Charles F. Shuler

The Role of Microbial Biofilms in Osteonecrosis of the Jaw Associated with Bisphosphonate Therapy

Microbial biofilms have been observed and described in bone specimens of patients with bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) and investigators are more recently suggesting that this condition essentially represents an osteomyelitis of the jaw clinically, with greater susceptibility in some patients on BP therapy. This article explains the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis, which include BP effects on bone remodeling, anti-angiogenesis, matrix necrosis, microcracks, soft tissue toxicity, and inflammation and wound healing. Recent findings suggest a key role for microbial biofilms in the pathogenesis of BONJ; this has important therapeutic implications because biofilm organisms represent a clinical target for prevention and treatment efforts aimed at reducing the significant morbidity and costs associated with this condition.

Hydrostatic pressures promote initial osteodifferentiation with ERK1/2 not p38 MAPK signaling involved.

Mechanical stress has been considered to be an important factor in bone remodeling and recent publications have shown that mechanical stress can regulate the direction of stem cell differentiation. The exact mechanobiological effects of pressure on initial osteodifferentiation of mesenchymal stem cells (MSCs) have not been determined. These mechanobiological mechanisms may be important both in biological responses during orthodontic tooth movement and in the development of new mechanobiological strategies for bone tissue engineering. We investigated the effects of static (23 kPa) or dynamic (10–36 kPa and at 0.25 Hz frequency) pressure on MSCs during the initial process of osteoblastic differentiation following treatment with dexamethasone, β‐glycerophosphate and ascorbic acid (for 0, 3, and 7 days, respectively). The following parameters were analyzed in the ALPase activity, mRNA level of osteogenesis‐related transcription factors (Runx2, Osterix, Msx2, and Dlx5), and phosphorylation of ERK1/2 and p38 MAPK. The results showed that exposure to either dynamic or static pressure induced initial osteodifferentiation of MSCs. Particularly both types of pressure strongly stimulated the expression of osteogenesis‐related factors of totally undifferentiated MSCs. ERK signaling participated in early osteodifferentiation and played a positive but non‐critical role in mechanotransduction, whereas p38 MAPK was not involved in this process. Furthermore, the undifferentiated MSCs were highly sensitive to pressure exposure; whereas after osteoinduction MSCs reacted to pressure in a lower response state. The findings should lead to further studies to unveil the complex initial biological mechanisms of bone remodeling and regeneration upon mechanical stimuli. J. Cell. Biochem. 107: 224–232, 2009.

Smad2 is involved in the apoptosis of murine gingival junctional epithelium associated with inhibition of Bcl-2

OBJECTIVEnGingival junctional epithelium (JE) actively contributes to the homeostasis of the periodontium. Altered activation of TGF-β signalling is implicated in the epithelium from chronic periodontitis. However, little is known about the effects of TGF-β signalling on the JE. In this study, we investigated the relationship between Smad2, which plays an important role in mediating TGF-β signal, and induction of apoptosis in the JE.nnnMETHODSnK14-Smad2 transgenic mice were used to observe the effect of over-expression of Smad2 driven by CK14 promoter in the JE. We performed TUNEL technique to evaluate the epithelial apoptosis. Expression of apoptosis related genes was examined using real-time PCR and immunofluorescence.nnnRESULTSnK14-Smad2 mice showed an increased number of phospho-Smad2 positive JE cells associated with an increase in TGF-β1 expression. K14-Smad2 mice have a significantly higher percentage of TUNEL positive cells in the JE. Immunofluorescence double labelling revealed that TUNEL positive cells showed immunoreactivity to phospho-Smad2. Real-time PCR analysis of apoptosis related gene expression provided evidence of lower expression of Bcl-2 in the gingival tissue from K14-Smad2 mice. There was a strong positive reaction for Bcl-2 protein in the junctional epithelium of wild type mice, while the gingival tissue of K14-Smad2 transgenic mice had only a faint signal for Bcl-2.nnnCONCLUSIONSnThe present study provided evidence that Smad2 plays a crucial role in the induction of apoptosis in gingival JE through inhibition of Bcl-2.

Spatiotemporal Localization of Periostin and Its Potential Role in Epithelial-Mesenchymal Transition during Palatal Fusion

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

Multi‐Layered hypertrophied MEE formation by microtubule disruption via GEF‐H1/RhoA/ROCK signaling pathway

Background: Formation of the secondary palate is complex and disturbance during palatal fusion may result in cleft palate. The processes of adhesion, intercalation, and disappearance of medial edge epithelia (MEE) are characterized by morphological changes requiring dynamic cytoskeletal rearrangement. Microtubules are one of the cytoskeletal elements involved in maintenance of cell morphology. Microtubule‐disrupting drugs have been reported to cause craniofacial malformations including cleft palate. The mechanisms underlying the failure of palatal fusion remain poorly understood. We evaluated the effect of nocodazole (NDZ), a drug that disrupts microtubules, on palatal fusion in organ culture. Results: NDZ caused failure of palatal fusion due to the induction of a multi‐layered hypertrophied MEE in the mid‐region of the secondary palatal shelves. Microtubule disruption increased RhoA activity and stress fiber formation. Pharmacological inhibition of the RhoA/ROCK pathway blocked multi‐layered MEE formation. Partial prevention of hypertrophied MEE was observed with Y27632 and cytochalasin, but not with blebbistatin. NDZ induced re‐localization of GEF‐H1 into cytoplasm from cell–cell junctions. Conclusions: The present study provided evidence that the GEF‐H1/RhoA/ROCK pathway plays a pivotal role in linking microtubule disassembly to the remodeling of the actin cytoskeleton, which resulted in a multi‐layered hypertrophied MEE and failure of palatal fusion. Developmental Dynamics 241:1169–1182, 2012.

Smad2 Overexpression Reduces the Proliferation of the Junctional Epithelium

The overexpression of the intracellular signaling molecule of the transforming growth factor–beta family (TGF-β) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE). Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild-type (WT) mice selected as the control group. Samples were stained with hematoxylin and eosin stains and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-Myc as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) μm2 for the WT and 24,937.25 (± 1,965) μm2 for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-Myc expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice. Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-Myc and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest.

The expression of TGF-β3 for epithelial-mesenchyme transdifferentiated MEE in palatogenesis

The fate of the palatal medial edge epithelial (MEE) cells undergoes programming cell death, migration, and epithelial-mesenchymal transdifferentiation (EMT) coincident with the process of palatal fusion and disappearance of MEE. Mesenchymal cells in the palate have both cranial neural crest (CNC) and non-CNC origins. The objectives of this study were to identify the populations of palatal mesenchymal cells using β-galactosidase (β-gal) and DiI cell lineage markers, and to determine whether MEE-derived cells continued to express transforming growth factor-β3 (TGF-β3) and transforming growth factor-β type III receptor (TβR-III), which were specific for MEE. A model has been developed using Wnt1 tissue specific expression of Cre-recombinase to activate β-gal solely in the CNC. The expressions of TGF-β3 and TβR-III in MEE were temporally correlated with critical events in palatogenesis. Three cell populations could be distinguished in the palatal mesenchymal CNC-derived, non-CNC derived and MEE-derived. After fusion, β-gal (−) and DiI (+) mesenchymal cells continued to express TGF-β3, however TβR-III was expressed only in the epithelial MEE, as well as keratin expression. In addition, we performed laser capture microdissection to identify mRNA expression of isolated DiI (+) MEE cells. Both epithelial and transdifferentiated MEE have expressed TGF-β3, however, TβR-III was only expressed in epithelium. Extracellular matrix, especially MMP13 has been expressed coincident with fused stage which can be strongly associated with TGF-β3. These results demonstrate that combining a heritable marker and a cell lineage dye can distinguish different populations of mesenchymal cells in the developing palate. Furthermore, TGF-β3 and MMP13 could be strongly associated with EMT in palatogenesis.

Smad2 overexpression induces alveolar bone loss and up regulates TNF-α, and RANKL

OBJECTIVEnThe aim of the current study was to investigate whether Smad2 overexpression in JE cells induced alveolar bone loss, and to understand the mechanisms regulating the bone loss.nnnMETHODSnA mouse line was created that used a cytokeratin 14 (K14) promoter to overexpress Smad2 in the epithelium of the transgenic mice (K14-Smad2). Micro CT radiographs (μCT) were used to assess bone loss, bone volume, and bone density. The expression of Tnfα, Il1-β, Ifγ, Rankl, and Opg were assessed by RT-PCR. Western blots were used to detect the protein levels of TNF-α and IL1-β. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for osteoclasts. Wild type (WT) mice were used as controls in all steps of the current study.nnnRESULTSnK14-Smad2 mice had 52.5% (±4.2) root exposed compared to 32.4%(±3.2) in the WT mice. There was a significant difference in alveolar bone volume in the K14-Smad2 mice when compared to WT mice 2.65mm3 (±0.3) and 4.3mm3 (±0.35) respectively. K14-Smad2 mice also had reduced bone density 696.8mg/cc (±70) at 12 months when compared to WT mice 845.9mg/cc(±10). The mRNA levels of Tnfα and Rankl increased by 3.26- and 2.5-fold respectively in the K14-Smad2 mice when compared to controls. The protein level of TNF-α was also significantly increased to 2.8-fold in K14-Smad2 mice when compared to WT mice. Smad2 overexpression increased the total numbers of osteoclasts in K14-Smad2 mice (3.4±0.2)-fold when compared to WT mice.nnnCONCLUSIONnSmad2 overexpression induces alveolar bone loss and increases the numbers of osteoclasts. Also, Smad2 overexpression up-regulates TNF-α and RANKL.

Comparisons in Basic Science Learning Outcomes Between Students in PBL and Traditional Dental Curricula at the Same Dental School

Comparison of student achievement on external assessment measures constitutes an important criterion for program evaluation. Standardized national dental board exams exist to permit comparisons between different dental curriculum structures. In the United States, the National Board Dental Examination (NBDE) Part 1 is one of the required steps for licensure. The NBDE Part 1 assesses dental students with respect to their knowledge of the basic sciences that establish the foundation for clinical dentistry. Some US dental schools have incorporated successful completion of NBDE Part 1 as a requirement for promotion to the clinical patient care component of their dental curriculum. The outcomes assessment processes that are required for accreditation of US dental schools have also incorporated NBDE scores as an external metric of the effectiveness of the dental curriculum. Thus NBDE Part 1 achievement can be a useful criterion for measuring the effectiveness of different pedagogies to provide the basic science content of a dental curriculum. The University of Southern California School of Dentistry (USCSD) began using a problem-based learning (PBL) pedagogy to provide the dental curriculum in 1995 to a subset of students learning in parallel to their peers enrolled in a traditional lecture-based system. Between 1997 and 2002, six classes of the PBL pilot and the traditional lecture-based students completed the same NBDE Part 1 examination. In 2001 the entire dental school adopted the PBL pedagogical approach for all students enrolled in the dental education program. This chapter compares the achievement of dental students on NBDE Part 1 at USCSD prior to the beginning of the PBL pilot program, during the 6 years when PBL and traditional ran in parallel and after the entire dental education program became PBL-based.

Functional role of TGF-β receptors during palatal fusion in vitro

OBJECTIVEnReported expression patterns for TGF-β receptors (TβR-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three TβRs during palatal fusion.nnnMETHODSnUsing organ culture of mouse palatal shelves, expression levels of TβR-I, -II, and -III were suppressed by transfecting the siRNAs siTβR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each TβR. Linkage between TGF-β signalling and critical events in palatal fusion led to the use of, MMP-13 expression as an outcome measure for the function of the TGF-β receptors.nnnRESULTSnThe siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTβR-I or -II, palatal shelves at E13+72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siTβR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTβR-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siTβRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTβR-I or -II, but not -III, showed altered MMP-13 expression levels.nnnCONCLUSIONnThe ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each TβR during palatogenesis.