Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments.

BACKGROUND: The thymus, responsible for T cell-mediated adaptive immune system, has a structural and functional complexity that is not yet fully understood. Until now, thymic anatomy has been studied using histological thin sections or confocal microscopy 3D reconstruction, necessarily for limited volumes. METHODS: We used Phase Contrast X-Ray Computed Tomography to address the lack of whole-organ volumetric information on the microarchitecture of its structural components. We scanned 15 human thymi (9 foetal and 6 postnatal) with synchrotron radiation, and repeated scans using a conventional laboratory x-ray system. We used histology, immunofluorescence and flow cytometry to validate the x-ray findings. RESULTS: Application to human thymi at pre- and post-natal stages allowed reliable tracking and quantification of the evolution of parameters such as size and distribution of Hassall's Bodies and medulla-to-cortex ratio, whose changes reflect adaptation of thymic activity. We show that Hassall's bodies can occupy 25% of the medulla volume, indicating they should be considered a third thymic compartment with possible implications on their role. Moreover, we demonstrate compatible results can be obtained with standard laboratory-based x-ray equipment, making this research tool accessible to a wider community. CONCLUSIONS: Our study allows overcoming the resolution and/or volumetric limitations of existing approaches for the study of thymic disfunction in congenital and acquired disorders affecting the adaptive immune system.

The thymus is the organ responsible for programming the immune system. It consists of two main compartments, named medulla and cortex. The medulla contains onion-shaped parts known as "Hassall's bodies". By imaging thymi at different stages of development with advanced x-ray methods, we gain understanding of changes that occur over time in 3D. We quantified how much of the thymus was occupied by these different components as they change with age, showing that Hassall's bodies can take up 25% of the medulla, and should therefore be considered a proper part of the thymus with a purpose. Having a better understanding of the thymus can prove important in targeting conditions such as Down syndrome and thymic tumours, as well as provide information about structure.

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

X-ray phase-contrast microtomography of soft tissues using a compact laboratory system with two-directional sensitivity.

X-ray microtomography is a nondestructive, three-dimensional inspection technique applied across a vast range of fields and disciplines, ranging from research to industrial, encompassing engineering, biology, and medical research. Phase-contrast imaging extends the domain of application of x-ray microtomography to classes of samples that exhibit weak attenuation, thus appearing with poor contrast in standard x-ray imaging. Notable examples are low-atomic-number materials, like carbon-fiber composites, soft matter, and biological soft tissues. We report on a compact and cost-effective system for x-ray phase-contrast microtomography. The system features high sensitivity to phase gradients and high resolution, requires a low-power sealed x-ray tube, a single optical element, and fits in a small footprint. It is compatible with standard x-ray detector technologies: in our experiments, we have observed that single-photon counting offered higher angular sensitivity, whereas flat panels provided a larger field of view. The system is benchmarked against known-material phantoms, and its potential for soft-tissue three-dimensional imaging is demonstrated on small-animal organs: a piglet esophagus and a rat heart. We believe that the simplicity of the setup we are proposing, combined with its robustness and sensitivity, will facilitate accessing quantitative x-ray phase-contrast microtomography as a research tool across disciplines, including tissue engineering, materials science, and nondestructive testing in general.

Technical note: Cartilage imaging with sub-cellular resolution using a laboratory-based phase-contrast x-ray microscope.

BACKGROUND: Microscopic imaging of cartilage is a key tool for the study and development of treatments for osteoarthritis. When cellular and sub-cellular resolution is required, histology remains the gold standard approach, albeit limited by the lack of volumetric information as well as by processing artifacts. Cartilage imaging with the sub-cellular resolution has only been demonstrated in the synchrotron environment. PURPOSE: To provide a proof-of-concept demonstration of the capability of a laboratory-based x-ray phase-contrast microscope to resolve sub-cellular features in a cartilage sample. METHODS: This work is based on a laboratory-based x-ray microscope using intensity-modulation masks. The structured nature of the beam, resulting from the mask apertures, allows the retrieval of three contrast channels, namely, transmission, refraction and dark-field, with resolution depending only on the mask aperture width. An ex vivo equine cartilage sample was imaged with the x-ray microscope and results were validated with synchrotron tomography and histology. RESULTS: Individual chondrocytes, that is, cells responsible for cartilage formation, could be detected with the laboratory-based microscope. The complementarity of the three retrieved contrast channels allowed the detection of sub-cellular features in the chondrocytes. CONCLUSIONS: We provide the first proof-of-concept of imaging cartilage tissue with sub-cellular resolution using a laboratory-based x-ray microscope.

Long-Term Results of Femorodistal Sequential Composite-Bypass Combining Heparin-Bonded PTFE-Prosthesis and Autologous Vein Using the Deutsch Bridge Technique in Critical Limb-Threatening Ischemia.

BACKGROUND: Autologous vein bypass provides excellent long-term results in critical limb-threatening ischemia (CLTI), but a substantial portion of patients have insufficient vein length. In limbs with two distal outflow vessels and limited vein length, a vascular prosthesis may be combined with autologous vein for a sequential composite bridge bypass (SCBB). Results regarding graft function, limb salvage and reinterventions are presented. METHODS: Between January 2010 and December 2019, 47 consecutive SCBB operations with a heparin-bonded PTFE-prosthesis and autologous vein were performed. Grafts were followed with a duplex scan with prospective documentation in a computerized vascular database. Retrospective analysis of graft patency, limb salvage and patient survival was performed. RESULTS: Mean follow-up was 34 months (range 1-127 months). 30-day mortality was 10.6% and 5-year patient survival 32%. Postoperative bypass occlusion occurred in 6.4% and late occlusions or graft stenoses in 30%. Two prostheses developed late infection and seven legs were amputated. Primary, primary assisted, secondary patency and limb salvage rate were 54%, 63%, 66% and 85% after 5 years, respectively. CONCLUSIONS: SCBB patency and limb salvage were good despite a high early postoperative mortality. Combination of a heparin-bonded PTFE-prosthesis and autologous vein appears to be a valuable tool in CLTI in case of insufficient vein.

Utility of knife-edge position tracking in cycloidal computed tomography.

Cycloidal computed tomography provides high-resolution images within relatively short scan times by combining beam modulation with dedicated under-sampling. However, implementing the technique relies on accurate knowledge of the sample's motion, particularly in the case of continuous scans, which is often unavailable due to hardware or software limitations. We have developed an easy-to-implement position tracking technique using a sharp edge, which can provide reliable information about the trajectory of the sample and thus improve the reconstruction process. Furthermore, this approach also enables the development of other innovative sampling schemes, which may otherwise be difficult to implement.

Monitoring tissue engineered constructs and protocols with laboratory-based x-ray phase contrast tomography.

Tissue engineering (TE) aims to generate bioengineered constructs which can offer a surgical treatment for many conditions involving tissue or organ loss. Construct generation must be guided by suitable assessment tools. However, most current tools (e.g. histology) are destructive, which restricts evaluation to a single-2D anatomical plane, and has no potential for assessing constructs prior to or following their implantation. An alternative can be provided by laboratory-based x-ray phase contrast computed tomography (PC-CT), which enables the extraction of 3D density maps of an organ's anatomy. In this work, we developed a semi-automated image processing pipeline dedicated to the analysis of PC-CT slices of oesophageal constructs. Visual and quantitative (density and morphological) information is extracted on a volumetric basis, enabling a comprehensive evaluation of the regenerated constructs. We believe the presented tools can enable the successful regeneration of patient-specific oesophagus, and bring comparable benefit to a wide range of TE applications. STATEMENT OF SIGNIFICANCE: Phase contrast computed tomography (PC-CT) is an imaging modality which generates high resolution volumetric density maps of biological tissue. In this work, we demonstrate the use of PC-CT as a new tool for guiding the progression of an oesophageal tissue engineering (TE) protocol. Specifically, we developed a semi-automated image-processing pipeline which analyses the oesophageal PC-CT slices, extracting visual and quantitative (density and morphological) information. This information was proven key for performing a comprehensive evaluation of the regenerated constructs, and cannot be obtained through existing assessment tools primarily due to their destructive nature (e.g. histology). This work paves the way for using PC-CT in a wide range of TE applications which can be pivotal for unlocking the potential of this field.

Improved visualization of X-ray phase contrast volumetric data through artifact-free integrated differential images.

Artifacts arising when differential phase images are integrated is a common problem to several X-ray phase-based experimental techniques. The combination of noise and insufficient sampling of the high-frequency differential phase signal leads to the formation of streak artifacts in the projections, translating into poor image quality in the tomography slices. In this work, we apply a non-iterative integration algorithm proven to reduce streak artifacts in planar (2D) images to a differential phase tomography scan. We report on how the reduction of streak artifacts in the projections improves the quality of the tomography slices, especially in the directions different from the reconstruction plane. Importantly, the method is compatible with large tomography datasets in terms of computation time.

Contemporary results with the biosynthetic glutaraldehyde denatured ovine collagen graft (Omniflow II) in femoropopliteal position.

OBJECTIVE: The objective of this study was to analyze the outcome of a contemporary series of femoropopliteal bypass operations with the glutaraldehyde denatured polyester mesh-reinforced ovine collagen prosthesis (OCP; Omniflow II [LeMaitre Vascular, Inc, Burlington, Mass]). The experience of two tertiary centers regarding long-term graft function, secondary reinterventions, and biodegeneration of the OCP prosthesis is presented. METHODS: Between January 2006 and January 2014, a series of 205 consecutive operations with the OCP in the femoropopliteal position (54 above knee and 151 below knee) were performed in 194 patients in 202 limbs for disabling claudication (72), chronic critical ischemia (105), acute ischemia (18), popliteal artery aneurysm (4), degeneration of a venous or prosthetic graft (5), and infection of a synthetic bypass graft (1). Grafts were observed with duplex ultrasound scan supplemented by additional angiography in case of recurrent ischemia with prospective documentation of follow-up data in a computerized vascular database. Retrospective analysis of graft patency, limb salvage, and diagnosis of aneurysmal graft degeneration was performed. RESULTS: The 30-day mortality was 3.9%. Early thrombotic bypass occlusion occurred in 8.2% of cases. Four early graft infections could be successfully managed by local treatment with graft preservation. After a mean (median) follow-up of 56 (55) months (range, 1-135 months), primary patency, primary assisted patency, secondary patency, and limb salvage were 71%, 78%, 78%, and 91% for above-knee bypass and 40%, 50%, 63%, and 87% for below-knee bypass at 5 years. Biodegeneration in the form of graft aneurysm or graft stenosis was detected in 26 grafts (12.6%), resulting in secondary open or endovascular procedures in 16 cases. CONCLUSIONS: The OCP provides satisfactory medium- and long-term patency and limb salvage in the femoropopliteal position. Aneurysmal degeneration or graft stenosis may develop over time, demanding lifelong duplex ultrasound surveillance and secondary intervention if needed. Its possible infection-resistant behavior in a contaminated field combined with an acceptable graft patency and limb salvage justifies the use of this graft in the absence of autologous vein.

External polytetrafluoroethylene reinforcement of varicose autologous vein grafts in peripheral bypass surgery produces durable bypass function.

OBJECTIVE: Use of autologous veins as peripheral bypass graft may become critical in the presence of significant varicose degeneration of the harvested vein. External support of such dilated veins with standard polytetrafluoroethylene (PTFE) prostheses was recommended as an option to use these veins for peripheral bypass. A single-center experience with this technique regarding long-term graft function, secondary reinterventions, and potential graft degeneration is presented. METHODS: Between January 1995 and January 2006, there were 54 patients with varicose veins who underwent 57 consecutive infrainguinal vein bypass operations with PTFE reinforcement in 57 limbs. Indications for surgery consisted of disabling claudication (5), chronic critical ischemia (40), popliteal aneurysm (11), and acute ischemia (1). Grafts were observed with duplex ultrasound scan supplemented by additional angiography in case of recurrent ischemia, with prospective documentation of follow-up data in a computerized vascular database. Graft patency, limb salvage, and possible degeneration of the vein grafts were retrospectively analyzed. RESULTS: Mean follow-up was 79 months (range, 1-219 months). The 30-day mortality was 2%. Secondary procedures to maintain or to restore bypass patency were necessary in 12 grafts (21%). Primary, primary assisted, and secondary patency rates were 54%, 73%, and 73% after 5 years for all bypasses, with a limb salvage rate for limbs operated on for chronic critical or acute ischemia of 83%. Significant stenosis of a reinforced vein segment was detected in one case after 56 months, with subsequent replacement of the vein graft with a biologic vascular prosthesis. CONCLUSIONS: Good late graft patency and limb salvage combined with a low rate of late vein graft degeneration justify the use of external PTFE reinforcement of varicose vein segments in infrainguinal bypass surgery.

Very distal vein bypass in patients with thromboangiitis obliterans.

BACKGROUND: Surgical revascularization for chronic critical limb ischaemia in patients with thromboangiitis obliterans (TAO) still remains controversial. Generally, besides cessation of smoking, conservative treatment supported by intravenous administration of vasoactive agents is regarded as the treatment of choice, in combination with local wound therapy or minor amputation. PATIENTS AND METHODS: In four male patients (42-47 years) surgical revascularization was chosen as therapy for established gangrene or non-healing ulceration after unsuccessful conservative treatment and cessation of smoking. Angiography was able to identify a suitable distal arterial segment for the bypass which was revascularized by means of an autologous vein graft. Grafts were followed with repetitive duplex ultrasound. Revision of the bypass graft was initiated if indicated by pathological duplex findings. RESULTS: In all cases a bypass could be constructed with either the ipsilateral greater saphenous vein or arm veins. A distal origin configuration was possible in three cases with popliteo-pedal or cruro-pedal bypasses. In the fourth case the distal superficial femoral artery was used for inflow. Two early graft thromboses underwent successful revision. During follow-up, duplex ultrasound identified graft stenoses in three bypasses which were successfully treated with endovascular techniques. All grafts are patent with complete resolution of ischaemic symptoms after 46, 42, 32, and 29 months. The patients remained non-smokers and returned to a professional life. CONCLUSIONS: Surgical therapy with distal vein bypass for persistent ischaemic symptoms after definitive cessation of smoking seems feasible in selected cases with TAO and a suitable distal artery. Close follow-ups of the patients with duplex ultrasound are necessary to identify developing vein graft stenoses. Angioplasty seems to be an important part of the long-term therapeutic concept.

Outcomes of complex femorodistal sequential autologous vein and biologic prosthesis composite bypass grafts.

OBJECTIVE: Femorodistal autologous vein bypass proves to be the preferred surgical therapy for long arterial occlusions and provides excellent early and long-term results in critical lower limb ischemia. Whenever vein length was insufficient and two distal outflow arteries were present, a sequential composite bypass configuration was chosen with human umbilical vein (HUV) or ovine collagen prosthesis (Omniflow II; Bio Nova International Pty Ltd, North Melbourne, Australia) as the proximal prosthetic part of the bypass. Single-center experience with this technique regarding limb salvage, graft function, secondary reinterventions, and biodegeneration is presented. METHODS: Between January 1998 and January 2009, 122 consecutive sequential composite bypass operations were performed on 116 patients for short-distance claudication (2), chronic critical ischemia (117), or acute ischemia (3) in the absence of sufficient autologous vein length. HUV was used in 90 cases and Omniflow II in 32 cases. Grafts were followed by duplex scan supplemented by angiography in case of recurrent ischemia with prospective documentation of follow-up data in a computerized vascular database. Retrospective analysis of graft patency, limb salvage, and aneurysmal degeneration of the biologic prosthesis was performed. RESULTS: Mean follow-up was 59 ± 45.5 months (range, 1-161 months). The 30-day mortality was 4.1%. Early postoperative complete or partial bypass thrombosis developed in 16% (20 cases) and required successful revision in 16 cases. During follow-up, 30 complete and 12 partial bypass occlusions occurred, necessitating selective surgical or interventional revision. Primary, primary assisted, and secondary patency rates and the limb salvage rate were 48%, 62%, 71%, and 87%, respectively, after 5 years and 26%, 46%, 54%, and 77%, respectively, after 10 years for all bypasses. Late biodegeneration of HUV prostheses was detected in four instances. CONCLUSIONS: Late graft patency and limb salvage were good. These factors, combined with a tolerable rate of late aneurysmal degeneration, justify the use of biologic vascular conduits and autologous vein for complex femorodistal reconstructions.

ß-Blockers in patients with intermittent claudication and arterial hypertension: results from the nebivolol or metoprolol in arterial occlusive disease trial.

The use of ß-receptor blockers in peripheral arterial disease is controversial for their impact on vasomotor tone. The ß-blocker nebivolol possesses vasodilating, endothelium-dependent, NO-releasing properties that might be beneficial in peripheral arterial disease. The aim of the study was to evaluate the effects and tolerability of nebivolol in comparison with metoprolol in these patients. A total of 128 patients with intermittent claudication and essential hypertension were included and double-blind randomized to receive 5 mg of nebivolol (N=65) or 95 mg of metoprolol (N=63) once daily. End points were changes in ankle-brachial index, initial and absolute claudication distance, endothelial function assessed by flow-mediated dilatation of the brachial artery, blood pressure, and quality of life using the claudication scale questionnaire. End point analysis was possible in 109 patients (85.2%). After the 48-week treatment period, ankle-brachial index and absolute claudication distance improved significantly in both patient groups (P<0.05 for both), with no difference across treatments. A significant increase of initial claudication distance was found in the nebivolol group. Adjusted mean change of initial claudication distance was 33.9% after nebivolol (P=0.003) and 16.6% after metoprolol (P=0.12) treatment. Quality of life was not influenced by either treatment, and there was no relevant change in flow-mediated dilatation in patients treated with nebivolol or metoprolol (P=0.16). Both drugs were equally effective in lowering blood pressure. In conclusion, ß-blocker therapy was well tolerated in patients with intermittent claudication and arterial hypertension during a treatment period of ≈1 year. In the direct comparison, there was no significant difference between nebivolol and metoprolol.

Different calculations of ankle-brachial index and their impact on cardiovascular risk prediction.

BACKGROUND: An ankle-brachial index (ABI; ratio of ankle and brachial systolic blood pressure) <0.9 indicates peripheral arterial disease (PAD) and is a strong predictor of cardiovascular events. The aim of the present study was to address the prognostic value of different methods of ABI calculation. METHODS AND RESULTS: In 831 patients admitted with chest pain for diagnostic heart catheterization, blood pressure of both anterior and posterior tibial arteries was measured. ABI was calculated for each leg with the higher of the 2 ankle pressures (current definition of the American Heart Association) or with the lower of the 2 ankle pressures (modified definition) in relation to the higher of the left or right brachial systolic blood pressure. For each patient, the lower ABI from both legs was used for further evaluation. Fifteen patients (1.8%) with ABI >1.5 were excluded. We compared patients with ABI <0.9 according to the current definition (with PAD, n=204 [25.0%]), those with ABI >or=0.9 according to the modified definition (without PAD, n=524 [64.2%]), and those with ABI <0.9 according to the modified definition and >or=0.9 according to the current definition (suspected PAD, n=88 [10.8%]). Follow-up data (median 6.6 years) were available for 812 patients (99.5%); 157 patients (19.3%) experienced cardiovascular events (cardiovascular death, myocardial infarction, or stroke). Patients without PAD had the lowest cardiovascular event rate, whereas event rates were comparable for patients with PAD and those with suspected PAD (14.8% versus 28.4% versus 25.0%, respectively). In a fully adjusted Cox regression analysis that included patients without PAD as the reference group, the hazard ratio (95% CI) was 1.56 (0.97 to 2.53) for patients with suspected PAD and 1.67 (1.16 to 2.40) for patients with PAD. CONCLUSIONS: When the higher ankle pressure is used for ABI calculation, a group of patients at high risk for cardiovascular events is overlooked. With a simple modification of ABI (use of the lower instead of the higher ankle pressure), more patients at risk could be identified.

Questionable value of adjuvant arteriovenous fistula in pedal bypass at high risk for early failure.

Results of an adjuvant arteriovenous fistula (AVF) in pedal bypass surgery in the presence of poor status of the recipient artery, severely impaired intraoperative runoff, or revision for early failure and flow restitution were analyzed in a retrospective study. From January 1998 to December 2006, 24 adjuvant AVFs were constructed in autologous vein or composite pedal bypasses with low intraoperative bypass flow, poor status of the pedal artery, or during successful early bypass revision to prevent graft failure. All infrainguinal bypass operations were registered in a computerized database and prospectively followed. Pedal bypasses with adjunctive AVF were reviewed for fistula function, graft patency, limb salvage, and patient survival. Primary and secondary bypass patency rates at 1 year were 59% and 77%, respectively, with an AVF patency of 36%. Four legs were amputated despite a patent bypass with patent AVF on three occasions. The corresponding limb salvage rate was 65% at 1 year. Patient survival was 50% at 3 years. Adjuvant AVF constructed in grafts considered at high risk for early failure in pedal vein graft or composite bypass does not seem to prevent future graft failure. In patent bypasses the fistula has a significant tendency for spontaneous occlusion. It may be considered in the use of prosthetic composite pedal grafts in selected cases.

[Prevention of thromboembolism with low molecular weight heparin (Dalteparin-Na) in risk pregnancy]. / Thromboembolieprophylaxe mit niedermolekularem Heparin (Dalteparin-Na) bei Risikoschwangerschaften.

BACKGROUND: Venous thrombotic events still remain the leading cause of maternal morbidity and mortality. During pregnancy as well as post partum, hemostasis changes also in normal pregnant women. Coagulation is activated and fibrinolysis suppressed, the concentration of particular coagulation factors is increased, while inhibitor potential is decreased. Additionally, the venous blood stream is mechanically hampered by the gravid uterus. As a result of these physiologic changes, the risk of thromboembolism is elevated. The risk increases frequently in women with previous thromboembolic episodes, a family history of thromboembolism, hereditary or acquired thrombotic disorders as well as the appearance of additional exposure prothrombogenic factors such as immobilization, inflammation, and operation. Simultaneous presence of combined prothrombogenic factors conducts a potentiation of the risk of thromboembolism. To avoid thromboembolism or rethromboembolism during pregnancy or puerperium, an individual risk-adapted heparin prophylaxis is indicated. PATIENTS AND METHODS: 17 pregnant women with inherited and/or acquired prothrombogenic disorders, eleven of them with previous thromboembolism episodes, were treated with low molecular weight heparin (LMWH; dalteparin-Na). The daily dose of 5,000-10,000 IU LMWH was applied subcutaneously by self-injection during pregnancy and up to 8 weeks post partum. Every 4 weeks clinical and laboratory monitoring was performed. Basic parameters as well as the special coagulation marker TAT (thrombin-antithrombin complex) and D-dimer were analyzed. RESULTS: Under the therapy with LMWH (dalteparin-Na), no thromboembolic events during pregnancy or post partum could be observed. No serious bleeding complications, except small subcutaneous local hematomas, occurred. Bolus applications of LMWH by self-injection were easy to practice, gained a good acceptance and high compliance. Increased TAT values above normal at the actual state of pregnancy could be suppressed to normal values by raising the LMWH dose. CONCLUSION: The individual thromboembolic prophylaxis with LMWH represents an effective and safe therapy in risk pregnancy with previous thromboembolic events and/or thrombotic disorders. TAT seems to be an effective marker for monitoring of the coagulation activity during pregnancy and puerperium. Under this management, thromboembolic prophylaxis can be optimized.