Radiographic diagnoses in 80 cats before and 73 cats after unobstructing the urethra.

OBJECTIVE: The aim of this study was to investigate causes for feline urethral obstruction and determine whether the frequency of radiographic diagnoses differs between cats radiographed before or after unobstruction of the urethra. MATERIALS AND METHODS: A retrospective cross-sectional study of cats with naturally occurring urethral obstruction was performed. Only cats presenting for their first urethral obstruction in which radiography was integrated in the initial evaluation were included. The diagnosis frequency (overall and for each disease type) was compared between cats radiographed before or after unobstruction of the urethra. RESULTS: Eighty cats (52%) had radiographs obtained before unobstructing the urethra and 73 cats (48%) had radiographs taken after unobstructing the urethra. Cats radiographed before unobstruction had a greater frequency of radiographic diagnoses than those radiographed after unobstruction (61% versus 45%). This difference was largely due to a greater frequency of urethral plugs detected before unobstruction versus after unobstruction (45% versus 5.5%). CLINICAL SIGNIFICANCE: Radiographs obtained before unobstructing the urethra provided a diagnostic advantage for detecting a cause for urethral obstruction compared to radiographs obtained after unobstructing the urethra. Urethral plugs were the most common diagnosis.

Temporal regulation of the Mre11-Rad50-Nbs1 complex during adenovirus infection.

Adenovirus infection induces a cellular DNA damage response that can inhibit viral DNA replication and ligate viral genomes into concatemers. It is not clear if the input virus is sufficient to trigger this response or if viral DNA replication is required. Adenovirus has evolved two mechanisms that target the Mre11-Rad50-Nbs1 (MRN) complex to inhibit the DNA damage response. These include E4-ORF3-dependent relocalization of MRN proteins and E4-ORF6/E1B-55K-dependent degradation of MRN components. The literature suggests that degradation of the MRN complex due to E4-ORF6/E1B-55K does not occur until after viral DNA replication has begun. We show that, by the time viral DNA accumulates, the MRN complex is inactivated by either of the E4-induced mechanisms and that, with E4-ORF6/E1B-55K, this inactivation is due to MRN degradation. Our data are consistent with the conclusion that input viral DNA is sufficient to induce the DNA damage response. Further, we demonstrate that when the DNA damage response is active in E4 mutant virus infections, the covalently attached terminal protein is not cleaved from viral DNAs, and the viral origins of replication are not detectably degraded at a time corresponding to the onset of viral replication. The sequences of concatemeric junctions of viral DNAs were determined, which supports the conclusion that nonhomologous end joining mediates viral DNA ligation. Large deletions were found at these junctions, demonstrating nucleolytic procession of the viral DNA; however, the lack of terminal protein cleavage and terminus degradation at earlier times shows that viral genome deletion and concatenation are late effects.

Solid Cherenkov detector for studying nucleosynthesis in inertial confinement fusion.

Measuring gamma rays emitted from nuclear reactions gives insight into their nuclear structure. Notably, there are several nuclear reactions that produce gamma rays at ∼1 MeV-3 MeV energies such as T(4He, γ)7Li, 4He(3He, γ)7Be, and 12C(p, γ)13N, which may solve questions lingering about big-bang nucleosynthesis and stellar nucleosynthesis. To observe 1 MeV-3 MeV gamma rays in an inertial confinement fusion system, a new style of the Cherenkov detector was developed using aerogel and fused silica as a Cherenkov medium. Utilizing the OMEGA laser facility, both aerogel and fused silica media were compared with the existing gas-medium Cherenkov detector to validate the concept. Gamma ray measurements from high yield inertial confinement fusion implosions (deuterium-tritium and deuterium-3He) demonstrated that aerogel and fused silica were viable Cherenkov media, paving the way for a potential optimized detector to make these cross section measurements on OMEGA or the National Ignition Facility.

Progress on next generation gamma-ray Cherenkov detectors for the National Ignition Facility.

Fusion reaction history and ablator areal density measurements for Inertial Confinement Fusion experiments at the National Ignition Facility are currently conducted using the Gamma Reaction History diagnostic (GRH_6m). Future Gas Cherenkov Detectors (GCDs) will ultimately provide ∼100x more sensitivity, reduce the effective temporal response from ∼100 to ∼10 ps, and lower the energy threshold from 2.9 to 1.8 MeV, relative to GRH_6m. The first phase toward next generation GCDs consisted of inserting the existing coaxial GCD-3 detector into a reentrant well which puts it within 4 m of the implosion. Reaction history and ablator gamma measurement results from this Phase I are discussed here. These results demonstrate viability for the follow-on Phases of (II) the use of a revolutionary new pulse-dilation photomultiplier tube to improve the effective measurement bandwidth by >10x relative to current PMT technology; and (III) the design of a NIF-specific "Super" GCD which will be informed by the assessment of the radiation background environment within the well described here.

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

Wnt-1 induces growth, cytosolic beta-catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts.

Genetic evidence suggests that regulation of beta-catenin and regulation of Tcf/Lef family transcription factors are downstream events of the Wnt signal transduction pathway. However, a direct link between Wnt activity and Tcf/Lef transcriptional activation has yet to be established. In this study, we show that Wnt-1 induces a growth response in a cultured mammalian cell line, Rat-1 fibroblasts. Wnt-1 induces serum-independent cellular proliferation of Rat-1 fibroblasts and changes in morphology. Rat-1 cells stably expressing Wnt-1 (Rat-1/Wnt-1) show a constitutive up-regulation of cytosolic beta-catenin, while membrane-associated beta-catenin remains unaffected. Induction of cytosolic beta-catenin in Rat-1/Wnt-1 cells is correlated with activation of a Tcf-responsive transcriptional element. We thus provide evidence that Wnt-1 induces Tcf/Lef transcriptional activation in a mammalian system. Expression of a mutant beta-catenin (beta-CatS37A) in Rat-1 cells does not result in a proliferative response or a detectable change in the cytosolic beta-catenin protein level. However, beta-CatS37A expression in Rat-1 cells results in strong Tcf/Lef transcriptional activation, comparable to that seen in Wnt-1-expressing cells. These results suggest that Wnt-1 induction of cytosolic beta-catenin may have functions in addition to Tcf/Lef transcriptional activation.

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

Nonhomologous recombination in human cells.

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

Catalase enrichment using recombinant adenovirus protects alphaTN4-1 cells from H(2)O(2).

Since oxidative stress has been implicated in the development of numerous diseases including cataract, this laboratory has created and investigated the stress response of murine immortal lens epithelial cell lines (alphaTN4-1) conditioned to withstand lethal peroxide concentrations. Two of a group of antioxidative defense (AOD) enzymes found in such cells to have markedly enhanced activity are catalase (CAT) and GSH S-transferase alpha2 (GST). In order to determine if enrichment of one or both of these AODs is sufficient to protect alphaTN4-1 cells from lethal H(2)O(2) levels, these cells were infected with adenovirus vectors capable of expressing these AODs at a high level. With this system, gene enrichment and increased enzyme activity were observed with both CAT and GST vectors. The percentage of cells infected ranged from about 50 to 90% depending on the multiplicity of infection (MOI). CAT but not GST protected the cells from H(2)O(2) stress. The CAT activity was increased from 15- to 150-fold and even at the lower levels protected the cells from H(2)O(2) concentrations as high as 200 microM or more (H(2)O(2) levels which rapidly kill non-enriched cells). Even when only about 50% of the cell population is infected as judged by GFP infection, the entire population appeared to be protected based on cell viability. The CAT enrichment appears to protect other intracellular defense systems such as GSH from being depleted in contrast to non-enriched cell populations where GSH is rapidly exhausted. The overall results suggest that enriching the cellular CAT gene level with an appropriate recombinant viral vector may be sufficient to protect in vivo systems from peroxide stress.

Aerogel Cherenkov detector for characterizing the intense flash x-ray source, Cygnus, spectrum.

An aerogel Cherenkov detector is proposed to measure the X-ray energy spectrum from the Cygnus-intense flash X-ray source operated at the Nevada National Security Site. An array of aerogels set at a variety of thresholds between 1 and 3 MeV will be adequate to map out the bremsstrahlung X-ray production of the Cygnus, where the maximum energy of the spectrum is normally around 2.5 MeV. In addition to the Cherenkov radiation from aerogels, one possible competing light-production mechanism is optical transition radiation (OTR), which may be significant in aerogels due to the large number of transitions from SiO2 clusters to vacuum voids. To examine whether OTR is a problem, four aerogel samples were tested using a mono-energetic electron beam (varied in the range of 1-3 MeV) at NSTec Los Alamos Operations. It was demonstrated that aerogels can be used as a Cherenkov medium, where the rate of the light production is about two orders magnitude higher when the electron beam energy is above threshold.

Next generation gamma-ray Cherenkov detectors for the National Ignition Facility.

The newest generation of Gas Cherenkov Detector (GCD-3) employed in Inertial Confinement Fusion experiments at the Omega Laser Facility has provided improved performance over previous generations. Comparison of reaction histories measured using two different deuterium-tritium fusion products, namely gamma rays using GCD and neutrons using Neutron Temporal Diagnostic (NTD), have provided added credibility to both techniques. GCD-3 is now being brought to the National Ignition Facility (NIF) to supplement the existing Gamma Reaction History (GRH-6m) located 6 m from target chamber center (TCC). Initially it will be located in a reentrant well located 3.9 m from TCC. Data from GCD-3 will inform the design of a heavily-shielded "Super" GCD to be located as close as 20 cm from TCC. It will also provide a test-bed for faster optical detectors, potentially lowering the temporal resolution from the current ∼100 ps state-of-the-art photomultiplier tubes (PMT) to ∼10 ps Pulse Dilation PMT technology currently under development.

Ionizing radiation modulates vascular endothelial growth factor (VEGF) expression through multiple mitogen activated protein kinase dependent pathways.

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcription factor complex.

The structure and function of the adenovirus major late promoter.

The adenovirus major late promoter (MLP) has played a pre-eminent role in the analysis of transcription initiation in mammalian cells, and is an outstanding example of the ways in which the study of adenovirus has led to fundamental insights into general cellular processes. The aim of this chapter is to give a comprehensive review of the structure and function of this model mammalian promoter. After a brief description of late transcription in the adenovirus replication cycle, the experimental evidence for the current consensus on the genetic structure of the MLP, including a consideration of non-primate adenovirus MLPs, will be reviewed. Next, the functions of the MLP in the viral life cycle will be examined, and some of the problems that remain to be resolved will be addressed. The review ends with some ideas on how the knowledge of the structure and function of the MLP can be used in designing virus vectors for specific experimental purposes.

Developmental analysis and computer modelling of bioengineered teeth.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

Sequence of the mouse adenovirus serotype-1 DNA encoding the precursor to capsid protein VI.

The nucleotide sequence predicted to encode the precursor to virion structural protein VI (preVI) of mouse adenovirus (Ad) serotype-1 (MAV-1) was determined. The 237-amino-acid sequence has 45% identity and 66% similarity to the human Ad serotype-2 preVI sequence. There is a marked conservation at the C terminus, the last eleven residues of which may be necessary for activating the Ad endoproteinase, and at the N terminus, including the consensus endoproteinase cleavage site.

Biochemical studies of homologous and nonhomologous recombination in human cells.

Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.

Studies on the mechanism of histamine-induced release of noradrenaline and 5-hydroxytryptamine from slices of rat cerebral cortex.

The effect of histamine on the release of endogenous noradrenaline and 5-hydroxytryptamine (5-HT) has been examined in slices of rat cerebral cortex. Histamine was found to produce a marked release of both amines from rat cerebral cortex at concentrations between 0.1 and 1 mM. This response to histamine was relatively resistant to removal of calcium ions from the incubation medium when compared to the release evoked by depolarising potassium stimuli. The response to 1 mM histamine was not, however, significantly inhibited by the H1-antagonist mepyramine (1 microM) or the H2-antagonist cimetidine (100 microM). Furthermore, impromidine which is both a potent H2-agonist and a potent H3-antagonist was without effect on the basal and histamine-stimulated release of endogenous noradrenaline and 5-HT. The response to histamine was, however, significantly attenuated by nisoxetine, fluoxetine and imipramine which are inhibitors of the amine uptake systems. The results of this study show that high concentrations (0.1 to 1 mM) of histamine can produce a marked increase in the release of endogenous 5-HT and noradrenaline from rat cerebral cortex, apparently via a non-receptor mechanism. This effect will need to be borne in mind in interpreting biochemical and behavioral responses to histamine in this concentration range.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Bioengineered teeth from cultured rat tooth bud cells.

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.