Kathy K.H. Svoboda

Chondrocyte Survival and Differentiation In Situ Are Integrin Mediated

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum‐free organ culture sternal model that recapitulates normal development and maintains the three‐dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (α2, α3, or β1). In the presence of anti‐β1 integrin (25 μg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti‐α2 (10 μg/ml, clone P1E6) or anti‐ α3 (10 μg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation. Dev. Dyn. 1997;210:249–263.

The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Epithelial-Mesenchymal Transformation during Craniofacial Development

Epithelial to mesenchymal phenotype transition is a common phenomenon during embryonic development, wound healing, and tumor metastasis. This transition involves cellular changes in cytoskeleton architecture and protein expression. Specifically, this highly regulated biological event plays several important roles during craniofacial development. This review focuses on the regulation of epithelial-mesenchymal transformation (EMT) during neural crest cell migration, and fusion of the secondary palate and the upper lip. Abbreviations used in this paper: BMP, bone morphogenic protein; CCFSE, 5 (and 6) carboxy 2,7′ dichlorofluorescein diacetate succinimidyl ester; CNC, cranial neural crest; DiI, 1,1-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate; EMT, epithelial-mesenchymal transformation; FGF, fibroblast growth factor; ILK, integrin-linked kinase; LEF1, Lymphoid enhancer factor-1; MEE, medial edge epithelia; MFS, mean fusion score; MMP, matrix metalloproteinase; PDK, 3-phosphoinostide-dependent protein kinase; Pax, paired box-1 to -9; PI-3 kinase, phosphatidylinositol-3 kinase; Ptc, patched; PTEN, phosphatase and tensin homolog deleted on chromosome ten; Shh, Sonic hedgehog; Tbx, T-box family; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase.

CHONDROCYTE-MATRIX ATTACHMENT COMPLEXES MEDIATE SURVIVAL AND DIFFERENTIATION

Integrin mediated cell‐extracellular matrix interactions are required for survival and differentiation of many cell types. In this review, the cell‐matrix attachment complex (CMAX) is described for chondrocytes. The evidence that integrin‐mediated signal transduction is necessary for normal chondrocyte differentiation and survival in various culture conditions and in vivo are reviewed. The possible signal transduction pathways stimulated by the extracellular matrix components are discussed with a review of current data from chondrocyte experiments. In addition, the influence of parathyroid hormone and transforming growth factor β on chondrocyte survival has been included as they may function in concert with integrin mediated signal transduction. Finally, specific changes in gene expression preceding apoptosis are discussed. The current understanding of how integrin‐mediated signals prevent apoptosis and implications of anchorage‐dependent survival for development and differentiation of the chondrocyte phenotype are discussed. Microsc. Res. Tech. 43:111–122, 1998.

PI‐3 kinase activity is required for epithelial‐mesenchymal transformation during palate fusion

Epithelial‐mesenchymal transformation (EMT) is the primary mechanism for the disappearance of medial edge epithelia (MEE) during palate fusion. This phenotype transition is highly regulated by growth factors, extracellular matrix, cell surface receptors, and a variety of intracellular signaling. Phosphatidylinositol‐3 (PI‐3) kinase regulates cytoskeleton reorganization, cell migration, and transforming growth factor (TGF) β–regulated EMT. Therefore, we investigated the role of PI‐3 kinase in EMT during palatal fusion in vitro. Palatal shelves from embryonic (E) 13.5 day mouse embryos were collected and cultured for up to 72 hr. A specific PI‐3 kinase inhibitor, LY294002, was added to the medium at concentrations of 100 ηM, 1 μM, and 10 μM. The fate of midline epithelia was traced by carboxyfluorescence labeling and analyzed by confocal microscopy. Harvested tissues were also processed for immunohistochemical analysis of a specific marker for basal lamina (laminin). Palatal fusion stages were scored on a scale of 1 to 5, with 1 equal to complete nonfusion and 5 equal to complete fusion. The mean fusion score (MFS) was calculated for each treatment group. Palatal shelves fused after 72 hr of culture in control and 100 ηM LY294002 inhibitor‐treated groups, with MFS of 4.67 and 4.5, respectively. Laminin was absent in the midline and epithelia transformed into mesenchyme. However, when cultured palates were treated with 1 and 10 μM LY294002, MEE persisted in the midline and the basal lamina remained intact after 72 hr. The MFS was significantly less in the 1 and 10 μM LY294002‐treated tissues at 2.08 and 1.33, respectively. Our results demonstrate that EMT during palatal fusion in vitro is dependent on PI‐3 kinase activity.

The role of twist during palate development

In palatogenesis, the MEE (Medial Edge Epithelium) cells disappear when palates fuse. We hypothesize that the MEE cells undergo EMT (Epithelial‐Mesenchymal Transition) to achieve mesenchyme confluence. Twist has an important role in EMT for tumor metastasis. The purpose of this study was to analyze Twist function during palatal fusion. Twist protein was expressed in palatal shelves and MEE both in vivo and in vitro just prior to fusion. Twist mRNA increased in chicken palates 3 and 6 hr after TGFβ3 treatment. Palatal fusion was decreased when cultured palatal shelves were treated with 200 nM Twist siRNA and the subcellular localization of β‐catenin was altered. Twist mRNA decreased in palatal shelves treated with TGFβ3 neutralizing antibody or LY294002, a specific phosphatidylinositol‐3 kinase (PI‐3K) inhibitor. In summary, Twist is downstream of TGFβ3 and PI‐3K pathways during palatal fusion. However, decreasing Twist with siRNA did not completely block palate fusion, indicating that the function of Twist may be duplicated by other transcription factors. Developmental Dynamics 237:2716–2725, 2008.

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non‐smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF‐β1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of α‐smooth muscle actin (α‐SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real‐time polymerase chain reaction (real‐time PCR). Phosphorylated p38 mitogen‐activated protein kinase (Phospho‐p38 MAPK) activity was analyzed by Western blots. TGF‐β1 induced an increase of α‐SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF‐β1‐induced expression of α‐SMA but not β‐actin. Nicotine treatment down‐regulated TGF‐β1‐induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone.

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.

Comparison of Vibringe, EndoActivator, and Needle Irrigation on Sealer Penetration in Extracted Human Teeth

INTRODUCTION Vibringe is a new device that allows continuous sonic irrigation of the canal system during endodontic treatment. The aim of this study was to compare the effect of different irrigation systems on sealer penetration into dentinal tubules of extracted single-rooted teeth. METHODS Fifty single-rooted human teeth were instrumented and randomly divided into 4 groups: group 1 (control), saline; group 2 (conventional irrigation), 17% EDTA followed by 6% NaOCl; group 3 (EndoActivator), same irrigants as group 2; group 4 (Vibringe), same irrigants as group 2. Obturation of all teeth was done with gutta-percha and SimpliSeal labeled with fluorescent dye. Transverse sections at 1 mm and 5 mm from the root apex were examined by using confocal laser scanning microscopy. Percentage and maximum depth of sealer penetration were measured by using NIS-Elements Br 3.0 imaging software. RESULTS Groups 3 and 4 had a significantly greater percentage of the canal wall penetrated by sealer at the 5-mm level than group 1 (P < .0125), but not group 2. No other differences were found between the groups at either section level for both the percentage of sealer penetration and maximum depth. The 5-mm sections in each experimental group had a significantly higher percentage and maximum depth of sealer penetration than did the 1-mm sections (P < .0125). CONCLUSIONS The use of sonic activation with either the EndoActivator or Vibringe did not significantly improve the sealer penetration when compared with conventional irrigation.

Regulation of epithelial-mesenchymal transition in palatal fusion.

During palatal fusion, the midline epithelial seam between the palatal shelves degrades to achieve mesenchymal confluence. Morphological and molecular evidence support the theory that the epithelial-mesenchymal transition is one mechanism that regulates palatal fusion. It appears that transforming growth factor (TGF)-β signaling plays a role in palatal EMT. TGFβ3 is the main inducer in palatal fusion and activates both Smad-dependent and -independent signaling pathways, including the key EMT transcription factors, Lef1, Twist, and Snail1, in the MEE prior to the palatal EMT program. The roles and interactions among these transcription factors will be discussed.