Cryopreservation with a twist - towards a sterile, serum-free surface-based vitrification of hESCs.

Human embryonic stem cells (hESCs) play an important role in the fields of regenerative medicine, basic scientific research, tissue engineering and toxicology. Their unique morphology however makes them very sensitive to cryopreservation procedures. We recently introduced a surface dependent, enzyme- and serum-free method for the effective cryopreservation of bulk quantities of hESC colonies using direct immersion into liquid nitrogen (Beier et al., 2011 [5]). However, direct contact with liquid nitrogen risks contamination and cell infection and severely limits clinical application. This work introduces a modified method and a new combined cultivation and cryopreservation device to facilitate the surface dependent vitrification without contact with (possibly unsterile) liquid nitrogen. The technique allows the culture, cryopreservation, storage and post-thawing cultivation in the same device without detaching cell samples from the cultivation surface. Successful vitrification of bulk quantities of hESCs without direct liquid nitrogen contact is an important step towards automated cryopreservation processes for clinical applications of stem cells and other colony forming cell types.

In vivo measurement of shoulder joint loads during walking with crutches.

BACKGROUND: Following surgery or injury of the lower limbs, the use of walking aids like crutches can cause high loads on the shoulder joint. These loads have been calculated so far with computer models but with strongly varying results. METHODS: Shoulder joint forces and moments were measured during crutch-assisted walking with complete and partial unloading of the lower limbs. Using telemeterized implants in 6 subjects axillary crutches and forearm crutches were compared. A force direction was more in the direction of the long humeral axis, and slightly lower forces were assumed using axillary crutches. Similar force magnitudes as those experienced during previously measured wheelchair weight relief tasks were expected for complete unloading. The friction-induced moment was hypothesized to act mainly around the medio-lateral axis during the swing phase of the body. FINDINGS: Maximum loads of up 170% of the bodyweight and 0.8% of the bodyweight times meter were measured with large variations among the patients. Higher forces were found in most of the patients using forearm crutches. The hypothesized predominant moment around the medio-lateral axis was only found in some patients. More often, the other two moments had larger magnitudes with the highest values in female patients. The assumed different load direction could only be found during partial unloading. INTERPRETATION: In general the force magnitudes were in the range of activities of daily living. However, the number of repetitions during long-lasting crutch use could lead to shoulder problems as a long-term consequence. The slightly lower forces with axillary crutches could be caused by loads acting directly from the crutch on the scapula, thus bypassing the glenohumeral joint. The higher bending moments in the female patients could be a sign of lacking muscle strength for centring the humeral head on the glenoid.

Effective surface-based cryopreservation of human embryonic stem cells by vitrification.

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing. Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.

Measurement of shoulder joint loads during wheelchair propulsion measured in vivo.

BACKGROUND: Recent in vivo measurements show that the loads acting in the glenohumeral joint are high even during activities of daily living. Wheelchair users are frequently affected by shoulder problems. With previous musculoskeletal shoulder models, shoulder joint loading was mostly calculated during well-defined activities like forward flexion or abduction. For complex movements of everyday living or wheelchair propulsion, the reported loads vary considerably. METHODS: Shoulder joint forces and moments were measured with telemeterized implants in 6 subjects. Data were captured on a treadmill at defined speeds and inclinations. Additional measurements were taken in 1 subject when lifting the body from the wheelchair, using his arms only, and in 2 subjects when rapidly accelerating and stopping the wheelchair. The influence of the floor material on shoulder joint loading was accessed in 2 subjects. In general, the maximum shoulder loads did not exceed those during daily living but the time courses and magnitudes of the loads intra-individually varied much. FINDINGS: The highest forces acted during maximum acceleration and lifting from the wheelchair (128% and 188% of body weight). Grass was the only surface which led to a general load increase, compared to a smooth floor. INTERPRETATION: The increased incidence of overuse injuries in wheelchair users are probably not caused by excessive load magnitudes during regular propulsion. The high number of repetitions is assumed to be more decisive.

In vivo gleno-humeral joint loads during forward flexion and abduction.

To improve design and preclinical test scenarios of shoulder joint implants as well as computer-based musculoskeletal models, a precise knowledge of realistic loads acting in vivo is necessary. Such data are also helpful to optimize physiotherapy after joint replacement and fractures. This is the first study that presents forces and moments measured in vivo in the gleno-humeral joint of 6 patients during forward flexion and abduction of the straight arm. The peak forces and, even more, the maximum moments varied inter-individually to a considerable extent. Forces of up to 238%BW (percent of body weight) and moments up to 1.74%BWm were determined. For elevation angles of less than 90° the forces agreed with many previous model-based calculations. At higher elevation angles, however, the measured loads still rose in contrast to the analytical results. When the exercises were performed at a higher speed, the peak forces decreased. The force directions relative to the humerus remained quite constant throughout the whole motion. Large moments in the joint indicate that friction in shoulder implants is high if the glenoid is not replaced. A friction coefficient of 0.1-0.2 seems to be realistic in these cases.

Loading of the knee joint during activities of daily living measured in vivo in five subjects.

Detailed knowledge about loading of the knee joint is essential for preclinical testing of implants, validation of musculoskeletal models and biomechanical understanding of the knee joint. The contact forces and moments acting on the tibial component were therefore measured in 5 subjects in vivo by an instrumented knee implant during various activities of daily living. Average peak resultant forces, in percent of body weight, were highest during stair descending (346% BW), followed by stair ascending (316% BW), level walking (261% BW), one legged stance (259% BW), knee bending (253% BW), standing up (246% BW), sitting down (225% BW) and two legged stance (107% BW). Peak shear forces were about 10-20 times smaller than the axial force. Resultant forces acted almost vertically on the tibial plateau even during high flexion. Highest moments acted in the frontal plane with a typical peak to peak range -2.91% BWm (adduction moment) to 1.61% BWm (abduction moment) throughout all activities. Peak flexion/extension moments ranged between -0.44% BWm (extension moment) and 3.16% BWm (flexion moment). Peak external/internal torques lay between -1.1% BWm (internal torque) and 0.53% BWm (external torque). The knee joint is highly loaded during daily life. In general, resultant contact forces during dynamic activities were lower than the ones predicted by many mathematical models, but lay in a similar range as measured in vivo by others. Some of the observed load components were much higher than those currently applied when testing knee implants.

In vivo measurement of shoulder joint loads during activities of daily living.

Until recently the contact loads acting in the glenohumeral joint have been calculated using musculoskeletal models or measured in vitro. Now, contact forces and moments are measured in vivo using telemeterized shoulder implants. Mean total contact forces from four patients during eight activities of daily living are reported here. Lifting a coffee pot (1.5kg) with straight arm caused an average force of 105.0%BW (%body weight) (range: 90-124.6%BW), while setting down the coffee pot in the same position led to higher forces of 122.9%BW on the average (105.3-153.4%BW). The highest joint contact forces were measured when the straight arm was abducted or elevated by 90 degrees or more, with a weight in the hand. Lifting up 2kg from a board up to head height caused a contact force of 98.3%BW (93-103.6%BW); again, setting it down on the board led to higher forces of 131.5%BW (118.8-144.1%BW). In contrast to previously calculated high loads, the contact force during passive holding of a 10kg weight laterally was only 12.3%BW (9.2-17.9%BW), but when lifting it up to belt height it increased to 91.5%BW (87-95%BW). The moments transferred inside the joint at our patients varied much more than did the forces both inter and intra-individually. Our data suggest that patients with shoulder problems or during the first post-operative weeks after shoulder fractures or joint replacements should avoid certain activities encountered during daily living e.g. lifting or holding a weight with an outstretched arm. Some energy-related optimization criteria used in the literature for analytical musculoskeletal shoulder models must now be reconsidered.

Total dura substitute in the spine: double layer dural substitute made from polylactide layer and bovine pericardium.

When there is significant loss of spinal dura mater, dural substitution with synthetic or allogenic materials is essential. In the case of laminectomy, mechanical protection and reformation of the dorsal spinal canal may be useful. This is a report on a patient with total dura loss through tumour atrophy of the dura and laminae. In order to reconstruct the dorsal face of the spinal canal a polylactide sheet was cut and shaped to fit the physiological contour. A bovine dura substitute was firmly attached and sutured to the inner surface of the polylactide shield. The implant was wedged in between the pedicles and the facet joints and resulted in a water-tight dura substitute maintaining the shape of the spinal canal and protecting it against mechanical forces and intradural scar formation.

[Is the application of CAD useful for radiologists for passing the official tests required for outpatient breast imaging?]. / Inwieweit verbessert der Einsatz eines CAD-Systems das Prüfungsergebnis bei der Beurteilung einer kassenärztlichen Mammographiefallsammlung?

PURPOSE: To analyze whether currently available CAD systems meet the diagnostic requirements for passing screening tests (first reader CAD) and to analyze whether the additional usage of CAD systems provides significant support for the diagnosing radiologist on the basis of official screening test cases (second reading by CAD). MATERIAL AND METHODS: 200 images of 100 mammographies of 50 patients of an official screening test case collection were analyzed double-blind with and without CAD printouts (iCAD, USA ) by three radiologists: one experienced in breast analysis and CAD application, one experienced in mammography analysis but inexperienced in CAD usage, one with minimal experience with breast analysis and CAD application. All radiologists measured the largest diameter of any malignant mass. The mean value of these calculations was correlated to the largest diameter given by CAD prompts. RESULTS: The mean sensitivity and specificity increased slightly as a result of the additional usage of CAD (1 and 0.6 %, resp.). Both values are not statistically significant. The highest effect was measured for the radiologist with CAD experience, while no effect was measured for the inexperienced radiologist. CAD met the sensitivity requirements but not the specificity criteria (96 and 20.3 %, resp.). The sizes given by CAD prompts corresponded significantly with the real sizes (r = 0.45, p < 0.05). CONCLUSION: A statistically significant effect of the use of CAD could not been detected dependent on the radiological experience or the CAD experience. The effect of CAD on radiologists is still subtle despite the highly sensitive CAD performance due to the high number of false positive prompts and thus does not reach statistical significance. Sizes given by CAD prompts correlate significantly with the real sizes of malignant lesions.

Auto fluorescence of intervertebral disc tissue: a new diagnostic tool.

The paper reports on auto fluorescence phenomena of inter-vertebral human discs. It systematically investigates the auto fluorescence effects of ex vivo disc specimen and reports on surgical cases to demonstrate the potential value of the new method. The paper offers biologic explanations of the phenomenon and discusses the potential value of the UV auto fluorescence technique as a diagnostic tool. Intra- and postoperative observations are made by a surgical microscope with an integrated UV light source. Quantitative measurements were carried out using a photon counter and a spectrometer ex vivo. The auto fluorescence phenomenon allows the differentiation of traumatized and degenerated disc tissue intraoperatively in some cases, it allows the differentiation of bony and collagen endplate in cervical disc surgery. The source of the auto fluorescent light emission are amino acids of the collagen molecules. The proteoglycan components and the liquid components of the disc do not show relevant auto fluorescence. Emission wavelength of disc material is equivalent to color perception. It differs due to different collagen composition of the intervertebral disc components from yellow-green to blue-green and can be visualized in situ by naked eye.UV-auto fluorescence of inter-vertebral discs is a new clinical tool that has the potential to differentiate disc material from the anatomical surrounding, to distinguish between different fractions of the disc and to give information on the quality and status of the disc material. Since the technology has just emerged, it needs further investigations to quantify the clinical observations reported in this paper.

Cortical venous aneurysm isolated cerebral varix.

SUMMARY: Venous aneurysms so-called isolated cerebral varix, are known as a related pathology in arteriovenous malformations (AVM) due to the arterial pressure on venous drainage (16). They are also observed in combination with developmental venous anomalies (DVA) (2,4,8,15). However, isolated varix is a rare entity (1,7,11,13). They appear in most cases without neurological deficits. Some of the cases mimic a meningioma due to their manifestation in CT and MR imaging and their axial cortical localization. The case presented here is a isolated varix of a cortical vein located rostral to the motor strip. The patient was operated on successfully. The MRI and the histology of the case are presented.

[Emergency situations in the North Caucasus and role of specific immunoprophylaxis in eradication and prevention of epidemiological consequences]. / Chrezvichainye situatsii na Severnom Kavkaze i rol' spetsificheskoi immunoprofilaktiki v likvidatsii i preduprezhdenii ikh épidemiologicheskikh posledstvii.

Information on emergency situations (ES) on the territory of the North Caucasus from the 90s of the last century till the present moment is presented. The importance of the specific immunoprophylaxis of infections for the liquidation of epidemiological aggravations in ES and for their prevention is shown. Information on the scope of specific immunoprophylaxis in the zones of ES and on different approaches to its carrying out. The conclusion was made on the expediency of making corrections in the normative documents of the Ministry of Health of the Russian Federation, regulating planned vaccinations and vaccinations made on epidemic indications in connection with different kinds of ES.

[Current state of epidemiologic surveillance of natural foci of plague of North Caucasus]. / Sovremennoe sostoianie épidemiologicheskogo nadzora v prirodnykh ochagakh chumy Severnogo Kavkaza.

Information on the epizootic situation in plague in the natural foci of North Caucasus and on the influence of a number of anthropogenic and natural factors on this situation is presented. The data given in this work indicate that under the conditions of the anthropogenic transformation of landscapes the character of the epizootic manifestations of plague is changed and new factors, capable of aggravating epidemiological situation, appear. In addition, some other factors must be considered, such as the insufficient financing of reliable field surveys at present, the impossibility of making reliable epizootological studies due to causes of the social character (armed conflicts), thus making it impossible to evaluate, with a sufficient degree of reliability, the real epizootic state of a number of territories and, therefore, the risk of human infection. In this connection the necessity to carefully plan prophylactic measures and measures aimed at the localization and liquidation of the probable foci of infection arises.

Temporary cardiac asystolia induced by intraoperative irritation of the eighth right sided anterior cervical nerve root.

BACKGROUND: The 60 year old women with no prior history of arrhythmia or other cardiac symptoms was operated on for a cervical disc herniation at the level C7/D1. The C8 nerve root was visualized via a posterior approach. FINDINGS: Removal of the sequestrum and irritation of the anterior root with surgical instruments triggered cardiac arrest. INTERPRETATION: We offer the opinion that irritation of the anterior root led to diminished activity of the supraspinal sympathetic control system and consecutive hyper-activation of the parasympathetic system.

Oxidative stress in chronic lymphoedema.

BACKGROUND: Chronic lymphoedema is one of the most frequent and debilitating complications after surgical and radiological tumour treatment. Prevention and therapy of lymphoedema is therefore an important problem of the rehabilitation of those patients. AIM: To investigate whether chronic lymphoedema results in increased oxidative stress. DESIGN: Prospective case-control study. METHODS: We obtained venous blood samples from patients (n=38) with chronic lymphoedema and determined biomarkers of prooxidative reactions and of antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Healthy volunteers (n=90) and patients who had undergone surgical and/or radiotherapeutic treatment of tumours without consequent lymphoedema (n=20) acted as controls. RESULTS: The blood of patients with chronic lymphoedema contained lower concentrations of GSH and higher levels of GSSG and of MDA and HNE, compared with the control group. MDA was increased by about three-fold in the serum of the lymphoedema patients. Accelerated free radical formation and lipid peroxidation processes were further demonstrated by the liberation of MDA and HNE into the blood serum after manual lymph drainage. DISCUSSION: Our data demonstrate enhanced formation of reactive oxygen species (ROS) and accelerated lipid peroxidation processes in chronic lymphoedematous tissue. The strengthening of antioxidative defense mechanisms could be useful in the therapy of chronic lymphoedema.