Efficacy of Sodium-Glucose Cotransporter 2 Inhibitors on Outcomes After Catheter Ablation for Atrial Fibrillation.

BACKGROUND: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have recently been a significant focus of attention because of their multiple pleiotropic effects. However, the impact of SGLT2i on atrial fibrillation (AF) remains unclear. OBJECTIVES: The goal of this study was to examine the effects of SGLT2i on AF after catheter ablation (CA). METHODS: This prospective, randomized controlled study compared the suppressive effect of SGLT2i vs dipeptidyl peptidase-4 inhibitors on AF recurrence after CA. Eighty AF patients with type 2 diabetes mellitus were randomized (by a computer-generated random sequence) to the tofogliflozin group (20 mg/d) or the anagliptin group (200 mg/d) stratified according to left atrial diameter and AF type (paroxysmal AF [PAF] or non-paroxysmal atrial fibrillation [PAF]) at screening. The primary outcome was AF recurrence at 12 months after CA. RESULTS: Seventy patients were analyzed (mean age 70.3 ± 8.1 years; 48 male; 30 with paroxysmal AF; 38 tofogliflozin treated). Recurrent AF was detected in 24 (34.3%) of 70 patients, and the AF recurrence ratio was higher in the anagliptin group than in the tofogliflozin group (15 of 32 patients [47%] vs 9 of 38 patients [24%]; P = 0.0417). Moreover, univariate analysis revealed that compared with the nonrecurrence group (n = 46), the recurrence group (n = 24) had a higher prevalence rate of non-PAF, elevated brain natriuretic peptide, higher urinary albumin-creatinine ratio, lower rate of SGLT2i use, larger left atrial diameter, elevated E wave, lower left ventricular ejection fraction, and lower rate of cryoballoon pulmonary vein isolation. CONCLUSIONS: Compared with anagliptin, tofogliflozin achieved greater suppression of AF recurrence after CA in patients with type 2 diabetes mellitus.

Increase in heart rate-dependent left atrial pressure is associated with symptoms in patients with paroxysmal atrial fibrillation.

BACKGROUND: It remains unclear why some patients with the same heart rate during an atrial fibrillation (AF) have subjective symptoms, whereas others do not. We assessed the hypothesis that different responses of arterial and left atrial blood pressures to rapid stimulation may be associated with the symptoms of AF. METHODS: A total of 110 patients who underwent catheter ablation for paroxysmal AF were retrospectively studied. Asymptomatic AF was defined as a European Heart Rhythm Association score of Ⅰ for AF-related symptoms. The left atrial pressure (LAP) was measured during sinus rhythm (SR), in 10 pacing per minute (ppm) increments from 100 ppm to the Wenckebach block rate in high right atrial pacing. RESULTS: Asymptomatic AF was observed in 19/110 patients (17%). Patients with symptomatic AF showed higher E/e' ratio and gradual LAP increase that was dependent on the pacing rate. Patients with asymptomatic AF had decreased LAP at 100 ppm compared that at SR, and thereafter, LAP gradually increased depending on the pacing rate. The rate of LAP change compared to that at SR was significantly lower in patients with asymptomatic AF than that in patients with symptomatic AF. The rate of LAP change was independently associated with AF symptoms. CONCLUSION: Patients with asymptomatic AF showed lower E/e' ratio and decreased LAP at 100 ppm to rapid stimulation, followed by a steady increase in LAP afterwards. Factors other than left ventricular diastolic dysfunction may be involved in AF symptoms.

Left ventricular stiffness assessed by diastolic Wall strain predicts asymptomatic atrial high-rate episodes in patients with pacemaker implantation.

BACKGROUND: Cardiac implantable electronic device-detected atrial high-rate episodes (AHREs) have been reported to be associated with thromboembolic risks. The present study aimed to investigate the association of echocardiographic and clinical parameters with the occurrence of AHREs in patients with a dual-chamber pacemaker (PMI). METHODS: One hundred forty-seven patients (76 males, 75.2 ± 8.9 years) who did not show atrial tachyarrhythmia before the implantation of the PMI were studied. Diastolic wall strain (DWS) and other measurements were assessed during sinus rhythm using transthoracic echocardiography before the PMI. DWS was calculated from the M-mode echocardiographic measurement of the left ventricular (LV) posterior wall thickness at end-systole (PWs) and end-diastole (PWd), and DWS was defined as (PWs-PWd) / PWs. RESULTS: AHREs (defined as AHREs duration >6 min and atrial rate >180 bpm) were detected in 50 / 147 patients during follow up (38.3 ± 13.8 months). Patients in the AHREs group had reduced DWS (0.29 ± 0.07 vs. 0.39 ± 0.06, p < 0.0001), larger left atrial volume index, thicker LV posterior diameter, higher rate of patients taking ß-blocker / diuretics, and higher prevalence of sinus node dysfunction. On multivariable analysis, only DWS was independently associated with AHREs. Patients with reduced DWS (<0.33) had a higher risk of incidences of AHREs. CONCLUSION: LV stiffness assessed by DWS was associated with AHREs in patients with a PMI.

Predictors for reduced flow velocity in left atrial appendage during sinus rhythm in patients with atrial fibrillation.

Discontinuation of anticoagulation therapy after catheter ablation (CA) for atrial fibrillation (AF) remains controversial. While decreased left atrial appendage flow velocity (LAAFV) during AF leads to left atrial appendage thrombus and embolic events, some AF patients show decreased LAAFV even during sinus rhythm (SR). We studied 392 patients (256 males, 68 ± 10 years) who exhibited SR during transesophageal echocardiography (TEE) before CA for AF. Clinical factors, transthoracic echocardiography, and blood samples were obtained before TEE. Reduced LAAFV was defined as < 35 cm/s of LAAFV. Reduced LAAFV was observed in 72/392 patients (18%). Reduced LAAFV was significantly associated with high prevalence of non-paroxysmal AF, elevated brain natriuretic peptide (BNP), prior heart failure, high CHADS2 score, high CHA2DS2-VASc score, no beta blocker administration, increased left atrial volume index (LAVI), elevated E/e' ratio, reduced left ventricular ejection fraction and high prevalence of left ventricular hypertrophy. On multivariate analysis, BNP (P = 0.0005, OR 1.045 for each 10 pg/ml increase in BNP, 95% CI 1.018-1.073) and LAVI (P = 0.0045, OR 1.044 for each 1 increase in LAVI, 95% CI 1.013-1.077) were associated with decreased LAAFV. The elevated BNP levels and large LAVI predict decreased LAAFV during SR in patients with AF.

Is the abnormal conduction zone of the left atrium a precursor to a low voltage area in patients with atrial fibrillation?

BACKGROUND: The abnormal conduction zone (ACZ) in the left atrium (LA) has attracted attention as an arrhythmia source in atrial fibrillation (AF). We investigated the hypothesis that the ACZ is related to the low voltage area (LVA) or the LA anatomical contact areas (CoAs) with other organs. METHODS AND RESULTS: We studied 100 patients (49 non-paroxysmal AF, 66 males, and 67.9 ± 9.9 years) who received catheter ablation for AF. High-density LA mapping during high right atrial pacing was constructed. Isochronal activation maps were created at 5-ms interval setting, and the ACZ was identified on the activation map by locating a site with isochronal crowding of ≥3 isochrones, which are calculated as ≤27 cm/s. The LVA was defined as the following; mild ( < 1.3 mV), moderate (<1.0 mV), and severe LVA (<0.5 mV). The CoAs (ascending aorta-anterior LA, descending aorta-posterior LA, and vertebrae-posterior LA) were assessed using computed tomography. The ACZ was linearly distributed, and observed in 95 patients (95%). The ACZ was most frequently observed in the anterior wall region (77%). A longer ACZ was significantly associated with a larger LA size and a prevalence of non-PAF. The 51.2 ± 36.2% of ACZ overlapped with mild LVA, 32.9 ± 32.8% of ACZ with moderate LVA, and 14.6 ± 22.0% of ACZ with severe LVA. In contrast, only 25.6 ± 28.0% of ACZ matched with the CoAs. CONCLUSION: The ACZ reflects LA electrical remodeling and may be a precursor finding of the low voltage zone and not the LA CoAs in patients with atrial fibrillation.

Predictors of movable type left atrial appendage thrombi in patients with atrial fibrillation.

Left atrial appendage thrombi (LAAT) are the main cause of thromboembolic events. Especially, movable type LAAT is high-risk for thromboembolic events. We aimed to investigate the predictors of the movable type LAAT in patients with atrial fibrillation (AF). We retrospectively studied 827 consecutive patients who underwent transthoracic echocardiography (TEE) prior to cardioversion or catheter ablation for AF. Sixty-nine patients who underwent cardiac surgery or significant valvular disease were excluded. The remaining 758 patients (age 67.6 ± 9.3, 535 males) were included in this study. Clinical data were evaluated at the time of TEE. The LAAT were classified into movable and fixed type LAAT by three independent observers who did not know clinical data. LAAT were detected in 57 (11 with movable and 46 with fixed type) of 758 patients (7.5%). Patients with movable type LAAT had an elevated E/e' ratio, lower left ventricular ejection fraction (LVEF), larger left atrial volume index, elevated C-reactive protein, higher prevalence of non-paroxysmal AF, patients taking warfarin (73% vs. 21%, P < 0.0001), and structural heart disease than control group (fixed type LAAT and without LAAT). On multivariate analysis, E/e' ratio, LVEF, and taking warfarin were significantly associated with movable type LAAT. The rate of movable type LAAT was the highest (7 of 49 patients, 14.3%) in patients with elevated E/e' ratio (> 12.7) and decrease LVEF (< 44%). E/e' ratio and LVEF could predict movable type LAAT in patients with AF. High-risk patients might need powerful antithrombotic therapy or taking early TEE.

Prediction of sudden cardiac death in chronic heart failure patients with reduced ejection fraction by ADMIRE-HF risk score and early repolarization pattern.

BACKGROUND: AdreView myocardial imaging for risk evaluation in heart failure (ADMIRE-HF) risk score is a novel risk score to predict serious arrhythmic risk in chronic heart failure patients with reduced ejection fraction (HFrEF). Moreover, early repolarization pattern (ERP) has been shown to be associated with an increased risk of sudden cardiac death (SCD) in HFrEF patients. We sought to investigate the prognostic value of combining ADMIRE-HF risk score and ERP to predict SCD in HFrEF patients. METHODS: We studied 90 HFrEF outpatients with LVEF< 40% in our prospective cohort study. In cardiac MIBG imaging, the heart-to-mediastinum (H/M) ratio was measured on the delayed planar image. ADMIRE-HF risk score was derived from the sum of the point values of LVEF, H/M ratio, and systolic blood pressure. We also assessed ERP on the standard electrocardiogram. RESULTS: During a median follow-up of 7.5(4.5-12.0) years, 22 patients had SCD. At multivariate Cox analysis, ADMIRE-HF risk score and ERP were independently associated with SCD. Patients with both intermediate/high ADMIRE-HF score and ERP had a higher SCD risk than those with either and none of them. CONCLUSION: The combination of ADMIRE-HF risk score and ERP would provide the incremental prognostic information for predicting SCD in HFrEF patients.

The association between left atrial stiffness and low-voltage areas of left atrium in patients with atrial fibrillation.

The low-voltage areas of left atrium (LA-LVA) have recently been of significant focus. However, very few studies have focused on the association between LA function and LA-LVA, and the mechanism of appearance of LA-LVA remains unclear. We investigated the marker for the existence of LA-LVA using automated 3-D mapping system. We studied 92 patients (75 males, 68 ± 9 years, 47 non-paroxysmal AF) who received CA for AF and 40 control patients without AF. Echocardiography was performed before the CA, and high-density voltage mapping during sinus rhythm after pulmonary isolation was performed in AF patients. LA-LVA was defined as < 0.5 mV, and LA stiffness index (LASI) was defined as the ratio of E/e' to LA peak strain. LA-LVA (LVA burden > 10%) was detected in 19/92 AF patients (21%). Patients with LA-LVA were associated with higher LASI (1.64 ± 1.70 vs. 0.61 ± 0.46, p < 0.0001), larger LA volume, non-paroxysmal AF, higher brain natriuretic peptide, structural heart disease, and older age. On multivariate analysis, LASI, LA volume, and age were independently associated with the existence of LA-LVA. Of these markers, the highest area under curve was obtained with LASI. The rate of high LASI (≥ 0.552) was highest in AF patients with LA-LVA. Moreover, the existence of LVA in anterior LA wall was associated with higher LASI. High LA stiffness index was associated with the presence of LA-LVA. The LA-LVA might be attributed to LA functional remodeling rather than LA anatomical remodeling.

Predictors of Left Atrial Thrombi and Spontaneous Echocardiographic Contrast in the Acute Phase After Cardioembolic Stroke in Patients With Atrial Fibrillation.

INTRODUCTION: The underlying mechanism of the residual left atrial thrombus (LAT)/spontaneous echo contrast (SEC) after the onset of cardioembolic stroke (CES) is unknown. This study aims to investigate the utility of CHADS2 and CHA2DS2-VASc scores for predicting LAT/SEC, and to investigate the risk factors of residual LAT/SEC after CES onset. METHODS: This retrospective study included 124 patients who were admitted with the acute phase of CES at our center. The clinical, echocardiographic variables, the CHADS2/CHA2DS2-VASc scores, and National Institutes of Health Stroke Scale score were retrospectively assessed on admission. RESULTS: Of 124 patients, LAT or SEC was detected in 39 patients (31.5%, 17 LAT and 38 SEC). Univariate analysis showed that the LAT/SEC group had a higher prevalence of nonparoxysmal atrial fibrillation (AF), left ventricular (LV) hypertrophy, hypertension, the rate of anticoagulation before admission, higher National Institutes of Health Stroke Scale score, larger left atrial diameter, and elevated E wave. In contrast, the CHADS2 and CHA2DS2-VASc scores were not associated with LAT/SEC. LAT/SEC was associated with nonparoxysmal AF and LV hypertrophy on multivariate analysis. Moreover, all patients were divided into 4 groups based on the combination between non-paroxysmal AF and LV hypertrophy. The rate of LAT/SEC was the highest (87.5%) in patients with nonparoxysmal AF and LV hypertrophy. CONCLUSIONS: Nonparoxysmal atrial fibrillation and left ventricular hypertrophy were associated with residual left atrial thrombus/spontaneous echo contrast in the acute phase after cardioembolic stroke that was independent of the CHADS2 and CHA2DS2-VASc scores.

Predictors of left atrial appendage stunning after electrical cardioversion in patients with atrial fibrillation.

The transient left atrial appendage (LAA) dysfunction after electrical cardioversion (CV), which is called as LAA-stunning, was found to be an important etiology of thrombus formation. The aim of the present study was to investigate the risk factors of LAA-stunning. This study included 134 patients who underwent catheter ablation for non-paroxysmal, non-valvular, and symptomatic atrial fibrillation (AF). Internal-CV was performed, and LAA emptying fraction (LAA-EF) was assessed using LAA-angiogram before and just after CV. LAA-stunning (defined as 10% reduction of LAA-EF after CV) was observed in 45/134 patients (34%). Patients in LAA-stunning group had longer duration of AF prior to CV, higher brain natriuretic peptide (BNP), higher prevalence of patients taking calcium blocker, larger left atrial (LA) diameter, elevated E wave, and larger LA volume than those in non LAA-stunning group. Multivariate analysis showed that longer duration of AF prior to CV (p = 0.015, OR 1.033 for 1 month extend, 95% CI 1.006-1.073) and elevated BNP (p = 0.038, OR 1.041 for each 10 pg/mL increase, 95% CI 1.001-1.009) were associated with LAA-stunning. In addition, all patients were divided into four groups based on the combination between duration of AF prior to CV and BNP; group 1 (low BNP/short-lasting AF), group 2 (high BNP/short-lasting AF), group 3 (low BNP/long-lasting AF), and group 4 (high BNP/long-lasting AF). The rate of LAA-stunning was the highest in the group 4 (55.6%). Elevated BNP and long duration of AF were associated with LAA stunning after electrical cardioversion.

Prediction of sudden cardiac death in patients with chronic heart failure by regional washout rate in cardiac MIBG SPECT imaging.

BACKGROUND: The sympathetic nervous system provides an important trigger for major arrhythmic events through regional heterogeneity of sympathetic activity, which could be evaluated by SPECT imaging as the regional MIBG washout rate (WR). There is little information available on the prognostic value of regional WR in SPECT imaging for the prediction of sudden cardiac death (SCD) in patients with chronic heart failure (CHF). METHODS: We studied 73 CHF outpatients with LVEF < 40%. At study entry, the regional WR was measured in 17 segments on the polar map. We defined abnormal regional WR as both the regional WR range (maximum - minimum regional WR) and maximum regional WR > mean value + 2SD obtained in 15 normal controls. RESULTS: During a mean follow-up of 7.5 ± 4.1 years, 15 of 73 patients had SCD. The abnormal regional WR and abnormal global WR on planar images were significantly and independently associated with SCD. Patients with both the abnormal regional WR and global WR had a significantly higher risk of SCD than those with none of these criteria. CONCLUSIONS: The analysis of regional MIBG WR on SPECT imaging provides additional prognostic value to global WR on planar images for SCD prediction in CHF patients.

A novel protocol for initial heparin administration during catheter ablation for atrial fibrillation in patients taking direct oral anticoagulants.

Thromboembolism and bleeding complications remain a major limitation of the catheter ablation (CA) for atrial fibrillation (AF). This study aimed to evaluate the association between achieving target activated clotting time (ACT) and clinical factors, and to develop an appropriate protocol for early achievement of target ACT in patients with direct oral anticoagulants (DOACs). At the initiation cohort, 190 patients (127 males, age 68 ± 9) taking DOACs who underwent CA for AF were studied. All patients underwent transthoracic echocardiography/transesophageal echocardiography/blood sampling before the CA. The ACTs were measured before heparin administration (pre-ACT) and in 30 min (30-min ACT) after initial heparin administration (100 U/kg +3000 U). At the validation cohort, the indicator obtained from the first study was reassessed in the subsequent 138 patients (94 males, age 68 ± 10). At the initiation cohort, 30-min ACT reached the target ACT in 79/190 patients (42%). Univariate analysis showed that longer pre-ACT, elevated aPTT, higher PT-INR, antiplatelet medication, and dabigatran were associated with achieving the target 30-min ACT. On multivariate analysis, only longer pre-ACT was independently associated with achieving the target 30-min ACT (P = 0.0396, the optimal cutoff value; 130 s). As a novel protocol, we added 2000 U of initial heparin dose (total 100 U/kg +5000 U) in patients with low pre-ACT (< 130); then, the achievement rate to target 30-min ACT improved from 41.6 to 80.5% without increasing bleeding complications. Our novel protocol of initial heparin administration based on pre-ACT is useful for an appropriate systemic anticoagulation in patients taking DOACs during the CA for AF.

Serial Change in Serum Chloride During Hospitalization Could Predict Heart Failure Death in Acute Decompensated Heart Failure Patients.

BACKGROUND: Although hyponatremia predicts morbidity and mortality in acute decompensated heart failure (ADHF), hypochloremia is also independently associated with poor prognosis in ADHF. Little is known, however, about the prognostic value of serial change in serum chloride during hospitalization in ADHF patients.Methods and Results:We prospectively studied 208 ADHF survivors after discharge and divided them into 4 groups according to serum chloride on admission and at discharge: (1) persistent hypochloremia group (n=12), hypochloremia both on admission and at discharge; (2) progressive hypochloremia group (n=42), development of hypochloremia after admission; (3) improved hypochloremia group (n=14), hypochloremia only on admission; and (4) no hypochloremia group, no hypochloremia during hospitalization (n=140). During a mean follow-up period of 1.86±0.76 years, 20 of 208 patients had heart failure death (HFD). In a model adjusted for hyponatremia, hypochloremia both on admission and at discharge was still significantly associated with HFD. Hyponatremia, however, was not significantly associated with HFD after adjustment for hypochloremia. Patients with persistent hypochloremia (HR, 9.13; 95% CI: 2.56-32.55) and with progressive hypochloremia (HR, 4.65; 95% CI: 1.61-13.4) had a significantly greater risk of HFD than those without hypochloremia during hospitalization. CONCLUSIONS: Both persistent hypochloremia and progressive hypochloremia during hospitalization are associated with HFD in ADHF patients.

Tolvaptan Reduces the Risk of Worsening Renal Function in Patients With Acute Decompensated Heart Failure and Preserved Left Ventricular Ejection Fraction　- Prospective Randomized Controlled Study.

BACKGROUND: Although the mainstay of treatment for acute decompensated heart failure (ADHF) is decongestion by diuretic therapy, it is often associated with worsening renal function (WRF). The effect of tolvaptan, a selective V2 receptor antagonist, on WRF in ADHF patients with preserved left ventricular ejection fraction (LVEF) is unknown.Methods and Results:We enrolled 50 consecutive ADHF patients whose LVEF on admission was ≥45%. Patients were randomly assigned to either tolvaptan add-on (n=26) or conventional diuretic therapy (n=24). The primary endpoint was the incidence of WRF, defined as an increase in serum creatinine (Cr) ≥0.3 mg/dL or 50% above baseline within 48 h of randomization. There was no significant difference between the 2 groups in the change in body weight or the total urine volume during 48 h. However, the change in Cr (∆Cr) at 24 and 48 h after randomization and the incidence of WRF (12% vs. 42%, P=0.0236) were significantly lower, and the fractional excretion of urea (FEUN) at 24 and 48 h after randomization was significantly higher in the tolvaptan group. There was an inverse correlation between ∆Cr and FEUN at 48 h after randomization. CONCLUSIONS: Tolvaptan can alleviate congestion with a significantly lower risk of WRF in ADHF patients with preserved LVEF, presumably through maintenance of renal perfusion.

Usefulness of Cardiac MetaIodobenzylguanidine Imaging to Improve Prognostic Power of the Model for End-Stage Liver Disease Scoring System in Patients With Mild-to-Moderate Chronic Heart Failure.

Liver dysfunction has a prognostic impact on the outcomes of patients with advanced heart failure (HF). The model for end-stage liver disease (MELD) score is a robust system for rating liver dysfunction, and a high score has been shown to be associated with a poor prognosis in ambulatory patients with HF. In addition, cardiac metaiodobenzylguanidine (MIBG) imaging provides prognostic information in patients with chronic HF (CHF). However, the long-term predictive value of combining the MELD score and cardiac MIBG imaging in patients with CHF has not been elucidated. To prospectively investigate whether cardiac MIBG imaging provides additional prognostic value to the MELD score in patients with mild-to-moderate CHF, we studied 109 CHF outpatients (New York Heart Association: 2.0 ± 0.6) with left ventricular ejection fraction <40%. At enrollment, an MELD score was obtained, and the heart-to-mediastinal ratio on delayed imaging and MIBG washout rate (WR) were measured using cardiac MIBG scintigraphy. During a follow-up period of 7.5 ± 4.2 years, 36 of 109 patients experienced cardiac death (CD). On multivariate Cox analysis, MELD score and WR were significantly independently associated with CD, although heart-to-mediastinal ratio showed an association with CD only on univariate Cox analysis. Patients with abnormal WR (>27%) had a significantly greater risk of CD than those with normal WR in both those with high MELD scores (≥10; hazard ratio 4.0 [1.2 to 13.6]) and with low MELD scores (<10; hazard ratio 6.4 [1.7 to 23.2]). In conclusion, cardiac MIBG imaging would provide additional prognostic information to the MELD score in patients with mild-to-moderate CHF.

Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells.

The present study was designed to evaluate the effects of neuropeptide substance P (SP) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the SP receptor, neurokinin-1 receptor (NK(1)-R). The addition of SP stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by SP or NK(1)-R antagonists. Increased expression of the receptor activator of nuclear factor kappaB ligand (RANKL) in synovial fibroblastic cells after the addition of SP was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium.

In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue.

Many studies have examined mineralized-tissue formation on titanium in vivo and in vitro; however, no precise method for measuring the mineralized tissue produced by cultured osteoblastic cells on titanium in vitro has been established. This study developed a method for measuring mineralized-tissue formation by cultured rat osteoblastic cells on titanium in vitro, and re-evaluated the effects of modifying the titanium surface. We used calcein blue, which binds to mineralized tissue, and measured the resulting fluorescence under ultraviolet light. A 1-h incubation with 3.1x10(-3)M calcein blue resulted in sufficient fluorescence of bone-like nodules on the titanium. Consequently, we found that osteoblastic cells produced larger bone-like nodules on titanium than in culture dishes. Fewer bone-like nodules formed on calcium-ion impregnated titanium than on pure titanium. However, when bisphosphonate was immobilized on the calcium-ion impregnated titanium, more bone-like nodules formed than on pure titanium. The results suggest that bisphosphonate immobilization on titanium is useful for stimulating mineralized-tissue formation on titanium implants.