More than 23 million people worldwide suffer from heart failure.1 Late-stage heart failure is best treated with heart transplantation; however, the demand for suitable donor organs outnumbers the supply by approximately 50-fold.2 The only promising alternatives to heart transplantation are mechanical circulatory assist devices, such as ventricular assist devices (VADs). These devices have areas of low and high fluid flow shear stresses.3 Very high shear stresses lead to destruction of von Willebrand factor (vWF), leading to bleeding complication rates of up to 65% in the first year after VAD placement.4,5 Despite the risk of life-threatening bleeding in 5–10% of VAD recipients per year,6,7 anticoagulation with warfarin is still needed to prevent thrombotic complications of VADs, specifically in the area of stagnant and low shear stresses (<10 dynes/cm2).8–10
The risk of thrombosis of VADs was addressed by modifying the blood-contacting surface with sintered titanium (Ti) microspheres of approximately 50–75 μm in diameter.11 Sintered surfaces create a “pseudointima” of adherent collagen and fibrin deposits and thus reduce the buildup of large thrombi and the corresponding risk of distal embolization.11,12 Despite these advances, thrombosis still occurs in approximately 7.5% of adult patients per year, and the risk is even higher in pediatric patients.13 Pediatric patients are in dire need of devices that can operate at low flow and are at an even higher risk of pump thrombosis.14,15
The ideal approach to reduce the risk of thrombosis of circulatory assist device application is to coat the blood-contacting Ti surfaces with endothelial cells (ECs) to reproduce the native antithrombotic lining of the blood vessels and the heart. The benefit of artificial surface endothelialization has been previously demonstrated, but its clinical application has been hindered by the need for an invasive procedure to surgically harvest ECs from native vessels.16,17 Further, the need for prolonged ex vivo culture of ECs on circulatory assist devices for surface endothelialization has made this method clinically impractical. Hence, the preferred approach to endothelialize artificial surfaces would be to rapidly seed devices with ECs obtained by a minimally invasive method, such as a peripheral blood draw. Peripheral blood is a source of late outgrowth endothelial progenitor cells, also known as endothelial colony-forming cells, which display all typical EC characteristics.18 Our previous work has shown that these cells protect against thrombosis in vivo in a porcine model utilizing smooth Ti implantable devices.19
The method that we term “rapid seeding” allows the cells to be added to a device at the point of care, just minutes before implantation without prior culture on the device. We have already demonstrated the efficacy of this procedure in a porcine model in our previous studies.19 Any prolonged culture of the cells on the device ex vivo would be less practical for translation into clinical practice, because it would require additional and expensive incubation facilities.20 In contrast, our rapid-seeding technology can be performed inside the operating room and reduces the time a patient would have to wait after cell harvest and before implantation of the endothelialized devices.
The overall goal of the this study is to investigate the feasibility of rapidly seeding sintered Ti with patients’ own blood-derived ECs minutes before VAD implantation to create a living antithrombotic surface, specifically in the thrombosis-prone regions (low shear stress areas). Our previous studies demonstrated the efficacy of rapid-seeded ECs on smooth Ti surfaces, which we now extend to sintered Ti with the same surface structure and composition as the blood-contacting surface of the most commonly implanted VAD, HeartMateII (Thoratec Corporation, Pleasanton, CA).21
Pediatric circulatory assist devices are at an especially high risk of thrombosis, and we previously investigated the outgrowth of ECs from pediatric patients. Our results established that colonies of highly functional ECs can be grown out as quickly as 8.3 ± 1.2 days after blood collection from human umbilical cord blood. We are utilizing these human cord blood derived endothelial cells (hCB-ECs) for the present proof-of-concept studies.22,23 This technology may generate a personalized and living antithrombotic coating on VADs to minimize the risk of thrombosis and potentially eliminate the need for anticoagulation.
Materials and Methods
See Supplemental Methods for further details (Supplemental Digital Content, http://links.lww.com/ASAIO/A91).
Isolation and Characterization of hCB-ECs
Human cord blood-derived endothelial cells were isolated from human umbilical cord blood and characterized for EC phenotypic markers by flow cytometry and immunocytochemistry, as previously described.20 The isolated hCB-ECs were utilized at passages 6–9 for all experiments.
Rapid Seeding of hCB-ECs onto Sintered Ti
Sintered Ti materials were provided by Thoratec Corporation. For rapid seeding of sintered Ti, Ti tubes (outer radius: 0.91 cm, inner radius: 0.63 cm) were seeded with hCB-ECs suspended in 1.5 ml serum-free Leibovitz’s L-15 medium (Life Technologies, NY) at various concentrations and then placed in a rotating seeding device (10 revolutions/hr for 45 min at 37°C, 5% CO2).19 Cell adherence was determined by quantifying the number of cells in the remaining hCB-EC suspension and subtracting this number from the total number initially seeded. The cells were stained with 1 mM Cell-Tracker Green (Life Technologies) and then imaged with a confocal microscope (Zeiss 780 upright, Zeiss, Oberkochen, Germany) and scanning electron microscope (Philips XL30 Environmental Scanning Electron Microscope, Phillips, Netherlands).
Rapid-seeded sintered Ti tubes were exposed to steady flow to mimic the continuous flow conditions of current generation VADs, specifically at low shear stresses in the range of 4.4 dynes/cm2 for 20 hr with a modified cardiopulmonary bypass circuit, as described previously.19
Cell Adherence and Function Tests
To quantify the number of hCB-ECs retained on the Ti surface after flow exposure, Cell Counting Kit-8 Assay (Sigma-Aldrich, Missouri, United States) was utilized. To assess the functions of hCB-ECs on Ti surface after fluid flow exposure, we quantified nitric oxide (NO) secretion and performed platelet adhesion assay, as previously described.21
Characterization of hCB-ECs
The isolated hCB-ECs exhibited characteristic EC cobblestone morphology (Figure 1A). Flow cytometry indicated positive expression of EC surface markers CD31, CD105, and CD146 (Figure 1B) and absence of leukocyte markers CD14 and CD45 (Figure 1C). Immunocytochemistry further confirmed EC-like properties of the isolated hCB-ECs. The cells stained positive for EC markers CD31 and vWF (Figure 2, A and B) and took up Dil-acetylated low-density lipoprotein (Dil-Ac-LDL; Figure 2C).
Rapid Seeding of hCB-ECs on Sintered Ti
The density of rapid-seeded hCB-ECs on sintered Ti (cells per square centimeter) linearly correlated with the initial concentration of hCB-ECs (per milliliter) used for rapid seeding (Figure 3). Immediately after rapid seeding, hCB-ECs were initially concentrated in the valleys between Ti microspheres. The distribution of hCB-ECs over sintered Ti microspheres was not affected by increasing seeding concentrations from 0.9 × 105 cells/cm2 up to 4.5 × 105 cells/cm2 (Figure 3). Despite this initial distribution in valleys, hCB-ECs grew over the Ti microspheres and formed a confluent monolayer after ex vivo static culture (37°C, 5% CO2, full EC medium) for 12 hr without any surface precoating (Figure 4).
Rapid Seeding of Sintered Ti Inhibits Platelet Adhesion
As shown in Figure 5, significantly fewer platelets adhered on rapid-seeded sintered Ti compared with control sintered Ti without hCB-ECs (n = 3, p = 0.02, paired t-test).
Adherence of Rapid-Seeded Cells After Exposure to Flow Shear Stresses
Exposure of the rapid-seeded sintered Ti to fluid flow shear stresses of 4.4 dynes/cm2 for 20 hr resulted in 67.5 ± 0.9% cell adherence (n = 3, rapid-seeding density: 3.3 ± 0.7 × 105 cell/cm2). When the rapid-seeded sintered Ti was preincubated under ex vivo static culture (37°C, 5% CO2, full EC medium) for 6 hr before flow exposure, the cell adherence on sintered Ti was similarly high (69.1 ± 3.0%; n = 3, rapid-seeding density: 4.0 ± 0.7 × 105 cells/cm2).
Monolayer Cell Coating on Rapid-Seeded Sintered Ti After Flow Exposure
The hCB-ECs that withstood 4.4 dynes/cm2 for 20 hr on sintered Ti were able to spread and grow to a monolayer under flow (Figure 6A). This finding was observed when the rapid-seeded sintered Ti was either preincubated or not preincubated under ex vivo static culture before flow exposure. As shown in Figure 6, previous incubation of rapid-seeded sintered Ti (6 hr static ex vivo culture) did not increase the extent of hCB-EC coverage on sintered Ti surface (Figure 6B). Therefore, these findings demonstrate the ability of rapid-seeded sintered Ti to withstand low fluid flow shear stresses without the need for additional ex vivo culture on the Ti surface to create an endothelial coating.
Function of Rapid-Seeded hCB-ECs on Sintered Ti After Flow Exposure
The function of rapid-seeded sintered Ti after exposure to 4.4 dynes/cm2 for 20 hr was investigated with a platelet adhesion and NO secretion assay. Significantly fewer platelets adhered on rapid-seeded sintered Ti compared with unseeded bare metal Ti controls without hCB-ECs (Figure 7, n = 3, p = 0.01, paired t-test). Further, hCB-ECs on rapid-seeded sintered Ti significantly increased the secretion of NO by >150-fold after flow exposure compared with hCB-ECs on static controls (Figure 8, n = 3, p < 0.01, paired t-test).
The Function of Rapid-Seeded hCB-ECs on Sintered Ti After Long-Term Maintenance
To examine EC function on rapid-seeded sintered Ti after long-term maintenance, we tested the metabolic activity of the rapid-seeded hCB-ECs after 15 days of ex vivo static culture. hCB-ECs retained their metabolic activity as shown by increased absorbance readings on a Cell Counting Kit-8 metabolic assay from 0.35 ± 0.02 (at 0 hr) to 1.25 ± 0.04 (at 3 hr) by hCB-ECs (n = 3; comparable to increased absorbance readings by hCB-ECs after 1 day of ex vivo static culture). In addition, the hCB-ECs also retained the EC characteristic properties, i.e., Dil-Ac-LDL uptake and thrombomodulin expression (Figure 9). Furthermore, rapid-seeded sintered Ti remained antithrombotic as shown by a persistent significant reduction in the number of adherent platelets, compared with control sintered Ti surfaces without hCB-ECs (1.4 ± 0.3 vs. 4.7 ± 0.9 × 103 platelets/mm2; n = 3, p = 0.039, paired t-test).
We have previously demonstrated that blood-derived ECs adhere well to smooth Ti surfaces, a finding that can be attributed to the naturally formed TiO2 film.21,24 This study shows that hCB-ECs are also able to adhere to sintered Ti without any surface precoatings, which may otherwise render the blood-contacting surface prothrombotic.21,24
In addition to showing excellent cell adherence on sintered Ti under static conditions, our study demonstrates that hCB-EC seeding on sintered Ti could be performed within minutes to create a monolayer that withstands low fluid flow shear stresses (4.4 dynes/cm2) resulting in approximately 70% cell adherence. Cell coverage within the low shear stress areas of mechanical circulatory assist devices is clinically relevant because these regions are especially prone to platelet adhesion and thrombus formation.8–10
Longer ex vivo static culture after rapid seeding did not increase cell adherence or the cells’ ability to form a monolayer on sintered Ti under shear stresses. Without the need for additional ex vivo culture, this rapid-seeding technology may be practical to apply in the operating room, e.g., on the back table while the patient is placed on cardiopulmonary bypass in preparation for VAD insertion.
Rapid-seeded hCB-ECs appeared to be functional under flow on sintered Ti as evidenced by the cells’ ability to secrete significantly more of the antithrombotic mediator NO (>150-fold) under flow than under static conditions, indicating a normal physiologic response characteristic of healthy ECs.23,25,26 Further, consistent with our previous work showing a dramatic reduction in platelet adhesion on EC-coated smooth Ti,21 there was a remarkable reduction in the number of platelets adhering on hCB-EC-covered sintered Ti, compared with control sintered Ti surfaces without hCB-ECs.
The function of hCB-ECs on sintered Ti was retained after 15-day ex vivo static culture. Human cord blood-derived endothelial cells retained platelet inhibition capacity on sintered Ti, as well as metabolic activity and expression of characteristic markers of functional ECs, such Dil-Ac-LDL uptake and thrombomodulin expression.
This study has several limitations. First, because this study was primarily aimed at assessing the feasibility of rapid-seeding ECs onto sintered Ti to prevent thrombosis in the thrombosis-prone low shear stress regions (<10 dynes/cm2), the properties of rapid-seeded hCB-ECs >4.4 dynes/cm2 were not investigated. Second, the function of hCB-ECs on sintered Ti was tested within relatively short-time periods because long-term flow experiments do not adequately reproduce in vivo environment in which blood-derived circulating cells can potentially replace sheared-off ECs. Third, ECs were derived from human umbilical cord because hCB-ECs have been shown to behave similarly with peripheral blood-derived ECs.24,27 Future works should assess adult blood-derived ECs and also perform in vivo studies for a proof of concept in the long term.
Our rapid-seeding technology could potentially be applied to both pediatric and adult patients. In pediatric patients, for whom congenital heart disease can be diagnosed before birth, a small amount of umbilical cord blood can be harvested at birth, and this will be sufficient to generate enough cells for surface coatings because of hCB-ECs’ high proliferative capacity.28 In adult patients, the source of ECs can be from peripheral blood. Despite not being enriched with ECs, with even 30% less yield in heart failure patients,29 peripheral blood can yield a sufficient number of ECs, e.g., with a mobilizing agent (Food and Drug Administration-approved plerixafor), in combination with apheresis.30–32
The presented rapid-seeding technology has shown promise that warrants future investigation to develop a novel technology utilizing patients’ own blood-derived ECs to create living personalized antithrombotic coatings in thrombosis-prone low-flow regions of VADs and artificial hearts. With less thrombotic complications, future devices could be specifically designed to operate at lower shear stresses, which will prevent vWF destruction. Combined with the possibility of reducing or eliminating the use of anticoagulation, life-threatening bleeding complications may be prevented.13 Such advances utilizing a rapid-seeding technology could broaden device indications, e.g., for elective placement or as destination therapy in less sick patients.
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