UV surface modification of a new nanocomposite polymer to improve cytocompatibility.

A novel modified nanocomposite was studied for the adhesion and proliferation of the human umbilical vein endothelial cell (HUVEC) line EA.hy926. The nanocomposite under investigation was poly(carbonate-urea)urethane with silsesquioxane nano-cages, here in the form of a mixture of two polyhedral oligomeric silsesquioxanes. The nanocomposite surfaces were exposed to ultraviolet (UV) light of a Xe(*)(2)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. The effects of the irradiation were characterized by atomic force and scanning electron microscopy (AFM, SEM), X-ray photo-electron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR) using an attenuated total reflection (ATR) device and measurements of advancing water contact angle (CA). The irradiation resulted in the introduction of new hydrophilic N- and O-containing groups into the surface, which was initially amphiphilic, while surface morphology remained mainly unchanged. Slight chemical changes were also observed for the silsesquioxane nano-cages at the surface. Onto the untreated and irradiated samples HUVECs were seeded and grown for various durations in culture. Standard tissue-culture polystyrene (PS) was employed as a positive control to check the efficiency of the cell-culture methods. Viability and proliferation of the cells were then assessed using a non-radioactive assay. Compared to the untreated nanocomposite polymer, irradiation times of at least 5 min resulted in a significantly increased cell proliferation between 3 and 8 days after seeding with the HUVEC line EA.hy926.

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.

Formation and role of plasma S-nitrosothiols in liver ischemia-reperfusion injury.

Plasma S-nitrosothiols (RSNOs) may act as a circulating form of nitric oxide that affects vascular function and platelet aggregation. Their role in liver ischemia/reperfusion (I/R) injury is largely unknown. The aim of the present study was to investigate the changes in plasma RSNOs following liver I/R injury. Two groups of New Zealand white rabbits were used (n=6, each): the I/R group underwent 60 min lobar liver ischemia and 7 h reperfusion, while the sham group underwent laparotomy but no liver ischemia. Serial RSNO levels were measured in plasma by electron paramagnetic resonance (EPR) spectrometry, nitrite/nitrates by capillary electrophoresis, hepatic microcirculation by laser Doppler flowmetry, redox state of hepatic cytochrome oxidase by near-infrared spectroscopy, liver iNOS mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) and the oxidation of dihydrorhodamine to rhodamine by fluorescence. The effect of the antioxidant N-acetylcysteine (NAC) on RSNOs formation and DHR oxidation was tested in a third group of animals (n=6) undergoing lobar liver I/R. Hepatic I/R was associated with a significant increase in plasma RSNOs, plasma nitrites, hepatic iNOS mRNA expression, impairment in hepatic microcirculation, decrease in the redox state of cytochrome oxidase, and significant production of rhodamine. The changes were more obvious during the late phase of reperfusion (>4 h). NAC administration decreased plasma RSNOs and oxidation of DHR to RH (P<0.05, 5 and 7 h postreperfusion, respectively). These results suggest that significant upregulation of nitric oxide synthesis during the late phase of reperfusion is associated with impairment in microcirculation and mitochondrial dysfunction. Plasma S-nitrosothiols are a good marker of this nitric oxide-mediated hepatotoxicity.

Incorporation of a lauric acid-conjugated GRGDS peptide directly into the matrix of a poly(carbonate-urea)urethane polymer for use in cardiovascular bypass graft applications.

Gly-Arg-Gly-Asp-Ser (GRGDS) was modified by conjugation to lauric acid (LA) to facilitate incorporation into the matrix of a poly(carbonate-urea)urethane (PCU) used in vascular bypass grafts. GRGDS and LA-GRGDS were synthesized using solid phase Fmoc chemistry and characterized by high performance liquid chromatography and Fourier transform infrared spectroscopy. LA-GRGDS was passively coated and incorporated as nanoparticle dispersion on the PCU films. Biocompatibility of the modified surfaces was investigated. Endothelial cells seeded on LA-GRGDS coated and incorporated PCU showed after 48 h and 72 h a significant (p < 0.05) increase in metabolism compared with unmodified PCU. The platelet adhesion and hemolysis studies showed that the modification of PCU had no adverse effect. In conclusion, LA-conjugated RGD derivatives, such as LA-GRGDS, that permit solubility into solvents used in solvent casting methodologies should have wide applicability in polymer development for use in coronary, vascular, and dialysis bypass grafts, and furthermore scaffolds utilized for tissue regeneration and tissue engineering.

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 (Student's t-test, p < 0.05) with no difference between POSS-PCU and control cell culture plates. POSSPCU was also capable of sustaining good cell proliferation for up to 14 d even from low seeding densities (1.0 x 10(3) cells/cm(2)) 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.

Review paper: principles and applications of surface analytical techniques at the vascular interface.

Surface properties have been found to be one of the key parameters which cause degradation and of thrombogenicity in all polymers used in biomedical devices, thus signifying the importance and the necessity for quantitative and accurate characterization of the polymer surface itself as used in the construction of the device. The characterization techniques employed generally involve thermal and spectroscopic measurements, in which class the electrochemical investigations and scanning probe microscopies can also be included. Current hypotheses on the correlations that exist between surface parameters and hemocompatibility and degradation of polymers are examined herein, but concentrating on the field of clinically utilized polymeric materials as used within medical devices themselves. Furthermore, this review provides a brief but complete synopsis of these techniques and other emerging ones, which have proven useful in the analysis of the surface properties of polymeric materials as used in the construction of cardiovascular devices. Statements and examples are given as to how specific information can be acquired from these differing methodologies and how it aids in the design and development of new polymers for usage in biomedical device construction.

Polyhedral oligomeric silsequioxane-polyurethane nanocomposite microvessels for an artificial capillary bed.

Fabricating artificial vascularised tissue would involve tissue-engineering techniques, but current technology limits this as cultured cells depend on growth media in vitro and on diffusion in vivo. Therefore, there is a need to construct a synthetic microvascular network, which would sustain these cultured cells in a similar manner to normal tissue. This is again hampered by the poor patency rates of current microvascular grafts. Based on our previous work on polyhedral oligomeric silsesquioxane-polyurethane nanocomposites, which have shown the unique ability to repel coagulant proteins whilst still allowing endothelialisation, we have now developed a new generation of microvascular prosthesis using this polymer. Using these dip-coated nanocomposite microvessels, we have shown that it is possible to mimic the hydraulic conductivity and pressure-responsive radial compliance characteristics of biological microvessels. This would allow nutrient exchange across its walls as well as minimise compliance mismatch throughout the physiological pressure range thus reducing intimal hyperplasia in the long term. This microvessel would have the following implications: (1) as a microvascular substitute to vein grafts and (2) in the future as a component of a microvascular network.

The antithrombogenic potential of a polyhedral oligomeric silsesquioxane (POSS) nanocomposite.

We have developed a nanocomposite using a silica nanocomposite polyhedral oligomeric silsesquioxane (POSS) and poly(carbonate-urea)urethane (PCU) for potential use in cardiovascular bypass grafts and the microvascular component of artificial capillary beds. In this study, we sought to compare its antithrombogenicity to that of conventional polymers used in vascular bypass grafts so as to improve upon current patency rates, particularly in the microvascular setting. Using atomic force microscopy (AFM) and transmission electron microscopy (TEM), surface topography and composition were studied, respectively. The ability of the nanocomposite surface to repel both proteins and platelets in vitro was assessed using thromboelastography (TEG), fibrinogen ELISA assays, antifactor Xa assays, scanning electron microscopy (SEM), and platelet adsorption tests. TEG analysis showed a significant decrease in clot strength (one-way ANOVA, p < 0.001) and increase in clot lysis (one-way ANOVA, p < 0.0001) on the nanocomposite when compared to both poly(tetrafluoroethylene) (PTFE) and PCU. ELISA assays indicate lower adsorption of fibrinogen to the nanocomposite compared to PTFE (one-way ANOVA, p < 0.01). Interestingly, increasing the concentration of POSS nanocages within these polymers was shown to proportionately inhibit factor X activity. Platelet adsorption at 120 min was also lower compared to PTFE and PCU (two-way ANOVA, p < 0.05). SEM images showed a "speckled" morphologic pattern with Cooper grades I platelet adsorption morphology on the nanocomposite compared to PTFE with grade IV morphology. On the basis of these results, we concluded that POSS nanocomposites possess greater thromboresistance than PTFE and PCU, making it an ideal material for the construction of both bypass grafts and microvessels.

The degradative resistance of polyhedral oligomeric silsesquioxane nanocore integrated polyurethanes: an in vitro study.

Polymer biostability is one of the critical parameters by which these materials are selected for use as biomedical devices. This is the major rationale for the use of polymers which are highly crystalline and stiff namely expanded polytetrafluoroethylene (ePTFE) and Dacron in particular, as arterial bypass grafts. While this is immaterial in high-flow states, it becomes critically important at lower flows with a greater need for more compliant vessels. Polyurethanes being one of the most compliant polymers known are as such, the natural choice to build such constructs. However, concerns regarding their resistance to degradation have limited their use as vascular prostheses and in order to augment their strength, herein a novel polyhedral oligomeric silsesquioxane integrated poly(carbonate-urea)urethane (POSS-PCU) nanocomposite was synthesised by our group. In the following series of experiments, the POSS-PCU nanocomposite samples were exposed to accelerated degradative solutions, in an 'in-house' established model in vitro for up to 70 days before being subjected to infra-red spectroscopy, scanning electron microscopy, stress-strain studies and differential scanning calorimetry. Our results demonstrate that these silsesquioxane nanocores shield the soft segment(s) of the polyurethane, responsible for its compliance and elasticity from all forms of degradation, principally oxidation and hydrolysis. These nanocomposites hence provide an optimal method by which these polymers may be strengthened whilst maintaining their elasticity, making them ideal as vascular prostheses particularly at low flow states.

Cardiovascular tissue engineering: state of the art.

In patients requiring coronary or peripheral vascular bypass procedures, autogenous arterial or vein grafts remain as the conduit of choice even in the case of redo patients. It is in this class of redo patients that often natural tissue of suitable quality becomes unavailable; so that prosthetic material is then used. Prosthetic grafts are liable to fail due to graft occlusion caused by surface thrombogenicity and lack of elasticity. To prevent this, seeding of the graft lumen with endothelial cells has been undertaken and recent clinical studies have evidenced patency rates approaching reasonable vein grafts. Recent advances have also looked at developing a completely artificial biological graft engineered from the patient's cells with surface and viscoelastic properties similar to autogenous vessels. This review encompasses both endothelialisation of grafts and the construction of biological cardiovascular conduits.

Polyhedral oligomeric silsesquioxane nanocomposites: the next generation material for biomedical applications.

The unique properties of nanocomposites have seen them creating the next revolution in materials science. Their quantal properties as a result of their size have given them unique physical characteristics, previously not possible because of classical physical laws. There is now evidence that these may also extend into the world of biology and medicine. In this Account, we look at the birth of a new generation of silica nanocomposites using polyhedral oligomeric silsesquioxanes, a promising nanoscale silica particle with particular use in cardiovascular interventional devices.

Single stage cell seeding of small diameter prosthetic cardiovascular grafts.

Small-diameter prosthetic cardiovascular bypass grafts have high occlusion rates. Thrombogenicity caused by the lack of endothelial cells (ECs) on the luminal surface of the grafts is one of the main reasons for its occlusion. One strategy to improve the clinical performance of cardiovascular prosthetic grafts has been to seed its luminal surface with a monolayer of the patient's own ECs. In this strategy a "two stage" seeding procedure is utilized whereby cells obtained from a vein are amplified in cell culture, then seeded onto a fibrin-arginine-glycine-aspartate (RGD) tripeptide-enriched expanded polytetrafluoroethylene (ePTFE) graft in a rotating bioreactor for one week, after which it is surgically implanted. This achieves patency rates approaching those of vein grafts. The disadvantage of two stage seeding is that it requires culture facilities, a large amount of RGD, which is expensive and is confined to elective cases because of the delay between cell cultivation, seeding, and graft implantation. A single stage seeding using freshly extracted ECs that is transplanted onto the graft at the same time frame of the bypass operation without the need for cell cultivation would be an ideal answer for the disadvantages of two stage seeding. Animal trials have been successful but human trials of single stage seeding have been disappointing. It has been hypothesized that extracted ECs are scarce, furthermore, they are washed off the graft surface once exposed to blood flow. This review examines the various techniques/technologies to improve endothelial cell extraction from various sources and retention onto the luminal surface of prosthetic cardiovascular grafts in order to develop a clinically applicable strategy for single stage seeding.

Advancing cartilage tissue engineering: the application of stem cell technology.

The treatment of cartilage pathology and trauma face the challenges of poor regenerative potential and inferior repair. Nevertheless, recent advances in tissue engineering indicate that adult stem cells could provide a source of chondrocytes for tissue engineering that the isolation of mature chondrocytes has failed to achieve. Various adjuncts to their propagation and differentiation have been explored, such as biomaterials, bioreactors and growth hormones. To date, all tissue engineered cartilage has been significantly mechanically inferior to its natural counterparts and further problems in vivo relate to poor integration and deterioration of tissue quality over time. However, adult stem cells--with their high rate of proliferation and ease of isolation--are expected to greatly further the development and usefulness of tissue engineered cartilage.

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.

Advancing vascular tissue engineering: the role of stem cell technology.

Atherosclerosis and heart disease are still the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties. This, in turn, is limited by the availability of suitable cells from which to construct the vessels. The development of prosthesis using progenitor cells and switching these into endothelial cells is an important and exciting advance in the field of tissue engineering. Here, we describe recent developments in the use of stem cells for the development of replacement vessels. These paradigm shifts in vascular engineering now offer a new route for effective clinical therapy.

Current status of prosthetic bypass grafts: a review.

Polymers such as Dacron and polytetrafluoroethylene (PTFE) have been used in high flow states with relative success but with limited application at lower flow states. Newer polymers with greater compliance, biomimicry, and ability to evolve into hybrid prostheses, suitable as smaller vessels, are now being introduced. In view of the advances in tissue engineering, this makes possible the creation of an ideal off-the-shelf bypass graft. We present a broad overview of the current state of prosthetic bypass grafts.

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.

Interactions between endothelial cells and a poly(carbonate-silsesquioxane-bridge-urea)urethane.

We have recently developed a polymer which contains silsesquioxane in the form of nano-bridges poly(carbonate-silsesquioxane-bridge-urea)urethane (PCBSU) for cardiovascular device applications. The polymer has been characterised and the durability has been confirmed with long-term in vivo tests. The aim of this study was to test the cytocompatibility of the new polymer and to investigate any potential cytotoxic effects. To assess the effect of direct contact with PCBSU sections of polymer material were cut and placed into a 24-well plate. Six discs were seeded with 2 x 10(5) human umbilical vein cells (HUVEC). As a positive control, six wells were seeded with the same number of HUVEC. In a further experiment to assess indirect contact with PCBSU a sample of the polymer was powdered using a Micro-Dismembrator. Cell culture medium was exposed to powdered polymer (1-100 mg/ml) for a period of 7 days. HUVEC seeded as above were then exposed to the treated cell culture medium for 24 and 96 h. Finally, cell proliferation was studied over 16 days by seeding 2 x 10(5) HUVEC on films of PCBSU cast in glass Petri dishes. Cell viability and growth were assessed using Alamar blue, lactate dehydrogenase and Pico green assays and morphology was studied by Toluidine blue staining and scanning electron microscopy. Viable cells were demonstrated to be present after 16 days seeded on PCBSU. Exposing cells to PCBSU-treated cell culture medium resulted in no apparent damage to the cells at concentrations of 1 or 10 mg/ml, and only a slight reduction at 100 mg/ml after 96 h exposure. This study demonstrates that PCBSU can support the growth of endothelial cells for a prolonged period and does not demonstrate any significant toxic effects to cells. Thus it has the potential to be used both as a medical device and as scaffolding in tissue engineering applications.

Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts.

To date no reliable method has been developed for the isolation of RNA from cells seeded onto cylindrical vascular grafts. This study was performed in order to develop a reliable methodology for isolating RNA from cylindrical conduits made from poly(carbonate-urea)urethane (PU). Human umbilical vein EC were seeded onto PU vascular grafts and an Alamar blue assay performed to assess cell viability. Cells were prepared for RNA extraction by trypsinisation, cell scraping and direct application of cell lysis buffer. In all cases RNA was extracted using a "Qiagen RNeasy" kit. Alamar blue showed viable cells were present on all of the seeded PU vascular grafts. Levels of RNA extracted from the cells removed from the graft by the trypsinisation yielded 0.130 microg/microl, by scraping 0.078 microg/microl and by direct lysing 0.093 microg/microl of RNA, respectively. RTPCR was conducted successfully for GAPDH and TGF-beta1. Trypsinisation prior to RNA extraction provided the highest RNA yield and attained near complete cell removal ensuring that gene expression obtained was representative.

The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review.

The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors. Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential. The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues. Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link. In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.