A Prosthodontic Approach as a Complementary Solution for a Complicated Orthodontic Treatment of a Patient with Cleidocranial Dysplasia.

This clinical report describes a prosthodontic rehabilitation of a 29-year-old patient with cleidocranial dysplasia (CCD), who, after completing an orthodontic treatment, was not satisfied with the aesthetic outcome. Besides aesthetics, the patient complained about mastication muscles pain, and clicking while eating but was not aware about her unilateral open bite on the right side. The aim of this treatment was to improve smile appearance and patient's well-being, as well as to restore the proper occlusal vertical dimension (OVD) along with complete intercuspation and to establish masticatory function. The first phase of the treatment concentrated on eliminating the muscle pain and temporomandibular joint (TMJ) clicking with a repositioning splint. During the second phase, the functional and aesthetic rehabilitation was obtained using adhesive prosthesis overlays and veneers.

An initial evaluation of cytotoxicity, genotoxicity and antibacterial effectiveness of a disinfection liquid containing silver nanoparticles alone and combined with a glass-ionomer cement and dentin bonding systems.

BACKGROUND: Bacterial reinfection of dental cavities remains an unsolved clinical problem. The search for methods enabling the limitation of the bacterial factor has become the fundamental goal of the dental materials research. Silver nanoparticles (AgNPs) are used as disinfection agents. An incomplete polymerization of the polymer resins combined with AgNPs, along with the increase of the release of the unbound monomers, have been found. OBJECTIVES: The aim of this study was to evaluate the vitality of the human dental pulp stem cells (DPSCs) in response to a disinfection agent containing silver and gold nanoparticles (NPs), different bonding systems, glass-ionomer cement (GIC), and their combinations with the disinfection agent. Also, the influence of these materials both on the secretory function of DPSCs and on their antibacterial properties was established. MATERIAL AND METHODS: Cytotoxicity (MTT assay) and genotoxicity (enzyme-linked immunosorbent assay - ELISA) assays were used in the study. Antibacterial features were assessed with the optical density (OD) measurement of the bacteria (Streptococcus mutans, Streptococcus salivarius and Lactobacillus acidophilus) kept in dental materials. RESULTS: The disinfection liquid proved to be biocompatible. However, it relevantly interfered with the total-etch bonding system in terms of vitality, which may have serious clinical implications. Its combination with the self-etching system was biocompatible, yet it impaired the antibacterial action of the system. An enhancement of antibacterial action of GIC with AgNPs was found. CONCLUSIONS: The disinfection liquid and GIC were biocompatible toward the DPSCs in terms of cytotoxicity and genotoxicity. Simultaneous usage of AgNPs with other dental materials did not affect the biocompatibility of the used materials. The disinfection liquid and GIC acted as antibacterial agents against all studied bacteria species. Used together with GIC and the total-etch bonding system, the disinfection liquid seemed to be efficient toward bacteria, yet it relevantly impaired the antibacterial action of self-etching systems.

The Level of TWIST1 expression determines the response of colon cancer cells to mitogen-activated protein kinases inhibitors.

Background/Aim: Currently, it has been proposed that combination of 5-fluorouracil (5FU) with inhibitors of the mitogen-activated protein kinases (MAPKs) signaling pathway might enhance the efficacy of 5FU-based chemotherapy in colon cancer. Our study aimed to investigate an impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU and selected MAPK inhibitors. Materials and Methods: The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-sh TWIST1). The statistical calculation was performed with analysis of variance or Dunnett's test for comparison to control group. Paired Student's t-test was performed when two groups were analyzed. Results: Suppression of TWIST1 reduced the proliferation rate of colon cancer cells and enhanced their sensitivity to 5FU and MAPKs inhibitors. The sensitivity of HT29 cells to examined compounds was more dependent on TWIST1 expression level compared to HCT116 cells. The most noticeable effect of TWIST1 suppression on sensitivity of both colon cancer cell lines to combined treatment of 5FU and the MAPKs inhibitors was observed for inhibitors of p38α/ß and JNK1-3. We also noted that the suppression of TWIST1 significantly sensitized both cell lines to combined treatment of 5FU and Rac inhibitor. Conclusions: Our observations point to TWIST1 expression level as a marker of colon cancer sensitivity to combined treatment of 5FU and MAPKs inhibitors.

Overexpression of ID1 reverses the repression of human dental pulp stem cells differentiation induced by TWIST1 silencing.

Multiple studies showed that the cessation of TWIST1 expression is the prerequisite for osteoblasts' maturation. However, recent reports revealed that the function of TWIST1 is different in the dental pulp stem cells (DPSCs), where a high level of TWIST1 expression promoted DPSCs' differentiation. The aim of the study was to investigate the impact of TWIST1 and ID1 on the differentiation process in the human DPSCs. METHODS: TWIST1 and ID1 expression in the DSPCs was modulated by lentivirus transduction. Genes expression was assessed with qRT-PCR. The proteins level was evaluated by Western blot. The DPSCs differentiation was assessed with the proliferation, alkaline phosphatase (ALP) activity, and calcium concentration assays. RESULTS: TWIST1 silencing suppressed the expression of ID1 and both the early and late markers of odontoblasts' differentiation detected at the transcript and protein level. The forced overexpression of ID1 increased the expression of the late markers of odontoblasts differentiation but diminished the expression of the early markers. DPCSs with the silenced TWIST1 and subsequent ID1 overexpression displayed an increase in the expression of the late markers of odontoblasts differentiation. Cells with silenced TWIST1 and overexpressing ID1 had increased activity of ALP, higher calcium concentration and decreased proliferation rate. The high level of ID1 expression might be a critical factor stimulating DPSCs differentiation and it might compensate the repressed differentiation of DPSCs caused by TWIST1 silencing. CONCLUSION: The mutual correlation between the expression level of TWIST1 and ID1 might be a critical factor driving the process of the human odontoblasts' differentiation.

Suppression of ID1 expression in colon cancer cells increases sensitivity to 5-fluorouracil.

Adjuvant chemotherapy with 5-fluorouracil remains the basic treatment for patients with advanced colorectal carcinoma. The major obstacle in successful treatment is the ability of CRC cells to acquire chemoresistance. Here we examined the impact of ID1 silencing on the sensitivity of CRC cells to 5-FU. To suppress ID1 expression in HT-29 and HCT-116 cells the cells were transduced with a lentiviral vector carrying the ID1 silencing sequence. Cells with silenced ID1 showed altered expression of epithelial and mesenchymal markers and exhibited increased proliferation rate compared to the parental cells. HCT-116 cells with suppressed ID1 became sensitized to 5-FU and this was not observed in HT-29 cells. Silencing ID1 resulted in altered expression of genes encoding enzymes metabolizing 5-FU. HT-29 cells with suppressed ID1 had significantly reduced mRNA level for thymidine phosphorylase, uridine-cytydine kinase 2 and dihydropyrimidine dehydrogenase. ID1 suppression in HCT-116 cells resulted in an increase of mRNA level for thymidine phosphorylase, thymidine kinase and uridine-cytydine kinase 2 with concurrent drop of dihydropyrimidine dehydrogenase and thymidylate synthetase mRNA levels. In conclusion, ID1 expression impacts the sensitivity of colon cancer cells to 5-FU and may be considered as a potential predictive marker in CRC treatment.

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil.

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.

Differential effect of adenosine receptors on growth of human colon cancer HCT 116 and HT-29 cell lines.

The study aimed to evaluate the impact of adenosine receptors (ARs) on human colon tumor cells (HCT 116, HT-29) growth and sensitivity to 5-Fluorouracil (5-FU) an anticancer chemotherapeutic drug. The exposure of cancer cells to a selective A(3)-AR agonist (IB-MECA) resulted in an increase in HT-29 cells number, whereas the number of HCT 116 cells decreased significantly. In the presence of IB-MECA (1 µM) the percentage of apoptotic HT-29 cells significantly decreased, whereas the number of apoptotic and necrotic HCT 116 cells increased by 3- and 2,5-fold, respectively. The application of a selective A(2A)-AR agonist resulted in the increased survival of HCT 116 cells, but not HT-29 cells. The blockade of A(2A)-AR with ZM 241385 (0.1 µM) significantly increased the cytotoxicity of 5-FU (1 µM) in HCT 116 cells but not in HT-29 cells. The suppression of A(3)-AR with MRS 1523 (1 µM) increased the sensitivity of HT-29 cells to 5-FU while response of HCT 116 cells to 5-FU decreased. The growth promoting effect of IB-MECA in HT-29 cells was associated with the decreased intracellular cAMP level, whereas IB-MECA growth inhibitory effect in HCT 116 cells was abolished by okadaic acid (2 nM) indicating the involvement of protein phosphatase PP2A.

High glucose impairs ATP formation on the surface of human peripheral blood B lymphocytes.

Diabetes-associated lymphocyte dysfunction may be attributed to the direct effect of hyperglycemia, but the impact of glucose concentration on B cell functionality is not fully resolved. Since, adenosine 5'-triphosphate (ATP) and its metabolite adenosine are the core constituents of the purinergic signaling network involved in regulation of immune response we aimed to investigate the impact of high glucose concentration on ATP outflow and metabolism on B cell surface. Purified human peripheral blood B cells cultured at high glucose (25 mM) concentration released significantly less ATP (~60%) comparing to cells cultured in low glucose (5mM) concentration. We observed that high glucose altered ATP hydrolysis on B cell surface due to increased activity of nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39). In the presence of 10 µM [(3)H]AMP and 100 µM ATP significant quantities of [(3)H]ADP and [(3)H]ATP were generated, although the AMP to ADP phosphorylation potential of B cells cultured in high glucose decreased significantly. The flow cytometry analysis revealed that the level of ecto-adenylate kinase 1ß (AK1ß) on surface of B cells cultured in high glucose decreased significantly. Inhibition of NTPDase1/CD39 activity with 100 µM ARL67156 resulted in decreased cell viability, although significantly more viable cells retained in the culture media containing low glucose compared to high glucose media. Selective inhibition of P2X7 purinergic receptor irrespective of glucose concentration completely protected B cells against the ARL 67156-induced cell death. We assume that high glucose-induced alteration of ATP handling on B cell surface might contribute to impaired functionality of B cells in diabetes.

High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: involvement of P2X7 receptor.

AIMS/HYPOTHESIS: Patients with diabetes are more prone to bacterial infections mostly due to hyperglycemia-induced suppression of immune cells function. B lymphocytes by secreting antibodies inhibit microbial replication, but the impact of high glucose concentration on humoral immune response is not fully resolved. The aim of this work was to investigate the effect of high glucose concentration on B cells response to stimulation with a bacterial antigen and autocrine regulation. METHODS: Purified human peripheral blood B cells were cultured at different glucose concentrations and stimulated in vitro with Staphylococcus aureus Cowan I (SAC) plus IL-2. B cells proliferation, differentiation and IgM expression were analyzed by flow cytometry. B cell ATP release and involvement of P2 purinergic receptors in regulation of IgM secretion was assessed. RESULTS: B cells cultured at 25 mM glucose in response to SAC stimulation released significantly less (≈ 55%) IgM comparing to cells maintained in 5mM glucose. Under resting and stimulatory conditions B cells released significant quantities of ATP to the culture media, but ATP level decreased when B cells were maintain in high glucose. SAC-induced B cell IgM release was totally blocked by highly selective antagonist (Az11645373) of P2X7 receptor. IgM secretion increased in the presence of potent P2X7 receptor agonist (BzATP), but this effect was abolished by high glucose concentration. CONCLUSIONS/INTERPRETATION: High glucose concentration impairs B cell function by suppression of P2X7 receptor-dependent IgM release in response to in vitro bacterial antigen stimulation. This alteration may greatly contribute to the impaired humoral immune response in diabetics.

Antoni Cieszynski: a pioneering dentist.

Cieszynrski was a Polish dentist who formulated the rules of isometry (Cieszyrnski's isometry) in dental radiology, which enables precise dental X-ray pictures that show the true dimensions of teeth. Cieszynski also developed an extra-oral technique of anesthetizing the mandibular alveolar nerve, and a treatment for trigeminal neuralgia which involved injections of alcohol to the Gasser's ganglion. The clinical activity of Antoni Cieszynski included periodontal surgery (Cieszynski-Widmann-Neumann's operation) and surgery of abscesses and phlegmones of head and neck. He was also a member of numerous scientific societies (ADA, ASI, FDI) and a founder of the first Polish dental journal Polska Dentystyka (Polish Dentistry). On the 70th anniversary of Doctor Antoni Cieszynski's tragic death, the authors recall his outstanding and fruitful clinical career.

Hereditary dentine diseases resulting from mutations in DSPP gene.

OBJECTIVES: This review groups the newest results of molecular analyses of DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and tries to link the phenotypes with specific mutations in the DSPP gene. DATA: The review includes biochemical data introducing a specificity of DSPP protein which justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia. SOURCES: An electronic search was conducted in the databases of Pub Med and supplemented by manual study of relevant references. STUDY SELECTION: 52 out of 108 references were finally selected for the review based on the novelty and/or originality of data. CONCLUSION: Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 3' terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required.

Transcriptional repression of the Dspp gene leads to dentinogenesis imperfecta phenotype in Col1a1-Trps1 transgenic mice.

Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro-computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI.

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

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

Distinct compartmentalization of dentin matrix protein 1 fragments in mineralized tissues and cells.

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH(2)- and COOH-terminal fragments. The NH(2)-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH(2)- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH(2)- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH(2)-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH(2)-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH(2)-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.

Identification of full-length dentin matrix protein 1 in dentin and bone.

Dentin matrix protein 1 (DMP1) has been identified in the extracellular matrix (ECM) of dentin and bone as the processed NH(2)-terminal and COOH-terminal fragment. However, the full-length form of DMP1 has not been identified in these tissues. The focus of this investigation was to search for the intact full-length DMP1 in dentin and bone. We used two types of anti-DMP1 antibodies to identify DMP1: one type specifically recognizes the NH(2)-terminal region and the other type is only reactive to the COOH-terminal region of the DMP1 amino acid sequence. An approximately 105-kDa protein, extracted from the ECM of rat dentin and bone, was recognized by both types of antibodies; and the migration rate of this protein was identical to the recombinant mouse full-length DMP1 made in eukaryotic cells. We concluded that this approximately 105-kDa protein is the full-length form of DMP1, which is considerably less abundant than its processed fragments in the ECM of dentin and bone. We also detected the full-length form of DMP1 and its processed fragments in the extract of dental pulp/odontoblast complex dissected from rat teeth. In addition, immunofluorescence analysis showed that in MC3T3-E1 cells the NH(2)-terminal and COOH-terminal fragments of DMP1 are distributed differently. Our findings indicate that the majority of DMP1 must be cleaved within the cells that synthesize it and that minor amounts of uncleaved DMP1 molecules are secreted into the ECM of dentin and bone.

Distribution of SIBLING proteins in the organic and inorganic phases of rat dentin and bone.

The SIBLING protein family is a group of non-collagenous proteins (NCPs) that includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). In the present study, we compared these four proteins in different phases of rat dentin and bone. First, we extracted NCPs in the unmineralized matrices and cellular compartments using guanidium-HCl (G1). Second, we extracted NCPs closely associated with hydroxyapatite using an EDTA solution (E). Last, we extracted the remaining NCPs again with guanidium-HCl (G2). Each fraction of Q-Sepharose ion-exchange chromatography was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Stains-All stain, and with western immunoblotting. In dentin, the NH(2)-terminal fragment of DSPP and its proteoglycan form were primarily present in the G1 extract, whereas the COOH-terminal fragment of DSPP was present exclusively in the E extract. The processed NH(2)-terminal fragment of DMP1 was present in G1 and E extracts, whereas the COOH-terminal fragment of DMP1 existed mainly in the E extract. Bone sialoprotein was present in all three extracts of dentin and bone, whereas OPN was present only in the G1 and E extracts of bone. The difference in the distribution of the SIBLING proteins between organic and inorganic phases supports the belief that these molecular species play different roles in dentinogenesis and osteogenesis.

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study.

UNLABELLED: Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.