Han-Wool Choung

NFI-C regulates osteoblast differentiation via control of osterix expression.

In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age‐related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I‐C (NFI‐C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI‐C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI‐C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic‐overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator‐activated receptor gamma expression in Nfic−/− mice showing an age‐related osteoporosis‐like phenotype. Finally, NFI‐C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein‐2‐Runx2 pathway. These results suggest that NFI‐C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI‐C expression in BMSCs could be a novel therapeutic approach for treating age‐related osteoporosis. Stem Cells 2014;32:2467–2479

Nucleotide biosynthesis arrest by silencing SHMT1 function via vitamin B6-coupled vector and effects on tumor growth inhibition.

Serine hydroxymethyltransferase isoforms (SHMT1 & SHMT2α), which serve as scaffold protein for the formation of a multi-enzyme complex and generate one-carbon unit for the de novo thymidylate biosynthesis pathway during DNA synthesis, are vitamin B6 (VB6)-dependent enzyme. Cancer cells with high proliferation intensity need increased SHMT activation which enforces the facilitated-diffusion of VB6 for the continuous functioning of thymidylate synthase cycle. Therefore, SHMT knockdown presents an alternative approach to prevent DNA synthesis in cancer cells; however, its potential to inhibit cancer growth remains unknown so far. Here we demonstrated that VB6 coupled to poly(ester amine) (VBPEA) enforces a high level of VTC (VB6-transporting membrane carriers)-mediated endocytosis of the complexed SHMT1 siRNA (siSHMT1) to interrupt the thymidylate biosynthesis pathway of cancer cells. The detrimental effect of SHMT1 knockdown on the disintegration of multi-enzyme complex resulted in cell cycle arrest and a decrease in cells genomic DNA content, leading to enhanced apoptotic events in cancer cells. A reduction in tumor size was observed with constant SHMT1 suppression in xenograft mice. This study illustrates how silencing the SHMT1 expression inhibits cancer growth and the increased VB6 channeling for sustenance of cancer cells promotes VB6-coupled vector to elicit enhanced delivery of siSHMT1.

CPNE7, a preameloblast-derived factor, regulates odontoblastic differentiation of mesenchymal stem cells

Tooth development involves sequential interactions between dental epithelial and mesenchymal cells. Our previous studies demonstrated that preameloblast-conditioned medium (PA-CM) induces the odontogenic differentiation of human dental pulp cells (hDPCs), and the novel protein Cpne7 in PA-CM was suggested as a candidate signaling molecule. In the present study, we investigated biological function and mechanisms of Cpne7 in regulation of odontoblast differentiation. Cpne7 was expressed in preameloblasts and secreted extracellularly during ameloblast differentiation. After secretion, Cpne7 protein was translocated to differentiating odontoblasts. In odontoblasts, Cpne7 promoted odontoblastic markers and the expression of Dspp in vitro. Cpne7 also induced odontoblast differentiation and promoted dentin/pulp-like tissue formation in hDPCs in vivo. Moreover, Cpne7 induced differentiation into odontoblasts of non-dental mesenchymal stem cells in vitro, and promoted formation of dentin-like tissues including the structure of dentinal tubules in vivo. Mechanistically, Cpne7 interacted with Nucleolin and modulated odontoblast differentiation via the control of Dspp expression. These results suggest Cpne7 is a diffusible signaling molecule that is secreted by preameloblasts, and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts.

Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling

Background: Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Results: JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. Conclusion: ODAM mediates JE attachment to healthy teeth. Significance: We investigate ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontal disease. Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β3- and β6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

Preameloblast-Derived Factors Mediate Osteoblast Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Runx2-Osterix-BSP Signaling.

Epithelial-mesenchymal interaction occurs during development of various tissues, including teeth and bone. Recently, a preameloblast-conditioned medium (PA-CM) from mouse apical bud cells (ABCs), a type of dental epithelial cell, was found to induce odontogenic differentiation of dental pulp stem cells and promote dentin formation. The aims of the present study were to investigate the effects of PA-CM on human bone marrow mesenchymal stem cells (hBMSCs) in vitro, and to investigate the bone regenerative capacity in vivo through epithelial-mesenchymal interactions of developmental osteogenesis. Coculturing with ABCs and PA-CM treatment upregulated osteoblast differentiation markers of hBMSCs compared to cells cultured alone. PA-CM accelerated mineralized nodule formation and also increased bone sialoprotein promoter activity in hBMSCs. PA-CM facilitated the migration of hBMSCs, but did not significantly influence proliferation. PA-CM promoted bone formation of hBMSCs in vivo. Radiographic and histologic findings showed that PA-CM induced the bony regeneration at calvarial defects in rat. Taken together, these data show that PA-CM enhances the migration and osteogenic differentiation of hBMSCs in vitro and induces bone formation in vivo.

Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

Tertiary Dentin Formation after Indirect Pulp Capping Using Protein CPNE7.

If there is a partial loss of dentin, the exposed dentinal surface should be protected by an indirect pulp capping (IPC) procedure to preserve pulp vitality and prevent symptoms of dentin hypersensitivity. In our previous study, copine7 (CPNE7) induced odontoblast differentiation in vitro and promoted dentin formation in vivo. The aim of this study was to investigate the possibility of IPC therapy using the CPNE7 protein at the exposed dentinal surface and the resulting effects on tertiary dentin formation in a beagle model. CPNE7 promoted mineralization of odontoblasts and had high calcium ion-binding capacity. The in vivo IPC model with canine teeth showed that regeneration of physiologic reactionary dentin with dentinal tubule structures was clearly observed beneath the remaining dentin in the CPNE7 group, whereas irregular features of reparative dentin were generated in the mineral trioxide aggregate (MTA) group. The CPNE7+MTA group also showed typical reactionary dentin without reparative dentin, showing synergistic effects of CPNE7 with MTA. A scanning electron microscopy analysis showed that dentinal tubules beneath the original dentin were occluded by the deposition of peritubular dentin in the CPNE7 and CPNE7+MTA groups, whereas those in the control group were opened. Therefore, CPNE7 may be able to serve as a novel IPC material and improve symptoms of dentin hypersensitivity.

Scaffolds for Human Dental Stem Cells to Regenerate Cementum

The basic strategy of tooth bioengineering involves the utilization of artificial extracellular matrix as scaffolds, in combination with specific cells under the stimulation of growth factors. Dental stem cells can successfully regenerated dental hard tissue according to the properties of scaffolds. The authors made synthetic hydroxyapatite (HA) from human tooth and named it as “toothapatite (TA)” because it was almost composed of HA and whitelockite. Through our research, biocompatibility, biodegradability and osteoconductivity of TA have been proven acceptable in vitro and in vivo. Therefore, TA can be anticipated as one of the adequate scaffold sources for culturing dental stem cells/dental cells. As degradable ‘bioceramics’, TA and β-tricalciumphosphate (TCP) were used with dental stem cells, in particular periodontal ligament stem cells (PDLSCs) and dental follicle stem cells (DFSCs) to regenerate cementum. The PDLSCs showed cellular cementum and DFSCs showed cementum–like mineralized tissues.

Per-oral cross-facial sural nerve graft for facial reanimation

BackgroundCross-facial nerve graft is considered the treatment of choice for facial reanimation in patients with unilateral facial palsy caused by central facial nerve damage. In most cases, a traditional parotidectomy skin incision is used to locate the buccal and zygomatic branches of the facial nerve.MethodsIn this study, cross-facial nerve graft with the sural nerve was planned for three patients with facial palsy through an intraoral approach.ResultsAn incision was made on the buccal cheek mucosa, and the dissection was performed to locate the buccal branch of the facial nerve. The parotid papillae and parotid duct were used as anatomic landmarks to locate the buccal branch.ConclusionsThe intraoral approach is more advantageous than the conventional extraoral approach because of clear anatomic marker (parotid papilla), invisible postoperative scar, reduced tissue damage from dissection, and reduced operating time.

Maximal strength and endurance scores of the tongue, lip, and cheek in healthy, normal Koreans

Objectives The purpose of this study was to establish normative data for healthy Korean adults by measuring the maximal strength and endurance scores of the tongue, lip, and cheek, and to examine correlations between these measurements. Materials and Methods This study included 120 subjects that were divided into three groups according to age: young (20-39 years), middle-aged (40-59 years), and older (over 60 years); and by gender. Measurements were taken using the Iowa Oral Performance Instrument (IOPI). Results The mean maximal tongue strengths were as follows: young men (46.7±10.2 kPa) and women (32.1±7.9 kPa), middle-aged men (40.9±9.3 kPa) and women (36.9±8.6 kPa), and older men (35.2±9.0 kPa) and women (34.5±6.9 kPa). The mean tongue endurance scores were: young men (28.8±12.6 seconds) and women (20.8±13.5 seconds), middle-aged men (17.0±8.5 seconds) and women (15.3±5.2 seconds), and older men (15.8±6.7 seconds) and women (17.9±8.1 seconds). The mean maximal lip strengths were: young men (11.6±3.0 kPa) and women (11.4±3.8 kPa), middle-aged men (11.4±4.2 kPa) and women (11.1±5.1 kPa), and older men (14.5±3.9 kPa) and women (11.7±2.6 kPa). The mean lip endurance scores were: young men (41.1±23.9 seconds) and women (22.4±21.7 seconds), middle-aged men (24.3±10.3 seconds) and women (30.5±13.4 seconds), and older men (24.9±11.0 seconds) and women (12.8±7.6 seconds). The mean maximal cheek strengths were: young men (24.5±4.6 kPa) and women (20.5±4.3 kPa), middle-aged men (25.2±6.4 kPa) and women (21.2±5.5 kPa), and older men (22.4±5.3 kPa) and women (18.0±4.8 kPa). The mean cheek endurance scores were: young men (47.8±24.4 seconds) and women (43.9±25.0 seconds), middle-aged men (27.3±11.3 seconds) and women (20.0±14.6 seconds), and older men (21.7±14.5 seconds) and women (17.2±11.4 seconds). Conclusion The data collected in this study will provide an important database of standardized measurements for maximal strength and endurance scores of the tongue, lip and cheek in healthy, normal Koreans.