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

这项工作得到了日本促进科学促进会 (26861689 和 17K11813) 资助的部分支持。作者感谢纳撒尼尔. 格林先生校对。

本协议旨在创建人牙龈组织当量, 牙龈伤口模型, 牙龈炎模型。人皮肤表皮角质细胞 (HaCaT) 是亲切提供的 Fusenig 教授 e. 德意志 Krebsforschungszentrum (德国海德堡)7。根据先前发布的协议8, 人牙龈成纤维细胞 (HGFs) 与牙龈组织分离。事先获得知情同意, 并根据道德委员会制定的指导方针, 东京牙科大学生活牙科学院 (授权号码: NDU-T2012-35) 批准了这项研究。协议步骤1–3应在单元格培养罩中执行。
1. 制备3D 人牙龈组织当量 (GTE) (图 1A)
<ol><li>混合胶原 i 型-一凝胶与10% 浓缩的内存α和10% 重建缓冲器 (2.2 克 NaHCO3和4.47 克 HEPES 100 毫升 0.05 N 氢氧化钠) 在15毫升不育管由吹打。将混合胶原蛋白溶液放在冰上, 直到使用。</li>
	<li>安置24井文化插入物 (孔大小3.0 µm) 入24井板材。</li>
	<li>
使用0.25% 胰蛋白酶-EDTA 溶液从培养基表面去除 HGFs。悬浮细胞在10毫升培养基 A (记忆α + 20% 血清 + 1% GlutaMAX)。通过 Bürker 细胞计数器计数单元格。
	<ol>
<li>提取所需数量的细胞 (1-5 x105细胞/毫升), 然后离心机为4分钟在 190 x g。</li>
		<li>在混合胶原溶液中悬浮细胞。把混合物放在冰上, 直到下一步。</li>
	</ol>
</li>
	<li>将0.5 毫升的细胞胶原混合物 (0.5–2.5 x 105细胞/井) 添加到区域性插入, 而不产生任何气泡。在 5% CO2在37摄氏度的湿润气氛中孵化混合物, 以使混合物凝结成凝胶, 10–30。 注: 将胶原蛋白混合物加入培养基的中心, 避免胶原凝胶的不均匀形成。</li>
	<li>加入1毫升培养基到井中, 0.5 毫升插入。在湿润气氛中孵育24小时或一夜的培养板, 5% CO2在37摄氏度。</li>
	<li>使用0.25% 胰蛋白酶-EDTA 溶液从培养表面去除 HaCaT 细胞。将细胞悬浮在中等 B 的10毫升内 (DMEM + 10% 只血清 + 1% GlutaMAX) 并计数细胞。提取所需数量的细胞 (1–5 x 105细胞/毫升) 与吸管, 离心机为4分钟在 190 x g. 暂停中 B 中的单元格。</li>
	<li>从培养物中移除培养基, 其中含有肝细胞生长因子胶原混合物, 由吸入。添加0.5 毫升的 HaCaT 细胞悬浮 (0.5–2.5 x 105细胞/井) 的顶部的 HGF-胶原蛋白混合物。培养24小时或一夜之间的文化。 注: 在这个时间点, 培养基在文化中应该是中等的, 而培养基在培养基中插入应该是中等的 B。</li>
	<li>用 KSR 培养基 (共培养培养基) 取代培养基 (GlutaMAX + 15% 剔除血清替代 + 1%) + 5%。增加1毫升培养基入文化井和0.5 毫升入文化插入物。培养的 24–48 h。</li>
	<li>用 KSR 培养基 + 1% 的血清取代培养基。增加1毫升入文化井和0.5 毫升入文化插入物。培养24小时或一夜之间的文化。</li>
	<li>用0.7–1毫升 KSR 培养基将培养基中的培养基替换好。从区域性插入中完全移除介质 (该培养基应暴露于空气中)。培养几个星期的文化。每星期两次在文化中改变培养基。 注意: 不要让中等水平上升以上的文化表面 (顶层组成的角质细胞)。始终保持文化表面干燥。</li>
</ol>2. 准备受伤的 GTE (图 1B)
<ol><li>准备一个1–3毫米直径的组织冲床。用高压釜消毒121摄氏度, 20 分钟。</li>
	<li>将组织冲床垂直推入 GTE 约300µm 深, 并在组织中打孔以模拟伤口。</li>
	<li>在这一步, 使用伤员 GTE 调查伤口愈合和组织再生。或者, 用10% 福尔马林中性缓冲液固定 GTE 进行组织学分析。</li>
</ol>3. 炎症 GTE 的制备 (iGTE) (图 1B)
<ol><li>根据上一个报告9培养 THP-1 细胞。</li>
	<li>混合胶原 i 型-一凝胶与10% 浓缩 RPMI 1640, 10% 重建缓冲器 (2.2 克 NaHCO3和 4.47 g HEPES 在100毫升 0.05 N 氢氧化钠) 和 10 ng/毫升。将混合胶原蛋白溶液放在冰上, 直到使用。</li>
	<li>计算 106 THP-1 细胞和离心机4分钟 190 x g. 在混合胶原溶液的1毫升中悬浮细胞。把混合物放在冰上, 直到下一步。</li>
	<li>将10–30µL THP-1-collagen 混合物添加到 GTE 受伤地区。将培养基替换为含有 10 0.7–1毫升的 KSR 培养基。</li>
	<li>在 5% CO2在37摄氏度的湿润气氛中孵化混合物72小时。</li>
	<li>使用模型来调查牙龈炎或牙周疾病 (例如, 添加潜在的抗炎药物 iGTE, 以观察其对细胞因子释放的影响10)。或者, 省略72小时的治疗, 加入2µg/毫升的 LPS 进入培养, 并孵化组织在一个湿润的气氛与 5% CO2在37摄氏度 48 h11,12。</li>
</ol>4. GTE 和 iGTE 文化的固定和全芒染色
<ol><li>
文化的固定。
	<ol>
<li>从24井板中取出培养基。用磷酸盐缓冲盐水 (PBS) 冲洗组织2次。</li>
		<li>添加1毫升10% 福尔马林中性缓冲液到插入和1毫升的文化良好。把24井板放在冰箱里, 一夜4摄氏度。</li>
		<li>第二天取出10% 福尔马林中性缓冲液。</li>
		<li>对于整个染色, 固定后用 PBS 清洗组织3-4 次。</li>
		<li>对于苏木精和伊红 (H & E) 染色和常规染色, 进行脱水, 石蜡嵌入和切片。 注: 对于整个装载染色, 可以省略此步骤。</li>
	</ol>
</li>
	<li>
全芒染色 注意: 此协议是从参考13中的一个修改的。<ol>
<li>在 PBS 0.5–1% 10–30, 每一次清洗组织3x。</li>
		<li>在室温下, 在阻塞缓冲液中孵化两次组织30分钟 (PBS 1% 海卫二 + 10% BSA)。</li>
		<li>每2×在阻塞缓冲液中清洗组织, 5–10分钟。</li>
		<li>添加50µL 的主要抗体溶液在所需的稀释/浓度的插入。使用薄塑料保鲜膜包装的插入物, 以避免干燥的组织。</li>
		<li>在4摄氏度孵育组织1到2天。</li>
		<li>清洗组织3次, 在 PBS 1% 海卫30分钟 + 10% BSA。再洗3次, 在 PBS 1% 海卫5分钟。</li>
		<li>添加50µL 的二级抗体在阻塞缓冲 (PBS 1% 海卫两 + 10% BSA) 和5µL 的赫斯特33342解决方案 (NucBlue 活细胞染色) 每个插入。使用薄塑料保鲜膜包装的插入物, 以避免干燥的组织。</li>
		<li>在4摄氏度孵育1到2天。将24井板裹在湿纸巾上, 然后放入塑料袋中, 用于整个孵化过程。</li>
		<li>清洗组织3次, 在 PBS 1% 海卫10分钟</li>
		<li>用锋利的针头切开培养物的膜以获得组织。将薄膜放到幻灯片上。 注: 组织应在膜上。</li>
		<li>添加2–3滴荧光贴装培养基。用盖玻璃覆盖组织, 在黑暗中贮存4摄氏度直到分析。</li>
		<li>用共聚焦激光扫描显微镜 (LSM) 查看组织。</li>
	</ol>
</li>
</ol>

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作者声明没有利益冲突。

该协议是基于创建牙龈组织当量和皮下脂肪组织等价的方法, 以前的报告中描述的8,21,22。虽然这是一个简单而简单的方法, 但有些步骤需要特别注意。例如, 胶原混合物应保持在冰上, 直到使用, 以避免凝胶形成的解决方案。当将胶原蛋白混合物添加到区域性插入中时, 请确保将该溶液注入插入中心, 以避免形成有偏见的凝胶 (如<...

牙周疾病是成人牙齿丢失的主要原因。牙龈炎和牙周炎是最常见的牙周疾病。牙龈中存在生物膜介导的急性或慢性炎症变化。牙龈炎的特点是急性炎症, 而牙周炎通常呈现为慢性炎症。在组织学层面上, 细菌成分会触发免疫细胞的活化, 如巨噬细胞、淋巴细胞、血浆细胞和桅杆单元1,2。这些免疫细胞, 特别是巨细胞, 与局部细胞 (包括牙龈上皮细胞、成纤维细胞、血管内皮和成骨细胞) 相互作用, 导致牙周组织的炎症性病变3,4。实验模型的牙周疾病已建立在各种类型的动物, 如大鼠, 仓鼠, 兔, 雪貂, 犬, 和灵长类动物。然而, 动物模型的生理病理与人类不同, 因此很难分析细胞和分子机制, 并评估牙周病的新药 5.利用牙周细菌和单层人口腔上皮细胞的共同培养, 研究了牙周感染的机制6。然而, 口腔细胞的单层培养缺乏完整组织的三维 (3D) 细胞结构;因此, 它们无法模拟体外情况。
在这里, 3D 炎症人的牙龈组织等效物 (iGTE) 是用来表示牙周疾病的在体外。这种3D 模型的牙周疾病在单层细胞培养和动物模型之间占据中间位置。三种类型的人类细胞, 包括 HaCaT 角质形成细胞, 牙龈成纤维细胞, 和 THP-1 巨噬细胞, 是由胶原凝胶共同培养, 并刺激炎症启动器, 以建立 iGTE。IGTE 严密模拟了牙龈中细胞分化、细胞相互作用和巨噬细胞活化的体内条件。该模型有许多可能的应用, 药物筛选和测试新的药理方法, 牙周疾病, 以及分析细胞和分子机制的伤口愈合, 炎症, 和组织再生。

<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

牙周疾病 (如牙龈炎和牙周炎) 是成人牙齿丢失的主要原因。牙龈炎症是牙周疾病的基本生理病理。目前在各种类型的动物中建立了牙周疾病的实验模型。然而, 动物模型的生理病理与人类不同, 因此很难分析细胞和分子机制, 并对牙周疾病新药进行评价。在这里, 我们提出了一个详细的协议, 以重建牙龈炎症组织等值的龈 (iGTE)在体外。我们首先使用两种类型的人类细胞, 包括人牙龈成纤维细胞 (HGF) 和人表皮角质细胞 (HaCaT), 在三维条件下, 构建牙龈 (GTE) 的人体组织当量。我们创建一个伤口模型, 使用组织冲床打孔在 GTE。然后, 将人 THP-1 单核细胞与胶原凝胶混合, 注入 GTE 的孔内。通过 adimistration 10 佛波 12-酯 13-醋酸盐 (PMA) 为72小时, THP-1 细胞分化成巨细胞形成炎症灶在 GTE (iGTE) (iGTE 也可以 stumilated 与2µg/毫升脂多糖 (LPS) 48 h 启动炎症).IGTE 是第一个体外模型的炎症牙龈使用人类细胞与三维体系结构。IGTE 反映了牙周疾病的主要病理变化 (免疫 activition、fibryoblasts、上皮细胞、单核和巨噬细胞之间的细胞间相互作用)。GTE, 受伤的 GTE 和 iGTE 可作为多功能工具, 研究伤口愈合, 组织再生, 炎症, 细胞相互作用, 和屏幕潜在药物的牙周疾病。

HaCaT 细胞在相对比显微镜观察下显示典型的角质形成形态 (图 2A)。扫描电镜 (SEM) 图像表明, HaCaT 细胞表面被许多微绒毛覆盖。HaCaT 细胞之间的细胞间连接由膜过程介导 (图 2B)。HaCaT 细胞表达牙龈上皮标记 K8/1814, 表明 HaCaT 细胞适合牙龈重建 (图 2C)。
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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

该协议的目标是建立一个炎症的人牙龈模型在体外。该组织模型共培养三种类型的人细胞, HaCaT 角质形成细胞, 牙龈成纤维细胞, THP-1 巨噬细胞, 在三维条件下。该模型可用于牙周疾病的研究, 如牙龈炎和牙周炎。

牙龈三维炎症人体组织当量

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

Research

JoVE Journal

Developmental Biology

9.7K 次观看.  The Nippon Dental University. 该协议的目标是建立一个炎症的人牙龈模型在体外。该组织模型共培养三种类型的人细胞, HaCaT 角质形成细胞, 牙龈成纤维细胞, THP-1 巨噬细胞, 在三维条件下。该模型可用于牙周疾病的研究, 如牙龈炎和牙周炎。

视频: 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.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

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

科研

生物工程

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.

小鼠牙龈细胞的分离，处理和分析

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

免疫与感染

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.

三维全层皮肤的生成等效和自动伤人

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.

三维组织工程人食管黏膜型号生产，表征和潜在用途

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.

工程三维上皮组织细胞外基质中嵌入

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.

分离，鉴定和牙龈免疫细胞网络的功能性考试

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.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

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.

一种监测人牙龈移植新生血管的新方法

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

医学

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.

用于研究体外宿主-病原体相互作用的器官型组织模型系统

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

使用新型 Mouth-Gag 创建牙髓炎症小鼠模型 

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.

作者聚焦：通过改进的小鼠模型增强牙髓研究

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

牙龈三维炎症人体组织当量

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一个人的3D组织工程