Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

The role of O-GlcNAcylation mediated by OGT during tooth development.

To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of ß-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.

Developmental function of Piezo1 in mouse submandibular gland morphogenesis.

Mechanically activated factors are important in organogenesis, especially in the formation of secretory organs, such as salivary glands. Piezo-type mechanosensitive ion channel component 1 (Piezo1), although previously studied as a physical modulator of the mechanotransduction, was firstly evaluated on its developmental function in this study. The detailed localization and expression pattern of Piezo1 during mouse submandibular gland (SMG) development were analyzed using immunohistochemistry and RT-qPCR, respectively. The specific expression pattern of Piezo1 was examined in acinar-forming epithelial cells at embryonic day 14 (E14) and E16, which are important developmental stages for acinar cell differentiation. To understand the precise function of Piezo1 in SMG development, siRNA against Piezo1 (siPiezo1) was employed as a loss-of-function approach, during in vitro organ cultivation of SMG at E14 for the designated period. Alterations in the histomorphology and expression patterns of related signaling molecules, including Bmp2, Fgf4, Fgf10, Gli1, Gli3, Ptch1, Shh, and Tgfß-3, were examined in acinar-forming cells after 1 and 2 days of cultivation. Particularly, altered localization patterns of differentiation-related signaling molecules including Aquaporin5, E-cadherin, Vimentin, and cytokeratins would suggest that Piezo1 modulates the early differentiation of acinar cells in SMGs by modulating the Shh signaling pathway.

Resveratrol enhances bone formation by modulating inflammation in the mouse periodontitis model.

OBJECTIVE: To evaluate the effect of resveratrol on periodontal bone regeneration after local delivery and to determine its effect on inflammatory mediators. BACKGROUND: Resveratrol is considered an anti-inflammatory polyphenolic stilbene involved in the modulation of inflammation. MATERIALS AND METHODS: Periodontitis was induced in mouse molars using a 5-day ligature model followed by the left second molar extraction and 50 µM resveratrol treatment for 1 and 2 weeks. We then examined specimens treated for 1 week histologically and with immunostaining. Microfocus-computed tomography (micro-CT) was used to examine the bone volume formation. RESULTS: After 1 week of treatment, proinflammatory cytokine levels (TNF-alpha and IL6), cells exhibiting neutrophil and macrophage marker (MPO), cell proliferation marker (Ki67), and preosteoblastic marker (RUNX2) reactivity decreased in the resveratrol-treated specimens compared to the control group. In contrast, we observed a higher number of CD31-, F4/80-, and osteocalcin- (OCN-) positive cells in the resveratrol-treated specimens. After 2 weeks, micro-CT confirmed an increased bone mass in the region of the extraction socket in the resveratrol-treated group. CONCLUSION: After 1 week, the resveratrol-treated specimens revealed evidence of inflammation modulation compared to the control group. These data suggest that resveratrol not only affects inflammation control but also is useful for treating periodontitis-related tissue defects and bone regeneration.

Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket.

BACKGROUND AND OBJECTIVE: After tooth extraction, the extraction socket undergoes several steps of soft and hard tissue healing. The healing process of the extraction socket is modulated by a range of signaling factors and biochemical agents. It has been reported that resveratrol, a polyphenolic compound, exhibits various biological effects, including anti-inflammatory, anti-carcinogenic, antioxidant, and anti-aging effects, and protects cardiovascular and bone tissues. In this study, we examined the cellular effects of resveratrol on human periodontal ligament (hPDL) cells and osteoblast-like (MC3T3-E1) cells and evaluated the bone-healing capacity of tooth extraction sockets in mice. MATERIAL AND METHODS: Resveratrol was applied to hPDL and MC3T3-E1 cells to detect cell proliferation and alkaline phosphatase (ALP) activity, and qPCR was employed to understand the gene expression level in vitro. For in vivo experiment, six-week-old C57BL/6 male mice were randomly divided into control (n = 15) and experimental (n = 15) groups and maxillary first molars were extracted by surgery. Experimental groups received 50-µM resveratrol on extraction sockets and analyzed the degree of new bone formation. RESULTS: Treatment of hPDL and MC3T3-E1 cells with resveratrol increased the cell proliferation and ALP activity and enhanced the expression of ALP, BMP-2, BMP-4, and OC genes. Resveratrol enhanced new bone formation in the lingual extraction socket in mice. CONCLUSION: These results suggest that resveratrol increases the cellular physiology of PDL and osteoblast including their proliferation and differentiation and may play an important role in bone-healing capacity after tooth extraction.

Signaling Modulations of miR-206-3p in Tooth Morphogenesis.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer; however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development-related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule-calcified teeth. Especially, mislocalization of ß-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson's trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1ß, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

An endoplasmic reticulum stress regulator, Tmbim6, modulates secretory stage of mice molar.

To understand the role of endoplasmic reticulum (ER)-stress in mice molar development, we studied Tmbim6 that antagonizes the unfolded protein response, using Tmbim6 knockout (KO) mice and in vitro organ cultivation with knocking down using small interfering RNA. During molar development, Tmbim6 is expressed in developing tooth at E14-E16, postnatal0 (PN0), and PN6. Mineral content in Tmbim6 KO enamel was reduced while dentin was slightly increased revealing ultrastructural changes in pattern formation of both enamel and dentin. Moreover, odontoblast differentiation was altered with increased Dspp expression at PN0 followed by altered AMELX localizations at PN5. These results were confirmed by in vitro organ cultivation and showed altered Bmp signaling, proliferation, and actin rearrangement in the presumptive ameloblast and odontoblasts that followed the altered expression of differentiation and ER stress-related signaling molecules at E16.5. Overall, ER stress modulated by Tmbim6 would play important roles in patterned dental hard tissue formation in mice molar within a limited period of development.

Effects of oleanolic acid acetate on bone formation in an experimental periodontitis model in mice.

OBJECTIVE: We evaluated the role of oleanolic acid acetate (OAA), a triterpenoid commonly used in the treatment of liver disorders, inflammatory diseases, and metastasis, in bone formation after tooth loss by periodontitis. BACKGROUND: Periodontitis causes the sequential degradation of the alveolar bone and associated structures, resulting in tooth loss. Several studies have attempted to regenerate the bone for implantation following tooth loss. METHODS: Maxillary left second molar was extracted from 8-week-old male mice following induction of periodontitis by ligature for 5 days. The extraction socket was treated with 50 ng/µL OAA for 1, 2, and 3 weeks. Detailed morphological changes were examined using Masson's trichrome staining, and the precise localization patterns of various signaling molecules, including CD31, F4/80, interleukin (IL)-6, and osteocalcin, were observed. The volume of bone formation was examined by Micro-CT. Osteoclasts were enumerated using tartrate-resistant acid phosphatase (TRAP) staining. For molecular dissection of signaling molecules, we employed the hanging-drop in vitro cultivation method at E14 for 1 day and examined the expression pattern of transforming growth factor (TGF)-ß superfamily and Wnt signaling genes. RESULTS: Histomorphometrical examinations showed facilitated bone formation in the extraction socket following OAA treatment. In addition, OAA-treated specimens showed the altered localization patterns of inflammatory and bone formation-related signaling molecules including CD31, F4/80, IL-6, and osteocalcin. Also, embryonic tooth germ mesenchymal tissue cultivation with OAA treatment showed the significant altered expression patterns of signaling molecules such as transforming growth factor (TGF)-ß superfamily and Wnt signaling. CONCLUSIONS: Oleanolic acid acetate induces bone formation and remodeling through proper modulation of osteoblast, osteoclast, and inflammation with regulations of TGF-ß and Wnt signaling.

Regulation of mesenchymal signaling in palatal mucosa differentiation.

Epithelial differentiation is thought to be determined by mesenchymal components during embryogenesis. In mice, palatal mucosa showed the region-specific keratinization pattern along antero-posterior axis. However, developmental mechanisms involved in oral mucosa differentiation with fine tuning of keratinization are not elucidated yet. To reveal this developmental mechanism, first, we conducted tissue recombination assay of the palate at E16 for 2 days which revealed that epithelial differentiation with specific localization of CK10 is modulated by mesenchymal components. Based on the results, we propose that mesenchymal signaling would determine the presumptive fate of developing palatal epithelium in spatiotemporal manner. Genome-wide screening analysis using laser micro-dissection to collect spatiotemporal specific molecules between anterior and posterior palate suggested Meox2 in the posterior mesenchymal tissue to be a candidate regulator controlling epithelial differentiation. To examine the detailed spatiotemporal function of Meox2, we employed in vitro organ cultivation with the loss- and gain-of-function studies at E14.5 for 2 and 4 days, respectively. Our results suggest that posteriorly expressed Meox2 modulates non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.

Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development.

Grainyhead-like 3 (Grhl3) is a transcription factor involved in epithelial morphogenesis. In the present study, we evaluated the developmental role of Grhl3 in structural formation of the circumvallate papilla (CVP), which undergoes dynamic morphological changes during organogenesis. The specific expression pattern of Grhl3 was examined in the CVP-forming region, specifically in the apex and epithelial stalk from E13.5 to E15.5 using in situ hybridization. To determine the role of Grhl3 in epithelial morphogenesis of the CVP, we employed an in vitro tongue culture method, wherein E13.5 tongue were isolated and cultured for 2 days after knocking down of Grhl3. Knockdown of Grhl3 resulted in significant changes to the epithelial structure of the CVP, such that the apical region of the CVP was smaller in size, and the epithelial stalks were more deeply invaginated. To define the mechanisms underlying these morphological alterations, we examined cell migration, proliferation, and apoptosis using phalloidin staining, immunohistochemistry against Ki67, ROCK1, and E-cadherin, and a TUNEL assay, respectively. These results revealed an increase in proliferation, a reduction in apoptosis, and an altered pattern of cytoskeletal formation in the CVP-forming epithelium, following Grhl3 knockdown. In addition, there were changes in the specific expression patterns of signaling and apoptosis-related molecules such as Axin2, Bak1, Bcl2, Casp3, Casp8, Ctnnb1, Cnnd1, Gli3, Lef1, Ptch1, Rock1, Shh, and Wnt11, which could explain the altered cellular and morphological events. Based on these results, we propose that developmental stage-specific Grhl3 plays a significant role in CVP morphogenesis not by just disruption of epithelial integrity but by regulating epithelial cell proliferation, apoptosis, and migration via Shh, Wnt, and apoptosis signaling during mouse embryogenesis.

Effects of vascular formation during alveolar bone process morphogenesis in mice.

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-ß. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

The role of APCDD1 in epithelial rearrangement in tooth morphogenesis.

Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE.

Mesenchymal signaling in dorsoventral differentiation of palatal epithelium.

After palatal fusion, the dorsal and ventral epithelia of the palatal shelf differentiate into the nasal and oral mucosa, respectively. The tissue-specific differentiation of palatal epithelia along the dorsal-ventral axis is regulated by the signaling molecules expressed in the underlying mesenchyme. Thus, as in many other epithelial organs, differentiation relies on epithelial-mesenchymal interactions. To screen for region-specific mesenchymal signaling molecules that determine the fate of the palatal epithelia, we employed a laser microdissection (LMD) method. LMD allowed us to collect region-specific mesenchymal tissues at E13, prior to palatal fusion and the development of distinct dorsal and ventral epithelial morphology. Genome-wide screening was performed on the tissues collected using LMD to identify candidate mesenchymal signaling molecules. The microarray results were validated using real-time quantitative (qPCR) and in situ hybridization methods. The developmental role and interactions of the candidate genes were evaluated in in vitro-cultivated E13 palates using an anti-sense oligodeoxynucleotide (AS-ODN)-based loss-of-function approach. Apparent changes in the expression patterns of Runt-related transcription factor 2 (Runx2) and LIM homeobox 8 (Lhx8) were observed after knocking down each gene. Knock-down of Runx2 and Lhx8 also altered the immunolocalization pattern of cytokeratin18 (CK18), an established marker for nasal epithelium. These results were confirmed using Runx2 heterozygote mice. The mesenchymal signaling molecules Runx2 and Lhx8, which possess region-specific expression patterns along the dorsoventral axis, functionally interact to regulate the cellular and molecular characteristics of dorsal and ventral epithelia, suggesting that mesenchymal signaling molecules determine the dorsoventral fate of epithelial structures in the developing palate.

Morphological and cellular examinations of experimentally induced malocclusion in mice mandibular condyle.

Occlusal alignment is known clinically to have a widespread influence on the stomatognathic system, including the temporomandibular joint and masticatory muscles. However, while occlusion is still an important determinant of most dental treatments, the exact effect of occlusal alignment is unclear because of a lack of conclusive scientific evidence. In this study, a malocclusion model system is used to examine the cellular and histologic alterations in the contralateral condyle of mice after a malocclusion was induced by a build-up of resin on the left maxillary molars. A significant decrease in the thickness of the condylar cartilage was found in the 1-week experimental group, together with increased apoptosis and decreased proliferation in the condylar head, which included cartilage and subchondral bone. Additionally, the number of TRAP-positive osteoclasts and MPO- and F4/80-positive inflammatory cells in the subchondral bone were significantly higher in the 1-week experimental group. Unbalanced malocclusion caused increased bone remodeling, as evidenced by increased osteoclastic activity and inflammatory responses (macrophages and neutrophils, respectively). However, these alterations in the 1-week experimental group were subsequently attenuated and restored almost to the baseline at 3 weeks after the induction of the malocclusion.

Experimental lens capsular bag model for posterior capsule opacification.

An in vitro culture model enabling posterior capsule opacification (PCO) to be investigated was developed and established by using low-melting-point (LMP)-agarose gel to support the capsular bag. After removal of the cornea from rodent and porcine eyeballs, the lens zonules were dissected. Whole lens explants were embedded into 2 % (37 °C) LMP-agarose gel solution. As performed routinely in cataract surgery, capsulotomy and lens fiber removal were carried out in the solidified LMP-agarose gel as sham cataract surgery. The LMP-agarose-gel-supported capsular bag/lens epithelial cell (CB-LEC) complexes were maintained in Dulbecco's modified Eagle medium supplemented with 10 % fetal bovine serum in an anterior face-down position. The proliferation and migration of LECs into the posterior capsule were observed every 12 h by phase-contrast microscopy. Epithelial cells were observed at the central portion of the CB-LEC complexes after 56.57 ± 16.56 h (n = 7) and 106 ± 14.03 h (n = 6) of culture, for rodent and porcine lenses, respectively. The solidified gel allowed clear microscopic observations and whole-mount immunostaining evaluations of the whole area of the capsular bag. Histological examinations revealed the proliferation, migration, and transdifferentiation of LECs related to posterior capsule opacification. This new in vitro culture model provides experimental benefits by maintaining the natural contour of the capsule without implants inside or outside of the capsule. In addition, this model system allows pharmacological and histological evaluations of the cultured CB-LEC complexes without additional manipulations.

Developmental regulations of Perp in mice molar morphogenesis.

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.

Unraveling the structure, chemical composition, and conserved signaling in leech teeth.

Unlike vertebrates, the number of toothed taxa in invertebrates is very few, with leeches being the only tooth-bearing organisms in the phylum Annelida. Copious studies have been conducted regarding vertebrate teeth; however, studies regarding the structure and function of invertebrate teeth are limited. In this study, the tooth structure of leeches, specifically Hirudo nipponia and Haemadipsa rjukjuana, was revealed, which showed sharp and pointed teeth along the apex of three jaws. Understanding conserved signaling regulations among analogous organs is crucial for uncovering the underlying mechanisms during organogenesis. Therefore, to shed light on the evolutionary perspective of odontogenesis to some extent, we conducted de novo transcriptome analyses using embryonic mouse tooth germs, Hirudo teeth, and Helobdella proboscises to identify conserved signaling molecules involved in tooth development. The selection criteria were particularly based on the presence of tooth-related genes in mice, Hirudo teeth, and Helobdella proboscis, wherein 4113 genes were commonly expressed in all three specimens. Furthermore, the chemical nature of leech teeth was also examined via TEM-EDS to compare the chemical composition with vertebrate teeth. The examination of tissue-specific genetic information and chemical nature between leeches and mice revealed chemical similarities between leech and mice teeth, as well as conserved signaling molecules involved in tooth formation, including Ptpro, Prickle2, and Wnt16. Based on our findings, we propose that leech teeth express signaling molecules conserved in mice and these conserved tooth-specific signaling for dental hard tissue formation in mice would corresponds to the structural formation of the toothed jaw in leeches.