In vivo characterisation of the inflammatory reaction following mesh implantation in transgenic mice models.

INTRODUCTION: Hernia repair with prosthetic meshes represents one of the most common surgical procedures in the field of surgery. This intervention is always associated with an ensuing inflammatory response, angiogenesis and fibrotic encapsulation forming a foreign body granuloma (FBG) around the mesh fibres. Several studies have described this inflammatory reaction by characterising inflammatory cell infiltrate around the FBG after mesh explantation. However, very little is known about the real-time progression of such an inflammatory response. The aim of this study was to investigate the feasibility of monitoring the ongoing inflammatory response to mesh implantation using bioluminescence in vivo. MATERIALS AND METHODS: Three luciferase transgenic mice strains (FVB/N-Tg(Vegfr2-luc)-Xen, BALB/C-Tg(NFκB-RE-luc)-Xen and Tg(INS/EpRE-Luc)T20Rbl) were used. Mice were anaesthetized with 2 % isoflurane, and two incisions were made on the left and right sides of the abdomen of the mice. A 1-cm(2) propylene mesh was implanted subcutaneously in the right incision wound of each mouse, and the left wound served as control. Two hundred microliters of D-luciferin was injected into the mice, and bioluminescence measurements were done prior to the surgical intervention and subsequently every 3 days. After mesh explantation, histological analysis was done. Statistical analysis was done using prism GraphPad software. RESULTS: Bioluminescence results revealed different time points of maximum signal for the different mice strains. VEGFR2 gene expression peaked on day 6, NFkB on day 12 and ARE on day 3 post mesh implantation. We also observed much higher bioluminescent signal around the FBG surrounding the mesh as compared to the control wound, with p < 0.05 for all the different mice strains. CONCLUSION: Our results prove the possibility of monitoring the inflammatory reaction after mesh implantation in vivo using bioluminescence signal release. This provides a novel method of accessing and accurately describing the ongoing inflammatory response over a given period of time.

A novel organ preservation for small partial liver transplantations in rats: venous systemic oxygen persufflation with nitric oxide gas.

The prognosis for recipients of small liver grafts is poor. The aim of this study was to determine the impact of venous systemic oxygen persufflation (VSOP) with nitric oxide (NO) gas for 30% partial liver preservation and transplantation in rats. After we determined optimal NO concentration as 40 ppm in vitro with the isolated perfused rat liver model, we assessed liver injury and regeneration in vivo at 1, 3, 24 and 168 h after transplantation in the following three groups after 3 h-cold storage (n = 20 per group): control group = static storage; VSOP group = oxygen persufflation and VSOP+NO group = oxygen with NO persufflation. The liver graft persufflation was achieved with medical gas via the suprahepatic vena cava; In comparison with control group after transplantation, VSOP+NO preservation (1) increased portal circulation, (2) reduced AST and ALT release, (3) upregulated hepatic endothelial NO synthase, (4) reduced hepatocyte and bileductule damage and (5) improved liver regeneration. These results suggest that gaseous oxygen with NO persufflation is a novel and safe preservation method for small partial liver grafts, not only alleviating graft injury but also improve liver regeneration after transplantation.

Meloxicam, a COX-2 inhibitor, ameliorates ischemia/reperfusion injury in non-heart-beating donor livers.

BACKGROUND/AIMS: Chronic organ donor shortage has led to the consideration to expand the donor pool with livers from non-heart-beating donors (NHBD), although a higher risk of graft dys- or nonfunction is associated with these livers. We examined the effects of selective cyclooxygenase-2 (COX-2) inhibition on hepatic warm ischemia (WI) reperfusion (I/R) injury of NHBD. METHODS: Male Wistar rats were used as donors and meloxicam (5 mg/kg body weight) was administered into the preservation solution. Livers were excised after 60 min of WI in situ, flushed and preserved for 24 h at 4°C. Reperfusion was carried out in vitro at a constant flow for 45 min. During reperfusion (5, 15, 30 and 45 min), enzyme release of alanine aminotransferase and glutamate lactate dehydrogenase were measured as well as portal venous pressure, bile production and oxygen consumption. The production of malondialdehyde was quantified and TUNEL staining was performed. Quantitative PCR analyzed COX-2 mRNA. COX-2 immunohistochemistry and TxB(2) detection completed the measurements. RESULTS: Meloxicam treatment led to better functional recovery concerning liver enzyme release, vascular resistance and metabolic activity over time in all animals. Oxidative stress and apoptosis were considerably reduced. CONCLUSION: Cold storage using meloxicam resulted in significantly better integrity and function of livers retrieved from NHBD. Selective COX-2 inhibition is a new therapeutic approach achieving improved preservation of grafts from NHBD.

Pulsatile perfusion preservation of warm ischaemia-damaged experimental kidney grafts.

BACKGROUND: Cold storage using histidine-tryptophan-ketoglutarate (HTK) solution is used widely in clinical practice for the preservation of warm ischaemia-damaged kidney grafts. This study assessed the efficacy of pulsatile machine perfusion in combination with Polysol for the preservation of warm ischaemia-damaged kidney grafts. METHODS: After induction of warm ischaemia by clamping of the left renal pedicle for 30 min, pigs were subjected to left nephrectomy. Thereafter, grafts were preserved for 20 h by cold storage with HTK (CS-HTK) or Polysol (CS-PS), or machine preservation with Polysol (MP-PS). Subsequently, contralateral kidneys were removed and preserved kidneys were transplanted. Control pigs underwent unilateral nephrectomy. Renal function was assessed daily for 1 week. Kidney biopsies were analysed for morphology and proliferative response. RESULTS: Renal function of warm ischaemia-damaged grafts preserved using MP-PS was comparable to that of non-ischaemic controls. MP-PS and CS-PS groups showed improved renal function compared with the CS-HTK group, with more favourable results for MP-PS than for CS-PS. The proliferative response of tubular cells in the CS-HTK group was higher than in all other groups. CONCLUSION: This study demonstrated that the function of warm ischaemia-damaged kidney grafts after pulsatile perfusion preservation was comparable to that of non-ischaemic controls.

Improved renal function of warm ischemically damaged kidneys using Polysol.

OBJECTIVE: We sought to assess the efficacy of POLYSOL, a low-viscosity, colloid-based organ preservation solution, for the preservation of warm ischemically damaged kidney grafts compared with histidine-tryptophane-ketoglutarate (HTK) solution. METHODS: Pigs (25-30 kg) underwent a left nephrectomy after clamping the renal vessels for 30 minutes. Kidney grafts washed out with Polysol (n = 6) or HTK (n = 6) were cold stored (CS) for 20 hours at 4 degrees C. After the preservation period, the contralateral kidney was removed and the preserved kidney implanted heterotopically. Renal function was assessed daily for 7 days. Thereafter, animals were killed and the kidney grafts removed for histologic analysis. RESULTS: All animals survived for 7 days. All Polysol CS-preserved grafts showed immediate function, as demonstrated by urine production within 24 hours after reperfusion as compared with 3/6 grafts in the HTK CS group. Overall, the Polysol CS group showed improved renal function compared with HTK CS. Also, peak serum creatinine and blood urea values were lower in the Polysol CS group compared with HTK-preserved grafts. Histologic evaluation of warm ischemically damaged grafts showed less glomerular shrinking, less tubular damage, less edema, less inflammatory infiltration, and less necrosis in Polysol compared with HTK-preserved grafts. CONCLUSION: Application of Polysol solution for washout and CS preservation of warm ischemically damaged kidney grafts resulted in improved renal function and structural integrity when compared with HTK.

Influence of temporary abdominal wall repair on the intestinal integrity: an experimental study in the rat.

BACKGROUND AND AIMS: The aim of this study was to analyze intestinal integrity after temporary abdominal wall repair with absorbable mesh. METHODS: Rats underwent abdominal wall repair with absorbable mesh or sham operation. Myeloperoxidase-positive cells in the intestinal muscularis were histochemically quantified. Intestinal transit was visualized 48 h after surgery. Local and systemic inflammatory response was measured with TNF-alpha and IL-6 ELISA as well as malondialdehyde (MDA) expression in serum and peritoneal fluid. RESULTS: Neutrophil count of the intestinal muscularis revealed that infiltration in the mesh-implanted and in the mesh-free animals 48 h postoperatively was similar. Gastrointestinal transit was similarly unaffected 48 h after surgery, with or without mesh implantation. TNF-alpha, IL-6 and MDA concentration in serum and peritoneal fluid showed no significant differences between the two groups. CONCLUSION: Intestinal contractility and local and systemic inflammatory response remained unaffected. Therefore, absorbable mesh augmentation is a safe and reliable method for temporary repair of the abdominal wall without affecting the intestinal integrity.

Exogenous superoxide dismutase prevents peroxynitrite-induced apoptosis in non-heart-beating donor livers.

OBJECTIVE: To investigate the role of oxygen free radicals in the induction of apoptosis in non-heart-beating donor (NHBD) livers, and if superoxide dismutase (SOD) ameliorates these alterations. METHODS: Rat livers were perfused via the portal vein with histidine/tryptophan/alpha-ketoglutarate solution from heart-beating donors (HBD) or 60-min warm ischemia from NHBD, with or without the addition of SOD. After 24 h, cold storage livers were evaluated by isolated reperfusion. RESULTS: NHBD showed significantly higher enzyme leakage and elevated portal venous pressure (PVP) versus HBD. Bile and total adenine nucleotides (TAN) were significantly decreased. Apoptosis was prominent in sinusoidal lining cells, coupled with strong nitrotyrosine staining (NTR). The concentrations of nitric oxide and lipoperoxides were largely increased. SOD medication reduced hepatic enzyme release by 30% and lipoperoxides by nearly 50%. Apoptosis and NTR were significantly decreased, and PVP was strikingly reduced to normal values. A 3-fold enhancement in bile production and 1.5-fold increase in TAN of the liver tissue were also observed. CONCLUSION: NHBD livers are prone to severe reoxygenation injury promoted by oxygen free radicals, massive nitrite oxide production and peroxynitrite-induced apoptosis within the sinusoids. Antioxidant medication with SOD should be considered as a useful means of preserving NHBD livers.

The sheep as a knee osteoarthritis model: early cartilage changes after meniscus injury and repair.

The purpose of this study was to analyse cartilage changes after traumatic meniscal lesions and to provide a detailed description of the model used with a view to reducing the number of animals used in future studies. The sheep's knee was chosen, as ovine biomechanics are similar to that of humans. In two groups of 10 animals each, a radial tear in the medial meniscus was either sutured with polydioxanone (PDS) or left untreated (sham-operated). Half of the animals in each group were sacrificed after six months, the other half after one year. The time periods to achieve weight bearing, meniscus healing, joint effusion (magnetic resonance imaging scan) and knee cartilage in the medial, lateral and patellofemoral compartments were evaluated in comparison to the opposite knee (control). Osteoarthritis (OA) was assessed by a modified Outerbridge classification and confirmed by scanning electron microscopy. Only one sutured meniscus remained completely adapted. In all other meniscus lesions, the rupture healed with a scar. In the PDS and sham-operated groups, OA was significantly higher in the medial knee compartment than in the lateral compartment and in controls (P < 0.001). In the operated groups, joint effusion was higher in the right hindlimb knee than in the normal left hindlimb knee (control) after six and 12 months (P < 0.001). Non-treated, displaced and even adapted sutured radial ovine meniscus tears produced intense OA within less than six months. Therefore, this animal model is suitable for assessment of new therapeutic regimens in meniscal surgery.

Reduced liver apoptosis after venous systemic oxygen persufflation in non-heart-beating donors.

Graft injury caused by warm ischemia in livers from non-heart-beating donors (NHBDs) strongly affects posttransplantation outcome and is associated with liver apoptosis, which is mediated by death receptors, such as Fas, a surface receptor of the tumor necrosis factor (TNF)-alpha family. The aim of this study was to test the ability of venous systemic oxygen persufflation (VSOP) to reduce apoptotic changes and Fas activation in the liver after warm ischemic insult in vivo. Livers of male Wistar rats were harvested 30 min after cardiac arrest from non-heart-beating donors (NHBD) with (NHBD + O2) or without (NHBD) application of gaseous oxygen during the cold storage period via the suprahepatic caval vein. After 24 h of storage in University of Wisconsin solution at 4 degrees C, viability of the livers was assessed upon isolated reperfusion in vitro. Conventional signs of tissue damage like enzyme release and bile production showed a significantly elevated nonspecific cell injury in the NHBD group. TUNEL staining revealed increased DNA fragmentation of sinusoidal endothelial cells in the NHBD group and more apoptotic hepatocytes than in the control group. All these alterations could be almost abrogated by the use of VSOP in the NHBD + O2 group. The immunohistochemical staining of Fas antigen expression showed a significantly elevated Fas receptor expression in the NHBD and NHBD + O2 groups, in accord with an eightfold increase of Fas receptor mRNA detected by real-time reverse-transcription polymerase chain reaction (RT-PCR). These results demonstrate that the postischemic apoptotic rate of sinusoidal endothelial cells in NHBD livers can be reduced by the use of VSOP. A significant improvement in liver integrity and viability was obtained with this technique, without influencing the expression of Fas expression.

Potential in two types of collagen scaffolds for urological tissue engineering applications - Are there differences in growth behaviour of juvenile and adult vesical cells?

The aging society has a deep impact on patient care in urology. The number of patients in need of partial or whole bladder wall replacement is increasing simultaneously with the number of cancer incidents. Therefore, urological research requires a model of bladder wall replacement in adult and elderly people. Two types of porcine collagen I/III scaffolds were used in vitro for comparison of cell growth of two different pig breeds at different growth stages. Scaffolds were characterised with scanning electron and laser scanning microscopy. Urothelial and detrusor smooth muscle cells were isolated from 15 adult Göttingen minipigs and 15 juvenile German Landrace pigs. Growth behaviour was examined in cell culture and seeded onto the collagen scaffolds via immunohistochemistry, two-photon laser scanning microscopy and a viability assay. The collagen scaffolds showed different structured surfaces which are appropriate for seeding of the two different cell types. Moisturisation of the scaffolds resulted in a change of the structure. Cell growth of German Landrace urothelial cells and smooth muscle cells was significantly higher than cell growth of the Göttingen minipig cells. Seeding of scaffolds with both cell types from both pig races was possible which could be shown by immunohistochemistry and two-photon laser scanning microscopy. Growth behaviour on the scaffolds was significantly increased for the German Landrace compared to Göttingen minipig. Nevertheless, seeding with the adult Göttingen minipig cells resulted in a closed layer on the surface and urothelial cells and smooth muscle cells showed increasing growth until day 14. The results show that these collagen scaffolds are adequate for the seeding with vesical cells. Moreover, they seem appropriate for the use as an in vitro model for the adult or elderly as the cells of the adult Göttingen minipig too, show good growth behaviour.

Recommendation for severity assessment following liver resection and liver transplantation in rats: Part I.

Score sheets were first introduced 30 years ago to assess pain, distress and suffering in animals. To date, however, there is still no general agreement on their use in research practice, and only a few publications can be found on this topic. In the present work, we demonstrate the use of a special score sheet for severity assessment in the first three postoperative days in two showcased studies performed on Wistar and Lewis rats undergoing liver resection or orthotopic liver transplantation, respectively. Scoring of different criteria and the total score were evaluated within each intervention. Additionally, both procedures were compared regarding their degree of severity. Suitability of these score sheets was evaluated for assessing severity of the procedures and these showed a minor severity within each investigated study. A comparison of both studies showed slightly higher scores involving liver transplantation. In contradiction to the common classification of these procedures as a moderate severity grade the score sheets applied here indicates a minor severity grade within each investigated study. Also, limitations and possible improvements in the design of our score sheets for defined interventions are reconsidered.

Severity assessment in rabbits after partial hepatectomy: Part II.

Although the recognition of pain, distress and discomfort has already been described in 1985 by Morton and Griffiths there is still very little known about the establishment of score sheets especially, regarding post-surgical pain and severity assessment for laboratory animals such as rabbits. In this paper we describe the estimation of severity and recovery status of 36 female New Zealand White rabbits (NZW) in a standardized liver resection model using two different adhesive treatments and one control group. Welfare was assessed at 3-4 consecutive days after surgery using a scoring system which included the following criteria: body weight, general state, clinical results, spontaneous behavior and clinical examination. Values could range from 0 to 20 where increasing values indicated increasing severity with a predefined humane endpoint for a score ≥20 points. Documented score points were almost exclusively a result of body weight loss, whereas clinical signs and general health status had no influence on the overall sum of points scored. Behavioral variation was solely observed postoperatively, within the first 24 h, with an average score ≤1. In contrast to the classification of a laparotomy as a moderate procedure in the EU Directive 2010/63 (annex VIII) the assessment herein presented showed a mild burden in all groups according to the scoring system used. The partial hepatectomy itself, as well as the adhesive treatment using either synthetic glue VIVO-107 or fibrin glue, were well tolerated.

Micturition in Göttingen minipigs: first reference in vivo data for urological research and review of literature.

One possible symptom of overactive bladder (OAB) is urinary incontinence, which is generally considered to be an age-associated disease and which is rapidly increasing with demographic changes. Rodent models are commonly used for the investigation of lower urinary tract functions, although the use of these species has limitations in several translational aspects. In biomedical research and preclinical toxicology, Göttingen minipigs are used increasingly. But in urological research, only few data are available for Göttingen minipigs. To the best of our knowledge, this study is one of the first to provide reference data of micturition in female Göttingen minipigs. Micturition frequency and volumes were monitored and analyzed in five female Göttingen minipigs. Voided volume was 520 ± 383 mL (mean ± standard deviation of mean [SD]) and micturition frequency 6.17 ± 3.68 (mean ± SD). We also performed a review of the literature to compare our data with data from different species (humans, pigs, rats and mice). Our findings revealed that micturition volume and frequency of Göttingen minipigs are more comparable with that of humans, leading to the conclusion that Göttingen minipigs may be the better choice for translational research in different research fields, such as urology, neurology and nephrology, etc. The provision of in vivo reference values meets with the 3R concept of 'reduction, refinement and replacement' of laboratory animals, because they allow comprehensive statistical power calculations (reduction), planning of telemetric approaches (refinement), and generation of computer-based modulation for the development of intravesical drug delivery systems (replacement).

Induction of experimental obstructive cholestasis in mice.

The induction of experimental obstructive cholestasis is a reliable model for cholestatic liver diseases in rodents. Bile duct ligation (BDL) in mice provokes typical time-dependent morphological and structural changes in the liver, ranging from liver cell injury and elevated serum enzyme levels after several days, to a severe inflammatory response in the liver after 5-7 days, up to an advanced hepatic fibrosis as soon as three to four weeks after surgical ligation of the common biliary duct. Upon BDL induction, hepatic stellate cells become activated and transdifferentiate into myofibroblasts that produce extracellular matrix proteins such as collagen. In principle, the periportal fibrosis induced by BDL in rat livers is reversible. After the relief of a biliary obstruction, the liver has the capacity to revert to a nearly normal histological architecture and a fully normal biochemical function. When BDL surgery is performed by an experienced scientist, this model has very high reproducibility among all fibrotic models. All these factors corroborate the outstanding value of this model for basic and translational research in biomedicine and hepatology. Nevertheless, this model can result in significant variations when surgery is carried out by untrained personnel or when unconscious modifications are implemented that affect the quality of the intervention. A detailed protocol is provided here for the provision of reliable and reproducible BDL in mice.

Synergistic value of fibrinolysis and hypothermic aerobic preservation with oxygen in the protection of livers from non-heart-beating donors: an experimental model.

The chronic organ shortage has led to the reconsideration of marginal donor pools such as non-heart-beating donors (NHBD). The use of these livers is limited due to their minimal tolerance for cold preservation. The aim of this study was to examine the combination of two different therapeutic strategies for the preservation of livers from NHBD. The livers of male Wistar rats were harvested after the induction of cardiac arrest via phrenotomy (30, 90 minutes). Livers were perfused with 10 mL of UW solution (UW), followed by hypothermic preservation with or without insufflation of gaseous oxygen (O2). In one group a fibrinolytic preflush (10 mL of Ringer's containing 7500 IU of streptokinase) was performed with subsequent preservation with O2 (O2+SK). After storage (24 h/4 degrees C/UW) livers were reperfused in vitro. Livers retrieved from heart beating donors served as controls. The results showed that even after only 30 minutes of warm ischemia livers displayed a serious disturbance in vascular perfusion (portal venous pressure, PVP = 7.4 +/- 0.2* versus control: 4.1 +/- 0.5 mmHg), associated with a more than 10-fold increase in enzyme release (ALT: 26819 +/- 513* versus control 683 +/- 152 mU/g/L), which was consistent with a significant depression in bile synthesis (1.21 +/- 0.35* versus 19.36 +/- 2.16 microL/g/45 min). However, these impairments could be prevented with O2. Even after 90 minutes of WI, the function was significant better using aerobic preservation (ALT: 3204 +/- 549 mU/g/L). With a supplementary fibrinolytic preflush, we effectively preserved livers up to 90 minutes of WI with results comparable to livers from heart beating donors with no WI (ALT: 1623 +/- 432 mU/g/L). The combination of these two techniques represents a new therapeutic approach for livers with extended or unclear WI periods in non-heart-beating donors (*P <.05 versus control).

Promising future for the transgenic rat in transplantation research.

The rat is the most widely used animal species in surgical research and offers distinct advantages over the mouse in transplantation models due to its size and close genetic similarity to humans. Sequencing of the rat genome and successful application of transgenic technologies which had only been available for mice have since led to a resurgence of the use of rat models. Transplantation provides the possibility to deliver transgenes through a variety of routes which can potentially offer treatment modalities for post-transplant dysfunction and rejection. Moreover, the use of genetically encoded fluorescent light probes has enabled in vivo visualization of organs and tissue in living animals. In recent years, generation of gene knockout rats via the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies has offered alternatives to the sophisticated embryonic stem cell based gene-targeting. In this review, we aim to provide an overview of transplantation studies involving transgenic techniques using rat models and recent advances in methods to modify the rat genome. Through novel gene modification techniques, precise, complete and conditional knockout and knockin rat models have become available which can provide promising new treatment options and opportunities for studying human transplant-related pathophysiology.

A lipid anchor improves the protective effect of ectoine in inflammation.

Others and we have shown in several studies that the natural tetrahydropyrimidine ectoine protects mammalian cells and tissues against various stress factors including ischemia/reperfusion injury, UV-irradiation, and inflammation. Since little is known about the molecular mechanism of this protective effect, which was ascribed exclusively to an extracellular action of this small water-soluble molecule, we asked whether and how a hydrophobic anchor modulates the inflammation protective properties of ectoine. We therefore investigated the influence of ectoine and of its semi-synthetic derivative lauryl-ectoine on inflammation in RAW 264.7 macrophages and primary cultured rat intestinal smooth muscle (RISM) cells. Both, ectoine and lauryl-ectoine considerably decreased lipopolysaccharide (LPS)-induced interleukin (IL)- 1, IL-6, tumor necrosis factor (TNF)- α, and cyclooxygenase (COX)-2 gene expression in macrophages as well as TNF-α- induced IL-1, IL-6 and COX-2 expression in RISM cells. This reduction of inflammatory agents was accompanied on the one hand by a significant decrease of nuclear translocation of nuclear factor (NF)-κB and on the other hand by a reduction of cellular ceramide content. Interestingly, lauryl- ectoine was much more active exerting its effect at about 10-fold lower concentrations than its natural counterpart. Note that ectoine was almost completely recovered in the medium whereas lauryl-ectoine was found to be cell-associated. Together our data indicate that a lipid anchor considerably improves a possible preventive and/or therapeutic implementation of ectoine in inflammatory processes.

Finite element modelling of stapled colorectal end-to-end anastomosis: advantages of variable height stapler design.

The impact of surgical staplers on tissues has been studied mostly in an empirical manner. In this paper, finite element method was used to clarify the mechanics of tissue stapling and associated phenomena. Various stapling modalities and several designs of circular staplers were investigated to evaluate the impact of the device on tissues and mechanical performance of the end-to-end colorectal anastomosis. Numerical simulations demonstrated that a single row of staples is not adequate to resist leakage due to non-linear buckling and opening of the tissue layers between two adjacent staples. Compared to the single staple row configuration, significant increase in stress experienced by the tissue at the inner staple rows was observed in two and three rows designs. On the other hand, adding second and/or third staple row had no effect on strain in the tissue inside the staples. Variable height design with higher staples in outer rows significantly reduced the stresses and strains in outer rows when compared to the same configuration with flat cartridge.

A new approach of in vivo musculoskeletal tissue engineering using the epigastric artery as central core vessel of a 3-dimensional construct.

The creation of musculoskeletal tissue represents an alternative for the replacement of soft tissue in reconstructive surgery. However, most of the approaches of creating artificial tissue have their limitations in the size as the maximally obtainable dimension of bioartificial tissue (BAT) is limited due to the lack of supporting vessels within the 3-dimensional construct. The seeded myoblasts require high amounts of perfusion, oxygen, and nutrients to survive. To achieve this, we developed a 3-dimensional scaffold which features the epigastric artery as macroscopic core vessel inside the BAT in a rat model (perfused group, n = 4) and a control group (n = 3) without the epigastric vessels and, therefore, without perfusion. The in vivo monitoring of the transplanted myoblasts was assessed by bioluminescence imaging and showed both the viability of the epigastric artery within the 3-dimensional construct and again that cell survival in vivo is highly depending on the blood supply with the beginning of capillarization within the BAT seven days after transplantation in the perfused group. However, further studies focussing on the matrix improvement will be necessary to create a transplantable BAT with the epigastric artery as anastomosable vessel.

Resuscitation of warm ischaemia predamaged porcine kidneys by fibrinolytic preflush with streptokinase: reduction of animal experiments.

In order to reduce the number of animal experiments, the use of non-heart-beating donors (NHBDs) from a commercial abattoir has been proposed. Since the use of slaughterhouse organs is legally not defined as animal experiment, this would fulfil international standards as an alternative to animal experiments. The development of intravascular thrombosis after cardiac arrest negatively impacts organ preservation and thus viability during ischaemic storage and reperfusion. A fibrinolytic preflush with streptokinase might overcome these limitations. Therefore, the functional and histomorphological integrity of kidneys preserved immediately with intact circulation (control group A) and kidneys preserved after cardiac arrest with a 30 min period of warm ischaemia (WI) (group B) was compared with kidneys preflushed with 12.5 kU/L (group C) or 50 kU/L streptokinase (group D) after 30 min WI prior to preservation. We could demonstrate that kidneys preflushed with 12.5 kU/L streptokinase (group C) performed better than those without streptokinase pretreatment after WI (group B). Parameters like oxygen consumption, perfusion pressure, laboratory values, lactate dehydrogenase level and lipidperoxidation were closer to that of the control (group A) than in groups B and D. The higher streptokinase concentration of 50 kU/L (group D) resulted histologically in a more pronounced tissue damage and an attenuated renal function, indicating toxic effects. On the basis of our results we believe streptokinase preflushed slaughterhouse kidneys to be an adequate alternative to organs from laboratory animals with the potential to further reduce the number of animal experiments.