Comparative stability of sodium tetradecyl sulphate (STD) and polidocanol foam: impact on vein damage in an in-vitro model.

OBJECTIVES: To compare the half-life of STD and polidocanol air-based foams and the damage they inflict upon human great saphenous vein in an in-vitro model. METHODS: The time for the volume of 3% STD and polidocanol foams to reduce by 10% (T(90)) and 50% (T(50)) was recorded in an incubator at 37 °C. Segments of proximal GSV harvested during varicose vein surgery were filled with foam for 5 or 15 min. Histological analysis determined percentage endothelial cell loss and depth of media injury. RESULTS: Median (±IQR) T(90) and T(50) for polidocanol were 123.3 s (111.7-165.6) and 266.3 s (245.6-383.1) versus 102.03 s (91.1-112) and 213.13 s (201-231.6) for STD (T(90)p = 0.008, T(50)p = 0.004). Median endothelial loss with polidocanol was; 63.5% (62.2-82.8) and 85.9% (83.8-92.5) versus 86.3% (84.8-93.7) and 97.64% (97.3-97.8) for STD after 5 and 15 min (p = 0.076 and p = 0.009). The median depth and % media thickness injured were 0 µm (0-0 µm) and 0% for both assessments with polidocanol versus 37.4 µm (35.3-45.8 and 43.4 µm (42.1-46.7) and 3.5% (3.1-3.6) and 5.3% (3.7-6.0) after 5 and 15 min for STD (p < 0.01 for all comparisons). CONCLUSION: Although polidocanol foam shows greater stability than STD foam perhaps remaining in the vein for longer, endothelial cell loss and damage to the media were significantly greater with STD.

Incidence and costs of severe hypoglycaemia requiring attendance by the emergency medical services in South Central England.

AIMS: The aim was to estimate the incidence of severe hypoglycaemia requiring emergency ambulance assistance, its management and associated costs. METHODS: A retrospective observational study used routinely collected data for a 1-year period from December 2009 to November 2010 from the South Central Ambulance Service National Health Service Trust, UK. The main outcome was episodes reported by ambulance personnel and costs were estimated from published data. RESULTS: During the 1-year study period, 398,409 emergency calls were received, of which 4081 (1.02%) were coded as hypoglycaemia. The overall numbers (and annual rate) of hypoglycaemia recorded among people ≥ 15 years with presumed diabetes was 3962 (2.1%), but for those aged 15-35 years was 516 (7.5%) and for those aged ≥ 65 years was 1886 (1.9%). Of those attended, 1441 (35.3%) were taken to hospital. The estimated total cost of initial ambulance attendance and treatment at scene was £553,000; if transport to hospital was necessary, the additional ambulance costs were £223,000 plus emergency department costs of £140,000; and the cost of primary care follow-up was estimated as £61,000. The average cost per emergency call was £263. The estimated annual cost of emergency calls for severe hypoglycaemia is £13.6m for England. CONCLUSIONS: Our estimates suggest prevalence of severe hypoglycaemia attended by the emergency services is high in younger age groups and lower for older age groups, although the absolute numbers of severe events in older age groups contribute substantially to the overall costs of providing emergency assistance for hypoglycaemia.

Cryopreservation of porcine aortic heart valve leaflet-derived myofibroblasts.

Cryopreservation by freezing is usually employed for storage of allogeneic valves destined for clinical use; however, disruption of leaflet extracellular matrices by ice may occur. This study was performed to determine the effects of cryoprotectants employed in VS55 for ice-free, vitreous cryopreservation upon adherent porcine heart valve leaflet-derived myofibroblasts in culture. Low-passage myofibroblasts exhibiting strong actin and myosin staining were employed for these experiments. Three cryoprotectants, dimethylsulfoxide, formamide, and propanediol, were tested individually and in combination. Exposure experiments demonstrated that the individual cryoprotectants (0-5 M) were generally cytotoxic in a dose-dependent manner with little if any loss of cells as determined by measuring metabolic activity and DNA content. Exposure to formamide resulted in the greatest loss of cells and reduction in viability. Combination of the three cryoprotectants demonstrated that the cytotoxic effects of each cryoprotectant were not cumulative. Cell viability and DNA content were equivalent to dimethylsulfoxide and propanediol and higher than formamide alone over most of the 0-5 M dose-response curve. After cryopreservation by slow-rate freezing, the benefits of the combination of cryoprotectants over individual cryoprotectants were demonstrated at the 4 M concentration range for both cell viability and cell retention. In conclusion, these studies demonstrated that, with the exception of the lower concentrations of propanediol, the combination of cryoprotectants employed in these studies result in equivalent or better cell viability and attachment than individual cryoprotectants.

Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Preservation of embryonic kidneys for transplantation.

BACKGROUND: Long-term storage of embryonic kidneys is crucial for the organization of transplantation and organ banking. In this study, we investigated the effects of controlled-rate freezing and ice-free vitrification on metanephroi (MN) viability. METHODS: Metanephroi isolated from 15-day (E15) timed pregnant Lewis rats were either: (i) frozen, using a DMSO/FCS/RPMI solution and a controlled freezing rate of -0.3 degrees C/min, from -10 degrees to -40 degrees C; or (ii) cryopreserved in an ice-free state by rapid cooling to -100 degrees C in cryoprotectant (VS55), followed by vitrification to -120 degrees C. After cryopreservation, the metanephroi were stored at -135 degrees C for 48 hours. After storage the MN were rewarmed, resuspended in culture media, and their viability was assessed using the AlamarBlue assay and histology (light microscopy, TEM, and cryosubstitution). RESULTS: There was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved MN groups relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing, yet in contrast the amount of ice was significantly reduced by vitrification. This was confirmed by TEM, where vacuolation of the cytoplasm of controlled-rate frozen metanephroi was observed, whereas vitrified metanephroi had little cytoplasmic disruption. However, vitrified metanephroi showed mitochondrial and nuclear injury at the cellular level. CONCLUSIONS: There is a need for long-term storage of organs to make MN transplantation a reality. This study demonstrates that standard freezing methods are unsuitable for this purpose. Vitrification yielded more promising results, but further development is required.

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.

Enhanced tissue strength in cryopreserved, collagen-based blood vessel constructs.

Traditional cryopreservation methods do not adequately preserve complex natural or engineered multicellular tissues due to the ice formation in the extracellular matrices. Vitrification is an alternate ice-free method for cryopreservation. This study compares the effects of vitrification and conventional cryopreservation on an engineered blood vessel construct. Collagen-based vascular constructs were used as models in this study. Tubular constructs were cut into rings and distributed into fresh, frozen, and vitrified groups for evaluation of mechanical properties and cell viability. Passive mechanical tests revealed enhanced tissue strength after both freezing and vitrification. Cryosubstitution studies of frozen and vitrified constructs revealed negligible ice in the vitrified specimens and extensive ice formation in the extracellular matrix of frozen specimens. Morphological changes associated with ice formation were visible within tissues preserved using traditional cryopreservation but not in tissue preserved using vitrification. The metabolic assay results indicated that vitrified tissue had similar viability to fresh controls. These results suggest that the increased tissue strength after cryopreservation may relate to thermal property change during preservation that cross-link collagen in tissue-engineered blood vessels. Further development of this cryopreservation method is necessary to minimize the alteration in material property and maintain cell viability of the constructs.

Patient-controlled analgesia and postoperative nausea and vomiting: efficacy of a continuous infusion of ondansetron.

A continuous infusion of ondansetron was compared with a placebo infusion in 80 patients undergoing major breast reconstructive surgery. All patients received a standard anaesthetic and a bolus dose of ondansetron after induction. They were then randomly allocated to receive an intravenous infusion of ondansetron or a placebo infusion for 24 h in a double-blind fashion. Postoperative analgesia was provided by patient-controlled subcutaneous diamorphine. In the ondansetron group, the severity of nausea, measured by a 10-point verbal rating scale, was reduced (p = 0.01) and fewer patients stated at postoperative interview that nausea and vomiting was a problem (p = 0.01).

Evaluation of a xenogeneic acellular collagen matrix as a small-diameter vascular graft in dogs--preliminary observations.

Autogenous veins are the materials of choice for arterial reconstruction. In the absence of autogenous material, prosthetic materials are used. However, vascular prostheses of less than 0.4 cm in diameter have low long-term patency. This study was designed to determine if cells would infiltrate an engineered xenogeneic biomaterial used as a small diameter arterial graft in dogs and, if so, to determine the phenotype of the infiltrating cells. Nine acellular xenogeneic grafts (0.4 cm in diameter, 5 cm long), composed of porcine collagen derived from the submucosa of the small intestine and type I bovine collagen, were implanted as end to-end interposition grafts in femoral arteries of five male mongrel dogs (total of nine grafts). All dogs received daily aspirin (325 mg). Patency of implanted grafts was monitored weekly by Duplex ultrasonography. After 9 weeks, or earlier in case of blood flow reduction by at least 75%, grafts were explanted and prepared for light or electron microscopy to evaluate cellularization. Eight of nine grafts remained patent up to 9 weeks. At explant, diameters were 0.31 +/- 0.02 cm at the midgraft, and 0.14 +/- 0.01 and 0.19 +/- 0.01 cm at the proximal and distal anastomoses. At explant, cells of mesenchymal origin (endothelial cells, smooth muscle cells, myofibroblasts) were embedded in the extracellular matrix of the graft scaffold. Minimal evidence of cellular inflammatory reaction and no aneurysmal dilatation or thrombus formation was detected. Variable degrees of hyperplasia were present at proximal and distal anastomoses. This preliminary study demonstrates that a collagen-based xenogeneic biomaterial provides a scaffold for cellularization when used for arterial reconstruction in dogs.

In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts.

Conventionally cryopreserved vascular grafts have performed poorly as arterial grafts. One possible mechanism that causes the poor function is the extracellular ice damage in tissue. We used a novel new ice-free cryopreservation (namely, vitrification) method for prevention of ice formation in cryopreserved venous grafts. This study was designed to evaluate the in vivo effects of the vitrification process on autologous vascular grafts using a short-term transplantation model and to examine the morphology and patency of vitrified grafts in correlation with control grafts. New Zealand White rabbits underwent a right common carotid interposition bypass graft. Fresh and vitrified reversed ipsilateral external jugular veins were used as autologous grafts. Animals were sacrificed at either 2 or 4 weeks after implantation, and fresh and vitrified vein grafts were harvested for histology studies. The results, comparing the patency of fresh and vitrified grafts, demonstrated similar short-term patency rates (approximately 90%). There were no signs of media disruption, aneurysm, or graft stenosis in vitrified vein grafts. Vitrification had not altered the pathophysiological cascade of events that occur when a vein graft is inserted into the arterial system. The vitrification process had no adverse effects locally or systemically in vivo. In addition, vitrification has preserved endothelial cell and smooth muscle cell integrity posttransplantation. In conclusion, this study, using an autologous animal model, clearly demonstrated a significant benefit of vitrification for preservation of graft function, and vitrification may be an acceptable approach for preservation of blood vessels or engineered tissue constructs.

Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo.

BACKGROUND AND AIM OF THE STUDY: Cryopreserved valve homografts often fail in infants. Controversies are ongoing concerning the relative contributions of cryopreservation variables, immune responses, cellular viability, and durability of the extracellular matrix to the mode of tissue failure. METHODS: Tissues to be examined for patterns of ice crystal distribution were cryopreserved. Tissue water was substituted with methanol and the tissues cryopreserved conventionally using dimethylsulfoxide, after which they were warmed and processed for light or electron microscopy. Selected specimens were vitrified to prevent ice crystal formation, cryopreserved, and subsequently warmed and processed for light and electron microscopy. RESULTS: Cryosubstitution of conventionally cryopreserved heart valves, while still frozen, demonstrated extensive extracellular ice formation, with smaller crystals in the ventricularis than in either the spongiosa or fibrosa. Extracellular ice formation was prevented by vitrification, a process in which the biological system is stabilized as an amorphous solid in the absence of crystalline ice. CONCLUSION: It is proposed that the extensive ice formation observed in conventionally cryopreserved heart valves may cause extracellular matrix damage that predisposes the valves to calcification. Future studies will assess the above hypothesis by comparison of conventional and ice-free (vitrification) cryopreservation methods in animal models of calcification.

Vitreous cryopreservation maintains the function of vascular grafts.

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.

Remodeling of an acellular collagen graft into a physiologically responsive neovessel.

Surgical treatment of vascular disease has become common, creating the need for a readily available, small-diameter vascular graft. However, the use of synthetic materials is limited to grafts larger than 5-6 mm because of the frequency of occlusion observed with smaller-diameter prosthetics. An alternative to synthetic materials would be a biomaterial that could be used in the design of a tissue-engineered graft. We demonstrate that a small-diameter (4 mm) graft constructed from a collagen biomaterial derived from the submucosa of the small intestine and type I bovine collagen has the potential to integrate into the host tissue and provide a scaffold for remodeling into a functional blood vessel. The results obtained using a rabbit arterial bypass model have shown excellent hemostasis and patency. Furthermore, within three months after implantation, the collagen grafts were remodeled into cellularized vessels that exhibited physiological activity in response to vasoactive agents.

Improving patients' postoperative sleep: a randomized control study comparing subcutaneous with intravenous patient-controlled analgesia.

One hundred female patients undergoing major reconstructive plastic or gynaecological surgery were randomized to either receive subcutaneous patient-controlled analgesia (PCA) (bolus dose 2.5 mg diamorphine in 1 ml with a 20-minute lockout) or intravenous PCA (bolus dose 0.5 mg diamorphine in 1 ml with a 5-minute lockout). Data were collected by questionnaire and interview to evaluate the intervention on pain scores, quality of sleep on the first postoperative night, postoperative nausea and vomiting (PONV) and overall patient acceptability. The subcutaneous PCA group experienced less 'worse pain' (P < 0.01) and less sleep disturbance due to pain (P < 0.001). Subcutaneous PCA would appear to offer patients a safe and effective means of analgesia and may offer significant advantages over the intravenous route of administration.

Increased platelet aggregation due to chilling to 20 degrees C is not related to increased sensitivity to agonists.

BACKGROUND: Previous studies suggested that chilled platelets have a greater sensitivity to agonists than do platelets aggregated at 37 degrees C. STUDY DESIGN AND METHODS: Washed platelets were aggregated at 20 or 37 degrees C with ADP (0-20 microM), arachidonic acid (0-200 microM), or the thromboxane mimetic U46619 (0-9 nM). RESULTS: Chilling caused a significant (p < or = 0.05) increase in spontaneous platelet aggregation (> 27% at 20 degrees C vs < 5% at 37 degrees C) and spontaneous dense granule release (> 0.01 nM of ATP at 20 degrees C vs. 0 nM of ATP at 37 degrees C), ADP and U46619 caused a significantly greater aggregation response and dense granule release at 20 degrees C, although there was no change in agonist sensitivity of platelets (effective dose of agonist necessary to induce 50% aggregation [ED50]: 1.00 +/- 0.35 microM ADP at 20 degrees C and 1.63 +/- 0.47 microM ADP at 37 degrees C; 8.26 +/- 3.65 pM U46619 at 20 degrees C and 18.97 +/- 4.82 pM U46619 at 37 degrees C). Platelets aggregated with arachidonic acid showed a significant decrease in aggregation and agonist sensitivity at 20 degrees C (ED50 118.7 +/- 44.4 microM) from those at 37 degrees C (ED50 25.6 +/- 7.2 microM), possibly as a result of the reduced enzymatic activity of cyclooxygenase and thromboxane synthase at the lower temperature. CONCLUSION: The data suggested that washed platelets chilled to 20 degrees C and aggregated are not more sensitive to agonists than are 37 degrees C controls, but rather that chilled platelets undergo greater spontaneous aggregation.