We acknowledge the UCLA Advanced Light Microscopy/Spectroscopy Laboratory and Leica Microsystems Center of Excellence at the California NanoSystems Institute (RRID:SCR_022789) for providing two-photon microscopy. AS was supported by ISF 604-21 from the Israel Science Foundation. JH was supported by R03DE030205 and R01DE030471 from the NIH/NIDCR. AS and JH were also supported by grant 2021007 from the United States-Israel Binational Science Foundation (BSF).

All mice were maintained in pathogen-free animal facilities at the University of California Los Angeles (UCLA) or the Hebrew University of Jerusalem (HUJI). All experiments involving mice were performed according to regulations and protocols approved by the respective Institutional Animal Care and Use Committee (IACUC) (ARC-2019-013; UCLA) or (MD-23-17184-3; HUJI). A general workflow of the experimental steps is shown in Figure 2A. See the Table of Materials for details rela...

Live Tracking of Dental Cells via Two-Photon Microscopy

The authors have no conflicts of interest to disclose.

Live tissue imaging is an important technique that allows us to study the dynamic processes and behaviors of cells when they are maintained in their niche environment41. Ideally, live imaging is performed in vivo with high spatiotemporal resolution. However, in vivo imaging for mammalian organs can be challenging due to tissue inaccessibility, optical opaqueness, and difficulty in immobilizing the animal or the organ for a prolonged period42. Tissue explan...

Over the past two decades, the mouse incisor has emerged as an invaluable platform for investigating the principles of adult stem cell regulation and tooth regeneration1,2. The mouse incisor grows continuously and renews itself throughout the animal&#39;s life. It does so by maintaining both epithelial and mesenchymal stem cells, which can self-renew and differentiate into different cell types of the tooth1,2. While dental epithelial stem cells give rise to ameloblasts, which secrete the enamel matrix, dental mesenchymal stem cells give rise to odontoblasts, cementoblasts, and fibroblasts, which form dentin, cementum, and periodontal ligament, respectively3,4,5,6. This constant supply of new cells maintains tissue homeostasis and allows the replacement of old cells that are lost due to masticatory wear or injuries7,8. Elucidating the cellular and molecular mechanisms that regulate the maintenance and differentiation of dental stem cells is therefore central to understanding dental regeneration, an area of growing interest.
Anatomically, a large portion of the adult mouse incisor is encased in the jawbone. While the incisal edge of the tooth is exposed, the apical end of the incisor fits within a socket and is firmly attached to the surrounding bone through periodontal ligaments and connective tissues (Figure 1A,B). The incisor&#39;s apical end is also the growth region of the tooth and maintains dental stem and progenitor cells in both the epithelial layer and the mesenchymal pulp9,10,11,12,13. Specifically, dental epithelial stem cells are maintained at the bulbous end of the epithelium, known as the apical bud, also referred to as the labial cervical loop (Figure 1C). Similar to the intestinal epithelium and the epidermis, epithelial renewal in the incisor is primarily supported by actively cycling stem cells and their highly proliferative intermediate descendants, called transit-amplifying cells14,15,16,17, both residing in the inner part of the cervical loop. However, whether the incisor epithelium contains and utilizes quiescent stem cells during regeneration remains to be determined. In contrast, both active and quiescent dental mesenchymal stem cells have been identified in the apical pulp, and the quiescent stem cells function as a reserve population that becomes activated during injury repair13,18.
Many of the discoveries on the biology of the mouse incisor renewal and regeneration have resulted from histological investigations, in which samples are obtained at distinct temporal junctures, fixed, processed, and then sectioned into micron-thin slices along a particular plane. Through detailed analysis of histological sections from different mouse models that enable lineage tracing or genetic perturbations, scientists have identified the cell lineages of different progenitor populations, as well as the genetic and signaling pathways that control incisor homeostasis and injury repair19,20,21. However, the static two-dimensional (2D) images of non-vital cells in sections cannot capture the full spectrum of cellular behaviors and spatial organizations in living tissue, such as cell shape changes, movements, and cellular kinetics. Detecting and measuring these rapid cellular changes, which occur at a timescale that is unresolvable through tissue sectioning, require a different strategy. Moreover, acquiring such information is also critical for understanding how dental cells interact with each other, react to different signaling stimuli, and self-organize to maintain tissue structures and functions.
The advent of four-dimensional (4D) deep tissue imaging using two-photon microscopy22, a technology that integrates three spatial dimensions with temporal resolution, overcomes the inherent limitations of histological analysis by enabling spatiotemporal examination of cultured tissue explants, organoids, or even tissues in situ23,24,25,26. For instance, 4D live imaging of the developing tooth epithelium has unveiled the spatiotemporal patterns of cell divisions and migrations that coordinate tissue growth, signaling center formation, and dental epithelial morphogenesis27,28,29,30,31,32. In the adult mouse incisor, 4D imaging has been recently adapted to study cellular behaviors during dental epithelial injury repair. Live imaging revealed that stratum intermedium cells in the suprabasal layer can be directly converted into ameloblasts in the basal layer to regenerate the damaged epithelium, challenging the traditional paradigm of epithelial injury repair15.
Here, we describe the dissection, culturing, and imaging of the adult mouse incisor, focusing on epithelial cells in the labial cervical loop (Figure 1). This technique preserves dental cell vitality for more than 12 h and permits live tracking of fluorescently labeled cells at single-cell resolution. This approach allows investigation of cell motion and migration as well as dynamic changes in cell shape and division orientation under normal culture conditions, or in responses to genetic, physical, and chemical perturbations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>24 well, flat bottom tissue culture plate</td>
			<td>Olympus plastics</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>25x HC IRAPO motCORR water dipping objective</td>
			<td>Leica</td>
			<td>11507704</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid (Vitamin C)</td>
			<td>Acros Organics</td>
			<td>352685000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose bioxtra&#160;</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Delta T system&#160;</td>
			<td>Bioptechs</td>
			<td>0420-4</td>
			<td>Including temperature control, culture dishes, and perfusion setup</td>
		</tr>
		<tr>
			<td>Dissection microscope- LEICA S9E</td>
			<td>Leica</td>
			<td>LED300 SLI</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Scientific</td>
			<td>11039047</td>
			<td>Basal media without phenol red</td>
		</tr>
		<tr>
			<td>Feather surgical blade (#15)</td>
			<td>Feather</td>
			<td>72044-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T</td>
			<td>11252-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>Thermo Scientific</td>
			<td>35050-061</td>
			<td>Glutamine substitute</td>
		</tr>
		<tr>
			<td>Leica SP8-DIVE equipped with a 25X HC IRAPO motCORR water dipping objective&#160;</td>
			<td>Leica</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>low-melting agarose</td>
			<td>NuSieve</td>
			<td>50080</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids (100x)</td>
			<td>Thermo Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin&#8211;streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td>10,000 U/mL&#160;</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Gen Clone</td>
			<td>32-107G</td>
			<td>90 mm&#160;</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Valley Biomedical</td>
			<td>AS3061SC</td>
			<td>Processed for live imaging</td>
		</tr>
		<tr>
			<td>Razor blade #9</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
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			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12</td>
			<td></td>
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			<td>Scissors</td>
			<td>F.S.T</td>
			<td>37133</td>
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			<td>serrated forceps</td>
			<td>F.S.T</td>
			<td>11000-13</td>
			<td></td>
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			<td>spring scissors</td>
			<td>F.S.T</td>
			<td>91500-09</td>
			<td></td>
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using ex vivo live imaging to investigate cell divisions and movements during mouse dental renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

To maintain the correct cellular organization during organ renewal, tissue approaching the cells move and dividing specific orientations and patterns. It is therefore important to understand the rules and regulations of these dynamic cellular events. The mouse incisor is an emerging model for adult stem cell research, and many genetic tools are available in the system to study gene functions, and to label cells for lineage tracing.Most studies to date use adult dental samples collected and fixed its specific time points, and this prevents us from examining life cell behavior. In this protocol, we describe a method to track lifestyles in the adult mouse incisor, and this technique will enable future studies that aim to understand how dynamic cell behaviors contribute to the renewal and repairment of dental tissues.

The apical region of the adult mouse incisor is encased within the mandible (Figure 1) and hence, not directly accessible for visualizing and live-tracking the progenitor cells residing within the growth region. Therefore, we have developed a method to extract the whole incisor from the jawbone and maintain it in an explant culture system for two-photon timelapse microscopy (Figure 2). Here we describe representative results that capture the dynamic process of c...

Watch this Scientific Journal Video about Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment at JoVE.com

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

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Research

JoVE Journal

Developmental Biology

Dissection and Isolation of Mouse Mandibular Incisors and Associated Periodontal Tissues to Expose the Incisor Epithelial Cervical Loop

To begin, turn the euthanized mouse over so its ventral side faces up and the mandibles are easily accessible. Secure the mouse's head gently between the thumb and the index finger. Using a razor blade, starting from the lower lip toward the neckline, make a mid-sagittal incision on the lower jaw skin.During the incision, use the thumb and index finger to open the cut skin, exposing the underneath muscles and jawbone. Next, sever the masseter muscles on the buccal side of the lower jaw. Sever the mylohyoid muscles along the inner side of the mandible and remove muscle attachments.Make another incision at the mandibular synthesis, connecting the two hemi-mandibles, separating them into the left and right halves. Wedge the razor blade between the mandibular condyle and the temporal mandibular joint to carefully dissect out the hemi mandible from the rest of the head. Immediately transfer the dissected mandibles to a Petri dish containing pre-warmed dissection medium.Next, under a brightfield dissection microscope, use a number 15 surgical blade to remove muscle tissues. Identify the oval region of the mandible that covers the incisor socket and houses the apical portion of the incisor. Position the mandibles such that the inner or lingual surface is facing upward.While holding the mandible with a pair of serrated forceps, using a number 15 surgical blade, shave off the overlying membrane bone from the condyle towards the molars to generate a window at the oval. This exposes the soft tissue of the apical incisor on the inner surface. Next, turn the mandible so the outer or buccal surface faces upwards.As demonstrated for the inner mandible, generate a window at the oval region on the outer mandible. Using a pair of forceps, pick away the remaining bone fragments at the edge, ensuring that the apical end of the tooth is visible from both sides. To isolate the entire tooth, make a clean cut at a plane immediately adjacent to the apical incisor to remove the condylar process.Then make a second cut just posterior to the third molar, but dorsal to the incisor, without damaging the tooth, to remove the coronary process. Serially cut from the tip of the angular process towards the incisor to gradually remove the ventral mandible. Next, cut away the alveolar bone with molars and any remaining bones that are still attached to the incisor.Transfer the fully dissected incisor to a dish containing warm dissection media. Turn on the fluorescent microscope and use the fluorescent signal from tissues to guide the removal of periodontium. Position the inner incisor on its lingual side, and using serrated forceps hold the tooth in place.Then using a pair of number five fine forceps, start tugging on the periodontal tissues, covering the apical incisor, and the cervical loop region. Carefully peel off periodontal tissues from the apical bud, such that the lateral side of the cervical loop is visible.

Periodontal Tissue Embedding and Timelapse Microscopy of Mouse Incisor Explant Cultures

To begin, add 500 microliters of warm culture gel to a well in a 24-well plate. Quickly transfer the dissected whole incisors isolated from the mouse to the well. Swirl the plate a few times to rinse the incisors.Add 400 microliters of warm culture gel to a culture dish and to transfer the rinsed incisors to the dish. Orient the incisor to position the cervical loop region at the center of the dish and adjust the tilt of the apical incisor for the desired imaging plane. Once the gel is set, using a pair of fine forceps, remove any gel on top of the region of interest.Add approximately 150 microliters of warm culture media against the edge of the dish to cover the sample. Place the explant culture at 37 degrees Celsius to allow the tissue to settle. After one hour, turn on the microscope and the two-photon laser.Secure the culture dish to the stage adapter and place the profusion adapter ring on top. Connect the stage adapter to a temperature controller to maintain the culture at 37 degrees Celsius. Connect the inlet and the outlet of the adapter ring to a microprofusion pump.Set the profusion speed to 20 and initiate the profusion of the medium over the sample. Place an ACBR on top of the adapter ring and raise the stage so the objective fits through the ACBR to make contact with the culture medium. Turn the laser wavelength to 920 nanometers to visualize GFP.After locating the sample through an eyepiece, visualize it with a microscope software, then set up Z-step size of four micrometers and a time interval of five minutes for 14 hours. Initiate the time-lapse imaging and save the subsequent files for downstream data analysis. In time-lapse microscopy of the K14-Cre;R26-rtTA;tetO-H2B-GFP cervical loop, the H2B-GFP signals were predominantly observed in the trans-amplifying region of the cervical loop, indicating active cell divisions.Further, the condensation and alignment of chromosomes at the metaphase plate in mitotic cells followed by their segregation during anaphase and into two daughter cells was observed. Most cell divisions were perpendicular or at an oblique angle relative to the basement membrane with fewer horizontal divisions parallel to the basement membrane. Cell division events were also apparent in K14-Cre;R26-mT/mG cervical loops where all epithelial cell membranes were labeled green.Mitotic cells were identified by their cell rounding and subsequent cytokinesis. In K14-Cre;R26-mT/mG cervical loops, both horizontal and vertical cell divisions were also observed.

770 Views.  University of California Los Angeles.  Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

Live Tracking of Dental Cells via Two-Photon Microscopy

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