Vanderbilt University’s Institutional Animal Care and Use Committee approved the operative protocol for hemofilter cartridge implantation in class A dogs. The animals were anesthetized with isoflurane. A total of 100 international unit (IU)/kg unfractionated heparin was administered as a bolus at the time of vascular isolation to achieve adequate levels of anticoagulation throughout the surgery. An upper midline laparotomy incision was made to gain entry into the peritoneal cavity. A combination of blunt and sharp dissection was performed to medially rotate the abdominal viscera to expose the left retroperitoneum, aorta, and inferior vena cava (IVC). The aorta and IVC were dissected free of surrounding tissue. Before placing a vascular clamp on the aorta to perform the atrial anastomosis, an additional 1,000 IU of heparin was administered. Seven millimeter-ringed polytetrafluoroethylene (PTFE) grafts were used to create the venous and arterial anastomosis to the IVC and aorta, respectively. The graft length was approximately 2.5–3.0 cm for each vascular anastomosis. The arterial inflow and venous outflow PTFE grafts were connected to the hemofilter. Blood flow was established through the hemofilter. The hemofilter was secured to the psoas muscle adjacent to the inferior pole of the left kidney in the retroperitoneum. Two separate effluent reservoir bags that collect filtered fluid from across each membrane were placed in the upper abdomen (Figure 3). Hemostasis was ensured, and the midline incision was reapproximated and sutured.
Postoperatively, the animals were housed without restrictions. Each animal received 1.5 mg/kg of acetylsalicylic acid orally twice each day. Blood velocities in the grafts were measured with Doppler ultrasound to estimate blood flow through the hemofilter. Centerline velocities were recorded over multiple cardiac cycles and integrated to estimate time-averaged blood velocity, and assuming Poiseuille flow, volumetric flow rate. On postoperative day 3 (n = 2), 4 (n = 2), 5 (n = 1), or 8 (n = 1), the animal was placed under general anesthetic and the hemofilter and effluent reservoir bags were retrieved. At the completion of the explantation, the animal was euthanized with pentobarbital (Fatal-Plus Solution, Vortech Pharmaceuticals, Dearborn, MI) while under anesthesia.
After explantation, all membranes were examined by light microscopy for defects. Albumin, hemoglobin, and lactate dehydrogenase (LDH) were measured before surgery and at explant in animals 4, 5, and 6 by drawing samples from intravenous catheters. Albumin sieving coefficient was calculated from the ratio of albumin concentration in the filtrate divided by the albumin concentration in the blood. Random samples of each lobe of both lungs were excised after sacrifice and placed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Nano- and microfabrication processes resulted in 1 × 1 cm silicon chips bearing 430 nm-thick nanopore membranes with total membrane area 3.6 × 10−5 m2 (36 mm2) (Figure 1). Estimated pore sizes in PEG-coated silicon nanopore membranes were similar to design targets of 5–7 nm (Table 1). In vitro sieving measurements demonstrated size-dependent retention of macromolecules (Table 1). Benchtop filtration rates at 103 mm Hg averaged 1.0 µl/min. All experimental animals tolerated the procedure well. Mean preoperative weight was 25.3 ± 1.86 kg, and mean weight gain over the course of the experiment was −0.2 ± 0.2 kg. One animal limped for a day after the surgery and was thought to have a transient nerve compression related to positioning during surgery. The limp spontaneously resolved. There was no evidence for severe hemolysis or high-output heart failure, such as volume overload, in any animal. In the three animals in which membranes remained intact, there was a trend toward increase in serum LDH from before the implant to time of explant (65.3 ± 4.7 IU vs. 175 ± 56 IU, p > 0.05 by t-test) and a trend toward decrease in hemoglobin (17.5 ± 0.4 g/L vs. 13.9 ± 1.0 g/L, p > 0.05). Histologic examination of wedge biopsies of each lobe of each animal’s lungs revealed normal lung architectures with occasional atelectasis. One biopsy showed an organizing thrombus that was several days old at the time of explant. This thromboembolus was in the same animal that evinced the transient limp, raising the possibility that the thromboembolus arose from that limb.
All cartridges had continuous blood flow without thrombosis. Peak blood flow velocities in the cartridge as measured by Doppler ultrasound were between 300 cm/sec, corresponding to a time-averaged volumetric flow of approximately 900 ml/min. There was no clear evidence of a time trend in blood flow rate from postoperative day 1 to time of explant.
At explant, animals 1–3 with experimental durations 4–8 days had much larger total volumes of filtered fluid (30–50 ml) than predicted from in vitro measurements (3–8 ml). Filtrate albumin concentrations in these experiments were higher than predicted (θ ~ 0.5 − 0.8) and higher than the 4 nm Ficoll sieving coefficients measured in vitro (Table 1). In animals 1–3 where UF rates and albumin sieving coefficients were higher than predicted, small areas where the silicon membrane appeared to have delaminated from its supporting structure were identified by light microscopy. We hypothesized that some time-dependent factor, as yet unknown, was leading to device failure after multiple days. Silicon nanopore membranes were implanted over shorter 2 to 3 day durations in animals 3–6. Albumin sieving coefficients in animals 3–6 (θ ~ 0.23 − 0.30) were similar to predictions from preimplant filtration tests using globular polysaccharides (Table 1).
The major barriers to clinical success of permanently implanted hemofiltration are thrombosis and fouling. In vitro, some protein fouling does occur immediately and appears self-limited.4,9 The technique for blood biocompatibility in these experiments, PEG, is a highly hydrated polymer that blocks protein adsorption to surfaces and does not promote thrombosis or complement activation.7 Polyethylene glycol was chosen for this set of experiments because there is a simple solution-phase procedure available to bond PEG to silicon.10,13 Unfortunately, PEG undergoes hydrolysis in aqueous solutions, so experiments are limited to days to weeks at most. Other polymer coatings appear to provide stable interfaces between blood and materials at the cost of more complex coating procedures.7,14
The observed UF rates and pore sizes calculated from observed hydraulic permeability generally were in good agreement with design goals and sieving coefficients of albumin and globular polysaccharides. Peritoneal patients on dialysis tolerate dialytic albumin losses on the order of 4 g per day or 2.7 mg/min.15 For an implanted hemofilter device, assuming no tubular uptake of albumin, an albumin sieving coefficient around 0.1% will be necessary. In prior work, albumin sieving coefficients below the level of detection of the instrumentation have been observed from silicon nanopore membranes with smaller pore sizes than those tested here.16
The observation that in a series of experiments, membranes suffered localized mechanical failures that had not been previously observed in bench testing is noteworthy. Silicon film membranes are brittle ceramic-like materials that do not appear to undergo progressive fatigue-based failure, but instead are best described by a probabilistic failure analysis.17,18 The concern that a small failure, presumably attributable to a manufacturing process variation, would result in catastrophic failure and exsanguination is not supported by the results here. The consequence of membrane fracture appears to be limited in the short term by a self-sealing microscopic thrombus. The mechanisms by which the first implanted hemofilters failed remain unclear but are thought to be an undetected flaw in the silicon processing. The pattern of damage, namely, localized punch through between adjacent pores at apparently random locations in the membrane, was unlike damage patterns observed with overpressure or rough handling that are characterized by large fractures, typically at membrane edges. The mechanisms by which the failures were related to implant duration are unknown.
Nevertheless, the use of cardiac perfusion pressure to enable continuous and selective hemofiltration by silicon nanopore membranes without external connections or implanted power supply is a significant achievement. Pump-free operation decreases the overall size of the implantable device and increases the likelihood of success of long-term operation by eliminating the need for bulky (and toxic) batteries or transcutaneous (and infection-prone) connections. Just as significant is the ability of the hemofilter to operate without chronic, long-term systemic anticoagulation, which is generally required with any implanted blood-contacting device. Longer implantation periods and larger devices are required to definitively establish whether the hemofilter can operate without any anticoagulation whatsoever. A range of 5–7 mm PTFE grafts used in vascular surgery have excellent patency rates, whereas those used in hemodialysis access may be more problematic. Other groups have used similar diameter vascular grafts to engineer vascularized implants, highlighting the long-term feasibility of this strategy.19,20 The possibility of avoiding systemic anticoagulation minimizes the risks such as hemorrhage and thrombocytopenia associated with renal replacement therapy.
The key findings of this manuscript are that in a small pilot and feasibility study in animals, the hemofiltration cartridge remained patent and thrombus free with only aspirin as an anticoagulant. The silicon membranes that remained intact during the experiments retained albumin, matching preimplantation measurements with Ficoll. The minimum diameter of a blood conduit, between 4 and 5 mm, and the perfusion pressure of the animal, around 100 mm Hg, caused high blood flow rates through the cartridge. The very high blood flow rate and consequently high shear rates at the membrane surface achieved by this cartridge design are not representative of the operating point of a full-scale cartridge, which will have lower total blood flow and lower shear rates at the membrane face. This feasibility study of an implanted hemofiltration membrane for renal replacement therapy based on a novel biomimetic silicon membrane justifies design engineering of cartridge designs scaled to deliver clinically relevant filtration volumes.
Preliminary results regarding the work in this manuscript were presented in abstract form at the American Society of Nephrology Kidney Week meeting in Philadelphia, Pennsylvania, on November 2014.
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Keywords:Copyright © 2016 by the American Society for Artificial Internal Organs
hemofiltration; nanotechnology; biocompatibility