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Laser capture microdissection (LCM) of dental epithelia, adjacent dental mesenchyme, odontoblasts and cementoblasts from sagittal sections of mouse mandibular incisors and molars. (a) Schematic illustration of a mouse mandible (sagittal plane) during tooth development. Rectangular frame shows the incisor cervical loop and surrounding area used for LCM (b). Dotted vertical line indicates frontal section of the first molar for LCM (c). Solid vertical lines indicate the orientation and size of the embedded tissues used in this study. Note the excess bones out of this border was trimmed off to make the sectioning more efficient and with high quality. (b) Undifferentiated epithelial and mesenchymal cells associated with incisor root formation. Cell nuclei are stained with cresyl violet (purple) on sagittal sections of the 7-day postnatal mouse mandible. The following three cell populations were captured: LCL (epithelia,red dotted line outlined),DP (mesenchyme) and DF (mesenchyme). Left panel: ×40; middle panel: ×200; right panel: ×200. (c) Terminally differentiated cells associated with molar root formation,including: OD (dentin forming cells,not captured yet) and CB (cementum forming cells). Left panel: ×100; right panel: ×200. Alv,alveolar bone; CEJ,cemento-enamel junction; CB,cementoblasts; DF,dental follicle; DP,dental papilla; LCL,lingual cervical loop; OD,odontoblasts.
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Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered b...
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Context 1
... shown in Figure 1, lingual cervical loop (LCL) epithelial cells, adja- cent apical dental papilla (DP) and apical DF cells were collected from mandibular incisors of three CD-1 mice on P7. Odontoblasts (OD) and CB were collected from roots of mandibular first molars from three mice on P14 when molar roots are actively developing. ...Context 2
... equipment used for surgical dis- section of mandibles was cleaned with RNaseZap (Applied Biosystem/ Ambion, Austin, TX, USA). After the erupted portion of mandibular incisors and excess bones were cut off (Figure 1a), the mandibles were embedded in an optimal cutting temperature (Tissue-Tek; Sakura Finetek USA, Torrance, CA, USA) block using standard cryomolds (Sakura Finetek USA) and stored at 280 6 C until use. ...Context 3
... capture microdissection for root study JX Sun et al 8 allowed to dry at room temperature for 5 min and immediately used for laser capture microdissection using a Veritas Microdissection sys- tem (Arcturus Bioscience, Mountain View, CA, USA). Figure 1b and c provides a visual example to demonstrate the locations from which samples were taken. As CB and OD line the root surface as a single-cell layer, the underlying cementum and attached CB, as well as the under- lying dentin and attached OD, were dissected out together to minimize cellular damage caused by placing a laser beam directly onto the cells. ...Similar publications
The goal of the study was to characterize the transcriptome profiles of human ameloblasts and odontoblasts, evaluate molecular pathways and advance our knowledge of the human "dentome". Laser capture microdissection was used to isolate odontoblasts and ameloblasts from human tooth buds (15-20week gestational age) from 4 fetuses. RNA was examined us...
Citations
... 'Domain III' or 'FC domain': A spherical domain present at the carboxyl terminal, which is a common feature of the FBLN family. [7][8][9] 'Domain II': Variable number of calcium-binding epithelial growth factor modules. 'Domain I': Variable region at the end of the amino group. FBLN s are classified into two subgroups based on the overall protein structure. ...
... 16 And it has been reported to improve the osteogenic signalling activity of Bmp-2 during osteogenesis, impacting the expression of the essential osteogenic transcription factor OSX. FBLNs expression pattern is similar to DMP1, suggesting that FBLN1 is crucial in cellmatrix and cell-to-cell interactions during dentin mineralization. 9,17 Given its critical role in mediating cellular interactions and signalling pathways essential for osteoblast function and skeletal development, elucidating the precise mechanisms of FBLN1 in MSCs can lead to breakthroughs in bone tissue engineering and regenerative medicine. ...
To investigate the role and mechanism of FBLN1 in the osteogenic differentiation and bone regeneration by using umbilical cord mesenchymal stem cells (WJCMSCs). We found that FBLN1 promoted osteogenic differentiation of WJCMSCs and WJCMSC‐mediated bone regeneration. It was showed that there was an m⁶A methylation site in 3′UTR of FBLN1 mRNA, and the mutation of the m⁶A site enhanced the stability of FBLN1 mRNA, subsequently fostering the FBLN1 enhanced osteogenic differentiation of WJCMSCs. YTHDF2 was identified as capable of recognizing and binding to the m⁶A site, consequently inducing FBLN1 instability and repressed the osteogenic differentiation of WJCMSCs. Meanwhile, miR‐615‐3p negatively regulated FBLN1 by binding FBLN1 3′UTR and inhibited the osteogenic differentiation of WJCMSCs and WJCMSC‐mediated bone regeneration. Then, we discovered miR‐615‐3p was found to regulate the functions of FBLN1 facilitated by YTHDF2 through an m⁶A‐miRNA regulation mechanism. We demonstrated that FBLN1 is critical for regulating the osteogenic differentiation potentials of WJCMSCs and have identified that miR615‐3p mediated the decay of FBLN1 mRNA which facilitated by m⁶A reading protein YTHDF2. This provided a novel m⁶A‐miRNA epigenetic regulatory pattern for MSC regulation and bone regeneration.
... In order to obtain reliable data, very high quality of RNA as well as sufficient amounts of RNA are essential (231)(232)(233). There are several published RNA isolation protocols available but vary according to study design and mainly describe the extraction of RNA from soft tissues (215,(234)(235)(236)(237). To the best of my knowledge, a RNA-seq study using feline teeth has not been published and therefore an optimised protocol for feline tooth RNA extraction does not exist. ...
... Due to suboptimal temperatures during sample processing or post mortem apoptosis, RNA cannot be devoid of some degradation. The overall quality of RNA from feline teeth was lower than that achieved from soft tissue but as similar to comparable studies of murine teeth (237). The quality of RNA-seq data will guide which data can be useful for inclusion or exclusion for further data analysis. ...
... This new data could be combined with my existing data using paired samples. To overcome the technical challenges encountered using mineralised tissues I would propose to use specialised strategies for mineralised tissues processing, and laser capture microdissection, a precise sample dissection technique utilised in mouse and human tooth studies (201,237). ...
Feline tooth resorption (TR) is a common and painful disease characterised by loss of mineralised tissues of the tooth. Due to the progressive nature of the disease the only available treatment is to extract affected teeth. Odontoclasts are specialised cells that resorb teeth and they share many similarities with osteoclasts, the bone resorbing cells. The main physiological role of these cells is to resorb deciduous teeth to allow for the permanent tooth replacement. In some cats these cells become dysregulated and attack the permanent teeth later in life. However, the aetiology of tooth resorption is still unclear. The aim of this PhD project was to identify transcriptomic changes between TR negative and TR positive teeth, set up a TR model to study odontoclast dysregulation in vitro and to use this system to test a novel therapeutic target for the treatment of TR. In this study, a successful RNA isolation procedure for feline teeth collected in a clinical setting and at post-mortem was developed. To elucidate the role of inflammation and vitamin D involvement in TR, quantitative PCR was performed but there was no statistically significant difference in the expression of any inflammatory cytokines, vitamin D receptor or receptor activator of nuclear factor kappa-B ligand (RANKL) between TR negative and TR positive teeth. Feline osteoclasts were successfully generated from feline bone marrow in vitro. I also found that primary feline periodontal ligament cells could induce osteoclast formation. Osteoclast precursors from TR positive cats formed higher numbers of osteoclasts when co-cultured with periodontal ligament cells compared to TR negative cats. Transcriptome analysis by RNA sequencing identified many differentially expressed genes between TR negative and TR positive teeth from the same cat. I also found that up-regulated genes in TR positive teeth, from the literature, are known to be associated with osteoclast differentiation and calcium signalling pathways. Apart from the genes related to osteoclast biology, I also identified up-regulated genes involved in muscle and tooth development, which suggests TR positive teeth undergo repair or bone formation in response to tooth resorption. Based on RNA sequencing data and quantitative PCR, I was able to identify a list of genes which may be involved in odontoclast dysregulation in TR. Among those, matrix metalloproteinase 9 (MMP9) was of great interest due to its potential role in osteoclast biology. High MMP9 expressing odontoclasts were found in tooth sections undergoing tooth resorption by immunohistochemistry. In functional studies, MMP9 inhibitor reduced both osteoclast formation and resorption activity in vitro. Furthermore, feline MMP9 siRNA inhibited osteoclast differentiation but showed little effect on dentine resorption activity. These results confirm the hypothesis that the transcriptome of TR positive teeth was locally different from TR negative teeth. The results revealed an upregulation of genes in pathways associated with osteoclast formation in TR positive teeth. Further testing of candidate genes as potential therapeutic targets in feline TR could be performed using the feline tooth resorption model system developed in this study.
... The expression of tenascin-W in a subset of skeletal muscles is evident both from immunohistochemistry (Scherberich et al., 2004) and microarray studies (Fischer et al., 2005). In the mouse, tenascin-W is expressed in developing teeth (Scherberich et al., 2004), and this has been confirmed by microarray analysis of RNA from laser-captured tissues (Sun et al., 2012). ...
Tenascins are a family of multifunctional glycoproteins found in the extracellular matrix of chordates. Two of the tenascins, tenascin-C and tenascin-W, form hexabrachions. In this review, we describe the discovery and domain architecture of tenascin-W, its evolution and patterns of expression during embryogenesis and in tumors, and its effects on cells in culture. In avian and mammalian embryos tenascin-W is primarily expressed at sites of osteogenesis, and in the adult tenascin-W is abundant in certain stem cell niches. In primary cultures of osteoblasts tenascin-W promotes cell migration, the formation of mineralized foci and increases alkaline phosphatase activity. Tenascin-W is also prominent in many solid tumors, yet it is missing from the extracellular matrix of most adult tissues. This makes it a potential candidate for use as a marker of tumor stroma and a target for anti-cancer therapies.
... There was no obvious outliers studies using soft tissues. However other studies extracting RNA from mineralised tissues reported similar limitations where RNA from hard tissues was more degraded than RNA extracted from soft tissues (Sun et al. 2012;Carter et al. 2012). We also investigated if there were any correlation between RNA purity as measured by the A 260:280 and A 230:260 ratios and RNA integrity as measured by RIN e values. ...
Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RINe) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A260:280 or A230:260 ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RINe values, while RPL17 was stable at all RINe values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RINe values. However one low yield sample with a high RINe value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes.
... The common research of highly mineralized tooth tissues is fraught with difficulties in preparing intact, unfixed, undecalcified sections of highly mineralized tissues, such as dentin, tooth cementum, and enamel. A modified procedure of sectioning unfixed and undecalcified frozen mouse mandibles was described to have been used for dental hard tissue-associated cells (19). More recently, use of LCM in oral biosciences has been thoroughly reviewed in a study by THENNAVAN et al. (20). ...
The dentin–enamel junction (DEJ) is the border where two different mineralized structures – enamel and dentin – meet. The protein‐rich DEJ, together with the inner enamel region of mature teeth, is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the DEJ and the adjacent enamel organic matrix (EOM). We studied proteomes of the DEJ and EOM of healthy human molars and compared them with dentin and enamel proteomes from the same teeth. These tissues were cut out of tooth sections by laser capture microdissection, proteins were extracted and cleaved by trypsin, then processed by liquid chromatography coupled to tandem mass spectrometry to analyze the proteome profiles of these tissues. This study identified 46 proteins in DEJ and EOM. The proteins identified have a variety of functions, including calcium ion‐binding, formation of extracellular matrix, formation of cytoskeleton, cytoskeletal protein binding, cell adhesion, and transport. Collagens were identified as the most dominant proteins. Tissue‐specific proteins, such as ameloblastin and amelogenin, were also detected. Our findings reveal new insight into proteomics of DEJ and EOM, highly mineralized tissues that are obviously difficult to analyze.
... Dental pulp tissue is a promising source of stem cells, which are widely used to test biomaterials, reviewed in [13,14]. In this context, we recently established a protocol for isolating and identifying a new subpopulation of pluripotent-like stem cells from dental pulp, named Dental Pulp Pluripotent-like Stem Cells (DPPSCs) [15], that express pluripotent markers and show genetic stability [16,17]. ...
The aim of this study is to compare the osteogenic differentiation capacity of the dental pulp pluripotent-like stem cells (DPPSCs) using conditional media pretreated with ProRoot-MTA, Biodentine (BD) or the newly manufactured pure Portland cement Med-PZ (MZ). DPPSCs, isolated from human third molars, are the most relevant cell model to draw conclusions about the role of biomaterials on dental tissue regeneration. Cytotoxicity, alkaline phosphatase (ALP) activity, and calcium deposition analysis were evaluated at different differentiation time points. Gene expression of key osteogenic markers (RUNX2, Collagen I and Osteocalcin) was determined by qRT-PCR analysis. The osteogenic capacity of cells cultured in conditioned media prepared from MZ or MTA cements was comparable. BD conditioned media supported cell proliferation but failed to induce osteogenesis. Relative to controls and other cements, high osteogenic gene expression was observed in cultures pre-treated with the novel endodontic cement MZ. In conclusion, the in vitro behavior of a MZ- endodontic cement was evaluated, showing similar enhanced cell proliferation compared to other commercially available cements but with an enhanced osteogenic capacity with prospective potential as a novel cement for endodontic treatments.
... Moreover, the limited access to the mixture of several types of cells within the developing periodontal tissues makes it difficult to extract mRNA from the designated cells and thus to assign expressed genes to specific cell populations. LCM technology allows precise isolation of either single cells or small groups of cells under high-magnification [16], which permits the precise isolation of DF during periodontal development. Here, we utilized LCM to isolate DF tissues at the prenatal stage (E17) and post-natal stage (PN2). ...
... The novel approach of LCM [16] was used to obtain homogenous populations of DF tissue from frozen sections in mouse mandible first molar. The samples were also analyzed using a microarray to compare the gene expression profiles during periodontal development. ...
Objectives: Odontogenesis is dependent on serial temporal and spatial epithelial-mesenchymal inter- Q2
action. Multiple signaling networks have been identified in the inductive interactions of odontogenesis at Q3
the early stage. Technical obstacles and heterogeneity of the dental follicle (DF), which is the origin of the
majority of periodontal tissues, have hindered the clarification of the molecular blueprint in early periodontal tissue development. This has in turn hindered studies of new/effective periodontal regeneration
therapy. In this study, we comparatively analyzed the gene expression profiles of DF at E17 (when DF cells
are histologically recognized) and PN2 (the initiation of periodontal development).
Methods: Gene expression profiles of DF at E17 and PN2 were assessed by the combined use of laser
capture microdissection and microarray.
Results: Comparative gene expression analysis of DF at E17 and PN2 during periodontal development
revealed 4 2-fold up-regulation and down-regulation of 2519 and 5060 genes, respectively. Bioinformatic analysis of the selected genes revealed that the temporally changed genes were mostly enriched in
GO terms relative to the vasculature system, and were sometimes linked to multiple processes. RT-qPCR
was used to verify the microarray data.
Conclusions: The delineation of the differential gene expressions between pre- and post-natal developmental stages of DF in vivo will increase the understanding of periodontal tissue development
... The common obstacle, in mineralized tissue research, is the difficulty in preparing intact unfixed, undecalcified sections of highly mineralized tissues such as dentin and enamel. Another impediment is the means to obtain high-quality RNA from such tissues (Sun et al, 2012). A modified procedure of sectioning unfixed and undecalcified frozen mouse mandibles, for microarray processing and subsequent gene expression analyses, has been described in recent times to be specifically used for dental hard tissue-associated cells (Sun et al, 2012). ...
... Another impediment is the means to obtain high-quality RNA from such tissues (Sun et al, 2012). A modified procedure of sectioning unfixed and undecalcified frozen mouse mandibles, for microarray processing and subsequent gene expression analyses, has been described in recent times to be specifically used for dental hard tissue-associated cells (Sun et al, 2012). ...
... Several studies have established the importance of LCM to generate sufficient genomic material for analysis (Hoffmann et al, 2001;Ohyama et al, 2002;Salmon et al, 2012;Sun et al, 2012). Indeed, single nucleotide polymorphism (SNP) array analysis of genotype, copy number, and LOH on the DNA content obtained from microdissected areas of FFPE samples of oral cancer has been successfully accomplished; however, it is still not precise enough to be used for diagnosis or treatment planning (Stokes et al, 2011). ...
Laser capture microdissection (LCM) is a high end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell or lesion specific genomic and proteomic analysis. Literature search on the application of LCM in oral tissues was made through PUBMED. There is ample evidence to substantiate the utility of LCM in understanding the underlying molecular mechanism involving an array of oral physiological and pathological processes, including odontogenesis, taste perception, eruptive tooth movement, oral microbes, and cancers of the mouth and jaw tumors. This review is aimed at exploring the potential application of LCM in oral tissues as a high-throughput tool for integrated oral sciences. The indispensable application of LCM in the construction of lesion specific genomic libraries with emphasis on some of the novel molecular markers thus discovered is also highlighted. This article is protected by copyright. All rights reserved.
... This finding is consistent with Papke et al. (2015), who suggest a unique and very particular role for fibulin-4 in ECM organization. Interestingly, a study on the tooth root development in mouse incisors and molars reveals that all members of the fibulin family except for fibulin-4 are expressed in cementoblasts of developing teeth (Sun et al., 2012). The present study was performed on adult mouse molars; supposedly, there is a switch in the expression pattern of fibulins during tooth root development that needs to be clarified by future studies. ...
Periodontitis refers to inflammatory disease of the periodontal structures (the gingiva, dental cementum, periodontal ligament (PDL) and alveolar bone) that ultimately leads to their destruction. Whereas collagens are well-examined main components of the periodontium, little is known about the other structural proteins that make up this tissue. The aim of this study was to identify new extracellular matrix (ECM) components, including fibulins and matrilins, in the periodontium of mice.
After sacrificing 14 mice (Sv/129 strain), jaws were prepared. Each tissue sample contained a molar and its surrounding alveolar bone. Immunohistochemistry was carried out on paraffin-embedded sections.
Our results show that mice exhibit fibulin-3, −4 and −5 and matrilin-1, −2, −3 and −4 in PDL and in blood vessels of alveolar bone and PDL as well as in the pericellular matrix of osteocytes and cementocytes. In dental cementum, only fibulin-4 is expressed.
For the first time, we show that fibulin-3, −4 and −5 and matrilin-1, −2, −3 and −4 are essential components of the periodontal tissues. Our findings indicate an association of these proteins with collagens and oxytalan fibers that might be of future interest in regenerative periodontitis therapy.
... Thus, more precise method should be employed like laser capture microdissection (LCM) technology to capture the dental epithelial cells only, then the interference from the mesenchyme could be avoided. LCM collection of intact cells with wellpreserved RNA allows subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth development (Sun et al. 2012). ...
Caveolin-1 is a scaffolding protein involved in the formation of cholesterol-rich caveolae lipid rafts within the plasma membrane and is capable of collecting signaling molecules into the caveolae and regulating their activity, including extracellular matrix metalloproteinase inducer (EMMPRIN). However, detailed expression patterns of caveolin-1 and EMMPRIN in the developing dental germ are largely unknown. The present study investigated the expression patterns of caveolin-1 and EMMPRIN in the developing mouse tooth germ by immunohistochemistry and real-time polymerase chain reaction. At the bud stage, caveolin-1 expression was initiated in the epithelium bud and mesenchymal cells, while EMMPRIN was weakly expressed at this stage. At the cap stage, caveolin-1 protein was located in the lingual part of the tooth germ; however, EMMPRIN protein was located in the labial part. From the bell stage to 2 days postnatal, caveolin-1 expression was detected in the ameloblasts and cervical loop area; with EMMPRIN expression in the ameloblasts and odontoblasts. Real-time polymerase chain reaction results showed that both caveolin-1 and EMMPRIN mRNA levels increased gradually with progression of developmental stages, and peaked at day two postnatal. The current finding suggests that both caveolin-1 and EMMPRIN take part in mouse tooth development, especially in the differentiation and organization of odontogenic tissues.
