Alkali-treated titanium dioxide promotes formation of proteoglycan layer and altered calcification and immunotolerance capacity in bone marrow stem cell.

Introduction: In this study, we report that a proteoglycans (PGs)-layer between the bone and titanium dioxide (TiO2) surface after osseointegration improved the calcification capacity and immunotolerance of human bone marrow mesenchymal stem cells (hBMSCs) on TiO2. Alkaline treatment of TiO2 is a method for promoting osteogenesis in hBMSCs. We hypothesized that promotion of osteogenesis due to alkaline treatment was caused by changing PGs-layer on TiO2. Objective: This study aimed to analyze whether alkaline treatment of TiO2 affects PGs-layer formation and immunotolerance in hBMSCs. Methods: The topology and wettability of the alkaline-treated titanium (Ti-Al) and unprocessed titanium (Ti-MS) surfaces were characterized. Initial cell attachment, cell proliferation, calcification capacity, alkaline phosphatase activity, PGs-layer formation, PGs function, and the expression of osteogenic and immunotolerance-related genes were analyzed. The conditioned medium (CM) from hBMSCs grown on Ti-Al and Ti-MS was added to macrophages (hMps) and Jurkat cells, and immunotolerance gene expression in these cells was analyzed. Results: hBMSCs cultured on Ti-Al showed increased initial cell attachment, cell proliferation, PG-layer formation, and osteogenic capacity compared with hBMSCs on Ti-MS. Gene expression of indoleamine 2,3-dioxygenase (IDO) in the hBMSCs cultured on Ti-Al was higher than that in the hBMSCs on Ti-MS. CM from hBMSCs did not affect markers of M1 and M2 macrophages in hMps. CM from hBMSCs cultured on Ti-Al altered the gene expression of Foxp3 in Jurkat cells compared to that of CM from hBMSCs on Ti-MS. Significance: These results suggest that alkaline treatment of TiO2 altered PGs-layer formation, and changed the osteogenesis and immunotolerance of hBMSCs.

Efficacy of central extracorporeal life support for patients with fulminant myocarditis and cardiogenic shock.

OBJECTIVES: Fulminant myocarditis with cardiogenic shock requires extracorporeal life support (ECLS) and has poor outcomes. To improve outcomes, we have converted patients with severely impaired cardiac and multiorgan function from peripheral to central ECLS. In this study, we reviewed these patients' clinical outcomes and investigated associated factors. METHODS: We retrospectively studied 70 consecutive patients with fulminant myocarditis under peripheral support from 2006 to 2020. Forty-eight patients underwent surgical conversion to central support, and the remaining patients continued peripheral support. The end point was survival and ventricular assist device-free survival. RESULTS: More severe pulmonary congestion and multiorgan failure were present in patients with central than peripheral support. Weaning from ECLS was achieved in 95% and 62% of patients with peripheral and central support, respectively. Five-year survival was not significantly different between patients with central and peripheral support (71.2% vs 87.5%, respectively; P = 0.15). However, the ventricular assist device-free survival rate was significantly higher in patients with central than peripheral support (82.2% vs 52.0%, respectively; P = 0.017). A peak creatine kinase-MB level of >180 IU/l, rhythm disturbance and aortic valve closure were detrimental to functional recovery in patients with central support. CONCLUSIONS: Conversion to central ECLS is feasible and safe in patients with fulminant myocarditis. Patients with severe myocardial injury as shown by a high creatine kinase-MB level, rhythm disturbance and aortic valve closure should be converted to a durable left ventricular assist device.

Chondroitin-4-sulfate transferase-1 depletion inhibits formation of a proteoglycan-rich layer and alters immunotolerance of bone marrow mesenchymal stem cells on titanium oxide surfaces.

Successful osseointegration is essential for dental implants. However, the complete molecular mechanism of osseointegration remains to be elucidated. In this study, we focused on the proteoglycan (PG)-rich layer between titanium oxides (TiOx) and bone, and chondroitin-4-sulfate transferase-1 (C4ST-1), which forms the sugar chain in PGs. Human bone marrow mesenchymal stem cells (hBMSCs) depleted of C4ST-1 were cultured on titanium (Ti) plates, and the interface between hBMSCs and TiOx was analyzed using transmission electron microscopy. Immunotolerance, proliferation, initial adhesion, and calcification of the cells were analyzed in vitro. At 14 days of cultivation, a PG-rich layer was observed between hBMSCs and TiOx. However, the PG-rich layer was reduced in C4ST-1-depleted hBMSCs on TiOx. Real-time RT-PCR showed that conditioned media increased the levels of expression of M1-macrophage markers in human macrophages. However, depletion of C4ST-1 did not affect calcification, cell proliferation, or initial cell adhesion on Ti plates. These results suggested that C4ST-1 in hBMSCs affects their immunotolerance and alters the formation of PG-rich layer formation on TiOx.

The effect of macrophages on an atmospheric pressure plasma-treated titanium membrane with bone marrow stem cells in a model of guided bone regeneration.

Guided bone regeneration (GBR) is an established treatment. However, the mechanisms of GBR are not fully understood. Recently, a GBR membrane was identified that acts as a passive barrier to regenerate bone via activation and migration of macrophages (Mps) and bone marrow stem cells (BMSCs). Atmospheric pressure plasma treatment of the titanium membrane (APP-Ti) activated macrophages. The purpose of this study was to analyze whether macrophages attached to an APP-Ti membrane affected differentiation of BMSCs in a GBR model. Human THP-1 macrophages (hMps) were cultured on non-treated Ti (N-Ti) and APP-Ti membrane. Macrophage polarization was analyzed by RT-PCR and immunocytochemistry. Secreted proteins from hMps on N-Ti and APP-Ti were detected by LC/MS/MS. hBMSCs were co-cultured with hMps on N-Ti or APP-Ti and analyzed by osteogenic differentiation, Alizarin red S staining, and alkaline phosphatase (ALP) activity. N-Ti and APP-Ti membrane were also implanted into bone defects of rat calvaria. hMps on APP-Ti were polarized M2-like macrophages. hMps on N-Ti secreted plasminogen activator inhibitor-1 and syndecan-2, but hMps on APP-Ti did not. hBMSCs co-cultured with hMps on APP-Ti increased cell migration and gene expression of osteogenic markers, but suppressed mineralization, while ALP activity was similar to that of hMps on N-Ti in vitro. The volume of newly formed bone was not significantly different between N-Ti and APP-Ti membrane in vivo. M2 polarized hMps on APP-Ti suppressed osteogenic induction of hBMSCs in vitro. The indirect role of hMps on APP-Ti in newly formed bone was limited.

Effect of Therapeutic Modification on Outcomes in Heart Transplantation Over the Past Two Decades　- A Single-Center Experience in Japan.

BACKGROUND: During these 2 decades (1999-2019), many therapeutic strategies have been developed in the field of heart transplant (HTx) to improve post-HTx outcomes. In the present study, 116 consecutive HTx adults between 1999 and 2019 were retrospectively reviewed to evaluate the influences of a therapeutic modification on post HTx outcomes.Methods and Results:Patient survival, functional status and hemodynamics after HTx and modification of therapeutic strategies were reviewed. The overall cumulative survival rate at 10 and 20 years post-HTx was 96.4 and 76.7%, respectively. There were no significant differences in survival rate or exercise tolerance after HTx between extracorporeal and implantable continuous flow-LVAD. Post-HTx patient survival in patients, irrespective of the donor risk factors such as donor age, low LVEF, history of cardiac arrest, was equivalent across cohorts, while longer TIT and higher inotrope dosage prior to procurement surgery were significant risk factors for survival. In 21 patients given everolimus (EVL) due to renal dysfunction, serum creatinine significantly decreased 1 year after initiation. In 22 patients given EVL due to transplant coronary vasculopathy (TCAV), maximum intimal thickness significantly decreased 3 years after initiation. CONCLUSIONS: The analysis of a 20-year single-center experience with HTx in Japan shows encouraging improved results when several therapeutic modifications were made; for example, proactive use of donor hearts declined by other centers and the use of EVL in patients with renal dysfunction and TCAV.

Experience of morphine therapy for refractory dyspnea as palliative care in advanced heart failure patients.

BACKGROUND: In the field of palliative care, morphine is known to be effective for alleviating dyspnea in cancer patients. However, little is known regarding the safety and efficacy of morphine therapy for refractory dyspnea as palliative care in advanced heart failure (HF) patients. METHODS: We retrospectively reviewed consecutive advanced HF patients who were referred to the Palliative Care Team at our institution and administered morphine for refractory dyspnea during hospitalization between September 2013 and December 2018. We investigated the details of morphine usage, vital signs, an 11-point quantitative symptom scale, and adverse events at baseline, 24 h, and 72 h after the start of treatment. RESULTS: Morphine was administered for refractory dyspnea in 43 advanced HF patients [mean age: 73.5 years, male: 28 (65%), New York Heart Association functional class IV: 43 (100%), median left ventricular ejection fraction: 25%, median B-type natriuretic peptide level: 927 pg/ml, concurrent intravenous inotrope: 33 (77%)]. Median initial dose of morphine was 5 mg/day in both oral and intravenous administration and median duration of administration was 5 days. Significant decreases in an 11-point quantitative symptom scale [7 (5, 9) vs. 2 (1, 6); p < 0.01, (data available in 8 patients)] and respiratory rate (22.2 ± 6.1 vs. 19.7 ± 5.2 breaths per minute; p < 0.01) were observed 24 h after the start of morphine administration. Meanwhile, oxygen saturation, blood pressure, and heart rate were not significantly altered after treatment (NS). Common adverse events were delirium (18%) and constipation (8%); however, no lethal adverse event definitely related to morphine therapy occurred during treatment. CONCLUSIONS: This single-center retrospective study revealed the clinical practice of morphine therapy and suggested that morphine therapy might be feasible for refractory dyspnea as palliative care in advanced HF patients.

Experience of the use of octreotide for refractory gastrointestinal bleeding in a patient with Jarvik2000® left ventricular assist device.

Gastrointestinal bleeding (GIB) is among the major complications affecting implantable continuous-flow left ventricular assist device (iLVAD) recipients and is the major cause of re-hospitalization. GIB in iLVAD recipients is sometimes critical, and controlling bleeding using conventional approaches is difficult. A 35-year-old woman developed refractory GIB from multiple gastric polyps and de novo angiodysplasia after Jarvik2000® iLVAD implantation. Discontinuation of anticoagulation and antiplatelet therapies had little effect on GIB; thus, multiple endoscopic hemostatic therapies were performed. However, bleeding recurred several times, and red blood cell (RBC) transfusion in large volumes was required for progressive anemia. Furthermore, the von Willebrand factor (VWF) multimer analysis revealed loss of the high-molecular weight multimer, which may have resulted from the high-speed rotation of the axial-flow LVAD pump. To supplement VWF, cryoprecipitate was administered, but it was effective for only several days. Finally, the patient was treated with octreotide, a somatostatin analog, on post-operative day 58. After starting octreotide, tarry stool gradually decreased, and progression of anemia slowed down within the first 14 days of treatment; thus, the total RBC transfusion volume was reduced without additional hemostatic interventions, including cryoprecipitate administration. The patient developed mediastinitis on post-operative day 68 and died of sepsis on post-operative day 72. There was no adverse effect associated with octreotide use. Although the observation period was short, octreotide appears to be useful for resolving recurrent GIB after iLVAD implantation and reducing blood transfusions.

Survey of Palliative Sedation at End of Life in Terminally Ill Heart Failure Patients　- A Single-Center Experience of 5-Year Follow-up.

BACKGROUND: Little is known about palliative sedation in terminally ill heart failure (HF) patients.Methods and Results:We retrospectively reviewed terminally ill HF patients who received palliative sedation from September 2013 to August 2018. Among 95 terminally ill HF patients, 25 were prescribed dexmedetomidine and 12 were prescribed midazolam at the end of life. Richmond Agitation-Sedation Scale was significantly reduced (P<0.01), but blood pressure and heart rate were unaltered after treatment in both the dexmedetomidine and midazolam groups. CONCLUSIONS: Prescription of dexmedetomidine and/or midazolam might be feasible in selected terminally ill HF patients.

De novo malignancy in heart transplant recipients: A single center experience in Japan.

BACKGROUND: Because of aggressive immunosuppression, heart transplant recipients have a high risk of de novo malignancy, which is a major cause of death and worse prognosis, regardless of the type. However, the impact of de novo malignancy on Japanese heart transplant recipients is unknown. METHODS: We analyzed 103 Japanese heart transplant recipients over 18-years-old at the time of transplantation between April 1999 and April 2017. Patient characteristics and prognosis were compared between heart transplant recipients with or without de novo malignancy after heart transplantation (HTx). Additionally, univariate and multivariate analyses for the risk factors of de novo malignancy after HTx were performed. RESULTS: De novo malignancy developed in 7 patients (6.8%; post-transplant lymphoproliferative disorders, n=3; Bowen's disease, n=1; colon cancer, n=2; bladder cancer, n=1). Follow-up time and previous antibody mediated rejection (AMR)≥grade 1 were risk factors of de novo malignancy after HTx in multivariate analysis (OR: 1.19, 95% CI: 1.00-1.42, p=0.043; and OR: 10.7, 95% CI: 1.37-83.68, p=0.038, respectively). History of malignancy was a potential risk factor, albeit not significant (OR: 23.05, 95% CI: 0.99-534.53, p=0.071). The survival rates in patients with de novo malignancy was significantly lower than in those without de novo malignancy (3-year survival rate: 100% versus 67%, p=0.0025). CONCLUSIONS: Long follow-up time and previous AMR≥grade 1 were risk factors of de novo malignancy after HTx. Japanese heart transplant recipients with de novo malignancy have worse prognosis; therefore, screening examinations are important for early diagnosis.

Kaempferol-immobilized titanium dioxide promotes formation of new bone: effects of loading methods on bone marrow stromal cell differentiation in vivo and in vitro.

BACKGROUND: Surface modification of titanium dioxide (TiO2) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO2 implants. METHODS: There were four experimental groups. Alkali-treated TiO2 samples (implants and discs) were used as a control and immersed in Dulbecco's phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50 µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50 µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO2 implants placed on rat femur bones were analyzed for bone-implant contact by histological methods. RESULTS: Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. CONCLUSION: TiO2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants.

Impact of mitral valve intervention with left ventricular assist device implantation on postoperative outcomes and morphologic change.

Although mitral regurgitation (MR) is prevalent in patients with end-stage heart failure, the impact of mitral valve (MV) surgery on outcomes after left ventricular assist device (LVAD) implantation and morphologic changes of MV remains unclear. We retrospectively reviewed 74 patients who underwent LVAD implantation as a bridge to transplant. Of these, 11 (15%) underwent MV repair concomitant with or prior to LVAD implantation, while 27 patients with preoperative significant (moderate or greater) MR did not undergo concomitant MV surgery. The mean interval between LVAD implantation and the last echocardiographic examination was 913 days. Irrespective of MV surgery, significant LV reverse remodeling including decreased LV and left atrial dimension and improved MR severity was observed in all patients except for patients with prior MV surgery. Histopathological examination of explanted hearts removed at heart transplantation (n = 69) or autopsy (n = 5) revealed that the MV annulus was still dilated (mean perimeter 11.7 cm) in the patients with preoperative significant MR and no concomitant MV surgery. Concomitant MV surgery at the time of LVAD implantation for significant MR might not be always necessary for bridge to transplant or destination therapy cases. However, it might be required in patients having potential for cardiac recovery or patients with severe pulmonary hypertension and depressed right ventricle.

A new application of cell-free bone regeneration: immobilizing stem cells from human exfoliated deciduous teeth-conditioned medium onto titanium implants using atmospheric pressure plasma treatment.

INTRODUCTION: Surface modification of titanium (Ti) implants promotes bone formation and shortens the osseointegration period. The aim of this study was to promote bone regeneration and stability around implants using atmospheric pressure plasma (APP) pretreatment. This was followed by immobilization of stem cells from human exfoliated deciduous teeth-conditioned medium (SHED-CM) on the Ti implant surface. METHODS: Ti samples (implants, discs, powder) were treated with APP for 30 seconds. Subsequently, these were immobilized on the treated Ti surface, soaked and agitated in phosphate-buffered saline or SHED-CM for 24 hours at 37 °C. The surface topography of the Ti implants was observed using scanning electron microscopy with energy dispersive X-ray spectroscopy. In vivo experiments using Ti implants placed on canine femur bone were then conducted to permit histological analysis at the bone-implant boundary. For the in vitro experiments, protein assays (SDS-PAGE, Bradford assay, liquid chromatography-ion trap mass spectrometry) and canine bone marrow stromal cell (cBMSC) attachment assays were performed using Ti discs or powder. RESULTS: In the in vitro study, treatment of Ti implant surfaces with SHED-CM led to calcium phosphate and extracellular matrix protein immobilization. APP pretreatment increased the amount of SHED-CM immobilized on Ti powder, and contributed to increased cBMSC attachment on Ti discs. In the in vivo study, histological analysis revealed that the Ti implants treated with APP and SHED-CM stimulated new bone formation around implants. CONCLUSIONS: Implant device APP pretreatment followed by SHED-CM immobilization may be an effective application to facilitate bone regeneration around dental implants.