Data from: Matrix stiffness governs fibroblasts' regulation of gingival immune homeostasis
Data files
Mar 16, 2026 version files 318.63 KB
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Figure_1D.csv
161.77 KB
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Figure_1I.csv
195 B
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Figure_2C.csv
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Figure_2D_storage_modulus.csv
168 B
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Figure_2D_tan_delta_.csv
179 B
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Figure_3A.csv
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Figure_3B.csv
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Figure_3C_CCL2.csv
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Figure_3C_IL6.csv
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Figure_3C_IL8.csv
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Figure_3D.csv
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Figure_3E.csv
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Figure_3F.csv
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Figure_4B.csv
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Figure_4D.csv
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Figure_4E_CCL2.csv
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Figure_4E_IL6.csv
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Figure_4E_IL8.csv
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Figure_4H.csv
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Figure_4I_CCL2.csv
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Figure_4I_IL6.csv
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Figure_4I_IL8.csv
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Figure_5C.csv
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Figure_5D.csv
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Figure_5E_IL6_Soft_.csv
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Figure_5E_IL6_Stiff.csv
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Figure_5E_IL8_Soft.csv
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Figure_5E_IL8_Stiff.csv
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Figure_6C___Live_CD45__CD11b__HLA-DR_.csv
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Figure_6C_gMFI_PD-L1.csv
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Figure_6D.csv
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Figure_6E.csv
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Figure_7B_Storage_Modulus.csv
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Figure_7B_tan_delta_.csv
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Figure_7C_CCL2.csv
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Figure_7C_IL6.csv
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README.md
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Abstract
Periodontal disease is characterized by inflamed gingival tissues and degradation of the gingival extracellular matrix (ECM), yet the role of mechanical cues remains poorly understood. Gingival ECM in periodontal disease showed reduced fibrillar collagen compared to healthy samples. We hypothesized that ECM softening in periodontal disease contributes to inflammation by dysregulating gingival fibroblasts (GFs). A mechanically tunable hydrogel model of the gingival ECM was developed to investigate the mechano-immune crosstalk. Stiff and soft collagen-alginate hydrogels matched the rheological properties of healthy and diseased gingival biopsies, respectively. Human donor GFs encapsulated in these stiff hydrogels showed significantly suppressed toll-like receptor-mediated inflammatory responses compared to those in soft hydrogels. The non-canonical NFκB pathway and epigenetic nuclear organization directed stiffness-dependent inflammatory responses of GFs. The direct impact of mechanical cues on immune responses was investigated ex vivo by co-culture of donor-derived human GFs with myeloid cells and in human gingival explants. Myeloid progenitors co-cultured with GFs in stiff hydrogels differentiated into immunomodulatory dendritic cells. Ex vivo crosslinking of human gingival tissue increased stiffness and reduced the production of inflammatory cytokines. Gingival mechano-immune regulation offers a novel approach to biomaterial-based treatments for periodontitis.
Dataset DOI: 10.5061/dryad.xksn02vw6
Description of the data and file structure
For detailed information on methods, please refer to supporting information: adma72486-sup-0001-SuppMat.pdf
Files and variables
File: Figure_1D.csv Description: Heatmap data showing the distribution of collagen fiber orientation as a function of Z-layer depth in native human gingival tissue.
- Z-layer: Numerical identifier for individual tissue layers through the depth of the biopsy
- Orientation (0°-180°): Pixel percentage counts representing the frequency of collagen fibers oriented at specific angular bins.
File: Figure_1I.csv Description: Quantification of collagen density in human gingival tissue biopsies measured via Mean Fluorescence Intensity (MFI) of the Second Harmonic Generation (SHG) signal.
Variables
- Healthy: SHG MFI values (arbitrary units) for healthy donor tissue
- Moderate Disease: SHG MFI values for Periodontal Disease (PD) tissue.
- Severe Disease: SHG MFI values for Severe Periodontal Disease tissue.
File: Figure_2C.csv Description: Data representing the distribution of collagen fiber orientation throughout the Z-depth of the developed soft and stiff hydrogels.
File: Figure_2D_storage_modulus.csv Description: Rheological characterization showing the Storage Modulus (G') in Pascals (Pa) for developed biomimetic hydrogels compared to native healthy and diseased human gingiva.
Variables
- SoftHydrogel: Storage modulus values for the soft hydrogel formulation.
- StiffHydrogel: Storage modulus values for the stiff hydrogel formulation.
- HealthyGingiva: Storage modulus values for native healthy human gingival tissue.
- Periodontitis: Storage modulus values for human gingival tissue with periodontal disease.
File: Figure_2D_tan_delta_.csv Description: Rheological characterization showing the Tan Delta values (ratio of loss modulus G'' to storage modulus G'), representing the viscoelasticity of hydrogels and native tissue.
Variables
- SoftHydrogel: Tan delta values for the soft hydrogel.
- StiffHydrogel: Tan delta values for the stiff hydrogel.
- HealthyGingiva: Tan delta values for native healthy gingival tissue.
- Periodontitis: Tan delta values for gingival tissue with periodontal disease.
File: Figure_3A.csv Description: Gene set enrichment analysis of biological pathways significantly upregulated in GFs cultured on stiff hydrogels (2 kPa) compared to soft hydrogels (0.75 kPa). Variables
- Pathway: Name of the significantly enriched Reactome pathway.
- Gene Ratio: Proportion of differentially expressed genes in the dataset relative to the total genes in the pathway.
- P-value: Benjamini-Hochberg corrected P-value for enrichment significance.
- Counts: Number of genes in the dataset associated with the pathway.
File: Figure_3B.csv Description: Heatmap data showing the relative expression of differentially expressed genes (z-scored) in GFs encapsulated in stiff versus soft hydrogel environments under baseline conditions. Variable
- rows: gene symbols for the differentially expressed genes.
- STIFF-1, 2, 3: Z-score normalized gene expression for three biologically independent stiff hydrogel replicates.
- SOFT-1, 2, 3: Z-score normalized gene expression for three biologically independent soft hydrogel replicates.
File: Figure_3C_CCL2.csv / IL6.csv / IL8.csv Description: ELISA quantification of secreted pro-inflammatory mediators from TLR2-activated GFs cultured in hydrogels of varying stiffness (0.75, 1.5, and 2 kPa).
Variables
- G' (0.75, 1.5, 2): Matrix stiffness categories in kPa.
- Rep 1-4: Normalized cytokine concentration values
File: Figure_3D.csv Description: Cell viability analysis using LDH assay for GFs in soft and stiff hydrogels with or without TLR2 agonist stimulation.
Variables
- Group: Treatment conditions (Untreated, TLR2 agonist).
- Soft1-4 / Stiff1-4: Percentage viability values for individual biological replicates.
File: Figure_3E.csv Description: Gene set enrichment of inflammatory signaling pathways regulated in TLR2-stimulated GFs cultured in stiff versus soft hydrogels.
Variables
- Pathway: Enriched inflammatory or signaling pathway name.
- Gene Ratio: Proportion of differentially expressed genes in the pathway.
- PP-value Statistical significance for pathway enrichment.
- Count: Number of genes associated with the pathway.
- Class: Direction of regulation
File: Figure_3F.csv Description: Heatmap data illustrating z-scored relative expression of genes in GFs cultured in soft versus stiff hydrogels following TLR2 activation. Variables
- rows: gene symbols.
- STIFF-1, 2, 3 / SOFT-1, 2, 3: Z-score normalized gene expression for TLR2-stimulated biological replicates.
File: Figure_4B.csv Description: Quantification of nuclear sphericity for individual nuclei of GFs cultured on hydrogels of increasing stiffness.
Variables
- Soft: Sphericity index for individual nuclei in soft hydrogels
- Stiff: Sphericity index for individual nuclei in stiff hydrogels
File: Figure_4D.csv Description: Quantification of cell length in GFs following treatment with cytoskeletal inhibitors (Blebbistatin or Y-27632) in soft and stiff matrices.
Variables
- Soft / Stiff: Control cell length measurements.
- Soft/Stiff + Blebb: Cell length after myosin II inhibition.
- Soft/Stiff + Y-27632: Cell length after ROCK inhibition.
File: Figure_4E_CCL2.csv / IL6.csv / IL8.csv Description: ELISA quantification of cytokines from TLR2-activated GFs treated with cytoskeletal inhibitors. Variables
- Control / Blebbistatin / Y27632: Experimental treatment groups.
- Soft 1-4 / Stiff 1-4: Normalized cytokine concentration values across replicates.
File: Figure_4H.csv Description: Mean Fluorescence Intensity (MFI) of nuclear-localized phosphorylated RelB (Ser552) in GFs cultured in soft versus stiff hydrogels.
Variables
- Soft: Phospho-RelB nuclear MFI values (arbitrary units).
- Stiff: Phospho-RelB nuclear MFI values (arbitrary units).
File: Figure_4I_CCL2.csv / IL6.csv / IL8.csv Description: ELISA quantification of cytokines from TLR2-activated GFs cultured in soft or stiff matrices with or without IKK inhibition (IKKi).
Variables
- Soft / Stiff: Baseline stiffness conditions.
- Soft/Stiff + IKKi: Cytokine levels following non-canonical NFκB pathway inhibition.
File: Figure_5C.csv Description: Quantification of GF cell length in stiff hydrogels following treatment with increasing concentrations of a DNMT inhibitor.
Variables
- Soft / Stiff: Baseline cell lengths.
- Stiff + DNMTi (0.5μM / 1μM): Cell lengths following dose-dependent inhibition
File: Figure_5D.csv Description: Nuclear volume analysis of GFs in stiff hydrogels with and without DNMT inhibitor treatment.
Variables
- Stiff: Nuclear volume of untreated cells.
- Stiff+DNMTi: Nuclear volume of inhibitor-treated cells.
File: Figure_5E_IL6_Soft.csv / IL6_Stiff.csv / IL8_Soft.csv / IL8_Stiff.csv Description: ELISA quantification of IL-6 and IL-8 secretion from GFs treated with varying doses of DNMT inhibitor.
Variables
- Control / 0.5μM / 1μM: DNMT inhibitor dosage groups.
- Replicate values: Normalized cytokine concentrations
File: Figure_6C_% Live CD45+ CD11b+ HLA-DR+.csv Description: Flow cytometry quantification showing the percentage of myeloid cells expressing maturation markers HLA-DR and CD11b.
Variables
- Soft / Stiff: Matrix stiffness conditions.
- mono culture 1-3: Values for myeloid cells cultured without GFs.
- coculture 1-3: Values for myeloid cells co-cultured with GFs.
File: Figure_6C_gMFI PD-L1.csv Description: Flow cytometry quantification of PD-L1 expression (gMFI) on myeloid cells in mono- and co-cultures.
Variables
- Group (Soft/Stiff): Matrix stiffness conditions.
- mono culture 1-3 / coculture 1-3: Geometric Mean Fluorescence Intensity values for PD-L1.
File: Figure_6D.csv Description: Quantification of phagocytic activity (normalized MFI) in myeloid cells derived from soft versus stiff hydrogel environments.
Variables
- Soft: Normalized MFI for cells in soft hydrogels
- Stiff: Normalized MFI for cells in stiff hydrogels
File: Figure_6E.csv Description: Heatmap data representing concentrations of secreted cytokines IL-6, IL-8, and CCL2 in GF-monocyte mono- and co-cultures.
Variables
- Cytokines: Pro-inflammatory mediators measured (IL6, IL8, CCL2).
- SOFT/STIFF MONOCULTURE/CO-CULTURE 1-3: Concentration values for individual biological replicates across all experimental conditions.
File: Figure_7B_tan_delta_.csv and Figure 7B Storage Modulus Description: Rheological characterization (Tan Delta and storage modulus) of human gingival tissue explants with and without Transglutaminase-mediated enzymatic crosslinking.
Variables
- Transglutaminase-: Storage Modulus/Tan delta values for untreated tissue explants.
- Transglutaminase+: Storage Modulus/ Tan delta values for crosslinked tissue explants.
File: Figure_7C_CCL2.csv / IL6.csv Description: ELISA quantification of cytokines secreted by TLR2-stimulated human gingival explants following mechanical modulation via Transglutaminase.
Variables
- Transglutaminase-: Cytokine concentrations from control explants.
- Transglutaminase+: Cytokine concentrations from stiffened explants.
Code/software
Statistical analyses and graphing were performed using Prism (Dotmatics). Additional data processing was conducted using FCS Express or Flow Jo for flow cytometry, Fiji (NIH) and Imaris (Oxford Instruments) for image analysis, and Reactome or NIH DAVID for bioinformatics analysis.