This study investigated the characteristics of arm elevation via principal component analysis in symptomatic overhead athletes with scapular dyskinesis. One hundred-thirty-four overhead athletes with scapular dyskinesis  were evaluated by three-dimensional electromagnetic motion and electromyography to record the scapular kinematics (upward rotation/posterior tipping/exterior rotation) and muscle activation  during lowering phase of arm elevation. The results showed: (1) for pattern I and II, the first 3 principal component (PCs) explained 41.4% and 42.6% of total variance of movement; (2) the first PCs were correlated with MT, LT activity (r = 0.41~0.61) and upward rotation, posterior tipping (r = −0.59~−0.33) in pattern I, and UT, MT, SA (r = 0.30~0.70) activity in pattern II; (3) contour plots of muscle activity demonstrated that muscle activities varied with dyskinesis patterns. In summary, for the pattern I, the major characteristics are coactivation of MT and LT and corresponding scapular posterior tipping and upward rotation. For the pattern II, the major characteristics are coactivation of UT, MT and SA without corresponding scapular external rotation. Previous research has found that the prevalence of scapular asymmetry in scapular plane elevation and flexion did not differ in asymptomatic and symptomatic participants 3. Other studies have demonstrated that various shoulder disorders, including rotator cuff injuries, glenohumeral instability, and labral tears, are associated with scapular dyskinesis, with prevalence rates of 33 to 100%7. One of the reasons for these non-specific results may be the methods used to assess scapular dyskinesis/asymmetry. With only video/visual observation of dyskinesis, lack of adequate reliability and validity has been reported10. Recently, the classification of patterns of dyskinesis based on visual observation and palpation has been shown to have sufficient reliability11. It is likely that patterns of scapular kinematics are specific to shoulder disorder mechanisms.Scapular dyskinesis has been recognized as a non-specific response to a painful condition in the shoulder12. Excessive protraction during arm elevation may result in decreased subacromial space and impingement13. Investigation of scapular kinematics in subjects with and without shoulder injuries identified decreased scapular upward rotation, decreased scapular posterior tilt, and increased scapular elevation in the subjects with shoulder injuries16. It was proposed that sufficient scapular movements may prevent the greater tuberosity of the humeral head from passing smoothly under the acromion during humeral elevations and lead to impingement syndrome19. Additionally, excessive activity of the upper trapezius (UT) muscle combined with reduced activity of the lower trapezius (LT) muscle and serratus anterior (SA) muscle have been observed in patients with shoulder impingement21. Moreover, lack of control of the functional kinetic chain of the scapula can result in an imbalance of force transmission from the lower extremities and trunk to the upper extremities. This imbalance can also influence skilled shoulder function, as occurs in athletic performance24. Therefore, scapular movement strategies specific to patterns of dyskinesis should be identified to facilitate the assessment of clinical pathologies and physiological adaptions, as well as that of treatment or rehabilitation effects.Several mechanisms have been proposed to explain shoulder injuries in terms of scapular dyskinesis. Unlike a retracted position of the scapula , a protracted position of the scapula  decreases the subacromial space19. Understanding the movement characteristics in patients performing overhead sports may help the treatment and prevention of shoulder disorders in overhead athletes with scapular dyskinesis. The aim of this study was to assess the scapular kinematics and associated muscle activities in overhead athletes with different patterns of scapular dyskinesis to determine whether scapular characteristics were specific to patterns of dyskinesis.Lack of appropriate scapular kinematics and muscle imbalance may significantly influence movement in overhead athletes such as throwing in baseball or spiking in volleyball because of the cumulative effect of repetitive overhead motions and forces on shoulder complexThe scapular dyskinesis pattern distribution in raising and lowering phases were demonstrated as follow: 1) raising phase ; (2) lowering phase . Because most of participants did not show scapular dyskinesis in raising phase, we would only focus on lowering phase in further analyses. As mentioned above, data with pattern I + II dyskinesis were included and analyzed in both the pattern I and the pattern II groups. As a result, the classification of dyskinesis patterns identified 88 participants with inferior angle prominence of the scapula in pattern I and mixed pattern dyskinesis  and 110 participants with medial border of the scapula prominence in pattern II and mixed pattern dyskinesis  in lowering phase. There were no differences in the demographic data among the 2 patterns. Kinematics and EMG data are presented in Table  raising For pattern I dyskinesis, 3 PCs explained a total of 41.4% of the variance of movement (KMO = 0.579). The first PC (17.5%) was correlated with MT, LT activity (r = 0.41~0.61) and upward rotation, posterior tipping (r = −0.59~−0.33); the second PC (13.4%), with MT activity and scapular external rotation (r = 0.40~0.67); and the third PC (10.5%), with LT activity (r = 0.44~0.57). For pattern II dyskinesis, 3 PCs explained a total of 42.6% of the variance of movement (KMO = 0.632). The first PC (17.0%) was correlated with UT, MT, SA activity (r = 0.30~0.70); the second PC (14.3%), with posterior tipping and LT (r = 0.55~0.72); and the third PC (11.3%), with MT activity and scapular upward rotation and external rotation (r = −0.6~0.47)  of the UT, MT, LT, SA shared the major components. This finding implies that these muscles are activated together as a force couple during arm movement.Principle component analysis of the scapular kinematics and muscle activities in different patterns of dyskinesis has not been reported previously. Theoretically, scapular kinematics and associated muscle activities during arm movements should share similar variance. However, our findings indicated that the first PC was described as primary mover/movement characteristic  in pattern I versus stability characteristic  for pattern II. External rotation was described as accessory movement characteristics in third PC in pattern II dyskinesis. Thus, change of muscle activation may not be obviously corresponding to scapular movement during arm elevation in pattern II dyskinesis. The results indicated that activation of the scapular muscles plays primary roles as stabilizers and secondary roles as movers of the scapula in patients with pattern II dyskinesis. This may explain why specific muscle activities and theoretically associated scapular kinematics were not found to be highly related in past research30. For medial border of scapula prominence (pattern II), UT, MT and SA were demonstrated as principal component without scapular kinematics included in the first PC, which indicated the role of stabilizers were essential in the UT, MT and SA in pattern II dyskinesis. For inferior angle prominence of scapula (pattern I), muscle function of MT, LT and shared variance in the first PC with upward rotation and posterior tipping can be explained to stabilize the axis for scapular upward rotation by MT and to function posterior tipping of the scapula by LT during arm elevation. However, the activation of LT cannot generate adequate scapular posterior tipping against the scapular inferior angle in pattern I dyskinesis. On the other hand, activated SA was not associated with generating scapular external rotation against the scapula medial border in pattern II dyskinesis.Scapular muscle activations are unique to each pattern of dyskinesis. MT and LT activities should be considered as characteristics of pattern I dyskinesis while UT, MT, SA activities can be considered as characteristics of pattern II dyskinesis. Additionally, the contour lines and plots expressed the functions of the observed or hypothetical muscle activations on two kinematic variables (upward rotation and posterior tipping). The UT has previously been shown to elevate the scapula and extend the neck, while the MT acts to retract the scapula. Additionally, the SA functions as scapular upward rotator and external rotatorIn clinical implication, evaluation of MT and LT muscle activations, as shared the same major variance with scapula kinematics, should be considered to correct inferior angle prominence of pattern I scapula dyskinesis subjects. On the other hand, without shared the variance with scapular kinematics in medial border prominence pattern II dyskinesis subjects, UT, MT and SA activations are likely to play major roles in stabilization of scapula instead of movement of the scapula. Instead of scapular muscle training, correction of medial border prominence dyskinesis may consider other factors, like soft tissue tightness or posture. Validation of this assumption should be further investigated.32. Third, movement artifacts and crosstalk cannot be excluded with the use of surface electrodes during dynamic movements. However, the 6-Hz filter and less than 10 kΩ of skin resistance of the EMG data reduced the possible impact33. Placing electrode pairs 2 cm apart can minimize the effect of crosstalk from other muscles34. Third, results from testing arm elevation in scapular plane may not represent results during functional movement or overhead sport activity. Additionally, participants were generally young and participated in overhead sports in this study. Sedentary or elderly people may have different scapular movement and muscle activation.The limitations of this study should be noted. First, the KMO values in both dyskinesis patterns are at the borderline of acceptance for PCA analysis. Further studies need to confirm our findings of movement characteristics in different scapular dyskinesis patterns. Second, the kinematics data were measured with humeral elevation of less than 120 degrees to reduce the error of the skin-based method. Validation studies have demonstrated that scapular motion can be accurately estimated for humeral elevation inferior to 120° for elevation in the sagittal planePCA demonstrated that the three PCs accounted for 41% and 43% of variance for pattern I and II dyskinesis, respectively. For the inferior angle prominence dyskinesis, the major characteristics are coactivation of middle and lower trapezius and corresponding scapular posterior tipping and upward rotation. For the medial border prominence dyskinesis, the major characteristics are coactivation of upper/middle trapezius and serratus anterior without corresponding scapular external rotation.This study was a cross-sectional study. All participants performed arm elevation in the scapular plane. The characteristics of the scapular kinematics and associated muscle activities specific to patterns of dyskinesis were identified.One hundred thirty-four subjects with unilateral shoulder pain and scapular dyskinesis were recruited for this study. The inclusion criteria were (1) age of 20 to 40 years old, (2) with unilateral shoulder pain of less than 5 on a 10-point visual analog scale during arm elevation and (3) demonstration of inferior angle or medial border of scapula prominence during arm elevation. All of the participants performed recreational overhead sports . Exclusion criteria were a history of stroke, diabetes mellitus, rheumatoid arthritis, rotator cuff tear, surgical stabilization of the shoulder, osteoporosis, or malignancies in the shoulder region. Participants who had pain or disorders of the cervical spine, elbow, wrist, or hand, who had pain radiating from the shoulder to the arm, or who could not elevate their arms to 150 degrees were also excluded. They received written and verbal explanations of the purposes and procedures of the study. All subjects gave written informed consent to the Research Ethics Committee of the National Taiwan University Hospital  following a complete explanation of this study, and in accordance with the Declaration of Helsinki.et al. validated scapular kinematics between skin-based sensor and bone-pinned methods and confirmed that the skin-based method is valid when arm elevation is below 120 degrees31. The details of the methodology can be found in a previous paper26. Three sensors were placed in locations where the skin motion artifact was minimized . Anatomic landmarks  were palpated and used for subsequent receiver mounting and landmark digitization. The investigator with 7 years’ clinical experience in musculoskeletal palpation did the anatomic landmarks palpation.The Polhemus 3Space FASTRAK system , an electromagnetic-based motion analysis system, was used for collecting 3-dimensional kinematic data of the scapula. Karduna The sEMG assembly comprised pairs of silver chloride circular (recording diameter of 10 mm) surface electrodes  with an interelectrode (center- to center) distance of 20 mm, and a Grass AC/DC amplifier  with a gain of 1000, a common mode rejection ratio of 86 dB at 60 Hz, and a bandwidth (−3 dB) of 10 to 500 Hz. The sEMG data were collected at 1,000 Hz/channel using a 16-bit analog to digital converter . Surface EMG electrodes were placed on the upper trapezius , middle trapezius , lower trapezius  and serratus anterior  of the involved shoulder. The referenced electrode was placed on the ipsilateral clavicle. Maximal voluntary isometric contraction (MVIC) was tested and used to normalize the sEMG data during the task . The MVICs were collected for 5 seconds in each of 3 trials, with 1 minute of rest separating trials.11. The inter-rater reliability of the classification test was moderate to substantial 11. Two single patterns with inferior angle of the scapula prominence (pattern I) and medial border of the scapula prominence (pattern II) and the mixed pattern with combination of the two single patterns were selected in this study.Visual combined palpation was used to classify scapular position and movement patterns (single patterns or mixed patterns) in both the raising and the lowering phases, modified by Kibler’s method35. Scapular orientation relative to the thorax was described using a Euler angle sequence of rotation about Zs (protraction/retraction), rotation about Y’s (downward/upward rotation), and rotation about X’s (posterior/anterior tipping). Full bandwidth sEMG data captured by the data acquisition software  were reduced using a root mean square (RMS) algorithm to produce sEMG envelopes with an effective sampling rate of 50 samples. Then the data were normalized to the MVIC trials. The EMG data of each muscle were the average of the middle 3 trials. The mean sEMG amplitude of each muscle, reported as a percentage of MVIC, was used to assess the activity of the muscle. For the analysis, 4 levels at 30°, 60°, 90°, and 120° of arm elevation for kinematics and 5 levels during 0–30°, 30–60°, 60–90°, 90–120° and above 120° of arm elevation for EMG data were used.Kinematics and sEMG data were collected during arm elevation in the scapular plane. Participants were asked to elevate the arms that the dumbbells in each hand weighed 2.3 kg (5 lb) or 1.4 kg (3 lb), depending on each participant’s ability to elevate the arm. In general, male participants lifted 5 lb and female participants used 3 lb of resistance. Raw kinematic data were low-pass filtered at a 6-Hz cutoff frequency and converted into anatomically defined rotations. In general, we followed the ISB guidelines for constructing a shoulder joint coordinate system36. Additionally, the characteristics of movements of the dyskinesis patterns were demonstrated by contour plots of 4-muscle EMG with scapular upward rotation and posterior tipping in the X and Y axes, respectively.The Statistical Package for the Social Sciences (SPSS) 17.0 was used for data analysis. Descriptive statistics of kinematics and EMG data were calculated. Data of participants with pattern I + II dyskinesis were included and analyzed in both the pattern I and the pattern II groups. To identify the characteristics of each dyskinesis pattern, principal component analysis (PCA) was performed on all data  for each dyskinesis pattern with varimax transformation. This analysis yielded the amount of variance explained by each principal component, and also the correlations between principal components (PCs) and the 3-dimensional scapular kinematics and 4 muscles EMG. The Kaiser-Meyer-Olkin (KMO) Measure of sampling adequacy was used to test the appropriateness of using PCA


Osteosarcomas (OSs) represent a huge challenge to improve the overall survival, especially in metastatic patients. Increasing evidence indicates that both tumor-associated elements but also on host-associated elements are under a remarkable effect on the prognosis of cancer patients, especially systemic inflammatory response. By analyzing a series prognosis of factors, including age, gender, primary tumor size, tumor location, tumor grade, and histological classification, monocyte ratio, and NLR ratio, a clinical predictive model was established by using stepwise logistic regression involved circulating leukocyte to compute the estimated probabilities of metastases for OS patients. The clinical predictive model was described by the following equations: probability of developing metastases = ex/(1 + ex), x = −2.150 +  (1.680 × monocyte ratio) + (1.533 × NLR ratio), where is the base of the natural logarithm, the assignment to each of the 2 variables is 1 if the ratio >1 (otherwise 0). The calculated AUC of the receiver-operating characteristic curve as 0.793 revealed well accuracy of this model . The predicted probabilities that we generated with the cross-validation procedure had a similar AUC . The present model could be used to improve the outcomes of the metastases by developing a predictive model considering circulating leukocyte influence to estimate the pretest probability of developing metastases in patients with OS. Nevertheless, metastatic OS, no more than 20%, still faces enormous challenges in increasing the low survival rate. Despite significant advances in multimodality treatment, the survival rate of patients with metastatic OS is just similar as 2 decades ago.Osteosarcomas (OSs), constituting nearly 1% of newly diagnosed malignancies annually, come from almost any embryonic mesodermal tissues.,6 Circulating monocyte count and neutrophil/lymphocyte ratio (NLR), as meaningful indicators inflammation status of patients, was proved to be prognosis predictor in various carcinomas.–13 Increasing neutrophils and/or decreasing counts of lymphocytes might suppress lymphocyte-activation, which would be deleterious to prognosis and survival.Nowadays, increasing evidence indicates that both tumor-associated elements but also on host-associated elements are under a remarkable effect on the prognosis of cancer patients, especially systemic inflammatory response.In OS patients, evaluating the high risk factor of developing metastases would be an important early step. It is necessary to select proper candidates with high risk of developing metastases and carefully evaluates more aggressive measures.Current study was designed to improve the outcomes of the metastases by developing a predictive model considering circulating leukocyte influence to estimate the pretest probability of developing metastases in patients with OS.2The institutional board of the first affiliated hospital of Anhui Medical University has approved the current study protocol. All patients have signed written informed consents: including tissue analysis and potential therapeutic research.Medical records from 290 consecutive OS patients between July 2001 and May 2013 were reviewed. Characteristics of patients and tumors at initial diagnosis of OS and development of metastases were collected. Metastases were definitively diagnosed through pathological examinations for tissues obtained via operations or biopsies. The following factors were studied: patient age, gender , primary tumor size, tumor location , tumor grade (1–4), and histological classification . WHO classification was used for the determination of pathological diagnosis and tumor grade. All pathological diagnoses were established after examining and confirming by 2 independent experienced pathologists.The calculation of monocyte ratio was the absolute monocyte count after initial treatment divided by the absolute monocyte count before initial treatment. The equation of NLR was described as the neutrophil count divided by lymphocyte count. NLR ratio was calculated as NLR after initial treatment divided by NLR before initial treatment.2.12 test were used. Accordingly, the Mann–Whitney U test or independent sample t test were employed.For categorical data, the Fisher exact test or Pearson χP value of 0.10, similar results would be observed. After tested all potential clinical interactions, since no statistically significant results were found, all of them were eliminated in the final model. Furthermore, all predictors entered in the final model were reported their odds ratios (ORs) and 95% confidence intervals (CIs).To develop the predictive model, stepwise logistic regression was used, where the final diagnosis was set as the dependent variable and the following characteristics as independent variables: patient age, gender, primary tumor size, tumor location, tumor grade, histological classification, monocyte ratio, and NLR ratio. The final model was established through eliminating variables by backward selection, where the selective criterion was statistically significant level of 0.05. If using a relevantly more liberal P > 0.05) was used. The cross-validation procedure was selected in model validation procedure, whose advantages were allowing to access the entire dataset for model validation. P values < 0.05 was defined as the criterion of statistical significant. Data analysis was performed using IBM SPSS Statistics 22.0 for Windows .The final model could be applied to compute the estimated probabilities of metastases for study individuals. To construct the receiver-operating characteristic curve, the predicted probabilities and definitive diagnoses of metastases were used. Then, in order to describe the accuracy of the model, the AUCs and their 95% CI were reported. To estimate model fit, the Hosmer–Lemeshow goodness-of-fit statistic ; 180 patients were female (62.1%) and 110 were male (37.9%). The tumor pathological subtypes included osteoblastic in 131 patients (45.2%), chondroblastic in 68 patients (23.4%), and others in 91 patients (31.4%) . The mean tumor size at diagnosis was 6.51 cm .Victims with metastatic OS would establish the following features: higher tumor grade, monocyte ratio >1, and NLR ratio >1. All the 3 features were tested to be statistically significant.Finally, under multivariate logistic regression analysis, 2 independent variables were identified as predictors of metastases , x = −2.150 +  (1.680 × monocyte ratio)  + (1.533 × NLR ratio). In this equation, e is the base of the natural logarithm, and the assignments of monocyte ratio and NLR ratio are 1 if the ratio >1 (otherwise 0). In addition, the Hosmer–Lemeshow test revealed a The calculated AUC of the receiver-operating characteristic curve as 0.793 revealed well accuracy of this model  Fig. . Accordi4,17 This is the reasons that we look for the methods to improve the outcomes of the metastases prediction and select the proper candidates with high risk of developing metastases to carefully evaluate for more aggressive measures.Current model summarized a single institutional experience of OS from cancer institution. Despite continuous advances in treatment, the prognosis of OS is still unsatisfying in the past decades if developing metastases. Moreover, extensive inflammatory response could be triggered and followed by tissue breakdown and disintegration. Evidence has also showed the relationship between prognosis, progressive nutritional decline of cancer patients, and systemic inflammatory response, which might be partly interpreted by insidious cancer aggressiveness activating innate immunity.Previous studies indicated that invasion and metastatic ability were affected by both the intrinsic characteristics and microenvironment of tumor.–25Decreasing lymphocytes, and increasing monocytes, neutrophils, as consequences of these inflammatory processes, have already been proved to promote inflammation-induced tumor growth through various proangiogenic cytokines, although widely existence of immune suppressive status in cancer population.–28 Additionally, many investigations have illustrated peripheral blood neutrophils and lymphocytes as prognosis predictors of cancer patients.–31 What is more, elevating NLR was associated with poor prognosis in ovarian cancer and gastric cancer by innumerable evidences.,33 This association might be due to suppress antitumor immune activity via natural killer cells and lymphocytes. Previous evidences on circulating leukocyte influenced us to pay attention on the significance of clinical circulating monocytes, neutrophils, and lymphocytes. Previous predictive models have been designed to compute the probability of clinical outcomes in other cancers.,35 They could estimate the probability of exact clinical outcomes with appropriate accuracy. Differently, relatively large number of patients with OS were enrolled in our study to establish a clinical predicting model on metastases. Furthermore, monocyte and NLR ratio, instead of cells counts, could evaluate the response to treatment, were used in our analysis.Peripheral blood monocytes have been shown to be an independent prognosis factor in neck and head, gastric, and cholangiocarcinoma cancers.Several limitations remained in this study. First, a relatively small number of clinical predictors were examined in this study, regardless of the fact that the statistical analysis indicates well and sufficient model fit. Second, monocyte phenotype or molecular information was not analyzed, which were caused by lack of this information in our retrospective data.In the current study, the model could be used to compute pretest probabilities of developing metastases in OS patients, and the equation might also be incorporated into a formal decision analysis. Despite the fact that the accuracy of the model was satisfactory, we accentuate that the model would not be devoted as a stand-alone test, but rather as a tool to guide the selection.


The aim of the present study was to monitor implant stability after sinus floor elevation with two biomaterials during the first six months of healing by resonance frequency analysis (RFA), and how physico-chemical properties affect the implant stability quotient (ISQ) at the placement and healing sites. Bilateral maxillary sinus augmentation was performed in 10 patients in a split-mouth design using a bobine HA (BBM) as a control and porcine HA (PBM). Six months after sinus lifting, 60 implants were placed in the posterior maxilla. The ISQ was recorded on the day of surgery from RFA at T1 (baseline), T2 (three months), and T3 (six months). Statistically significant differences were found in the ISQ values during the evaluation period. The ISQ (baseline) was 63.8 ± 2.97 for BBM and 62.6 ± 2.11 for PBM. The ISQ (T2) was ~73.5 ± 4.21 and 67 ± 4.99, respectively. The ISQ (T3) was ~74.65 ± 2.93 and 72.9 ± 2.63, respectively. All of the used HAs provide osseointegration and statistical increases in the ISQ at baseline, T2 and T3 (follow-up), respectively. The BBM, sintered at high temperature with high crystallinity and low porosity, presented higher stability, which demonstrates that variations in the physico-chemical properties of a bone substitute material clearly influence implant stability. The edentulous ridge in the posterior maxilla often presents a limited bone volume due to both a lack of alveolar bone after ridge remodeling and maxillary sinus pneumatization . AdequatSeveral factors influence maxillary sinus floor grafting results: specific surgical techniques, a simultaneous versus a delayed procedure, using barrier membranes over the lateral window, implant surface characteristics, the length and width of implants, and selecting the graft material . As regards the last factor/variable, researchers have not reached an agreement about the most suitable material for sinus augmentation . In the Today not many controlled research works evaluate the use of different bone grafting materials for sinus augmentation. Sinus elevation, performed with a wide variety of different graft materials, has been used. However, it still remains unclear which is the most suitable bone grafting material for enhancing bone regeneration in the augmented sinus ,14. DeprThe ultra-structural interface of the graft bone tissue interface, as well as the ideal time point of placing implants, have not yet been described .Typical surgical protocols are simultaneous one-stage lateral or crestal antrostomy when the residual crestal bone is greater than 3–6 mm, or a two-stage delayed procedure, which is recommended when the residual bone is less than 3–6 mm. Furthermore, the risk of implant failure is halved when the two-stage technique is used . The graft consolidation gradient reflects the features of each bone substitute at sinus augmentation sites . AccordiAn undisturbed healing period of at least 3–6 months at surgical sites is the generally accepted protocol after implant placement. This can ensure uneventful healing and improve osseointegration between the implant and bone. The reason for this approach is based on the fact that the functional force around the bone-implant interface causes implant micromotion during wound healing, while implant micromotion may induce fibrous tissue rather than bone contact, which results in clinical failure. All of these concerns about waiting periods have long since been a challenge for both patients and clinicians. Changing trends and demands have rendered the introduction of early loading techniques necessary as a result of searching for faster dental function restoration using implants . SuccessImplant stability can be defined as the combination of both mechanical and biological stability. While mechanical stability appears as the result of bone tissue compression during implantation, biological stability is obtained as a result of the formation of new bone cells on the implant surface during the osseointegration process. Hence implant stability is associated with the quality and quantity of local bone . NowadayOptimal outcomes in implant survival terms have been demonstrated for implants placed in the maxillary sinus filled with deproteinized bovine bone mineral, and this material can be considered a safe predictable graft material for sinus floor augmentation . HoweverAfter a six-month follow-up period of our ten partially-edentulous patients treated with xenograft materials for sinus floor augmentation, the success rate was 100%. No sinus membrane perforation or other clinical complications, such as sinusitis or pain, resulted from surgery. The increased volumes produced by the xenograft procedures were stable by the end of the healing period, as seen in Bone density at the implant site could be crucial as it has been reported to correlates with failure rates and primary stability. The bone density in the grafted area with both biomaterials was evaluated radiologically as a routine diagnostic approach. Color thermal graduation was used to observe changes in radiopacity in the intrasinus bone grafted area. At the time of implant insertion, and after a six-month healing period, the augmentation sites treated with the PBM showed denser new bone formation, which was achieved along the inner surface of the replaced bony window than the area on the bone graft A,B. The Although the radiopacity of the augmented volume increased with time for both xenograft materials, the receiving control sites showed a higher density of radiopacity compared with the PBM. Maxillary sinus membrane preservation is important to avoid the displacement of graft materials into the sinus cavity. However, nonobserved small perforations can imply a risk if left untreated.Primary implant stability in relation with bone density can also be evaluated by the implant stability quotient (ISQ). Detailed distributions for the implants and ISQ values over the investigated time periods are depicted in p < 0.0001).The ISQ (Baseline) averaged values were 63.8 ± 2.97 for a sintered BBM and 62.6 ± 2.11 for a non-sintered PBM, and differences were statistically significant. The ISQ (Stage 2) average values were 73.5 ± 4.21 for the BBM and 67 ± 4.99 for the PBM, and differences were statistically significant. The ISQ (Stage 3) average values were 74.65 ± 2.93 for the BBM and 72.9 ± 2.63 for the PBM, and differences were statistically significant. The detailed distributions for the groups are depicted in In the vestibule-lingual direction, the mean and standard deviation ISQ values measured at Baseline, Stage 2, and Stage 3 were 63.4 ± 2.88, 73.9 ± 4.11, and 73.8 ± 2.99, respectively, for the BBM, and were, respectively, 62.4 ± 2.92, 66.9 ± 2.67, and 72.6 ± 7.67 for the measured PBM ISQ values.In the mesio-distal direction, the mean and standard deviation ISQ values measured at Baseline, Stage 2, and Stage 3 were 64.2 ± 3.18, 75.2 ± 4.29, and 74.2 ± 3.01, respectively, for the BBM, and were, respectively, 62.8 ± 3.23, 67.1 ± 2.33, and 74.2 ± 7.59 for the measured PBM ISQ values.p = 0.0120; r = −0.13), gender , group , position , and time .The multivariate regression analysis  demonstrated significant influences on the ISQ values in relation to age  . The aimThe present study examined the same implants three and six months after their installation for the implants inserted by a two-step procedure. The RFA values after three months showed that the grafted sites provided good implant stability and, on average, the BBM were better than the PBM group sites. This difference was still present after 6 months and was also statistically significant. The importance of bony quality in relation to implant stability has been previously highlighted. Given the relative lack of bone, there is some concern about the initial stability of the implants placed in grafted bone. An RFA enables the stability under load to be qualitatively measured, and has been advocated as a means of assessing implant stability at the time of placement and in later phases when providing restorations . This is the first study to compare ISQs with density values after sinus lift procedures six months after healing and primary stability. The primary initial stability (IS) is a crucial factor to establish osseointegration ,43,44, aThe above studies have demonstrated a strong correlation between implant displacement and bone properties, and have concluded that better bone quality leads to better implant stability. Al-Khaldi has also proven that implants with a high degree of density placed in bone have higher initial stability values than those placed in soft bone , accordiA previous study on the same pool of implants used in our study (tapered implants) demonstrated that grafted bone can offer good primary stability to implants and that, during the surgical procedure, only a few mechanical characteristics of implants (length and diameter) were able to influence ISQ values . Degidi This should be taken into account for alveolar ridges with deficiencies in bone quality and quantity, which is the reason why almost every company in the sector has introduced tapered-design implants.In our study we used a small particle size of two different deproteinized bone grafts. Jensen et al. evaluated the influence of the particle size of DBBM on bone formation and implant stability when used for sinus floor elevation in a mini-pig model. In the initial healing phase, small particle size DBBM showed marginally higher osteoconductive capacity than large particle size DBBM. However, no differences were observed in the amount and speed of bone formation, BIC or implant stability between the two test groups. However at the baseline and at six and 12 weeks, the BIC values were comparable, or even higher, than in our study in humans. Primary implant stability is dependent on not only the thickness of the bone into which the implant is placed, but also on bone density, thread configuration, and implant shape, and the presence of an implant neck. Therefore, all these factors should be taken into account when making the clinical decision to perform a one-stage or a two-stage procedure . As evidenced in the review of Browaeys et al. on using biomaterials in sinus augmentation, within the limitation of the animal studies examined, and based only on histological examinations, the biomaterial used in the grafting procedure does not influence the initial osseointegration of dental implants . The correlation between the RFA values and good bone quality reported herein seemed to confirm the different importances of the factors that determine RFA values upon implant insertion and if it is able to maintain this stability after three or six months. In fact, good quality bone probably reacts better to implant insertion, and implant stability after bone remodeling could be greater.Long-term stability has also been reported by Hallman et al. In their study 108 dental implants were placed six months after sinus floor augmentation with a mixture of autogenous and deproteinized bovine bone. After three years of loading, implant stability was recorded using an Osstell instrument. The mean reported RFA values were 67.4 ± 14.5 for residual bone and 65.6 ± 13.8 for the augmented sites. Unfortunately, no more data are currently available about the importance of bone quality in determining long-term RFA values, so more studies have to be conducted .Healing times of 6–9 months before implant placement are usually recommended for sinus elevation in combination with grafting material , and an Our results also indicate that regardless of the substitute material, the implants inserted into a grafted sinus can be predictably loaded as the implants inserted into a grafted area. Previous studies have concluded that the prognosis of implants inserted into augmented sinuses and the fixed restoration supported by these implants do not appear to be influenced by factors, such as graft material, restoration type, residual bone height, and time of implant placement. Within the limits of this review, the prognosis of implants and fixed restorations did not seem to be influenced by the restoration type, graft material, residual bone height, and time of implant placement. However, the conclusions of this review are based on studies that provide a low level of evidence. Therefore, careful interpretations are required. Multicenter randomized controlled clinical trials with sufficient statistical power that concentrate on a few factors are needed to draw sound conclusions . The differences between the two HAs, in porosity, crystallinity, density, surface area and composition terms, may determine different behaviors of this material, and might affect early implant stability in this clinical situation. The HA of a bovine origin sintered with high crystallinity, low porosity, high density, and a larger granule size presents better stability, which demonstrates that variations in the physico-chemical properties of a bone substitute material clearly influence implant stability. A profound knowledge of the graft material characteristics is of utmost importance when assessing their clinical outcomes. The physico-chemical properties of bone substitute materials can influence osseointegration and shorten healing times from implant placement to restoration. Understanding not only the biomaterial properties, but also their relation and influence on bone healing, seems crucial.® ; deproteinized bovine hydroxyapatite (BBM), called Endobon® . The physico-chemical and morphological characterizations of both xenograft materials are found in a previous study [The commercial graft materials used in this study were two different types of bone deprotenized hydroxyapatite materials of diferent origins employed in dentistry: deproteinized porcine bone mineral hydroxyapatite (PBM), called OsteoBiolus study .Ten partially-edentulous patients , whose ages ranged from 37 to 60 years, attended the Department of Oral and Maxillofacial Surgery. Patients who demanded fixed restorative appliances in the posterior maxilla were selected for maxillary sinus augmentation because sufficient bone tissue was lacking to place endosseous dental implants. The protocol for harvesting bone samples was approved by the University Ethics Committee and informed consent was obtained from all the patients . The study was designed following the Declaration of Helsinki guidelines for experimentation on human subjects. Any possible complications that could arise from surgical therapy were treated following standard dental management protocols.Atrophy of the lateral-posterior maxilla and residual crestal bone height was classified according to Cawood et al.’s subsummizing classes I–VI. All of the patients underwent CBCT before surgery as a routine diagnostic approach to carefully evaluate the available bone at the intended surgical site and for planning the grafting procedure. The inclusion criteria were as follows: maxillary partial bilateral edentulism that involves premolar-molar areas. The cases with a crestal bone height between 7 mm and 0 mm, and with high postero-lateral atrophy (Cawood V–VI), are most likely to undergo a two-stage lateral antrostomy. The exclusion criteria were: patients who suffer from an uncontrolled systemic disease or a condition known to alter bone metabolism ; subjects who were taking/had taken medications known to modify bone metabolism; e.g., bisphosphonates, corticosteroids, etc.; women who were pregnant or trying to get pregnant at the time of screening; patients who presented existing sinus conditions, sepsis, a history of cancer and/or radiation to the oral cavity; or complications derived from any of these conditions that affect the sinus area.The study was performed in two surgical phases. In the first phase all the patients took 875/125 mg of amoxycillin/clavulanic acid every 8 h starting one day before surgery. A 300-mg dose of clindamycin every 8 h was prescribed to penicillin-allergic patients. This medication was maintained for seven days. All of the surgical procedures were performed under local anesthesia . The basic surgical procedure was represented in all the patients by maxillary sinus floor elevation via a lateral approach, as described by Boyne and James . ®, Tecnoss Dental S.R.L., Torino, Italy). Sinuses were allocated to the non-sintered HAs (PBM) or to the sintered HAs (BBM) group via a standard randomization protocol (http://www.randomization.com). On one side, sinus cavities were grafted with the PBM . After grafting, an absorbable collagen membrane  was placed over the window to minimize soft tissue invasion. On the other side, sinus cavities were grafted with the BBM . The grafting materials were mixed with venous blood from the defect area and were carefully packed in the created volume following mucous membrane elevation. After bone grafting, a short-term absorbable collagen membrane  was placed over the window. Primary closure was achieved in both cases by suturing with 3–0 silk suture . Sutures were removed two weeks after surgery. During the postoperative period, patients were clinically and radiologically followed up at monthly intervals  Cone Beam 3D and were analyzed using the I-Cat vision software. The obtained images were processed by Image J software , developed by the National Institute of Health (NIH) of the USA; a 3D plug-in with a thermal LUT and a grid size of 128 × 128, smoothing of 6.0, and a perspective of 0.2 on a 1:1 scale. Color thermal graduation was used to observe the changes in radiopacity in the grafted area (intrasinus bone graft). The scale was graduated with values from 0 to 240, where 0–20 were the values assigned to air, 20–100 were the values assigned to water and soft tissues, 100–140 were the values assigned to lower density bone, and 140–220 were the values assigned to higher density bone  placed six months after augmentation. All of the patients took 875 mg/125 mg units of amoxycillin/clavulanic acid every 8 h starting one day before surgery. A dose of 300 mg of clindamycin every 8 h was prescribed to penicillin-allergic patients. This medication was maintained for seven days. All of the surgical procedures were performed under local anesthesia  in an outpatient setting by the same surgeon, who was familiar with the implant system. For the procedure, a full thickness mucoperiosteal flap was elevated on the sides. The osteotomy using a conical drill, with copious irrigation using saline solution, was done on the crest of the bone. Then osteotomies for fitting implants were produced using the initial drill to determine the depth and direction of the site. Afterward, implants were positioned in the local area, predetermined at the crestal bone level. Sixty conical implants with an internal hexagon connection were applied, which were all 11.5 mm long with a 4.0 mm diameter. Implants were selected according to the prior evaluation of each case. All the implants were fitted using surgical guides, and wounds were sutured in a tension-free state. Antibiotics and analgesics were given for one week. All of the patients took 875/125 mg of amoxycillin/clavulanic acid every 8 h starting one day before surgery, and 300 mg of Clindamycin every 8 h werre prescribed to penicillin-allergic patients. This medication was maintained for seven days. Patients were asked to rinse with 0.12% chlorhexidine three times daily for two postoperative weeks. Sutures were removed two weeks after surgery. All of the implants were prepared with a healing abutment until rehabilitation commenced.The second surgical phase was performed after the healing period. Functional implants were placed on each side. Each side received three implants  to measure the implant’s primary stability. A Smartpeg™  was placed in each implant and was tightened to approximately 4–5 Ncm. The transducer probe was aimed at the small magnet at the top of the Smartpeg and at a distance of 2–3 mm, and was held stable during pulsing until the instrument beeped and displayed the ISQ value . The RF All of the data were recorded, reviewed, and inputted into a computing system. Analyses were performed using specific software . The influence of age, gender , group , ISQ measurement direction , and the evaluation time period, (T1) baseline, (T2) three months, abnd (T3) six months, on the ISQ values was evaluated by an analysis of variance (ANOVA) and multiple regression at the 5% significance level (backward method) with the help of appropriate software .The differences between the two HAs found in porosity, crystallinity, density, surface area, and composition terms may determine the different behaviors of this material and might affect early implant stability in this clinical situation. The HA of a bovine origin (BBM) sintered with high crystallinity and low porosity presents better stability, which demonstrates that variations in the physico-chemical properties of a bone substitute material clearly influence implant stability. Detailed information about the graft material’s characteristics is crucial to evaluate its clinical outcomes. The influence of the physico-chemical properties of bone graft materials on osseointegration has led to shorter healing times from implant placement to restoration. A sound understanding of various aspects of biomaterial properties, and their relation to and influence on bone healing, is of utmost importance.


Patients on antiplatelet therapy following percutaneous coronary intervention can become coagulopathic due to infection. Performing regional anesthesia for bilateral surgery in such cases is challenging. We report a case of successful combined inguinal femoral and subgluteal sciatic nerve blocks (CFSNBs) for simultaneous bilateral below-knee amputations in a coagulopathic patient on antiplatelet therapy.A 70-year-old male patient presented with pain in both feet due to diabetic foot syndrome. The condition could not be managed by open amputations of the toes at the metatarsal bones and subsequent antibiotic therapy. Computed tomographic angiography showed significant stenosis in the arteries supplying the lower limbs, indicating atherosclerotic gangrene in both feet. Balloon angioplasty and bypass surgery with subsequent debridements with application of negative-pressure wound therapy and additional open amputations did not improve the patient's clinical condition: his leukocyte counts and C-reactive protein levels were above the normal range, and his prothrombin and activated partial thromboplastin times were increased.Simultaneous bilateral below-knee amputations were performed under ultrasound-guided CFSNBs. Following left CFSNBs using 45 mL of a local anesthetic mixture (1:1 ratio of 1.0% mepivacaine and 0.75% ropivacaine), the left below-knee amputation was performed for 76 minutes. Subsequently, under right CFSNBs using 47 mL of the local anesthetic mixture, the right below-knee amputation proceeded for 85 minutes. Throughout each surgery, dexmedetomidine was continuously administered, and a sensory blockade was well maintained in both limbs. The patient did not complain of pain due to regression of the first CFSNBs during the second surgery. The CFSNBs successfully prevented tourniquet pain. Local anesthetic systemic toxicity (LAST) and hemodynamic instability due to tourniquet deflation and administration of dexmedetomidine did not occur. No additional analgesic was required to supplement insufficient surgical anesthesia. Postoperatively, no neurologic complications related to the CFSNBs were reported.The timely placement of bilateral CFSNBs immediately before the corresponding limb surgery, which lasted for less than 2 hours, provided successful surgical anesthesia in both lower limbs without LAST or pain due to regression of the CFSNBs that were performed during the first surgery. Theoretically, due to its relatively compact topographical arrangement, the brachial plexus could be easily blocked by a single local anesthetic injection that provides anesthesia to nearly the entire area of the upper limb. In contrast, the entirety of the lumbosacral plexus that innervates a lower limb cannot be anesthetized with a single injection. Therefore, the lumbar and sacral plexuses should be separately blocked for complete unilateral lower limb anesthesia, necessitating a larger dose of local anesthetics than that used for brachial plexus blocks. If the blocks are performed bilaterally, LAST cannot be easily averted. For this reason, neuraxial or general anesthesia has been preferred to bilateral peripheral nerve blocks. However, neuraxial or general anesthesia is frequently contraindicated in patients undergoing major lower extremity amputation due to the need for concomitant administration of anticoagulants and/or antiplatelet agents or because of comorbidities that cause anesthesia-related hemodynamic instability. In addition, major lower extremity amputation for peripheral arterial disease that is complicated by gangrene carries a 2-year mortality rate of approximately 30%. Herein, we report our success in performing combined inguinal femoral and subgluteal sciatic nerve blocks (CFSNBs) immediately before corresponding limb amputations (to reduce the risk of LAST) for bilateral diabetic gangrene in a patient on antiplatelet therapy who became coagulopathic due to infection. The gangrene was unresponsive to a previously performed peripheral arterial angiography and bypass surgery.The placement of peripheral nerve blocks for bilateral limb surgery is challenging because increases in the required dose of a local anesthetic lead to increases in the risk of local anesthetic systemic toxicity (LAST) and other side effects related to regional anesthesia. Furthermore, this procedure prolongs anesthesia preparation time, leading to a delay in surgery. To date, several reports have been published on the use of bilateral brachial plexus blocks for surgical anesthesia2A 70-year-old male patient  presented with pain in both feet due to diabetes mellitus. He underwent open amputations of the right 3rd to 5th and left 4th and 5th metatarsal bones in a local medical clinic. However, due to refractory abscesses around the operation sites that were unresponsive to antibiotic therapy, the patient was transferred to our hospital 13 days after the operations for further evaluation and treatment. On admission, he was given 60 mg of gliclazide and 1000 mg of metformin per day for diabetes mellitus that had been diagnosed 40 years previously. He received 50 mg of atenolol, 12.5 mg of chlorthalidone, 4 mg of benidipine, and 100 mg of aspirin per day for hypertension that had been diagnosed 30 years previously and was complicated with chronic ischemic heart disease, as demonstrated by total occlusion of the left anterior descending artery on coronary angiography. The occlusion was treated with a percutaneous coronary intervention 3 years previously. The above medications were maintained during the admission period. A complete blood cell count revealed a leukocyte count of 14,800 cells/μL, a hemoglobin level of 9.8 g/dL, and a platelet count of 452,000 cells/μL. Liver function tests, prothrombin time, activated partial thromboplastin time, serum electrolytes, sodium levels, and creatinine levels were within normal ranges. Transthoracic echocardiography indicated apical septal hypokinesia, left ventricular concentric hypertrophy, mild diastolic dysfunction of the left ventricle, decreased mobility of the noncoronary and left coronary cusps of the aortic valve, and a left ventricular ejection fraction of 61%. Computed tomographic angiography showed significant stenosis in the bilateral tibial, dorsalis pedis, and plantar arteries and diffuse atherosclerosis with calcified plaques in the aorta and pelvic and femoral arteries, indicating atherosclerotic gangrene in both feet. Daily administration of alprostadil (10 μg/d) was started to dilate the stenotic vessels and maintain their blood flow and continued until the bilateral below-knee amputations were performed. Under epidural anesthesia, balloon angioplasties in the left posterior tibial and peroneal arteries and a right above-knee popliteal artery to right posterior tibial artery bypass with a reversed left great saphenous vein were respectively performed 2 and 7 days after the admission. Despite subsequent debridements with application of negative pressure and additional open amputations of the right 2nd and 3rd metatarsal and 1st proximal phalangeal bones and the left 2nd metatarsal bone and 1 metatarsophalangeal joint under spinal anesthesia, the status of the wound did not improve. Therefore, simultaneous bilateral below-knee amputations were planned 49 days after the admission. The leukocyte count and C-reactive protein level were maintained above normal ranges  during admission, and the prothrombin time and activated partial thromboplastin time were increased to 15.3 and 55.6 seconds, respectively, from 1 month before the bilateral below-knee amputations.Upon arrival in the operating room, electrocardiography, pulse oximetry, and noninvasive blood pressure monitoring were instituted. After draping the left inguinal region, the femoral nerve was identified lateral to the femoral artery using a 5- to 13-MHz linear phased array transducer  equipped on an ultrasound machine . Under ultrasound guidance, a 23-ga Tuohy needle was introduced toward the femoral nerve parallel to the ultrasound beam, and 21 mL of a local anesthetic mixture containing 30 mL of 1.0% mepivacaine and 30 mL of 0.75% ropivacaine was injected through the needle near the femoral nerve  and patients receiving general anesthesia. and 667.2 ± 61.2 mg (mean ± standard deviation) of mepivacaine were administered via a single-shot technique for CFSNBs, no signs or symptoms of LAST were detected. However, an average of 667.2 mg of mepivacaine increased the maximum plasma concentration of mepivacaine (5.1 μg/mL on average) to the threshold needed for toxic symptoms to appear (5–6 μg/mL), whereas 700 mg of mepivacaine did not (3.91 ± 0.95 μg/mL) have the same effect. In addition, a threshold concentration of ropivacaine for LAST (1–2 μg/mL) was achieved in the absence of suggestive signs and symptoms of LAST following the administration of 300 mg of 0.75% ropivacaine mixed with 1:200,000 epinephrine for CFSNBs. Therefore, a dose of ropivacaine higher than that used in the above study and the additive effect of mepivacaine  would considerably increase the possibility of LAST. Moreover, the interval between the 2 block sessions (115 minutes) did not appear to help prevent the development of LAST because the local anesthetic concentration was maintained near the peak value until 90 to 120 minutes after the end of local anesthetic injection.,20 Fortunately, our patient did not experience signs and symptoms of LAST.The total doses of 1% mepivacaine and 0.75% ropivacaine used for the 2 sessions of the CFSNBs were 460 and 345 mg, respectively. However, the total doses were administered at an interval of 115 minutes between the end of the local anesthetic injection for the 1st and 2nd CFSNBs (225 mg of 1% mepivacaine and 168.75 mg of 0.75% ropivacaine for the 1st CFSNBs and 235 mg of 1% mepivacaine and 176.25 mg of 0.75% ropivacaine for the 2nd CFSNBs). Despite the separate administration of the local anesthetics at an interval, the possibility of LAST remained. In previous studies, when 700 In addition, real-time ultrasound guidance might prevent vascular injury, which commonly leads to intravascular injection of a local anesthetic. Ultrasound imaging could also detect inadvertent intravascular entry of a block needle, thereby minimizing the amount of local anesthetic injected into a blood vessel.,23 Furthermore, the compression of veins resulting from ultrasound transducer use reduces the risk of vascular puncture and local anesthetic injection into vessels. When a fixed dose of mepivacaine was administered for various types of nerve blocks, CFSNBs resulted in the lowest maximum plasma concentration of the anesthetic and prolonged the time to reaching the maximum plasma concentration than other types of regional anesthesia, such as lumbar epidural, caudal, intercostal-nerve , and supraclavicular-brachial-plexus blocks. Finally, dexmedetomidine used for intraoperative sedation has an anticonvulsive effect by increasing the convulsive dose of local anesthetics. Therefore, it appears that our patient, who did not experience LAST, benefited from the above factors contributing to the prevention of LAST despite the excessive local anesthetic dose.Additional factors may have contributed to the prevention of LAST. For example, the use of a local anesthetic mixture has advantages over a single local anesthetic because the mixture compensates for the short durations of action of rapidly acting anesthetics (mepivacaine in this case) and the long latencies of longer acting anesthetics (ropivacaine in this case). The mixture could also decrease the incidence of cardiac events by decreasing the doses of more potent local anesthetics, the convulsion doses of which can easily cause a cardiac event. and lateral femoral cutaneous nerve blocks for the prevention of LAST, the patient did not complain of tourniquet pain during the 2 consecutive surgeries. This was presumably due to the intravenous administration of dexmedetomidine before the beginning of the surgery, as dexmedetomidine reduces sympathoadrenal responses to tourniquet pain as well as opioid requirements to relieve the pain.Even if a concern existed regarding tourniquet pain due to the omission of the obturatorIn conclusion, the timely placement of bilateral CFSNBs immediately before corresponding limb surgery that lasts for less than 2 hours provides successful surgical anesthesia in the bilateral lower limbs without resulting in LAST, hemodynamic instability, or tourniquet pain and avoids the regression of CFSNBs performed earlier during the surgery.


Autoimmune cytopenias (AICs) are frequently associated with chronic lymphocytic leukemia (CLL). The most common of these AICs is autoimmune hemolytic anemia (AIHA); the second most is immune thrombocytopenia (ITP). Here, we report on a patient with CLL-associated ITP, with thrombocytopenia refractory to corticosteroids and intravenous immunoglobulins, in which continuous oral treatment with Eltrombopag allowed initiation and maintenance of an oral anticoagulation treatment with Acenocoumarol that was indicated because of a severe arrhythmogenic cardiomyopathy. While other autoimmune disorders have been reported in CLL, autoimmune cytopenias (AICs) are by far the most frequent immune complications  . CLL patITP occurs in 2–5% of all CLL patients, although it may be underestimated unless a bone marrow examination is performed in all patients with thrombocytopenia . In CLL The mechanism of ITP is similar to that of AIHA as an antibody-mediated process. However, ITP is less well understood than AIHA as key experiments are not usually performed in ITP. The pathogenic autoantibody is not easy to detect on platelets and less is known about isotype .The goal of ITP treatment is to prevent or treat bleeding complications. Among the therapeutic arsenal, the second-generation agonists of the thrombopoietin (TPO) receptor, Romiplostim and Eltrombopag, have been introduced in clinical practice for the treatment of chronic ITP patients as a second-line therapy during the last 7 years , 7. CurrWidely used for primary ITP, Eltrombopag has shown responses in CLL-associated ITP in retrospective case series . Since TWe report the case of a patient with CLL and moderate thrombocytopenia refractory to corticosteroids and intravenous immunoglobulins, in which continuous oral treatment with Eltrombopag has allowed us to initiate and maintain an oral anticoagulant treatment with Acenocoumarol that was indicated due to a severe arrhythmogenic cardiomyopathy.A 69-year-old male patient with a personal history of hypertension, type 2 diabetes, and chronic auricular fibrillation under treatment with Propafenone since January 2003 but without ventricular dysfunction in echocardiogram. He was diagnosed with a Binet A/Rai 0 stage CLL, with 13q14 deletion in November 2003, and remained stable without treatment until 2011.9/L, and WBC 124 × 109/L (90% lymphocytes). Three cycles of Bendamustine treatment as monotherapy (70 mg/m2/day) × 2 days every 28 days followed by 2 cycles of Bendamustine plus Rituximab were administered, achieving a partial response after treatment. In January 2012, treatment was interrupted due to poor hematologic tolerance and permanent cytopenias . In the following weeks, hemoglobin levels and neutrophils were normalized, with remaining platelets under 50 × 109/L. A bone marrow biopsy was performed; the report described areas of moderate infiltration by small and mature lymphocytes cells, with conserved numbers of megakaryocytes of heterogeneous aspect.In June 2011, the patient developed B symptoms and a CT scan revealed multiple abdominal lymphadenopathy and splenomegaly (17 cm), due to disease progression (Binet C/Rai IV stage) with Hb 9.95 g/dl, platelets 91.4 × 109/L despite the treatment but without signs of bleeding.In this context, a diagnosis of immune thrombocytopenia (ITP) was made. The patient commenced oral prednisone at a dose of 1 mg/kg and he received 1 g/kg/day of intravenous immunoglobulins in two consecutive days. The platelet count remained < 40 × 109/l with Hb 12,8 g/dl, WBC 9.11 × 109/L, and neutrophils 7.8 × 109/L. We decided to ask for an off-label use of Eltrombopag to increase platelet counts to safer levels for anticoagulation. The patient's written informed consent was requested. Eltrombopag, at an initial dose of 50 mg daily, was given, and, after an initial platelet recovery to 120 × 109/l, Acenocoumarol treatment was started. After twelve months of treatment with Eltrombopag, the patient persisted with adequate platelet count (80 × 109/L), allowing continued treatment without complications and starting intermittent doses of oral Chlorambucil.In October 2012, the patient required hospitalization due to symptoms and signs of congestive cardiac failure, with left ventricular ejection fraction of 25% by echocardiogram in addition to dilated myocardiopathy signs. Anticoagulant therapy for thromboembolic risk prevention was proposed, which could not be initiated because the patient's platelet count was below 50 × 10Eltrombopag treatment was well tolerated with no evidence of adverse effects related to its administration without thrombotic complications during the two years of continuous treatment. Finally, in October 2014, the patient died of respiratory complications due to pneumonia with pleural effusion.We show  the evolUp to 8% of patients with chronic lymphocytic leukemia (CLL), including those with stable diseases, develop secondary autoimmune disorders, which can confound cytopenias, complicate the course of their CLL, compromise their quality of life, and, in some cases, be fatal . The mosThe second most common among autoimmune disorder associated with CLL is immune thrombocytopenic purpura (ITP), with an incidence of approximately 2–5% of the CLL population . The ITPUnlike AIHA, there are no ready markers for platelet destruction. Commercially available antiplatelet antibody testing is generally unhelpful and has not been expressly studied in CLL patients. CLL-associated thrombocytopenia may be due to bone marrow infiltration by the disease and the splenomegaly usually found in these patients. Thrombocytopenia in a patient with CLL can be considered immune-mediated when there is a sudden large fall in platelets in the absence of splenomegaly, infection, or chemotherapy and with plentiful megakaryocytes in the bone marrow .9/L [Due to the risk of bleeding, it has been recommended to initiate therapy in those patients with platelet counts of <30 × 109/L . ITP con9/L and at least a twofold increase in the baseline count without evidence of bleeding can be considered as an ITP treatment good response [In our case, we highlight the difficulty of treating the autoimmune manifestations associated with CLL. A platelet count > 30 × 10response . Conventresponse . Our patAlthough splenectomy continued to be an effective treatment, particularly for ITP, this procedure does not ensure a successful response. Provan et al. have reported success rates approach 90% with two-thirds of patients having a durable response . And immTPO receptor agonists (Romiplostim and Eltrombopag) represent a new option in the ITP treatment. These agents act by increasing the production of platelets rather than by avoiding their premature destruction. Both analogues have been reported to be successful in case reports of patients with CLL-associated ITP , 16, 17.The case, we report, illustrates the effectiveness of Eltrombopag in cases with refractory thrombocytopenia associated with CLL. The thrombopoietic mimetic agent allowed initiating and maintaining an oral anticoagulant treatment with Acenocoumarol that was indicated due to a severe arrhythmogenic cardiomyopathy. The platelet response was achieved quickly and platelet counts remained stable during 24 consecutive months under Eltrombopag therapy.Other authors have communicated their experiences with the use of TPO mimetic drugs during short periods of time to increase platelet counts in patients with ITP before surgical procedures, to optimize the procedure and to reduce bleeding episodes. Our case illustrates how TPO mimetic drugs can be safely administered over long periods of time, helping to keep platelet counts above life-threatening levels and preserving the immune system because they are nonimmunosuppressive drugs.The possibility of administering TPO mimetic drugs in severely thrombocytopenic patients who need to initiate or maintain an anticoagulant therapy is of great interest. Our clinical experience demonstrates its feasibility in patients with ITP associated with CLL.


The use of natural products (NPs) as possible alternative biocidal compounds for use in antifouling coatings has been the focus of research over the past decades. Despite the importance of this field, the efficacy of a given NP against biofilm (mainly bacteria and diatoms) formation is tested with the NP being in solution, while almost no studies test the effect of an NP once incorporated into a coating system. The development of a novel bioassay to assess the activity of NP-containing and biocide-containing coatings against marine biofilm formation has been achieved using a high-throughput microplate reader and highly sensitive confocal laser scanning microscopy (CLSM), as well as nucleic acid staining. Juglone, an isolated NP that has previously shown efficacy against bacterial attachment, was incorporated into a simple coating matrix. Biofilm formation over 48 h was assessed and compared against coatings containing the NP and the commonly used booster biocide, cuprous oxide. Leaching of the NP from the coating was quantified at two time points, 24 h and 48 h, showing evidence of both juglone and cuprous oxide being released. Results from the microplate reader showed that the NP coatings exhibited antifouling efficacy, significantly inhibiting biofilm formation when compared to the control coatings, while NP coatings and the cuprous oxide coatings performed equally well. CLSM results and COMSTAT analysis on biofilm 3D morphology showed comparable results when the NP coatings were tested against the controls, with higher biofilm biovolume and maximum thickness being found on the controls. This new method proved to be repeatable and insightful and we believe it is applicable in antifouling and other numerous applications where interactions between biofilm formation and surfaces is of interest. Biofouling, the accumulation of marine organisms on a surface, severely affects manmade structures such as ship hulls, energy systems, environmental sensors, aquaculture settings, etc. A number of technological advances focusing mainly on coating these structures with antifouling agents, have been utilised over the last few decades, with the use of biocides being the most successful. However, the biocide with the best antifouling (AF) efficacy against a wide range of target species, known as tributyltin (TBT), has been found to be detrimental to the marine environment ,2. SinceCobetia marina) on coated surfaces using universally available equipment. This was achieved by utilising 24-well plates combined with nucleic acid staining, high-throughput plate reader technology, and confocal laser scanning microscopy (CLSM). Microplate readers have the capacity to rapidly screen bacterial responses  under different conditions and have been routinely used for the discovery of novel antimicrobials. Furthermore, CLSM allows three-dimensional (3D) visualisation of the biofilm, providing new insights into biofilm development. A few recent studies have reported the use of plate readers (microtiter plate assays) in conjunction with biological stains to measure biofilm inhibition of adhesion and/or growth within an antifouling context [The first objective of the current work was to develop a bioassay for the rapid in situ assessment of biofilm formation  in order to establish the wavelength at which the compound has maximum absorption via UV-Vis, which was found to be at 520 nm. A calibration curve was generated at this wavelength. To establish the extent of juglone leaching from the two juglone-containing coatings, tests were performed over 48 h. This was done in order to determine whether juglone actually leached from the coated surfaces, and if so, to quantify the concentrations within the 24-well fluid volumes.During the leaching tests, two samples were taken at 24 h and 48 h. Overall, juglone leaching was detected from both coating formulations at concentrations ranging from 1.70 ppm to 1.99 ppm after 24 h, and 1.96 ppm to 2.35 ppm after 48 h, for the coating containing juglone (JUG)+poly(methyl methacrylate) (PMMA)+Rosin (ROS) and the coating JUG+PMMA, respectively see . OverallPMMA is a common acrylic polymer material also used in orthopaedic surgery as part of bone cement. Antibiotics are often incorporated into these cements, where subsequent elution produces high local concentrations of antibiotics while simultaneously minimising systemic toxicity ,18,19. ACobetia marina (ATCC 25374). C. marina has previously been used as model species for marine biofilm related studies [C. marina at 16 ppm, while the EC50 was found to be at 5 ppm, C. marina was found to be at 16 ppm. The measured concentration of released juglone, as shown in the previous section, falls within the same range as the concentration for the EC50 for this species.The growth-inhibiting activity of juglone was examined against planktonic growth of the model species  ATCC 2534. C. mar studies ,15,26,272O-containing coatings when compared to their controls, PMMA and PMMA + Rosin which acquired significantly higher growth (p < 0.004). For the “dead” (propidium iodide stained cells) wavelength measurements (“dead” = cells with compromised membranes), there was no significant difference between coatings and there was an overall low number of compromised cells for the duration of the experiment.The in situ evaluation of biofilm growth on coated surfaces was achieved through the use of a plate reader utilising nucleic acid staining. In 2O) with p < 0.042 and p < 0.003, respectively . This indicates that PMMA alone as a binder did not facilitate sufficient compound release and therefore did not perform as well as the binder with the rosin additive. The leaching tests  and the biocidal coating , indicating a clear antibiofilm activity against C. marina.When the juglone coatings were compared against the Cuas found b,c. In tp = 0.039) and Cu2O (p = 0.044 and p = 0.019) when compared to the controls (PMMA ROS and PMMA), with the exception of the Juglone–PMMA combination. As shown earlier, the rosin additive performed better in releasing juglone into the solution, therefore its higher availability could account for lower bacterial growth in these wells.The planktonic growth measured at the 48 h end-point within the wells containing the coated coupons can be seen in i) confirm the plate reader results and allow direct comparisons between the two techniques and (ii) to utilise the high resolution of the CLSM to assess the biofilm microstructures that formed on the various coatings. 2O), no biofilm formation can be seen and the cell morphology appears to be different, with maybe a few rod-like cells and more undefined shaped particles, leading to doubts as to whether these bacterial cells are intact  using COMSTAT was undertaken in order to: (e intact .p < 0.001 for both parameters) on the control coatings PMMA + Rosin when compared to the juglone and Cu2O coatings. These results are in good agreement with the data obtained from the plate reader (p < 0.002) of the PMMA controls on biofilm morphology was also observed for the JUG PMMA vs. Cu2O PMMA vs. PMMA (control), i.e., greater biofilm thickness  were found on the PMMA control coatings  on cell division and membrane transport ,30 may aThe use of CLSM was found to be informative on various additional biofilm characterisation parameters corroborating/adding to the less sensitive but high-throughput analysis by the plate reader. Overall, there was good consistency between the two techniques, underlining a good antibiofilm performance of the juglone-containing coatings. The few discrepancies between the two datasets may be attributed to the nature of the measurement methods, i.e., CLSM uses laser light which provides better controllability since emitted wavelengths can usually be optimised to limit overlapping of the coatings’ potential autofluorescence and the nucleic acid stains.Circular glass coupons  were abraded using a lapping machine  with 25 μm diamond microparticles for 20 min to produce rough surfaces. This was done to improve adhesion between the smooth glass surface and the coatings. Each coupon was permanently numbered and weighed. The main binder for these model coatings was poly(methyl methacrylate) (PMMA); however, the sole use of this hard acrylic polymer as an inert binder could potentially limit the release of the NP. Therefore, rosin was also added, since it is a common binder ingredient in paint formulations. The composition (matrix) of these paints must contain a material sufficiently soluble in seawater to maintain adequate leaching of the toxin, but not so soluble as to result in the rapid deterioration of the film. Rosin is commonly used for this purpose since its solubility appears to be within the limits required . Detail2O)  was dispersed in the polymer solutions at ambient temperature by placing the mixtures in glass laboratory bottles (Duran) and rotating these on a laboratory roller mixer .Polymer solutions for the coatings were prepared by dissolving solid polymer (PMMA at 30 wt. %) in toluene solvent  under constant stirring. Rosin  was dissolved rapidly in toluene under mild agitation at ambient temperature, while dissolution of PMMA  was slower and was facilitated by heating the mixture of polymer powder and toluene at 50 °C in a reaction flask fitted with a reflux condenser and stirring with a paddle stirrer at 60 rpm. To make the 80:20 wt./wt. PMMA:rosin polymer blend, solutions of each polymer having the same solid content (30 wt. %) were mixed in this weight ratio. Juglone was readily dissolved in the polymer solutions and the common AF biocidal pigment cuprous oxide  was used to achieve uniform layers with predictable and reproducible thicknesses. Specifically, the settings for the spin coating were as follows: acceleration: 0–2000 rpm in 10 s, speed: 7500 rpm, time: 1 min, paint viscosity: 500 mPa. To maintain the viscosity, toluene solvent was added as required. Following the spin-coating procedure, the coupons were placed in sterile Petri dishes where they were transferred under sterile conditions (laminar flow hood) to allow solvent evaporation (drying) for 4 days. Following drying, the coupons were carefully partially immersed in 70% EtOH so that the coated surface stayed exposed to air .n = 8 per dilution, optical bottom plated with polymer base, clear, Nunc™) and the UV-Vis absorbance spectrum function of a microplate reader  was utilised over a scan wavelength range of 200–700 nm at a 2 nm resolution.To establish calibration curves for juglone, the compound was dissolved in dimethyl sulfoxide , and serial dilutions of 64 ppm, 32 ppm, 16 ppm, 8 ppm, 4 ppm, 2 ppm, 1 ppm, and 0.5 ppm were prepared in sterile ASW  [TM was used to seal the lid of the 24-well plate. ParafilmTM is a material that is known to allow gas exchange, but not water evaporation. The plate was then incubated in the dark at 28 °C on a rotary shaker at 100 rpm agitation. Each coating formulation was replicated 12 times. The leaching of materials was assessed at two experimental time points, 24 h and 48 h. At each time point, the 24-well plate was removed from the incubator and an aliquot of 200 μm taken from each coupon-containing well was placed in 96-well plates (n = 12 per coupon) and UV-Vis optical density measurements were then made at λ = 520 nm using the microplate reader.In order to assess the leaching of juglone from the current experimental paint matrix, a pilot assay was conducted over 48 h for the coatings 1, 2, 5, and 6 listed in Cobetia marina (ATCC 25374). C. marina was grown in sterile sea salts and peptone media . Juglone serial dilutions were prepared as described in the previous section, i.e., 64 ppm, 32 ppm, 16 ppm, 8 ppm, 4 ppm, 2 ppm, 1 ppm, and 0.5 ppm in 0.5% DMSO. There were two controls, one with SSP media plus 0.5% DMSO and one with just SSP media. Each dilution and control was replicated five times while uninoculated wells for each concentration served as blanks and were replicated three times. Measurements were taken every 15 min for 24 h at 28 °C. The MIC was determined as the lowest concentration of juglone at which there was no growth [50) was determined as the concentration at which 50% of bacterial growth was inhibited by juglone. Following the 24 h experimental period for the MICs, 5 μL from each well (in triplicates) were transferred onto marine agar to establish the minimum bactericidal concentrations (MBC). Each concentration tested for bacterial growth without the inhibitor (juglone) was replicated three times. The marine agars were then incubated for 24 h and 48 h at 28 °C. Where bacterial colonies were not formed, the MBC was established.To establish the effect of juglone in solution, experiments to measure the minimum inhibitory concentration (MIC) were conducted against the marine biofilm-forming species one, SSP ) overnigEX = 485 nm and λEM = 510 nm, DEAD: λEX = 540 nm and λEM = 610 nm, excitation and emission, respectively) to account for any potential autofluorescence deriving from the coatings and correct for any nonbiological signal. After that, the 24-well plate cells containing the coatings were inoculated with 50 μL of C. marina grown overnight in SSP (measured OD595 = 0.1 at the starting point), as shown in the experimental set up in TM and placed in the dark at 28 °C under agitation (100 rpm) for a total of 48 h.The coated coupons were placed in a 24-well plate (Nunclon™ D Multidishes). The experimental set-up can be seen in 595 measurements were followed. A fluorescent staining method using LIVE/DEAD  was performed to assess biofilm growth and cell integrity at the end-point. The LIVE/DEAD kit is comprised of two nucleic acid stains: (1) Syto9, a green fluorescent intercalating membrane permeable stain which is expected to stain all cells and (2) propidium iodide (PI), which is membrane impermeable and is known to stain cells with compromised membranes. The LIVE/DEAD kit was freshly prepared according to the manufacturer’s instructions, i.e., 2 μL of each stain component per 1 mL of phosphate buffer saline, pH 7.4. An aliquot of 500 μL of the LIVE/DEAD mastermix was added to each well ensuring full immersion of the coating in the LIVE/DEAD mixture. The plate was then incubated for 20 min in the dark at 28 °C. At the end of the incubation period, the coupons were gently rinsed twice with ASW to remove excess stain. Finally, 1 mL of ASW was added in order to keep the samples (and subsequently the biofilms) hydrated during the data-acquisition time. The microplate reader  was utilised for direct end point measurements  to allow high-resolution analysis of fluorescence intensity which was conducted using λEX = 485 nm (for excitation) and λEM = 510 nm (for emission). Coatings were scanned by the microplate reader and produced a heat map of fluorescence signals from the LIVE/DEAD wavelengths emitted from the coated surfaces using a 30 × 30 well scanning matrix . The software used was Mars Data Analysis Software, Version 2.00 .Following incubation for 48 h, the coupons were removed from the wells and gently rinsed with sterile ASW via pipetting to remove any nonattached cells. To establish planktonic growth at the end-point, ODn > 6). In all experiments, images were acquired from random positions on the coupons. The z-stacks were constructed using the Volocity software .CLSM was performed using a Leica TCS SP2 AOBS  microscope. The excitation wavelength used for Syto9 was 480 nm, and the emitted fluorescence was measured in the range of 500–600 nm. The red fluorescent nucleic acid stain PI was excited at 488 nm, and the emitted fluorescence was recorded in the range of 650–700 nm. Images were collected through a Leica water immersion ×63 objective and acquired at 0.502 μm intervals down through the biofilm, and therefore, the number of images in each stack varied according to the thickness of the biofilm. For each coating 1–6, see , at leasCLSM images were analysed by the computer program COMSTAT . A fixed3 μm–2). It is obtained by multiplying the sum of the biomass pixels in all cross-sectional images of a stack with the voxel size ((pixel size)x × (pixel size)y × (pixel size)z), and dividing the result by the area of the substratum. Apart from the overall volume of the biofilm, the biovolume also provides an estimate of the biomass within the biofilm [Biovolume. The biovolume is defined as the average volume of biomass present per unit of substratum area  pixel in the bottom layer containing biomass. Hence, thickness is defined as the maximum thickness over a given location, ignoring pores and voids inside the biofilm. The thickness distribution can be used to calculate a range of variables, including biofilm roughness and mean biofilm thickness [Thickness distribution and mean thickness. This function locates the highest point (μm) above each (hickness .p < 0.05). Statistical analysis was performed using SPSS version 17.0 and Minitab version 16.Differences between biocide-containing coatings and controls (for both plate reader and CLSM data) were assessed by applying one-way ANOVA. To establish the homogeneity of variances, Levene’s test of equal variances was applied. Where homogeneity of variances was not met, the nonparametric Kruskal–Wallis test was applied. All conclusions are based on 5% level of confidence , therefore epifluorescence microscopy can be used as a complementary method in future experiments. Model coatings using inert polymer binders were designed containing the NP juglone and the biocide Cueriments . Howevereriments , as welleriments . For suc