Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (26861689 and 17K11813). The authors would like to thank Mr. Nathaniel Green for proofreading.

This protocol is designed to create human gingival tissue equivalents, gingival wound models, and gingivitis models. Human skin epidermal keratinocytes (HaCaT) were kindly provided from Professor Norbert E. Fusenig of Deutsches Krebsforschungszentrum (Heidelberg, Germany)7. Human gingival fibroblasts (HGFs) were isolated from gingival tissues according to the previously published protocols8. Informed consent was obtained beforehand, and the study was approved according to the guidelines set by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo (Authorization Number: NDU-T2012-35). Protocol steps 1&#8211;3 should be performed in a cell culture hood.
1. Preparation of 3D Human Tissue Equivalents of Gingiva (GTE) (Figure 1A)
<ol>
	<li>Mix collagen type I-A gel with 10% concentrated MEM-alpha and 10% reconstruction buffer (2.2 g of NaHCO3 and 4.47 g HEPES in 100 mL 0.05 N NaOH) in a 15 mL sterile tube by pipetting. Keep the mixed collagen solution on ice until used.</li>
	<li>Place 24-well culture inserts (pore size 3.0 &#181;m) into a 24-well plate.</li>
	<li>Remove HGFs from the culture surface using 0.25% trypsin-EDTA solution. Suspend the cells in 10 mL of culture medium A (MEM-alpha + 20% FBS + 1% GlutaMAX). Count the cells by a B&#252;rker-T&#252;rk cell counter.
	<ol>
		<li>Extract the desired quantity of cells (1-5 x105 cells/mL), then centrifuge for 4 min at 190 x g.</li>
		<li>Suspend the cells in the mixed collagen solution. Keep the mixture on ice until the next step.</li>
	</ol>
	</li>
	<li>Add 0.5 mL of the cell-collagen mixture (0.5&#8211;2.5 x 105 cells/well) into the culture insert without producing any bubbles. Incubate the mixture for 10&#8211;30 min in a humidified atmosphere with 5% CO2 at 37 &#176;C to coagulate the mixture into a gel. 
	NOTE: Add the collagen mixture into the center of the culture insert to avoid unequal formation of collagen gel.</li>
	<li>Add 1 mL of the culture medium into the well and 0.5 mL into the insert. Incubate the culture plate for 24 h or overnight in a humidified atmosphere with 5% CO2 at 37 &#176;C.</li>
	<li>Remove HaCaT cells from the culture surface using 0.25% trypsin-EDTA solution. Suspend the cells in 10 mL of medium B (DMEM + 10% FBS + 1% GlutaMAX) and count the cells. Extract the needed quantity of cells (1&#8211;5 x 105 cells/mL) with a pipette, centrifuge for 4 min at 190 x g. Suspend the cells in medium B.</li>
	<li>Remove the medium from the culture inserts, which contain the HGF-collagen mixture, by aspiration. Add 0.5 mL of HaCaT cell suspension (0.5&#8211;2.5 x 105 cells/well) on the top of the HGF-collagen mixture. Incubate the cultures for 24 h or overnight. 
	NOTE: At this time point, the medium in the culture well should be medium A, while the medium in the culture insert should be medium B.</li>
	<li>Replace the culture medium with KSR medium (co-culture medium) (MEM-alpha + 15% KnockOut Serum Replacement + 1% GlutaMAX) + 5% FBS. Add 1 mL of the culture medium into the culture well and 0.5 mL into the culture insert. Incubate the cultures for 24&#8211;48 h.</li>
	<li>Replace the culture medium with KSR medium + 1% FBS. Add 1 mL into the culture well and 0.5 mL into the culture insert. Incubate the cultures for 24 h or overnight.</li>
	<li>Replace the culture medium in the culture well with 0.7&#8211;1 mL KSR medium. Remove the medium completely from the culture insert (the culture surface should be exposed to air). Incubate the cultures for 1&#8211;2 weeks. Change the medium in the culture well two times per week. 
	NOTE: Do not let the medium level rise above the culture surface (the top layer composed of keratinocytes). Always keep the culture surface dry.</li>
</ol>
2. Preparation of Wounded GTE (Figure 1B)
<ol>
	<li>Prepare a 1&#8211;3 mm diameter tissue puncher. Sterilize it with an autoclave at 121 &#176;C for 20 min.</li>
	<li>Push the tissue puncher perpendicularly into GTE about 300 &#181;m deep, and punch a hole in the tissue to simulate a wound.</li>
	<li>At this step, use the wounded GTE for investigating wound healing and tissue regeneration. Alternatively, fix GTE using 10% formalin neutral buffer solution to perform histological analysis.</li>
</ol>
3. Preparation of Inflammatory GTE (iGTE) (Figure 1B)
<ol>
	<li>Cultivate THP-1 cells according to the previous report9.</li>
	<li>Mix collagen type I-A gel with 10% concentrated RPMI 1640, 10% reconstruction buffer (2.2 g of NaHCO3 and 4.47 g HEPES in 100 mL 0.05 N NaOH) and 10 ng/mL PMA. Keep the mixed collagen solution on ice until use.</li>
	<li>Count 1&#8211;2 x 106 THP-1 cells and centrifuge for 4 min at 190 x g. Suspend the cells in 1 mL of the mixed collagen solution. Keep the mixture on ice until the next step.</li>
	<li>Add 10&#8211;30 &#181;L THP-1-collagen mixture into the wounded area of GTE. Replace the culture medium to 0.7&#8211;1 mL KSR medium containing 10 ng/mL of PMA.</li>
	<li>Incubate the mixture in a humidified atmosphere with 5% CO2 at 37 &#176;C for 72 h.</li>
	<li>Use the model for investigating gingivitis or periodontal disease (for example, add potential anti-inflammatory medicine to iGTE to see its effects on cytokine release10). Alternatively, omit 72 h PMA-treatment, add 2 &#181;g/mL of LPS into the culture, and incubate the tissue in a humidified atmosphere with 5% CO2 at 37 &#176;C for 48 h11,12.</li>
</ol>
4. Fixation and Whole Mount Immunostaining of GTE and iGTE Cultures
<ol>
	<li>Fixation of the cultures.
	<ol>
		<li>Remove the culture medium from the 24-well plate. Wash the tissues 2 times with phosphate buffered saline (PBS).</li>
		<li>Add 1 mL of 10% formalin neutral buffer solution to the insert and 1 mL to the culture well. Leave the 24-well plate in a refrigerator at 4 &#176;C overnight.</li>
		<li>Remove the 10% formalin neutral buffer solution the following day.</li>
		<li>For whole mount immunostaining, wash the tissues 3-4 times with PBS after fixation.</li>
		<li>For hematoxylin and eosin (H&#38;E) staining and regular immunostaining, proceed with dehydration, paraffin embedding, and sectioning. 
		NOTE: For whole mount immunostaining, this step can be omitted.</li>
	</ol>
	</li>
	<li>Whole mount immunostaining 
	NOTE: This protocol was modified from the one in reference 13.
	<ol>
		<li>Wash the tissues 3x in PBS 0.5&#8211;1% Triton, 10&#8211;30 min each time.</li>
		<li>Incubate the tissues twice for 30 min in blocking buffer (PBS 1% Triton + 10% BSA), at room temperature.</li>
		<li>Wash the tissues 2&#215; in blocking buffer, 5&#8211;10 min each time.</li>
		<li>Add 50 &#181;L of primary antibody solution at the required dilution/concentration to the inserts. Use a thin plastic cling film to wrap the inserts to avoid drying out the tissue.</li>
		<li>Incubate the tissue for 1 to 2 days at 4 &#176;C.</li>
		<li>Wash the tissues 3 times, for 30 min in PBS 1% Triton + 10% BSA. Wash again 3 times, for 5 min in PBS 1% Triton.</li>
		<li>Add 50 &#181;L of secondary antibody in blocking buffer (PBS 1% Triton + 10% BSA) and 5 &#181;L of Hoechst 33342 solution (NucBlue Live Cell stain) to each insert. Use a thin plastic cling film to wrap the inserts to avoid drying out the tissue.</li>
		<li>Incubate for 1 to 2 days at 4 &#176;C. Wrap the 24-well plate in a wet paper towel, and then put it into a plastic pack to be used throughout incubation.</li>
		<li>Wash the tissues 3 times, for 10 min in PBS 1% triton</li>
		<li>Use a sharp needle to cut the membrane of the culture insert to obtain the tissue. Place the membrane onto a slide. 
		NOTE: The tissue should be on the membrane.</li>
		<li>Add 2&#8211;3 drops of fluorescence mount medium. Cover the tissue with a cover glass and store at 4 &#176;C in the dark until analysis.</li>
		<li>View tissues with a confocal laser scanning microscopy (LSM).</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

This protocol is based on methods of creating gingival tissue equivalents and subcutaneous adipose-tissue equivalents described by previous reports8,21,22. Although this is a simple and easy method, some steps require special attention. For example, the collagen mixture should be kept on ice until use to avoid gel formation in the solution. When adding the collagen mixture into the culture insert, make sure the solution was injected in the center of the insert to avoid forming a biased gel (like in Figure 3B). Punching a hole in GTE also takes skill. The tissue puncher sometimes cannot pick up the tissue if the tissue is too wet. A tissue biopsy system or a pair of forceps would be helpful; however, the dimensions of the wounded location will be much changed. It is also possible to use a syringe with sharp needle to inject the THP-1 cell-collagen mixture without punching a hole (make sure the gel does not clog the needle).
The limitation of this method is that it is unable to perfectly represent the complex biological phenomena of gingivitis and periodontitis, because iGTE lacks blood vessels, neuronal cells, and other immune cells, and the inflammation is not caused by the host-biofilm interactions. However, it is the only in vitro gingivitis tissue model made by human cells that partly presents the pathology of inflammatory gingiva. The model can be modified to be more similar to the real gingivitis and periodontal disease by 1) co-cultivating GTE with biofilm to create host-biofilm interactions or stimulating inflammatory responses in iGTE by using LPS (the compounds in the outer membrane of Gram-negative bacteria and a strong inflammatory initiator as we suggested in the protocol), 2) seeding the collagen-HGF solution on extracted teeth to form gingiva-tooth complexes and observing how inflammation in gingiva influences hard tissue, and 3) using human gingival epithelial cells to form the epidermis of GTE and iGTE.
With the methods presented here, GTE and iGTE cultures can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and to screen potential medicines for gingivitis and periodontal disease.

Periodontal diseases are the leading cause of tooth loss in adults. Gingivitis and periodontitis are the most common periodontal diseases. Both present biofilm-mediated acute or chronic inflammatory changes in gingiva. Gingivitis is characterized by acute inflammation, whereas periodontitis usually presents as chronic inflammation. On the histological level,bacterial components trigger the activation of immune cells, such as macrophages, lymphocytes, plasma cells, and mast cells1,2. These immune cells, especially macrophages, interact with local cells (including gingival epithelial cells, fibroblasts, endothelial cells, and osteoblasts) resulting in inflammatory lesions in periodontal tissue3,4. Experimental models of periodontal diseases have been established in various types of animals, such as rats, hamsters, rabbits, ferrets, canines, and primates. 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 of periodontal diseases5. Co-cultivation of periodontal bacteria and monolayer human oral epithelial cells has been used to investigate the mechanism of periodontal infections6. Nevertheless, monolayer cultures of oral cells lack the three-dimensional (3D) cellular architecture of intact tissue; therefore, they cannot mimic the in vitro situation.
Here, 3D inflammatory human tissue equivalents of gingiva (iGTE) are established to represent periodontal diseases in vitro. This 3D model of periodontal diseases occupies an intermediate position between monolayer cell cultures and animal models. Three types of human cells, including HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, are co-cultivated on collagen gel, and stimulated by inflammatory initiators to build iGTE. IGTE closely simulates the in vivo conditions of cell differentiation, cell-cell interaction, and macrophage activation in gingiva. This model has many possible applications for drug screening and testing new pharmacological approaches in periodontal diseases, as well as for analyzing cellular and molecular mechanisms in wound healing, inflammation, and tissue regeneration.

<table><tbody><tr><td>Collagen type I-A</td><td>Nitta Gelatin Inc</td><td></td><td>For making dermis of GTE</td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>11900073</td><td>Cell culture medium</td></tr><tr><td>Cell Culture Insert (for 24-well plate), pore size 3.0 &amp;mu;m</td><td>Corning, Inc.</td><td>353096</td><td>For tissue culture</td></tr><tr><td>GlutaMAX</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Cell culture reagent</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>31600034</td><td>Cell culture medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828028</td><td>Cell culture reagent</td></tr><tr><td>Tissue puncher</td><td>Shibata system service co., LTD</td><td>SP-703</td><td>For punching holes in GTE</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>31800022</td><td>Cell culture medium</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A3294</td><td>For immunostaining</td></tr><tr><td>Hoechst 33342 (NucBlue Live Cell stain)</td><td>Thermo Fisher Scientific</td><td>R37605</td><td>For labeling nuclei</td></tr><tr><td>Fluorescence mount medium</td><td>Dako</td><td></td><td>For mounting samples after immunostaining</td></tr><tr><td>Anti-Cytokeratin 8+18 antibody [5D3]</td><td>abcam</td><td>ab17139</td><td>For identifying epithelium</td></tr><tr><td>Scaning electron microscope</td><td>Hitachi, Ltd.</td><td>HITACHI S-4000</td><td>For observing samples&#039; surface topography and composition</td></tr><tr><td>Confocal laser scanning microscopy</td><td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td><td>LSM 700</td><td>For observing samples&#039; immunofluorescence staining</td></tr><tr><td>Anti-Cytokeratin 19 antibody</td><td>abcam</td><td>ab52625</td><td>For identifying epithelium</td></tr><tr><td>Anti-vimentin antibody</td><td>abcam</td><td>ab92547</td><td>For identifying fibroblasts and activated macrophages</td></tr><tr><td>Anti-TE-7 antibody</td><td>Millipore</td><td>CBL271</td><td>For identifying fibroblasts in the dermis</td></tr><tr><td>Anti-CD68 antibody</td><td>Sigma-Aldrich</td><td>SAB2700244</td><td>For identifying macrophages</td></tr><tr><td>Human CD14 Antibody</td><td>R&amp;amp;D Systems</td><td>MAB3832-SP</td><td>For identifying macrophages</td></tr><tr><td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td><td>Thermo Fisher Scientific</td><td>A11012</td><td>For immunofluorescence staining</td></tr><tr><td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td><td>Thermo Fisher Scientific</td><td>A11001</td><td>For immunofluorescence staining</td></tr><tr><td>EVOS FL Cell Imaging System</td><td>Thermo Fisher Scientific</td><td></td><td>For observing the sample&#039;s immunofluorescence staining</td></tr><tr><td>THP-1 cells</td><td>Riken BRC cell bank</td><td>RCB1189</td><td>For making iGTE</td></tr><tr><td>PMA(Phorbol 12-myristate 13-acetate)</td><td>Sigma-Aldrich</td><td>P8139</td><td>For differentiatting THP-1 cells</td></tr></tbody></table>

three-dimensional inflammatory human tissue equivalents of gingiva

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 &#181;g/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.

HaCaT cells displayed typical keratinocyte morphology under phase-contrast microscopic observation (Figure 2A). Scanner electron microscopic (SEM) images showed that HaCaT cell surfaces were covered by many microvilli. Intercellular connections between HaCaT cells were mediated by membrane processes (Figure 2B). HaCaT cells expressed gingival epithelium marker K8/1814, indicating that HaCaT cells are suitable for gingiva reconstruction (Figure 2C).
Figure 3A shows a successful example of GTE, and Figure 3B shows a failed example of GTE. The unsuccessful trial (Figure 3B) is caused by the collagen gel mixture not being added in the center of the culture insert. Figure 3C showed that a wound model of GTE can be made by punching a hole in the tissue with a tissue puncher. H&#38;E staining (Figure 3D) and SEM images (Figure 3E) demonstrated that at 19 days of cultivation, about 10&#8211;20 layers of HaCaT cells form epithelium, and collagen gel-embedded HGF cells form lamina propria. In the lamina propria of the reconstructed GTE, HGF cells exhibited characteristic morphology of fibroblasts (Figure 3F), and were surrounded by well-defined arrays of collagen fibers (Figure 3G). Immunofluorescence staining showed that vimentin and TE-7 (both of them are dermal fibroblast markers15) are expressed in the lamina propria of GTE (Figure 4A and 4B). K19, a specific marker to junctional and pocket epithelium in periodontitis16, is weakly expressed in the epithelium of GTE (Figure 4C), and is strongly expressed in the epithelium of iGTE (Figure 4D). The data suggested that HaCaT cells can differentiate into key epithelial cells in periodontitis, especially after inflammatory stimulation of the lamina propria.
Inflammatory gingival diseases, such as gingivitis and periodontitis, are histologically characterized by the presence of lymphocytes and macrophages, and result in fibrosis and tissue necrosis17,18. In this protocol, THP-1 cells were used as the inflammatory cells for iGTE. As shown in Figure 5, without PMA-stimulation, THP-1 cells presented a monocytic morphology and most of them floated in the medium (Figure 5A). In contrast, after being exposed to PMA 10 ng/mL for 72 h, more than 50% of THP-1 cells differentiated into macrophage-like cells and adhered onto the culture surface (Figure 5B). Similar to HaCaT and HGF cells8, THP-1 cells grew well inside the collagen gel in the co-culture medium (KSR medium), and differentiated into macrophage-like cells after PMA-treatment (Figure 5C). Immunostaining showed that the adhered THP-1 cells expressed CD68 (a marker of macrophages19) (Figure 5D) and CD14 (another marker of macrophages) (Figure 6). To form iGTE, THP-1 monocytes were embedded in the collagen gel and injected into the hole simulating a wound (Figure 5E). At 72 h after PMA (10 ng/mL)-stimulation, THP-1 cells differentiated into macrophages (Figure 5F) inside the hole simulating a wound and expressed CD68 (Figure 5G), indicating they are able to form inflammatory foci in the wounded GTE.
Whole mount immunohistochemistry (Figure 6) showed that THP-1 cells in the hole simulating a wound not only expressed CD14 (another marker of macrophages), but also expressed vimentin as well as the fibroblasts in the edge of the hole. Vimentin is expressed by active macrophages20. The data confirmed that THP-1 macrophages are functional.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57157/57157fig1.jpg" /> 
Figure 1: Experimental designs. Experimental setup of 3D human tissue equivalents of gingiva (GTE) (A) and inflammatory GTE (iGTE) (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57157/57157fig2.jpg" /> 
Figure 2: Characterization of HaCaT keratinocytes. (A) Phase-contrast microscopic image of HaCaT cells in culture. Scale bar = 50 &#181;m. (B) SEM image of HaCaT cells. (C) Immunohistochemical staining of HaCaT cells for gingival epithelial cell marker K8/18. Green, K8/18. Blue, DAPI. Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57157/57157fig3.jpg" /> 
Figure 3: Characterization of 3D human tissue equivalents of gingiva (GTE). (A) Example of a successful GTE in culture. The tissue is in the middle of the culture insert. (B) Example of a failed GTE: Part of the GTE is separated from the tissue and biased toward the wall (Red arrow). (C) Wounded GTE: Yellow arrow indicates a hole punched by the tissue puncher. (D) H&#38;E staining of GTE cultivated for 19 days (14 days air exposure). Scale bar = 50 &#181;m. (E) SEM image of a vertical section of GTE cultivated for 19 days. (F) Phase-contrast microscopic image of HGF cells in collagen gel that formed the lamina propria of GTE. Scale bar = 25 &#181;m. (G) SEM image of massive collagen fibers around HGF cells in the lamina propria of GTE. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57157/57157fig4.jpg" /> 
Figure 4: Expression of vimentin, TE-7 and K19 in GTE. Immunofluorescence staining of GTE for lamina propria markers vimentin (A and B) and TE-7 (B) in GTE, and epidermis marker K19 in GTE (C) and iGTE (D). Red, vimentin and K19. Green, TE-7. Blue, DAPI. E, epidermis; LP, lamina propria. Scale bar = 50 &#181;m (A, C and D), 20 &#181;m (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57157/57157fig5.jpg" /> 
Figure 5: Characterization of THP-1 cells. (A) Undifferentiated THP-1 cells in culture. Scale bar = 15 &#181;m. (B) Differentiated THP-1 macrophages. Scale bar = 15 &#181;m. (C) THP-1 cells were embedded in collagen gel in KSR medium and treated with PMA for 24 h. Scale bar = 50 &#181;m. (D) Immunohistochemical staining of THP-1 macrophages for macrophage marker CD68. Red, CD68. Blue, DAPI. Scale bar = 5 &#181;m. (E) THP-1-collagen mixture in the wounded hole of HGE at the beginning of PMA treatment. Scale bar = 25 &#181;m. (F) THP-1 cells differentiated into macrophages (red arrows) at 72 h after PMA treatment. Scale bar = 25 &#181;m. (G) THP-1 cells expressed macrophage marker CD68. Yellow dashed line indicates the area of the wounded hole in H&#38;E. Scale bar = 100 &#181;m. Red, CD68. Blue, hoechst 33342. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57157/57157fig6.jpg" /> 
Figure 6: Characterization of iGTE. Whole mount immunohistochemistry for CD14 (green) and vimentin (red) was performed on iGTE. Z-series images (C) captured through iGTE (416 &#181;m in total. Panel 1 is the first section LSM scanned, and panel 25 is the last. The interval is about 16.64 &#181;m between sections). Representative section (A) (No. 17 of the Z-series images), orthophoto and 3D image of Z-stack (B) presented the 3D distribution of CD14- and vimentin- positive THP-1 macrophages in the hole simulating a wound in the iGTE. The scanning position of LSM in iGTE is indicated in (D) and the left top corner in (A). Scale bar = 50 &#181;m. Yellow dotted lines indicate the area of the wounded hole in iGTE. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57157/57157fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.7K Views.  The Nippon Dental University. The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Video: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Bioengineering

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Immunology and Infection

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Medicine

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Creating a Pulp Inflammation Mouse Model Using a Novel Mouth-Gag 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Author Spotlight: Enhancing Dental Pulp Research with Improved Mouse Models

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Summary

Explore More Videos

Chapters in this video

Tissue Engineering of a Human 3D in vitro Tumor Test System

Isolation, Processing and Analysis of Murine Gingival Cells

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research