The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration.

Spinal cord injury is very complicated, as there are factors in the body that inhibit its repair. Although regeneration of the mammalian central nervous system (CNS) was once thought to be impossible, studies over the past two decades have shown that axonal growth after spinal cord injury can occur when provided with the correct substratum. Traditionally, tissue transplantation or peripheral nerve grafting are used to repair damaged or diseased regions of the CNS, but donor shortage and immunological problems associated with infectious disease are often encountered. Fortunately, recent advances in neuroscience, cell culture, and biomaterials provide optimistic future using new treatments for nerve injuries. Biomaterial scaffold creates substrate within which cells are instructed to form a tissue or an organ in a highly controlled way. The principal function of a scaffold is to direct cell behavior such as migration, proliferation, differentiation, maintenance of phenotype, and apoptosis by facilitating sensing and responding to the environment via cell-matrix and cell-cell communications. Therefore, having such abilities provides scaffolds seeded with a special type of cell as an important part of tissue engineering and regenerative medicine which spinal cord regeneration is an example of. Nevertheless, the vast number of biodegradable synthetic and natural biopolymers makes choosing the right one very difficult. In this review article, it was tried to provide an inclusive survey of biopolymers seeded with Schwann cells (SCs) to be used for axonal regeneration in the nervous system.

A new mathematical approach to modelling thrombin generation.

The plasma coagulation system is a biochemical chain reaction where inactive proenzymes are converted to active enzymes in a cascade pattern. One of the problems encountered in the modelling of thrombin generation in plasma is that neither the reaction mechanism nor the reaction constants and initial concentrations are precisely known. Therefore, these quantities are taken as unknown parameters in the theoretical model and are estimated by fitting experimental data. In the literature there are two different mathematical models for approaching a part of the blood coagulation mechanism. Both models comprise a stiff system of non-linear differential equations. We aimed to analyze both systems after linearization, to steer or influence the system and to calculate the period of time for it to attain a final equilibrium as a basis for model extension.

Effect of hirudin versus heparin on hemocompatibility of blood contacting biomaterials: an in vitro study.

Hirudin serves as an alternative anticoagulant for extracorporeal blood circulation. Comparing anticoagulation with hirudin (2.5 or 5.0 microg/mL) and heparin (2.0 or 4.0 IU/mL) human blood was circulated in a modified 'Chandler System' using PVC-tubes for 2 hours at 37 degrees C. Activation of coagulation (thrombin-antithrombin III-complex, prothrombin fragment 1+2 and D-Dimer), platelet (platelet factor 4 - PF4) and complement systems was analyzed. Both heparin concentrations and 5.0 microg/dL hirudin led to as significantly less activated plasmatic coagulation as 2.5 microg/dL hirudin. Decreased levels of PF4 and anaphylatoxin C5a (p<0.05) as well as terminal complement complex demonstrated improved hemocompatibility after anticoagulation with heparin in contrast to hirudin. Because initial coagulation cascade, platelet activation and complement activation is less influenced by hirudin than by heparin, hemocompatibility is more dependent on the characteristics of the biomaterials used. This predestines hirudin as anticoagulant for in vitro studies analyzing hemocompatibility of biomaterials or surface modifications.

Application of Taylor vortices in hemocompatibility investigations.

BACKGROUND: Artificial organs, implants and extracorporeal circulation affect the physiological flow characteristics of blood as a liquid organ. These artificial systems consist of a wide variety of biomaterials with different geometries and, therefore, with their own flow properties. Secondary flow also occurs in extra--as well as in intracorporeal circulation. METHODS: In order to investigate the influence of vortical flow conditions a modified Taylor-Couette system was introduced. It consisted of two coaxial cylinders whose surfaces were the target of investigation. The annular gap was filled with donor blood shear and secondary flows were produced by rotating the inner cylinder. Platelet activation and protein adsorption were investigated as markers for thrombogenicity. RESULTS: At shear rates high enough to establish stable Taylor vortices (G > or = 550 s(-1)) significant differences between vortical Taylor flow and steady laminar flow were detected. At shear rates of G > or = 550 s(-1) laminar flow caused a significantly higher platelet drop and PF4 release when compared to Taylor vortex flow. Also protein adsorption per square unit was significantly higher for laminar flow. CONCLUSIONS: Based on the present data we conclude that vortical flow patterns lead to an accumulation of platelets and plasma proteins in the vortex center and therefore to a decreased probability of contact between platelets and material surfaces. It can be concluded that a preactivation of the platelets circulating in extracorporeal circuits can be manifested downstream in other geometrical configurations and flow conditions.

In vitro blood damage by high shear flow: human versus porcine blood.

Devices for modern heart support are minimized to reduce priming blood volume and contact area with foreign surfaces. Their flow fields are partly governed by very high velocity gradients. In order to investigate blood damage, porcine and human blood was passed through a narrow Couette type shear gap applying defined high shear rates within the typical range for devices such as blood pumps or artificial heart valves (gamma = 1800/s to 110,000/s for 400 ms). Traumatization profiles of both blood species were recorded in terms of hemolysis and platelet count. Sublethal damage in terms of platelet (PF4) and complement activation (C5a) was additionally measured for human blood. Results for porcine and human blood were very similar. Hemolysis was not started until critical shear rates of about 80,000/s. Impact on platelets was severe with drops in cell count of up to 65% (at gamma = 55,000/s to 110,000/s) likely to set stronger limits to the design layout of devices than hemolysis. Concentrations of PF4 and C5a clearly increased with shear rate exhibiting stronger gradients where hemolysis started. Due to the similar results of porcine and human blood for hemolysis and platelet drop, porcine blood seems to be suitable for device testing. Selection of blood species would thus depend on handling, availability and analysis demands.

Investigation on the ability of an ultrasound bubble detector to deliver size measurements of gaseous bubbles in fluid lines by using a glass bead model.

Detectors based on ultrasonic principles are today's state of the art devices to detect gaseous bubbles that may be present in extracorporeal circuits (ECC) for various reasons. Referring to theoretical considerations and other studies, it also seems possible to use this technology to measure the size of detected bubbles, thus offering the chance to evaluate their potential hazardous effect if introduced into a patient's circulation. Based on these considerations, a commercially available ultrasound bubble detector has been developed by Hatteland Instrumentering, Norway, to deliver bubble size measurements by means of supplementary software. This device consists of an ultrasound sensor that can be clamped onto the ECC tubing, and the necessary electronic equipment to amplify and rectify the received signals. It is supplemented by software that processes these signals and presents them as specific data. On the basis of our knowledge and experience with bubble detection by ultrasound technology, we believe it is particularly difficult to meet all the requirements for size measurements, especially if these are to be achieved by using a mathematical procedure rather than exact devices. Therefore, we tried to evaluate the quality of the offered bubble detector in measuring bubble sizes. After establishing a standardized test stand, including a roller pump and a temperature sensor, we performed several sets of experiments using the manufacturers software and a program specifically designed at our department for this purpose. The first set revealed that the manufacturer's recommended calibration material did not meet essential requirements as established by other authors. Having solved that problem, we could actually demonstrate that the ultrasonic field, as generated by the bubble detector, has been correctly calculated by the manufacturer. Simply, it is a field having the strongest reflecting region in the center, subsequently losing strength toward the ECC tubing's edge. The following set of experiments revealed that the supplementary software not only does not compensate for the ultrasonic field's inhomogeneity, but, furthermore, delivers results that are inappropriate to the applied calibration material. In the last set of experiments, we were able to demonstrate that the signals as recorded by the bubble detector heavily depend upon the circulating fluid's temperature, a fact that the manufacturer does not address. Therefore, it seems impossible to resolve all these sensor related problems by ever-increasing mathematical intervention. We believe it is more appropriate to develop a new kind of ultrasound device, free of these shortcomings. This seems to be particularly useful, because the problem of determining the size of gaseous bubbles in ECC is not yet solved.

C1 inhibitor prevents capillary leakage after thermal trauma.

OBJECTIVE: In burned patients, activation of the complement and clotting systems is suggested to play an important role in the development of the capillary leak syndrome and inflammatory tissue destruction. In an animal model of thermal trauma, the possible protective effect of C1 inhibitor (C1Inh), a major control protein of both the complement and clotting systems, was investigated. DESIGN: Prospective, controlled study. SETTING: Animal model. SUBJECTS: Healthy pigs weighing 30 kg. INTERVENTIONS: Pigs were scalded for 25 secs with 75 degrees C hot water to achieve a 30% total body surface deep partial-thickness burn. The treatment group (n = 8) received C1Inh concentrate at an initial dose of 100 units/kg body weight immediately after thermal trauma, followed by three further applications every 12 hrs. Two control groups included animals that were either scalded (n = 8) or not scalded (n = 7) and treated with lactated Ringer's solution. MEASUREMENTS: Before and at various time points after trauma blood samples were analyzed for complement activation (APH50, CH50, SC5b-9, C3). Continuous monitoring of hemodynamic variables was performed and postmortem histologic examination of specimens from lung, heart, liver, kidney, stomach, duodenum, jejunum, ileum, and colon was carried out. Aseptically collected mesenteric lymph nodes were pooled and screened for bacterial translocation. For evaluation of the burn wound, biopsies from defined scalded and not scalded areas were taken daily. As a measure for edema formation, the weight of the animals was recorded every 2 hrs. RESULTS: After C1Inh treatment, which led to a significantly reduced complement activation, the clinical outcome was clearly improved, as indicated by vital signs and as demonstrated by reduced edema formation. Treated animals presented a diminished bacterial translocation. Pathologic alterations were clearly diminished in the burned skin, in shock-related organs, and in the intestines. CONCLUSION: Application of C1Inh appears to be an effective means to prevent capillary leakage and inflammatory tissue destruction after thermal trauma.

In vitro validation of gastric air tonometry using perfluorocarbon FC 43 and 0.9% sodium chloride.

Monitoring splanchnic perfusion by means of gastric intramucosal tonometry is carried out using saline, or more recently, air tonometry using the Tonocap. The objective of this study was the validation of the Tonocap in saline and perfluorocarbon FC 43. The two methods underestimated the predefined PCO2 value by an average of 10%, with clinically acceptable precision. Accuracy of the Tonocap improved at high PCO2 values (9.33 and 9.94 kPa), whereas saline tonometry was superior at low PCO2 values (3.99 and 3.75 kPa). The Tonocap provides a fast and reliable estimation of the PCO2, and with the revised software requiring only 10 min of equilibration, will increase the comparability of future studies.

Scale-independent shape analysis for quantitative cytology using mathematical morphology.

A system for automatic quantification of morphological changes of cell lines, proposed for cytotoxicity tests of biomaterials, is presented. Light-micrographs of cultured cells are segmented by adaptive thresholding within a local adaptive window. Connected cells in binarized micrographs are separated by a novel morphological multiscale method, treating cells in their size-specific scale and hence resulting in scale-independent separations. Significant shape descriptors correlating well with cell toxicity are extracted from single cells. Size and compactness distributions turned out to be reliable and useful parameters, providing an alternative to the common subjective grading of shape deformations by visual inspection. The system is evaluated for several standardized toxical reference substances and is now in use for clinical biocompatibility testing.

Application of a new dynamic flow model for investigating the biocompatibility of modified surfaces.

An in vitro model was developed to compare the biocompatibility of four different coating methods (three heparin and one nonheparin) under hemodynamic conditions. Fresh human donor blood (heparin 5 IU/ml) was recirculated in a standardized experimental circuit. All circuit components were either coated or remained uncoated for control purposes. The aim of the study was to investigate a wide spectrum of effects on blood; coagulation parameters (e.g., fibrinogen, ATIII, thrombin-antithrombin-complex), complement parameters (C1rsC1 Inh, C3b(Bb)P, SC5b-9, C5a), differential blood analyses, platelet activation (flow cytometric investigations), PF 4, and PMN-elastase release were examined by showing possible trends. All heparin coated systems reduced platelet stimulation in comparison to untreated biomaterials. Leukocyte activation was reduced to different degrees depending upon the coating method used. Complement activation was markedly reduced by all coated systems. The results obtained indicate that the pump driven, dynamic blood flow model is suitable to characterize the biocompatibility of surface modified biomaterials. Advantages lie in the integration of the different polymers as parts of the circuit, the low priming volume, and the generation of blood flow conditions similar to those that occur in clinical applications.

A novel method for quantifying shape deformation applied to biocompatibility testing.

Cytotoxicity tests are important for the screening and evaluation of biocompatibility of artificial organs. Morphologic changes of cells that were contacted biomaterials or biomaterial extracts indicate their toxicity. However, information on cytotoxic effects is still obtained by subjective visual inspection of microscopic samples. In this article, a novel computer assisted method is introduced. The automatic analysis of digitized micrographs is achieved in several stages: segmentation, separation, classification, and measurement. The segmentation of the image is provided by a new local adaptive thresholding technique, which adapts the threshold window sizes onto local gray level distribution and yields optimal window sizes. The actual threshold is obtained by maximizing interclass variances and minimizing intraclass variance. For the separation of connected cells, the binarized samples are cleaned from "false" markers by morphologic filtering. The subsequent separation is a two phase approach. Information levels are generated top-down by successively applying an enhanced erosion operator, which yields markers and filters noise usually evolving from multiple erosions. The converse bottom-up integration of the eroded markers is performed by successively applying an enhanced dilation operator, which reconstructs the cells and prevents merging of already separated objects. The subsequent measuring provides quantitative parameters of the distribution of size and compactness of the cells contained within the sample. The method was evaluated by L-929 fibroblasts that were in contact with 0%, 5%, and 10% concentrations of ethanol. For each concentration, 268 images of the cell populations were captured. The obtained quantitative parameters are highly correlated to the common verbal description of morphologic changes. Therefore, the proposed automatic method has several advantages compared with subjective examinations. The results allow an objective comparison of the quantification of phenomena; the subjective influence of the observer is eliminated; and the laboratory staff is relieved of time consuming routine work.

The therapeutic effect of C1-inhibitor on gut-derived bacterial translocation after thermal injury.

To test the effects of C1-esterase inhibitor in scald burns on bacterial translocation and intestinal damage, standardized deep partial-thickness burns were inflicted on domestic pigs, scalding 30% of the skin surface for 25 s with 75 degrees C hot water. The animals (n = 17; weight 25-35 kg) were divided into three groups: I) the control group (n = 5) without scald burn; II) the group (n = 6) with scald burn; and III) the group with C1-inhibitor (n = 6): scald burn and treatment with C1-inhibitor (C1-INH; BERINERT, Behring, Marburg, Germany). Parameters measured and compared in this model were activity of complement system, hemodynamics, body weight, pathological organ alterations including intestinal lesions, bacterial translocation, and skin damage. C1-INH administration significantly decreased the plasma levels of the specific soluble membrane attack complex (SC5b-9), bacterial translocation, and the degree of intestinal ischemia in the postburn period compared with untreated animals. Moreover, animals treated with C1-INH exhibited a minor degree of organ alterations including damage of the skin and development of edema. The favorable effects of C1-INH may be explained by the protection of the intestinal and dermal microcirculation in the acute phase of thermal injury.

[Determining the effectiveness of percutaneous cava filters: experimental studies]. / Bestimmung der Effektivität perkutaner Kavafilter: Experimentelle Untersuchungen.

PURPOSE: In vitro clot-trapping capacity of 16 different caval filters should be evaluated under varying experimental conditions. MATERIAL AND METHODS: In a flow model simulating in vivo conditions (soft latex tube, dextran solution at 37 degrees C, pulsatile flow at a mean rate of 3 1/min) the efficiency of 16 caval filters was evaluated in horizontal and vertical position by using 640 or 1280 clots/filter (8 sizes). Non-self centering filters were tested in centric and in tilted position. RESULTS: Efficiency of optimally centered caval filters varied between 97.8 and 69.4%. The largest thrombi were captured by all optimal centered filters. A change from vertical to horizontal position of the flow model resulted in a variation of filter efficiency by about 4.8%. Efficiency of non-self centering filters decreased significantly when placed in a tilted position (mean decrease 15.5%; range 2.7%-37.7%) resulting in a deterioration of the capture rate by as much as 43.2%. CONCLUSION: Under optimal study conditions efficiency of all evaluated caval filters was high. Tilting of caval filters resulted in a significant efficiency decrease.

Extracorporeal circulation: in vivo and in vitro analysis of complement activation by heparin-bonded surfaces.

Complement activation was analyzed during extracorporeal CO2 removal to compare heparin-coated with standard surfaces where systemic heparinization was required. In vivo studies were performed in adult sheep for up to 5 days under standardized conditions using a capillary membrane oxygenator. Applying assays for hemolytic complement function (CH50, APH50) and C3-derived split products, we found that complement activation was markedly reduced in sheep connected to an extracorporeal circuit where heparin was covalently bound by end-point attachment. In addition, incubation of human serum in a miniaturized circulation system revealed less complement activation by heparin-bonded surfaces, as evaluated by enzyme-linked immunosorbent assays for C3a and the activation-specific protein-protein complexes, C1rsC1 inhibitor (classical pathway) and C3b(Bb)P (alternative pathway). Our results provide further evidence that biocompatibility can be improved by end-point attachment of heparin to the surfaces of the extracorporeal circuit.

Progress in veno-venous long-term bypass techniques for the treatment of ARDS. Controlled clinical trial with the heparin-coated bypass circuit.

Extracorporeal CO2 removal combined with low-frequency positive pressure ventilation (ECCO2-R LFPPV) is a new therapeutic approach in treatment of ARDS. The main problem during long-term extracorporeal support is anticoagulation and related bleeding problems. We conducted a prospective, randomized and controlled clinical trial in 18 patients to compare the effect of the non-heparin-coated (Scimed = group 1) with the heparin-coated (Carmeda = group 2) extracorporeal circuit on clinical course and complication rate. In group 2 the daily blood loss, the amount of substituted red cells and the i.v. heparin dose were significantly lower than in group 1. Bleeding complications were less and more patients survived in group 2. The disadvantage of the hollow fiber oxygenators in the heparin-coated system was plasma leakage, which was more frequent in patients with pancreatitis and hyperbilirubinemia.

In vivo demonstration of the Haldane effect during extracorporeal gas exchange.

During the extracorporeal support (LFPPV-ECCO2R) of 11 patients suffering from severe lung failure (ARDS), we consistently noticed a higher arterial than mixed-venous PCO2 in blood samples drawn at the same time. Two explanations are possible: a) the Haldane effect (HE), b) CO2 from lung tissue metabolism. In order to distinguish changes in PCO2 due to the HE from those due to tissue CO2 production, CO2 content (CCO2) was calculated. The results were compared to animal experiments with hyperoxic apnea, after which arterial and mixed-venous samples were drawn simultaneously. All blood gas samples were analyzed for pH, PCO2, PO2, and O2-saturation, from which CCO2 was calculated. In both groups, PaCO2 was 2.15 mmHg (2.7 mmHg respectively) higher at a lower CaCO2 (-2.87 ml/l, -14.9 ml/l). Oxygen saturation increased by 8.1% in the human group and 17.8% in the animal group. A significant relationship was found between changes in PCO2 and changes in O2-saturation. This is a demonstration of the Haldane effect.

Heparin-coated versus non-coated surfaces for extracorporeal circulation.

Studies were made to compare completely heparin-bonded (HBS) and conventional extracorporeal circulation surfaces using capillary membrane oxygenators (CMO) in sheep and dogs for up to five days. The aims were: to investigate the need for systemic heparinization in the case of heparin-coated surfaces, to assess blood compatibility and gas exchange performance of both systems and the extent of complement activation, and to find solutions for plasma leakage by the use of CMO. All studies were performed under standardized conditions, such as drugs, surgery, priming, blood flow rate etc. For heparin-coated surface studies all blood interfaces (CMO, catheters, tubes, etc) were coated. It was possible to eliminate systemic heparinization totally when HBS were used. During the five-day non-heparin application period blood coagulation parameters were almost unchanged and in the physiological range, platelets did not drop below 80%, hemolysis was negligible and gas exchange performance was unaffected. Less complement activation occurred with HBS than with non-coated surfaces.

Right ventricular function assessed by thermodilution technique during apnea and mechanical ventilation.

OBJECTIVES: To evaluate strategies for thermodilution-based measurement of cardiac output and right ventricular (RV) ejection fraction and to assess the effects of controlled mechanical ventilation in patients. Furthermore, to compare strategy-associated reproducibility with reference values obtained during long-term apnea. DESIGN: Crossover trial in patients; reference values from apneic animals. SETTING: University ICU and physiology laboratory. PATIENTS: Six consecutive male ICU patients (48 to 70 yrs) after major abdominal vascular surgery. ANIMALS: two adult female sheep. INTERVENTIONS: Three ventilatory rates (8, 16, and 24 cycles/min) and 15-sec periods of apnea were selected for measurements in patients. In animals, continuous apnea was achieved with extracorporeal CO2 removal and apneic oxygenation. MEASUREMENTS: Measurements were performed using an appropriate pulmonary artery catheter and an ejection fraction/cardiac output computer prototype. The thermal indicator was injected automatically at four defined points of the ventilatory cycle, but triggered manually during apnea. MAIN RESULTS: At 8 cycles/min, there was a wide mean range of cyclic variable modulation, with a coefficient of variation of 11.6% and 23.2% for cardiac output and RV ejection fraction, respectively. Allowing for ventilatory phase or changing from 8 to 16 cycles/min reduced errors by half. Combining both procedures resulted in a coefficient of variation of 4.7% and 6.6% for cardiac output and RV ejection fraction, respectively. The best coefficient of variation values obtained during 15 secs of apnea in patients approached those variations in experimental apnea (coefficient of variation of 2.1% and 4.5% for cardiac output and RV ejection fraction, respectively). CONCLUSIONS: At low ventilatory rates, best results are achieved by averaging four phase-selected measurements. One-point measurements were less accurate and random point measurements less reproducible.

Effect of long-term hypoxia on oxygen transport properties of blood in pregnant guinea pigs.

Pregnant guinea pigs undergoing long-term hypoxia were studied and the results compared with those of control animals (pregnant, but non-hypoxic). Hypoxic animals demonstrated a decrease of O2 affinity (-7%) and an increase of O2 capacity (+35%). In addition, the HCT was found to be higher in the hypoxic group (+41%), causing haemorheological disadvantages; in a shear model study the blood of hypoxic animals had to be exposed to the gas compartment of the rheo-oxymeter up to 62% longer than that of the control group. We have postulated, that this rheological impairement is compensated, since no abnormalities in number and abortion rate of fetuses (due to a possible O2 delivery impairment) were found. Our morphological studies in fact support this opinion, showing e.g. more capillary branchings and loops and a reduction of diffusion distances between maternal and fetal blood in hypoxic guinea pig placentae. The results emphasize the importance of more detailed rheological studies in connection with other investigations for a complete description of compensatory mechanisms.