Henryk J. Salacinski

The contemporary role of antioxidant therapy in attenuating liver ischemia‐reperfusion injury: A review

Oxidative stress is an important factor in many pathological conditions such as inflammation, cancer, ageing and organ response to ischemia‐reperfusion. Humans have developed a complex antioxidant system to eliminate or attenuate oxidative stress. Liver ischemia‐reperfusion injury occurs in a number of clinical settings, including liver surgery, transplantation, and hemorrhagic shock with subsequent fluid resuscitation, leading to significant morbidity and mortality. It is characterized by significant oxidative stress but accompanied with depletion of endogenous antioxidants. This review has 2 aims: firstly, to highlight the clinical significance of liver ischemia‐reperfusion injury, the underlying mechanisms and the main pathways by which the antioxidants function, and secondly, to describe the new developments that are ongoing in antioxidant therapy and to present the experimental and clinical evidence about the role of antioxidants in modulating hepatic ischemia‐reperfusion injury. (Liver Transpl 2005;11:1031–1047.)

Development of a hybrid cardiovascular graft using a tissue engineering approach

Tissue engineering of endothelial cells (EC) and chemical engineering with anticoagulant moieties has been undertaken in order to improve prosthetic graft patency and thrombogenicity. This was done by covalently bonding a compliant poly(carbonate‐urea)urethane graft (MyoLink™) with with arginine‐glycine‐aspartate (RGD) or/and heparin (Hep) to ascertain whether EC retention could be improved. The retention of these moieties and EC was assessed after exposure to pulsatile flow. We covalently bonded RGD, Hep, and RGD/Hep onto the luminal surface of MyoLink using spacer arm technology. Narrow‐beam X‐ray photoelectron spectroscopy was carried out to check the efficiency of the bonding. EC were radiolabeled and seeded onto native MyoLink and with 1) RGD‐, 2) Hep‐, and 3) RGD/Hep‐bonded grafts and exposed to shear stress in a physiological flow circuit for 6 h, which reproduces femoral artery flow waveforms and pulsatility. Results were recorded on a gamma camera imaging system. Viability of cells was tested with a modified Alamar Blue assay (ABA) and scanning electron microscopy for morphological appearance of seeded cells. Experiments were repeated (n=6). RGD, Hep, and RGD/Hep were bonded together in a uniform distribution on the luminal surface of each graft type, and bioactivity of each moiety covalently bonded was very high. In the flow circuit, there was exponential cell retention for the first 60 min of flow for all the grafts, but after 6 h of exposure to pulsatile flow the RGD/Hep‐bonded graft had a significantly better cell retention rate than native MyoLink (75.7%±2.3 vs. 60.5±10.1, P<0.05). ABA test showed that all the seeded cells postexposure to flow were viable, and significantly higher metabolic activity was recorded on a RGD/Hep‐bonded graft than with MyoLink‐seeded graft (P<0.01). Using RGD/Hep covalently bonded onto graft surfaces improves cell retention and provides an antithrombogenic surface for initial blood flow in vivo until full EC activity develops postseeding. This would allow the development and further improvement of hybrid grafts.—Tiwari, A., Salacinski, H. J., Punshon, G., Hamilton, G., Seifalian, A. M. Development of a hybrid cardiovascular graft using a tissue engineering approach. FASEB J. 16, 791–796 (2002)

New prostheses for use in bypass grafts with special emphasis on polyurethanes.

Vascular bypass procedures using traditional prosthetic grafts such as polytetrafluoroethylen (PTFE) and polyethylene tetraphthlate (Dacron) are prone to failure when used in low flow states such as in below knee bypass and when the diameter of the graft is less than 6 mm. A major factor in this is compliance mismatch between the graft and the diseased vessel, which may cause intimal hyperplasia at the distal anastomosis. PTFE and Dacron are rigid grafts with poor compliance. By improving the compliance of the prosthetic graft it is hoped that patency will improve. Recent advances in polyurethane chemistry have developed materials that do not degrade and which allow compliance matching of the graft to the patients vasculature. It is now possible to manufacture biologically and haemodynamically compatible grafts with small diameter from these polyurethane graft materials. This review will focus on the lack of compliance in current vascular bypass grafts and the promise of the new polyurethane polymers in a new generation of small-bore bypass grafts.

The use of animal models in developing the discipline of cardiovascular tissue engineering: a review.

Cardiovascular disease remains one of the major causes of death and disability in the Western world. Tissue engineering offers the prospect of being able to meet the demand for replacement of heart valves, vessels for coronary and lower limb bypass surgery and the generation of cardiac tissue for addition to the diseased heart. In order to test prospective tissue-engineered devices, these constructs must first be proven in animal models before receiving CE marking or FDA approval for a clinical trial. The choice of animal depends on the nature of the tissue-engineered construct being tested. Factors that need to be considered include technical requirements of implanting the construct, availability of the animal, cost and ethical considerations. In this paper, we review the history of animal studies in cardiovascular tissue engineering and the uses of animal tissue as sources for tissue engineering.

Shear‐stress preconditioning and tissue‐engineering‐based paradigms for generating arterial substitutes

In situ tissue engineering using shear‐stress preconditioning and adhesive biomolecules is a new approach to autologous tissue engineering. In the present study, novel tissue‐engineering grafts (TEGs) were preconditioned within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), to establish whether low‐shear‐stress conditions promoted endothelial cell (EC) retention and differentiation. TEGs (n=24) were generated by the contraction and compaction of collagen(I) by porcine aortic smooth‐muscle cells (SMCs) on to a compliant polyester graft scaffold. ECs were radiolabelled with [111In]indium tropolonate and seeded on to the luminal surface of the TEGs. Following organ culture in a bioreactor (7 days), TEGs were split into four groups (n=six TEGs per group): Group A acted as controls with TEGs unmodified and seeded with radiolabelled ECs; Group B underwent luminal pre‐coating with FN (75 μg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 10–20 μN (1–2 dyn)/cm2 for 7 days prior to EC seeding, and Group D TEGs were preconditioned for 7 days at 1–2 dyn/cm2, followed by luminal pre‐coating with FN prior to EC seeding. The resistance to physiological shear stress of the seeded ECs was assessed using a γ‐radiation counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 93.2 μN (9.32 dyn)/cm2. Environmental scanning electron microscopy (ESEM) was used to determine the distribution and degree of differentiation of the attached Ecs, and tissue‐type‐plasminogen‐activator (tPA) assays provided a measure of function and viability. EC resistance to shear stress at 93.2 μN/cm2 was significantly enhanced by a period of preconditioning (Group C) at 10–20 μN/cm2, surface modification with FN (Group B), or both (Group D) when compared with control grafts (Group A). However, TEGs coated with FN whether preconditioned (Group D) or not (Group B) demonstrated the best results for EC retention. ESEM demonstrated near‐confluent differentiated flattened ECs in both these cases. EC function was demonstrated by a steady increase in tPA production. Low‐shear‐stress preconditioning of TEGs enhances EC retention in vitro with an additional advantage demonstrated by pre‐treatment with FN prior to endothelialization. These findings may be exploited in the development of tissue‐engineered constructs to maintain a confluent endothelial lining.

Silsesquioxane nanocomposites as tissue implants.

Background: Silicone implants are being used increasingly worldwide, especially in breast augmentation procedures. The most common morbidity observed is capsular contracture, which occurs in 15 percent of cases. To overcome this problem, the authors have developed a novel nanocomposite based on polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) for use as tissue implants. Methods: These polymers were implanted in six healthy sheep (n = 6) for 36 months and a siloxane served as the positive control. After explantation, these polymers were extracted, as was the surrounding capsule, if any. Attenuated total reflectance Fourier transform infrared spectroscopy analysis was performed to look for signs of surface degradation on the polymers and histopathologic and electron microscopic examinations were performed to study the interaction between the biomaterial and the host environment in greater detail. Results: After implantation, the authors observed minimal inflammation of the nanocomposite within the sheep model as compared with the siloxane control. Contact angle measurements and fibrinogen enzyme-linked immunosorbent assay tests were then conducted on the POSS-PCU nanocomposite to determine the reason for this behavior. The increased fibrinogen adsorption on POSS-PCU, its amphilicity, and large contact-angle hysteresis indicated that POSS-PCU inhibits inflammation by adsorbing and inactivating fibrinogen on its surface. In complete contrast, the control siloxane in the same setting demonstrated very significant inflammation and degradation, resulting in capsular formation. Naturally, there was no evidence of degradation of the nanocomposite compared with the siloxane control. Conclusions: POSS-PCU nanocomposites have enhanced interfacial biocompatibility and better biological stability as compared with conventional silicone biomaterials, thus making them safer as tissue implants.

Cellular engineering of vascular bypass grafts: Role of chemical coatings for enhancing endothelial cell attachment

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5–6 mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patients own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.

The endothelialization of polyhedral oligomeric silsesquioxane nanocomposites - An in vitro study

It has been recognized that seeding vascular bypass grafts with endothelial cells is the ideal method of improving their long-term patency rates. The aim of this study was to assess the in vitro cytocompatibility of a novel silica nanocomposite, polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) and hence elicit its feasibility at the vascular interface for potential use in cardiovascular devices such as vascular grafts. Using primary human umbilical vein endothelial cells (HUVEC), cell viability and adhesion were studied using AlamarBlue assays, whereas cell proliferation on the polymer was assessed using the PicoGreen dye assay. Cellular confluence and morphology on the nanocomposite were analyzed using light and electron microscopy, respectively. Our results showed that there was no significant difference between cell viability in standard culture media and POSS-PCU. Endothelial cells were capable of adhering to the polymer within 30 min of contact (Students t-test, p<0.05) with no difference between POSS-PCU and control cell culture plates. POSS-PCU was also capable of sustaining good cell proliferation for up to 14d even from low seeding densities (1.0×103 cells/cm2) and reaching saturation by 21 d. Microscopic analysis showed evidence of optimal endothelial cell adsorption morphology with the absence of impaired motility and morphogenesis. In conclusion, these results support the application of POSS-PCU as a suitable biomaterial scaffold in bio-hybrid vascular prostheses and biomedical devices.

Artificial nerve conduits in peripheral-nerve repair

Injuries to the nervous system are the result of mechanical, thermal, chemical or congenital pathologies and, if function is not restored, they lead to loss of muscle function, pain and impaired sensation. Current treatment modalities essentially coapt the two nerves ends together or place a nerve graft between the cut ends. However, clinical results have never been optimal, and therefore a quest for better options has taken place. In this review article we look at the synthetic and biomimetic options currently being tested as potential nerve grafts.

An assessment of covalent grafting of RGD peptides to the surface of a compliant poly(carbonate-urea)urethane vascular conduit versus conventional biological coatings: its role in enhancing cellular retention.

The aim of sodding prosthetic grafts with endothelial cells (EC) is to establish a functioning antithrombogenic monolayer of EC. Application of basement membrane proteins improves EC adherence on ePTFE grafts. Their addition to a biodurable compliant poly(carbonate-urea)urethane graft (CPU) was studied with respect to EC adherence. Preclot, fibronectin, gelatin, and collagen were coated onto CPU. RGD peptide, heparin, and both RGD and heparin were chemically bonded to CPU. Human umbilical vein EC (HUVEC) labeled with 111-Indium oxine were sodded (1.8 x 10(6) EC/cm(2)) onto native and the modified CPU. The grafts were washed after 90 min and EC retention determined. The experiments were repeated six times. EC retention on native CPU was 1.0 +/- 0.2 x 10(5) EC/cm(2). The application of preclot, fibronectin, gelatin, and collagen did not improve EC retention, which was 0.8 +/- 0.1, 0.4 +/- 0.1, 0.3 +/- 0.08, and 0.5 +/- 0.2 x 10(5) EC/cm(2), respectively. Bonding RGD, heparin, and both RGD and heparin significantly improved EC retention to 1.9 +/- 0.6, 1.7 +/- 0.5, and 2.6 +/- 0.6 x 10(5) EC/cm(2), respectively (p < 0.01). Bonding of RGD, heparin, and both RGD and heparin accelerates and enhances EC retention onto CPU. Simple coating of basement membrane proteins confers no advantage over native CPU.