Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.

Influence of pericardial suction blood retransfusion on memory function and release of protein S100B.

BACKGROUND: To study the influence of pericardial suction blood (PSB) on postoperative memory disturbances and release patterns of protein S100B during and after cardiopulmonary bypass (CPB). METHODS: Sixty male patients admitted for coronary artery bypass surgery were prospectively randomized to receive PSB either by using conventional cardiotomy suction retransfusion or after cell-saver processing. RESULTS: The concentration of S100B rose during the period of CPB from 0.065 +/- 0.004 to 0.24 +/- 0.001 microg/L (p < 0.001). PSB contained 18.0 +/- 1.7 microg/L of S100B. Direct retransfusion from the cardiotomy reservoir made the systemic level increase to 1.42 +/- 0.19 microg/L compared to 0.25 +/- 0.02 microg/L using a cell-saver. Signs of postoperative memory dysfunction ( > 1 SD) were discovered in one of three tests, but were unrelated to technique of retransfusion. No associations were found between serum concentrations of S100B and memory function. CONCLUSION: In this study, retransfusion of PSB during cardiac surgery appeared not to cause memory disturbances. PSB contained high concentrations of protein S100B making its use as a marker of cerebral injury unsuitable.

Use of heparin-bonded circuits in cardiopulmonary bypass improves clinical outcome.

OBJECTIVE: The use of heparin-coated surfaces in cardiopulmonary bypass has been shown to decrease the inflammatory response imposed by the contact between blood and artificial surfaces. One would expect this reaction to improve clinical outcome. However, this has been difficult to verify. This investigation is based on an aggregation of two randomized studies from our institution and highlights possible effects of heparin coating on a number of clinically oriented parameters. DESIGN: Departmental analysis of patients subjected to coronary artery bypass surgery using heparin-coated circuits. Cardiopulmonary bypass was employed using either the Carmeda or Duraflo heparin coatings compared with a control. The systemic heparin dose was reduced in the heparin-coated groups (ACT > 250 s) vs control group patients (ACT > 480 s). The effects of heparin coating related to clinical outcome were studied. RESULTS: The use of heparin-coated circuits reduced the mean length of stay in hospital from 7.8 +/- 2.5 to 7.3 +/- 1.8 days (p = 0.040) and postoperative ventilation time from 9.7 +/- 9.2 to 8.2 +/- 8.5 h (p = 0.018), blood loss 8 h post surgery from 676 +/- 385 to 540 +/- 245 ml (p = 0.001), individual perioperative change of haemoglobin loss (p = 0.001), leukocyte count (p = 0.000) and creatinine elevation (p = 0.000), proportion of patients exposed to allogenous blood transfusions 39.2 vs 23.9% (p = 0.001), postoperative coagulation disturbances 4.4 vs 0.4% (p = 0.006), postoperative deviations from the normal postoperative course 47.2 vs 36.7% (p = 0.035), neurological deviations 9.4 vs 3.9% (p = 0.021) and atrial fibrillation 26.4 vs 18.0% (p = 0.041). No effects were found with respect to perioperative platelet count, postoperative fever reaction and 5-year survival. CONCLUSION: Based on several indicators, the use of heparin coating in cardiopulmonary bypass is associated with improved clinical results.

Transventricular aortic cannulation for repair of aortic dissection.

We have used transventricular aortic cannulation as arterial inflow from the heart-lung machine in seven consecutive operations done in 1 year for acute aortic dissection. Satisfactory cardiopulmonary bypass was achieved in all patients.

Neurological and general outcome in low-risk coronary artery bypass patients using heparin coated circuits.

OBJECTIVE: The clinical significance of heparin coating in cardiopulmonary bypass has previously been investigated. However, few studies have addressed the possible influence on brain function and memory disturbances. METHODS: Three hundred low-risk patients exposed to coronary bypass surgery were randomised into three groups according to type of heparin coating: Carmeda Bioactive Surface, Baxter Duraflo II and a control group. Outcome was determined from a number of clinically oriented parameters, including a detailed registry of postoperative deviations from the normal postoperative course. Brain damage was assessed through S100 release and memory tests, including a questionnaire follow-up. RESULTS: Clinical outcome was similar for all groups. Blood loss (Duraflo only), transfusion requirements and postoperative creatinine elevation were reduced in the heparin-coated groups. A lower incidence of atrial fibrillation was noted in the Duraflo group. Heparin coating did not uniformly attenuate the release of S100 or the degree of memory impairment. CONCLUSIONS: Cardiopulmonary bypass (CPB) with heparin coating and a reduced dose of heparin seems to be safe. Clinical outcome and neurological injury seem not to be associated with type of heparin coating used for CPB. However, blood loss and transfusion requirements may be reduced.

TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis.

Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGFbeta, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinbetaA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.

Integration of FGF and TWIST in calvarial bone and suture development.

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.

Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema regions.

Rodents have a toothless diastema region between the incisor and molar teeth which may contain rudimentary tooth germs. We found in upper diastema region of the mouse (Mus musculus) three small tooth germs which developed into early bud stage before their apoptotic removal, while the sibling vole (Microtus rossiaemeridionalis) had only a single but larger tooth germ in this region, and this developed into late bud stage before regressing apoptotically. To analyze the genetic mechanisms of the developmental arrest of the rudimentary tooth germs we compared the expression patterns of several developmental regulatory genes (Bmp2, Bmp4, Fgf4, Fgf8, Lef1, Msx1, Msx2, p21, Pitx2, Pax9 and Shh) between molars and diastema buds of mice and voles. In diastema tooth buds the expression of all the genes differed from that of molars. The gene expression patterns suggest that the odontogenic program consists of partially independent signaling cascades which define the exact location of the tooth germ, initiate epithelial budding, and transfer the odontogenic potential from the epithelium to the underlying mesenchyma. Although the diastema regions of the two species differed, in both species the earliest difference that we found was weaker expression of mesenchymal Pax9 in the diastema region than in molar and incisor regions at the dental lamina stage. However, based on earlier tissue recombination experiments it is conceivable that the developmental arrest is determined by the early oral epithelium.

Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice.

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1-/- mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.

Appropriateness of referral of coronary angiography patients in Sweden. SECOR/SBU Project Group.

OBJECTIVE: To evaluate the appropriateness of referral following coronary angiography in Sweden. DESIGN: Prospective survey and review of medical records. PATIENTS: Consecutive series of 2767 patients who underwent coronary angiography in Sweden between May 1994 and January 1995 and were considered for coronary revascularisation. MAIN OUTCOME MEASURES: Percentage of patients referred for coronary artery bypass graft surgery (CABG) and percutaneous transluminal coronary angioplasty (PTCA) for indications that were judged necessary, appropriate, uncertain, and inappropriate by a multispecialty Swedish national expert panel using the RAND/University of California Los Angeles (UCLA) appropriateness method, and the percentage of patients referred for continued medical management who met necessity criteria for revascularisation. RESULTS: Half the patients were referred for CABG, 25% for PTCA, and 25% for continued medical therapy. CABG was judged appropriate or necessary for 78% of patients, uncertain for 12% and inappropriate for 10%. For PTCA the figures were 32%, 30% and 38%, respectively. Two factors contributed to the high inappropriate rate. Many of these patients did not have "significant" coronary artery disease (although all had at least one stenosis > 50%) or they were treated with less than "optimal" medical therapy. While 96% of patients who met necessity criteria for revascularisation were appropriately referred for revascularisation, 4% were referred for continued medical therapy. CONCLUSIONS: Using the RAND/UCLA appropriateness method and the definitions agreed to by the expert panel, which may be considered conservative today, it was found that 19% of Swedish patients were referred for coronary revascularisation judged inappropriate. Since some cardiovascular procedures evolve rapidly, the proportion of patients referred for inappropriate indications today remains unknown. Nevertheless, physicians should actively identify those patients who will and will not benefit from coronary revascularisation and ensure that they are appropriately treated.

Quality assurance in clinical perfusion.

OBJECTIVE: To investigate the properties and usefulness of prospective routine registration of incidents related to cardiopulmonary bypass and its clinical significance as a quality assurance instrument. METHODS: Incidents or deviations from the normal course observed during cardiopulmonary bypass procedures were registered in a computer database. Each incident was classified according to 14 pre-defined categories. The cause of each incident was evaluated, as well as patient outcome. Incidents leading to permanent or temporary injury were denoted accidents. The general- and category-related incidence rate was calculated for the observation period 1989-1997 encompassing 6918 cardiopulmonary bypass procedures. RESULTS: The general incidence rate varied between 4.5-7.6% per year during the registration period. Most incidents (57%) occurred during established, or start of, cardiopulmonary bypass, whereas the remaining proportion of incidents were detected either before (27%) or when terminating (16%). The most common category of incidents was oxygenator failure (1.6%), followed by mechanical (1.4%) and surgical (1.2%) incidents. Accidents and fatal outcomes occurred in 0.03% of the cases. CONCLUSIONS: Routine registration of incidents yields a clinically attractive instrument of controlling safety aspects and quality measures in cardiopulmonary bypass. The observed incidence rates are somewhat higher than previously reported, probably primarily related to the methodology implemented in this study.

Association of developmental regulatory genes with the development of different molar tooth shapes in two species of rodents.

While the evolutionary history of mammalian tooth shapes is well documented in the fossil record, the developmental basis of their tooth shape evolution is unknown. We investigated the expression patterns of eight developmental regulatory genes in two species of rodents with different molar morphologies (mouse, Mus musculus and sibling vole, Microtus rossiaemeridionalis). The genes Bmp-2, Bmp-4, Fgf-4 and Shh encode signal molecules, Lef-1, Msx-1 and Msx-2, are transcription factors and p21CIP1/WAF1 participates in the regulation of cell cycle. These genes are all known to be associated with developmental regulation in mouse molars. In this paper we show that the antisense mRNA probes made from mouse cDNA cross-hybridized with vole tissue. The comparisons of gene expression patterns and morphologies suggest that similar molecular cascades are used in the early budding of tooth germs, in the initiation of tooth crown base formation, and in the initiation of each cusp's development. Furthermore, the co-localization of several genes indicate that epithelial signalling centres function at the three stages of morphogenesis. The earliest signalling centre in the early budding epithelium has not been reported before, but the latter signalling centres, the primary and the secondary enamel knots, have been studied in mouse. The appearance of species-specific tooth shapes was manifested by the regulatory molecules expressed in the secondary enamel knots at the areas of future cusp tips, whilst the mesenchymal gene expression patterns had a buccal bias without similar species-specific associations.

A graphical WWW-database on gene expression in tooth.

We have constructed a WWW-site that presents gene expression in developing dental tissues (Gene expression in tooth, http://honeybee.helsinki.fi/toothexp). Our aim is to provide a tool for learning and for research, in particular to facilitate comparisons of the expression patterns of different genes and detection of their coexpression as a starting point for experimental studies. For this reason, we have included schematic illustrations of the gene expression in different stages of development when adequate information has been available. The site also contains a comprehensive list of references. Additionally phenotypic consequences of defective gene expression are indicated. The pages also provide links to other biological databases on the Internet, both as context-dependent and as general links. We welcome submissions from researches elsewhere to include their information of gene expression and function.

The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot.

The enamel knot, a transient epithelial structure, appears at the onset of mammalian tooth shape development. Until now, the morphological, cellular and molecular events leading to the formation and disappearance of the enamel knot have not been described. Here we report that the cessation of cell proliferation in the enamel knot in mouse molar teeth is linked with the expression of the cyclin-dependent kinase inhibitor p21. We show that p21 expression is induced by bone morphogenetic protein 4 (BMP-4) in isolated dental epithelia. As Bmp-4 is expressed only in the underlying dental mesenchyme at the onset of the enamel knot formation, these results support the role of the cyclin-dependent kinase inhibitors as inducible cell differentiation factors in epithelial-mesenchymal interactions. Furthermore, we show that the expression of p21 in the enamel knot is followed by Bmp-4 expression, and subsequently by apoptosis of the differentiated enamel knot cells. Three-dimensional reconstructions of serial sections after in situ hybridization and Tunel-staining indicated an exact codistribution of Bmp-4 transcripts and apoptotic cells. Apoptosis was stimulated by BMP-4 in isolated dental epithelia, but only in one third of the explants. We conclude that Bmp-4 may be involved both in the induction of the epithelial enamel knot, as a mesenchymal inducer of epithelial cyclin-dependent kinase inhibitors, and later in the termination of the enamel knot signaling functions by participating in the regulation of programmed cell death. These results show that the life history of the enamel knot is intimately linked to the initiation of tooth shape development and support the role of the enamel knot as an embryonic signaling center.

The relative safety of an oxygenator.

Analysis of prospectively registered incidents related to cardiopulmonary bypass (CPB) was initiated to establish the incident rate for 10 different oxygenator brands employed over a seven-year period in 5000 clinical perfusions. A general safety index (SI) was defined as the number of recorded incidents in a given series of oxygenators divided by its total number and multiplied by 100. Specific SI was calculated for each of the following categories: high-pressure drop, debris, gas exchange, leakage, material failure and oxygenator change-out. An SI of 0.2 was arbitrarily set as a reference and an acceptable safety level. An estimate of the relative risk for a particular oxygenator brand was compared with the reference by calculating the odd's ratio with a 95% confidence interval. The mean SI was determined to be 1.6, ranging from 0 for the Maxima CBAS and the Cobe CML to 2.92 for the Safe oxygenator. The dominating specific type of incident was HPD with an SI of 0.81 followed by debris, SI = 0.71. A systematic analysis of adverse events in CPB may be used to evaluate and to set standards, a method already employed in the pharmaceutical industry. Our results indicate that oxygenator safety margins may vary between different brands.

Clinical effects of the heparin coated surface in cardiopulmonary bypass.

OBJECTIVE: In a randomised study of 120 patients, undergoing primary operation for coronary heart decease, two groups were investigated as regards to the effects of heparin coated cardiopulmonary bypass on brain function parameters and general clinical outcome. The study group (n = 56) was perfused using an extra-corporeal circuit treated with covalent bonded heparin; the control group (n = 59) used an identical set-up without heparin treatment. Systemic heparin doses were calculated to achieve ACT levels of 250 and 500 s, respectively. Postoperative course was evaluated by examining a set of clinically relevant parameters including a detailed registry of postoperative deviations. Brain function was assessed by the biochemical marker S-100 and tests of memory performance. RESULTS: There were several signs of reduced operative trauma in the study group. Hospital stay was reduced by nearly 1 day (P < 0.05). Time on postoperative ventilatory support was approximately 4 h shorter (P = 0.009). Chest drain blood loss was decreased both at 8 (P = 0.01) and 24 h (P = 0.007) postoperatively. Body temperature was lower after surgery and especially on days 2 (P = 0.03) and 3 (P = 0.01). Perioperative creatinine elevation was significantly reduced (P = 0.03). Neurological deviations were fewer (P = 0.01). Brain function assessment revealed reduced plasma levels of S-100 both at termination of cardiopulmonary bypass (P = 0.008) and 7 h later (P = 0.04). However, no remediation of memory impairment could be demonstrated. CONCLUSIONS: Cardiopulmonary bypass with covalent bonded heparin attached to the extra-corporeal circuit in combination with a reduced systemic heparin dose seems to reduce safely and effectively the operative stress to the patient. There were also signs of improved cerebral protection.