Peritubular dentin, a highly mineralized, non-collagenous, component of dentin: isolation and capture by laser microdissection.

We demonstrate the capability and technique to perform microdissection and isolation of select regions of untreated, mineralized dentin using laser capture. Dentin is a complex, non-homogeneous tissue comprised of a mineralized collagenous matrix (intertubular dentin [ITD]), odontoblastic processes (ODPs), a void space (tubules) that forms within the ITD left behind by the retraction of ODPs during dentin maturation, and a highly mineralized non-collagenous component that exists at the interface between the tubules and ITD known as peritubular dentin (PTD). PTD forms as the dentin matures. The ODPs retract toward the direction of the pulp; leaving very little PTD at either the DEJ or near the pulp. Statistical analysis of thin cross-sections of coronal bovine dentin imaged by light microscopy reveal that the area occupied by PTD >50%. To examine the nature of PTD and its relation to both the tubules and ITD, we devised a series of steps to carefully prepare sections of coronal bovine dentin so that areas of the dentin tissue could be cut and isolated for further analysis. We demonstrate that it is possible to selectively isolate targeted regions of dentin for analysis and that high resolution analysis of such sections can be performed using electron microscopy. Results show that the mineralized PTD has a different texture than mineralized ITD and that there is a distinct boundary between the PTD and the ITD. Selective isolation of mineralized tissue components for further analytical study opens the door for the investigation of similar enigmatic mineralized structures.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

Identification of differentially expressed genes in mandibular condylar and tibial growth cartilages using laser microdissection and fluorescent differential display: chondromodulin-I (ChM-1) and tenomodulin (TeM) are differentially expressed in mandibular condylar and other growth cartilages.

Mandibular condylar cartilage can be distinguished from articular and growth cartilages of long bones based on several differences in morphology, physiology, and function between these structures. However, there is almost no information available on the types of genes that contribute to these differences. In this study, genes that were differentially expressed in mandibular condylar and growth cartilages in 1-week-old rats were investigated using fluorescent differential display (FDD) and laser microdissection (LMD). A number of genes were identified by FDD including chondromodulin-1 (ChM-1), which is known to be an angiogenesis inhibitor of endochondral ossification. ChM-1 expression was then compared with that of tenomodulin (TeM) in mandibular condylar and tibial cartilages of 1- and 5-week-old rats using real time PCR (RT-PCR), immunohistochemistry, and in situ hybridization. There was negligible detection of ChM-1 mRNA and protein in mandibular condylar cartilages compared to tibial cartilages of 1- and 5-week-old rats. On the other hand, TeM mRNA was more abundant in mandibular condylar cartilage than in tibial. These observations demonstrated that gene expression in mandibular condylar cartilage differed from other types of cartilage such as articular and growth ones.

[Comparison of gene expression profile of cementoblasts with periodontal ligament cells in mouse mandible with laser capture microdissection].

Cementum is an essential tissue to maintain tooth function and should be closely correlated to tooth root development and periodontal tissue regeneration. However, detailed features of the periodontium including cementum and specific markers for cementoblasts are unknown. Moreover, the molecular mechanism of periodontal tissue development, homeostasis and regeneration remains unknown. Previous studies have usually examined cementum or periodontalligament (PDL) tissue obtained by manual curettage, resulting in difficulties in isolating pure cementum or PDL. We employed laser capture microdissection (LCM) to isolate cementoblasts and PDL cells from undecalcified frozen sections of murine mandible and to obtain RNA of good quality for subsequent genetic analysis. Over 500 cementoblasts and PDL cells were separately laser captured under microscopy. A bioanalyzer detected peaks of 18S and 28S rRNA both in the laser-dissected cementoblasts and in PDL cells, suggesting that the RNA was of sufficient quality. The RNA samples were amplified due to their small amount and a comparative analysis of mRNA expression by GeneChip showed that about 2,000 genes were differentially expressed between cementoblasts and PDL cells. Both cementoblast-positive and PDL cell-negative genes were serially analyzed by quantitative RT-PCR using RNA samples obtained from mandibles and femurs. Several genes were expressed at higher levels in the mandible than in the femur, suggesting that some might be cementoblast-specific markers. We established a novel experimental system with which to isolate target tissues from single cells in undecalcified frozen sections and to obtain intact RNA. These methodologies could be useful for further investigation of mineralized tissues and to explore tissue-specific factors.

The Use of Semitranslucent Rubber Pledgets During Microsurgical Dissection of Cerebellopontine Angle Tumors: Technical Note.

BACKGROUND AND IMPORTANCE: Dissection of cerebellopontine angle (CPA) tumors that abut or adhere to the brainstem or cranial nerves can be a challenging surgical endeavor. We describe the use of semitranslucent latex rubber pledgets in the tumor-brain interface as a method to improve visualization and protection of vital tissue during microsurgical dissection of CPA masses. The rubber pledgets are fashioned by cutting circular discs out of the cuff portion of talc-free, partially opaque latex gloves. These pledgets provide a semitranslucent, nonadherent membrane that can be placed between vital neural tissues and a tumor capsule to minimize trauma during dissection. The semitranslucent latex enables visualization of the underlying anatomical structures while also providing a protective surface onto which a suction device can be rested to facilitate clearance of the surgical field. CLINICAL PRESENTATION: A 56-yr-old woman with left ear tinnitus presented with a 3-cm CPA meningioma. During microsurgical dissection, rubber pledgets were used to preserve the interface between the brain stem, cranial nerves, and tumor capsule. The use of the rubber pledgets appeared to secure the interface between to tumor and the brain while at the same time protecting the cranial nerves, brainstem, and cerebellum. CONCLUSION: Semitranslucent rubber pledgets may facilitate microsurgical dissection of CPA tumors.

Laser capture microdissection.

Laser capture microdissection (LCM) is a technique for isolating pure cell populations from a heterogeneous tissue section or cytological preparation via direct visualization of the cells. This technique is applicable to molecular profiling of diseased and disease-free tissue, permitting correlation of cellular molecular signatures with specific cell populations. DNA, RNA, or protein analysis can be performed with the microdissected tissue by any method with adequate sensitivity. The principle components of LCM technology are (1) visualization of the cells of interest via microscopy, (2) transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods, and tissue-preservation protocols that allow microdissection of fresh or archival specimens. LCM platforms are available as a manual system (PixCell; Arcturus Bioscience) or as an automated system (AutoPix).

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography.

Plasminogen activation is believed to be critical to the progression of oral squamous cell carcinoma by facilitating matrix degradation during invasion and metastasis, and high levels of urokinase plasminogen activator (uPA) and plasminogen activator (PA) inhibitor-1 (PAI-1) in tumors predict poor disease outcome. We describe the development of a novel method for studying PA in oral cancer that combines the sensitivity and specificity of zymography with the spatial resolution of immunohistochemistry. Laser capture microdissection (LCM) was combined with plasminogen-casein zymography to analyze uPA, tissue PA (tPA), uPA-PAI-1 complexes, and tPA-PAI-1 complexes in 11 tumors and adjacent non-malignant epithelium from squamous cell carcinomas of the tongue, floor of mouth, larynx, and vocal cord. uPA was detectable in all tumor samples analyzed, uPA-PAI-1 complexes in three samples, and tPA in nine. PA was detectable in as little as 0.5 microg protein lysate from microdissected tumors. In all specimens, uPA expression was highly increased in tumor tissue compared to adjacent non-malignant tissue. In conclusion, LCM combined with zymography may be excellently suited for analyzing the prognostic significance and causal involvement of the plasminogen activation system in oral cancer.

In vivo expression of RANKL in the rat dental follicle as determined by laser capture microdissection.

Tooth eruption is a localized event in which many of the genes required for eruption are expressed in the dental follicle. A major function of the follicle is to recruit mononuclear cells for osteoclastogenesis such that the alveolar bone can be resorbed. Osteoclastogenesis is primarily regulated by receptor activator of nuclear factor-kappa B ligand (RANKL), colony-stimulating factor-one (CSF-1) and osteoprotegerin (OPG). In the rat first mandibular molar, osteoclastogenesis is maximal at day 3 and CSF-1 is maximally expressed in the follicle at this time whereas OPG expression is reduced. Whether or not RANKL is expressed in vivo in the follicle is controversial, however. It is critical to determine this because others have shown that in partially-rescued mice null for RANKL, teeth do not erupt. This suggests that RANKL should be expressed in the follicle for eruption to occur. Thus, to precisely determine if RANKL is expressed in the follicle in vivo, laser capture microdissection (LCM) was used to excise dental follicle tissue from frozen sections followed by RNA isolation and RT-PCR. The results show that RANKL is expressed in the dental follicle at days 1-9 postnatally. The technique was confirmed by controls showing that LCM isolates of the follicle, and alveolar bone, express OPG. Also, LCM isolates of alveolar bone were positive for RANKL. Thus, RANKL has now been shown to be expressed in the follicle and it is probable that interactions between it, CSF-1 and OPG regulate locally the osteoclastogenesis needed for tooth eruption.

Piezo-power microdissection of mature human dental tissue.

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1alpha and collagen-3alpha were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

Inferior alveolar nerve transposition and reposition for dental implant placement in edentulous or partially edentulous mandibles: a multicenter retrospective study.

The aim of this study was to evaluate the success and complications following inferior alveolar nerve (IAN) transposition/reposition for dental implant placement in edentulous or partially edentulous mandibles. This was a multicenter retrospective study; patients who had undergone IAN transposition/reposition at four surgical clinics were retrospectively evaluated. Adverse effects, especially neural disturbances, were recorded and followed. Overall, 68 IAN reposition and 11 nerve transposition procedures were performed in 57 patients (only three patients reported on smoking). The residual bone above the IAN was an average 3.88±1.98mm. A total of 232 dental implants were inserted in the area after transposition/reposition of the nerve. The average follow-up time was 20.62±9.79 months, ranging from 12 to 45 months. One implant loss was observed during the follow-up period. Four patients reported prolonged transient neural disturbances immediately following surgery (5% of the operations). The duration of neural disturbances after the surgery ranged from 1 to 6 months. No permanent neural damage was reported. Thus, within this study's limitations, it can be concluded that IAN transposition and reposition are useful adjunct techniques for managing severely atrophic edentulous or partially edentulous mandibles with dental implants. The risk of neural dysfunction appears to be low.

Expression microdissection adapted to commercial laser dissection instruments.

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.

Nucleic acids extraction from laser microdissected FFPE tissue sections.

Tissue heterogeneity is a common source of unsuccessful experiments. Laser capture microdissection is a tool to prepare homogeneous tissue and cell areas as starting material for reliable and reproducible results as it allows the defined investigation of spatially different tissue areas.Nearly all samples allow the extraction of DNA. Fresh or fresh frozen samples are an ideal source for getting access to high-quality RNA. But also the large archives of formalin-fixed, paraffin-embedded (FFPE) tissue specimens are a valuable source of sample material for RNA extraction. Optimized protocols may help to make the RNA from FFPE material suitable for expression studies.

Gene expression dynamics during bone healing and osseointegration.

BACKGROUND: Understanding the molecular features of bone repair and osseointegration may aid in the development of therapeutics to improve implant outcomes. The purpose of this investigation is to determine the gene expression dynamics during alveolar bone repair and implant osseointegration. METHODS: An implant osseointegration preclinical animal model was used whereby maxillary defects were created at the time of oral implant placement, while a tooth extraction socket healing model was established on the contralateral side of each animal. The surrounding tissues in the zone of the healing defects were harvested during regeneration for temporal evaluation using histology, immunohistochemistry, laser capture microdissection, and quantitative reverse transcription-polymerase chain reaction for the identification of a panel of 17 putative genes associated with wound repair. RESULTS: In both models, three distinct expression patterns were displayed: 1) genes that are slowly increased during the healing process, such as bone morphogenetic protein 4, runt-related transcription factor 2, and osteocalcin; 2) genes that are upregulated at the early stage of healing and then downregulated at later stages, such as interleukin and chemokine (C-X-C motif) ligands 2 and 5; and 3) genes that are constitutively expressed over time, such as scleraxis. Although some similarities between osseointegration and tooth extraction socket were seen, distinct features developed and triggered a characteristic coordinated expression and orchestration of transcription factors, growth factors, extracellular matrix molecules, and chemokines. CONCLUSIONS: Characterization of these events contributes to a better understanding of cooperative molecular dynamics in alveolar bone healing, and highlights potential pathways that could be further explored for the enhancement of osseous regenerative strategies.

Clinical and patient-centered outcomes after minimally invasive non-surgical or surgical approaches for the treatment of intrabony defects: a randomized clinical trial.

BACKGROUND: The present study aims to compare the performance of minimally invasive non-surgical and surgical approaches for the therapy of intrabony defects. METHODS: Twenty-nine patients who presented with intrabony defects were randomly assigned to: 1) a minimally invasive non-surgical technique (MINST) group, or 2) minimally invasive surgical technique (MIST) group. The chair time of each therapeutic procedure was calculated. The probing depth (PD), position of the gingival margin (PGM) and relative clinical attachment level (RCAL) were evaluated at 3 and 6 months after treatments. The patient perception of discomfort/pain experienced during and after therapy and patient satisfaction regarding treatments were also evaluated. RESULTS: Significant PD reductions, RCAL gains, and no changes in the PGM were obtained at 3 and 6 months in MINST and MIST groups (P <0.05). No differences were observed between groups at any time points (P >0.05). Patient-oriented outcomes did not demonstrate differences between therapeutic approaches (P >0.05). Significant higher chair times were required in the MIST group than in the MINST group (P <0.05). CONCLUSIONS: Minimally invasive non-surgical and surgical approaches were successfully used for the treatment of intrabony defects and achieved periodontal health in association with negligible morbidity and suitable patient satisfaction. However, non-surgical therapeutic modality presented an advantage in terms of a reduction of treatment chair time.

Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples.

Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish. To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.

Heparanase, heparan sulfate and perlecan distribution along with the vascular penetration during stellate reticulum retraction in the mouse enamel organ.

OBJECTIVE: Immunohistochemical and gene expression profiles of heparanase were determined in murine molar tooth germs from their embryonic to postnatal stages, paying special attention to neovascularization within the enamel organ, which is poorly vascularized before birth. DESIGN: Protein and gene expression profiles of heparanase, heparan sulfate (HS), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and perlecan were comparatively examined by immunohistochemistry and in-situ hybridization, respectively, in mouse mandibular molar tooth germs from embryonic day 11.5 to postnatal day 6. At the same time, their mRNA expression levels were also confirmed by reverse transcriptase-polymerase chain reaction using laser-captured microdissection of enamel organ tissues. RESULTS: Stellate reticulum cells highly expressed perlecan but only slightly expressed heparanase and HS in their embryonic days. On and after postnatal day 1, the expressions of heparanase became dramatically higher in the stellate reticulum, while HS disappeared leaving the immunopositivity for perlecan core protein. Immunohistochemically, HS was enhanced around blood vessels which were newly formed after birth within the enamel organs, whose volume was also regressive. Similar expression patterns were obtained for VEGF and TGF-beta1. CONCLUSIONS: Such synchronized expression modes among the HS metabolism-related molecules suggested that heparanase plays an important role in degradation of HS chains, which is closely related to vascular penetration into the stellate reticulum, which may be one of the driving forces for the postnatal regression of the enamel organ.

Laser microdissection microscopy and single cell PCR of avian hemosporidians.

Avian malaria parasites (Plasmodium spp.) and related species of Haemoproteus constitute a remarkably diverse and species rich group of blood parasites. Analyses of the mitochondrial cytochrome b gene of these hemosporidians have demonstrated unexpected patterns of host distribution, host shifts, and host sharing. However, deeper insights into these patterns require access to multiple genetic markers and genetic analyses of single parasite cells. In the present study, we demonstrate the potential of laser microdissection microscopy (Olympus/MMI CellCut microdissection system) for solving these 2 problems. This technique was used for isolation of single blood stages and ookinetes of avian Haemoproteus and Plasmodium spp., which were then successfully used for DNA isolation, amplification, and sequencing. The methods of single cell dissection of hemosporidian parasites and PCR-based analyses with dissected single cells are described. These methods can be used to isolate substantial quantities of pure hemosporidian parasite DNA for large-scale sequencing, essential information when designing primers for developing multiple nuclear genetic markers. Such markers can then be applied to isolated single parasite cells for identification of parasites in mixed infections and deciphering mechanisms behind apparent reproductive isolation between parasite lineages. This method can be used in the molecular investigation of blood parasites of birds, reptiles, and fish because it enables removing the parasite DNA from the overpowering host DNA, which is present in red blood cells.

Immuno-laser capture microdissection of frozen prolactioma sections to prepare proteomic samples.

Laser capture microdissection (LCM) technology combined with immunohistochemistry (immuno-LCM) is a valuable tool to obtain specific target cell populations and therefore this technique enables more accurate proteomic profile. In this study, we optimized the regular immuno-LCM technique to isolate and stain pure prolactin cells from either normal human pituitary (n=6) or prolactioma (n=11). Compared with the routine procedure, more intense and specific staining could be obtained when sections were pretreated with 0.2% Triton X-100 for 4 min. Interestingly, longer pretreatment (0.2% Triton X-100 for 10 min) or higher concentration (2% Triton X-100 for 4 and 10 min) greatly impaired labeling intensity and cell shape. Further scanning electron microscope study revealed that the component extracted from the cell surface by Triton X-100 was lipid. Using the optimized immuno-LCM technique, more pure prolactin cells could be isolated and prepared for further proteomic analysis. Taken together, we reported an optimized immuno-LCM technique that could effectively dissect pure target cells in different type pituitary adenomas for further proteomics analysis.

Lateral approach to the levator veli palatini: a preliminary report.

The abnormal anatomy in the cleft palate has been of interest to surgeons for a long time. Different authors have independently evolved the techniques of radical reconstruction of the palatal musculature and have suggested the medial approach to dissect the levator. We hereby report the technique in which the levator is identified through the lateral incision of the soft palate. This lateral approach helps in the complete release of the levator from all abnormal attachments and ensures reconstruction of an effective sling. This technique is of particular benefit in a palate re-repair.