Reduced extracellular matrix stiffness increased pro-inflammatory signaling in gingival fibroblasts, whereas restoring stiffness suppressed cytokine production and was consistent with gingival immune homeostasis.
In a study, researchers developed a tunable three-dimensional collagen-alginate hydrogel system to replicate the mechanical properties of healthy and diseased gingival tissue. Healthy gingiva exhibited a storage modulus of approximately 2,000 Pa compared with approximately 1,000 Pa in periodontal disease, reflecting extracellular matrix softening associated with degradation of the collagen network and extracellular matrix components.
Human gingival fibroblasts cultured in stiff hydrogels (approximately 2 kPa) demonstrated suppressed toll-like receptor–mediated inflammatory responses compared with those in soft hydrogels (approximately 0.75 kPa). Following Toll-like receptor 2 stimulation, fibroblasts in softer matrices produced higher levels of interleukin (IL)-6, IL-8, and CCL2, whereas increasing stiffness progressively reduced the secretion of these mediators.
Bulk RNA sequencing showed that stiff matrices upregulated extracellular matrix synthesis pathways, including extracellular matrix organization pathways with about 200 associated genes. In contrast, soft matrices upregulated chemokines such as CXCL12, CXCL5, CXCL6, CSF3, and CCL8, along with matrix-degrading enzymes including MMP3 and MMP12, indicating increased inflammatory signaling and matrix breakdown.
Mechanistically, stiffness-dependent regulation of inflammation was mediated by downregulation of the noncanonical nuclear factor kappa–light-chain–enhancer of activated B cells pathway in stiff matrices. Protein analysis confirmed reduced nuclear-localized phosphorylated RelB signaling in fibroblasts cultured in stiff hydrogels. Pharmacologic inhibition of upstream signaling reduced cytokine production in soft matrices to levels comparable with stiff conditions, indicating pathway dependence.
In co-culture experiments, stiff matrices promoted differentiation of myeloid progenitors into immunomodulatory dendritic cells, with approximately 40% of cells expressing CD11b-positive and HLA-DR–positive and increased PD-L1 expression and phagocytic activity. Soft matrices, particularly in co-culture, produced the highest levels of IL-6, IL-8, and CCL2.
Ex vivo human gingival explants further supported these findings. Enzymatic crosslinking increased tissue stiffness and reduced inflammatory mediator release following toll-like receptor stimulation, including decreases in IL-6 and CCL2.
The researchers reported that the experimental system used Toll-like receptor 2 stimulation and was designed to isolate mechanical properties rather than incorporate the broader biochemical features of the gingival extracellular matrix.
“[R]estoring the mechanical integrity of the gingival [extracellular matrix] impairs downstream inflammatory cascades,” wrote lead study author Hardik Makkar, of the Center For Innovation & Precision Dentistry as well as the Department of Preventive and Restorative Sciences in the School of Dental Medicine at the University of Pennsylvania, and colleagues.
The study authors reported no conflicts of interest.
Source: Advanced Materials
