wound healing, crucially preventing ECM and collagen breakdown during remodeling[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Collagen I interacts with different pro-fibrotic tissue remodeling proteins to form the ECM. Other ECM molecules, like FN1 and TIMP-1, integrate into the network to balance the construction and breakdown of damaged tissues[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. COL8A1 is a secreted protein found in many rapidly growing cells and promotes smooth muscle cell migration and proliferation during vascular remodeling[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Col8a1 genes may be biomarkers for heart remodeling[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Endothelial-derived Col8a1 is a key factor in endothelial cell proliferation and may positively influence endothelial repair and the integrity of new intimal layers[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Fibronectin-1 (FN1) is a glycoprotein found on cell surfaces, in extracellular fluids, and in connective tissues, interacting with collagens, fibronectins, and integrins. FN1 primarily supports cell adhesion and is involved in cytoskeletal organization, cell migration, and important physiological processes like wound healing, thrombosis, and aging[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. High levels of FN1 interact with integrin ITGB3, regulating gene activity that influences various signaling pathways and is involved in ovarian follicle development, maturation, ovulation, and corpus luteum formation[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. During tissue injury, mRNA levels of ITGB3 and FN1 increase under low oxygen conditions, allowing them to stay in local areas, stimulate cell proliferation, speed up blood vessel formation, and enhance wound healing[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Adding stem cells increases ITGB3 protein expression. Integrins are transmembrane receptors that help cells stick to the ECM, bind to various growth factors, generate different intracellular signals, and regulate cell proliferation, differentiation, and fusion[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Phosphorylation of ITGB3 can stimulate angiogenesis and VEGF expression via the MAPK/ERK pathway, regulating vascular formation and stability[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Vimentin is a type III intermediate filament protein that is important for cell functions like migration, proliferation, and division. It is also linked to cell adhesion and migration during epithelial-mesenchymal transition, ECM remodeling, and inflammatory responses, which are essential for normal wound healing[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Vimentin promotes nerve sprout growth and sensory recovery, providing neuroprotective functions[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Vimentin is essential for regulating vascular contraction, tension, endothelial integrity, and barrier function. It participates in vascular remodeling and smooth muscle cell differentiation, which are important for tissue homeostasis and repair[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Fibromodulin (FMOD) binds to ECM structural components, regulating collagen cross-linking, assembly, and fiber structure. It also interacts with signaling molecules, influencing cell adhesion, proliferation, migration, invasion, differentiation, and metastasis. Therefore, FMOD promotes migration, angiogenesis, anti-inflammation, and anti-fibrosis, playing important roles in determining cell fate and tissue regeneration[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. FMOD is involved in wound healing and the assembly of ECM components, such as collagen, and is closely related to muscle regeneration and myoblast proliferation and differentiation[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. FMOD can induce a more \"fetal-like\" migratory and contractile phenotype in adult dermal fibroblasts, which reduces scar formation without weakening tensile strength in adult wound models[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. During vaginal reconstruction, increased expression of collagen, fibronectin, and vimentin offers support and stability for new tissues, while MMPs, TIMPs, and FMOD aid in regeneration and repair by degrading and remodeling ECM components. The balance between these factors is essential for successful vaginal tissue reconstruction.\u003c/p\u003e \u003cp\u003eWe observed increased expression of CCN family members in the scaffold group. CCNs can promote tissue regeneration through various mediators like growth factors, matrix proteins, and integrins[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Studies show that CCN2 speeds up the healing rate of burns and diabetic wounds[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. CCN2 regulates keratinocyte migration, promoting re-epithelialization during skin wound healing[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Dynamic expression of CCNs during tissue regeneration drives epithelial regeneration and mediates fibrogenesis and anti-fibrotic activity during repair. FMOD coordinates the timing and location of TGF-β ligands and receptors in vivo, regulating collagen fiber generation and reducing scar formation in the extracellular matrix. In oral mucosal wound healing, FMOD's location and abundance in the wound epithelium are regulated over time and space, promoting wound re-epithelialization and connective tissue regeneration[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. As a hollow organ, the vagina shows different contractile behaviors, such as peristalsis, tension maintenance, and spasms, due to its smooth muscle components. Beta-3 integrin mediates myogenesis by regulating myogenic gene expression and satellite cell migration[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Myosin heavy chain subtype I (MYH1) is part of the MYH gene family and encodes the MyHC-IIX protein, which is expressed in intermediate muscle fibers and helps form skeletal muscle fibers[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. MYH1 may be linked to SMSC differentiation and muscle fiber hypertrophy, and it is highly expressed in the late stages of C2C12 cell differentiation[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. THBS1 promotes axon growth in neurons in vitro and aids axon regeneration through intrinsic mechanisms in retinal ganglion cells[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. In addition, VIM, ITGB1 and CTSS can also regulate axon regeneration[\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Vaginal epithelial cells produce glycogen, which converts to lactate when shed, creating a unique microenvironment that prevents pathogenic bacteria from invading[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. We found that PYGM, a protein involved in glycogen breakdown metabolism, is downregulated in the reconstructed vaginal tissue with added cells. PYGM mainly provides energy for muscle contraction but is also expressed in other tissues and plays a key role in the early steps of glycogen breakdown through phosphorylation[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Inhibiting PYGM reduces glycogen breakdown, promoting glycogen storage within epithelial cells, beneficial for maintaining the vaginal microenvironment. The upregulation of CCN2, FMOD, MYH1, THBS1, VIM, and CTSS, along with the downregulation of PYGM, suggests that bioengineered scaffolds promote vaginal epithelialization, muscle regeneration, vaginal smooth muscle contraction, axon regeneration, and increased glycogen storage, aiding in the formation and recovery of vaginal tissue structure.\u003c/p\u003e \u003cp\u003eCompared to the scaffold-only group, the cell-added group showed increased levels of proteins related to the coagulation cascade and anticoagulant systems, such as FGA, PLG, and SERPIND1, as well as proteins associated with the complement cascade and immune responses, including C9, C5, C3, C1Q1, HP, and ORM1. The coagulation, fibrinolysis, and complement systems are essential for maintaining tissue homeostasis, with many interactions between these cascades that regulate fibrinolysis and complement activation during injury and acute inflammation[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Tissue injury, trauma, or systemic inflammation trigger complement activation and the coagulation and fibrinolysis pathways, where coordinated enzymatic cascades repair damaged vessels and regulate the inflammatory process, despite their opposing functions[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The final step of the coagulation cascade involves converting fibrinogen alpha chain (FGA) to fibrin, which then polymerizes to form a blood clot[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Elevated FGA in inflammation-driven acute responses promotes hemostasis by activating platelets, which help distribute red blood cells, macrophages, and fibroblasts around wounds. Controlling these processes is essential for hemostasis, wound healing, and tissue regeneration[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].In addition to its role in hemostasis, FGA regulates leukocyte function during inflammation, with soluble fibrinogen influencing neutrophil adhesion and affecting leukocyte recruitment in vivo, thereby promoting inflammatory responses[\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. FGA and its degradation products regulate vascular generation potential[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. PLG is one of the most abundant proteins involved in hemostasis and is activated by t-PA and u-PA to produce plasmin, which starts fibrinolysis[\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Plasminogen has functions beyond the fibrinolysis system, such as removing misfolded or aggregated proteins, triggering other enzyme cascades like complement activation, regulating cell behavior, and affecting immune and inflammatory processes, enhancing thrombolysis and speeding up wound repair[\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. FGA and PLG work together to maintain balance between the coagulation cascade and the fibrinolysis system, promoting tissue structure and functional reconstruction, especially in epithelial injury and reshaping[\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The complement system is a key part of the innate immune system, serving as the first line of defense against invading pathogens. It also removes apoptotic and necrotic cells and regulates the adaptive immune system and inflammatory processes[\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Complement has been shown to enhance coagulation by inhibiting fibrinolysis, while proteases from the coagulation and fibrinolysis systems cleave and activate C3 and generate C5a[\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. During infection, local complement-mediated coagulation activation boosts clotting, creating a barrier against bacterial spread and aiding in the production of antimicrobial peptides while supporting the inflammatory response[\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. In neovaginal formation, the coagulation system, fibrinolysis system, and complement system are essential for maintaining tissue homeostasis. They ensure vascular integrity, prevent thrombus formation, resist foreign pathogens, and maintain tissue stability through cooperation and precise regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed the critical roles of genes and proteins related to extracellular matrix remodeling, vascular regeneration, inflammatory response, epithelialization, and muscle formation in vaginal reconstruction through combined transcriptomic and proteomic analysis. This research offers a new perspective on vaginal tissue regeneration and highlights the potential of personalized bio-scaffolds for promoting tissue repair and functional recovery. In the future, further exploration of these molecular mechanisms and improvements in 3D printing and cell therapy technologies aim to achieve better clinical outcomes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eExperimental Animals and Grouping.\u003c/b\u003e Female New Zealand White rabbits aged 1\u0026ndash;2 months, weighing 1.0-1.5 kg, were used to harvest BMSCs. Mature female New Zealand White rabbits aged 7\u0026ndash;8 months, weighing 3.5-4.0 kg, were used to create a vaginal defect model for testing vaginal bioengineered scaffolds. All animals were bought from Tonghui Breeding Co., Ltd. in Wangdu County, Hebei Province, China. All procedures involving these animals strictly adhered to the Guidelines for Care and Use of Laboratory Animals of Hebei Medical University and received approval from the Animal Ethics Committee of the Second Hospital of Hebei Medical University(Approval Letter No.2024-AE011). All methods are reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org).Eac\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org).Eac\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eh animal was housed separately in the Animal Experimental Center of the Second Hospital of Hebei Medical University. There were three experimental groups: 1) Normal group (n\u0026thinsp;=\u0026thinsp;4): sham-operated group; 2) 3D cell-free biomimetic scaffold group (n\u0026thinsp;=\u0026thinsp;4): each rabbit received a bioengineered scaffold; 3) 3D cell-seeded biomimetic scaffold group (n\u0026thinsp;=\u0026thinsp;4): each rabbit received a scaffold seeded with rabbit BMSCs. Following the completion of the experiments, to ensure the comfort of the rabbits and alleviate their suffering, the researchers adhered to the guidance of the Animal Ethics Committee and employed euthanasia through overdose anesthesia. This method is considered a humane and effective means of euthanasia for experimental animals.\u003c/p\u003e \u003cp\u003ePreparation of BMSCs. Rabbit BMSCs were prepared by density gradient centrifugation and adherent culture. P3 cells were used for the experiments. When the cell fusion rate reached 80%-90%, the cells were detached with trypsin and resuspended as a single-cell suspension for further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of vECM-GelMA-SF Bioink.\u003c/b\u003e ECM was prepared as described previously[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The lyophilized ECM was ground into a powder and mixed with 3 ml of 0.01 mol/L HCl solution containing 45 mg of pepsin. Digestion was carried out at 37\u0026deg;C, 80 rpm for 12 hours to prepare vECM hydrogel. The pH was adjusted from acidic to neutral using 0.1 mol/L NaOH. GelMA (8% concentration) and photoinitiator (5%) were dissolved in 3 ml of sterile distilled water at 60\u0026deg;C for 40 minutes, filtered through a 0.22 \u0026micro;m sterile filter, and sterilized by autoclaving. This solution was mixed with the prepared 3 ml vECM solution and 5% SF (50 mg/ml) to obtain vECM-GelMA-SF bioink. P3-BMSCs were added to the bioink at a concentration of 1x10^7 cells/ml, loaded into sterile syringes, and kept at 4\u0026deg;C until use. The 3D biomimetic vaginal scaffold was printed using a JNOV Life 3D bioprinting system. An adapter needle (diameter 0.34 mm) was installed and fixed on the bioprinter's low-temperature nozzle. The scaffold parameters were set to X\u0026thinsp;=\u0026thinsp;10 mm, Y\u0026thinsp;=\u0026thinsp;10 mm, Z\u0026thinsp;=\u0026thinsp;15 mm, with a layer thickness of 0.34 mm and 30 layers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiocompatibility Testing of vECM-GelMA-SF Bioink.\u003c/b\u003e P3-BMSCs loaded scaffolds were cultured in vitro for 7 days. Cell viability was assessed with a live/dead cell staining kit, and fluorescence confocal microscopy was used to observe cell survival.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScaffold In Situ Transplantation.\u003c/b\u003e Experimental animals were anesthetized with 1% pentobarbital sodium (0.4 ml/100 g) via ear marginal vein injection. After anesthesia, the abdominal area was prepared and disinfected with iodine, and a midline incision was made in the lower abdomen to expose the uterus and vagina. Partial vaginal excision was performed by cutting the upper end of the vagina 5 mm below the cervix and the lower end 1 cm above the urethral meatus. The prepared bioengineered scaffold was fixed at the upper end to the external uterine orifice and aligned with the remaining vaginal end, then the abdominal cavity was closed layer by layer. All surgeries were performed by the same surgical team. To prevent infection, 1\u0026nbsp;million units of sodium penicillin were injected into the abdominal cavity once a day for 3 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample Collection and Evaluation.\u003c/b\u003e Three months post-surgery, newly formed vaginal tissues were collected for histological examination. The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 mm thick sections. Sections were stained with HE, Masson, VG, and PAS stains to observe epithelialization, vascularization, smooth muscle formation, and glycogen synthesis in the regenerated vaginal epithelium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA Sequencing.\u003c/b\u003e Vaginal tissues from the three groups, with three samples per group, were collected. Total RNA was extracted using TRIzol Reagent. RNA concentration and purity were assessed using an Agilent 2100 Bioanalyzer and RNA-specific agarose gel electrophoresis. mRNA enriched with polyA structure was selected using Oligo(dT) magnetic beads. RNA was then fragmented to approximately 300 bp using ion disruption, and 300 bp fragments were synthesized into cDNA using RNA as a template. After library construction, PCR amplification was performed to enrich the library fragments. Library size selection was set to 450 bp. The quality and concentration of the libraries were evaluated using the Agilent 2100 Bioanalyzer. The libraries were then subjected to next-generation sequencing (NGS) using the Illumina sequencing platform for paired-end (PE) sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDEGs Analysis.\u003c/b\u003e Initially, the expression level correlation among samples was examined using Pearson correlation coefficien. PCA based on expression levels was conducted using the DESeq package in R. Differential gene expression analysis was performed using DESeq package in R. volcano plots were generated using ggplot2 package in R to display the distribution of genes, fold change differences, and significance results. DEGs specific to and shared among the comparison groups were statistically analyzed. Heatmaps were constructed using the pheatmap package in R to perform bidirectional clustering analysis of DEGs across all comparison groups, based on Euclidean distance and complete linkage method. DEGs were annotated using GO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GO annotation analysis was conducted using Goatools software. DEGs were also mapped to KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for pathway enrichment analysis using KOBAS software.\u003c/p\u003e \u003cp\u003eProteomic Analysis\u003c/p\u003e \u003cp\u003e \u003cb\u003eDIA Analysis.\u003c/b\u003e Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptide sequences (n\u0026thinsp;=\u0026thinsp;3) from vaginal tissues of each group, which were then used for proteomic analysis. Adequate protein samples were used to construct a spectral library. Protein samples from each group were subjected to SDS-PAGE to assess sample consistency. Trypsin digestion was performed using FASP method. Peptide concentration was determined by OD280, and peptides were fractionated using HPRP method. Each fraction was analyzed by data-dependent acquisition (DDA) mass spectrometry after addition of iRT standard peptides. Nano-flow HPLC system Nanoelute was used for chromatographic separation. Peptides were analyzed by timsTOF Pro mass spectrometer after liquid chromatography separation. Maxquant software was used for data processing, and Mus musculus_uniprot database was employed for database searching with the following parameters: trypsin enzyme, maximum missed cleavage sites set to 2, fixed modification of Carbamidomethyl (C), and dynamic modifications of Oxidation (M) and Acetyl (Protein N-term). Identified proteins were filtered based on a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;1%. Spectral libraries were constructed by importing raw files and search results into Maxquant software. DIA mass spectrometry was performed on all samples, with chromatographic separation using Nanoelute and the same liquid chromatography gradient and DDA testing conditions. Peptides separated by nano-flow high-performance liquid chromatography were analyzed by timsTOF Pro mass spectrometer using DIA data acquisition mode. Data were processed using Maxquant software with the same database used for library construction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDEPs Analysis.\u003c/b\u003e Annotation information corresponding to species was downloaded from uniprot. Direct and indirect interactions among DEPs were investigated based on STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://string-db.org/\u003c/span\u003e\u003cspan address=\"http://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) information for PPI network analysis. Results from PPI network analysis were imported into Cytoscape (v3.7.1) to analyze and select proteins with top 30 Degree values. These proteins were further subjected to GO functional enrichment analysis and KEGG pathway enrichment analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntegration Analysis of Transcriptomic and Proteomic Data.\u003c/b\u003e Transcriptomic and proteomic data were integrated to identify genes and proteins that were differentially expressed and regulated in the same way between the two sequencing methods. Venn diagrams were used to display the intersection of upregulated/downregulated genes and proteins. Correlation analysis was performed to examine the relationship between differentially expressed proteins and genes. GO functional enrichment analysis and KEGG pathway enrichment analysis were conducted for differentially expressed genes and proteins. Direct and indirect interactions between differentially expressed genes and proteins were explored using STRING database, followed by PPI analysis to identify proteins with high connectivity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis.\u003c/b\u003e GraphPad Prism 8.0 software was used for data processing. Continuous variables with a normal distribution were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). Student's t-test was used to analyze the significance of differential gene and protein expression, with fold change (FC) criteria typically set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FC\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.5. GO and KEGG database analyses were conducted using Fisher's exact test for functional and pathway enrichment, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating significant enrichment, where smaller values indicated more significant enrichment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXuemei Zhang and Jiahua Zheng, contributed to conception, design, acquisition, analysis, and interpretation of data, drafed manuscript, revised manuscript. Xianghua Huang and Lin Zhang, contributed to acquisition, analysis, and interpretation of data, critically revised manuscript, gave fnal approval. Liye Zhang and Jingkun Zhang, acquisited and analyzed the datas. All authors edited and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe extend our gratitude to all members of the Hebei Key Laboratory of Regenerative Medicine of Obstetrics and Gynecology and Hebei Medical University's Core Facilities and Centers for their valuable discussions regarding this work and the manuscript. The authors extend their gratitude to\u0026nbsp;technicist from Shiyanjia Lab (www.shiyanjia.com) for the sequencing services and bioinformatic analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data that support the findings of this study have been deposited in the NCBI with the primary accession code PRJNA1175434. Web Links：ID 1175434 - BioProject - NCBI.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcQuillan, S.K. \u0026amp; Grover, S.R. 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The contact system--a novel branch of innate immunity generating antibacterial peptides. \u003cem\u003eEmbo j\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 5569-5578 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vaginal reconstruction, Biological scaffold, Bone marrow-derived mesenchymal stem cells, Proteomics, Transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-5146586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5146586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e3D bioprinting technology has been applied to vaginal reconstruction with satisfactory results. Understanding the transcriptome and proteome of regenerated vaginas is essential for knowing how biomaterials and seed cells contribute to vaginal regeneration. There are no reports on the systemic analysis of vaginal regeneration transcriptomes or proteomes. This study aims to explore the transcriptomic and proteomic features of vaginal tissue reconstructed with 3D bioprinted scaffolds. The scaffolds were made with biomaterials and bone marrow-derived mesenchymal stem cells (BMSCs) and then transplanted into a rabbit model.RNA sequencing was used to analyze the transcriptomes of reconstructed and normal vaginal tissues, identifying 11,956 differentially expressed genes (DEGs). Proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and data-independent acquisition (DIA) identified 7,363 differentially expressed proteins (DEPs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on DEGs and DEPs. Results showed that DEGs and DEPs were involved in extracellular matrix remodeling, angiogenesis, inflammatory response, epithelialization, and muscle formation. This study shows that 3D bioprinted scaffolds are feasible for vaginal reconstruction and offers new insights into the molecular mechanisms involved.\u003c/p\u003e","manuscriptTitle":"Transcriptomic and Proteomic Integrated Analysis Reveals Molecular Mechanisms of 3D Bioprinted Vaginal Scaffolds in Vaginal Regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 15:40:12","doi":"10.21203/rs.3.rs-5146586/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-19T07:13:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-19T09:23:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168170133547819422229256893073974971400","date":"2024-12-09T01:30:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T07:47:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96932584950169202535374000497445405014","date":"2024-11-17T03:36:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-16T01:29:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T08:15:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-07T07:32:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-04T11:51:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-24T16:04:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2da06180-3e1b-4430-bb90-86200b9c636f","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40325682,"name":"Biological sciences/Biological techniques"},{"id":40325683,"name":"Biological sciences/Biotechnology"}],"tags":[],"updatedAt":"2025-06-02T16:03:14+00:00","versionOfRecord":{"articleIdentity":"rs-5146586","link":"https://doi.org/10.1038/s41598-025-00507-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-28 15:57:51","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2024-11-15 15:40:12","video":"","vorDoi":"10.1038/s41598-025-00507-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-00507-3","workflowStages":[]},"version":"v1","identity":"rs-5146586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5146586","identity":"rs-5146586","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}