The free vascularized lateral femoral trochlea osteochondral graft: a reliable alternative for Stage III Kienböck's disease.

Lunate reconstruction using a lateral femoral trochlea osteochondral graft was carried out in 27 patients with Stage III Kienböck's disease from 2012 to 2019. Twenty-three of these patients could be followed-up in this retrospective study. Ten were women and 13 men. Nine were Lichtman Stage IIIA, seven Stage IIIB and seven Stage IIIC. The mean follow-up was 39 months (range 12-86). Bony consolidation was found in 18 of the 23 patients, with no graft loss. The mean Disabilities of the Arm, Shoulder and Hand score (DASH score) was 11 and the Modified Mayo Wrist Score was 83. There were only two radiological deteriorations, with the same or improved Lichtman classifications in the other patients and a significant reduction in pain. Postoperative extension of the wrist (52°) and flexion (48°) were comparable with preoperative values and, respectively, 81% and 72% of the contralateral side. Grip strength and pinch grip were 32 kg and 12 kg, 88% and 94% of the other hand, respectively, and an insignificant increase compared with the preoperative values. The vascularized lateral femoral trochlea osteochondral graft yields good short- and mid-term results in Grade III Kienböck's disease.Level of evidence: IV.

Ultrashort single-pulse laser ablation of stainless steel, aluminium, copper and its dependence on the pulse duration.

In this work, we investigate single-pulse laser ablation of bulk stainless steel (AISI304), aluminium (Al) and copper (Cu) and its dependence on the pulse duration. We measured the reflectivity, ablation thresholds and volumes under the variation of pulse duration and fluence. The known drop of efficiency with increasing pulse duration is confirmed for single-pulse ablation in all three metals. We attribute the efficiency drop to a weakened photomechanically driven ablation process and a stronger contribution of photothermal phase explosion. The highest energetic efficiency and precision is achieved for pulse durations below the mechanical expansion time of 3-5 ps, where the stress confinement condition is fulfilled.

Anterior open bite - diagnostics and therapy.

An anterior open bite (AOB) is an occlusal disorder that causes the patient both an aesthetic and functional handicap. The lower third of the face is disproportionately larger. Patients are unable to properly occlude with the anterior part of dental arch and occlusion only happens in the premolar and/or molar regions. An anterior open bite may be the result of anatomical anomalies. Long term stability as well as an immediate outcome of the surgery depends on the choice of a suitable treatment strategy. In this article, we review options of AOB treatment, from classical orthodontic treatment to current combined orthodontic and surgical approach with a benefit of an anchor system.

INDICATION AND IMPORTANCE OF RECONSTRUCTIVE SURGERIES OF FACIAL SKELETON IN MAXILLOFACIAL SURGERY: REVIEW.

Malignant head and neck tumors belong among common diseases and their incidence constantly rises. In the Czech Republic, the proportional representation of orofacial tumors ranges around 2% of the total number of malignancies. Rational treatment of these tumors is complex and long. In the course of therapeutic planning, you have to consider the age of the patient and the stage of the disease including the presence of distant metastases. Removal of the tumor with a sufficient safety margin and an eventual treatment of the relevant lymphatic system according to the type of the tumor is an important prerequisite for the success of the surgical therapy. Reconstructive procedures in maxillofacial oncosurgery presume good interdisciplinary cooperation and a high professional preparedness of the surgical and nursing team. Selection of the right patient is also very important with regard to the risks of both local and systemic postoperative complications. Use of the free flap techniques is currently the gold standard, but it is also necessary to master pedicled flap techniques, whose advantages lie in simpler technique and often better aesthetic results. At the same time, we have to realize that even traditional, classical reconstructive procedures using prosthetic replacements can still represent the ideal solution in many cases.

SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication.

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

HIRUDOTHERAPY IN RECONSTRUCTIVE SURGERY: CASE-REPORTS AND REVIEW.

Medicinal leeches (Hirudo medicinalis, Hirudo verbana) have been used in the field of medicine to treat various diseases for thousands of years. Popularity of their use changed over time and in Europe, it peaked at the beginning of the 19th century. In modern medicine, application of leeches on flaps with venous congestion was first used and described by Deganc and Zdravic in 1960. A certain renaissance of leech use is currently taking place, especially in the field of reconstructive surgery. In general, use of leeches is indicated during critical post-operative period, in which the microcirculation and veins are incapable of sufficient drainage of venous blood, which can lead to stagnation of circulation in tissues at all levels, clinically manifested as a change in color and turgor of the flap. If this venostasis is not recognized in time and treated adequately, tissue necrosis can develop. Medicinal leeches can be used in venous drainage disorders after a replantation of fingers, auricles, lips and parts of the nose. In head and neck reconstructive surgery, there are many studies that confirm the success rate of hirudotherapy in hematoma evacuation or in dealing with complications after scalp replantation and transfers of free and pedicled flaps. Leech application therapy can also be indicated as a part of non-surgical methods that improve conditions of the venous system.

Pedicled pectoralis major flap in head and neck reconstruction - technique and overview.

The pedicled pectoralis major flap was the original workhorse flap for head and neck reconstruction. Over time, it became the secondary choice for oropharyngeal reconstruction with the implementation of free-soft tissue transfers. Nowadays, a polymorbid patient is primarily indicated for pedicled pectoralis major flap reconstruction, other indications include combinations of pedicled pectoralis major flap with free microvascular flap, salvage reconstruction due to complications, salvage reconstruction due to free flap failure and salvage reconstruction due to recurrent or extended primary disease. Pedicled pectoralis major flap can be successfully used for specific oropharyngeal defects, even primary resections, especially for less cooperative patients and patients after extensive neck dissection. Improving the flap harvesting techniques can reduce undesired complications in specific cases of oropharyngeal reconstruction. Flap morbidity in these cases remains comparable to morbidity of patients who had undergone free flap reconstruction. Pedicled pectoralis major flap remains valid reconstruction tool that should be included in the armamentarium of each surgeon dealing with reconstruction of the head and neck.

Pedicled pectoralis major flap in head and neck reconstruction - our experience.

INTRODUCTION: The pedicled pectoralis major flaps are still harvested and mainly indicated for reconstruction in the polymorbid patient. Other indications are combinations of pedicled pectoralis major flaps with free microvascular flap, salvage reconstruction following complications, free flap failure and recurrent or extended primary disease. MATERIALS AND METHODS: We describe the pedicled pectoralis major flap in 18 patients operated on at the Department of Oral and Maxillofacial Surgery, Olomouc from 1st January 2014 to 1st December 2016. RESULTS: Fifteen oropharyngeal defect reconstructions were performed using pedicled pectoralis major flap (including 1 submandibular defect of the neck). Indications were primary resection in polymorbid patients in 10 cases and recurrent diseases after previous neck dissection and radiotherapy in 5 cases. Pedicled pectoralis major flap was used for secondary reconstruction in 3 cases. Complications occurred in 50 % of patients, 28 % were major and 22 % minor. Major complications included a total flap failure (defect was successfully treated with free tissue transfer of latissimus dorsi myocutaneous flap) in 1 case, plate exposure in 2 cases, large dehiscence and large hemorrhage 1 case each. Minor complications included only small dehiscences (22 %). One was associated with fluidothorax after rib harvesting (6%). There were no cases of neck contracture or supraclavicular bulge. CONCLUSION: Even today, usage the pedicled pectoralis major flap in head and neck reconstruction surgery cannot be considered as an obsolete reconstructive procedure that has been completely replaced by a free microvascular flap. Innovations of flap harvesting techniques and high rate of flap survival are the main reasons why pedicled pectoralis major flap can still be primarily indicated for high-risk patients, non-cooperative patients and also for patients with extensive neck dissection. The pedicled pectoralis major flap has been the first choice in salvage surgery, in cases of a complication or free flap failure or a recurrence of a primary disease.

Reconstruction Strategies of the Ureter and Urinary Diversion Using Tissue Engineering Approaches.

IMPACT STATEMENT: Tissue Engineering (TE) approaches are needed to advance the field of reconstructive urology. We indicate that regeneration of ureteral tissue and the formation of a urinary diversion using TE approaches are possible, although it is currently very time-consuming and complex to achieve well-developed neotissue. Faster regeneration approaches using novel scaffolds are desirable. The findings of this review may help to develop smart hybrid scaffolds and enhance the design of future studies, which may ultimately lead to improved care for patients with ureteral defects as well as to curb complications associated with urinary diversion.

Ultrafast dynamics of hard tissue ablation using femtosecond-lasers.

Several studies on hard tissue laser ablation demonstrated that ultrafast lasers enable precise material removal without thermal side effects. Although the principle ablation mechanisms have been thoroughly investigated, there are still open questions regarding the influence of material properties on transient dynamics. In this investigation, we applied pump-probe microscopy to record ablation dynamics of biomaterials with different tensile strengths (dentin, chicken bone, gallstone and kidney stones) at delay times between 1 picosecond and 10 microseconds. Transient reflectivity changes, pressure and shock wave velocities and elastic constants were determined. The result revealed that absorption and excitation show the typical well-known transient behavior of dielectric materials. We observed for all samples a photomechanical laser ablation process, where ultrafast expansion of the excited volume generates pressure waves leading to fragmentation around the excited region. In addition, we identified tensile-strength-related differences in the size of ablated craters and ejected particles. The elastic constants derived were in agreement with literature values. In conclusion, pressure-wave-assisted material removal seems to be a general mechanism for hard tissue ablation with ultrafast lasers. This photomechanical process increases ablation efficiency and removes heated material, thus ultrafast laser ablation is of interest for clinical application where heating of the tissue must be avoided.

Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses.

Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation.

Clinical protocol levels are required in laboratory animal surgery when using medical devices: experiences with ureteral replacement surgery in goats.

It is common to test medical devices in large animal studies that are or could also be used in humans. In this short report we describe the use of a ureteral J-stent for the evaluation of biodegradable tubular constructs for tissue reconstruction, and the regeneration of ureters in Saanen goats. Similarly to a previous study in pigs, the ureteral J-stent was blindly inserted until some resistance was met. During evaluation of the goats after three months, perforation of the renal cortex by the stent was observed in four out of seven animals. These results indicated that blind stent placement was not possible in goats. In four new goats, clinical protocols were followed using X-ray and iodinated contrast fluids to visualize the kidney and stent during stent placement. With this adaptation the stents were successfully placed in the kidneys of these four new goats with minimal additional effort. It is likely that other groups in other fields ran into similar problems that could have been avoided by following clinical protocols. Therefore, we would like to stress the importance of following clinical protocols when using medical devices in animals to prevent unnecessary suffering and to reduce the number of animals needed.

Intraindividual Comparison of 18F-PSMA-1007 PET/CT, Multiparametric MRI, and Radical Prostatectomy Specimens in Patients with Primary Prostate Cancer: A Retrospective, Proof-of-Concept Study.

68Ga-prostate-specific membrane antigen (PSMA)-11 PET/CT represents an advanced method for the staging of primary prostate cancer (PCa) and diagnosis of recurrent or metastatic PCa. However, because of the narrow availability of 68Ga the development of alternative tracers is of high interest. The objective of this study was to examine the value of the new PET tracer 18F-PSMA-1007 for the staging of local disease by comparing it with multiparametric MRI (mpMRI) and radical prostatectomy (RP) histopathology. Methods: In 2016, 18F-PSMA-1007 PET/CT was performed in 10 men with biopsy-confirmed high-risk PCa. Nine patients underwent mpMRI in the process of primary diagnosis. Consecutively, RP was performed in all 10 men. Agreement analysis was performed retrospectively. PSMA staining was added for representative sections in RP specimen slices. Localization and agreement analysis of 18F-PSMA-1007 PET/CT, mpMRI, and RP specimens was performed by dividing the prostate into 38 sections as described in the prostate imaging reporting and data system (PI-RADS) (version 2). Sensitivity, specificity, positive predictive values, negative predictive values (NPVs), and accuracy were calculated for total and near-total agreement. Results:18F-PSMA-1007 PET/CT had an NPV of 68% and an accuracy of 75%, and mpMRI had an NPV of 88% and an accuracy of 73% for total agreement. Near-total agreement analysis resulted in an NPV of 91% and an accuracy of 93% for 18F-PSMA-1007 PET/CT and 91% and 87% for mpMRI, respectively. Retrospective combination of mpMRI and PET/CT had an accuracy of 81% for total and 93% for near-total agreement. Conclusion: Comparison with RP histopathology demonstrates that 18F-PSMA-1007 PET/CT is promising for accurate local staging of PCa.

Tubular collagen scaffolds with radial elasticity for hollow organ regeneration.

Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs. STATEMENT OF SIGNIFICANCE: In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs.

Pesticides drive risk of micropollutants in wastewater-impacted streams during low flow conditions.

Micropollutants enter surface waters through various pathways, of which wastewater treatment plants (WWTPs) are a major source. The large diversity of micropollutants and their many modes of toxic action pose a challenge for assessing environmental risks. In this study, we investigated the potential impact of WWTPs on receiving ecosystems by describing concentration patterns of micropollutants, predicting acute risks for aquatic organisms and validating these results with macroinvertebrate biomonitoring data. Grab samples were taken upstream, downstream and at the effluent of 24 Swiss WWTPs during low flow conditions across independent catchments with different land uses. Using liquid chromatography high resolution tandem mass spectrometry, a comprehensive target screening of almost 400 organic substances, focusing mainly on pesticides and pharmaceuticals, was conducted at two time points, and complemented with the analysis of a priority mixture of 57 substances over eight time points. Acute toxic pressure was predicted using the risk assessment approach of the multi-substance potentially affected fraction, first applying concentration addition for substances with the same toxic mode of action and subsequently response addition for the calculation of the risk of the total mixture. This toxic pressure was compared to macroinvertebrate sensitivity to pesticides (SPEAR index) upstream and downstream of the WWTPs. The concentrations were, as expected, especially for pharmaceuticals and other household chemicals higher downstream than upstream, with the detection frequency of plant protection products upstream correlating with the fraction of arable land in the catchments. While the concentration sums downstream were clearly dominated by pharmaceuticals or other household chemicals, the acute toxic pressure was mainly driven by pesticides, often caused by the episodic occurrence of these compounds even during low flow conditions. In general, five single substances explained much of the total risk, with diclofenac, diazinon and clothianidin as the main drivers. Despite the low predicted acute risk of 0%-2.1% for affected species, a significant positive correlation with macroinvertebrate sensitivity to pesticides was observed. However, more effect data for pharmaceuticals and a better quantification of episodic pesticide pollution events are needed for a more comprehensive risk assessment.

Time-resolved microscopy reveals the driving mechanism of particle formation during ultrashort pulse laser ablation of dentin-like ivory.

In dental health care, the application of ultrashort laser pulses enables dental tissue ablation free from thermal side effects, such as melting and cracking. However, these laser types create undesired micro- and nanoparticles, which might cause a health risk for the patient or surgeon. The aim of this study was to investigate the driving mechanisms of micro- and nanoparticle formation during ultrashort pulse laser ablation of dental tissue. Time-resolved microscopy was chosen to observe the ablation dynamics of mammoth ivory after irradiation with 660 fs laser pulses. The results suggest that nanoparticles might arise in the excited region. The thermal expansion of the excited material induces high pressure in the surrounding bulk tissue, generating a pressure wave. The rarefaction wave behind this pressure wave causes spallation, leading to ejection of microparticles.

Comparison of 68Ga-DOTATOC-PET/CT and PET/MRI hybrid systems in patients with cranial meningioma: Initial results.

BACKGROUND: (68)Ga-DOTATOC-PET/CT is a well-established method for detecting and targeting the volume definition of meningiomas prior to radiotherapy. Moreover, there is evidence that this method is able to detect meningiomas with higher sensitivity than the goldstandard MRI. Since the hybrid PET/MRI scanner became available in the past few years, the next stage of development could consequently evolve by evaluating the feasibility of a hybrid PET/MRI scanner using (68)Ga-DOTATOC for detecting meningiomas. METHODS: Fifteen patients received (68)Ga-DOTATOC-PET/CT (0.5 h post injection [p.i.]) followed by PET/MRI 2 hours p.i. Both investigations were analyzed separately and then compared with respect to image quality, detection of intracranial meningiomas, and radiotracer uptake values (RUVs). In addition, ratios between radiotracer uptake in meningiomas and pituitary glands were compared between both PET/CT and PET/MRI. RESULTS: Overall, 33 intracranial meningiomas were detected. All were visible with high contrast in both PET/CT and PET/MRI. (68)Ga-DOTATOC-PET/MRI provided flawless image quality without artefacts. Calculated RUV in meningiomas, as well as the ratios of RUVs in meningiomas to those of pituitary glands, were higher in PET/CT. As a result, meningiomas can be distinguished from pituitary glands better in early images. CONCLUSIONS: (68)Ga-DOTATOC-PET/MRI provided flawless image quality and presented an ideal combination of high sensitivity/specificity (PET) and the best possible morphological visualization of meningiomas (MRI). In addition, excellent detection of meningiomas is already possible at 0.5 hours p.i. Later images do not improve the distinction between pituitary gland and adjacent meningiomas. However, RUVs need to be carefully compared between both imaging modalities.

Progression types after antiangiogenic therapy are related to outcome in recurrent glioblastoma.

OBJECTIVE: This retrospective study analyzed whether the type of radiologic progression, classified according to contrast enhancement on MRI T1-weighted sequences and changes in T2-hyperintense signal, is relevant for outcome in patients with progressive glioblastoma (pGB) treated with bevacizumab. METHODS: MRI scans of 83 patients with pGB treated with bevacizumab were evaluated prior to and at disease progression. Based on initial decrease in and subsequent flare-up of contrast enhancement in T1 and 2 patterns of T2-hyperintense tumor progression, progression types (PTs) were categorized as cT1 flare-up, T2-diffuse, T2-circumscribed, or primary nonresponder. Overall survival (OS), survival from start of bevacizumab therapy (OS_Bev), survival after bevacizumab failure (OS_PostBev), time from initial diagnosis until initiation of bevacizumab therapy (StartBevT), and time to bevacizumab progression were evaluated using Kaplan-Meier curves, log-rank test, and Cox regression analyses. RESULTS: The time observed for development of a T2-diffuse (n = 15) or a cT1 flare-up (n = 35) progression was longer than for progression in primary nonresponders (n = 16) or T2-circumscribed progression (n = 17). The T2-diffuse PT showed longer OS, OS_Bev, OS_PostBev, and StartBevT compared to the other PTs. Postprogression therapy tended to be relevant only for patients with a T2-circumscribed PT. CONCLUSIONS: Radiologic PTs following bevacizumab treatment failure show differences in time to development and are related to outcome. We therefore hypothesize that these PTs reflect a different glioma biology, including differential resistance mechanisms to bevacizumab, and may be associated with different responses to postprogression therapy.

Ion Prostate Irradiation (IPI) - a pilot study to establish the safety and feasibility of primary hypofractionated irradiation of the prostate with protons and carbon ions in a raster scan technique.

BACKGROUND: Due to physical characteristics, ions like protons or carbon ions can administer the dose to the target volume more efficiently than photons since the dose can be lowered at the surrounding normal tissue. Radiation biological considerations are based on the assumption that the α/ß value for prostate cancer cells is 1.5 Gy, so that a biologically more effective dose could be administered due to hypofractionation without increasing risks of late effects of bladder (α/ß = 4.0) and rectum (α/ß = 3.9). METHODS/DESIGN: The IPI study is a prospective randomized phase II study exploring the safety and feasibility of primary hypofractionated irradiation of the prostate with protons and carbon ions in a raster scan technique. The study is designed to enroll 92 patients with localized prostate cancer. Primary aim is the assessment of the safety and feasibility of the study treatment on the basis of incidence grade III and IV NCI-CTC-AE (v. 4.02) toxicity and/or the dropout of the patient from the planned therapy due to any reason. Secondary endpoints are PSA-progression free survival (PSA-PFS), overall survival (OS) and quality-of-life (QoL). DISCUSSION: This pilot study aims at the evaluation of the safety and feasibility of hypofractionated irradiation of the prostate with protons and carbon ions in prostate cancer patients in an active beam technique. Additionally, the safety results will be compared with Japanese results recently published for carbon ion irradiation. Due to the missing data of protons in this hypofractionated scheme, an in depth evaluation of the toxicity will be created to gain basic data for a following comparison study with carbon ion irradiation. TRIAL REGISTRATION: Clinical Trial Identifier: NCT01641185 (clinicaltrials.gov).