This work was funded by the University Paris-Diderot, the French National Institute of Health and Medical Research (INSERM), and the French Institute for Odontological Research (IFRO).

All animals used in the present study were maintained in accordance with guidelines for the care and use of laboratory animals from the French Ministry of Agriculture (A-75-06-12).
1. Animal Exposure to Toxicants
<ol>
	<li>Prior to performing this protocol, obtain necessary institutional approval and be sure to comply with all animal care guidelines.</li>
	<li>Apply the design of the research protocol allowing the constitution of different experimental groups in order to test the impact of the molecule(s) investigated on ameloblast secretion stage and the ameloblast maturation stage. Here, four groups of male Wistar rats were constituted depending on their exposure to fluoride (NaF) in combination or not with BPA (Figure 1A)2. All animals were observed and dissected on day 65 (Figure 1B).</li>
</ol>
2. Dissection of Hemi-Mandibles from Adult Rats
<ol>
	<li>Euthanize rats by CO2 asphyxiation, optionally followed by decapitation to the separate head from the rest of the body. Expose rats to CO2 for 5 min in a dedicated box16.</li>
	<li>Remove all the skin from the lower lips using a #11 scalpel in order to get easy access to the lower incisors.</li>
	<li>With the same scalpel, make an incision between the two lower incisors with a slight pressure and the mandible will be split into two halves.</li>
	<li>Cut the temporal mandibular joint with a scalpel to detach the jaw, and hold the hemi-mandible with fine tweezers.</li>
	<li>Cut the surrounding soft tissues with a scalpel and remove all muscles, tendons, and ligaments using a scraper until the bone is completely clean.</li>
</ol>
3. Isolation of the Incisor17,18
<ol>
	<li>Carefully shave off the bone by placing the blade parallel to the major longitudinal axis of the incisor. Start at the bony ridge near the tips of the incisor (proximal end) until the end of the incisor near the cervical loop. The incision motion proceeds from the proximal to the distal direction.</li>
	<li>Make a first cut distally to the cervical loop with the scalpel to remove the gonion of the hemi-mandible and permit taking off the incisor easily without damaging the cervical loop or the secretory stage of ameloblasts (red line in the Figure 2).</li>
	<li>Make a second cut below the second molar, and insert the scalpel between the bone and the medial surface of the incisor. When all the basal bone is lifted, rotate outwards the incisor and take it off carefully to avoid enamel organ tissue impair using fine tweezers.</li>
</ol>
4. Micro-Dissection of Enamel Organ under Binocular Lens
<ol>
	<li>Drop 200 &#181;L of Saline Phosphate Buffer (PBS 1X) on the incisor using a pipette. Take the incisor with fine tweezers with the labial surface facing up. Make a scalpel mark between the colorless and the orange parts of the tissue. This mark corresponds to the underlying white spot which is observable afterwards (Figure 2).</li>
	<li>Scrape, with an excavator or equivalent tool, the cell surface from the scalpel mark to the apical end corresponding to secretion stage (colorless) ameloblasts, and from the scalpel mark to the tip of the incisor for the maturation stage (orange) ameloblasts.</li>
	<li>Remove the 2-mm tissue corresponding to the transition stage, as earlier described19. Do not open the incisor during enamel organ dissection to avoid contamination by the mesenchyme.</li>
	<li>Cut the cervical loop situated immediately at the apical part of the incisor (Figure 2) as previously described20.</li>
	<li>Collect it in 10% formalin or lysis solution for further investigations (see Table of Materials).</li>
</ol>
5. Collection of Separated Enamel Organ Tissues for Further Investigations
<ol>
	<li>Drop 200 &#181;L PBS 1X buffer on the incisor.</li>
	<li>With a #11 scalpel blade, carefully detach the dental epithelial cells gradually in the buffer, separately for each stage with the help of the scalpel mark made previously.</li>
	<li>For cell RNA and protein extractions, put the tissue in a lysis solution that allows the extraction of both proteins and RNAs (see Table of Materials). 
	NOTE: Prepare high quality RNAs by turning the tissue-excavator in the tube, and then grinding the cells until obtaining a homogeneous mixture. The mixture is ready for RNA and protein extractions according to the manufacturer&#39;s procedure, which was previously described2,8,17. Usually, about 5-10 &#181;g total RNAs and 150 &#181;g total proteins were obtained for each preparation.</li>
	<li>For histological analyses, immunohistochemistry (IHC), and in situ hybridization (ISH), put the cell layer in 10% formalin for 2 h, then store at 4 &#176;C in PBS 1X. The tissue can then be embedded in wax/paraffin or tissue OCT for frozen sections.</li>
	<li>For EMP extractions, take the incisor with fine tweezers. After a short exposure to air (slight dehydration), a white spot will be visible around the middle part of the labial surface of the incisor, which represents the initiation of the enamel mineralization. This white spot corresponds to the transition-/early maturation-stage of ameloblasts, so use it as an indicator for extraction of EMPs21.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Altered ameloblast activity and/or disrupted ameloblast proliferation, differentiation, and maturation processes lead to irreversible enamel defects and, in turn, the characterization of enamel defects may help develop understanding of the altered ameloblast activity during amelogenesis. Thus, the studies on isolated enamel organ are determinant to elucidate the pathological events leading to enamel defects whatever their origin, environmental or genetic.
This technique has originally been described by Hiller et al.17 and Robinson et al.18, and is used to demonstrate specific effects on each stage of enamel organ differentiation. It has then been adapted for EMP extractions19,24,25. We presently use this procedure to separate the main parts of the enamel organ and collect the biological samples in different environmental conditions to analyze RNAs and proteins (possibly intra- and extra-cellular proteins) using quantitative and qualitative techniques.
The precision of the micro-dissection requires substantial training before its application, to allow the collection of the preserved enamel organ and to avoid mesenchymal contamination and any tissue destruction. Moreover, it is quite difficult to succeed with mice or small rats. Indeed, the trickiest part of the procedure is to preserve the tissue morphology, histology, and orientation in IHC and ISH, as opposed to classical techniques commonly performed on incisors for these two approaches.
One of the main advantages of using the micro-dissected enamel organ compared to the demineralized whole hemi-mandible is the maintenance of enamel RNAs and proteins thanks to the lack of any treatment post-collection. The micro-dissected enamel organ can directly be used for all the techniques of molecular biology and biochemistry without selecting or culturing cells. The advantage of this procedure is the direct measurements of RNA and protein levels that reflects their quantities in specific ameloblasts in vivo in physiological or pathological conditions. In addition, some toxicants act on one differentiation stage specifically, as is the case with fluoride, which acts on maturation-stage ameloblasts and not on secretion-stage ones23,24. The micro-dissection of the different regions of the enamel organ allows the study of the mechanism of action of some toxicants or specific genes whose effects may be undetectable on the whole enamel organ or on isolated cells. Indeed, ameloblasts, especially differentiated ones, are difficult to collect and culture in vitro, which is attested to by the lack of studies reported on the subject. The only dental epithelial cells that can be isolated are pre-ameloblasts and stem cells from the cervical loop20. In addition, the reported ameloblastic cell lines, mainly rat HAT-729, mouse LS830, ALC31, and human AM-132, often lose the differentiation characteristics of in vivo ameloblasts: they express extracellular matrix proteins (EMPs) such as ameloblastin similarly to secretion-stage ameloblasts, and also KLK4 or SLC26A4/pendrin, such as maturation-stage ameloblasts8, but are more or less unable to express amelogenin and to mineralize the matrix to produce enamel. The isolation of dental epithelial stem cells and embryonic ameloblasts may constitute an option to study the first stages of ameloblast differentiation, but the studies on the terminal enamel mineralization process can&#39;t be carried out differently than with in vivo models. This last part of amelogenesis is determinant for enamel quality. Whereas key factors involved in tooth development, such as Bmp, Msx, Fgf, Notch, Shh, Wnt are well described33,34, those tightly involved in enamel quality are still poorly understood. The characterization of the key factors in enamel quality is necessary to decipher dental decay and discover innovative curative or preventive treatment for dental decay, which constitute a major public health problem as 92% of 20 to 64 years old adults in the world have or have had at least one caries35,36,37.
Rodents exposed to environmental toxicants able to disrupt amelogenesis and alter enamel quality may constitute a good model for studies on environmental factors. Similar procedures could also be used for functional studies on genes of interest directly or indirectly involved in amelogenesis. The micro-dissected enamel organ is a suitable material to characterize the mechanisms of action of toxicants and their target genes in vivo avoiding any experimental bias. When developmental enamel defects will be characterized, such an approach could be used as a predictive model of novel pollutants&#39; potential pathological impacts.

Many developmental enamel defects may result from exposure to environmental toxicants and/or inappropriate life-style1,2,3,4. Characterization of disrupting events and molecules of amelogenesis using the presently described procedure will promote the use of resulting enamel defects as early markers of exposure to several toxicants, and may help to reconstitute the history of health of each patient during the perinatal period when enamel is synthetized1,2. Enamel synthesis can be divided into four main stages depending on ameloblast activity5. The first step regroups precursor cell and pre-ameloblast proliferation. During the second step, differentiated ameloblasts secrete enamel matrix proteins (EMPs), mainly amelogenin, enamelin and ameloblastin, which determine the thickness of the final enamel. Thus, any disruption of EMP synthesis leads to quantitative defects of enamel. After the deposition of the full enamel thickness, the maturation stage begins. During this stage, apatite crystallite growth in width and thickness allows the enamel to reach the highest mineralization ratio found in a biological tissue, with up to 96% by weight. Disrupting events that occur during the maturation stage lead to qualitative enamel defects. Finally, ameloblasts enter a phase of post-maturation, also called pigmentation in rodents, and undergo apoptosis during tooth eruption making enamel defects (if any) irreparable and irreversible, thus defects provide potential retrospective recording of ameloblast stresses. In rodents, amelogenesis follows a similar sequence of events with the particularity that their incisors are continuously growing, which makes them a suitable model to study the general process of amelogenesis. Thus, any disruption of amelogenesis results in alterations of enamel quality and/or quantity, depending on the time-window of the disrupting event. In that sense, exposure to dioxin, lead, and endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), genistein, and vinclozolin, have been shown to generate enamel hypomineralizations1,2,3,6,7,8. Asymmetric white opaque spots were identified on the incisors of rats exposed to a low-dose BPA dose during the fetal period and the first month after birth1. These enamel defects in rats, and those of human molar incisor hypomineralization (MIH), share similar clinical, structural, and biochemical characteristics. MIH is a recently described dental enamel pathology, for which the etiology still remains unclear9,10 despite many causal factors having been hypothesized9,10,11,12.
Another important enamel hypomineralization pathology due to environmental factors is dental fluorosis (DF), which is the consequence of excessive fluoride absorption (&#62;0.1 mg/kg/day)13,14. The main source of fluoride is drinking water that is either supplemented or naturally enriched with fluoride. Fluoride is also often prescribed to prevent dental caries, but the prophylactic dose is only 50% lower than the toxic one (&#8804;0.05 mg/kg/day). MIH and DF, two frequent pathologies resulting from exposure to environmental factors, may present common features that need to be characterized due to the potentiation of hypomineralizing effects of fluoride combined with other toxicants such as EDCs2 or amoxicillin15.
Micro-dissection of rat enamel organ containing ameloblasts at different differentiation stages will help to understand the mechanism of action of molecules able to disrupt ameloblast activity and cause enamel defects to be diagnosed after tooth eruption. In other words, the characterization of changes of enamel gene expression and enamel matrix composition due to environmental toxicants allows the reconstitution of the history of exposure to toxicants, and facilitates environmental safety monitoring for public health.

<table><tbody><tr><td>Bisphenol A</td><td>Sigma Aldrich, Saint Louis MO</td><td>239658</td><td></td></tr><tr><td>formalin 10%</td><td>Sigma-Aldrich, Saint Louis, MO</td><td>HT5012</td><td></td></tr><tr><td>Tri-Reagent</td><td>Euromedex, France</td><td>TR118</td><td></td></tr><tr><td>RLT buffer</td><td>Qiagen, Les Ulis, France</td><td>74126</td><td>RNeasy Protect Mini Kit</td></tr><tr><td>Androgen receptor antibody</td><td>Santa Cruz Biotechnology, Santa Cruz, CA)</td><td>sc-816</td><td>rabbit polyclonal antibody</td></tr><tr><td>PBS 10x</td><td>EUOMEDEX</td><td>ET330.A</td><td></td></tr><tr><td>Sodium fluoride (NaF)</td><td>Sigma-Aldrich, Saint Louis, MO</td><td>S-1504</td><td></td></tr><tr><td>paraplast regular</td><td>Leica microsystems, Nanterre cedex,&amp;nbsp;France</td><td>39601006</td><td>called was/parafin in the text</td></tr><tr><td>tissue OCT</td><td>VWR, Fontenay-sous-Bois, France</td><td>411243</td><td></td></tr><tr><td>Extra Fine Bonn Scissors - Straight/8.5 cm</td><td>PHYMEP , Paris, France</td><td>14084-08</td><td></td></tr><tr><td>Handle for Scalpel Blades - 12.5 cm</td><td>PHYMEP, Paris, France</td><td>10035-12</td><td></td></tr><tr><td>Curved Scalpel Blade</td><td>PHYMEP , Paris, France</td><td>10035-20</td><td></td></tr><tr><td>Dissecting Knife - Fine/Straight Tip</td><td>PHYMEP , Paris, France</td><td>10055-12</td><td></td></tr><tr><td>Circle Knife</td><td>PHYMEP, Paris, France</td><td>10059-15</td><td></td></tr><tr><td>scalpel blades n&amp;deg;11 Swann-Morton</td><td>VWR, Fontenay-sous-Bois, France</td><td>233-0024</td><td></td></tr><tr><td>binocular lens</td><td>Leica biosystems, Nanterre cedex,&amp;nbsp;France</td><td>MZFLIII</td><td></td></tr></tbody></table>

micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Many enamel defects, such as dental fluorosis12,13, may result from environmental conditions due to excessive fluoride absorption or enamel hypomineralization similar to MIH due to exposure to some EDCs1,7,22. These developmental enamel defects may be experimentally reproduced on rats (Figure 1)1,2,7,23,24. The rat hemi-mandible contains a single incisor separated from three molars by a gap called the diastema. The continually growing incisor contains two mineralized tissues, the dentin produced by odontoblasts, and the enamel produced by ameloblasts present in enamel organ with other epithelial cells (Figure 2). The rodent incisor appears to be a suitable model to study the amelogenesis as, contrary to the molars, it contains all the ameloblast proliferation/differentiation stages throughout the animal&#39;s life.
Micro-dissected enamel organ from rat incisor can be separated according to ameloblast differentiation stage with the help of a white spot that appears after a slight dehydration, and this may be used as an indicator of the transition-stage between secretion- and maturation-stages19,21,25. The quality of the micro-dissected enamel tissues can be checked by Trichrome Masson&#39;s staining (Figure 3A). Microscopic observation showed typical enamel epithelial cells and ameloblast palisade. The absence of mesenchymal contamination was attested by the absence of collagen green staining (Figure 3A) and expression (Figure 3B). These micro-dissected tissues may be used for qualitative analyses such as IHC (Figure 3C) and ISH, and quantitative analyses such as RT-qPCR (Figure 3B), RNA-seq, and Western blotting. For example, the androgen receptor was specifically localized in the maturation-stage ameloblasts using a specific antibody directed against this protein (see Table of Materials) (Figure 3C)8. The main ameloblast differentiation stages are characterized by specific gene expression patterns26 that can be identified on micro-dissected enamel organ. For example, secretion-stage ameloblasts express enamelin2 and maturation-stage ameloblasts express Kallikrein 4 (KLK4) (Figure 3B). They contain high levels of ferritin that confer the ability to store high amounts of iron, which is responsible for the orange color of the enamel27.
Fluoride disruption of amelogenesis can easily be followed by observation of cell lysates for RNA extractions: Control maturation-stage lysates were orange, whereas fluoride-treated ones were light yellow to colorless (Figure 4A). Analysis of RNA levels extracted from micro-dissected maturation-stage enamel organ showed KLK4 down-regulation upon fluoride treatment (Figure 4B), which was also evidenced by a large-scale transcriptomic analysis reported recently2. Specific proteins expressed either by secretion- or maturation-stage ameloblasts may also be localized using micro-dissected enamel organ28. For example, the androgen receptor was specifically localized using micro-dissected enamel organ8,28.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57081/57081fig1.jpg" /> 
Figure 1: Enamel defects observed on rats exposed to fluoride (NaF) in combination or not with bisphenol A (BPA). (A) Schematic representation of the four experimental groups of Wistar rats constituted for the present study, groups which depended on the different environmental conditions applied to the rats. From gestational day 1 (fecondation) until weaning day 21 (P21), 5 &#181;g/kg BPA in 0.5 mL of corn oil was orally administered by gavage daily to pregnant and lactating females, whereas corn oil alone was administered to the control group. After weaning, each dam was identified and randomly distributed in one of the corresponding two groups. Male rats were selected for this study and exposed to 5 &#181;g/kg/day BPA alone, 5 mM NaF alone, or both at the same dose until 65 days after birth (P65). Control group was given solvent alone. (B) At postnatal day 30 (P30), rats chronically exposed to low-dose BPA exhibited white opaque spots and a no longer evident dental phenotype was identified for adult rats (P65). Alternating white and orange bands of typical dental fluorosis (DF) were observed in rats treated with NaF. A more severe phenotype characterized by an important incisor discoloration was identified in rats exposed to both agents. Homogeneous orange incisal enamel typified control rats. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57081/57081fig1large.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/57081/57081fig2.jpg" /> 
Figure 2: Schematic representation of the procedure. Isolation of ameloblasts from secretion- and maturation-stages from micro-dissected enamel organ. The rat hemi-mandible contains three molars and a continually growing incisor with two mineralized tissues, the dentin produced by odontoblasts (in yellow), and the enamel produced by ameloblasts (in orange). When the incisor is isolated and slightly dehydrated, a white spot near the apical part of the labial surface is observable. It helps to identify ameloblast stage of differentiation, as it covers the transition stage between the secretion- and maturation-stages. The enamel organ is carefully micro-dissected, separated in 3 parts depending on the ameloblast differentiation stages, and used either for qualitative (histological) analysis or quantitative experimental approaches (mRNA and protein analyses by RT-qPCR and Western blotting, respectively).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57081/57081fig3.jpg" /> 
Figure 3: Quality control of micro-dissected secretion- and maturation-stages of enamel organ. Rat enamel organ is separated in 3 parts containing the cervical loop, the secretion-stage ameloblasts, and maturation stage ameloblasts. (A) Trichrome Masson&#39;s staining of micro-dissected parts showed the different epithelial cells and the absence of mesenchymal cells. Scale bar, 100 &#181;m. (B) Gene expression analysis by RT-qPCR experiments of RNAs extracted from secretion- and maturation-stage enamel organ. A typical pattern of enamelin and KLK4 expression was observed, as well as the absence of collagen 1 expected in mesenchymal cells. (C) Fluorescent staining used an antibody (see Table of Materials) specifically directed against the androgen receptor expressed only by maturation-stage ameloblasts. Scale bar, 20 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57081/57081fig3large.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/57081/57081fig4.jpg" /> 
Figure 4: Representative results obtained with micro-dissected rat enamel organ. (A) When cells were resuspended in a lysis buffer for RNA extraction (see Table of Materials), maturation-stage tissue containing pigmented ameloblasts appeared orange in control- and BPA-treated conditions. The secretion-stage portion is yellow no matter the conditions. In NaF treatment, all lysates were light yellow to colorless. (B) Example of RT-qPCR data obtained with RNA preparations from enamel organs of rats submitted to various environmental conditions. NaF down-regulated KLK4 in the maturation-stage, and BPA increased enamelin expression in the secretion-stage. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57081/57081fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

micro-dissection-enamel-organ-from-mandibular-incisor-rats-exposed-to

Nanyang Technological University

Research

JoVE Journal

Medicine

10.1K Views.  Cordeliers Research Centre. Begrip glazuur vorming en mogelijk wijzigingen vereist de studie van ameloblast activiteit. Hier beschrijven we een betrouwbare en consistente methode voor micro-ontleden glazuur organen met secretie - en rijping-fase-ameloblasts die kunnen worden gebruikt voor verdere kwantitatieve en kwalitatieve experimentele procedures.

Video: Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Environment

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Developmental Biology

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Live Tracking of Dental Cells via Two-Photon Microscopy

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 

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 ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Bioengineering

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Biology

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

Immunology and Infection

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

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