Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and Osteoblast Cell Lines Retain Their Primary Characteristics.

Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines.

Novel, potent, selective and cellular active ABC type PTP1B inhibitors containing (methanesulfonyl-phenyl-amino)-acetic acid methyl ester phosphotyrosine mimetic.

Protein tyrosine phosphatase 1B (PTP1B) which plays an important role in the negative regulation of insulin and leptin pathway has emerged as a novel promising therapeutic target for the treatment of type 2 diabetes mellitus and obesity. Upon careful study, a series of novel scaffold and simple synthesis method inhibitors were discovered based on the analysis of X-ray crystal structures of PTP1B/inhibitor complexes and docking simulations. Among them, compound P7 exhibited high inhibitory activity (IC50=222 nM) with moderate selectivity (8-fold) over T-cell PTPase (TCPTP) through interacting with the A, B and C binding sites of PTP1B enzyme. Further studies on cellular activities revealed that compound P7 could enhance insulin-mediated IRß phosphorylation and insulin-stimulated glucose uptake.

Expression of Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs) in the reparative dentin of rat molars.

AIM: To analyze the expression and distribution of Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs) in reparative dentin (RepD). METHODOLOGY: Cavities on the mesial surfaces of rat molars were prepared to expose the pulp, and a calcium hydroxide agent was applied to cap the exposed pulp. The molars with pulp capping were extracted at postoperative 1, 2, and 4 weeks. The immunolocalization of four SIBLINGs, dentin matrix protein 1 (DMP1), dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteopontin (OPN) in RepD, was analyzed in comparison with reactionary dentin (ReaD) and primary dentin (PD). RESULTS: At two weeks after operation, the region of the exposed pulp formed a layer of reparative dentin bridge sealing the communication between the cavity and pulp chamber. Dentinal tubules in RepD were more irregular in shape and fewer in number than PD. At postoperative 2 and 4 weeks, RepD had lower levels of DMP1 and DSP than PD. BSP and OPN were present in RepD, but not in PD. RepD showed certain similarities to ReaD in the expression of SIBLINGs. CONCLUSIONS: The reduced levels of DMP1 and DSP may be associated with the decreased number of dentinal tubules in RepD. The expression of BSP and OPN in RepD indicates that the odontoblast-like cells were attempting to produce a hard tissue at a very rapid pace. These findings suggest that in response to the surgical injury, the newly differentiated odontoblast-like cells altered their synthesis of the dentinogenesis-related proteins and produced a hard tissue that is an intermediate between dentin and bone.

Ablation of FAM20C caused short root defects via suppressing the BMP signaling pathway in mice.

Short root defects are prone to cause various periodontal diseases and lead to tooth loss in some serious cases. Studies about the mechanisms governing the development of the root are needed for a better understanding of the pathogenesis of short root defects. The protein family with sequence similarity 20 group C (FAM20C) is a Golgi casein kinase that has been well studied in the development of tooth crown formation. However, whether FAM20C plays a role in the development of tooth root is still unknown. Thus, we generated Sox2-Cre;Fam20cfl/fl (cKO) mice, in which Fam20c was ablated in both the dental epithelium and dental mesenchyme, and found that the cKO mice showed severe short root defects mainly by inhibiting the development of dental mesenchyme in the root region. In this investigation, we found morphological changes and differentiation defects, with reduced expression of dentin sialophosphoprotein (DSPP) in odontoblasts of the root region in cKO mice. Furthermore, the proliferation rate of apical papillary cells was reduced in the root of cKO mice. In addition, the levels of bone morphogenetic protein 4 (BMP4) and phospho-Smad1/5/8, and that of Osterix and Krüppel-like factor 4 (KLF4), two downstream target molecules of the BMP signaling pathway, were significantly reduced in the root of cKO mice. These results indicate that FAM20C plays an essential role in the development of the root by regulating the BMP signaling pathway.

Transgenic expression of dentin phosphoprotein (DPP) partially rescued the dentin defects of DSPP-null mice.

Mutations in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta. After synthesis, DSPP is proteolytically processed into NH2- and COOH-terminal fragments. The NH2-terminal fragment of DSPP is highly glycosylated but not phosphorylated, whereas the COOH-terminal fragment (named "dentin phosphoprotein" or "DPP") is highly phosphorylated but not glycosylated. These two fragments are believed to perform distinct roles in dentin formation. To analyze the functions of DPP in dentinogenesis, we created "Dspp-/-;DPP Tg mice", which expressed transgenic DPP driven by a Type I collagen promoter but lacked the endogenous Dspp gene. We characterized the dentin of the Dspp-/-;DPP Tg mice using X-ray radiography, histology, scanning electron microscopy, double fluorochrome labeling, immunohistochemistry and in situ hybridization. Micro-computed tomography analyses revealed that at postnatal 6 months, the transgenic expression of DPP increased the dentin thickness of the Dspp-null mice by 97.1% and restored the dentin material density by 29.5%. Histological analyses showed that the Dspp-null mice manifested an abnormal widening of the predentin while the predentin in Dspp-/-;DPP Tg mice was narrower than in the Dspp-null mice. Scanning electron microscopy analyses showed that the dentinal tubules in the Dspp-/-;DPP Tg mice were better organized than in the Dspp-null mice. The double fluorochrome labeling analyses demonstrated that the dentin mineral deposition rate in the Dspp-/-;DPP Tg mice was significantly improved compared to that in the Dspp-null mice. These findings indicate that the transgenic expression of DPP partially rescued the dentin defects of the DSPP-null mice, suggesting that DPP may promote dentin formation and that the coordinated actions between DPP and the NH2-terminal fragment of DSPP may be necessary for dentinogenesis.

Arthroscopic-assisted inflatable bone tamp reduction for treatment of posterolateral tibial plateau fractures.

AIM: Our study aimed to assess the safety and efficacy of an innovative arthroscopic-assisted inflatable tamp reduction technique for the treatment of posterolateral tibial plateau fractures. PATIENTS AND METHODS: Twenty-six patients with posterolateral tibial plateau fractures were treated with arthroscopy through inflation reduction technique were enrolled. Arthroscopy was used to observe the reduction of articular surface to avoid over-reduction or de-reduction. An arthroscopic assessment of anatomic joint reduction completed the procedure. Inflatable bone tamp was used to reduce the fractures and calcium phosphate cement was used as bone substitute to augment the repairs. RESULTS: Under arthroscopy, the reduction was observed to be excellent without any residual deformity in all the cases. Cement overflow into the soft tissues or the knee joint was not observed. During the follow-up period, no obvious articular surface subsidence (>5 mm) was observed and no evidence of posttraumatic osteoarthritis could be detected. Radiographs under full weight bearing revealed neither loss of reduction nor any valgus deviation. Three months after surgery, the graft was almost completely replaced by new bone. The functional evaluation following the Rasmussen score yielded excellent results. CONCLUSIONS: This study provided a novel technique for the reduction of depressed and split-depressed pasterolateral tibial plateau fractures. Arthroscopic-assisted inflatable bone tamp reduction is an effective method for the treatment of posterolateral tibial plateau fractures.

Specific ablation of mouse Fam20C in cells expressing type I collagen leads to skeletal defects and hypophosphatemia.

FAM20C mutations in humans cause Raine syndrome and our previous studies showed that global inactivation of mouse Fam20C led to bone and dental defects. By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C flox/flox mice, we created 2.3 kb Col 1a1-Cre;Fam20C foxl/flox (cKO) mice, in which Fam20C was inactivated in cells expressing Type I collagen. This study showed that the long bones of cKO mice were shorter and had a lower level of mineralization compared to the normal mice. The collagen fibrils in Fam20C-deficient bone were disorganized and thicker while the growth plate cartilage in cKO mice was disorganized and wider compared to the normal mice. The Fam20C-deficient bone had a lower level of dentin matrix protein 1, and higher levels of osteopontin and bone sialoprotein than the normal. The blood of cKO mice had an elevated level of fibroblast growth factor 23 and reduced level of phosphorus. These findings indicate that inactivation of Fam20C in cells expressing type I collagen led to skeletal defects and hypophosphatemia. The altered levels of dentin matrix protein 1 and osteopontin in Fam20C-deficient bone may be significant contributors to the mineralized tissue defects in human patients and animals suffering from the functional loss of FAM20C.

Loss of epithelial FAM20A in mice causes amelogenesis imperfecta, tooth eruption delay and gingival overgrowth.

FAM20A has been studied to a very limited extent. Mutations in human FAM20A cause amelogenesis imperfecta, gingival fibromatosis and kidney problems. It would be desirable to systemically analyse the expression of FAM20A in dental tissues and to assess the pathological changes when this molecule is specifically nullified in individual tissues. Recently, we generated mice with a Fam20A-floxed allele containing the beta-galactosidase reporter gene. We analysed FAM20A expression in dental tissues using X-Gal staining, immunohistochemistry and in situ hybridization, which showed that the ameloblasts in the mouse mandibular first molar began to express FAM20A at 1 day after birth, and the reduced enamel epithelium in erupting molars expressed a significant level of FAM20A. By breeding K14-Cre mice with Fam20A(flox/flox) mice, we created K14-Cre;Fam20A(flox/flox) (conditional knock out, cKO) mice, in which Fam20A was inactivated in the epithelium. We analysed the dental tissues of cKO mice using X-ray radiography, histology and immunohistochemistry. The molar enamel matrix in cKO mice was much thinner than normal and was often separated from the dentinoenamel junction. The Fam20A-deficient ameloblasts were non-polarized and disorganized and were detached from the enamel matrix. The enamel abnormality in cKO mice was consistent with the diagnosis of amelogenesis imperfecta. The levels of enamelin and matrix metalloproteinase 20 were lower in the ameloblasts and enamel of cKO mice than the normal mice. The cKO mice had remarkable delays in the eruption of molars and hyperplasia of the gingival epithelium. The findings emphasize the essential roles of FAM20A in the development of dental and oral tissues.

Cyclosporine a inhibits apoptosis of rat gingival epithelium.

BACKGROUND: The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site-specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site-specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. METHODS: Forty Wistar rats were divided into CsA-treated and non-treated groups. Paraffin-embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl-2 and caspase-3, and the staining of terminal deoxynucleotidyl transfer-mediated dUTP nick-end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl-2- and caspase-3-positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. RESULTS: In CsA-treated rats, bcl-2 expression was significantly upregulated, whereas caspase-3 expression was downregulated, along with a reduced number of TUNEL-positive cells. The site-specific distribution of bcl-2 was consistent with the site-specific hyperplasia of the gingival epithelium in CsA-treated rats. CONCLUSIONS: CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl-2 might play a critical role in the pathogenesis of the site-specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.

Inactivation of Fam20C in cells expressing type I collagen causes periodontal disease in mice.

BACKGROUND: FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues. METHODS: By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20Cfl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20Cfl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches. RESULTS: The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice. CONCLUSION: Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.

Differential expression and localization of dentin matrix protein 1 (DMP1) fragments in mouse submandibular glands.

It has been demonstrated that dentin matrix protein 1 (DMP1) is an essential regulator in the formation of bone and tooth. In addition to the mineralized tissues, DMP1 is also expressed in the non-mineralized tissues such as kidney, brain and salivary glands. Some studies have shown that the expression of DMP1 is significantly elevated in cancerous glands, while details about the expression and localization patterns of DMP1 in these glandular tissues still remain largely unknown. In this study, with multiple approaches, we systematically analyzed the expression and localization of DMP1 in mouse submandibular glands (SMGs). The results showed that although DMP1 was expressed in both female and male mouse SMGs, the mRNA levels of DMP1 in male mice were higher than those in female mice after the appearance of granular convoluted tubule (GCT). In mouse SMGs, DMP1 was primarily present as the 46 kDa C-terminal fragment and the 37 kDa N-terminal fragment. The C-terminal fragment was mainly localized in the nuclei of acinar and ductal cells, while the N-terminal fragment was restricted to the cytoplasm of ductal cells. This study showed the expression of DMP1 in the GCT of male mice, a novel finding different from the result of previous reports. Collectively, the differential localization patterns of DMP1 fragments indicate that different forms of DMP1 may play distinct roles in the SMGs.

[The effects of nicotine on bone calcium and phosphorus content and alkaline phosphatase activity in different part of rat].

OBJECTIVE: To analyze the effects of nicotine on the bone calcium and phosphorus content and alkaline phosphatase (ALP) activity in rat alveolar bone and mandible. METHODS: Twenty health male Wistar rats of five weeks of age were randomly assigned to two groups and received daily intraperitoneal injections for three months as follows: Saline solution for control group, nicotine 0.73 mg.kg-l.d-1 for experimental group. The bone calcium phosphorus content were detected by concentrated acid digestion method and the ALP activity was examined by improved Reddi method. RESULTS: Compared to the control group, bone calcium and phosphorus content was lower in the experimental group (P<0.05), ALP activity had no statistical significance(P>0.05). Bone calcium phosphorus and ALP activity in different parts had no statistical significance (P>0.05). CONCLUSION: The nicotine reduces calcium phosphorus deposition of jaw bone, but has no obvious influence to ALP activity.

[Apoptosis and expression of Bcl-2 and caspase-3 in ciclosporin-induced gingival overgrowth of rats].

OBJECTIVE: To observe the effect of Ciclosporin (CsP) on apoptosis and expression of the associated protein Bcl-2, Caspase-3 in gingival epithelium of rats in order to approach the mechanism of CsP-induced gingival epithelium overgrowth. METHODS: Eighty SPF grade male 7-week-old Wistar rats were randomly divided into experimental group and control group, and each group was divided into 4 subgroups according to the duration of treatment (10, 20, 30 and 40 days). The experimental objects were given fresh milk including CsP intragastrically and the control ones were given only fresh milk. After perfusion of 4% paraform for internal fixation, the specimens' bucco-lingual paraffin sections at lower first molar were made. Apoptosis was detected using TdT-mediated dUT nick end labeling (TUNEL) and the expression of Bcl-2 and Caspase-3 using immunohistochemisty of PV. The apoptotic index, positive cell rate of Caspase-3 and average gray scale of Bcl-2 was measured with an image analysis system. Data were analyzed by two-way analysis of variance of factorial design. RESULTS: The apoptosis index and positive cell rate of Caspase-3 were down-regulated in the experimental group, and were significant difference from the control group (P < 0.05). The average gray scale of Bcl-2 was up-regulated in the experimental group, and was significant difference from the control group (P < 0.05). CONCLUSION: CsP-induced gingival epithelial overgrowth is likely to associated with interference to the path of mitochondrial apoptosis and inhibition apoptosis.

[Quantitative analysis the effect of Ciclosporin on gingival tissue modality of rats].

OBJECTIVE: Quantitative analysis the regional specificity and time dependence of Ciclosporin (CsP) on gingival tissue modality of rats. METHODS: Eighty SPF grade male Wistar rats of 7 weeks old were randomly divided into experimental group and control group, and each group was divided into 4 subgroups according to the duration of treatment (10, 20, 30 and 40 days). The experimental objects were given intragastric administration of fresh milk including CsP, and the control ones were given intragastric administration of fresh milk the same as the experimental objects. After giving perfusion 4% paraform trans-heart to internal fixation, the specimens were get and made bucco-lingual paraffin sections at lower first molar and made HE staining. The area of buccal and lingual gingival epithelial and connective tissue, the length of the longest rete pegs were measured with the image analysis system. Data were analyzed by two-way analysis of variance of factorial design. RESULTS: The rats of the experimental group took place gingival overgrowth, and the rete pegs of gingival epithelium of attached gingiva approach muco-gingival junction were prolonged. The area of buccal and lingual gingival epithelium and connective tissue, the length of buccal longest rete pegs of the experimental group were significantly higher than that of the control group rats (bucca P < 0.01, lingua 0.01 < P < 0.05). The length of lingual longest rete pegs of the experimental group were no difference than that of the control group rats. The area of buccal and lingual gingival epithelial, connective tissue, the length of longest rete pegs among experimental groups were no difference. CONCLUSION: CsP may have the regional specificity on the rats' gingival epithelium of buccal attached gingiva approach muco-gingival junction, but the effect of CsP on the rats' gingiva was no time dependence.