Helen Marie Nugent

Biological surface engineering: a simple system for cell pattern formation

Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.

Tissue Engineering Therapy for Cardiovascular Disease

Abstract— The present treatments for the loss or failure of cardiovascular function include organ transplantation, surgical reconstruction, mechanical or synthetic devices, or the administration of metabolic products. Although routinely used, these treatments are not without constraints and complications. The emerging and interdisciplinary field of tissue engineering has evolved to provide solutions to tissue creation and repair. Tissue engineering applies the principles of engineering, material science, and biology toward the development of biological substitutes that restore, maintain, or improve tissue function. Progress has been made in engineering the various components of the cardiovascular system, including blood vessels, heart valves, and cardiac muscle. Many pivotal studies have been performed in recent years that may support the move toward the widespread application of tissue-engineered therapy for cardiovascular diseases. The studies discussed include endothelial cell seeding of vascular grafts, tissue-engineered vascular conduits, generation of heart valve leaflets, cardiomyoplasty, genetic manipulation, and in vitro conditions for optimizing tissue-engineered cardiovascular constructs.

Endothelial Implants Inhibit Intimal Hyperplasia After Porcine Angioplasty

The perivascular implantation of tissue-engineered endothelial cells around injured arteries offers an opportunity to study fundamental vascular physiology as well as restore and improve tissue function. Cell source is an important issue because the ability to implant either xenogeneic or allogeneic cells would greatly enhance the clinical applications of tissue-engineered grafts. We investigated the biological and immunological responses to endothelial cell xenografts and allografts in pigs 4 weeks after angioplasty of the carotid arteries. Porcine or bovine aortic endothelial cells were cultured within Gelfoam matrices and implanted in the perivascular space of 42 injured arteries. Both porcine and bovine endothelial cell grafts reduced the restenosis index compared with control by 54% and 46%, respectively. Perivascular heparin release devices, formulated to release heparin at twice the rate of release of heparan sulfate proteoglycan from endothelial cell implants, produced no significant reduction in the restenosis index. Endothelial cell implants also reduced occlusive thrombosis compared with control and heparin release devices. Host immune responses to endothelial implants were investigated by immunohistochemical examination of explanted devices and by immunocytochemistry of serum samples. The bovine cell grafts displayed infiltration of leukocytes, consisting primarily of lymphocytes, and caused an increase in antibodies detected in serum samples. Reduced cellular infiltration and no humoral response were detected in animals that received allografts. Despite the difference in immune response, the biological effects of xenografts or allografts did not differ significantly.

Perivascular Endothelial Implants Inhibit Intimal Hyperplasia in a Model of Arteriovenous Fistulae: A Safety and Efficacy Study in the Pig

Vascular access complications are a major problem in hemodialysis patients. Native arteriovenous fistulae, historically the preferred mode of access, have a patency rate of only 60% at 1 year. The most common mode of failure is due to progressive stenosis at the anastomotic site. We have previously demonstrated that perivascular endothelial cell implants inhibit intimal thickening following acute balloon injury in pigs and now seek to determine if these implants provide a similar benefit in the chronic and more complex injury model of arteriovenous anastomoses. Side-to-side femoral artery-femoral vein anastomoses were created in 24 domestic swine and the toxicological, biological and immunological responses to allogeneic endothelial cell implants were investigated 3 days and 1 and 2 months postoperatively. The anastomoses were wrapped with polymer matrices containing confluent porcine aortic endothelial cells (PAE; n = 14) or control matrices without cells (n = 10). PAE implants significantly reduced intimal hyperplasia at the anastomotic sites compared to controls by 68% (p <0.05) at 2 months. The beneficial effects of the PAE implants were not due to differences in the rates of reendothelialization between the groups. No significant immunological response to the allogeneic endothelial cells that impacted on efficacy was detected in any of the pigs. No apparent toxicity was observed in any of the animals treated with endothelial implants. These data suggest that perivascular endothelial cell implants are safe and reduce early intimal hyperplasia in a porcine model of arteriovenous anastomoses.

Multicenter phase I/II trial of the safety of allogeneic endothelial cell implants after the creation of arteriovenous access for hemodialysis use: The V-HEALTH study

OBJECTIVES Vascular access dysfunction is the major cause of morbidity in patients on hemodialysis to treat end stage renal disease. Preclinical studies have demonstrated that the perivascular placement of implants containing allogeneic aortic endothelial cells (Vascugel) reduces thrombosis, inflammation, stenosis and increases lumen diameter in porcine models of arteriovenous fistulae (AVF) and arteriovenous grafts (AVG). We conducted a phase I/II clinical study to investigate the safety of Vascugel placement around the surgical anastomotic sites of newly constructed dialysis accesses. METHODS From July 2006 to August 2006, eight patients (4 AVG, 4 AVF) were treated with two Vascugel sponges at the venous anastomosis in the open-label phase I trial. From January 2007 to August 2007 57 patients (30 AVG and 27 AVF) were randomized in a 2:1 fashion to receive either Vascugel or control matrices (placebo) at surgery. The phase II AVG patients had sponges placed at both the venous and arterial anastomoses. All patients were followed for 24 weeks. The primary objective of the study was to demonstrate the safety (incidence of infection, intervention, and thrombosis) of Vascugel compared with placebo within 30 days post-surgery. Secondary endpoints included assessments of patency, lumen diameter, and immunologic sensitization to human leukocyte antigens (HLA) determined by measurement of panel reactive antibodies (PRA). RESULTS There was no difference in early complication rates between the Vascugel and placebo groups at 4 weeks (10.9% vs 21.1%, respectively). There were no statistically significant differences in primary or assisted primary patency between the intent to treat groups at 24 weeks. Vascugel treated AVG had a primary patency rate of 38% and an assisted primary patency rate of 72% (vs 23% and 58%, respectively, for placebo). Vascugel treated AVF had a primary patency rate of 60% at 24 weeks and an assisted primary patency rate of 96% (vs 62% and 88%, respectively, for placebo). A greater than 30% increase in PRA was detected in 9 of the 46 (19.5%) Vascugel treated patients and one of the 19 (5.2%) placebo patients (P = .26) and was not associated with any evidence of local or systemic complications. CONCLUSIONS Targeted local therapy with perivascular, allogeneic endothelial cells is a safe and novel therapeutic approach that may be ideally suited to control the response to injury at surgical anastomoses. Larger randomized trials are needed to determine if Vascugel can prolong AVG or AVF patency.

Matrix Embedding Alters the Immune Response Against Endothelial Cells In Vitro and In Vivo

Background—Endothelial cell (EC) dysfunction represents the first manifestation of atherosclerotic disease. Restoration of endothelium via seeding or transfection is hampered by local alterations in flow, inflammation, and metabolic activation. Perivascular EC matrix implants are shielded from these forces and still control vascular repair. The host immune response to such implants, however, remains largely unknown. We investigated the effect of embedding of ECs within 3-dimensional matrices on host immune responses in vitro and in vivo. Methods and Results—We compared expression of major histocompatibility complex (MHC), costimulatory, and adhesion molecules by free aortic ECs or ECs embedded in Gelfoam matrices by flow-cytometry. T-cell proliferation was assessed by [3H] thymidine incorporation. Humoral immune response (ELISA and FACS analysis) and cellular (histopathology) infiltration were investigated after subcutaneous injection of free porcine aortic ECs (PAEs) or of a Gelfoam/EC block, or after concomitant injection of PAEs adjacent to Gelfoam in rats. Aortic ECs embedded in Gelfoam expressed lower levels of MHC class II, costimulatory, and adhesion molecules compared with free ECs (P<0.001), and induced 3-fold less proliferation of human CD4+ T-cells (P<0.0005). Implantation of a Gelfoam/EC block in rats nearly abrogated the immune response with 1.75- to 9.0-fold downregulation in tumor necrosis factor-&agr;, interleukin-6, monocyte chemotactic protein-1, and PAE-specific immunoglobulin G (P<0.005) and 3.3- to 4.5-fold reduction in leukocytic tissue infiltration. Injecting PAEs adjacent to Gelfoam induced a significant response comparable to that of free implanted PAEs. Conclusions—Embedding ECs within 3-dimensional matrices alters the host immune response by inhibiting expression of MHC class II, costimulatory, and adhesion molecules, offering the rationale to develop novel therapies for vascular diseases.

Influence of diabetes and perivascular allogeneic endothelial cell implants on arteriovenous fistula remodeling

OBJECTIVES Arteriovenous fistula (AVF) is the preferred type of vascular access for hemodialysis to treat end-stage renal disease. A high proportion of AVF are never used for dialysis because the vein fails to mature adequately. We have previously described the safety and feasibility of Vascugel (Genzyme BioSurgery, Cambridge, Mass) (allogeneic aortic endothelial cells in a gelatin matrix) when placed around the anastomotic and venous outflow sites of AVFs (Vascular intimal Hyperplasia: Extending Arterial and venous patency, Limiting vascular Trauma, and inhibiting Hyperplasia while re-establishing vascular health [V-HEALTH] clinical study). In this retrospective analysis, we investigated factors that influenced AVF remodeling in patients from the V-HEALTH study. We hypothesized that providing healthy endothelial cells and their secreted factors immediately after surgery could enhance venous remodeling in the setting of vascular injury. METHODS Thirty-one AVF patients from the V-HEALTH study were randomized 2:1 to receive either Vascugel or control matrices (placebo) at surgery and were followed for 24 weeks. Venous lumen diameter was measured by ultrasound at 1, 3, and 5 cm from the anastomosis. Vein remodeling (change in lumen diameter at 4, 12, and 24 weeks compared with baseline diameter at 2 weeks) was analyzed using a multiple regression mixed model. RESULTS The results indicated that diabetes was a significant, negative predictor of venous remodeling over the 24-week study (P = .02). The model-predicted change in lumen diameter from 2 to 24 weeks was -0.7 mm in diabetic patients (n = 11) and +2.4 mm in nondiabetic patients (n = 15), a difference of 3.1 mm, 95% confidence interval [CI] (1.4-4.9), P = .0014. Patient race, baseline vein diameter, and time post-AVF creation were also significant factors that affected remodeling (P < .05). Compared with placebo, there was a strong suggestion that Vascugel treatment improved the rate of venous enlargement in diabetic patients (P = .05). The model-predicted change in lumen diameter at 24 weeks was -1.9 mm for placebo-treated diabetic patients and +0.4 mm for Vascugel-treated diabetic patients, a difference of 2.3 mm, 95% CI (-0.1-4.8), P = .06, suggesting that treatment with Vascugel may mitigate the negative influence of diabetes on AVF remodeling. CONCLUSIONS Diabetes negatively impacts AVF remodeling and targeted local therapy with perivascular, allogeneic endothelial cells may ameliorate this effect. A phase II trial designed specifically to evaluate AVF remodeling is needed to determine if Vascugel can increase AVF maturation and use and to support larger randomized trials.

Delivery Site of Perivascular Endothelial Cell Matrices Determines Control of Stenosis in a Porcine Femoral Stent Model

PURPOSE Endothelial cells, grown within gelatin matrices and implanted onto the adventitia of injured vessels, inhibit stenosis in experimental models. To determine if this technology could be adapted for minimally invasive procedures, the authors compared the effects of cells in an implantable sponge to that of an injectable formulation and investigated the importance of delivery site in a stent model. MATERIALS AND METHODS Stents were implanted in the femoral arteries of 30 pigs. This was followed by perivascular implantation of sponges or injection of particles containing allogeneic endothelial cells. Controls received acellular matrices or nothing. The effects of delivery site were assessed by injecting cellular matrices into or adjacent to the perivascular tissue or into the neighboring muscle. Animals were sacrificed after 28 days. Pre-sacrifice angiograms and tissue sections were evaluated for stenosis. RESULTS Arteries treated with cellular matrices had a 55%-63% decrease in angiographic stenosis (P < .05) and a 38%-43% reduction in histologic stenoses (P < .05) compared to controls. Intimal area was greatest when cellular matrices were delivered into the muscle (6.35 mm(2) +/- 0.95) rather than into or adjacent to the perivascular tissue (4.05 mm(2) +/- 0.56 and 4.73 mm(2) +/- 0.53, respectively; P < .05). CONCLUSIONS Perivascular endothelial cell matrices reduced stenosis after stent-induced injury. The effects were not dependent on the formulation but appeared to be dependent on delivery site. Minimally invasive injections of endothelial cell matrices to the adventitia of arteries following peripheral interventions may decrease restenosis rates.

Ultrasound-guided percutaneous delivery of tissue-engineered endothelial cells to the adventitia of stented arteries controls the response to vascular injury in a porcine model

OBJECTIVE High restenosis rates are a limitation of peripheral vascular interventions. Previous studies have shown that surgical implantation of a tissue-engineered endothelium onto the adventitia surface of injured vessels regulates vascular repair. In the present study, we developed a particulate formulation of tissue-engineered endothelium and a method to deliver the formulation perivascular to injured blood vessels using a percutaneous, minimally invasive technique. METHODS Stainless steel stents were implanted in 18 balloon-injured femoral arteries of nine domestic swine, followed by ultrasound-guided percutaneous perivascular injection of gelatin particles containing cultured allogeneic porcine aortic endothelial cells (PAE). Controls received injections of empty particles (matrix) or no perivascular injection (sham) after stent deployment. Animals were sacrificed after 90 days. RESULTS Angiographic analysis revealed a significantly greater lumen diameter in the stented segments of arteries treated with PAE/matrix (4.72 ± 0.12 mm) compared with matrix (4.01 ± 0.20 mm) or sham (4.03 ± 0.16 mm) controls (P < .05). Similarly, histologic analysis revealed that PAE/matrix-treated arteries had the greatest lumen area (20.4 ± 0.7 mm(2); P < .05) compared with controls (16.1 ± 0.9 mm(2) and 17.1 ± 1.0 mm(2) for sham and matrix controls, respectively) and the smallest intimal area (3.3 ± 0.4 mm(2); P < .05) compared with controls (6.2 ± 0.5 mm(2) and 4.4 ± 0.5 mm(2) for sham and matrix controls, respectively). Overall, PAE-treated arteries had a 33% to 50% decrease in percent occlusion (P < .05) compared with controls. Histopathological analysis revealed fewer leukocytes present in the intima in the PAE/matrix group compared with control groups, suggesting that the biological effects were in part due to inhibition of the inflammatory phase of the vascular response to injury. CONCLUSIONS Minimally invasive, perivascular delivery of PAE/matrix to stented arteries was performed safely using ultrasound-guided percutaneous injections and significantly decreased stenosis. Application at the time of or subsequent to peripheral interventions may decrease clinical restenosis rates.

Pilot safety study of perivascular injection of tissue-engineered allogeneic aortic endothelial cells in patients undergoing minimally invasive peripheral revascularization

OBJECTIVE Restenosis is a limitation of endovascular interventions performed in the superficial femoral artery (SFA). Preclinical studies have demonstrated that the perivascular delivery of tissue-engineered allogeneic aortic endothelial cells (PVS-10200) reduced stenosis in porcine models of SFA revascularization. The purpose of this study was to investigate the safety and feasibility of percutaneous PVS-10200 delivery after angioplasty and stenting in the SFA of patients with peripheral artery disease. METHODS In this phase I open-label trial, 21 patients (average lesion length of 10.10 ± 2.36 cm and ≥70% stenosis) were treated with PVS-10200: 11 in a low-dose cohort (cohort A) and 10 in a high-dose cohort (cohort B). The primary objective was to demonstrate the safety (incidence of major adverse events) of PVS-10200 within 4 weeks after surgery. Secondary end points included assessments of resting ankle-brachial index (ABI) in the treated leg, Fontaine class, and time to target lesion revascularization (TLR). RESULTS No patient had a major adverse event within 4 weeks. One patient required a limb amputation at 30 weeks. At 48 weeks, cohort A and cohort B patients maintained a 37% and 62% increase in ABI compared with baseline, respectively; 70% of cohort A and 78% of cohort B improved by ≥1 Fontaine classification stage, and the TLR rate was 39% for cohort A and 20% for cohort B. CONCLUSIONS Percutaneous local delivery of PVS-10200 is a well-tolerated and novel therapeutic approach that may be a suitable treatment for patients after endovascular intervention of the SFA. Larger randomized trials are needed to determine if PVS-10200 can improve ABI and reduce TLR rates.