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‐β1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG‐35 elicits osteoblast differentiation through TGF‐β1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF‐β1/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.
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 β‐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.
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.
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.
Tmbim6 is involved in regulating endoplasmic reticulum (ER)‐stress. It plays an important role in the formation of patterned dental hard tissue in mice molar via modulation of ER stress in developing tooth.
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.
Periodontitis is an excessive inflammatory event in tooth-supporting tissues and can cause tooth loss. We used erythropoietin (EPO), which has been reported to play an important role in bone healing ...and modulation of angiogenesis, as a therapeutic agent
in vivo
and
in vitro
experimental models to analyze its effect on periodontitis. First
,
EPO was applied to
in vitro
MC3T3-E1 cells and human periodontal ligament fibroblast (hPDLF) cells to examine its function in altered cellular events and gene expression patterns.
In vitro
cultivation of MC3T3-E1 and hPDLF cells with 10 IU/ml EPO at 24 and 48 h showed an obvious increase in cell proliferation. Interestingly, EPO treatment altered the expression of osteogenesis-related molecules, including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OC) in MC3T3-E1 cells but not in hPDLF cells. In particular, MC3T3-E1 cells showed increased expression of ALP, BMP-2, and OC on day 5, while hPDLF cells showed increased expression of BMP-2, and OC on day 14. Based on the
in vitro
examination, we evaluated the effect of EPO on bone formation using an experimentally-induced animal periodontitis model. After the induction of periodontitis in the maxillary left second M, 10 IU/ml of EPO was locally applied to the extraction tooth sockets. Histomorphological examination using Masson’s trichrome (MTC) staining showed facilitated bone formation in the EPO-treated groups after 14 days. Similarly, stronger positive reactions against vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) were detected in the EPO-treated group compared to the control. Meanwhile, myeloperoxidase, an inflammatory marker, was decreased in the EPO-treated group on days 1 and 5. Overall, EPO facilitates bone healing and regeneration through altered signaling regulation and modulation of inflammation in the osteoblast cell lineage and to a lesser extent in hPDLF cells.
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)‐β 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)‐β 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‐β and Wnt signaling.
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
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
during tooth morphogenesis using ex-vivo culture method. The expression pattern of
was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of
clearly altered expression patterns of dental-development-related signaling molecules, including
and
. 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 β-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of
and
. Overall, our data suggest that the
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.
During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs ...inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4-Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice.
We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The
cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis.
Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration.
In the present study, we examined the bone healing capacity of
, 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
in human periodontal ligament fibroblasts to examine the osteogenic potential of
. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of
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-1β, 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
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.
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