RESUMO
Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1CreERT2::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L-amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C ß2 (PLCß2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.
Assuntos
Envelhecimento/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Fosfolipase C beta/metabolismo , Papilas Gustativas/citologia , Papilas Gustativas/metabolismo , Animais , Biomarcadores/metabolismo , CamundongosRESUMO
Orthodontic patients complain of pain for the first few days after insertion of appliances. Mechanical force has been reported to produce oxidants in periodontal ligament (PDL) cells. It has not been studied whether orthodontic force-induced oxidative stress elicits nociception. Herein, we focused on the role of the oxidant-sensitive channel TRPA1 on nociception in orthodontic pain. In a rat model of loaded orthodontic force between the maxillary first molar and incisor, the behavioral signs of orofacial nociception, facial rubbing and wiping, increased to a peak on day 1 and gradually diminished to the control level on day 5. Administration of free radical scavengers (Tempol and PBN) and TRPA1 antagonist (HC-030031) inhibited nociceptive behaviors on day 1. In the PDL, the oxidative stress marker 8-OHdG was highly detected on day 1 and recovered on day 5 to the sham-operated level. The dental pulp showed similar results as the PDL. TRPA1 mRNA was abundantly expressed in the trigeminal ganglion relative to PDL tissue, and there were TRPA1-immunopositive neuronal fibers in the PDL and pulp. In dissociated trigeminal ganglion neurons, H2O2 at 5 mM induced a Ca2+ response that was inhibited by HC-030031. Although H2O2 at 100 µM did not yield any response, it enhanced the mechanically activated TRPA1-dependent Ca2+ response. These results suggest that oxidative stress in the PDL and dental pulp following orthodontic force activates and/or mechanically sensitizes TRPA1 on nociceptive fibers, resulting in orthodontic nociception. Later, the disappearance of nociception seems to be related to a decrease in oxidative stress, probably due to tissue remodeling.
Assuntos
Polpa Dentária , Nociceptividade , Animais , Humanos , Peróxido de Hidrogênio , Estresse Oxidativo , Ligamento Periodontal , Ratos , Canal de Cátion TRPA1/genéticaRESUMO
BACKGROUND/PURPOSE: Bone resorption and inhibition of bone formation occur on the compressed side during orthodontic tooth movement. Bone formation inhibitory factors such as sclerostin (encoded by SOST) are secreted on the compressed side by periodontal ligament (PDL) cells. PDL cells control bone metabolism, and compressed PDL cells inhibit bone formation during orthodontic tooth movement. The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. MATERIALS AND METHODS: Changes in SOST expression and subsequent protein release from human PDL (hPDL) cells were assessed using the real-time polymerase chain reaction (PCR), semiquantitative PCR, and immunofluorescence in hPDL cells subjected to centrifugal force (40g and 90g). To confirm the effects on bone formation, human alveolar bone-derived osteoblasts (hOBs) were grown with the addition of sclerostin peptide. In vivo, a compressive force was applied using the Waldo method in rats, and the distribution of sclerostin in PDL tissues was examined by immunohistochemistry. RESULTS: SOST expression was downregulated in vitro by centrifugation at 90g for 24 hours but upregulated by centrifugation at 40g based on real-time PCR, as was confirmed by immunofluorescence staining. The addition of sclerostin peptide significantly decreased the mineralized area in hOBs. However, slightly weakly sclerostin-positive PDL cells were observed on the compressed side in vivo. CONCLUSION: These results indicate that PDL cells subjected to light compressive force exhibit increased expression of SOST/sclerostin, which inhibits bone formation on the compressed side during orthodontic tooth movement.
RESUMO
OBJECTIVE: During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. DESIGN: In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 × g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry. RESULTS: In vitro, hPDL cells subjected to 90 × g for 24h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed. CONCLUSIONS: PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Osteogênese/fisiologia , Ligamento Periodontal/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas da Matriz Extracelular/biossíntese , Expressão Gênica , Marcadores Genéticos , Humanos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Pressão , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Técnicas de Movimentação DentáriaRESUMO
Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5 days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10â»7 M) treatment led to an increased number of osteoclasts, HK-1 (10â»7 M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1 compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs.
Assuntos
Maxila/metabolismo , Osteoclastos/metabolismo , Substância P/farmacologia , Taquicininas/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Masculino , Dente Molar/efeitos dos fármacos , Dente Molar/metabolismo , Osteoclastos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1/metabolismo , Taquicininas/farmacologiaRESUMO
Neurokinin B (NKB) is a neuropeptide in the tachykinin family that acts as a neurotransmitter and neuromodulator, primarily in the central nervous system. The distribution and role of NKB and its receptor, the neurokinin-3 receptor (NK-3R), in peripheral tissues are poorly understood. In this study, we investigated the distribution of NKB and NK-3R in peripheral tissues as well as the role of NKB in bone metabolism, especially in osteoclast formation and bone resorption activity through NK-3R. The distributions of NKB in intact rat neurons of the trigeminal ganglion (TG) and in axons of periodontal tissue were investigated by immunohistochemistry. Osteoclasts from cultured rat bone marrow cells were used to examine the distribution of NK-3R by immunocytochemistry and RT-PCR and to investigate the effects of NKB on the resorption activity of osteoclasts on ivory slices. We found that NKB immunopositive neurons were localized in the rat TG and that NKB immunopositive axons were distributed in periodontal tissues. Immunoreactivity for NK-3R was found in cultured osteoclasts, and NK-3R mRNA expression in the osteoclasts was confirmed by RT-PCR. The addition of NKB significantly increased the number of osteoclasts and the resorption area compared with the control. These findings suggest that NKB was localized in peripheral neurons and may involve the activation of osteoclast formation and bone resorption through NK-3R.