Testing of microencapsulated porcine hepatocytes in a new model of fulminant liver failure in baboons.

BACKGROUND: There is no standard therapy for acute liver failure. Hepatocyte transplantation has been proposed for temporary liver function support, while the injured liver regenerates or while waiting for transplantation. We have previously shown such efficacy for microencapsulated porcine hepatocytes in mice with fulminant liver failure. We aimed to establish a large animal model for fulminant liver failure to assess the efficacy of microencapsulated porcine hepatocytes in temporary liver function support. METHODS: The model was developed in baboons; for testing microencapsulated hepatocytes, the best condition was 75% hepatectomy and 60 min warm ischemia time. Fulminant liver failure was characterized by steep increases in liver biochemical parameters, severe steatosis, and massive hepatocyte necrosis during the first 10 days. Hepatocytes from miniature swine were microencapsulated in alginate-poly-l-lysine microspheres, and transplanted intraperitoneally immediately after hepatectomy and warm ischemia (80-120 mL packed hepatocytes in 200-350 mL microspheres, about 30%-50% of the baboon's native liver volume). RESULTS: In the control group, three of five animals were sacrificed after 6-10 days because of fulminant liver failure, and two of five animals recovered normal liver function and survived until elective euthanasia (28 days). In the treatment group of four animals, one animal developed liver failure but survived to 21 days, and three animals recovered completely with normal liver function. CONCLUSIONS: The results indicate that microencapsulated porcine hepatocytes provide temporary liver function support in baboons with fulminant liver failure. These data support development of this cell therapy product toward clinical trials in patients with acute liver failure.

Cardioscopic Tool-delivery Instrument for Beating-heart Surgery.

This paper describes an instrument that provides solutions to two open challenges in beating-heart intracardiac surgery - providing high-fidelity imaging of tool-tissue contact and controlling tool penetration into tissue over the cardiac cycle. Tool delivery is illustrated in the context of tissue removal for which these challenges equate to visualization of the tissue as it is being removed and to control of cutting depth. Cardioscopic imaging is provided by a camera and illumination system encased in an optical window. When the optical window is pressed against tissue, it displaces the blood between the camera and tissue allowing clear visualization. Control of cutting depth is achieved via precise extension of the cutting tool from a port in the optical window. Successful tool use is demonstrated in ex vivo and in vivo experiments.

Increased transfusion-free survival following auxiliary pig liver xenotransplantation.

BACKGROUND: Pig to baboon liver xenotransplantation typically results in severe thrombocytopenia and coagulation disturbances, culminating in death from hemorrhage within 9 days, in spite of continuous transfusions. We studied the contribution of anticoagulant production and clotting pathway deficiencies to fatal bleeding in baboon recipients of porcine livers. METHODS: By transplanting liver xenografts from α1,3-galactosyltransferase gene-knockout (GalT-KO) miniature swine donors into baboons as auxiliary organs, leaving the native liver in place, we provided the full spectrum of primate clotting factors and allowed in vivo mixing of porcine and primate coagulation systems. RESULTS: Recipients of auxiliary liver xenografts develop severe thrombocytopenia, comparable to recipients of conventional orthotopic liver xenografts and consistent with hepatic xenograft sequestration. However, baboons with both pig and native livers do not exhibit clinical signs of bleeding and maintain stable blood counts without transfusion for up to 8 consecutive days post-transplantation. Instead, recipients of auxiliary liver xenografts undergo graft failure or die of sepsis, associated with thrombotic microangiopathy in the xenograft, but not the native liver. CONCLUSION: Our data indicate that massive hemorrhage in the setting of liver xenotransplantation might be avoided by supplementation with primate clotting components. However, coagulation competent hepatic xenograft recipients may be predisposed to graft loss related to small vessel thrombosis and ischemic necrosis.

Tissue formation and host remodeling of an elastomeric biodegradable scaffold in an ovine pulmonary leaflet replacement model.

In pursuit of a suitable scaffold material for cardiac valve tissue engineering applications, an acellular, electrospun, biodegradable polyester carbonate urethane urea (PECUU) scaffold was evaluated as a pulmonary valve leaflet replacement in vivo. In sheep (n = 8), a single pulmonary valve leaflet was replaced with a PECUU leaflet and followed for 1, 6, and 12 weeks. Implanted leaflet function was assessed in vivo by echocardiography. Explanted samples were studied for gross pathology, microscopic changes in the extracellular matrix, host cellular re-population, and immune responses, and for biomechanical properties. PECUU leaflets showed normal leaflet motion at implant, but decreased leaflet motion and dimensions at 6 weeks. The leaflets accumulated α-SMA and CD45 positive cells, with surfaces covered with endothelial cells (CD31+). New collagen formation occurred (Picrosirius Red). Accumulated tissue thickness correlated with the decrease in leaflet motion. The PECUU scaffolds had histologic evidence of scaffold degradation and an accumulation of pro-inflammatory/M1 and anti-inflammatory/M2 macrophages over time in vivo. The extent of inflammatory cell accumulation correlated with tissue formation and polymer degradation but was also associated with leaflet thickening and decreased leaflet motion. Future studies should explore pre-implant seeding of polymer scaffolds, more advanced polymer fabrication methods able to more closely approximate native tissue structure and function, and other techniques to control and balance the degradation of biomaterials and new tissue formation by modulation of the host immune response.

Optically-guided instrument for transapical beating-heart delivery of artificial mitral chordae tendineae.

OBJECTIVE: We sought to develop an instrument that would enable the delivery of artificial chordae tendineae (ACT) using optical visualization of the leaflet inside the beating heart. METHODS: A delivery instrument was developed together with an ACT anchor system. The instrument incorporates an optically clear silicone grasping surface in which are embedded a camera and LED for direct leaflet visualization during localization, grasping, and chordal delivery. ACTs, comprised of T-shaped anchors and an expanded polytetrafluoroethylene chordae, were fabricated to enable testing in a porcine model. Ex vivo experiments were used to measure the anchor tear-out force from the mitral leaflets. In vivo experiments were performed in which the mitral leaflets were accessed transapically using only optical guidance and ACTs were deployed in the posterior and anterior leaflets (P2 and A2 segments). RESULTS: In 5 porcine ex vivo experiments, the mean force required to tear the anchors from the leaflets was 3.8 ± 1.2 N. In 5 porcine in vivo nonsurvival procedures, 14 ACTs were successfully placed in the leaflets (9 in P2 and 5 in A2). ACT implantation took an average of 3.22 ± 0.83 minutes from entry to exit through the apex. CONCLUSIONS: Optical visualization of the mitral leaflet during chordal placement is feasible and provides direct feedback to the operator throughout the deployment sequence. This enables visual confirmation of the targeted leaflet location, distance from the free edge, and successful deployment of the chordal anchor. Further studies are needed to refine and assess the device for clinical use.

Xenogeneic Heterotopic Auxiliary Liver transplantation (XHALT) promotes native liver regeneration in a Post-Hepatectomy Liver failure model.

The liver's regenerative capacity is unique, but too small a segment can overwhelm its ability to simultaneously regenerate and support the host, resulting in liver dysfunction and death. Here we tested a temporary Xenogeneic Heterotopic Auxiliary Liver Transplant (XHALT) from Gal-KO miniature swine in a baboon model of Post-Hepatectomy Liver Failure (PHLF) by 90%- hepatectomy. Immunosuppression consisted of CVF, ATG, FK 506 and steroids. 90%-hepatectomized animals died within 4-5 days with the clinical picture of PHLF, (high LFTs and bilirubin, ascites, encephalopathy and coagulopathy). The 10% remnants had macroscopic and histological evidence of severe steatosis and absence of hepatocyte replication. In contrast, the addition of XHALT prolonged survival up to 11 days, with the cause of death being sepsis, rather than liver failure. The remnant liver appeared grossly normal, and on histology, there was no evidence of fatty infiltration, but there was pronounced Ki-67 staining. In conclusion, temporary auxiliary xenografts have the potential to support a small for size liver graft while it grows to adequate size or provide an opportunity for organ recovery in acute liver failure.

In vivo tissue regeneration with robotic implants.

Robots that reside inside the body to restore or enhance biological function have long been a staple of science fiction. Creating such robotic implants poses challenges both in signaling between the implant and the biological host, as well as in implant design. To investigate these challenges, we created a robotic implant to perform in vivo tissue regeneration via mechanostimulation. The robot is designed to induce lengthening of tubular organs, such as the esophagus and intestines, by computer-controlled application of traction forces. Esophageal testing in swine demonstrates that the applied forces can induce cell proliferation and lengthening of the organ without a reduction in diameter, while the animal is awake, mobile, and able to eat normally. Such robots can serve as research tools for studying mechanotransduction-based signaling and can also be used clinically for conditions such as long-gap esophageal atresia and short bowel syndrome.

A low-cost bioprosthetic semilunar valve for research, disease modelling and surgical training applications.

OBJECTIVES: This paper provides detailed instructions for constructing low-cost bioprosthetic semilunar valves for animal research and clinical training. This work fills an important gap between existing simulator training valves and clinical valves by providing fully functioning designs that can be employed in ex vivo and in vivo experiments and can also be modified to model valvular disease. METHODS: Valves are constructed in 4 steps consisting of creating a metal frame, covering it with fabric and attaching a suture ring and leaflets. Computer-aided design files are provided for making the frame from wire or by metal 3D printing. The covering fabric and suturing ring are made from materials readily available in a surgical lab, while the leaflets are made from pericardium. The entire fabrication process is described in figures and in a video. To demonstrate disease modelling, design modifications are described for producing paravalvular leaks, and these valves were evaluated in porcine ex vivo (n = 3) and in vivo (n = 6) experiments. RESULTS: Porcine ex vivo and acute in vivo experiments demonstrate that the valves can replicate the performance of clinical valves for research and training purposes. Surgical implantation is similar, and echocardiograms are comparable to clinical valves. Furthermore, valve leaflet function was satisfactory during acute in vivo tests with little central regurgitation, while the paravalvular leak modifications consistently produced leaks in the desired locations. CONCLUSIONS: The detailed design procedure presented here, which includes a tutorial video and computer-aided design files, should be of substantial benefit to researchers developing valve disease models and to clinicians developing realistic valve training systems.

Cardioscopically Guided Beating Heart Surgery: Paravalvular Leak Repair.

PURPOSE: There remains a paucity of direct visualization techniques for beating-heart intracardiac procedures. To address this need, we evaluated a novel cardioscope in the context of aortic paravalvular leaks (PVLs) localization and closure. DESCRIPTION: A porcine aortic PVL model was created using a custom-made bioprosthetic valve, and PVL presence was verified by epicardial echocardiography. Transapical delivery of occlusion devices guided solely by cardioscopy was attempted 13 times in a total of three pigs. Device retrieval after release was attempted six times. Echocardiography, morphologic evaluation, and delivery time were used to assess results. EVALUATION: Cardioscopic imaging enabled localization of PVLs via visualization of regurgitant jet flow in a paravalvular channel at the base of the prosthetic aortic valve. Occluders were successfully placed in 11 of 13 attempts (84.6%), taking on average 3:03 ± 1:34 min. Devices were cardioscopically removed successfully in three of six attempts (50%), taking 3:41 ± 1:46 min. No damage to the ventricle or annulus was observed at necropsy. CONCLUSIONS: Cardioscopy can facilitate intracardiac interventions by providing direct visualization of anatomic structures inside the blood-filled, beating-heart model.

Soft robotic sleeve supports heart function.

There is much interest in form-fitting, low-modulus, implantable devices or soft robots that can mimic or assist in complex biological functions such as the contraction of heart muscle. We present a soft robotic sleeve that is implanted around the heart and actively compresses and twists to act as a cardiac ventricular assist device. The sleeve does not contact blood, obviating the need for anticoagulation therapy or blood thinners, and reduces complications with current ventricular assist devices, such as clotting and infection. Our approach used a biologically inspired design to orient individual contracting elements or actuators in a layered helical and circumferential fashion, mimicking the orientation of the outer two muscle layers of the mammalian heart. The resulting implantable soft robot mimicked the form and function of the native heart, with a stiffness value of the same order of magnitude as that of the heart tissue. We demonstrated feasibility of this soft sleeve device for supporting heart function in a porcine model of acute heart failure. The soft robotic sleeve can be customized to patient-specific needs and may have the potential to act as a bridge to transplant for patients with heart failure.

Monitoring radiofrequency renal lesions in real time using contrast-enhanced ultrasonography: a porcine model.

BACKGROUND AND PURPOSE: Ablation by cold (cryoablation) or radiofrequency energy (RFA), has been popularized for the treatment of small renal tumors. Regrettably, there currently is no reliable method of radiologically monitoring the propagation of RF lesions in real time. Ultrasonography enhanced by gas-filled microbubble contrast agents allows depiction of regions of tissue perfusion and has been described as a useful adjunct in diagnosing renal pseudotumors, improving prostate biopsy results, and confirming successful ablation of liver tumors. We hypothesized that contrast-enhanced ultrasonography (CEUS) would allow us to define, in real time, areas of cell death secondary to RFA and thus determine successful treatment. MATERIALS AND METHODS: Five female swine underwent initial laparoscopic exploration and creation of ipsilateral upper- and lower-pole renal RFA lesions. Lesion size was measured with standard gray-scale, Doppler, and microbubble CEUS. After 2 weeks, an identical procedure was performed on the contralateral kidney, including repeat sonographic measurements on the first kidney. All swine were then immediately sacrificed, and both kidneys (20 lesions) were harvested for pathologic analysis (hematoxylin-eosin and nicotinamide adenine dinucleotide stains). Radiographic lesion size was then compared with the gross and microscopic findings. RESULTS: The RFA lesions could not be imaged accurately in real time with standard gray-scale or Doppler sonography. However, microbubble CEUS was able to monitor parenchymal blood flow and, thus, the lesions (no blood flow) in real time. Hypoechoic lesions (no bubble enhancement) imaged during contrast sonography corresponded with regions of cell death as demonstrated on pathologic analysis. CONCLUSIONS: Microbubble CEUS is can monitor RFA lesions in real time. This novel imaging modality should allow more effective renal tumor ablation.

Time management in the operating room: an analysis of the dedicated minimally invasive surgery suite.

BACKGROUND: Dedicated minimally invasive surgery suites are available that contain specialized equipment to facilitate endoscopic surgery. Laparoscopy performed in a general operating room is hampered by the multitude of additional equipment that must be transported into the room. The objective of this study was to compare the preparation times between procedures performed in traditional operating rooms versus dedicated minimally invasive surgery suites to see whether operating room efficiency is improved in the specialized room. METHODS: The records of 50 patients who underwent laparoscopic procedures between September 2000 and April 2002 were retrospectively reviewed. Twenty-three patients underwent surgery in a general operating room and 18 patients in an minimally invasive surgery suite. Nine patients were excluded because of cystoscopic procedures undergone prior to laparoscopy. Various time points were recorded from which various time intervals were derived, such as preanesthesia time, anesthesia induction time, and total preparation time. A 2-tailed, unpaired Student t test was used for statistical analysis. RESULTS: The mean preanesthesia time was significantly faster in the minimally invasive surgery suite (12.2 minutes) compared with that in the traditional operating room (17.8 minutes) (P=0.013). Mean anesthesia induction time in the minimally invasive surgery suite (47.5 minutes) was similar to time in the traditional operating room (45.7 minutes) (P=0.734). The average total preparation time for the minimally invasive surgery suite (59.6 minutes) was not significantly faster than that in the general operating room (63.5 minutes) (P=0.481). CONCLUSION: The amount of time that elapses between the patient entering the room and anesthesia induction is statically shorter in a dedicated minimally invasive surgery suite. Laparoscopic surgery is performed more efficiently in a dedicated minimally invasive surgery suite versus a traditional operating room.

Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells.

BACKGROUND: Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies. METHODS: Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α(M)ß(2) integrin Ab) were tested for the ability to inhibit phagocytosis. RESULTS: None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01). CONCLUSIONS: Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.

Pediatric intestinal retransplantation: techniques, management, and outcomes.

BACKGROUND: Intestinal retransplantation (Re-ITx) has historically been associated with high morbidity and mortality. METHODS: The outcomes of all children receiving Re-ITx between 1990 and 2007 at our center were reviewed. RESULTS: One hundred seventy-two children received primary intestinal grafts. Fourteen children (8.1%) were retransplanted with 15 grafts. Causes of graft failure were acute cellular rejection (ACR, n=4), liver failure (n=2), chronic rejection (n=3), posttransplant lymphoproliferative disorder (n=1), graft dysmotility or dysfunction (n=3), ACR with severe infection (n=1), and arterial graft aneurysm (n=1). Initial transplants were isolated bowel in nine, liver-bowel in five, and one multivisceral. The mean time of initial graft survival was 34.2 months. Re-ITx was with isolated bowel in two, liver-bowel in four, and multivisceral in nine (four with kidney). Initial immunosuppression was Tac-Pred based in nine and rabbit antithymocyte globulin-Tac based in six cases. Re-ITx was carried out under Tac-Pred in six, rabbit antithymocyte globulin-Tac in eight, and alemtuzumab monoclonal anti-CD52 antibody in one. Ten (71.4%) patients are alive with functioning grafts at a mean current follow-up time of 55.9 months. Four patients died from posttransplant lymphoproliferative disorder, severe ACR, fungal sepsis, and bleeding from pseudoaneurysm, respectively, at a mean time of 5.7 months post-Re-ITx. All surviving patients weaned-off total parenteral nutrition at a median time of 32 days and 90% are off intravenous fluids. CONCLUSIONS: Improved long-term survival and outcome in pediatric Re-ITx may be attributed to improvements in initial immunosuppression protocols, technical modifications, proper timing, and improved infectious disease monitoring. Careful patient selection and posttransplant management are essential for successful long-term outcome.

Randomized comparison of standard laparoscopic trainer to novel, at-home, low-cost, camera-less laparoscopic trainer.

OBJECTIVES: To evaluate the utility of a portable, at-home, low-cost, camera-less laparoscopic trainer to provide laparoscopy training inexpensively and outside the constraints of the hospital environment. METHODS: Twenty-two urology residents (postgraduate year 1 to 7) were tested prospectively on four basic laparoscopic tasks on a standard video laparoscopic trainer (pretest). Objective and subjective data were collected for each participant. The subjects were then randomized to 5 hours of training on either a portable, camera-less trainer (group 1) or a standard video laparoscopic trainer (group 2). Each resident was then retested on the initial same four laparoscopic tasks (post-test). Efficiency ratios were calculated for each task, incorporating both time and accuracy. Improvements between the pretest and post-test evaluations were compared between the two groups using the two-group t test. RESULTS: All subjects significantly improved their performance with training. The average improvement in the time required to complete the laparoscopic tasks for all participants was 36%. Efficiency increased by 52%. However, no difference was found between the two groups. In the poststudy self-evaluations, 91% of participants in group 1 either agreed or strongly agreed that the at-home trainer was a helpful teaching modality. CONCLUSIONS: The improvements in laparoscopic skills on our inexpensive, portable, at-home, camera-less trainer were not significantly different from those gained using a traditional video laparoscopic trainer. Participants trained on our portable trainer were able to improve objectively and subjectively by both external and internal evaluations. Thus, our system will allow trainees to develop their laparoscopic skills in an inexpensive manner in the comfort of their own home.