CBCT in contemporary endodontics.

Cone-beam computed tomography (CBCT) is a three-dimensional imaging modality which can aid endodontic diagnosis and treatment planning. While there are guidelines available describing the indications, there are divergent philosophies on when this technology should be applied in clinical practice. This paper reviews the applications of CBCT including the clinical relevance of parameters which can be assessed, highlights the limitations of this technology, and provides guidance on how to maximise its clinical utility.

The growth of Medicare rebatable cone beam computed tomography and panoramic radiography in Australia.

BACKGROUND: There is a lack of objective data documenting the growth of cone beam radiology in Australia. Medicare rebates for cone beam computed tomography (CBCT) scans have been available since 1 July 2011. The aim of this study was to examine the Medicare data on the use of CBCT in order to quantify the growth of this technology over a three-year period and to assess if the growth of CBCT was impacting upon the use of dental panoramic radiology. METHODS: The Medicare dataset was accessed, and data relating to the number and distribution of CBCTs and panoramic radiographs (PRs) were extracted. The data were broken down by state and territory, as well as by age group and gender of the patient, for the financial year periods July 2011 to June 2012, July 2012 to June 2013 and July 2013 to June 2014. Items relating to PRs were also extracted for the periods 2005-2008 and 2008-2011. RESULTS: In the period July 2011 to June 2014, a total of 226 232 CBCTs and 2 881 351 PRs were rebated through Medicare. The rate of CBCT services provided per 100 000 population increased by 42.3% over the three-year period, whereas the rate of PRs remained fairly constant. From the age group 5-14 years through to 55-64 years, females received more CBCTs and PRs than males. The total number of PRs rebated through Medicare increased slightly over each previous three-year period (2005-2008 and 2008-2011). CONCLUSIONS: There has been rapid growth of the use of CBCT over a three-year period, most marked in the State of Victoria. The higher number of CBCT examinations in females across almost all age groups, but most notably in the 15-24 years age group, raises questions about clinical decision making in the selection of cone beam imaging. There was little evidence that CBCTs were being used as a substitute for PRs, except for a small but consistent drop in the number of PRs being requested for diagnosis of surgically related dental conditions.

Inadequate record keeping by dental practitioners.

BACKGROUND: When a dental practitioner is brought before a disciplinary body, a common finding is that dental records were poorly kept and inadequate to establish issues of consent for treatment or the nature of the treatment undertaken. Often this finding may be incidental to the actual issue that brought the practitioner before a regulatory body or the Courts. The aim of this study was to examine recent cases reported in the State of Victoria involving dental practitioners, specifically seeking those cases where the record keeping was found to be inadequate as an incidental finding. METHODS: Published rulings of formal complaints and notification cases brought before disciplinary hearings in the State of Victoria for the period January 2000 to June 2014 were analysed with regards to orders made in respect to record keeping. RESULTS: Complaints and notifications specific to dental record keeping accounted for less than 2% of formal complaints. And yet up to 75% of cases have made a finding of unprofessional conduct against a dental practitioner on the basis of inadequate record keeping, most often in combination with other breaches of conduct. CONCLUSIONS: Adherence to the traditional format of handwriting or typing entries into patient records may contribute to the problem of poor record keeping in a clinical dental setting. Newer technologies such as digital intraoral and extraoral photography and audio-recording of patient interactions may offer a solution to the problems of record keeping.

VEGF-A angiogenesis induces a stable neovasculature in adult murine brain.

Angiogenesis is a critical component of stroke, head injury, cerebral vascular malformation development, and brain tumor growth. An understanding of the mechanisms of adult cerebral angiogenesis is fundamental to therapeutic vessel modulation for these diseases. To study angiogenesis in the central nervous system, we injected an adenoviral vector engineered to express vascular endothelial growth factor (VEGF-A164) into adult murine striatum. Vector-infected astrocytes expressed VEGF-A164 resulting in vascular permeability, hemorrhage, and the formation of greatly enlarged "mother" vessels. Subsequently, endothelial cells and pericytes lining mother vessels proliferated and assembled into glomeruloid bodies, complex cellular arrays interspersed by small vessel lumens. As VEGF-A164 expression declined, glomeruloid bodies involuted through apoptotic processes to engender numerous small daughter vessels. Characterized by modestly enlarged lumens with prominent pericyte coverage, daughter vessels were distributed with a density greater than normal cerebral vessels. Daughter vessels remained stable and patent to 16 months and represented the final stage of VEGF-A-induced cerebral angiogenesis. Together, these findings provide a mechanistic understanding of angiogenesis in cerebral disease processes. Furthermore, the long-term stability of daughter vessels in the absence of exogenous VEGF-A164 expression suggests that VEGF-A may enable therapeutic angiogenesis in brain.

Increased Tie2 expression, enhanced response to angiopoietin-1, and dysregulated angiopoietin-2 expression in hemangioma-derived endothelial cells.

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745-752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.

Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism.

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro- angiogenic and anti-angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, we subjected TSP-2-deficient and wild-type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP-2 expression was strongly upregulated in the mesenchymal stroma of wild-type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP-2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre-malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP-2-deficient mice. While TSP-2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was significantly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP-2 expression as a novel host anti-tumor defense mechanism.

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery.

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.

Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

Angiogenesis is a key component of human cancer progression and metastasis. In an effort to recapitulate early events in tumor-induced angiogenesis, we have employed a subcutaneous Matrigel implant model using immunodeficient mice as hosts. Matrigel-containing fibroblast growth factor 2 (FGF-2; 1.2 microg/ml) induced stromal cell infiltration into the Matrigel/skin interface within 4 days and maximal neovascularization at 7 days. Cells staining positive for the endothelial cell marker, platelet-endothelial cell adhesion molecule 1 (PECAM-1), were present in neovessels and in isolated cells within the Matrigel matrix. Immunohistochemical analysis revealed high levels of vascular endothelial growth factor (VEGF) deposited in the stromal interface present only in the FGF-2-containing but not in control Matrigel implants. VEGF expression was confirmed with in situ hybridization. High VEGF mRNA levels were observed in the infiltrating stromal cells but not in endothelial or endothelial precursors as defined by PECAM-1 staining. In vitro analysis of FGF-2-treated embryonic fibroblasts, Balb/c 3T3 cells, showed an induction of VEGF transcription, mRNA synthesis, and protein secretion as defined by transcriptional reporter, Northern blot, and ELISA assays. The FGF-2-induced VEGF expression was not dependent on select matrix adherence or signaling components because VEGF mRNA expression induced by FGF-2 was equally activated on serum, basement membrane, and fibronectin matrix substrates. Systemic application of anti-VEGF antibodies significantly repressed FGF-2-induced angiogenesis over control antibody by 88% (p < 0.001). These data support an FGF-2 angiogenic model that is dependent on endothelial cell activation, stromal cell infiltration, and VEGF expression by the infiltrating stromal cell population.

Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.

VPF/VEGF acts selectively on the vascular endothelium to enhance permeability, induce cell migration and division, and delay replicative senescence. To understand the changes in gene expression during endothelial senescence, we investigated genes that were differentially expressed in early vs. late passage (senescent) human dermal endothelial cells (HDMEC) using cDNA array hybridization. Early passage HDMEC cultured with or without VPF/VEGF overexpressed 9 and underexpressed 6 genes in comparison with their senescent counterparts. Thymosin beta-10 expression was modulated by VPF/VEGF and was strikingly down-regulated in senescent EC. The beta-thymosins are actin G-sequestering peptides that regulate actin dynamics and are overexpressed in neoplastic transformation. We have also identified senescent EC in the human aorta at sites overlying atherosclerotic plaques. These EC expressed senescence-associated neutral beta-galactosidase and, in contrast to adventitial microvessel endothelium, exhibited weak staining for thymosin beta-10. ISH performed on human malignant tumors revealed strong thymosin beta-10 expression in tumor blood vessels. This is the first report that Tbeta-10 expression is significantly reduced in senescent EC, that VPF/VEGF modulates thymosin beta-10 expression, and that EC can become senescent in vivo. The reduced expression of thymosin beta-10 may contribute to the senescent phenotype by reducing EC plasticity and thus impairing their response to migratory stimuli.

The association between tumour progression and vascularity in myxofibrosarcoma and myxoid/round cell liposarcoma.

Angiogenesis is an important factor in the morphological progression and metastasis of many solid tumours. We studied two homogeneous series of myxofibrosarcoma (MFS) and myxoid/round liposarcoma (MRLS), characterised by distinct vascular patterns and correlated the intratumoral microvessel density (IMD) with morphologic progression in both types of sarcoma. In our study, 43 cases of MFS and 42 cases of MRLS were graded according to established diagnostic criteria. For evaluation of IMD, representative sections were stained immunohistochemically for CD31. After selection of "neovascular hot spots", IMD was calculated by measuring the endothelial surface within twenty 200x fields in relation to the total analysed area. In addition to the correlation of IMD with histological grades of malignancy, a correlation of IMD with the inflammatory infiltrate in MFS was done. To determine whether vascular endothelial growth factor (VEGF) and its receptors, KDR and flt-1, may play a role in the progression of both types of sarcomas, we used mRNA in situ hybridisation (ISH) to study VEGF, KDR and flt-1 expression in selected cases. In addition, the expression of thrombospondin-1, which has been reported to inhibit angiogenesis, and of collagen type I was studied using mRNA ISH. Cases of MFS varied histologically from hypocellular, mainly myxoid, neoplasms (low-grade malignant, 18 cases) to intermediate-grade malignant lesions with increased cellularity and mitotic activity (13 cases), and high-grade malignant cases with marked pleomorphism, high proliferative activity and areas of necrosis in many cases (12 cases). Cases of purely low-grade myxoid liposarcoma (16 cases) were characterised by low-cellularity, mucin pooling and plexiform vasculature. In combined MRLS, these hypocellular areas were admixed with hypercellular, round cell areas (5-80% of the analysed tumour area; 23 cases), and in round cell liposarcoma (three cases) rounded tumour cells predominated (>80% of the analysed tumour area). The average IMD in intermediate and high-grade malignant MFS (4.03 and 4.09, respectively) was significantly higher than in low-grade malignant MFS (2.73). Correlation of vascularity with the inflammatory infiltrate in MFS showed increased IMD only in cases with abundant neutrophils; most of these cases were high-grade malignant neoplasms. In contrast, no statistical correlation between morphological progression and IMD was seen in myxoid liposarcoma (6.08), MRLS (6.57) and round cell liposarcoma (4.07). VEGF mRNA was expressed by tumour cells in all histological grades of MFS and MRLS. VEGF receptor mRNA was weakly expressed by endothelia of newly formed blood vessels in both entities. Interestingly, tumour cells of all analysed cases of MFS strongly expressed collagen type I and thrombospondin-1, while these proteins were not detected in tumour cells of MRLS. In conclusion, morphologic tumour progression in MFS is associated with increased IMD, whereas, in MRLS, no such correlation is seen. Whereas VEGF and VEGF receptor mRNA were expressed in both entities, a characteristic expression profile of collagen type I and thrombospondin-1 in MFS emerged. Further studies are necessary to correlate vascularity and clinical course in MFS and MRLS.

Control of hair growth and follicle size by VEGF-mediated angiogenesis.

The murine hair follicle undergoes pronounced cyclic expansion and regression, leading to rapidly changing demands for its vascular support. Our study aimed to quantify the cyclic changes of perifollicular vascularization and to characterize the biological role of VEGF for hair growth, angiogenesis, and follicle cycling. We found a significant increase in perifollicular vascularization during the growth phase (anagen) of the hair cycle, followed by regression of angiogenic blood vessels during the involution (catagen) and the resting (telogen) phase. Perifollicular angiogenesis was temporally and spatially correlated with upregulation of VEGF mRNA expression by follicular keratinocytes of the outer root sheath, but not by dermal papilla cells. Transgenic overexpression of VEGF in outer root sheath keratinocytes of hair follicles strongly induced perifollicular vascularization, resulting in accelerated hair regrowth after depilation and in increased size of hair follicles and hair shafts. Conversely, systemic treatment with a neutralizing anti-VEGF antibody led to hair growth retardation and reduced hair follicle size. No effects of VEGF treatment or VEGF blockade were observed in mouse vibrissa organ cultures, which lack a functional vascular system. These results identify VEGF as a major mediator of hair follicle growth and cycling and provide the first direct evidence that improved follicle vascularization promotes hair growth and increases hair follicle and hair size.

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Expression of Tie1, Tie2, and angiopoietins 1, 2, and 4 in Kaposi's sarcoma and cutaneous angiosarcoma.

The angiopoietins are recently described growth factors for vascular endothelium. The Tie1 and Tie2 receptors are expressed by endothelium. Acquired immune deficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS) and cutaneous angiosarcoma are malignancies of endothelial origin. KS involves primarily the skin and mucosal surfaces and is common in AIDS patients. In an effort to determine whether the angiopoietins and Tie receptors play a role in the pathobiology of angiosarcoma and KS, we studied the expression of angiopoietin-1, angiopoietin-2, angiopoietin-4, Tie1, and Tie2 mRNAs in biopsies of KS from 12 AIDS patients, in biopsies of cutaneous angiosarcoma from two patients, and in control biopsies of normal skin from three volunteers by in situ hybridization. Strong expression of angiopoietin-2, Tie1, and Tie2 mRNAs was detected in the tumor cells of KS and cutaneous angiosarcomas, in contrast to the focal low-level expression in normal skin biopsies. Focal low-level expression of angiopoietin-1 was seen in KS, cutaneous angiosarcomas, and in normal skin. Focal low-level expression of angiopoietin-4 was identified in a minority of KS lesions. These findings suggest that the angiopoietins and Tie receptors may play an important role in the pathobiology of KS and cutaneous angiosarcoma and identify additional potential targets for therapeutic intervention in these vascular malignancies.

Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR-1 and VEGFR-2 in vivo.

Vascular endothelial growth factor (VEGF or vascular permeability factor) is an important angiogenic factor that is up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. To determine the functional role of VEGF in the development of endothelial tumors, we expressed primate VEGF 121 in an endothelial cell line, MS1, derived from primary murine cells by immortalization with a temperature-sensitive SV40 large T antigen. This cell line expresses the VEGFR-2 (Flk-1/Kdr) receptor for VEGF. Expression of VEGF 121 led to the development of slowly growing endothelial tumors, which were histologically well-differentiated angiosarcomas. The angiosarcomas generated from MS1 VEGF cells demonstrated up-regulation of the VEGF receptors VEGFR-2 and VEGFR-1 (Flt-1) in vivo compared with benign hemangiomas generated from MS1 cells. Treatment of these cells with the VEGFR-2 tyrosine kinase inhibitor SU 1498 led to decreased expression of ets-1, a transcription factor which has been shown to be stimulated by VEGF. These results suggest that high level expression of VEGF in endothelial cells may result in malignant transformation. This transformation process likely involves both autocrine and paracrine pathways.

Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (< or =3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels ("mother" vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.

Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.

Inhibition of the vascular endothelial growth factor (VEGF) receptor Flk-1 has been shown to prevent invasion of experimental squamous cell carcinomas (SCC). To directly investigate the role of VEGF in tumor invasion, we stably transfected human SCC-13 cells, which are characterized by a noninvasive phenotype in vivo, with expression vectors containing murine VEGF(164) in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec). SCC/vec cells formed slowly growing, well-differentiated tumors with well-defined borders between tumor and stroma, after intradermal or subcutaneous injection. In contrast, SCC/VEGF+ tumors were characterized by rapid tumor growth, with small cell groups and single cells invading into the surrounding tissue, and by admixture of blood vessels and tumor cells in areas of tumor invasion. We detected an increase in tumor vessel density and size in VEGF-overexpressing tumors, resulting in a more than fourfold increase in total vascular areas. In contrast, SCC/VEGF- clones formed noninvasive, sharply circumscribed tumors with reduced vascular density. These findings demonstrate that selective VEGF overexpression was sufficient to induce tumor invasiveness, and they provide further evidence for an active role of the tumor stroma in cancer progression.

Design considerations for piezoelectric polymer ultrasound transducers.

Much work has been published on the design of ultrasound transducers using piezoelectric ceramics, but a great deal of this work does not apply when using the piezoelectric polymers because of their unique electrical and mechanical properties. The purpose of this paper is to review and present new insight into seven important considerations for the design of active piezoelectric polymer ultrasound transducers: piezoelectric polymer materials selection, transducer construction and packaging requirements, materials characterization and modeling, film thickness and active area design, electroding selection, backing material design, and front protection/matching layer design. Besides reviewing these design considerations, this paper also presents new insight into the design of active piezoelectric polymer ultrasonic transducers. The design and fabrication of an immersible ultrasonic transducer, which has no adhesive layer between the active element and backing layer, is included. The transducer features direct deposition of poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] copolymer onto an insulated aluminum backing substrate. Pulse-echo tests indicated a minimum insertion loss of 37 dB and -6 dB bandwidth of 9.8 to 22 MHz (71%). The use of polymer wear-protection/quarter-wave matching layers is also discussed. Test results on a P(VDF-TrFE) transducer showed that a Mylar/sup TM/ front layer provided a slight increase in pulse-echo amplitude of 15% (or 1.2 dB) and an increase in -6 dB pulse-echo fractional bandwidth from 86 to 95%. Theoretical derivations are reported for optimizing the active area of the piezoelectric polymer element for maximum power transfer at resonance. These derivations are extended to the special case for a low profile (i.e., thin) shielded transducer. A method for modeling the non-linear loading effects of a commercial pulser-receiver is also included.