Decellularization combined with enzymatic removal of N-linked glycans and residual DNA reduces inflammatory response and improves performance of porcine xenogeneic pulmonary heart valves in an ovine in vivo model.

BACKGROUND: Limited availability of decellularized allogeneic heart valve substitutes restricts the clinical application thereof. Decellularized xenogeneic valves might constitute an attractive alternative; however, increased immunological hurdles have to be overcome. This study aims for the in vivo effect in sheep of decellularized porcine pulmonary heart valves (dpPHV) enzymatically treated for N-glycan and DNA removal. METHODS: dpPHV generated by nine different decelluarization methods were characterized in respect of DNA, hydroxyproline, GAGs, and SDS content. Orthotopic implantation in sheep for six months of five groups of dpPHV (n = 3 each; 3 different decellularization protocols w/o PNGase F and DNase I treatment) allowed the analysis of function and immunological reaction in the ovine host. Allogenic doPHV implantations (n = 3) from a previous study served as control. RESULTS: Among the decellularization procedures, Triton X-100 & SDS as well as trypsin & Triton X-100 resulted in highly efficient removal of cellular components, while the extracellular matrix remained intact. In vivo, the functional performance of dpPHV was comparable to that of allogeneic controls. Removal of N-linked glycans and DNA by enzymatic PNGase F and DNase I treatment had positive effects on the clinical performance of Triton X-100 & SDS dpPHV, whereas this treatment of trypsin & Triton X-100 dpPHV induced the lowest degree of inflammation of all tested xenogeneic implants. CONCLUSION: Functional xenogeneic heart valve substitutes with a low immunologic load can be produced by decellularization combined with enzymatic removal of DNA and partial deglycosylation of dpPHV.

Prediction of transplant outcome after 24-hour ex vivo lung perfusion using the Organ Care System in a porcine lung transplantation model.

Ex vivo lung perfusion (EVLP) has become routine practice in lung transplantation. Still, running periods exceeding 12 hours have not been undertaken clinically to date, and it remains unclear how the perfusion solution for extended running periods should be composed and which parameters may predict outcomes. Twenty-four porcine lungs underwent EVLP for 24 hours using the Organ Care System (OCS). Lungs were ventilated and perfused with STEEN's solution enriched with erythrocytes (n = 8), acellular STEEN's solution (n = 8), or low-potassium dextran (LPD) solution enriched with erythrocytes (n = 8). After 24 hours, the left lungs were transplanted into recipient pigs. After clamping of the contralateral lung, the recipients were observed for 6 hours. The most favorable outcome was observed in organs utilizing STEEN solution enriched with erythrocytes as perfusate, whereas the least favorable outcome was seen with LPD solution enriched with erythrocytes for perfusion. Animals surviving the observation period showed lower peak airway pressure (PAWP) and pulmonary vascular resistance (PVR) during OCS preservation. The results suggest that transplantation of lungs following 24 hours of EVLP is feasible but dependent on the composition of the perfusate. PAWP and PVR during EVLP are early and late predictors of transplant outcome, respectively.

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Treatment of infected lungs by ex vivo perfusion with high dose antibiotics and autotransplantation: A pilot study in pigs.

The emergence of multi-drug resistant bacteria threatens to end the era of antibiotics. Drug resistant bacteria have evolved mechanisms to overcome antibiotics at therapeutic doses and further dose increases are not possible due to systemic toxicity. Here we present a pilot study of ex vivo lung perfusion (EVLP) with high dose antibiotic therapy followed by autotransplantation as a new therapy of last resort for otherwise incurable multidrug resistant lung infections. Severe Pseudomonas aeruginosa pneumonia was induced in the lower left lungs (LLL) of 18 Mini-Lewe pigs. Animals in the control group (n = 6) did not receive colistin. Animals in the conventional treatment group (n = 6) received intravenous application of 2 mg/kg body weight colistin daily. Animals in the EVLP group (n = 6) had their LLL explanted and perfused ex vivo with a perfusion solution containing 200 µg/ml colistin. After two hours of ex vivo treatment, autotransplantation of the LLL was performed. All animals were followed for 4 days following the initiation of treatment. In the control and conventional treatment groups, the infection-related mortality rate after five days was 66.7%. In the EVLP group, there was one infection-related mortality and one procedure-related mortality, for an overall mortality rate of 33.3%. Moreover, the clinical symptoms of infection were less severe in the EVLP group than the other groups. Ex vivo lung perfusion with very high dose antibiotics presents a new therapeutic option of last resort for otherwise incurable multidrug resistant pneumonia without toxic side effects on other organs.

Decellularized mitral valve in a long-term sheep model.

OBJECTIVES: The objective of this study was to evaluate surgical handling, in vivo hemodynamic performance and morphological characteristics of decellularized mitral valves (DMVs) in a long-term sheep model. METHODS: Ovine mitral valves were decellularized using detergents and ß-mercaptoethanol. Orthotopic implantations were performed in 6-month-old sheep (41.3 ± 1.2 kg, n = 11) without annulus reinforcement. Commercially available stented porcine aortic valves [biological mitral valve (BMV), n = 3] were implanted conventionally and used as controls. Valve function was evaluated by transoesophageal echocardiography and explants were investigated by a routine bright field microscopy and immunofluorescent histology. RESULTS: During implantation, 2 DMVs required cleft closure of the anterior leaflet. All valves were competent on water test and early postoperative transoesophageal echocardiography. Six animals (DMV, n = 4; BMV, n = 2) survived 12 months. Six animals died within the first 4 months due to valve-related complications. At 12 months, transoesophageal echocardiography revealed severe degeneration in all BMVs. Macroscopically, BMV revealed calcification at the commissures and leaflet insertion area. Histological examination showed sporadic cells negative for endothelial nitric oxide synthase, von Willebrand factor and CD45 on their surface. In contrast, DMV showed no calcification or stenosis, and the regurgitation was trivial to moderate in all animals. Fibrotic hardening occurred only along the suture line of the valve annulus, immunostaining revealed collagen IV covering the entire leaflet surface and a repopulation with endothelial cells. CONCLUSIONS: Surgical implantation of DMV is feasible and results in good early graft function. Additional in vivo investigations are required to minimize the procedure-related complications and to increase the reproducibility of surgical implantation. Degenerative profile of allogeneic DMV is superior to commercially available porcine aortic prosthesis.

Irradiation before and donor splenocyte infusion immediately after transplantation induce tolerance to lung, but not heart allografts in miniature swine.

Solid organs may differ in their potential to induce and maintain a state of donor-specific tolerance. Previously, we induced stable immunological tolerance in a lung transplantation model in miniature swine. Here, we wished to transfer this established protocol into a heart transplantation model in miniature swine. Heterotopic heart transplantation (HTX) was performed in four and left-sided lung transplantation (LTX) in seven minipigs from gender- and SLA-mismatched donors. All recipients received nonmyeloablative irradiation, donor splenocyte infusion and intravenous pharmacologic immunosuppression for 28 postoperative days. All transplanted hearts were rejected within 95 days. In contrast, four animals of the LTX group developed stable tolerance surviving beyond 500 days, and three further animals rejected 119, 239 and 360 days post-transplantation. In both groups, peripheral blood donor leucocyte chimerism peaked 1 h after reperfusion of the allograft. Importantly, the early chimerism level in the LTX group was significantly higher compared to the HTX group and remained detectable throughout the entire observation period. In conclusion, lungs and hearts vary in their potential to induce a state of tolerance after transplantation in a protocol with pre-operative recipient irradiation and donor splenocyte co-transplantation. This could be due to differential early levels of passenger leucocyte chimerism.

Aortic valve replacement with sutureless prosthesis: better than root enlargement to avoid patient-prosthesis mismatch?

OBJECTIVES: Aortic valve replacement in patients with a small aortic annulus may result in patient-prosthesis mismatch (PPM). Aortic root enlargement (ARE) can reduce PPM, but leads to extended cardiac ischaemia times. Sutureless valves have the potential to prevent PPM while reducing cardiac ischaemia times. METHODS: Between January 2007 and December 2011, a total of 128 patients with a small aortic annulus underwent surgery for aortic valve stenosis at our centre. Thirty-six (17% male, n = 6) patients received conventional valve replacement with ARE and 92 (16% male, n = 18) subjects received sutureless valve implantation (Sorin Perceval). We conducted a comparative, retrospective study with follow-up. RESULTS: The sutureless group showed a significantly higher age (79 years) than the ARE patients (62 years, P < 0.001) and received significantly more concomitant cardiac procedures (33%, n = 30 vs 6%, n = 2, P = 0.001). The mean operation, cardiopulmonary bypass and cross-clamp times were significantly lower in sutureless patients (147 ± 42, 67 ± 26 and 35 ± 13 min, respectively) than in ARE patients (181 ± 41, 105 ± 29 and 70 ± 19 min, respectively, P < 0.001). The mean postoperative effective orifice area (EOA) indexed to the body surface area was 0.91 ± 0.2 cm(2)/m(2) in ARE patients and 0.83 ± 0.14 cm(2)/m(2) in sutureless patients (P = 0.040). The rate of patients with severe PPM was 6% (n = 2) in ARE patients and 11% (n = 8%) in sutureless patients (not significant, n.s.). The 30-day mortality rates were 2% (n = 2) in sutureless patients and 6% (n = 2) in ARE patients (n.s.). The 1- and 5-year survival rates of the sutureless group were 92 and 54% years, respectively, whereas the 1- and 5-year survival rates of the ARE group were 76% (n.s.). CONCLUSIONS: Although the sutureless valve patients received significantly more concomitant procedures, all operation-associated times were significantly shorter. Despite sutureless valve patients being older, the 30-day mortality and survival rates were comparable in the two groups. Since the indexed EOA was only slightly lower and the incidence of severe PPM was not significantly higher in the sutureless valve patients, we conclude that sutureless valve implantation is an alternative to conventional ARE to treat a small aortic annulus and avoid PPM, especially in geriatric patients who benefit from the quick implantation process.

Novel method for the generation of tissue-engineered vascular grafts based on a highly compacted fibrin matrix.

The generation of tissue-engineered blood vessel substitutes remains an ongoing challenge for cardiovascular tissue engineering. Full biocompatibility and immediate availability have emerged as central issues for clinical use. To address these issues, we developed a technique that allows the generation of highly stable tubular fibrin segments. The process is based on the compaction of fibrin in a custom-made high-speed rotation mold. In an automated process, fibrin is precipitated from plasma by means of the Vivostat® system. Following application to the rotating mold, the fibrin was compacted by centrifugal force and excess fluid was pressed out. This compaction results in increasing cross-links between the fibrin fibrils and a corresponding significant increase of biomechanical stability up to a burst strength of 230mm of mercury. The molding process allows for a simultaneous seeding procedure. In a first in vivo evaluation in a sheep model, segments of the carotid artery were replaced by tissue-engineered vascular grafts, generated immediately prior to implantation (n=6). Following subjection to the body's remodeling mechanisms, the segments showed a high structural similarity to a native artery after explantation at 6months. Thus, this technique may represent a powerful tool for the generation of biomechanically stable vascular grafts immediately prior to implantation. STATEMENT OF SIGNIFICANCE: Fibrin has previously been shown to be suitable as a matrix for the seeding of different celltypes and for that reason was widely used as scaffold in different fields of tissue engineering. Nevertheless, fibrin's lack of stability has strongly limited its application. Our study describes a novel moulding technique for the generation of a highly compacted fibrin matrix. Using this approach, it was possible to optimize the engineering process of tubular fibrin segments to provide bioartificial vascular grafts within one hour with sufficient stability for immediate implantation in the arterial system. Thus, this technique may represent a powerful tool to get closer to the ultimate aim of an optimal bioartificial vascular graft.

Aortic valve re-replacement after Bentall procedure with a biological valved conduit in a sheep model.

BACKGROUND AND AIM OF THE STUDY: The Bentall procedure is the 'gold standard' for the repair of a combined pathology of ascending aorta and aortic valve. Because there is no need for long-term anticoagulation, biological-valved conduits have become increasingly popular; however, the possible need for reoperation due to valve degeneration is a major disadvantage. The aim of this animal-based study was to prove the feasibility of an isolated replacement of the aortic valve prosthesis six months after a previous implantation of a biological valved conduit (BioValsalva) in a sheep model. A total aortic root replacement, using the BioValsalva conduit, was performed in 10 juvenile sheep. After six months, the surviving sheep were reoperated on, and the stentless valve was replaced with a stented biological valve placed inside the previously implanted vascular conduit. RESULTS: Five animals survived the initial implantation of a BioValsalva conduit. During reoperation, the triple-layered vascular graft with polytetrafluoroethylene on the outside showed only slight adhesions with the surrounding tissue. The stentless valve was removed in one piece, after which a new stented valve was implanted inside the conduit. An X-radiographic examination of the explanted valve showed moderate calcification of the leaflet, and severe calcification of the aortic wall. CONCLUSION: The results of this animal study confirmed that a degenerated stentless biological valve inside the BioValsalva conduit could be replaced with a new valve, without having to remove the entire conduit.

In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions.

The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.

Hypothermic circulatory arrest with and without cold selective antegrade cerebral perfusion: impact on neurological recovery and tissue metabolism in an acute porcine model.

OBJECTIVE: Clinically, selective antegrade cerebral perfusion (SACP) seems to be associated with a better neurological outcome compared to hypothermic circulatory arrest (HCA) alone, but the pathophysiological mechanisms are not well understood. Therefore, this study was undertaken to assess the effects of HCA with and without SACP on the cerebral integrity using multimodal neurophysiological monitoring. METHODS: 12 pigs were randomly assigned to 100 min HCA at 20 degrees C brain temperature with (n = 6) and without (n = 6) SACP. Haemodynamics, metabolics and neurophysiology (EEG, SSEP, ICP, spectroscopy, cerebral tissue monitoring) were monitored. Animals were sacrified 4 h after reperfusion and the brains perfused for histopathological assessment. RESULTS: There were no clinically relevant differences in hemodynamics between groups. During reperfusion, EEG and SSEP recovery was significantly faster in the SACP group (P < 0.05). The rise in ICP during reperfusion was markedly reduced in the SACP group (P < 0.01) for the trend). Three hours after reperfusion, median ICP was 130% compared to baseline in the SACP group and 225% in the HCA group (P < 0.01). Invasive as well as noninvasive cerebral monitoring indirectly indicates the occurrence of tissue acidosis in the HCA group even 4 h after HCA. CONCLUSIONS: Cold SACP is associated with better neurophysiological recovery and less cerebral edema, indicated by lower intracranial pressures during reperfusion. Neurophysiological recovery correlated well with the rise in ICP. HCA alone causes prolonged acidosis in the brain tissue during reperfusion. From these data, SACP appears to be superior to HCA alone, but further studies have to elucidate the optimal regimes for SACP.