In vitro and in vivo assessment of a novel ultra-flexible ventriculoamniotic shunt for treating fetal hydrocephalus.

Fetal aqueductal stenosis (AS) is one of the most common causes of congenital hydrocephalus, which increases intracranial pressure due to partial or complete obstruction of cerebrospinal fluid (CSF) flow within the ventricular system. Approximately 2-4 infants per 10,000 births develop AS, which leads to progressive hydrocephalus, which enlarges the head often necessitating delivery by cesarean section. Most babies born with AS are severely neurologically impaired and experience a lifetime of disability. Therefore, a new device technology for venticuloamniotic shunting is urgently needed and has been studied to ameliorate or prevent fetal hydrocephalus development, which can provide a significant impact on patients and their family's quality of life and on the decrease of the healthcare dollars spent for the treatment. This study has successfully validated the design of shunt devices and demonstrated the mechanical performance and valve functions. A functional prototype shunt has been fabricated and subsequently used in multiple in vitro tests to demonstrate the performance of this newly developed ventriculoamniotic shunt. The shunt contains a main silicone-nitinol composite tube, a superelastic 90° angled dual dumbbell anchor, and an ePTFE valve encased by a stainless-steel cage. The anchor will change its diameter from 1.15 mm (collapsed state) to 2.75 mm (deployed state) showing up to 1.4-fold diameter change in human body temperature. Flow rates in shunts were quantified to demonstrate the valve function in low flow rates mimicking the fetal hydrocephalus condition showing "no backflow" for the valved shunt while there is up to 15 mL/h flow through the shunt with pressure difference of 20 Pa. In vivo ovine study results show the initial successful device delivery and flow drainage with sheep model.

In-vivo assessment of a tissue engineered vascular graft computationally optimized for target vessel compliance.

Tissue engineered vascular grafts (TEVGs) have the ability to be tuned to match a target vessel's compliance, diameter, wall thickness, and thereby prevent compliance mismatch. In this work, TEVG compliance was manipulated by computationally tuning its layered composition or by manipulating a crosslinking agent (genipin). In particular, these three acelluluar TEVGs were compared: a compliance matched graft (CMgel - high gelatin content); a hypocompliant PCL graft (HYPOpcl - high polycaprolactone content); and a hypocompliant genipin graft (HYPOgen - equivalent composition as CMgel but hypocompliant via increased genipin crosslinking). All constructs were implanted interpositionally into the abdominal aorta of 21 Sprague Dawley rats (n=7, males=11, females=10) for 28 days, imaged in-vivo using ultrasound, explanted, and assessed for remodeling using immunofluorescence and two photon excitation fluorescence imaging. Compliance matched grafts remained compliance-matched in-vivo compared to the hypocompliant grafts through 4 weeks (p<0.05). Construct degradation and cellular infiltration was increased in the CMgel and HYPOgen TEVGs. Contractile smooth muscle cell markers in the proximal anastomosis of the graft were increased in the CMgel group compared to the HYPOpcl (p=0.007) and HYPOgen grafts (p=0.04). Both hypocompliant grafts also had an increased pro-inflammatory response (increased ratio of CD163 to CD86 in the mid-axial location) compared to the CMgel group. Our results suggest that compliance matching using a computational optimization approach leads to the improved acute (28 day) remodeling of TEVGs. To the authors' knowledge, this is the first in-vivo rat study investigating TEVGs that have been computationally optimized for target vessel compliance.

Acute In Vivo Functional Assessment of a Biodegradable Stentless Elastomeric Tricuspid Valve.

Degradable heart valves based on in situ tissue regeneration have been proposed as potentially durable and non-thrombogenic prosthetic alternatives. We evaluated the acute in vivo function, microstructure, mechanics, and thromboresistance of a stentless biodegradable tissue-engineered heart valve (TEHV) in the tricuspid position. Biomimetic stentless tricuspid valves were fabricated with poly(carbonate urethane)urea (PCUU) by double-component deposition (DCD) processing to mimic native valve mechanics and geometry. Five swine then underwent 24-h TEHV implantation in the tricuspid position. Echocardiography demonstrated good leaflet motion and no prolapse and trace to mild regurgitation in all but one animal. Histology revealed patches of proteinaceous deposits with no cellular uptake. SEM demonstrated retained scaffold microarchitecture with proteinaceous deposits but no platelet aggregation or thrombosis. Explanted PCUU leaflet thickness and mechanical anisotropy were comparable with native tricuspid leaflets. Bioinspired, elastomeric, stentless TEHVs fabricated by DCD were readily implantable and demonstrated good acute function in the tricuspid position.

Taking the Next Steps in Regenerative Rehabilitation: Establishment of a New Interdisciplinary Field.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

In vivo functional assessment of a novel degradable metal and elastomeric scaffold-based tissue engineered heart valve.

OBJECTIVE: Ideal heart valve solutions aim to provide thrombosis-free durability. A scaffold-based polycarbonate urethane urea tissue-engineered heart valve designed to mimic native valve microstructure and function was used. This study examined the acute in vivo function of a stented tissue-engineered heart valve in a porcine model. METHODS: Trileaflet valves were fabricated by electrospinning polycarbonate urethane urea using double component fiber deposition. The tissue-engineered heart valve was mounted on an AZ31 magnesium alloy biodegradable stent frame. Five 80-kg Yorkshire pigs underwent open tissue-engineered heart valve implantation on cardiopulmonary bypass in the pulmonary position. Tissue-engineered heart valve function was echocardiographically evaluated immediately postimplant and at planned study end points at 1, 4, 8, and 12 hours. Explanted valves underwent biaxial mechanical testing and scanning electron microscopy for ultrastructural analysis and thrombosis detection. RESULTS: All 5 animals underwent successful valve implantation. All were weaned from cardiopulmonary bypass, closed, and recovered until harvest study end point except 1 animal that was found to have congenital tricuspid valve dysplasia and that was euthanized postimplant. All 5 cases revealed postcardiopulmonary bypass normal leaflet function, no regurgitation, and an average peak velocity of 2 m/s, unchanged at end point. All tissue-engineered heart valve leaflets retained microstructural architecture with no platelet activation or thrombosis by scanning electron microscopy. There was microscopic evidence of fibrin deposition on 2 of 5 stent frames, not on the tissue-engineered heart valve. Biaxial stress examination revealed retained postimplant mechanics of tissue-engineered heart valve fibers without functional or ultrastructural degradation. CONCLUSIONS: A biodegradable elastomeric heart valve scaffold for in situ tissue-engineered leaflet replacement is acutely functional and devoid of leaflet microthrombosis.

Assessment of Thrombelastography and Platelet Life Span in Ovines.

Ovines are a common animal model for the study of cardiovascular devices, where consideration of blood biocompatibility is an essential design criterion. In the ovine model, tools to assess blood biocompatibility are limited and continued investigation to identify and apply additional assays is merited. Toward this end, the thrombelastograph, clinically utilized to assess hemostasis, was used to characterize normal ovine parameters. In addition, platelet labeling with biotin was evaluated for its potential applicability to quantify ovine platelet life span. Mean ovine thrombelastograph values were reaction-time: 4.9 min, K-time: 2 min, angle: 64.1°, maximum amplitude: 68.6mm, actual clot strength: 11.9 kd/s, and coagulation index: 1.5. Reaction time was significantly shorter and maximum amplitude, actual clot strength, and coagulation index were all significantly higher when compared to normal human thrombelastograph values suggesting some hypercoagulability of sheep blood. Biotinylation and reinfusion of ovine platelets allowed temporal tracking of the labeled platelet cohort with flow cytometry. These data indicated a mean ovine platelet life span of 188h with a half-life of 84h. The collection of these parameters for normal ovines demonstrates the applicability of these techniques for subsequent studies where cardiovascular devices may be evaluated and provides an indication of normal ovine values for comparison purposes.

Biocompatibility Assessment of the CentriMag-Novalung Adult ECMO Circuit in a Model of Acute Pulmonary Hypertension.

Extracorporeal membrane oxygenation (ECMO) is rarely used in patients with severe pulmonary hypertension (PH) as a bridge to lung transplantation. In this study, we assess the blood biocompatibility of the integrated CentriMag-Novalung ECMO system (venoarterial) in an acute model of PH. Severe PH (≥2/3 systemic) was induced in eight sheep through progressive ligation of the main pulmonary artery. System performance, platelet activation, thromboelastography (TEG) parameters, fibrinogen, plasma-free hemoglobin, and total plasma protein were measured at initiation, 3, and 6 hr of support in the ECMO (N = 4) and sham (N = 4) groups. A stable ECMO flow (2.2 ± 0.1 L/min), low transmembrane pressure gradient, and steady blood O2 and CO2 levels were maintained. Platelet activation was low (<4%) in both the groups, whereas platelet responsiveness to agonist (platelet activating factor) was reduced in the sham group when compared with the ECMO group. There were no differences in the TEG parameters, fibrinogen concentration, plasma-free hemoglobin (<10 mg/dl), and plasma total protein between the two groups. The findings of low levels of platelet activation and plfHb suggest adequate blood biocompatibility of the integrated CentriMag-Novalung circuit use for short-term support in a model of PH.

Biocompatibility assessment of the first generation PediaFlow pediatric ventricular assist device.

The PediaFlow pediatric ventricular assist device is a miniature magnetically levitated mixed flow pump under development for circulatory support of newborns and infants (3-15 kg) with a targeted flow range of 0.3-1.5 L/min. The first generation design of the PediaFlow (PF1) was manufactured with a weight of approximately 100 g, priming volume less than 2 mL, length of 51 mm, outer diameter of 28 mm, and with 5-mm blood ports. PF1 was evaluated in an in vitro flow loop for 6 h and implanted in ovines for three chronic experiments of 6, 17, and 10 days. In the in vitro test, normalized index of hemolysis was 0.0087 ± 0.0024 g/100L. Hemodynamic performance and blood biocompatibility of PF1 were characterized in vivo by measurements of plasma free hemoglobin, plasma fibrinogen, total plasma protein, and with novel flow cytometric assays to quantify circulating activated ovine platelets. The mean plasma free hemoglobin values for the three chronic studies were 4.6 ± 2.7, 13.3 ± 7.9, and 8.8 ± 3.3 mg/dL, respectively. Platelet activation was low for portions of several studies but consistently rose along with observed animal and pump complications. The PF1 prototype generated promising results in terms of low hemolysis and platelet activation in the absence of complications. Hemodynamic results validated the magnetic bearing design and provided the platform for design iterations to meet the objective of providing circulatory support for young children with exceptional biocompatibility.

Hemocompatibility assessment of carbonic anhydrase modified hollow fiber membranes for artificial lungs.

Hollow fiber membrane (HFM)-based artificial lungs can require a large blood-contacting membrane surface area to provide adequate gas exchange. However, such a large surface area presents significant challenges to hemocompatibility. One method to improve carbon dioxide (CO(2)) transfer efficiency might be to immobilize carbonic anhydrase (CA) onto the surface of conventional HFMs. By catalyzing the dehydration of bicarbonate in blood, CA has been shown to facilitate diffusion of CO(2) toward the fiber membranes. This study evaluated the impact of surface modifying a commercially available microporous HFM-based artificial lung on fiber blood biocompatibility. A commercial poly(propylene) Celgard HFM surface was coated with a siloxane, grafted with amine groups, and then attached with CA which has been shown to facilitate diffusion of CO(2) toward the fiber membranes. Results following acute ovine blood contact indicated no significant reduction in platelet deposition or activation with the siloxane coating or the siloxane coating with grafted amines relative to base HFMs. However, HFMs with attached CA showed a significant reduction in both platelet deposition and activation compared with all other fiber types. These findings, along with the improved CO(2) transfer observed in CA modified fibers, suggest that its incorporation into HFM design may potentiate the design of a smaller, more biocompatible HFM-based artificial lung.

In vivo assessment of a tissue-engineered vascular graft combining a biodegradable elastomeric scaffold and muscle-derived stem cells in a rat model.

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for bypass or replacement of small-diameter arteries remain a concern in the surgical community. These limitations could potentially be improved by a tissue engineering approach. We report here our progress in the development and in vivo testing of a stem-cell-based tissue-engineered vascular graft for arterial applications. Poly(ester urethane)urea scaffolds (length = 10 mm; inner diameter = 1.2 mm) were created by thermally induced phase separation (TIPS). Compound scaffolds were generated by reinforcing TIPS scaffolds with an outer electrospun layer of the same biomaterial (ES-TIPS). Both TIPS and ES-TIPS scaffolds were bulk-seeded with 10 x 10(6) allogeneic, LacZ-transfected, muscle-derived stem cells (MDSCs), and then placed in spinner flask culture for 48 h. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks. Angiograms and histological assessment were performed at the time of explant. Cell-seeded constructs showed a higher patency rate than the unseeded controls: 65% (ES-TIPS) and 53% (TIPS) versus 10% (acellular TIPS). TIPS scaffolds had a 50% mechanical failure rate with aneurysmal formation, whereas no dilation was observed in the hybrid scaffolds. A smooth-muscle-like layer of cells was observed near the luminal surface of the constructs that stained positive for smooth muscle alpha-actin and calponin. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of von Willebrand Factor-positive cells was observed in the lumen of MDSC-seeded constructs, whereas acellular controls showed platelet and fibrin deposition. This is the first evidence that MDSCs improve patency and contribute to the remodeling of a tissue-engineered vascular graft for arterial applications.

A three-dimensional gel bioreactor for assessment of cardiomyocyte induction in skeletal muscle-derived stem cells.

Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

Assessment of hydraulic performance and biocompatibility of a MagLev centrifugal pump system designed for pediatric cardiac or cardiopulmonary support.

The treatment of children with life-threatening cardiac and cardiopulmonary failure is a large and underappreciated public health concern. We have previously shown that the CentriMag is a magnetically levitated centrifugal pump system, having the utility for treating adults and large children (1,500 utilized worldwide). We present here the PediVAS, a pump system whose design was modified from the CentriMag to meet the physiological requirements of young pediatric and neonatal patients. The PediVAS is comprised of a single-use centrifugal blood pump, reusable motor, and console, and is suitable for right ventricular assist device (RVAD), left ventricular assist device (LVAD), biventricular assist device (BVAD), or extracorporeal membrane oxygenator (ECMO) applications. It is designed to operate without bearings, seals and valves, and without regions of blood stasis, friction, or wear. The PediVAS pump is compatible with the CentriMag hardware, although the priming volume was reduced from 31 to 14 ml, and the port size reduced from 3/8 to (1/4) in. For the expected range of pediatric flow (0.3-3.0 L/min), the PediVAS exhibited superior hydraulic efficiency compared with the CentriMag. The PediVAS was evaluated in 14 pediatric animals for up to 30 days, demonstrating acceptable hydraulic function and hemocompatibility. The current results substantiate the performance and biocompatibility of the PediVAS cardiac assist system and are likely to support initiation of a US clinical trial in the future.

Preclinical biocompatibility assessment of the EVAHEART ventricular assist device: coating comparison and platelet activation.

Thromboembolism and bleeding remain significant complications of ventricular assist device (VAD) support. Increasing the amount of biocompatibility data collected during preclinical studies can provide additional criteria to evaluate device refinements, while design changes may be implemented before entering clinical use. Twenty bovines were implanted with the EVAHEART centrifugal VAD for durations from 30 to 196 days. Titanium alloy pumps were coated with either diamond-like carbon or 2-methoxyethyloylphosphoryl choline (MPC). Activated platelets and platelet microaggregates were quantified by flow cytometry, including two new assays to quantify bovine platelets expressing CD62P and CD63. Temporally, all assays were low preoperatively, then significantly increased following VAD implantation, before declining to a lower, but still elevated level over 2-3 weeks. MPC-coated VADs produced significantly fewer activated platelets after implant trauma effects diminished. Three animals receiving no postoperative anticoagulation had similar amounts of circulating activated platelets and platelet microaggregates as animals receiving warfarin anticoagulation. Two new methods to quantify bovine activated platelets using antibodies to CD62P and CD63 were characterized and applied. These measures, along with previously described assays, were able to differentiate between two biocompatible coatings and assess effects of anticoagulation regimen in VAD preclinical testing.

Blood biocompatibility assessment of an intravenous gas exchange device.

To treat acute lung failure, an intravenous membrane gas exchange device, the Hattler Catheter, is currently under development. Several methods were employed to evaluate the biocompatibility of the device during preclinical testing in bovines, and potential coatings for the fibers comprising the device were screened for their effectiveness in reducing thrombus deposition in vitro. Flow cytometric analysis demonstrated that the device had the capacity to activate platelets as evidenced by significant increases in circulating platelet microaggregates and activated platelets. Thrombus was observed on 20 +/- 6% of the surface area of devices implanted for up to 53 h. Adding aspirin to the antithrombotic therapy permitted two devices to remain implanted up to 96 h with reduced platelet activation and only 3% of the surface covered with thrombus. The application of heparin-based coatings significantly reduced thrombus deposition in vitro. The results suggest that with the use of appropriate antithrombotic therapies and surface coatings the Hattler Catheter might successfully provide support for acute lung failure without thrombotic complications.

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx.

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.