George Truskey - Academia.edu (original) (raw)

Papers by George Truskey

Biomaterials, Aug 1, 2005

The development of a functional, adherent endothelium is one of the major factors limiting the su... more The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/ cm 2 for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.

Annals of Biomedical Engineering, Apr 19, 2013

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential ... more Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

Journal of Biomedical Materials Research, Jun 15, 1998

This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms ... more This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms around implants and how they are affected by implant porosity. The number of vessels and their permeability to rhodamine were measured by intravascular injection of the fluorophore tracer into Sprague-Dawley rats that hosted for 3-4 months polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) subcutaneous implants. Rats were implanted with four pore sizes of PVA--a nonporous PVA (PVA-skin), and 5, 60, and 700 micron mean pore sizes (PVA-5, PVA-60, and PVA-700, respectively)--and two pore sizes of PTFE: 0.50 (PTFE-0.5) and 5.0 (PTFE-5) mean micron pore sizes. Photodensitometric image analysis was used to quantify the local tracer extravasation and, hence the permeability coefficients of isolated vessels around the implants. The number of functional vessels within 100 microm of the implants highlighted by the lissamine-rhodamine tracer were counted with fluorescence microscopy and with H&E stained sections using brightfield microscopy. The permeability of vessels did not vary substantially with implant pore size but generally were lower than those measured for surrounding subcutis. Pore size, however, had a dramatic effect on the vascular density of tissue-encapsulating implants: the number of microvessels (under 10 microm in radius) within the tissue surrounding the porous implants was higher than the number around nonporous implants. Pore sizes on the order of cellular dimensions incited optimal neovascularization; the vascular density around PVA-60 implants was six times higher (p < .001) and three times higher (p < .001) than those around PVA-0 implants in the fluorescent images and in brightfield, respectively. Moreover, brightfield microscopy showed the number of vessels around PVA-60 implants was almost double those in normal subcutis. The results suggest that optimal vascular density around long-term implants, such as sensors, biofluid cell constructs, and immunoisolated cell systems, may be engineered with pore size.

Tissue Engineering Part A, Jun 1, 2010

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surfac... more Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC=smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and functi... more Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7 + cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Journal of Biomedical Materials Research, Jun 15, 1998

ABSTRACT

Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infus... more Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood–derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. Methods and Results—Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface 51 integrin heterodimers per cell (P0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdcscid/J mice. Two weeks postoperatively, vein grafts from hCB-EC–treated mice demonstrated approximately 55 % reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombos...

APL Bioengineering, 2021

Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the... more Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the United States and many other developed countries. Tissue engineered vascular systems (TEVS) can serve as grafts for CVD treatment and be used as in vitro model systems to examine the role of various genetic factors during the CVD progressions. Current focus in the field is to fabricate TEVS that more closely resembles the mechanical properties and extracellular matrix environment of native vessels, which depends heavily on the advance in biofabrication techniques and discovery of novel biomaterials. In this review, we outline the mechanical and biological design requirements of TEVS and explore the history and recent advances in biofabrication methods and biomaterials for tissue engineered blood vessels and microvascular systems with special focus on in vitro applications. In vitro applications of TEVS for disease modeling are discussed.

Scientific Reports, 2015

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an importan... more Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm2. The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vei...

Regenerative medicine, 2015

Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies an... more Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Annals of biomedical engineering, Jan 17, 2015

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present... more Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and simil...

Journal of visualized experiments : JoVE, Jan 17, 2012

The overall goal of this method is to describe a technique to subject adherent cells to laminar f... more The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other expe...

Tissue Engineering Part A, 2010

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surfac... more Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC=smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Tissue Engineering Part A, 2009

Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising s... more Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AFÔ-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cellspecific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of a 5 b 1 and a v b 3 integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of Eselectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-a stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.

Tissue Engineering Part A, 2013

Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent ... more Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood-derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.

Tissue Engineering Part A, 2009

The lack of easily isolated autologous endothelial cell (EC) sources is one of the major challeng... more The lack of easily isolated autologous endothelial cell (EC) sources is one of the major challenges with vascular tissue engineering interventions. This article examines the isolation and expansion of late-outgrowth endothelial progenitor cells (EPCs) from 50-mL samples of peripheral blood drawn from patients with significant coronary artery disease (CAD) and healthy young adult volunteers. In cases in which late-outgrowth EPCs were successfully isolated, the cells were assayed in vitro for their expression of EC markers, proliferation potential and ability to endothelialize synthetic materials, form new blood vessels, and produce nitric oxide. Late-outgrowth EPCs from patients with CAD and healthy volunteers exhibited critical EC markers and morphological characteristics that were analogous to a control population of human aortic ECs. To our knowledge, this is the first study to examine the suitability of late-outgrowth EPCs from patients with CAD for autologous endothelialization applications.

Journal of Biomedical Materials Research, 2004

In this study, we examined combinations of three approaches to improve the adhesion of endothelia... more In this study, we examined combinations of three approaches to improve the adhesion of endothelial cells (EC) onto expanded polytetrafluoroethylene (ePTFE) vascular grafts placed at the femoral artery of rats: (1) high-affinity receptor-ligand binding of RGD-streptavidin (SA) and biotin to supplement integrin-mediated EC adhesion; (2) cell sodding to pressurize the seeded EC into the interstices of the ePTFE grafts; and (3) longer postseeding attachment time from 1 to 24 h prior to implantation. An in vitro system, which accounts for cell loss due to both graft handling and shear stress, was designed to optimize conditions for in vivo experiments. Results suggest that longer in vitro attachment time enabled the adherent EC to endure mechanical stresses by forming strong adhesions to the underlying extracellular matrix substrates; cell sodding helped to retain the adherent EC by physically docking the cells against the graft interstices; and the SA-biotin interaction enhanced the early attachment of EC but did not lead to better cell retention or reduced surface coverage of blood clot in the current study. Mechanical manipulation of cells during implantation is a limiting factor in maintaining a confluent EC layer on synthetic vascular grafts.

Journal of Biomechanics, 2011

Journal of Biomaterials Science, Polymer Edition, 1998

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of ... more Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p &lt; 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.

Biomaterials, Aug 1, 2005

The development of a functional, adherent endothelium is one of the major factors limiting the su... more The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/ cm 2 for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.

Annals of Biomedical Engineering, Apr 19, 2013

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential ... more Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

Journal of Biomedical Materials Research, Jun 15, 1998

This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms ... more This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms around implants and how they are affected by implant porosity. The number of vessels and their permeability to rhodamine were measured by intravascular injection of the fluorophore tracer into Sprague-Dawley rats that hosted for 3-4 months polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) subcutaneous implants. Rats were implanted with four pore sizes of PVA--a nonporous PVA (PVA-skin), and 5, 60, and 700 micron mean pore sizes (PVA-5, PVA-60, and PVA-700, respectively)--and two pore sizes of PTFE: 0.50 (PTFE-0.5) and 5.0 (PTFE-5) mean micron pore sizes. Photodensitometric image analysis was used to quantify the local tracer extravasation and, hence the permeability coefficients of isolated vessels around the implants. The number of functional vessels within 100 microm of the implants highlighted by the lissamine-rhodamine tracer were counted with fluorescence microscopy and with H&E stained sections using brightfield microscopy. The permeability of vessels did not vary substantially with implant pore size but generally were lower than those measured for surrounding subcutis. Pore size, however, had a dramatic effect on the vascular density of tissue-encapsulating implants: the number of microvessels (under 10 microm in radius) within the tissue surrounding the porous implants was higher than the number around nonporous implants. Pore sizes on the order of cellular dimensions incited optimal neovascularization; the vascular density around PVA-60 implants was six times higher (p < .001) and three times higher (p < .001) than those around PVA-0 implants in the fluorescent images and in brightfield, respectively. Moreover, brightfield microscopy showed the number of vessels around PVA-60 implants was almost double those in normal subcutis. The results suggest that optimal vascular density around long-term implants, such as sensors, biofluid cell constructs, and immunoisolated cell systems, may be engineered with pore size.

Tissue Engineering Part A, Jun 1, 2010

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surfac... more Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC=smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and functi... more Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7 + cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Journal of Biomedical Materials Research, Jun 15, 1998

ABSTRACT

Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infus... more Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood–derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. Methods and Results—Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface 51 integrin heterodimers per cell (P0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdcscid/J mice. Two weeks postoperatively, vein grafts from hCB-EC–treated mice demonstrated approximately 55 % reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombos...

APL Bioengineering, 2021

Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the... more Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the United States and many other developed countries. Tissue engineered vascular systems (TEVS) can serve as grafts for CVD treatment and be used as in vitro model systems to examine the role of various genetic factors during the CVD progressions. Current focus in the field is to fabricate TEVS that more closely resembles the mechanical properties and extracellular matrix environment of native vessels, which depends heavily on the advance in biofabrication techniques and discovery of novel biomaterials. In this review, we outline the mechanical and biological design requirements of TEVS and explore the history and recent advances in biofabrication methods and biomaterials for tissue engineered blood vessels and microvascular systems with special focus on in vitro applications. In vitro applications of TEVS for disease modeling are discussed.

Scientific Reports, 2015

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an importan... more Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm2. The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vei...

Regenerative medicine, 2015

Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies an... more Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Annals of biomedical engineering, Jan 17, 2015

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present... more Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and simil...

Journal of visualized experiments : JoVE, Jan 17, 2012

The overall goal of this method is to describe a technique to subject adherent cells to laminar f... more The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other expe...

Tissue Engineering Part A, 2010

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surfac... more Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC=smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Tissue Engineering Part A, 2009

Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising s... more Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AFÔ-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cellspecific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of a 5 b 1 and a v b 3 integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of Eselectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-a stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.

Tissue Engineering Part A, 2013

Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent ... more Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood-derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.

Tissue Engineering Part A, 2009

The lack of easily isolated autologous endothelial cell (EC) sources is one of the major challeng... more The lack of easily isolated autologous endothelial cell (EC) sources is one of the major challenges with vascular tissue engineering interventions. This article examines the isolation and expansion of late-outgrowth endothelial progenitor cells (EPCs) from 50-mL samples of peripheral blood drawn from patients with significant coronary artery disease (CAD) and healthy young adult volunteers. In cases in which late-outgrowth EPCs were successfully isolated, the cells were assayed in vitro for their expression of EC markers, proliferation potential and ability to endothelialize synthetic materials, form new blood vessels, and produce nitric oxide. Late-outgrowth EPCs from patients with CAD and healthy volunteers exhibited critical EC markers and morphological characteristics that were analogous to a control population of human aortic ECs. To our knowledge, this is the first study to examine the suitability of late-outgrowth EPCs from patients with CAD for autologous endothelialization applications.

Journal of Biomedical Materials Research, 2004

In this study, we examined combinations of three approaches to improve the adhesion of endothelia... more In this study, we examined combinations of three approaches to improve the adhesion of endothelial cells (EC) onto expanded polytetrafluoroethylene (ePTFE) vascular grafts placed at the femoral artery of rats: (1) high-affinity receptor-ligand binding of RGD-streptavidin (SA) and biotin to supplement integrin-mediated EC adhesion; (2) cell sodding to pressurize the seeded EC into the interstices of the ePTFE grafts; and (3) longer postseeding attachment time from 1 to 24 h prior to implantation. An in vitro system, which accounts for cell loss due to both graft handling and shear stress, was designed to optimize conditions for in vivo experiments. Results suggest that longer in vitro attachment time enabled the adherent EC to endure mechanical stresses by forming strong adhesions to the underlying extracellular matrix substrates; cell sodding helped to retain the adherent EC by physically docking the cells against the graft interstices; and the SA-biotin interaction enhanced the early attachment of EC but did not lead to better cell retention or reduced surface coverage of blood clot in the current study. Mechanical manipulation of cells during implantation is a limiting factor in maintaining a confluent EC layer on synthetic vascular grafts.

Journal of Biomechanics, 2011

Journal of Biomaterials Science, Polymer Edition, 1998

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of ... more Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p &lt; 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.