dissection and isolation of mouse mandibular incisors and associated periodontal tissues to expose the incisor epithelial cervical loop

Live Tracking of Dental Cells via Two-Photon Microscopy

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.

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.

Watch this Scientific Journal Video about Dissection and Isolation of Mouse Mandibular Incisors and Associated Periodontal Tissues to Expose the Incisor Epithelial Cervical Loop at JoVE.com

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.

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Research

JoVE Journal

Developmental Biology

183 Görüntüleme.  University of California Los Angeles. Başlamak için, ötenazi fareyi ventral tarafı yukarı bakacak ve mandibulalara kolayca erişilebilecek şekilde ters çevirin. Farenin başını başparmak ve işaret parmağı arasına nazikçe sabitleyin. Bir tıraş bıçağı kullanarak, alt dudaktan başlayarak boyun çizgisine doğru, alt çene derisinde orta sagital bir kesi yapın.Kesi sırasında, kesilen cildi açmak için başparmak ve işaret parmağını kullanın, alt kasları ve çene kemiğini açığa çıkarın. Ar...