Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Abstract

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 µg/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

Hydrogen Nano-Bubble Water Suppresses ROS Generation, Adipogenesis, and Interleukin-6 Secretion in Hydrogen-Peroxide- or PMA-Stimulated Adipocytes and Three-Dimensional Subcutaneous Adipose Equivalents

“…Since PKC signaling activates inflammatory responses, PMA is often used as a potent inducer of inflammation in animal models [ 38 , 39 ]. In our previous study, we found that 10 ng/mL of PMA could efficiently induce the differentiation of THP-1 cells, as did other researchers [ 40 , 41 , 42 ]. We also discovered that a longer duration of treatment (more than 4 days) with PMA can promote maturation of THP-1 macrophages (data not shown).…”

Blockade of exosome generation by GW4869 inhibits the education of M2 macrophages in prostate cancer

“…Current studies have suggested that tumor cells regulate the polarization process of macrophages through a variety of mechanisms. To understand the role of prostate cancer exosomes in macrophage polarization, we first induced THP1 cells into macrophages with 10 ng/ml PMA for 72 h [ 20 ], and then these macrophages were treated with IFN-γ (50 ng/mL), IL-4 (25 ng/mL), 2B4-exos (100 μg/ml), and IE8-exos (100 μg/ml) [ 21 ]. After culturing them for 48 h, we observed that, similar to the macrophages induced by IL-4, macrophages treated by 2B4-exos or IE8-exos significantly over-expressed CD206, a molecular marker of the M2 phenotype, IL-10 (M0 vs. 2B4-exo-Mφ, p < 0.0001; M0 vs.1E8-exo-Mφ, p = 0.0004) and TGF-β1 (M0 vs. 2B4-exo-Mφ, p = 0.0001; M0 vs. 1E8-exo-Mφ, p = 0.0004) (Fig.…”

Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages

“…18 Xiao et al punched a hole in a full-thickness OME composed of primary gingival fibroblasts, topped with the HaCaT skin keratinocyte cell line and injected it with LPS-stimulated, PMA-differentiated THP-1 cells to mimic periodontal disease. 20 Bao et al 22 developed a similar periodontal model using HPV E6/7-immortalized gingival fibroblasts, keratinocytes, and MM6 cells to produce an immune model that was then challenged with multispecies bacterial biofilm to mimic subgingival plaque. To date, Holmström et al have developed the most characterized immune-responsive OME.…”

“…Previous studies have generated immune oral gingival models by incorporating primary monocytes, peripheral blood mononuclear cells, or myeloid cancer cell lines (MonoMac-6 [MM6], U937, or THP-1 cells) and by observing changes in inflammatory markers and proteases in response to bacterial LPS, [18][19][20][21] bacterial biofilms, 22 or X-ray treatment. 23 While the use of myeloid cancer cell lines presents fewer technical limitations and reproducibility compared with primary immune cells, there is good evidence that their phenotype and function is markedly altered compared with primary cells, with the often used THP-1 cells shown to express aberrant levels of key macrophage phenotypic markers and thus respond differently to stimuli.…”