Cellular ageing of oral fibroblasts differentially modulates extracellular matrix organization.

BACKGROUND AND OBJECTIVES: Ageing is associated with an impaired cellular function that can affect tissue architecture and wound healing in gingival and periodontal tissues. However, the impact of oral fibroblast ageing on the structural organization of the extracellular matrix (ECM) proteins is poorly understood. Hence, in this study, we investigated the impact of cellular ageing of oral fibroblasts on the production and structural organization of collagen and other ECM proteins. METHODS: Oral fibroblasts were serially subcultured, and replicative cellular senescence was assessed using population doubling time, Ki67 counts and expression of P21WAFI . The production and structural organization of ECM proteins were assessed at early (young-oFB) and late (aged-oFB) passages. The thickness and pattern of collagen produced by live cultures of young- and aged-oFB were assessed using a label-free and non-invasive second harmonic generation (SHG)-based multiphoton imaging. Expression of other ECM proteins (fibronectin, fibrillin, collagen-IV and laminins) was evaluated using immunocytochemistry and confocal microscopy-based depth profile analysis. RESULTS: Aged-oFB displayed a higher population doubling time, lower Ki67+ cells and higher expression of P21WAFI indicative of slower proliferation rate and senescence phenotype. SHG imaging demonstrated that young-oFB produced a thick, interwoven network of collagen fibres, while the aged-oFB produced thin and linearly organized collagen fibres. Similarly, analysis of immunostained cultures showed that young-oFB produced a rich, interwoven mesh of fibronectin, fibrillin and collagen-IV fibres. In contrast, the aged-oFB produced linearly organized fibronectin, fibrillin and collagen-IV fibres. Lastly, there was no observable difference in production and organization of laminins among the young- and aged-oFB. CONCLUSION: Our results suggest that oral fibroblast ageing impairs ECM production and more importantly the organization of ECM fibres, which could potentially impair wound healing in the elderly.

Fluid flow-induced modulation of viability and osteodifferentiation of periodontal ligament stem cell spheroids-on-chip.

Developing physiologically relevant in vitro models for studying periodontitis is crucial for understanding its pathogenesis and developing effective therapeutic strategies. In this study, we aimed to integrate the spheroid culture of periodontal ligament stem cells (PDLSCs) within a spheroid-on-chip microfluidic perfusion platform and to investigate the influence of interstitial fluid flow on morphogenesis, cellular viability, and osteogenic differentiation of PDLSC spheroids. PDLSC spheroids were seeded onto the spheroid-on-chip microfluidic device and cultured under static and flow conditions. Computational analysis demonstrated the translation of fluid flow rates of 1.2 µl min-1 (low-flow) and 7.2 µl min-1 (high-flow) to maximum fluid shear stress of 59 µPa and 360 µPa for low and high-flow conditions, respectively. The spheroid-on-chip microfluidic perfusion platform allowed for modulation of flow conditions leading to larger PDLSC spheroids with improved cellular viability under flow compared to static conditions. Modulation of fluid flow enhanced the osteodifferentiation potential of PDLSC spheroids, demonstrated by significantly enhanced alizarin red staining and alkaline phosphatase expression. Additionally, flow conditions, especially high-flow conditions, exhibited extensive calcium staining across both peripheral and central regions of the spheroids, in contrast to the predominantly peripheral staining observed under static conditions. These findings highlight the importance of fluid flow in shaping the morphological and functional properties of PDLSC spheroids. This work paves the way for future investigations exploring the interactions between PDLSC spheroids, microbial pathogens, and biomaterials within a controlled fluidic environment, offering insights for the development of innovative periodontal therapies, tissue engineering strategies, and regenerative approaches.

Differential immune responses of 3D gingival and periodontal connective tissue equivalents to microbial colonization.

Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.