Abnormal bone regeneration induced by FK506 in medaka fin revealed by in vivo imaging.

OBJECTIVES: Bone tissue in bony fish demonstrates a remarkable ability to regenerate, particularly evident following induction of extensive bone defects, such as fin amputation. This regenerative capacity has been reported to be promoted by the immunosuppressant FK506, yet its precise effects on bone cells during fin regeneration remains insufficiently elucidated. This study aims to investigate the effects of FK506 treatment on bone morphology, osteoblasts, and osteoclasts in the bony fin rays of osterix promoter-DsRed/TRAP promoter-EGFP double transgenic (Tg) medaka. METHODS: The caudal fin of double Tg medaka was amputated, followed by a 20-day treatment with FK506 (1.0 µg/ml) to observe its effects on fin regeneration. Additionally, the regenerated caudal fin area underwent evaluation using genetic analysis and cell proliferation assays. RESULTS: FK506 treatment significantly increased osterix-positive osteoblast formation, resulting in both a significantly longer fin length and fewer joints in the bony fin rays formed during fin regeneration. Notably, TRAP-positive osteoclast formation and bone resorption were observed to occur primarily during the latter stages of fin regeneration. Furthermore, while the expression levels of osteoblast-related genes in the regenerated area remained unchanged following FK506 treatment, a heightened cell proliferation was observed at the tip of the fin. CONCLUSIONS: Our findings suggest that treatment with FK506 promotes bone regeneration by increasing the number of osteoblasts in the amputated area of the fin. However, long-term treatment disrupts regular bone metabolism by inducing abnormal osteoclast formation.

Tacrolimus, FK506, promotes bone formation in bone defect mouse model.

OBJECTIVES: Some studies have reported that tacrolimus (FK506), an immunosuppressant, may have positive effects on bone formation. However, the precise effects of FK506 on bone repair or osteoblasts remain inadequately elucidated, and limited research has explored the outcomes of its use in an in vivo mouse model. This study aims to examine the effects of FK506 on bone repair and osteoblast functions using bone defect and BMP-2-induced ectopic ossification mouse models, as well as cultured primary mouse osteoblasts treated with FK506. METHODS: We established mouse models of femur bone defect and BMP-2-induced ectopic ossification to evaluate the effect of FK506 on new bone formation, respectively. Additionally, primary mouse osteoblasts were cultured with FK506 and examined for gene expressions related to osteoblast differentiation. RESULTS: While FK506 promoted the repair of bone defect areas in the femur of the bone defect mouse model, it also led to widespread abnormal bone formation outside the intended area. Additionally, following the implantation of a collagen sponge containing BMP-2 into mouse muscle tissue, FK506 was found to promote ectopic ossification and enhance BMP-2-induced osteoblast differentiation in vitro. Our findings also revealed that FK506 increased the number of immature osteoblasts in the absence of BMP-2 without affecting osteoblast differentiation. Furthermore, direct effects were observed, reducing the ability of osteoblasts to support osteoclastogenesis. CONCLUSIONS: These results indicate that FK506 increases new bone formation during bone repair and influences the proliferation of immature osteoblasts, as well as osteoblast-supported osteoclastogenesis.

Planar catechin increases bone mass by regulating differentiation of osteoclasts in mice.

OBJECTIVES: While catechins have been reported to exhibit potential to benefit osteoporosis patients, the effects of planar catechin (PCat), synthesized during the development of drugs for Alzheimer's disease, have not been clearly elucidated. Here, we examined the effects of PCat on mouse bone metabolism both in vivo and in vitro. METHODS: Six week old female mice were orally administered PCat (30 mg/kg) every other day for four weeks, and their femurs were analyzed using micro-computed tomography imaging. Osteoclasts and osteoblasts were collected from mice and cultured with PCat. Subsequently, osteoclast formation and differentiation and osteoblast differentiation were observed. RESULTS: Mice orally administered PCat displayed significantly increased femur bone mass compared to the control group. Quantitative polymerase chain reaction findings indicated that PCat addition to osteoclast progenitor cultures suppressed osteoclast formation and decreased osteoclast marker expression without affecting the proliferative potential of the osteoclast progenitor cells. Addition of PCat to osteoblast cultures increased osteoblast marker expression. CONCLUSIONS: PCat inhibits osteoclast differentiation and promotes osteoblast differentiation, resulting in increased bone mass in mice. These results suggest that PCat administration is a promising treatment option for conditions associated with bone loss, including osteoporosis.

Effects of anti-RANKL antibodies administered to pregnant mice on bone and tooth development in neonates.

OBJECTIVES: This study examined how the anti-bone resorptive agent denosumab, which comprises anti-receptor activator of nuclear factor kappa B ligand (anti-RANKL) monoclonal antibodies, administered during pregnancy affected neonatal development. Anti-RANKL antibodies, which are known to bind to mouse RANKL and inhibit osteoclast formation, were administered to pregnant mice. Following this, the survival, growth, bone mineralization, and tooth development of their neonates were analyzed. METHODS: Anti-RANKL antibodies (5 mg/kg) were injected into pregnant mice on day 17 of gestation. After parturition, their neonatal offspring underwent microcomputed tomography at 24 h and at 2, 4, and 6 weeks after birth. Three-dimensional bone and teeth images were subjected to histological analysis. RESULTS: Approximately 70% of the neonatal mice born to mice who received anti-RANKL antibodies died within 6 weeks after birth. These mice had a significantly lower body weight and significantly higher bone mass compared with the control group. Furthermore, delayed tooth eruption and abnormal tooth morphology (eruption length, enamel surface, and cusps) were observed. Conversely, while the tooth germ shape and mothers against decapentaplegic homolog 1/5/8 expression remained unchanged at 24 h after birth in the neonatal mice born to mice that received anti-RANKL antibodies, osteoclasts were not formed. CONCLUSIONS: These results suggest that anti-RANKL antibodies administered to mice in the late stage of pregnancy results in adverse events in their neonatal offspring. Thus, it is speculated that administering denosumab to pregnant humans will affect fetal development and growth after birth.

Cholesterol and fat in diet disrupt bone and tooth homeostasis in mice.

Dyslipidemia is a condition of high levels of triglycerides and cholesterol in the blood, and high levels of cholesterol is associated with a variety of systemic diseases. The effects of a high-fat diet on bone have been reported, however, it is not clear which components of a high-fat diet affect bone. This study was conducted to examine the effects of dietary lipids and cholesterol on bone homeostasis maintenance. Eight-week-old male mice (C57BL/6 J) were fed five types of feed with different amounts of fat (14 %, 36 %) and cholesterol (0.01 %, 1.25 %, 5 %) for 12 weeks. Blood, femur, tibia, and tooth samples were examined, and serum lipid markers and bone morphology were determined using µCT and histological analysis. Additionally, bone marrow cells were obtained and cultured, and osteoclast differentiation markers analyzed using qPCR. Mice fed a diet high in both fat (36 %) and cholesterol (1.25 %) showed increased total cholesterol and low-density lipoprotein levels in blood, and decreased bone volume fraction as compared to the standard diet group. However, bone mass was unaffected in the high fat only (36 %) and high cholesterol only (1.25 %, 5 %) groups. Mice given a high fat (36%) diet also demonstrated significantly narrowed incisor pulp. In contrast, osteoclast formation was not significantly different among the groups. These results suggest that a diet with high amounts of both fat and cholesterol induces bone loss.

Effects of Anti-Receptor Activator of Nuclear Factor Kappa B Ligand Antibody and Zoledronic Acid on Periapical Lesion Development in Mice.

INTRODUCTION: Antiresorptive drugs are widely used to treat osteoporosis and other systemic bone diseases, although their efficacy for local bone resorption after localized inflammation has not been fully elucidated. We examined the effects of an anti-receptor activator of nuclear factor kappa B ligand (RANKL) antibody and the bisphosphonate zoledronic acid (ZOL) on periapical lesion (PL) development in mice. METHODS: Dental pulp of lower first molars in mice was removed, with the exposed dental pulp chambers left open to the oral environment to induce apical periodontitis. An anti-RANKL antibody or ZOL was intraperitoneally injected once per week until postoperative day 21, and then micro-computed tomographic imaging and histologic analyses were performed. RESULTS: PL enlargement was inhibited by both the anti-RANKL antibody and ZOL in a dose-dependent manner, and a reduction of inflammatory cell infiltration in apical tissues inhibited periapical bone resorption. The anti-RANKL antibody decreased the number of osteoclasts in periapical tissues, whereas ZOL suppressed periapical bone resorption with osteoclast numbers maintained. Although the administration of each of the antiresorptive drugs increased femoral bone mass, femoral bone mineral density in the PL group was lower compared with the sham-operated group. CONCLUSIONS: These results suggest that an antiresorptive drug administered systemically is distributed to areas of local inflammation in the jaw can prevent PL development.

Neural crest-derived cells possess differentiation potential to keratinocytes in the process of wound healing.

Neural crest-derived cells (NCDCs), which exist as neural crest cells during the fetal stage and differentiate into palate cells, also exist in adult palate tissues, though with unknown roles. In the present study, NCDCs were labeled with EGFP derived from P0-Cre/CAG-CAT-EGFP (P0-EGFP) double transgenic mice, then their function in palate mucosa wound healing was analyzed. As a palate wound healing model, left-side palate mucosa of P0-EGFP mice was resected, and stem cell markers and keratinocyte markers were detected in healed areas. NCDCs were extracted from normal palate mucosa and precultured with stem cell media for 14 days, then were differentiated into keratinocytes or osteoblasts to analyze pluripotency. The wound healing process started with marginal mucosal regeneration on day two and the entire wound area was lined by regenerated mucosa with EGFP-positive cells (NCDCs) on day 28. EGFP-positive cells comprised approximately 60% of cells in healed oral mucosa, and 65% of those expressed stem cell markers (Sca-1+, PDGFRα+) and 30% expressed a keratinocyte marker (CK13+). In tests of cultured palate mucosa cells, approximately 70% of EGFP-positive cells expressed stem cell markers (Sca-1+, PDGFRα+). Furthermore, under differentiation inducing conditions, cultured EGFP-positive cells were successfully induced to differentiate into keratinocytes and osteoblasts. We concluded that NCDCs exist in adult palate tissues as stem cells and have potential to differentiate into various cell types during the wound healing process.

Novel gene Merlot inhibits differentiation and promotes apoptosis of osteoclasts.

Extended osteoclast longevity is deeply involved in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis, though the mechanisms that determine osteoclast lifespan are not fully understood. Here we present findings indicating that the newly characterized gene Merlot, which encodes a highly conserved yet uncharacterized protein in vertebrates, is an important regulator for termination of osteoclastogenesis via induction of apoptosis. Mice lacking Merlot exhibited low bone mass due to increased osteoclast and bone resorption. Furthermore, osteoclast precursors overexpressing Merlot failed to differentiate into mature osteoclasts, while Merlot deficiency led to hyper-nucleation and prolonged survival of osteoclasts, accompanied by sustained nuclear localization of nuclear factor of activated T cell c1 (NFATc1) and derepression of glycogen synthase kinase-3ß (GSK3ß) activity, known to regulate NFATc1 activity and induce apoptosis. Merlot-deficient osteoclasts were found to represent suppression of caspase-3-mediated apoptosis and Merlot deficiency caused transcriptional downregulation of a proapoptotic cascade, including Bax, Bak, Noxa, and Bim, as well as the executor caspase members Casp-3, -6, and -7, and upregulation of anti-apoptotic Bcl2, resulting in a low apoptotic threshold. Thus, Merlot regulates osteoclast lifespan by inhibition of differentiation and simultaneous induction of apoptosis via regulation of the NFATc1-GSK3ß axis.

Effects of N-methyl-d-aspartate receptor antagonist MK-801 (dizocilpine) on bone homeostasis in mice.

OBJECTIVES: To gain insight into the role of the N-methyl-d-aspartate (NMDA) receptor in bone metabolism by examining the effects of its noncompetitive antagonist, MK-801 (dizocilpine), on bone homeostasis and bone healing in mice. METHODS: MK-801 (2.5 mg/kg) or saline (in control groups) was intravenously administered to healthy mice and mice with bone-defects daily for seven to 14 days. Bone defects were artificially created in femurs using a drill and reamer. Following euthanasia, bones were extracted and processed for microcomputed tomography (µCT) and histological analyses. The effects of MK-801 on osteoclast differentiation by bone marrow macrophages (BMMs) were examined in vitro. mRNA expressionlevels of Grin3b levels were also examined using reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: Bone volume was significantly decreased in mice administered MK-801 for 14 days. Additionally, the number of osteoclasts was reduced, while number of osteoblasts and rate of bone formation were increased in these mice. MK-801 inhibited osteoclast differentiation dose-dependently in vitro. RT-PCR findings suggested expression of Grin3b, a subunit of the NMDA receptor, in BMMs. During the healing process of artificially created defects in femurs, no significant differences were found between the control and MK-801-treated groups, indicating no stimulatory or inhibitory effects by MK-801 administration. CONCLUSIONS: These results indicate that blockade of the NMDA receptor by MK-801 administration affects bone metabolism but not the healing process of artificial bone defects.

Effects of lipid metabolism on mouse incisor dentinogenesis.

Tooth formation can be affected by various factors, such as oral disease, drug administration, and systemic illness, as well as internal conditions including dentin formation. Dyslipidemia is an important lifestyle disease, though the relationship of aberrant lipid metabolism with tooth formation has not been clarified. This study was performed to examine the effects of dyslipidemia on tooth formation and tooth development. Dyslipidemia was induced in mice by giving a high-fat diet (HFD) for 12 weeks. Additionally, LDL receptor-deficient (Ldlr-/-) strain mice were used to analyze the effects of dyslipidemia and lipid metabolism in greater detail. In the HFD-fed mice, incisor elongation was decreased and pulp was significantly narrowed, while histological findings revealed disappearance of predentin. In Ldlr-/- mice fed regular chow, incisor elongation showed a decreasing trend and pulp a narrowing trend, while predentin changes were unclear. Serum lipid levels were increased in the HFD-fed wild-type (WT) mice, while Ldlr-/- mice given the HFD showed the greatest increase. These results show important effects of lipid metabolism, especially via the LDL receptor, on tooth homeostasis maintenance. In addition, they suggest a different mechanism for WT and Ldlr-/- mice, though the LDL receptor pathway may not be the only factor involved.

Myelination during fracture healing in vivo in myelin protein zero (p0) transgenic medaka line.

During the fracture healing process, osteoblasts and osteoclasts, as well as the nervous system are known to play important roles for signaling in the body. Glia cells contribute to the healing process by myelination, which can increase the speed of signals transmitted between neurons. However, the behavior of myelinating cells at a fracture site remains unclear. We developed a myelin protein zero (mpz)-EGFP transgenic medaka line for tracing myelinating cells. Mpz-enhanced green fluorescence protein (EGFP)-positive (mpz+) cells are driven by the 2.9-kb promoter of the medaka mpz gene, which is distributed throughout the nervous system, such as the brain, spinal cord, lateral line, and peripheral nerves. In the caudal fin region, mpz+ cells were found localized parallel with the fin ray (bone) in the adult stage. mpz+ cells were not distributed with fli-DsRed positive (fli+) blood vessels, but with some nerve fibers, and were dyed with the anti-acetylated tubulin antibody. We then fractured one side of the caudal lepidotrichia in a caudal fin of mpz-EGFP medaka and found a unique phenomenon, in that mpz+ cells were accumulated at 1 bone away from the fracture site. This mpz+ cell accumulation phenomenon started from 4 days after fracture of the proximal bone. Thereafter, mpz+ cells became elongated from the proximal bone to the distal bone and finally showed a crosslink connection crossing the fracture site to the distal bone at 28 days after fracture. Finally, the effects of rapamycin, known as a mTOR inhibitor, on myelination was examined. Rapamycin treatment of mpz-EGFP/osterix-DsRed double transgenic medaka inhibited not only the crosslink connection of mpz+ cells but also osterix+ osteoblast accumulation at the fracture site, accompanied with a fracture healing defect. These findings indicated that mTOR signaling plays important roles in bone formation and neural networking during fracture healing. Taken together, the present results are the first to show the dynamics of myelinating cells in vivo.

Biological Effects of Anti-RANKL Antibody and Zoledronic Acid on Growth and Tooth Eruption in Growing Mice.

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

Bone loss caused by dopaminergic degeneration and levodopa treatment in Parkinson's disease model mice.

Accumulating evidence have shown the association of Parkinson's disease (PD) with osteoporosis. Bone loss in PD patients, considered to be multifactorial and a result of motor disfunction, is a hallmark symptom that causes immobility and decreased muscle strength, as well as malnutrition and medication. However, no known experimental evidence has been presented showing deleterious effects of anti-PD drugs on bone or involvement of dopaminergic degeneration in bone metabolism. Here, we show that osteoporosis associated with PD is caused by dopaminergic degeneration itself, with no deficit of motor activity, as well as treatment with levodopa, the current gold-standard medication for affected patients. Our findings show that neurotoxin-induced dopaminergic degeneration resulted in bone loss due to accelerated osteoclastogenesis and suppressed bone formation, which was associated with elevated prolactin. On the other hand, using an experimental model of postmenopausal osteoporosis, dopaminergic degeneration did not result in exacerbation of bone loss due to estrogen deficiency, but rather reduction of bone loss. Thus, this study provides evidence for the regulation of bone metabolism by the dopaminergic system through both gonadal steroid hormone-dependent and -independent functions, leading to possible early detection of osteoporosis development in individuals with PD.

Treatment with synthetic glucocorticoid impairs bone metabolism, as revealed by in vivo imaging of osteoblasts and osteoclasts in medaka fish.

Glucocorticoids (GCs) play an important role in the stress reaction and function in the development of multiple tissues in our body. When given chronically in supraphysiologic doses, GCs are associated with orthodontic tooth movement, with serious side effects and particularly adverse effects on bone metabolism. However, the effects of steroids on bone cell dynamics are incompletely understood. Therefore, in this present study we examined the participation of osteoblasts and osteoclasts in osterix-DsRed/TRAP-EGFP double transgenic (Tg) medaka treated with synthetic GCs. Chronic continuous administration of prednisolone (PN) significantly reduced the fluorescence signals in the whole body of both osterix-DsRed and TRAP-EGFP medaka at 18 days, and those of the pharyngeal bone and tooth region at 32 days. To examine the capacity of the medaka for fracture healing during chronic administration of PN, we caused a fracture of a part of the bony fin ray at 18 days after the initiation of PN continuous administration. The bone fracture healing was significantly delayed by 32 days, accompanied by decreased signal area of both osterix-DsRed and TRAP-EGFP compared with that of the control. Next, to investigate the effect of acute administration of GC on the fracture healing, we started administration of dexamethasone (DX) immediately after the bone fracture, and this administration lasted during the 11 days of fracture healing. The results showed that the TRAP-EGFP-positive osteoclasts were reduced in area, but not the osterix-DsRed-positive osteoblasts. Lastly, to confirm the function of the glucocorticoid receptor in bone healing, we generated glucocorticoid receptor 2-deficient medaka (gr2-/-). The fluorescent signal area of osterix-DsRed and TRAP-EGFP were increased at bone fracture sites in these fish, and DX treatment of them decreased the TRAP-EGFP signal area compared with that for the control fish. Our results indicate that GRs negatively regulated osteoclast recruitment and the accumulation of osteoblasts in bone fracture healing.

In Vitro Study of the Effects of Denosumab on Giant Cell Tumor of Bone: Comparison with Zoledronic Acid.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that contains numerous osteoclasts formed from marrow-derived precursors through receptor activator of nuclear factor κ-B ligand (RANKL), an osteoclast differentiation factor expressed in neoplastic cells of GCTB. Denosumab, a fully human monoclonal antibody targeting RANKL, has recently been used for the treatment of GCTB, and superior treatment effects have been reported. The aim of this work was to elucidate the mechanism of action of denosumab, and the differences between denosumab and zoledronic acid at the level of GCTB cells. We isolated GCTB cells from 3 patients and separated them into osteoclasts, osteoclast precursors and proliferating spindle-shaped stromal cells (the true neoplastic component), and examined the action of denosumab on differentiation, survival and bone resorption activity of osteoclasts. Denosumab and zoledronic acid inhibited osteoclast differentiation from mononuclear cells containing osteoclast precursors. Zoledronic acid inhibited osteoclast survival, whereas an inhibitory effect of denosumab on osteoclast survival was not observed. The inhibitory effect on bone resorption by both agents was confirmed in culture on dentin slices. Furthermore, zoledronic acid showed dose-dependent inhibition of cell growth of neoplastic cells whereas denosumab had no inhibitory effect on these cells. Denosumab has an inhibitory effect on osteoclast differentiation, but no inhibitory effects on survival of osteoclasts or growth of neoplastic cells in GCTBs.

Anti-mouse RANKL Antibodies Inhibit Alveolar Bone Destruction in Periodontitis Model Mice.

Denosumab is an anti-bone resorptive drug consisting of complete human monoclonal antibodies that targets receptor activator of nuclear factor κB ligand (RANKL), which is responsible for osteoclast formation. The drug has been adapted for bone diseases, such as osteoporosis and bone metastasis related to cancer, but is not used for alveolar bone destruction related to periodontitis. In the present study, we aimed to clarify whether denosumab prevents bone destruction associated with lipopolysaccharide (LPS)-induced calvaria inflammation and experimental periodontitis in model mice. Denosumab does not bind to mouse RANKL, thus we used anti-mouse monoclonal RANKL antibodies. We also examined the inhibitory effects toward bone destruction of another anti-bone resorptive drug zoledronate, a nitrogen-containing bisphosphonate. Local administration of anti- RANKL antibodies into the calvaria area inhibited LPS-induced osteoclast formation and bone destruction, while zoledronate inhibited bone destruction but not osteoclast formation due to its different action mechanism. In periodontitis model mice, in which the second molars were ligated with a silk suture to induce inflammation, intraperitoneal administration of anti-RANKL antibodies significantly inhibited alveolar bone destruction and tooth root exposure. On the other hand, zoledronate only weakly repressed alveolar bone destruction and failed to inhibit root exposure. These results suggest that denosumab is a promising candidate to prevent alveolar bone destruction associated with periodontitis.

Biological effects of anti-RANKL antibody administration in pregnant mice and their newborns.

Denosumab, a fully human monoclonal antibody that neutralizes receptor activator of nuclear factor-κB ligand (RANKL) and blocks osteoclast differentiation, has received approval in Japan for use as an anti-resorptive drug for osteoporosis and skeletal-related events (SREs) in patients with solid cancer. Denosumab is contraindicated during pregnancy, though the effects of blocking RANKL activity on pregnant mothers and their newborns are unclear. We used mice to investigate the effects of an anti-RANKL antibody on maternal and newborn health. Mothers injected with the anti-RANKL antibody had increased bone mass as compared with the controls, while osteoclast number and the level of tartrate-resistant acid phosphatase (TRAP) in serum were increased at the end of pregnancy. Newborn mice exposed to the antibody in utero were normally born, but showed increased bone mass and died within 48 h after birth. None of the newborns were found to have milk in their stomachs, suggesting that they died due to a maternal defect in lactation. Consistent with this, anti-RANKL antibody-injected mothers displayed impaired mammary gland development. However, fostering by healthy surrogate mothers rescued only 33% of the antibody-exposed newborns, suggesting that neonatal mortality was due, at least in part, to an intrinsic defect in the newborns. Our findings show that anti-RANKL antibody administration during pregnancy results in not only an undesirable increase in bone mass, but also has harmful effects on newborn survival.

Effects of tetracycline hydrochloride on measurements with the laser fluorescence device DIAGNOdent: in vitro and in vivo studies.

Dental materials that fluoresce affect the reading of the laser fluorescence device DIAGNOdent. Tetracycline hydrochloride (TCH) shows fluorescence and is retained in teeth. The aim of this study was to evaluate the effects of TCH on the DIAGNOdent reading. Filter-paper discs that contained various amounts of TCH were prepared (0.16-10 mg per disc). One-day-old newborn rats were subcutaneously injected with TCH for 29 days, and their mandibles were then removed. The DIAGNOdent values (D-V) of the discs and first molars of the rats were measured before and after they were subjected to ultraviolet irradiation (UV). The D-V of discs containing TCH increased depending on the amount of TCH. The D-Vs of discs with lower amounts of TCH (0.16-1.25 mg) were approximately 10-15, and these values increased to 30-40 under UV. In addition, the D-Vs of molars after UV were twofold greater than those before UV. These results suggest that TCH might affect the readings obtained by DIAGNOdent.