Ketut Suardita

Alveolar Bone Marrow as a Cell Source for Regenerative Medicine: Differences Between Alveolar and Iliac Bone Marrow Stromal Cells

We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells.

Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages

The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-β or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-β and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation.

Efficacy of Smear Layer Removal at the Root Tip by Using Ethylenediaminetetraacetic Acid and Erbium, Chromium: Yttrium, Scandium, Gallium Garnet Laser

INTRODUCTION The purpose of this study was to compare the efficacy of laser-driven irrigation in removing the smear layer and debriding the apical region of the root canal (the root tip) with that of ultrasonic irrigation. METHODS Forty extracted human teeth with straight single roots were randomized into 4 groups (n = 10). The specimens were shaped by using hand instruments up to a size 30/.02 file (Control, Laser 1, and Laser 2 groups) or a size 20/.02 file (Laser 3 group). During instrumentation, each canal was irrigated with 3% NaOCl and 17% ethylenediaminetetraacetic acid alternately between the use of successive files. The 4 groups of 10 teeth were processed as follows. In the Control group, teeth were irrigated with 17% ethylenediaminetetraacetic acid, and the irrigant was activated with an ultrasonic device for 60 seconds. In the Laser 1 and Laser 3 groups, the irrigant was activated with the laser for 60 seconds. In the Laser 2 group, the irrigant was activated with the laser for 30 seconds. RESULTS There were significant differences between the smear layer and debris scores for the Laser 1 group and those for the Control (P < .001), Laser 2 (P = .002), and Laser 3 groups (P = .012 and P = .013, respectively). Completely clean root canals were found in the Laser 1 group. CONCLUSIONS Use of a laser with a plain fiber tip, which produces cavitation in the irrigant, has potential as an improved alternative method for removing of the smear layer from the apical region of a straight root canal.

Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A

Membrane-bound transferrin-like protein (MTf), a glycosylphosphatidylinositol-anchored protein, is expressed at high levels in many tumors and in several fetal and adult tissues including cartilage and the intestine, as well as in the amyloid plaques of Alzheimers disease, although its role remains unknown. MTf is one of the major concanavalin A-binding proteins of the cell surface. In this study, we examined the effects of anti-MTf antibodies and concanavalin A on cell shape and gene expression, using cultures of chondrocytes and MTf-overexpressing ATDC5 and C3H10T1/2 cells. In cultures expressing MTf at high levels, concanavalin A induced cell-shape changes from fibroblastic to spherical cells, whereas no cell-shape changes were observed with wild-type ATDC5 or C3H10T1/2 cells expressing MTf at very low levels. The cell-shape changes were associated with enhanced proteoglycan synthesis and expression of cartilage-characteristic genes, including aggrecan and type II collagen. Some anti-MTf antibodies mimicked this action of concanavalin A, whereas other antibodies blocked the lectin action. The findings suggest that the crosslinking of MTf changes the cell shape and induces chondrogenic differentiation. MTf represents the first identification of a plant lectin receptor involved in cell-shape changes and the differentiation of animal cells.

Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro.

Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-β as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 μg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype. These findings suggest that the expression of MTf on the cell surface facilitates the differentiation of prechondrogenic cells, although MTf overexpression alone seems to be insufficient to commit pluripotent mesenchymal cells to the chondrocyte lineage.

Extrusion of Irrigant in Open Apex Teeth with Periapical Lesions Following Laser-Activated Irrigation and Passive Ultrasonic Irrigation

Introduction: Sodium hypochlorite (NaOCl) irrigation is critical for the success of endodontic treatment and several agitation techniques have been developed to improve the efficacy of this irrigation. Using a combination of contrast medium and radiographic examination, this study evaluated NaOCl extrusion during agitation of irrigant. Development of pressure, which may result in apical extrusion of the irrigant, has been described during laser-activated irrigation (LAI) and passive ultrasonic irrigation (PUI). Methods and Materials: We examined 40 single root canals categorized as having open apices with apical lesions in 40 patients. For the final irrigation, the teeth were irrigated with a mixture of radiopaque contrast medium and 2.5% NaOCl in solution. The solution was activated for 60 sec in both groups [the Er, Cr: YSGG laser group (n=20) and the ultrasonic group (n=20)]. The teeth were imaged subsequently using radiography for the evaluation of contrast extrusion. Results: Radiopaque contrast medium was absent from the periapical tissues in all cases. Conclusion: Use of LAI or PUI appears to be safe as used currently in endodontic treatment.

Characterization of human dental pulp cells grown in chemically defined Serum-Free medium

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbeccos modified Eagles medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-βE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine.