Peter Zilla

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD‐containing peptides and provision of cell‐mediated remodeling by cross‐linking matrix metalloproteinase substrate peptides. By using a Michael‐type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix‐conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF‐modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft‐membrane contact. When implanted subcutaneously in rats, these VEGF‐containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience.

BACKGROUND Clinical in vitro endothelialization was assessed for its ability to improve the long-term patency of prosthetic femoropopliteal bypass grafts. METHODS Between June 1989 and May 1998, 100 patients received 113 in vitro endothelialized expanded polytetrafluoroethylene grafts (ePTFE). Bilateral implantations were performed in 13 patients. In phase 1 of the study, 24 patients received 27 endothelialized grafts and 16 patients received 17 untreated grafts. In phase 2, endothelialization was offered to all patients who did not have a suitable saphenous vein available. Phase 2 began in June 1993 and included 76 patients who received 86 endotheliazed ePTFE grafts. In all, 100 patients had autologous endothelial cells harvested from 4- to 5-cm segments of a subcutaneous vein. In phase 1, the external jugular vein was used. In phase 2, the cephalic vein was used. These cells were grown to first-passage mass cultures and were lined confluently onto 6-mm ePTFE grafts, pre-coated with fibrin glue. Patency assessment for Kaplan-Meier survivorship analysis was determined by using duplex sonography and angiography. RESULTS In phase 1, the Kaplan-Meier method revealed a primary 9-year patency rate for 65% for the endothelialized group, versus 16% for the control group (log-rank test, P = .002; Wilcoxon test, P = .003). In phase 2, the 5-year primary patency rate for all in vitro endothelialized infrainguinal reconstructions was 68% (66% for above-the-knee grafts and 76% for below-the-knee grafts). CONCLUSIONS Nine years of clinical in vitro endothelialization provided strong evidence that autologous endothelial cell lining improves the patency of small-diameter vascular grafts and that a cell culture-dependent procedure can be used in a clinical routine.

Clinical autologous in vitro endothelialization of 153 infrainguinal ePTFE grafts

BACKGROUND Over the past 17 years, our group has developed and clinically applied an in vitro endothelialization procedure whereby infrainguinal expanded polytetrafluoroethylene (ePTFE) prostheses are confluently lined with cultured autologous endothelial cells before implantation. After a successful randomized pilot study from 1989 to 1993, the procedure was adopted for routine operations. METHODS Since June 1993, 153 endothelialized ePTFE grafts were implanted in the infrainguinal position in 136 patients (102 above knee (AK) and 51 below knee (BK), 89 men and 47 women, mean age 64.7+/-9.4 years). Seventeen patients received an endothelialized prosthesis bilaterally. Autologous endothelial cells were harvested from 4- to 5-cm segments of a subcutaneous vein (in 86% the cephalic vein), grown to first-passage mass cultures and confluently lined onto 6- (n = 113) or 7-mm (n = 40) inner diameter (ID) ePTFE grafts, precoated with fibrin glue. The observation period for 6-mm grafts was 7 years, and for 7-mm grafts was 4 years. Patency assessment for Kaplan-Meier survivorship analyses was based on duplex sonography and angiography. RESULTS Kaplan-Meier survivorship function revealed a primary patency rate of 62.8% after 7 years (SE = 0.05) for all infrainguinal reconstructions (60% AK/70.8% BK). The primary patency for stage II and III patients was 64.4% after 7 years. The more recent group of 7-mm ID grafts showed a primary patency of 83.7% after 4 years. CONCLUSIONS Our data provide strong evidence that autologous endothelial cell lining distinctly improves the patency of small diameter vascular grafts.

Precoating substrate and surface configuration determine adherence and spreading of seeded endothelial cells on polytetrafluoroethylene grafts

Primary adherence and attachment area of seeded human endothelial cells (EC) were determined on differently coated polytetrafluoroethylene (PTFE) grafts. Cell counts and morphometric analyses were done immediately after 60 minutes of electronically controlled seeding of 3 x 10(4) EC/cm2, as well as after 3 hours of subsequent incubation. Cell adherence and cell spreading were distinctly superior on two surface-covering substrates: fibronectin-treated type I/III collagen and fibrinolytically inhibited fibrin glue. Uncovered, purely fibronectin- or laminin-coated PTFE or type IV collagen treated with the specifically binding glycoprotein laminin showed a far lower EC attachment rate and less pronounced cell spreading. It appears that not only a high surface content of fibronectin but also a smooth PTFE-covering matrix are prerequisites for optimal primary adherence and cell spreading. Because fibrin glue might be fibrinolytically degraded despite its plasmin-inhibiting epsilon-amino-caproic acid compound, type I/III collagen plus fibronectin could provide an optimal precoating substrate for EC lining of PTFE grafts.

Long-term experience in autologous in vitro endothelialization of infrainguinal ePTFE grafts

OBJECTIVE Based on a previous randomized study showing significantly superior patency rates for in vitro endothelialized expanded polytetrafluoroethylene (ePTFE) grafts we investigated whether it was feasible for a nontertiary institution to offer autologous in vitro endothelialization to all elective infrainguinal bypass patients who had no suitable saphenous vein available. METHODS Over a period of 15 years, 310 out of 318 consecutive nonacute patients (age 64.7 +/- 8.6) received 341 endothelialized ePTFE grafts (308 femoropopliteal: 153 above knee [AK] and 155 below knee [BK] and 33 femorodistal). Autologous endothelial cells were harvested from short segments (3.9 +/- 1.1 cm) of subcutaneous veins (80% cephalic, 11% basilic, 2% external jugular, and 7% saphenous) and grown to mass cultures within 18.9 +/- 4.5 days before being confluently lined onto fibrin glue-coated ePTFE grafts. The graft diameter was 6 mm (64%) or 7 mm (36%). The overall procedure-related delay for graft implantation was 27.6 + 7.8 days. Growth failure prevented 2.5% of patients from receiving an endothelialized graft. The mean observation period was 9.6 years. Primary patencies were obtained from Kaplan-Meier survivorship functions. Explants for morphological analysis were obtained from eight patients. RESULTS The overall primary patency rate of femoropopliteal grafts was 69% at 5 years (68% [AK] vs 71% [BK]) and 61% at 10 years (59% [AK] vs 64% [BK]). Primary patency of 7 mm vs 6 mm grafts was 78%/62% at 5 years and 71%/55% at 10 years. The difference between the two groups was statistically significant (log rank test P = .023; Breslow test P = .017). Stage I vs II/III patients showed 5-year patencies of 67% vs 73% (N.S.) and 10-year patencies of 61%% vs 53% (N.S.). The primary patency of femorodistal grafts was 52% at 5 years and 36% at 10 years. The limb salvage rate was 94% (fempop) vs 86% (femdistal) at 5 years and 89% vs 71% at 10 years. All retrieved samples showed the presence of an endothelium after 38.9 +/- 17.8 months. CONCLUSION Autologous in vitro endothelialization was shown to be a feasible routine procedure at a nontertiary hospital. Explants confirmed the presence of an endothelium years after implantation while the primary patency in the particularly challenging subgroup of patients without a suitable saphenous vein resembles that of vein grafts.

A Synthetic Non-degradable Polyethylene Glycol Hydrogel Retards Adverse Post-infarct Left Ventricular Remodeling

BACKGROUND Left ventricular remodeling after myocardial infarction is a key component of heart failure and it has long been postulated that it may result from increased wall stress. It has recently been suggested that an injectable, non-degradable polymer may limit pathological remodeling in a manner analogous to that of cardiac support devices. We have tested a non-degradable polyethylene glycol (PEG) gel in a rat infarction model. METHODS AND RESULTS After permanent ligation of the left anterior descending artery in male Wistar rats, PEG gel reagents were injected into the infarcted region and polymerized in situ. At 4 weeks, fractional shortening and infarct volume were unchanged relative to a saline injected control, but the infarct-induced left ventricular end-diastolic diameter (LVEDD) increase was substantially reduced (43%, P < .05) and wall thinning was completely prevented. At 13 weeks, the LVEDD were similar for both saline- and PEG-injected hearts. The non-degradable PEG gels did elicit a macrophage-based inflammatory reaction. CONCLUSIONS The injection of non-degradable synthetic gel was effective in ameliorating pathological remodeling in the immediate postinfarction healing phase, but was unable to prevent the dilation that occurred at later stages in the healed heart.

The Endothelium: A Key to the Future

The vascular endothelium is a complex modulator of a variety of biological systems and may well be the key to definitive success in the treatment of cardiovascular disorders. Surgically‐induced endothelial injury may occur preoperatively during cardiac catheterization and intraoperatively from mechanical manipulation, ischemia, hypothermia, and exposure to cardio‐plegic solutions. The normal endothelium is antithrombogenic and yet promotes platelet aggregation and coagulation if injured. Vasospasm, occlusive intimal hyperplasia, and accelerated arteriosclerosis can also all occur as a result of endothelial injury. Furthermore, endothelial injury is harmful even in the absence of disruption of its monolayer integrity. Thus, preservation of the endothelium should be an additional objective for all cardiovascular surgeons. Synthetic vascular grafts, cardiac valves, and artificial ventricles do not spontaneously endothelialize and thus usually require some form of anticoagulation to maintain patency. Hence, endothelialization of prosthetic implants became an attractive concept. A number of different methods of obtaining an endothelial lining of prosthetic material has since been developed; these include facilitated endothelial cell migration, and endothelial cell seeding by using either venous or microvascular endothelial cells. Manipulating the endothelium might well provide the next major advancement for therapeutic and preventative measures for cardiovascular disease.

Off-the-shelf human decellularized tissue-engineered heart valves in a non-human primate model

Heart valve tissue engineering based on decellularized xenogenic or allogenic starter matrices has shown promising first clinical results. However, the availability of healthy homologous donor valves is limited and xenogenic materials are associated with infectious and immunologic risks. To address such limitations, biodegradable synthetic materials have been successfully used for the creation of living autologous tissue-engineered heart valves (TEHVs) in vitro. Since these classical tissue engineering technologies necessitate substantial infrastructure and logistics, we recently introduced decellularized TEHVs (dTEHVs), based on biodegradable synthetic materials and vascular-derived cells, and successfully created a potential off-the-shelf starter matrix for guided tissue regeneration. Here, we investigate the host repopulation capacity of such dTEHVs in a non-human primate model with up to 8 weeks follow-up. After minimally invasive delivery into the orthotopic pulmonary position, dTEHVs revealed mobile and thin leaflets after 8 weeks of follow-up. Furthermore, mild-moderate valvular insufficiency and relative leaflet shortening were detected. However, in comparison to the decellularized human native heart valve control - representing currently used homografts - dTEHVs showed remarkable rapid cellular repopulation. Given this substantial in situ remodeling capacity, these results suggest that human cell-derived bioengineered decellularized materials represent a promising and clinically relevant starter matrix for heart valve tissue engineering. These biomaterials may ultimately overcome the limitations of currently used valve replacements by providing homologous, non-immunogenic, off-the-shelf replacement constructs.

Injectable living marrow stromal cell-based autologous tissue engineered heart valves: first experiences with a one-step intervention in primates

AIMS A living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates. METHODS AND RESULTS Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n = 6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118 ± 17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodelling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress-strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue. CONCLUSION Here, we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.

In vitro endothelialization of expanded polytetrafluoroethylene grafts: A clinical case report after 41 months of implantation

PURPOSE Forty-one months after we performed bilateral implantation of in vitro endothelialized femoropopliteal bypass grafts in a 69-year-old patient, we obtained a central graft segment for histologic and ultrastructural investigation. METHODS Before implantation the grafts were confluently lined with autologous first passage mass cultures of pure cephalic vein endothelial cells. The precoating of the expanded polytetrafluoroethylene prosthesis was done with fibrinolytically inhibited fibrin glue. Reoperation became necessary because of symptomatic unilateral atherosclerotic lesions located in the center of one of the two in vitro lined grafts. A 21 cm long graft segment was removed and replaced by a new in vitro endothelialized expanded polytetrafluoroethylene graft. RESULTS On scanning electron microscopy a confluently covering mature endothelium was found throughout the whole length of the removed prosthesis. The endothelial identity was confirmed by a positive immunohistochemical CD 34, von Willebrand factor-staining, and the ultrastructural demonstration of Weibel Pallade bodies. The endothelium rested on a collagen IV positive basement membrane. Histologic cross sections revealed uniformly developed subintimal tissue of 1.21 +/- 0.19 mm thickness, which was separated from the intima by a distinct internal elastic membrane. The cells of this cell-rich matrix stained strongly positive for actin. Ultrastructurally, this matrix was dominated by highly contractile myofibroblasts loaded with peripherally located well-developed actin fillaments. A number of these cells also showed signs of secretory cells with a distinct endoplasmic reticulum and a Golgi complex. In areas of atherosclerotic lesions the subendothelial matrix was partially exposed, and the internal elastic membrane had to a certain extent disintegrated. Only in these areas KP-1 and MG-M1 positive foamy macrophages and CD 34 positive capillaries were found. The myofibroblasts of this diseased part of the subintimal tissue contained large lipid vacuoles. CONCLUSIONS We conclude that the confluent in vitro lining of synthetic vascular grafts with pure autologous endothelial cells facilitates graft healing, which may result in a hybrid structure with features of a native vessel.