Despite the high biocompatibility of titanium and its alloys, the need to remove titanium implants is increasingly being debated due to the potential for adverse effects associated with long-term retention. Therefore, new solutions are being sought to enhance the biocompatibility of titanium implants. One of them is to increase the thickness of the passive layer of the implant made of titanium dioxide. We were the first to evaluate the effect of hard-anodized (type II) Ti-6Al-4V alloy discs on the cytotoxicity, mitochondrial function, and redox balance of fibroblasts mitochondria compared to standard-anodized (type III) and non-anodized discs. The study used fibroblasts obtained from human gingival tissue. The test discs were applied to the bottom of 12-well plates. Cells were cultured for 24 h and 7, 14, and 21 days and mitochondria were isolated. We demonstrated the occurrence of oxidative stress in the mitochondria of fibroblasts of all tested groups, regardless of the presence and type of anodization. Type II anodization prevented changes in complex II activity (vs. control). The lowest degree of citrate synthase inhibition occurred in mitochondria exposed to titanium discs with type II anodization. In the last phase of culture, the presence of type II anodization reduced the degree of cytochrome c oxidase inhibition compared to the other tests groups and the control group, and prevented apoptosis. Throughout the experiment, the release of titanium, aluminium, and vanadium ions from titanium discs with a hard-anodized passive layer was higher than from the other titanium discs, but decreased with time. The obtained results proved the existence of dysfunction and redox imbalance in the mitochondria of fibroblasts exposed to hard-anodized titanium discs, suggesting the need to search for new materials perhaps biodegradable in tissues of the human body. The dismutation reaction is the dismutation reaction of superoxide anion (O2•− to H2O2. This includes NADPH oxidase (NOX), which generates large amounts of O2•−, and, sometimes, H2O2 [The standard treatment for mandibular fractures is surgery with titanium plates and screws. The use of osteosynthesis allows for shorter intermaxillary fixation times and faster restoration of stomatognathic function. Despite the high biocompatibility of titanium and its alloys, the necessity of removing titanium implants is increasingly debated due to the possibility of distant side effects associated with leaving biomaterials in the body for a long time. As indicated in the literature, some patients with titanium implants made of anodized alloys have been observed to have metallosis as a standard phenomenon ,2,3,4. Ies, H2O2 . In a stes, H2O2 , brain [es, H2O2 , liver, es, H2O2 . Evaluates, H2O2 .In the case of biomaterials such as titanium alloys used in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation and, thus, oxidative stress (OS) are desirable. That is why researchers are looking for various solutions to increase the biocompatibility of titanium implants. Mieszkowska A. et al.  used fibThe introduction of titanium implants with a thickened titanium dioxide layer could reduce the harmful influence of titanium discs on redox balance and, consequently, on implant disintegration. This layer is expected to protect, to a large extent, against the effects of mechanical friction, and is expected to minimize the risk of ion migration from the implant alloy into the surrounding tissues. Given the lack of studies on the effect of hard anodized implants on cytotoxicity, or OS phenomenon, it is very reasonable to evaluate the effect of titanium discs made of Ti-6AI-4V alloy subjected to hard anodization (type II) on cytotoxicity, function, and redox balance of mitochondria in a fibroblast research model (ATCC-PCS-201-018) and to compare the results with those obtained in cultures of fibroblasts exposed to titanium discs constructed of Ti-6Al-4V alloy subjected to standard anodization (type III) and to titanium discs constructed of Ti-GAl-4V alloy not subjected to anodization, the so-called “raw discs”.There were no significant differences in the values of the parameters studied between the groups after 24 h of exposure, so the figures and description were omitted.p = 0.004 and p = 0.0004, respectively) and V(t) alloys  compared to V alloy (Fibroblast viability scores in the MTT assay after 14 and 21 days were significantly higher for V(st) , as well as compared to V alloy . After 14 days of the experiment, SOD activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs was significantly higher for V(st) and V(t) alloys compared to controls . SOD activity in the mitochondria of fibroblasts exposed to V(st) discs was significantly higher for V and V(t) alloys compared to control . SOD activity in mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days’ exposure was significantly higher for alloy V(st) compared to control (p < 0.0001). SOD activity in the mitochondria of fibroblasts exposed to V(st) discs was significantly higher compared to alloys V and V(t)  (SOD activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days’ exposure was significantly higher for V(st) and V(t) alloys compared to control (ctively) .p < 0.0001), as well as compared to alloys V(st) and V(t) .CAT activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for alloy V compared to control . The activity of this enzyme in the mitochondria of fibroblasts exposed to titanium–vanadium discs was significantly higher for alloy V compared to alloys V(st) and V(t)  , and V(t) alloys compared to controls (ctively) .p < 0.001), V(st) (p < 0.0001), and V(t) (p < 0.0001) alloys compared to controls. GPx activity was significantly higher for alloy V(st) compared to V(t) (p = 0.048) (GPx activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly higher for V (= 0.048) .p = 0.0006 and p < 0.0001, respectively) and to alloy V .The concentration of glutathione (GSH) in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly higher for alloys V(st) and V(t) compared to control .The concentration of GSH in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for alloy V(t) compared to control and alloys V and V(st) . The concentration of GSH in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for V(st) and V(t) alloys compared to controls   and V(t) (ctively) .p = 0.0003 and p = 0.008, respectively), moreover, the concentration of 4-HNE was higher for alloy V(st) compared to control (p = 0.03). The concentration of 4-HINE adducts in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for alloy V compared to control and alloys V(t) and V(st) , moreover, the concentration of 4-HNE was higher for alloy V(st) compared to control (p = 0.002). The concentration of 4-HNE adducts in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for alloy V compared to control and alloys V(t), V(st)   in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly higher for alloy V compared to control and alloy V(t) (ctively) .p = 0.0003 and p = 0.005, respectively). The concentration of disulfide groups in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly lower for alloy V compared to alloy V(st) (p = 0.03).The concentration of disulfide groups in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly lower for alloy V and V(t), compared to the control , V(st) , and V(t)  relative to controls. The concentration of disulfide groups in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 and 21 days of exposure was significantly lower for alloy V, relative to alloy V(t) , moreover, the concentration of disulfide groups in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for alloy V(st) compared to alloy V (p = 0.001)  .p = 0.005, p = 0.005, and p = 0.01, respectively) (Advanced protein glycation end products (AGE) levels in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure were significantly higher for V, V(st), and V(t) alloys compared to controls (ctively) .p = 0.0004, p = 0.02, and p = 0.01, respectively).NOX activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 days of exposure was significantly higher for V, V(st), and V(t) alloys compared to controls (p < 0.0001), as well as relative to the activity of this enzyme in the mitochondria of fibroblasts exposed to alloy V(t) (p = 0.03). NOX activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for V(st) alloy relative to control (p = 0.02).NOX activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for alloy V relative to the control . NOX activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for alloy V relative to V(t) (p = 0.013) , and V(t) alloys relative to controls (= 0.013) .p < 0.0001, p < 0.0001, and p < 0.0001, respectively). CASP-3 activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for V and V(st) alloys relative to controls  and relative to CASP-3 activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs from V(t) alloy  (Caspase -3 (CASP-3) activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for V, V(st), and V(t) alloys relative to controls (ctively) .p = 0.005, p = 0.003, and p = 0.007, respectively) (The concentration of peroxynitrite (ONOO-) in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for V, V(st), and V(t) alloys compared to controls (ctively) .p = 0.0005 and p = 0.02, respectively).The concentration of 3-nitrotyrosine (3-NT) in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for V and V(st) alloys compared to controls . The concentration of 3-NT in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was significantly higher for V and V(st) alloys compared to V(t) alloy ; it was also significantly higher for V alloy compared to V(st) (p = 0.008) , and V(t) relative to controls (= 0.008) .p < 0.0001, p < 0.0001, and p < 0.0001, respectively). After 7 days of exposure, complex I activity was significantly higher for alloys V(st) and V(t) compared to alloy V . Complex I activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs on day 14 of the experiment was significantly lower for alloys V and V(st) compared to controls . It was also significantly higher for alloy V(t) compared to alloys V(st) and V . Complex I activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs on day 21 of the experiment was significantly lower for alloy V compared to control and alloys V(st) and V(t)  , and V(t) compared to the control (ctively) .p < 0.0001 and p = 0.03, respectively). The activity of complex II in the mitochondria of fibroblasts exposed to titanium discs with vanadium after 7 days of exposure was significantly lower for alloy V compared to alloys V(st) (p = 0.025) and V(t) (p < 0.0001), as well as a significantly higher activity of the complex in question was noted for alloy V(t) compared to V(st) (p = 0.03). The activity of complex II in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly lower for V, V(st), and V(t) alloys compared to controls . Complex II activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was also significantly higher for V(st) and V(t) alloys compared to V . Complex II activity in the mitochondria of fibroblasts exposed to titanium discs after 21 days of exposure was significantly lower for V and V(st) alloys compared to controls . Complex II activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 21 days of exposure was also significantly higher for V(st) and V(t) alloys compared to V  (Complex II activity in the mitochondria of fibroblasts exposed to titanium discs after 7 days of exposure was significantly lower for V and V(st) alloys compared to controls (ctively) .p < 0.0001, p < 0.0001, and p < 0.0001, respectively) and 14  days of exposure was significantly lower for V, V(st), and V(t) alloys compared to controls. On day 21, COX activity in mitochondria of fibroblasts exposed to titanium–vanadium discs was significantly lower only for V and V(st) alloys compared to control . At day 7, the activity of the enzyme in question was significantly higher for V(st) alloy compared to V (p = 0.02) and, after 21 days, COX activity for V and V(st) alloys was significantly lower compared to COX activity in the mitochondria of fibroblasts exposed to V(t) alloy  (Cytochrome c oxidase (COX) activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 (ctively) .p < 0.0001, p < 0.0001, and p < 0.0001, respectively), 14 , and 21  days of exposure was significantly lower for V, V(st), and V(t) alloys compared to controls. CS activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 14 days of exposure was significantly higher for V(t) alloy compared to V (p = 0.015) and, after 21 days of exposure, CS activity in the mitochondria of V(st) and V(t) alloys was significantly higher compared to CS activity in the mitochondria of fibroblasts exposed to V alloy  (Citrate synthase (CS) activity in the mitochondria of fibroblasts exposed to titanium–vanadium discs after 7 (ctively) .p = 0.002 and p = 0.003, respectively), 6 , 15 , and 21  days of exposure of fibroblasts to titanium–vanadium discs was significantly higher for V(t) alloy compared to V and V(st) alloys. The titanium content of the medium taken after 6 (p = 0.04), 15 (p = 0.04), and 21 (p = 0.03) days of exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V compared to V(st) (The titanium content of the medium taken after 3 (to V(st) .p = 0.001 and p = 0.003, respectively) and 6  days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for V(t) alloy compared to V and V(st) alloys.Aluminum content in the medium taken after 3 (p = 0.03) and 15 (p = 0.04) days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V compared to alloy V(st). Aluminum content in the medium taken after 15 days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V(t) compared to alloy V(st) (p = 0.001). The aluminum content of the medium taken after 21 days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V(t) compared to alloy V (p = 0.008) (Aluminum content in the medium taken after 6 (= 0.008) .p = 0.001 and p = 0.001, respectively), 6 , 15 , 21  days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V(t) compared to alloys V and V(st). Vanadium content in the medium taken after 15 days’ exposure of fibroblasts to titanium–vanadium discs was significantly higher for alloy V compared to alloy V(st) (p = 0.02) (Vanadium content in the medium taken after 3 ( = 0.02) .p = 0.004, p < 0.0001, and p < 0.0001), V(st)  and V(t)  compared to controls (The concentration of fibroblast growth factor 2 (FGF 2) in the medium collected after 7, 14, and 21 days’ exposure of fibroblasts to titanium–vanadium discs was significantly lower for V alloys , V(st) , and V(t)  compared to control  in the medium collected after 7, 14, and 21 days’ exposure of fibroblasts to titanium–vanadium discs was significantly lower for V alloys ( control .p < 0.05).Day 7 showed a negative correlation between peroxynitrite concentration and complex I activity in the mitochondria of fibroblasts treated with all three titanium discs  manner and without a passive layer on the surface.The mere presence of a titanium implant, let alone the products of its wear, disrupts the body’s immune processes and causes OS and inflammation, which can lead to the need for further surgery and result in complications in distant organs ,10,11. IFor ethical reasons, the current study was conducted on cell culture of primary human gingival fibroblasts . It should be emphasized, the results obtained in this way cannot be translated directly to humans. The fact is that in studies evaluating titanium implants, mainly two types of cells are used, osteoblasts and fibroblasts, as both types show high adhesion to implants. As studies have shown, osteoblasts prefer rougher surfaces, while fibroblasts prefer smooth surfaces ,20. Sinc2 layer and two alloys with a passive layer: one standard anodized, the other with type II anodization on the implant surface. The metabolic activity of fibroblasts after exposure to titanium implants was evaluated. An MTT assay was performed after 24 h and 7, 14, and 21 days of culture with titanium discs. Differences in cell viability were observed only on the 14th and 21st day of the experiment; the metabolic activity of fibroblasts incubated with the disc devoid of the passive layer significantly decreased (the result is presented as % of control) compared to the other two test groups. The metabolic activity of fibroblasts exposed to discs with an anodized surface did not differ according to the type of anodization. Similarly, greater cytotoxicity of implants lacking the TiO2 layer compared to standard anodized implants was demonstrated by El-Shenawy N. et al. [In the study, titanium–vanadium alloy (Ti4AI16V) was used in three surface layer configurations: an alloy without a TiO. et al. , reduced2 layer on the surface of the implant is believed to be a protective layer and determines the biocompatibility of titanium implants. Bikondoa et al. [2 surface, mediate water dissociation. Evidence of the reactivity of titanium alloy is also evidenced by elevated titanium concentrations in the serum and distant organs of patients after implantation, as we have previously written. It is believed that the release of titanium may be the result of an anodic corrosion process, where the TiO2 layer is damaged. The cathodic part of the corrosion process involves oxygen reduction at physiological pH with the generation of ROS and non-radical compounds, such as hydrogen peroxide H2O2 [2 layer in biological solutions is due to the continuous corrosion process of titanium implants in the human body. Wear particles formed at the bone/implant interface can disturb the protective TiO2 layers, which further induces corrosion, leading to a synergistic fretting-wear process. In addition to the possible formation of ROS by the titanium alloy itself as a result of cathodic corrosion, titanium can be exposed to ROS produced by inflammatory cells coming into contact with titanium immediately after implantation or when cultured with stimulated phagocytic cells present in the culture medium. The phenomenon of “oxygen burst” and generation of huge amounts of O2•− and H2O2 then occurs.TiOa et al.  showed, ide H2O2 ,23,24. Lide H2O2  claim th2−), which undergoes a dismutation reaction catalyzed by SOD. Under physiological conditions, complexes I and III are responsible for the reduction of oxygen to O2− [One of the most important processes occurring in mitochondria is oxidative phosphorylation. This process is accompanied by the production of energy stored as ATP by the mitochondrial complex system (I–IV). About 0.1–1% of the oxygen consumed in the mitochondria is converted into superoxide anion  also has another role, as a structural and regulatory component of the mitochondrial ATP-sensitive K+ channel (mitoKATP) [Extremely important, in terms of the protective properties of the TiOitoKATP) . IncreasitoKATP) .2− generation. Surprisingly, there was no difference in the activity of this enzyme between the group of fibroblasts exposed to discs without a passive layer and the control group. The lack of change in CAT activity at day 7 in all groups of fibroblasts exposed to titanium implants compared to controls, with a concomitant increase in GPx activity , suggests a small increase in H2O2 generation. Indeed, GPx is known to have a much higher affinity for H2O2 than CAT and its activity increases with non-significant increases in H2O2 generation [2O2 generation at which CAT becomes active. High concentrations of H2O2 inhibit GPx activity [2O2 may have both negative and positive significance. Lee et al. [2 layer can interact with H2O2 leading to the formation of hydroxyl radicals, which are among the most dangerous ROS. On the other hand, it is known that high concentrations of H2O2 may be desirable in the process of indentation of a titanium implant and in wound healing in general. The generation of large amounts of H2O2 at the implant/wound site and the resulting gradient in its concentration are essential for rapid leukocyte recruitment and, thus, implant engraftment/wound healing [It can be seen that the behaviour of the antioxidant defence of fibroblasts exposed to titanium discs lacking the passive layer differs significantly from the changes in the antioxidant defence of fibroblasts exposed to anodized titanium discs, with more adverse changes seen in fibroblasts exposed to standard anodized discs. The intensification of the dismutation (↑SOD) reaction from day 7 of the experiment in the groups of fibroblasts exposed to anodized discs (V(st) and V(t)) vs. control persisted until the end of culture, which is most likely due to the increase in Oneration . Howeveractivity . The pute et al.  have sho healing .The reduction in GSH concentration, initiated on day 7 of the experiment, in the group of fibroblasts exposed to titanium discs without a passive layer vs. the other test groups, persists throughout the experiment. In the groups of fibroblasts exposed to anodized discs, we observed an increase in GSH concentration vs. the control group and the group exposed to discs without anodization. Given that the overriding function of GSH is to maintain the thiol groups of proteins in a reduced state, its deficiency/consumption could be a reason an increased reduction of protein thiol groups in V group. However, the observed increases in GSH pool concentrations in fibroblasts exposed to titanium discs with a passive layer anodized with standard and type II anodization vs. control appear insufficient to protect protein thiol groups from oxidation (↑SS concentration in V(st) and V(t)) vs. the control). Oxidation of disulfide groups can have a very negative reflection on all cellular processes, since keeping them in a reduced state is an essential factor for the activity of enzymes, messenger proteins, or transport proteins. An oxidation of the disulfide groups of lysyl oxidase causes impaired wound healing as a result of abnormal collagen synthesis .Changes in the activity of ROS-neutralizing enzymes, or changes in concentrations of small-molecule antioxidants observed in fibroblast cells, are not evidence of OS. They are, certainly, an adaptive response to the increase in ROS generation. ROS are capable of oxidizing any of the cellular components: proteins, lipids, DNA, RNA, or sugars, with a single biomarker of oxidative damage having little clinical value in diagnostic or prognostic terms. The most commonly measured indicators of oxidative modification include 4-HNE adducts with proteins, the concentration of disulfide groups, and AGE. 4-HNE adducts are simple to determine, and their concentration is directly proportional to the degree of cell membrane damage ,40.In the group of fibroblasts exposed to titanium discs lacking a passive layer, the concentrations of lipid peroxidation products, throughout the experiment, were higher compared to the other two groups of subjects and the control group. On days 7 and 14, elevated concentrations of 4-HNE adducts vs. control were observed in the group of fibroblasts exposed to standard anodized titanium discs. Of note is the absence of any change in the concentration of 4-HNE adducts in the group of fibroblasts exposed to passive layer discs with type II anodization vs. control. This result indicates the so-called reversibility of the process of lipid peroxidation in the situation of exposure to titanium discs with a passive layer with II type of anodization, as described in the case of salivary glands of rats exposed to a carbohydrate-rich diet . MoreoveAn increase in AGE concentrations in all groups of fibroblasts exposed to titanium discs vs. the control group was observed at the final stage of the experiment. Because AGE can activate a number of inflammatory reactions leading to accelerated vascular damage and reduced vascular permeability , an incrThe expression of specific growth factors, such as fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), transforming growth factor-beta (IGF-s), vascular endothelial growth factor (VEGF), and bone morphogenetic proteins (BMPs) in fibroblasts, osteoblasts, and endothelial cells during healing confirms the key role of these secreted factors in the bone repair process ,51. In fThroughout the experiment, we observed significantly lower concentrations of VEGF-A and FGF-2 in the medium taken from fibroblasts exposed to titanium discs regardless of the presence or absence of a passive layer. These results suggested that the presence of titanium implants interferes with the processes of angiogenesis and bone tissue regeneration. The observed deficiency of FGF-2 may result in a slower rate of mineralization and bone bridging in the fracture defect, as well as a reduced number of osteocytes in the bone healing after injury, as observed in the study of Murakami et al.  and PilmMetal content analysis was performed in mineralized medium samples by plasma ionization mass spectrometry (ICP-MS) using an external calibration method. Ion release was not determined in the cell lysate, for technical reasons. Therefore, the percentage of these ions that could penetrate the cells is not known, so it is difficult to look for cause-and-effect relationships between the concentration of ions in the medium and the parameters studied. The release of ions from titanium discs was highest at the beginning of the experiment and decreased with time. Throughout the experiment, the concentration of titanium, aluminum, and vanadium ions were highest in the medium taken from fibroblasts exposed to titanium discs with a hard-anodized passive layer.It is important to highlight the weaknesses of the current experiment. First, the research was conducted in a cellular model, which does not reflect the complex relationships and interactions at the implant–bone interface. Physiologically, the forces exerted on the implant material consist of tensile, compressive, and shear elements, and such conditions cannot be reproduced in the research model used. Secondly, we used only one type of cell, which does not allow us to reproduce the biological processes occurring in bone tissue. When analysing the results presented here, it should be taken into account that we also evaluated only some markers of oxidative modification and antioxidant barrier elements. Evaluation of other markers of oxidative stress may completely or partially change our observations and assumptions. We used one thickness of the anode layer; it is not known how other thicknesses of this layer would affect biological systems. It is also not known whether the release of ions would be at a similar level.The study was approved by the Bioethics Committee of the Medical University of Bialystok (resolution no. APK.002.280.2021).TM), which was enriched with antibiotics and antimycotics , streptomycin (10 µg/mL), and amphotericin B (25 ng/mL)) and a bovine serum fibroblast growth kit  (5 ng/mL), L-glutamine (7.5 mM), ascorbic acid (50 ug/mL), hydrocortisone (1 µg/mL), rh insulin (5 µg/mL), bovine serum (2%), and also red phenol solution ). Cell cultures were conducted at 37 °C in an atmosphere of 5% CO2 (Forma Steri-cycle i160). Cells were seeded into culture bottles  as well as plates  at a density of 3000 cells per cm2. Cells from the 3rd passage were used for the experiments. Cell morphology was assessed daily using a Delta Optical IB-100 inverted optical microscope . Culture medium  was changed every 2 days. The work was carried out under aseptic conditions, in a laminar airflow chamber . Cells were cultured in 150 cm2 bottles  until a confluence of 80% was reached (as recommended by the manufacturer). The medium was harvested, after which the cells were washed twice with sterile PBS buffer  , and heated to 37 °C . Then, cells were trypsinized with 3 mL of freshly diluted trypsin solution . After a 4 min incubation, trypsin was neutralized by adding 10 mL of medium. Then, the cells were sieved into 12-well plates. Cells were cultured by adding 1 mL of medium to each well. Once the cells reached 80% confluence (average 3–4 days), titanium/polystyrene discs were applied to the bottom of the plates. All variants of the experiment were carried out in 6 independent experiments. Cells were cultured for 24 h and 7, 14, and 21 days. After sufficient time , cells were trypsinized and centrifuged .The study used fibroblasts obtained from human gingival tissue , which were purchased from ATCC . Cells were cultured in fibroblast-dedicated basal medium , the supernatant fluid was discarded, and the cell pellet was resuspended in hypotonic buffer (5 mL per 1 g of pellet) containing 100 mM sucrose , 10 mM MOPS , 1 mM EGIA (ethyleneglycol-O-O’-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid, Sigma-Aldrich, M6768, Poznań, Poland), and allowed to swell on ice for 10 min. In the next step, the samples were homogenized with a glass up/down hand homogenizer, after which hypertonic buffer  was added and diluted with isolation buffer , 0.1% BSA without fatty acids ). The samples were then subjected to centrifugation , after which the homogenization process was repeated 6 times. After another centrifugation , the supernatant fluid was removed, and 5 mL of MiPO5 buffer , 20 mM HEPES , 10 mM KH2PO4 , 3 mM MgCl2 × 6H2O 0.5 mM EGTA , 20 mM taurine , 0.1% BSA without fatty acids), and, once again, the samples were subjected to centrifugation . After centrifugation, the supernatant liquid was discarded, and 50 µL of MiPO5 buffer was added to the pellet. The samples were mixed on a vortex and used immediately for the assays.Isolation of mitochondria was performed using the method described previously . After cThe discs were made to individual order by ChM sp. z o. o. . All discs were polished according to a standard polishing procedure to a surface roughness of Ra ≤ 0.63. Rosler Keramo-Finish Compound 629  was used for polishing.The study used discs with a diameter of 21 mm and a thickness of 1 mm, constructed of titanium alloy Ti-6AI-4V containing a minimum of 88 wt% titanium, as well as 6 wt% aluminum and 4 wt% vanadium. Other titanium alloys produced by ChM do not contain vanadium. They may contain an admixture of niobium or a higher carbon content. The titanium discs were constructed in three varieties depending on the process of formation of the passive layer: standard anodized discs (III type) (V(st)), cold anodized discs (II type) (V(t)), and raw discs not subjected to the anodizing process (V). The control group consisted of polystyrene discs of the same diameter and thickness as the titanium discs.The discs have undergone the following treatment:1. Discs without a passive layer, raw: they have not been electrochemically treated.3 acids, and subjected to a Type III anodizing process in a 0.5 M H2SO4 solution at voltages up to 100 V. The discs were washed again to remove residues of the electrochemical treatment bath.2. Type III (standard) anodization: the discs were subjected to a washing process to remove the polishing paste. Then, the discs were chemically deoxidized in a solution of a mixture of HF/HNO3 acids, and subjected to a Type Il anodizing process in a NaOH solution with a pH above 13 at voltages up to 100 V. The discs were washed again to remove residues of the electrochemical treatment bath.3. Type II (hard anodizing): the disks were anodized in accordance with AMS 2488D (“Anodic Treatment—Titanium and Titanium Alloys Solution pH 13 or Higher”), this specification describes the technical requirements related to the production of an electrolytic conversion (anodic) coating on titanium and titanium alloys. It describes the properties of the coating. The discs were washed to remove residues such as polishing paste. The disks were then chemically deoxidized in a solution of a mixture of HF/HNOThe metallographic microsection of the Ti6Al4V sample was analysed using the FEI Quanta 3D FEGSEM scanning electron microscope (The Netherlands) equipped with an EDAX X-ray spectrometer.Observations were conducted using backscattered electron (BSE) images at 2000×–10,000× magnification. Analysis of chemical composition—X-ray spectroscopy with energy dispersion. The thickness of the layer on the exterminations measured in an electron microscope was in the range of 1–2 micrometres; the average value was 1.5 +/− 0.5). It was not technically possible to prepare microsections for measuring the thickness of the standard—layer thickness was too small. The images obtained suggest that the surface topography of both types of layers was similar. Coating nanohardness tests determined by the instrumental method on the Micro Combi Tester device . The nanohardness value of the type II anodic layer was 160% of the value of the standard layer.After the treatment process, the discs were rinsed in water and then fully immersed in an aqueous detergent solution , followed by ultrasonic cleaning in an Elmasonic X-tra Pro 800  washer for 15 min at 130 kHz and a bath temperature of 55 ± 5 °C. The ultrasonic washing process (non-sterile) included inter-operative and final washing. After washing, the discs were dried in a Pol-Eko SLW 240 electric dryer  at 105 ± 5 °C for a minimum of 60 min. After this stage, the products did not leave the packaging room. The next stage in the production of discs is to subject them to washing again in a disinfector washer. The discs were subjected to disinfectant washing with detergent in a closed circuit at 55 °C. The final rinse with thermal disinfection was conducted in demineralized water at 90 °C. Finally, drying was carried out at the same temperature for 40 min. After washing and drying, the discs were packed in a double layer comprising paper–foil bags. Each sample was packaged separately. The final step in preparing the discs was steam sterilization of the packed discs in an ASL 100MSV sterilizer, at 134 °C and an exposure time of 15 min.Cell viability was assessed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay, which takes advantage of the presence of mitochondrial succinate dehydrogenase only in living cells. Cells from 12-well plates were washed with 1 mL of PBS . After pipetting off the buffer, 1 mL PBS and 25 µL MTT (5 mg MTT per 1 mL PBS) were added to the cells again. The plates were incubated for 10 min at 37 °C in a laboratory hothouse. Then, the liquid was pipetted off and the resulting crystals were dissolved in 1mL of DMSO solution . After 10 min of incubation at room temperature, 10 µL of Sorensen buffer  was added to the plates. The absorbance of the mixture was measured at 570 nm. The results for each group were expressed as a percentage of the control, which was considered 100%.Total protein concentration was assessed in the mitochondrial suspension, as well as the activity of superoxide dismutase , catalase , glutathione peroxidase , and the concentration of reduced glutathione (GSH). NADPH oxidase (NOX) activity, concentration of oxidative damage to lipids ) and proteins (content of disulfide groups (SS), end products of advanced protein glycation (AGE)) were also assessed. Nitrosative stress parameters were evaluated: peroxynitrite (ONOO-) and 3-nitrotyrosine (3-NT). In addition, respiratory chain function was assessed by measuring the activities of complex I (EC 1.6.5.3) and complex II (EC 1.3.5.1), cytochrome c oxidase , and citrate synthase , as well as caspase-3 (CASP-3) activity. In the medium taken from the cells, the concentration of fibroblast growth factor FGF-2, the concentration of vascular endothelial growth factor (VEGF-A), and the content of metals (titanium (Ti), aluminum (AI), and vanadium (V)) were determined.All assays were performed in duplicate. 96-well microplates were incubated in a DIS-4 Sky-Line shaker, Elmi, while Eppendorf-type tubes were incubated in a SalvisLab laboratory hothouse, . ELISA microplate washing was performed using a Biotek 50 TS automatic microplate washer. Absorbance/fluorescence of the samples was measured using a Tecan Infinite M200 PRO Multimode microplate reader . The results obtained were standardized per 1 mg of total protein.Total protein content was determined by the bicinchoninic colorimetric method using the Thermo Scientific PIERCE BCA Protein Assay commercial diagnostic kit . The composition of individual reagents is protected by the manufacturer’s secrecy.2+, which shows a maximum of absorption at 562 nm.The principle of the method is based on the production of a stable complex between a peptide band and bicinchoninic acid (BCA) and copper ions Cu2O2 at a wavelength of 240 nm. 1 unit of enzyme activity was defined as the amount of enzyme degrading 1 mol of H2O2 in 50 mM phosphate buffer pH 7.0 for 1 min at 25 °C. CAT activity was standardized to total protein and expressed in mol/min/mg of total protein.Catalase activity  was measured colorimetrically using the Aebi method . The pri+ [Glutathione peroxidase  activity was determined by a colorimetric method based on the reduction of glutathione with simultaneous oxidation of NADPH to NAD+ . The amoSuperoxide dismutase  activity was determined by a colorimetric method according to Misra et al. . The priReduced glutathione (GSH) concentration was determined colorimetrically based on the reduction of 5,5-dithio-bis-(2-nitrobenzoic) acid (DTNB) to 2-nitro-5-mercaptobenzoic acid under the influence of GSH . AbsorbaThe concentration of 4-hydroxynonenal adducts with proteins (4-HNE) was determined by immunoenzymatic ELISA using an off-the-shelf diagnostic kit . The composition of individual reagents is protected by manufacturer’s confidentiality. Absorbance was measured at 450 mm. The concentration of 4-HNE was calculated using a standard curve for HNE-BSA. The concentration of 4-HNE was standardized to total protein and expressed as nmol/mg of total protein.The total level of disulfide groups (SS) was measured colorimetrically using Ellman’s reagent in 0.1 M phosphate buffer, pH 8.0. Absorbance was measured at 412 nm, and thiol group content was calculated from the standard curve with GSH as standard .The content of advanced protein glycation end products (AGEs) was assessed using the fluorimetric method described by Kalousova et al. . This meAGE content was standardized to total protein content and expressed in arbitrary fluorescence units (AFU)/mg of total protein.2•− formation in a reaction catalyzed by NOX using lucigenin as a luminophore. The amount of enzyme that causes the release of 1 mM O2•− at 37 °C in 50 mM phosphate buffer pH 7.0 in 1 min was defined as 1 unit of NOX activity. NOX activity was standardized to total protein content and expressed as nM O2•−/min/mg protein total [The activity of NADPH oxidase  was determined using a chemiluminescent method. The principle of the method is to measure the rate of O−1 cm−1 was used. The concentration of ONOO- was standardized to total protein content and expressed in pmol/mg of total protein.Peroxynitrite concentration (ONOO-) was determined by fluorimetric method  based onThe concentration of 3-nitrotyrosine (3-NT) was determined by immunoenzymatic ELISA using a commercial diagnostic kit . The composition of the individual reagents is protected by manu facturer’s confidentiality. Absorbance was measured at 450 nm. The concentration of 3-NT was calculated using a standard curve for 3-NT. The concentration of 3-NT was standardized to total protein and expressed as pmol/mg of total protein.FGF-2 and VEGF-A concentrations were determined by immunoenzymatic ELISA using the commercial ELISA Kit for Human Heparin-binding growth Factor 2   and ELISA Kit for IT man Vascular endothelial growth factor A (VEGF-A), ElAab, E0143h. The composition of each reagent is protected by manu facturer’s confidentiality. Absorbance was measured at 450 nm.The activity of mitochondrial complex I (EC 1.6.5.3) was determined by a colorimetric method based on the reduction of 2,6-dichloro-diphenol (DCIP) by electrons derived from decylubiquinol . The dec1 unit of mitochondrial complex I activity was defined as 1 µmol of DCIP reduced in 1 min at 37 °C. Mitochondrial complex I activity was standardized to total protein and expressed as mU/mg of total protein.Mitochondrial complex II (EC 1.3.5.1) activity was determined by a colorimetric method based on measurement of succinate-ubiquinone reductase activity . The decCytochrome c oxidase  activity was determined by a colorimetric method by measuring the oxidation of reduced cytochrome c at 550 nm . CytochrCitrate synthase  activity was assessed colorimetrically using a method with 5-thio-2-nitrobenzoic acid, which is formed from 5,5′-dithiobis-2-nitrobenzoic acid during the CS synthesis reaction . AbsorbaCaspase-3 (CASP-3) activity was assessed by colorimetric assay using Ac-Asp-Glu-Val-Asp-p-nitroanilide as substrate . The pri3) and 0.35 mL of 30% H2O2 were added. The test tubes were capped with a stopper and placed in a water bath at 90 °C for 90 min. The samples were then allowed to cool and degassed in an ultrasonic bath for 30 min (100% power). After degassing, the samples were diluted with deionized water to a volume of 9 mL and stored at 4 °C until analysis. To control the accuracy of the measurements, a recovery experiment was performed. For this purpose, the medium samples were enriched with Al, Ti and V standards at three different concentrations: 3 ng mL−1, 5 ng mL−1, and 6 ng mL−1 (n = 3), and then digested in a manner analogous to the medium samples from the cells.In order to determine the metal content of the samples, cell medium was taken every 3 days for 3 weeks (the first medium was taken on the 3rd day after the titanium plates were applied), in which aluminum, titanium, and vanadium were determined by ICP-MS (inductively coupled plasma mass spectrometry). The medium from above the cells and the control medium were mineralized in the same way. In a 12-mL centrifuge test tube , 3 mL of cell medium was transferred and 0.65 mL of 65% nitric acid  equipped with an SPS4 sample feeder, a MicroMist nebulizer, a Scott-type mist chamber cooled by a Peltier system, nickel sampler and collector cones, and an ORS−1 in the control medium solution after mineralization. A Rh solution of 100 ng mL−1 was used as an internal standard to compensate for the influence of the matrix.Quantitative determinations in mineralized samples of cell medium were conducted using the calibration curve method. To prepare standard solutions of analytes: Al, Ti, and V, single element standard solutions were used, from which working standards were prepared by appropriate dilution. Calibration curves of Al, Ti, and V were made in the concentration range from 0.5 to 50 ng mLblank/a , were, respectively: 0.645 ng mL−1 for Al, 0.417 ng mL−1 for Ti, and 0.052 mg mL−1 for V. The accuracy of the method was confirmed each measurement day in a recovery experiment by analysing mineralized medium samples enriched with analytes. Metal recoveries were 102–106% for Al, 101–106% for Ti, and 91–102% for V.Instrument analyte limits of detection (LOD), calculated as 3SDStatistical analyses were performed using GraphPad Prism 9.0 software . Normality of distribution was checked using the Shapiro–Wilko test, while homogeneity of variance was checked using the Brown–Forsythe test. Quantitative variables were described by parameters of descriptive statistics, i.e., arithmetic mean and standard deviation. One-way ANOVA analysis of variance with Tukey’s HSD post-hoc test was used for group comparisons. A significance level of less than 0.05 was assumed for the statistical analyses performed.Person’s parametric correlation test was used to assess the relationship between quantitative variables.The II type of anodization of the titanium surface induced slight difference in the antioxidant response of human, non-genetically modified, primary gingival fibroblasts exposed to titanium discs;Type II anodization: prevented changes in complex II activity (vs. control); inhibited CS activity the least, as well as in the final phase of culture, reduced the degree of COX inhibition and prevented apoptosis in the mitochondria compared to the other test groups and the control group;The obtained results proved the existence of mitochondrial dysfunction and redox imbalance in the mitochondria of fibroblasts exposed to hard-anodized titanium discs, which suggests the need to search for new materials perhaps biodegradable in body tissues.


Histiocytoid cardiomyopathy is a rare infancy cardiac disorder manifesting as severe cardiac arrhythmias or dilated cardiomyopathy. There is no specific treatment for these arrhythmias. This is the first report of infantile histiocytoid cardiomyopathy whose refractory ventricular arrhythmias were successfully controlled by high-dose carvedilol.A 4-month-old girl presented with asystole, and recurrent ventricular tachycardias. From the histological findings and clinical symptoms, she was diagnosed as histiocytoid cardiomyopathy. Sedatives were the most effective therapy for her arrhythmia, but the cardiac sympathetic denervation was not effective enough. Finally, her ventricular arrhythmias were controlled with high-dose carvedilol, and she was discharged on hospitalization Day 393.2+ release (SOICR) in myocardium at high dosage. The arrhythmias did not disappear with bisoprolol, landiolol, or verapamil, but high-dose carvedilol was effective. This clinical course suggested that the arrhythmias in histiocytoid cardiomyopathy might be related with SOICR. High-dose carvedilol might be a key drug for patients with histiocytoid cardiomyopathy.Carvedilol is the only beta blocker that directly acts on the ryanodine receptor (RyR2) and inhibits store-overload-induced Ca The arrhythmia control in histiocytoid cardiomyopathy is challenging with no specific therapy.2+ release might be a key drug for patients with histiocytoid cardiomyopathy.High-dose carvedilol that directly inhibits store-overload-induced Ca1, is a rare, genetic infancy or childhood cardiac disorder that mainly affect females younger than 2 years, and manifest as severe cardiac arrhythmias or dilated cardiomyopathy.2–4 Autosomal recessive, X-linked, and maternal inheritance has been described in the previous reports.3 Extracardiac manifestations include abnormalities of the central nervous system, the eyes, the endocrine systems, or steatosis of the liver.3 The diagnosis is established based on these clinical findings and myocardial biopsy or autopsy.3 There is no specific treatment for arrhythmias of histiocytoid cardiomyopathy. Cataldo et al.5 and Hirano et al.6 reported the cases of histiocytoid cardiomyopathy treated with radiofrequency ablation but their outcomes were not satisfactory.Histiocytoid cardiomyopathy, first described by VothWe report a case of histiocytoid cardiomyopathy with refractory ventricular tachycardia that was successfully controlled by high-dose carvedilol.A 4-month-old Asian girl was transferred to the previous hospital due to fever and episodic seizure after vaccination. She was asystole on arrival, and after intravenous administration of adrenaline and cardiac resuscitation, she had ventricular fibrillation requiring several defibrillations. She was transferred to our hospital on suspicion of myocarditis. She had no family histories of arrhythmias, cardiomyopathies, or sudden cardiac death.Figure 1. Her heart rate was elevated to 140 beats per min (sinus rhythm), blood pressure was 84/56 mmHg, and percutaneous oxygen saturation (SpO2) was 96%. On physical examination, she had no external malformations, inspiratory retraction, nor heart murmur. The echocardiography showed no anomalies of the heart nor the great vessels. Her blood test showed no abnormalities of electrolytes including potassium, calcium, and magnesium. C-reactive protein was elevated to 7.03 mg/dL, but leucocyte count was normal and there was no elevation of creatinine kinase. For definitive diagnosis, myocardial biopsy was performed on Day 1. During biopsy ventricular tachycardia (VT) was easily induced by catheter manipulation, and intermittent sustained or non-sustained VT was observed in the critical care unit ; therefore, continuous intravenous infusion of amiodarone, landiolol, and deep sedation was started. Histological work-up using a light microscope showed large, round polygonal cells (histiocytoid cells) clustered under the endocardium, and the examination with electron microscope revealed numerous enlarged mitochondria in the histiocytoid cells . From these findings and clinical course, she was diagnosed as histiocytoid cardiomyopathy. Since VT disappeared on Day 15 with oral amiodarone and bisoprolol, an implantable cardioverter defibrillator (ICD) was implanted for secondary prevention on Day 63.The clinical course is shown in Figure 2B–D) therefore, oral verapamil was added on, and amiodarone was weaned off. On Day 132 with tapering the sedatives, however, VT recurred and required sedation and intravenous amiodarone again. Oral verapamil and bisoprolol were thought to be ineffective and were stopped. Under deep sedation, the arrhythmias were completely controlled with intravenous amiodarone and landiolol. Since sedatives appeared to be more effective than anti-arrhythmic drugs for her arrhythmias, cardiac sympathetic denervation therapy8 was scheduled.On Day 73, however, the ICD systems had to be removed due to infection of the device pocket. Frequent VT or fibrillation recurred during and after the operation, and intravenous amiodarone, landiolol, and sedatives were administered again. A month later with tapering the sedatives, various types of VT manifested, including junctional tachycardia, torsade de pointes, and short-coupled variant of torsade de pointes , the aorta and pulmonary artery were divided and each anastomosed,9 and the ICD system was re-implanted on Day 213 after approval from the hospital ethics committee. She developed frequent VT for 2 weeks after the surgery because of systemic or local inflammation. Intravenous magnesium sulfate and potassium chloride were partially effective but could not control her arrhythmias completely.Due to a lack of experience with ganglion blockade2+ release (SOICR) in the myocardium,10 was started and was increased gradually, while the sedatives were concurrently tapered. When carvedilol was increased to more than 0.8 mg/kg/day, VT decreased significantly, and no PVCs were seen at the dose of 1.5 mg/kg/day. Finally, oral carvedilol 1.5 mg/kg/day and amiodarone 13 mg/kg/day completely controlled her VT without ventricular dysfunction and bradycardia (with a help of AAI pacing).From Day 253, oral carvedilol 0.05 mg/kg/day, an inhibitor of store-overload-induced CaOther than arrhythmias, she suffered from severe hypoxic encephalopathy, epilepsy, and liver steatosis. After epilepsy was controlled with phenobarbital and levetiracetam, she was discharged on Day 393. She readmitted because of pneumonia several times but VT has not been recorded since discharge.2–4 Pathohistologically, histiocytoid cardiomyopathy is characterized by the development of abnormal Purkinje fibres, which result in subendocardial yellow-tan nodules built up of histiocytoid myocytes, which are filled with abnormal mitochondria.4 Generally, arrhythmia control in histiocytoid cardiomyopathy is challenging with no specific therapy.6 To our knowledge, this is the first case report of histiocytoid cardiomyopathy with refractory ventricular arrhythmias controlled by oral medication.Histiocytoid cardiomyopathy is a rare infancy or childhood disorder, ∼20% of which present as sudden death.8 Cataldo et al.5 reported a case of histiocytoid cardiomyopathy in which sympathetic denervation by removal of T2–T3 stellate ganglion had been performed but was ineffective. Without the experience of these techniques in our hospital, we chose open surgery in which the aorta and pulmonary artery were divided and anastomosed each (similar to heart transplant9). However, after denervation, VT was frequently seen with lower dose sedatives. This approach might be ineffective for this kind of arrhythmia.At first, cardiac denervation was thought to be effective because deep sedation was more effective than anti-arrhythmic drugs. Studies have shown that the autonomic nervous system has an important role in triggering and maintaining ventricular arrhythmia, and cardiac sympathetic intervention by stellate ganglion blockade or surgical resection of extrinsic sympathetic chain may reduce the burden of ventricular arrhythmias.2+ release (SOICR) in the myocardium.10 Ventricular tachycardia was observed with <0.8 mg/kg/day carvedilol, but disappeared with a higher dose, therefore high-dose carvedilol played an important role for control of her arrhythmias.The key drug of this case was carvedilol. In this case, bisoprolol, landiolol, and verapamil could not control ventricular arrhythmia. In order to control the arrhythmias with oral medication, we chose carvedilol that is the only beta blocker inhibiting store-overload-induced Ca11 Verapamil and other beta blockers can also suppress SOICR by acting on dihydropyridine receptor (DHPR) and beta-adrenergic receptor (BAR) each.12 In contrast, carvedilol directly acts on the cardiac ryanodine receptor (RyR2).10 However, the concentrations of carvedilol required to suppress SOICR are much higher than those for beta blockade, therefore, strong inhibition of SOICR requires high doses of carvedilol.10 The clinical course of our case suggests that the SOICR of RyR2 is one of the causes of her arrhythmias, consequently high-dose carvedilol was effective for her. RyR2 gene mutations are often reported in cases of CPVT but were negative based on her blood sample.The SOICR is a cause of triggered activity, therefore, carvedilol is thought to be more effective than the other beta blockers for catecholaminergic polymorphic ventricular tachycardia (CPVT).2+ concentration. The Ca2+ concentration in cardiomyocytes is regulated by some exchanger and receptors on cytomembrane, sarcoplasmic reticulum, and mitochondria .14 The abnormal mitochondria in cardiomyocytes in histiocytoid cardiomyopathy and resulting in abnormal regulation of intracellular Ca2+ concentration may be associated with their refractory ventricular tachycardias.Generally, SOICR of RyR2 is caused by elevation of intracellular CaTo our knowledge, there is no report of successful control of arrhythmias in histiocytoid cardiomyopathy with high-dose carvedilol. High-dose carvedilol that directly inhibits SOICR might be a key drug for patients with histiocytoid cardiomyopathy.


Learning in the operating room (OR) for residents in anesthesiology is difficult but essential for successful resident education. Numerous approaches have been attempted in the past to varying degrees of success, with efficacy often judged afterward using surveys distributed to participants. The OR presents a particularly complex set of challenges for academic faculty due to the pressures required by concurrent patient care, production pressures, and a noisy environment. Often, educational reviews in ORs are personnel specific, and instruction may or may not take place in this setting, as it is left to the discretion of the parties without regular direction.This study aims to determine if a structured intraoperative keyword training program could be used to implement a curriculum to improve teaching in the OR and to facilitate impactful discussion between residents and faculty. A structured curriculum was chosen to allow for the standardization of the educational material to be studied and reviewed by faculty and trainees. Given the reality that educational reviews in the OR tend to be personnel specific and are often focused on the clinical cases of the day, this initiative sought to increase both the time and efficiency of learning interactions between learners and teachers in the stressful environment of the OR.The American Board of Anesthesiology keywords from the Open Anesthesia website were used to construct a weekly intraoperative didactic curriculum, which was distributed by email to all residents and faculty. A weekly worksheet from this curriculum included 5 keywords with associated questions for discussion. The residents and faculty were instructed to complete these questions on a weekly basis. After 2 years, an electronic survey was distributed to the residents to evaluate the efficacy of the keyword program.A total of 19 teaching descriptors were polled for participants prior to and following the use of the intraoperative keyword program to assess the efficacy of the structured curriculum. The survey results showed no improvement in intraoperative teaching based on respondent perception, despite a slight improvement in teaching time, though this was statistically insignificant. The respondents reported some favorable aspects of the program, including the use of a set curriculum, suggesting that greater structure may be beneficial to facilitate more effective intraoperative teaching in anesthesiology.Although learning is difficult in the OR for residents, the use of a formalized didactic curriculum, centered on daily keywords, does not appear to be a useful solution for residents and faculty. Further efforts are required to improve intraoperative teaching, which is well known to be a difficult endeavor for both teachers and trainees. A structured curriculum may be used to augment other educational modalities to improve the overall intraoperative teaching for anesthesia residents. Education is a critical component of residency training; however, learning in the operating room (OR) for residents in anesthesiology is difficult and unstructured . VariousAnesthesia education efficacy is difficult to assess due to the subjective nature of teaching and the rare measurable data points of formal examinations. Survey-based assessments to ascertain efficacy have been deployed in the past. Wakatsuki et al  used thiAn important consideration when discussing intraoperative teaching is the maintenance of safety in patient care and vigilance for ongoing procedures. The practice of reading intraoperatively during periods of maintenance anesthesia  has beenIntraoperative teaching is also difficult for surgical services, in which faculty and residents spend the majority of their time engaging in patient care. A surgical study by Iwaszkiewicz et al  showed tAnother interesting approach to intraoperative education for residents is the “briefing, intraoperative teaching, debriefing” model . This moThe American Board of Anesthesiology keywords  were used for a new intraoperative learning curriculum for the Department of Anesthesiology at Rutgers New Jersey Medical School .Each week, 5 keywords were selected at random from the American Board of Anesthesiology keywords list, and a series of questions (3-5 per keyword) distributed based primarily upon the information on the Open Anesthesia website. The questions were open-ended to promote conversation between residents and faculty. The residents were instructed to choose a keyword each day to discuss with their assigned intraoperative faculty and to make that determination the night before so that both faculty and residents could study the topic ahead of time. Keywords were sent via email to residents and faculty on each Friday for the following week.The keyword program began in March of 2019, and instructions were given in detail both at the beginning of the program and at regular intervals. The program continued for 2 years prior to evaluation by resident surveys. The survey to assess the efficacy of the keyword program was a modified version of the Anesthesia Theater Education Environment Measure (ATEEM) questionnaire . The ATENote that despite the distribution of the weekly keywords to all members of the department, keyword discussions did not occur between the residents and faculty daily. This was due to changes in staffing, changes in cases or OR assignments, emergent cases, or an inability of the faculty member to remain in the OR during maintenance anesthesia, when most intraoperative teaching occurs.Institutional review board of experimental protocols was approved by Rutgers University (reference number Pro2019001411). All methods were carried out in accordance with the relevant guidelines and regulations. All participants signed informed consent for participation in this study. No compensation was provided for the study participants. Moreover, study data were deidentified prior to analysis.t test. No statistically significant changes were found between the 2 groups of responses, indicating no effect for the program. The residents were asked to rate each of these descriptors on the Likert scale for days when keywords were used and for days when keywords were not used for intraoperative teaching. Using an value of .05, no category demonstrated significant difference between the 2 groups.The program was initiated in July of 2019 and continued for 2 years until June 2021. Surveys were distributed to residents in June 2021, and 54 responses were recorded for the surveys, accounting for 90% of all residents during this time period. The results of the responses to the 19 questions —comparinThe teaching helps to develop my confidence.I receive effective supervision from the clinical teachers.Teaching is done at appropriate times not affecting vigilance.I receive teaching anesthetic specialty areas targeted at my learning needs.The teacher helps to develop my competence.My clinical teachers are accessible for advice.I experience friendly relations with my teachers in the operating room.The clinical teachers in this hospital interact well with trainees.My clinical teachers promote an atmosphere of mutual respect.I have an appropriate level of clinical responsibility.My clinical teachers are clear in their teaching.I am clear about the learning objectives of teaching sessions in the operating room.I receive the necessary clinical supervision.I have a good collaboration with anesthesia staff.I have the opportunity for on-the-job learning.My clinical teachers have established good rapport with me.I am encouraged to participate in the theatre setting.There is a systematic clinical training program.I feel able to ask the question I want.This investigation sought to improve intraoperative teaching by incorporating a structured curriculum with keywords for residents in anesthesiology and the faculty. Unfortunately, there was no demonstrable positive effect to this intervention. There was essentially no statistically significant difference in the responses by the residents to a survey when comparing intraoperative teaching with and without the use of keywords. This may demonstrate a failure on the part of the initiative to make meaningful improvements to intraoperative teaching. The survey used for the study was an ATEEM tool, which is a validated structure to assess education in anesthesiology intraoperatively, and it was modified to assess the efficacy of the keyword program in this study.In Nonetheless, the residents identified several characteristics of the keyword program that they believe contributed to a positive effect of keywords on intraoperative teaching in Intraoperative learning is a notoriously difficult task for educators of residents in medicine. Past studies have attempted to use such modalities as traditional lectures, problem-based learning discussions, and case debriefing, as well as the targeted assessment of residents -3. Our sLimitations of this study include a lack of assessment of the percentage of time when the intraoperative keyword curriculum was used by learners. The keywords were sent to both residents and faculty weekly, with no mechanism in place to ensure a successful adherence to the program. This was deemed too difficult due to clinical production pressure, patient emergencies, call burden, vacations, and off-site rotations. Nonetheless, this is a significant limitation, because survey respondents may be included who did not participate in the program or use the keywords in a meaningful manner during the study period.After using this intraoperative keyword teaching program for more than 2 years, this study revealed that it had a minimal effect on intraoperative teaching between the anesthesiologist resident trainees and faculty. Consideration should be given to alternate methodologies to improve intraoperative teaching for learners in the anesthesiology residency. The results presented in this study may suggest characteristics of a future intervention that may be more successful in improving intraoperative education. Nearly half of the respondents agreed that the use of a structured curriculum contributed positively to education. Perhaps the use of a structured curriculum that is custom tailored to a resident’s current rotation  may be more efficacious for residents’ educational enhancement. It was also noted by the survey respondents that the coordination of educational material between the residents and faculty was advantageous for learning. This could be incorporated into a policy in which the resident and faculty member plan their educational discussions ahead of time to provide both the teacher and the learner the opportunity to review a chosen topic before their review together in the OR.


In the rapidly evolving world of digital technology, few concepts have garnered as much attention and intrigue as non-fungible tokens (NFTs). Originating from the domain of art and collectibles, NFTs have transcended their initial use cases to impact various fields, from entertainment to real estate, and now, healthcare . While fNeurosurgery, with its intricate procedures and reliance on detailed imaging and data, offers a fertile ground for NFT applications . WhetherFor a clearer understanding of the specialized terminology used in this paper, readers are encouraged to refer to the glossary of key terms presented in Table The author (ALM) conducted a comprehensive scoping review to explore the potential applications of NFTs in neurosurgery. A systematic search was carried out using key electronic databases: PubMed, Embase, IEEE Xplore, and Google Scholar, without date restrictions. The search was executed in August 2023. To ensure a broad capture of relevant literature, the search terms utilized were “(“non-fungible token*” OR NFT OR NFTs OR blockchain OR “block chain”) AND .” Given the novelty of the topic, grey literature sources, including preprint repositories and conference proceedings, were also scrutinized to capture emerging research and concepts in the field. Articles not written in English, those that were merely abstracts, and case reports were excluded to maintain the specificity of the review. Articles that specifically discussed the applications, challenges, or innovations of NFTs within neurosurgical practices or related patient data management were included. Further pertinent articles were identified and sourced from the reference lists of the initial set of articles. Additionally, relevant texts from non-neurosurgical medical domains and ancillary fields were examined to gain a comprehensive understanding and to identify potential cross-disciplinary applications and insights. Data from these articles were extracted and synthesized to inform the main content of the review.At the foundation of NFT technology is the blockchain — a decentralized ledger system that supports cryptocurrencies and decentralized applications. But what exactly is a blockchain? In essence, a blockchain is a chain of blocks, where each block contains data, and each subsequent block carries a unique cryptographic signature, ensuring the integrity and immutability of the data stored within –9.NFTs are unique tokens minted on blockchains. Unlike cryptocurrencies like Bitcoin or Ethereum, which are fungible and interchangeable, NFTs are distinct and cannot be exchanged on a one-to-one basis . This unFor a detailed, sequential overview of how blockchain technology and NFT function, especially in the context of medical data management, refer to Table NFTs, with their unique ability to authenticate and provide undisputed digital ownership, can offer transformative solutions to longstanding issues in data management, procedure verification, and patient engagement , 13. ThiIn neurosurgery, the precision and clarity of diagnostic imaging are paramount . ProceduNeurological conditions often have underlying genetic components. As genomic sequencing becomes more commonplace, there’s an increasing amount of data about individual genetic predispositions to certain neurological conditions or even the genetic constitution of individual tumors . NFTs caClinical trials are the cornerstone of medical advancements, and the integrity of their records is paramount . NeurosuThe field of neurosurgery is constantly evolving, with new techniques and procedures being developed regularly . NFTs caNeurosurgical treatments often involve the use of specialized medications, for example, 5-aminolevulinic acid hydrochloride (Gliolan) for tumor resections, selumetinib (Koselugo) for neurofibromatosis type 1–related inoperable plexiform neurofibroma, or bevacizumab (Avastin) for neurofibromatosis type 2–related vestibular schwannomas –26. EnsuRefurbished medical devices present economic advantages, yet their reintroduction to the healthcare system often sparks concerns about quality and potential counterfeit risks , 30. An Virtual reality (VR) and augmented reality (AR) are becoming integral in surgical planning and patient education. Surgeons can use VR to simulate complex procedures, while patients can use AR to better understand their conditions and treatments , 33. NFTNFTs, celebrated for their unique value proposition around digital ownership and authenticity, introduce a myriad of monetization possibilities in neurosurgery . It’s crIn the current era of data-driven medical research, there’s an increasing interest in acquiring expansive datasets . With NFInnovative surgical techniques or proprietary surgical tools could be tokenized and licensed to other professionals or institutions. Instead of a one-time sale, neurosurgeons or institutions could charge a recurring fee for the continued use of their innovations.Neurosurgeons could create and tokenize detailed training modules, virtual surgeries, case studies, scientific manuscripts, and their supporting datasets. By doing so, they not only ensure the authenticity and originality of their work but also pave the way for a new era of digital dissemination and access. Medical students, institutions, researchers, or other professionals could purchase access to these educational and research-based NFTs, ensuring genuine content while simultaneously providing a potential revenue stream for the creators.In a more patient-centric approach, neurosurgical clinics could offer tokenized premium services. Patients holding a particular NFT could receive benefits such as priority scheduling, access to detailed digital reports, or virtual consultations.As an example, NFTs could be harnessed to facilitate access to specialized medical services, such as outpatient rehabilitation. Consider a patient who requires a tailored rehabilitation program following a complex neurosurgical procedure. Instead of traditional referral systems or insurance approvals, healthcare providers or insurers could issue NFTs that represent a package of specific rehabilitation services. Upon acquisition, patients could redeem these NFTs at partnered outpatient centers, ensuring they receive the exact suite of services tailored to their recovery needs. Not only could this streamline the patient’s access to care, but it also offers a transparent and verifiable method of ensuring service quality and authenticity. Furthermore, the decentralization aspect of blockchain, upon which NFTs are built, can empower patients by giving them direct control over their rehabilitation journey. They can choose when and where to redeem their NFT and even transfer or sell it should they decide on an alternative rehabilitation pathway.Tokenized research projects could attract funding from institutions, pharmaceutical companies, or even public grants. By purchasing an NFT associated with a research project, funders could receive periodic updates, early access to findings, or even acknowledgment in publications.One of the intrinsic features of NFTs is the ability to embed royalties . Every tWhile the integration of NFTs into neurosurgery offers promising avenues, it’s essential to approach this frontier with a balanced perspective. The implementation of any new technology, especially one as disruptive as NFTs, brings with it inherent challenges and potential limitations , 41. FroOne of the primary criticisms of blockchain technology, and by extension NFTs, is the energy consumption associated with maintaining and validating the blockchain, especially for proof-of-work systems . This enThough blockchain is praised for its security and immutability, it’s essential to differentiate between the security of the transaction history and the data to which an NFT might link . If an NThe ability to tokenize medical data, procedures, and even genomic information introduces a host of ethical questions . If patiWhile the concept of NFTs and blockchain might be familiar to tech-savvy individuals, it remains a complex topic for many. For widespread adoption in the neurosurgical community, there’s a need for user-friendly interfaces and platforms that abstract away the technical complexities while retaining the benefits of the technology. Training and education will also play a pivotal role in bridging this gap.The integration of NFTs into the medical field will undoubtedly face regulatory scrutiny . GovernmThe path ahead requires targeted research to validate applications, refine methodologies, and ensure optimal integration. The upcoming segments detail specific areas of interest and potential research directions that can shape the future role of NFTs in neurosurgery.The traditional proof-of-work blockchain model, with its high energy consumption, might not be sustainable for widespread healthcare applications. However, emerging consensus mechanisms, such as proof-of-stake and federated blockchains, offer more energy-efficient alternatives. Research into making these models compliant with healthcare requirements can open doors for more sustainable NFT use in neurosurgery.As the digitization of medical records and imaging continues to grow, there’s a clear need for secure platforms that allow for easy exchange of this data . IntegraWhile monetizing medical data has its pitfalls, it also offers opportunities. Future research can focus on developing models that prioritize patient welfare, ensuring that patients understand the implications of sharing their data and are fairly compensated for it. Ethical guidelines and best practices can be formulated to guide the process.For NFTs to be effectively integrated into neurosurgery, collaborations between blockchain experts, software developers, and neurosurgeons are crucial. Such collaborations can lead to tools and platforms specifically tailored for neurosurgical applications, ensuring that the technology meets the unique needs of the field.With the growing interest in NFTs in healthcare, there’s a clear need for standardized protocols and regulatory frameworks . ResearcBeyond patient data and procedures, there’s potential for NFTs to play a role in medical education. Tokenized modules, virtual surgeries, or even patient cases can be used for training purposes, ensuring that students and trainees are accessing authentic and approved educational material.The integration of NFTs into neurosurgery presents a blend of exciting opportunities and notable challenges. As with any technological advancement in healthcare, the primary focus remains the betterment of patient care, ensuring safety, authenticity, and precision in treatments and interventions.NFTs have the potential to reshape the way neurosurgeons interact with patient data, authenticate procedures, and even contribute to research and education. Their capacity to offer undisputed ownership and control over digital assets can redefine data sharing, research collaborations, and even the monetization of specific medical assets. However, it’s imperative to navigate the ethical, environmental, and technical challenges associated with this technology.While the current state of NFTs in neurosurgery is nascent, the trajectory suggests a growing interest and potential for wider adoption in the coming years . Collabo


BackgroundIt is important to learn interprofessional education (IPE) facilitation skills to promote interprofessional collaboration in healthcare. Nonetheless, to date, only a handful of IPE facilitation programs have been developed through research.ObjectiveThe objective of this study was to create an IPE facilitation program for healthcare professionals who wanted to promote interprofessional collaboration in their organizations based on the tenets of instructional design and evaluate its effectiveness.MethodsThis study’s methodology was a mixed method based on relative subjectivism. We developed a two-day IPE facilitation program to learn IPE facilitation skills and promote interprofessional collaboration in the participants’ own organizations. The program was developed based on the instructional design principles of the attention, relevance, confidence, and satisfaction (ARCS) model, measuring the participants’ Interprofessional Facilitation Scale (IPFS) scores at three time points: before the first day, after the second day, and approximately one year after the course was completed. A one-way analysis of variance test was used to compare IPFS means at the three time points, and open-ended statements were qualitatively analyzed using thematic analysis.Results = 0.008). In addition, qualitative analysis suggested that the knowledge and skills learned in the program could be applied in the participants’ workplaces, which helped them maintain their IPE facilitation skills.Twelve healthcare providers  participated in the completed IPE facilitation program. Their IPFS scores increased significantly from 17.4 ± 16.1 before the program to 38.1 ± 9.4 after the program and remained at 35.1 ± 11.7 for one year (pConclusionWe developed a two-day IPE facilitation program based on the ARCS instructional design model, and the participants’ IPE facilitation skills scores increased and were maintained one year later. Interprofessional collaboration among healthcare professionals is of the utmost importance, as it fosters team-based work, which results in superior communication and coordination of care ,2. This Interprofessional education (IPE) plays a crucial role in fostering collaboration among all professionals involved in patient care, ultimately leading to healthcare providers who are optimally prepared and primed for interprofessional teamwork . NonetheHowever, there is a lack of comprehensive evidence regarding IPE facilitation. Although there is literature analyzing the IPE facilitation process through qualitative research , there aThis study’s methodology was a mixed method based on relative subjectivism. We employed a mixed method approach in order to quantitatively measure the learning effects of the developed program on the participants and to explain the reasons for the changes through qualitative analysis.Program development through instructional designWe attempted to develop an IPE facilitation program based on the instructional design model. We designed a two-day IPE facilitation program that featured targeted footage of flawed interprofessional collaborations Figure  as a viaThe attention, relevance, confidence, and satisfaction (ARCS) model has been validated as effective in enhancing learner motivation and learning outcomes in numerous studies; thus, we adopted the ARCS model of instructional design to develop our IPE facilitation program ,13. ARCSWe designed the IPE facilitation program content to address the ARCS components.AttentionThis step involves capturing the learners’ attention and getting them interested in the course material. We generated interest from the participants by showing examples of failed interprofessional collaboration in video clips.RelevanceThis step focuses on demonstrating the significance of the material to the learners’ goals and interests. Our program targets professionals who want to become IPE facilitators in their organizations and enhance the interprofessional collaboration at their workplaces. The learning objectives are as follows: (1) to understand IPE facilitation skills and the barriers to interprofessional collaboration, (2) to analyze the barriers to interprofessional collaboration in the participants’ organizations, and (3) to design and facilitate an IPE program that can be applied in their organizations.ConfidenceThis step aims to build the learners’ confidence in their ability to learn and apply the material. Our program provided opportunities to practice facilitation through mock workshops and tutor feedback on IPE facilitation.SatisfactionThe final step entails providing opportunities for the participants to apply what they learned in the course and to receive feedback that increases their satisfaction with the learning experience. Our program was designed to help participants reflect on the challenges of interprofessional collaboration in their organizations and be able to apply the skills they learned in those contexts effectively.The pilot version of the two-day IPE facilitation program took place as follows: the first day consisted of (1) viewing and discussing a video of a case in which interprofessional collaboration did not go well, 2) a lecture on IPE facilitation skills and barriers to interprofessional collaboration, and (3) analyzing interprofessional collaboration in one’s organization using the interprofessional collaboration analysis worksheet  a discussion of the educational program designed by each participant, (2) a mock program to practice IPE facilitation, and (3) a feedback from the tutor on facilitation skills.The evaluation of the participants’ IPE facilitation skills was conducted primarily as a formative assessment, with the tutors providing feedback on the participants’ performance during the mock workshop.Participants and data collectionThe target participants of the program were healthcare professionals who wanted to promote interprofessional collaboration and learn IPE facilitation skills in their organizations. They were invited to contact acquaintances of the first author (DS) through Facebook and other social networking sites. As a pilot program (beta test), 21 professionals  participated for two days in July and September 2014. After implementing the pilot version of the program, the participants identified areas for improvement. The participating health professionals provided input on the program’s content in a post-program questionnaire on feasibility and implementation.To implement the final version of the IPE facilitation program, which was completed after content refinement, announcements were posted on social media sites such as Facebook and the mailing lists of related academic societies, and participants were recruited.The IPE facilitation program was conducted with the participants who applied, and their facilitation skills were evaluated. The participants were asked to complete an online questionnaire: T1, before the day 1 program; T2, after the day 2 program three weeks later, and T3, approximately one year later; they self-assessed using the Japanese version of the Interprofessional Facilitations Scale (IPFS) , becauseData analysisA nonparametric analysis, the Kruskal-Wallis one-way analysis of variance test, was performed to detect statistical differences in the means of the IPFS scores at the three time points. A Bonferroni analysis was performed as a post hoc analysis to identify significant differences between pairs at each time point. The significance level was set at 0.05. The Statistical Package for Social Sciences (SPSS) version 28  was the statistical software used. The T3 questionnaire asked the participants to respond in an open-ended format about the IPE facilitation activities they have conducted in their own organizations in the past year, the lessons they have learned in their practice, and the barriers to their practice. Free-text answers were analyzed qualitatively using thematic analysis . The theEthical considerationsThe participation in the program in this study was limited to those who were well-informed about the study’s purpose, significance, and protocol. The participants were duly informed of the protection of personal information. All participants understood and agreed to participate, and written informed consent was obtained from all the participants. This study was conducted with the approval of the Ethics Committee of the Japanese Primary Care Association . All participants consented to the publication of the results, as shown in Figure The final version of the IPE facilitation program was conducted in January and February 2015, three weeks apart. Twelve healthcare professionals  participated in the program. The participants were encouraged to use the worksheet to analyze the interprofessional collaboration challenges in their organizations during the three-week interval between days 1 and 2 and to try to implement IPE facilitation skills.The IPFS scores of the participants increased significantly after attending the program and were largely maintained after one year Table . Ten parThe results of the Kruskal-Wallis test for the difference in the means of the IPFS at the three time points showed a significant difference (p = 0.008), and a post hoc analysis using the Bonferroni method showed substantial differences from T1 to T2 and from T1 to T3, with both effect sizes being greater than 1.0 , Level 4a is organizational change, and Level 4b is the benefits to patients/clients . The leaThe limitations of the current study are, first, the small sample size and the limited types of professionals who participated, which restrict the generalizability of the study’s findings. Second, the two dropouts in the one-year survey may have been participants who were not practicing IPE facilitation. In this case, selection bias is possible in the IPFS scores after one year. Third, although the learning outcomes from this IPE facilitation program were considered to be Kirkpatrick’s Level 3, the participants’ performance in the workplace was self-assessed and not peer-assessed. Objective evaluations would have been possible through interviews with colleagues or work-based assessments.We developed a two-day IPE facilitation program based on the ARCS instructional design model and tested its effectiveness. The results showed that the participants’ IPE facilitation skills scores increased and were maintained one year later. The relevance of the knowledge and skills learned in the program, which were applied in the workplace, and the participants’ confidence in their ability to develop skills that would motivate other professionals and inspire them to develop initiatives were considered the keys to success.