Electrospun small-diameter polyurethane vascular grafts: ingrowth and differentiation of vascular-specific host cells.

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.

Rapid induction of hypothermia with a small volume aortic flush during cardiac arrest in pigs.

PURPOSE: The induction of deep cerebral hypothermia (15°C) via large-volume cold (4°C) saline aortic flush during cardiac arrest and resuscitation with cardiopulmonary bypass improves neurologic outcome in pigs. We hypothesized that induction of mild cerebral hypothermia (33°C) via smaller volume and resuscitation without bypass will improve survival and neurologic outcome after 15 minutes of cardiac arrest as compared with conventional resuscitation attempts. BASIC PROCEDURES: Twenty-four pigs (29-38 kg) underwent ventricular fibrillation cardiac arrest for 15 minutes. Conventional resuscitation (n=8) was compared with hypothermic (4°C, n=8) and normothermic (38.5°C, n=8) aortic flush (30 mL/kg) at the beginning of resuscitation efforts, with defibrillation attempts 2 minutes later. Outcomes after 9 days were compared. MAIN FINDINGS: In the hypothermic flush group, brain temperature decreased from 38.3°C±0.5°C to 33°C±0.5°C within 277±112 seconds. We observed considerably higher mean coronary perfusion pressures in the normothermic and hypothermic flush groups (hypothermic vs conventional, P=.023; normothermic vs conventional, P=.041). Three animals of each flush group, compared with none of the conventional group, achieved restoration of spontaneous circulation (P=.2); and 3 pigs of the hypothermic flush group and 2 pigs of the normothermic flush group survived to 9 days without differences in neurologic outcome. PRINCIPAL CONCLUSION: A smaller volume, cold saline aortic flush during prolonged cardiac arrest rapidly induces mild cerebral hypothermia to 33°C and improves coronary perfusion pressure but does not result in a significant improvement in outcome as compared with conventional resuscitation attempts.

Cold aortic flush and chest compressions enable good neurologic outcome after 15 mins of ventricular fibrillation in cardiac arrest in pigs.

OBJECTIVE: The induction of deep cerebral hypothermia via ice-cold saline aortic flush during prolonged ventricular fibrillation cardiac arrest, followed by hypothermic stasis and delayed resuscitation (emergency preservation and resuscitation), improved neurologic outcome after cardiac arrest in pigs, as compared to conventional resuscitation. We hypothesized that emergency preservation and resuscitation with chest compressions would further improve outcome in the same model. DESIGN: Prospective experimental study. SETTING: University research laboratory. SUBJECTS: : Twenty-four female, large, white breed pigs (27-37 kg). INTERVENTIONS: Fifteen minutes of ventricular fibrillation cardiac arrest were followed by 20 mins of resuscitation with chest compressions (control, n = 8), deep cerebral hypothermia via 200 mL/kg 4 degrees C saline aortic flush and hypothermic stasis (emergency preservation and resuscitation, n = 8), and emergency preservation and resuscitation combined with chest compressions (emergency preservation and resuscitation plus chest compressions, n = 8). At 35 mins after cardiac arrest, cardiopulmonary bypass was initiated, followed by defibrillation. Mild hypothermia was continued for 20 hrs. Pigs were evaluated after 9 days using a neurologic deficit (neurologic deficit score: 100% = brain dead; 0%-10% = normal) and an overall performance category score (overall performance category score: 1 = normal; 2 = slightly handicapped; 3 = severely handicapped; 4 = comatose; 5 = dead/brain dead). MEASUREMENTS AND MAIN RESULTS: Brain temperature decreased from 38.5 degrees C to 15.3 degrees C +/- 3.3 degrees C in the emergency preservation and resuscitation group, and to 11.3 degrees C +/- 1.2 degrees C in the emergency preservation and resuscitation plus chest compressions group. In the control group, restoration of spontaneous circulation was achieved in four out of eight pigs, and one survived to 9 days. In the emergency preservation and resuscitation group, restoration of spontaneous circulation was achieved in seven out of eight pigs and five survived; in the emergency preservation and resuscitation plus chest compressions group, all had restoration of spontaneous circulation and seven survived (restoration of spontaneous circulation, p = .08). Neurologic outcome for (median and interquartile range) the control group included overall performance category score of 3, neurologic deficit score of 45%; for the emergency preservation and resuscitation group, overall performance category score was 3 (2-5) and neurologic deficit score was 45% (36; 50) and in the emergency preservation and resuscitation plus chest compressions group, overall performance category score was 2 (1-3) and neurologic deficit score was 13% (5; 21) (overall performance category score, p = .04; neurologic deficit score emergency preservation and resuscitation vs. emergency preservation and resuscitation plus chest compressions, p = .003). CONCLUSIONS: Emergency preservation and resuscitation by deep cerebral hypothermia combined with chest compressions during prolonged cardiac arrest in pigs are feasible and improve neurologic outcome.

S-nitroso human serum albumin reduces ischaemia/reperfusion injury in the pig heart after unprotected warm ischaemia.

AIMS: Uncoupled endothelial nitric oxide synthase (eNOS) is a major contributor to vascular reactive oxygen species generation in ischaemia/reperfusion (I/R) injury. Supplementation of NO by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) may inhibit uncoupling of eNOS (feedback inhibition). METHODS AND RESULTS: Pigs (n = 14; 33.1 +/- 1.7 kg) were continuously monitored for heart rate (HR), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and coronary flow (CF). Infusion of either human serum albumin (n = 8; controls) or S-NO-HSA (n = 6) lasted 60 min (0.1 micromol/kg/h) starting 15 min prior to ischaemia. After clamping the aorta under cardiopulmonary bypass (CPB), the hearts underwent 15 min of warm, unprotected ischaemia (37 degrees C). Reperfusion lasted 150 min (30 min under CPB; 15 min weaning; additional 105 min reperfusion). In biopsies from non-ischaemic hearts and myocardial biopsies taken after 150 min of reperfusion, high-energy phosphates were measured and the calcium ionophore-stimulated release of NO, superoxide, and peroxynitrite (ONOO(-)) were monitored with nanosensors. Compared with non-ischaemic hearts, the NO level decreased from 930 +/- 25 to 600 +/- 15 nmol/L (P < 0.001) while the superoxide level increased from 45 +/- 5 to 110 +/- 10 nmol/L (P < 0.001) after ischaemia. S-NO-HSA restored the NO level to 825 +/- 20 nmol/L, shifted favourably the [NO]/[ONOO(-)] balance (a marker of eNOS uncoupling) from 1.36 +/- 0.06 (ischaemia) to 3.59 +/- 0.18, significantly improved CF (65 +/- 10 vs. control, 43 +/- 5 mL/min, P < 0.05), MAP (57 +/- 5 vs. 39 +/- 3 mm Hg, P < 0.01), LVSP (106 +/- 5 vs. 81 +/- 4 mm Hg, P < 0.01) and phosphocreatine (PCr) content (41.5 +/- 7.3 vs. 18.0 +/- 5.6 micromol/g protein; P < 0.01) at 150 min of reperfusion. CONCLUSION: Long-lasting release of NO by S-NO-HSA prevented uncoupling of eNOS and thereby improved systolic and diastolic function, myocardial perfusion, and the energetic reserve of the heart after I/R injury.

Therapeutic hypothermia with a novel surface cooling device improves neurologic outcome after prolonged cardiac arrest in swine.

OBJECTIVE: Devices for rapid induction of mild hypothermia after cardiac arrest are needed. We hypothesized that the Life Recovery Systems' ThermoSuit System provides effective core cooling by pumping ice water over the skin surface and improves neurologic outcome after prolonged cardiac arrest. DESIGN: Prospective experimental study. SETTING: University research laboratory. SUBJECTS: Large White breed pigs (29 to 35 kg). INTERVENTIONS: Swine were anesthetized and mechanically ventilated. Ten minutes of untreated ventricular fibrillation, 3 mins of basic life support, and 5 mins of advanced cardiac life support, including two 0.4 IU/kg doses of vasopressin, were followed by up to three countershocks. After restoration of spontaneous circulation, swine were randomized to two groups (normothermic control, hypothermia). The hypothermia group was cooled from a pulmonary artery temperature of 38.5 +/- 0.5 degrees C to 33.0 degrees C and kept for 14 hrs. At day 9 of the experiment, overall performance categories scores (1, normal; 2, slightly disabled; 3, severely disabled; 4, comatose; 5, dead, brain dead) and neurologic deficit scores (0%, normal; 100%, brain dead) were assessed. Data are presented as median and interquartile range; group comparison was done with a Mann-Whitney U test. MEASUREMENTS AND MAIN RESULTS: In total, 16 of 22 animals were randomized. Time to target temperature in the hypothermia group (n = 8) was 9.0 (5.3-11.9) mins (cooling rate 0.4 [0.3-0.8] degrees C/min), and all animals achieved an overall performance categories score of 1. In the control group, one swine achieved an overall performance categories score of 1, three achieved a score of 2, and four achieved a score of 3 (p = .002). Neurologic deficit score was 0% (0%-4%) in the hypothermia group and 39% (19%-55%) in the control group (p = .001). No harmful side effects could be observed. CONCLUSIONS: The Life Recovery Systems' ThermoSuit System rapidly and safely induced mild therapeutic hypothermia. Hypothermia improved neurologic outcome in swine after cardiac arrest as compared with normothermia. Further studies are warranted to compare the device with established cooling methods.

Rapid non-invasive external cooling to induce mild therapeutic hypothermia in adult human-sized swine.

AIM OF THE STUDY: Mild therapeutic hypothermia is a promising new therapy for patients resuscitated from cardiac arrest. Early and fast induction of hypothermia seems to be crucial for best results. The aim of the study was to investigate the feasibility and safety of a new surface cooling method using cold metal plates. SUBJECTS AND METHODS: Twelve adult human-sized swine (79+/-9 kg) were cooled from 38 to 33 degrees C brain temperature. The skin surface was covered with -20 degrees C metal plates (M), as compared to ice packs, alcohol rubs, and fans used in a control group (C). Each method was tested during spontaneous circulation and, after re-warming, during cardiac arrest. Temperatures were recorded continuously. Data are given as mean+/-standard deviation or as median (interquartile range), if not normally distributed. Comparisons between the treatment groups were performed with the independent samples t-test, or the Mann-Whitney rank-sum test. RESULTS: During spontaneous circulation, cooling rates were 9.3+/-1.4 degrees C/h (M), and 6.1+/-1.4 degrees C/h (C) (p=0.003); no skin lesions were observed. During cardiac arrest, cooling rates were 4.1 degrees C/h (1.8-4.8) (M), and 3.7 degrees C/h (3.1-5.3) (C) (p=0.9); no skin lesions were observed. CONCLUSION: Cooling with cold metal plates was an effective method for rapid induction of mild therapeutic hypothermia in adult human-sized swine during spontaneous circulation, without any signs of skin damage. This new surface-cooling device, independent of energy supply during use, should be further investigated.

In-vitro and in-vivo measurement of the animal's middle ear acoustical response by partially implantable fiber-optic sensing system.

The main obstacle in realization of a totally implantable hearing aid is a lack of reliable implantable microphone. In this paper we have described a potentially miniature fiber-optic vibrometer based on a modified Michelson interferometer, designed to serve as a middle-ear microphone for totally implantable cochlear- or middle-ear hearing aids. A model of the sensing system was used for in-vitro and in-vivo investigation of acoustical response of sheep's middle-ear ossicles. Surgical and implantation procedure of introducing the sensing optical fiber into the middle-ear and its aiming at the incus was investigated and described here in detail. The frequency responses of the incus was measured while a cadaver and living sheep was exposed to the sinusoidal acoustical excitation of 40-90dB SPL, in the frequency range from 100Hz to 10kHz. The amplitude of the incus vibration was found to be in the range between 10pm to 100nm, strongly depending on the frequency, with a lot of resonant peaks, corresponding mainly to the natural outer ear canal gain. The noise floor in the experiments was about 2pm/Hz1/2, but recently we have decreased it to < 0.5pm/Hz1/2, which corresponds to a minimal detectable sound level of 31-35dB(A) SPL for humans. The histological examination of temporal bones of cadaver animals and the intensity of in-vivo optical signal demonstrated that the aiming of the sensing fiber to the target has been preserved for five months after the implantation.

External cardiac defibrillation during wet-surface cooling in pigs.

OBJECTIVE: During surface cooling with ice-cold water, safety and effectiveness of transthoracic defibrillation was assessed. METHODS: In a pig ventricular fibrillation cardiac arrest model, once (n = 6), defibrillation was done first in a dry and then in a wet condition using the ThermoSuit System (Life Recovery Systems, HD, LLC, Kinnelon, NJ), which circulates a thin layer of ice-cold water (approximately 4 degrees C) over the skin surface. Another time (n = 6), defibrillation was done first in a wet and then in a dry condition. Success of defibrillation was defined as restoration of spontaneous circulation, and the current and voltage of the defibrillation signal was measured. RESULTS: There was a tendency toward less number of shocks needed for achieving restoration of spontaneous circulation in the wet condition as compared with the number of shocks needed in the dry condition. The energy delivered in both dry and wet conditions was 144 +/- 3 J. DISCUSSION: Transthoracic defibrillation is safe and effective in a wet condition after cooling with ice-cold water.

Bioelectrical signals improve cardiac function and modify gene expression of extracellular matrix components.

AIMS: Beyond the influence of stimulating devices on cardiac excitation, their use in treating patients with heart failure has positive effects on the myocardium at the molecular level. Electrical signals can induce a wide spectrum of effects in living tissue. Therefore, we sought to determine whether applying electrical microcurrent directly to failing hearts leads to functional improvement. METHODS AND RESULTS: Sixteen male spontaneously hypertensive rats (SHRs) with heart failure underwent application of a patch electrode to the left ventricular epicardium and placement of a subcutaneous counter electrode. The electrode delivered a 0.35 µA microcurrent to nine of the SHRs for 45 ± 3 days; the other seven SHRs were used as controls. At baseline and before the SHRs were humanely put to death, we measured the left ventricular ejection fraction (LVEF) and the thickness of the LV posterior wall during systole and diastole (LVPWs/d). We used quantitative PCR to determine extracellular matrix parameters [collagen I-III, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases 3 (TIMP3), TIMP4, connexins (Cxs) 40/43/45, transforming growth factor (TGF)-ß, and interleukin (IL)-6]. Among SHRs undergoing microcurrent application, LVEF normalized (mean decrease, 22.8%; P = 0.009), and LVPWs decreased (mean, 35.3%; P = 0.001). Compared with the control group, the SHRs receiving microcurrent exhibited a mean decrease in the gene expression of collagen I (10.6%, P = 0.003), TIMP3 (18.5%, P = 0.005), Cx43 (14.3%, P = 0.003), Cx45 (12.7%, P = 0.020), TGF-ß (13.0%, P = 0.005), and IL-6 (53.7%, P = 0.000). Microcurrent application induced no changes in the expression of collagen III, MMP-2, MMP-9, TIMP4, or Cx40. CONCLUSIONS: Applying microcurrent to the LV epicardium of SHRs leads to statistically significant functional improvement and alterations in the levels of inflammatory and extracellular matrix components.

Microcurrent stimulation promotes reverse remodelling in cardiomyocytes.

AIMS: It has been shown that electrical stimulation can improve tissue repair in patients. Imbalances in the extracellular matrix composition induce manifestation of heart failure. Here we investigated the application of microcurrent (MC) to modulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in cardiomyocytes in vitro and in vivo to reverse remodelling in the heart in spontaneous hypertensive rats (SHR). METHODS: Cardiomyocytes from young SHR (7 months) and old SHR (14 months) were stimulated in vitro and in vivo with MC. MMP and TIMP expression were analysed by qPCR and immunofluorescence to evaluate the modulation of MC treatment. RESULTS: Modulation of cardiomyocytes with MC enhances proliferation with no morphological changes in vitro. By electrical stimulation dual effects, increase and decrease, on MMP-2, MMP-9, TIMP-3, and TIMP-4 mRNA as well as protein expression were observed, depending on the age of the cardiomyocytes. In our in vivo study, MC down-regulated MMP-2, MMP-9, and TIMP-4 and increased TIMP-3 in young SHR. In old SHR MMP-2, MMP-9, and TIMP-4 were up-regulated, whereas TIMP-3 was unaffected. CONCLUSIONS: Our data indicate that treatment of MC can modulate the expression of MMPs and TIMPs in vitro and in vivo in SHR. Based on these results new treatments for heart failure could be developed.

Bone marrow stromal cells can provide a local environment that favors migration and formation of tubular structures of endothelial cells.

Findings suggest that mesenchymal progenitor cells can support the process of blood vessel formation, which may be relevant during granulation tissue formation at defect sites. The aim of this study was to investigate possible mechanisms of the angiogenic process that can be stimulated by mesenchymal progenitor cells. In the in vivo-like model of the chick embryo chorioallantoic membrane assay, we observed blood vessel ingrowth into collagen sponges containing conditioned medium from undifferentiated bone marrow stromal cells. In the Boyden chamber assay, the conditioned medium was chemotactic for human umbilical vascular endothelial cells and human uterus microvascular endothelial cells, and when cells were placed on Matrigel-coated culture dishes, formation of tubular structures was enhanced. The presence of vascular endothelial growth factor-neutralizing antibodies did not affect the outcome of the two in vitro assays. Bone marrow stromal cell-conditioned medium had no effect on proliferation of endothelial cells, as determined by measuring [3H]thymidine incorporation, and on matrix metalloproteinase 2 expression, as evaluated by reverse transcription-polymerase chain reaction and gelatin zymography. These data indicate that mesenchymal progenitor cells can provide a local environment that supports the ingrowth of blood vessels into a defect site.

Effect of laser perforation on the remodeling of acellular matrix grafts.

Autologous cells migrate only slightly into acellular matrix grafts. This study was carried out in small-diameter, allogeneic matrix grafts to investigate the effects on cell repopulation and remodeling caused by increased wall porosity induced by laser perforation. Allogeneic ovine carotid arteries were decellularized by dye-mediated photooxidation (Photofix). Matrix grafts (10 cm x 4 mm i.d.) were perforated with holes of 50 microm diameter at a density of 50 holes/cm(2) using a Ti-sapphire laser. The grafts were implanted in the carotid arteries of 10 sheep and were compared to nonperforated grafts implanted contralaterally. The prostheses were retrieved after 6 weeks or 3 or 6 months following implantation and were evaluated by histologic examination, immunohistochemical staining, and scanning electron microscopy. All grafts, except one of the perforated specimens, remained patent. Perforated implants, examined at 6 weeks, showed faster recellularization with endothelial cells than did the corresponding contralateral controls. Perforated grafts, examined at 6 months, showed a significantly thicker neointima and clear signs of neovascularization: endothelial cells, basal lamina, elastic fibers, circular and longitudinally orientated smooth muscle cells in comparison to nonperforated specimens. Repopulation of the decellularized matrix with host cells was higher in the perforated than in the nonperforated prostheses. These results suggest that the increased matrix porosity induced by laser perforation promotes graft remodeling and reconstitution with host cells.

Glycoprotein 130 ligand oncostatin-M induces expression of vascular endothelial growth factor in human adult cardiac myocytes.

OBJECTIVE: In murine and rat cardiac myocytes the gp130 system transduces survival as well as hypertrophic signals and via induction of the expression of the potent angiogenic factor VEGF in these cells also indirectly contributes to cardiac repair processes through the development of new blood vessels. There are, however, species differences in receptor specificity and receptor crossreactivity in the gp130-gp130 ligand system. We asked whether gp130 signaling is also involved in the regulation of VEGF in human cardiac myocytes and if so which gp130 ligands are critical for such an effect. METHODS: Human adult cardiac myocytes (HACMs) were isolated from myocardial tissue and characterised by positive staining for myocardial actin, troponin-I and cardiotin. HACMs were treated with the gp130 ligands CT-1, IL-6, LIF or OSM and VEGF-1 was determined by a specific ELISA in the conditioned media of these cells. RT-PCR and Western blot analysis was used in order to detect gp130, IL-6-receptor, LIF-receptor or OSM-receptor specific protein and mRNA in human adult cardiac myocytes and for detection of VEGF-1 specific mRNA in cardiac myocytes after incubation with OSM. Pieces of myocardial tissue were incubated ex vivo in the presence and absence of OSM and VEGF was determined in supernatants of these cultures and immunohistochemistry was performed on the tissue using specific antibodies for VEGF-1. Immunohistochemistry was also employed to detect VEGF in sections from a healthy human heart and in a heart from a patient suffering from acute myocarditis. RESULTS: OSM, but not CT-1, IL-6 or LIF increased VEGF-1 production in human adult cardiac myocytes dose-dependently derived from five different donors. This selective stimulation of VEGF by gp130 ligands was also reflected by a specific receptor expression on these cells. We detected high levels of mRNA for gp130 and the OSM receptor in freshly isolated human cardiac myocytes but only low amounts of mRNA for the IL-6 receptor whereas mRNA for the LIF receptor was hardly detectable by RT-PCR. OSM receptor and IL-6 receptor were also detectable by Western blotting whereas LIF receptor was only present as a faint band. OSM also increased the expression of VEGF-1 mRNA in cardiac myocytes. When pieces of human myocardial tissue were incubated with the gp130 ligands in an ex vivo model only OSM resulted in an increase in VEGF-1 in the supernatants of these cultures. Furthermore, VEGF increased in tissue samples treated with OSM in cardiac myocytes as evidenced by immunohistochemistry. In addition, we found increased VEGF-1 expression in myocardial tissue from a patient suffering from acute myocarditis. CONCLUSION: The gp130-gp130 ligand system is also involved in VEGF regulation in human cardiac myocytes and OSM is the gp130 ligand responsible for this effect in the human system whereas LIF and CT-1 which had been shown to regulate VEGF expression in mouse and rat cardiac myocytes had no effect. Thus we have added OSM, which is produced by activated T lymphocytes and monocytes, to the list of regulatory molecules of VEGF production in the human heart. Our results lend further support to the notion that besides hypoxia, inflammation via induction of VEGF through autocrine or paracrine pathways plays a key role in (re)vascularisation of the myocardium.

Prosthetic replacement of the aorta is a risk factor for aortic root aneurysm development.

BACKGROUND: Noncompliant prostheses are used in aortic replacement. We hypothesized that this leads to increased distension and wall stress in the aortic root because of the loss of ventriculo arterial coupling. METHODS: Pressure relations in the aortic root caused by changes of aortic elasticity simulating prosthetic aortic replacement were tested in a computer model. We then developed an in vitro model using porcine aortas and performed in vivo validation. RESULTS: Findings in vitro and in vivo confirmed the predicted changes of the computer model. Pressure amplitude increased significantly by 17% after prosthetic replacement (p < 0.01). Pressure-time differential (Dp/dt) and dicrotic notch pressure amplitude both increased significantly. Echocardiography demonstrated systolic aortic root distension with percentage area change increasing in vitro from 28.2%+/-9.7% to 35.9%+/-10% (p < 0.05) and in vivo from 13.3%+/-3.1% to 24.3%+/-3.1% (p < 0.0001). Aortic root wall stress increased markedly. CONCLUSIONS: Replacement of the aorta with vascular prostheses causes important negative alterations of hemodynamics and increases in wall stress.

Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering?

It was the aim of this study to evaluate chitosan-thioglycolic acid (chitosan-TGA) conjugate as scaffold material in tissue engineering. Chitosan was modified by the introduction of thiol groups. Briefly, TGA was introduced to chitosan via amide bond formation mediated by a carbodiimide. The properties of the resulting polymer were thereby altered in regard to water solubility, mucoadhesion, biodegradability and in situ gelling compared to the original polymer. Due to the immobilised thiol groups (240+/-30 micromol thiol groups per gram polymer), the viscosity of a 1.5% chitosan-TGA solution was improved 4.3-fold. This can be explained by the formation of disulphide bonds within this polymeric network. The conjugate was tested as scaffold material in form of a gel and sheets. Furthermore, the influence of the thiol groups on the viability of L-929 mouse fibroblasts was evaluated. It was shown that the L-929 mouse fibroblasts grew on both scaffolds despite the thiol groups, although the different surface conditions seemed to have an influence on the growing rate. Chitosan-TGA sheets seemed to be the more preferred layer. The improved in situ gelling may be important for ongoing developments. Direct injectable matrices at the site of tissue damage mimicking the tissue being restored may be a future trend on this topic. Hence, chitosan-TGA is a promising candidate as scaffold material in tissue engineering.

The mandatory CAM testing of cells and scaffolds for tissue engineering: benefits for the three Rs of cooperation with the vaccine industry.

In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell-scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.

Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses.

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.

New conventional long-term survival normovolemic cardiac arrest pig model.

AIM OF THE STUDY: A reproducible long-term intensive care and outcome cardiac arrest model for exploring new cerebral preservation strategies is needed. We tried to determine effects and limitations of current therapies after different 'no-flow' times. METHODS: Thirty-five female Large White Breed pigs (26-37kg) were included in the study. Three pigs served as sham animals without cardiac arrest (CA). Ventricular fibrillation (VF) CA was induced in 32 animals for 0, 7, 10 and 13min (each group consisting of 8 animals), followed by 8min of chest compressions, mechanical ventilation and vasopressors. Thereafter, up to 3 defibrillations were delivered. After restoration of spontaneous circulation (ROSC), the animals underwent intensive care for 20h. Neurologic examination was performed at designated time points using a neurologic deficit (ND) and an overall performance category (OPC) score. RESULTS: Restoration of spontaneous circulation was achieved in 8 of 8 animals in the 0min-group, 6 of 8 in the 7min-group, 7 of 8 in the 10min-group and 0 of 8 in the 13min-group. All animals of the sham-group and 0min-group were neurologically intact survivors; the 7 and 10min-groups showed a median ND of 55%(26;94) and 73%(58;78), respectively. There were no significant differences between the 7 and 10min-groups regarding OPC and NDS. Coronary perfusion pressure during CPR decreased concordantly with 'no-flow' times with a tendency towards significance. CONCLUSION: This study established a reproducible cardiac arrest and resuscitation model in pigs which will be used to test novel resuscitation strategies to improve neurologic outcome after cardiac arrest.

The importance of surface area for the cooling efficacy of mild therapeutic hypothermia.

AIM OF THE STUDY: Mild hypothermia after cardiac arrest should be induced as soon as possible. There is a need for improved feasibility and efficacy of surface cooling in ambulances. We investigated which and how much area of the body surface should be covered to guarantee a sufficient cooling rate. METHODS: Each of five adult, human-sized pigs (88-105kg) was randomly cooled in three phases with pads that covered different areas of the body surface corresponding to humans (100% or 30% [thorax and abdomen] or 7% [neck]). The goal was to quickly lower brain temperature (Tbr) from 38 to 33°C within a maximum of 120min. Linear regression analysis was used to test the association between cooling efficacy and surface area. Data are presented as mean±standard deviation. RESULTS: The 100% and 30% cooling pads decreased the pigs' Tbr from 38 to 33°C within 33±7min (8.2±1.6°C/h) and 92±24min (3.6±1.1°C/h). The 7% achieved a final Tbr of 35.8±0.7°C after 120min (1.1±0.4°C/h). The 30% and 7% cooling surface areas achieved 37±11% and 15±7% of the cooling rate compared to the 100% cooling pads. For every additional percent of surface area cooled, the cooling rate increased linearly by 0.07°C/h (95% CI 0.05-0.09, p=0.001). No skin lesions were observed. CONCLUSIONS: The cooling pads were effective and safe for rapid induction of mild hypothermia in adult, human-sized pigs, depending on the percentage of body surface area covered. Covering only the neck, chest, and abdomen might achieve satisfactory cooling rates.

Comparison of surface cooling and invasive cooling for rapid induction of mild therapeutic hypothermia in pigs--effectiveness of two different devices.

AIM OF THE STUDY: The effectiveness and safety of non-invasive surface cooling was compared to invasive endovascular cooling in an animal model. METHODS: Eight healthy pigs (29-38 kg) were cooled twice, starting in the first 4 pigs with unique surface cooling pads followed by endovascular cooling. In the second 4 pigs the order was reversed. The goal was to quickly lower pulmonary artery temperature from 38 to 33°C. A paired t-test was used to compare cooling rates (°C/h, mean±standard deviation) between both cooling techniques. RESULTS: Mean non-invasive surface cooling rate (11.9±3.8°C/h) significantly exceeded mean invasive cooling rate (3.9±0.7°C/h; p<0.001). The mean difference in cooling rates was 8.0±3.6°C/h. No surface cooling related adverse skin reactions were observed. CONCLUSIONS: Surface cooling is a simple method for achieving fast cooling rates. In our animal model, non-invasive cooling was three times faster than rapid endovascular cooling without overshoot.