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ORIGINAL ARTICLE

The Endothelialization of Polyhedral Oligomeric Silsesquioxane Nanocomposites An In Vitro Study

Ruben Y. Kannan,1 Henryk J. Salacinski,1 Kevin M. Sales,1 Peter E. Butler,2 and Alexander M. Seifalian 1,* 1Biomaterials

& Tissue Engineering Centre, Academic Division of Surgery and Interventional Sciences, University College London, Hampstead Campus, London NW3 2PF, United Kingdom; 2Department. of Plastic and Reconstructive Surgery, Royal Free Hospital Hampstead NHS Trust, London NW3 2QG, United Kingdom

Abstract 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 × 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. Index Entries: Polyhedral oligomeric silsesquioxanes (POSS); endothelial cells; bypass graft; cytocompatibility; tissue engineering, nanocomposite.

have limited its clinical potential, particularly at lower flow rates at which patency has been shown to bear an inverse relationship to the internal diameter of the grafts (6,7). This may be reversed by lining these bypass grafts with endothelial cells (EC) (8,9). Randomized clinical trials using endothelialized 7-mm internal diameter ePTFE vascular grafts have shown primary patency rates of 83.7% in lower limb bypass grafts at four years (10,11). Its success, however, depends on the initial seeding density, duration of culture and the capacity of the cells to proliferate on the biopolymer (12). At lower flow rates, the compliance mismatch between current bypass grafts and the native vessels has

INTRODUCTION Vascular grafts can be constructed by using biopolymers coated with heparin, endothelial cells, or as truly biological grafts tissue-engineered in vitro before reimplantation (1–3). Polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) have been the biopolymers of choice in the construction of synthetic vascular grafts to date clinically. However, poor elastic properties (4,5) and graft thrombogenicity * Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected] Cell Biochemistry and Biophysics

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been hypothesized to be a cause of intimal hyperplasia; a major cause of long-term graft occlusion (13,14). Polyurethanes (PU) are a newer generation of polymers with similar radial compliance characteristics to native vessels (15). Of these, carbonate-based polyurethanes, unlike earlier generations of PU, do not possess ether or ester linkages and are hence less susceptible to degradation. Our own study using poly(carbonate-urea) urethane (PCU) grafts on the aorta-iliac vessels of four beagle dogs older than 36 mo showed 100% patency, minimal intimal hyperplasia, and no signs of aneurysmal dilatation. This preliminary study suggests that improved radial compliance will reduce intimal hyperplasia and hence improve long-term patency. Siloxane molecules have been reported to reduce platelet and thrombin adsorption (16) and hence the addition of such groups have been postulated to confer an antithrombogenic effect (17). Copolymers of polyurethanes and siloxanes used in intra-aortic balloons have relative hemocompatibility that is the result of the dispersive surface force field on its surface, which maintains any adsorbed protein in an unstable state (18). However, silicon-based vascular grafts have not been successful as the formation of an EC lining on these constructs is suboptimal (19). Silsesquioxanes (SQS) are silicon-based nanostructures that exist as chain-or ladder-type structures (20). In 1995, a closedcage SQS called polyhedral oligomeric silsesquioxane (POSS) (21) was developed. This molecule is composed of two cyclic rings composed of silicon and oxygen linked together. By altering the number of side-groups it possesses, POSS-nanocomposites can function as part of a linear polymeric backbone or a crosslinked system. Based on these data, we hypothesized that the addition of POSS as a pendant side chain to PCU would form nanocages on the surface and improve cross-linking within. While increased crosslinkage within the copolymer would impart greater resistance to biodegradation, its surface nanostructure consisting of alternating areas of POSS and PCU would confer the dual properties of anti-thrombogenicity (anti-platelet activity and coagulation cascade inhibition) and ability to sustain an EC lining. These are ideal characteristics for biomaterials used in vascular tissue engineering and in general, for biomedical interventional devices. In this study, we assessed whether POSS-PCU was safe and compatible with in vitro cell cultures. Apart from indicating its safety as a biomaterial at the cellular level, such information would also serve as a measure of its potential for developing bio-hybrid vascular grafts. As shown in Fig. 1, developing a bio-hybrid vascular graft would require the EC to undergo four phases. These cells have first to remain viable within the culture medium exposed to POSS-PCU and on direct contact

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Fig. 1. (A) Schematic representation of the stages of cell seeding on biomaterials. (B) Molecular structure of the POSS nanocore/filler integrated with the hard urethane segment of poly(carbonate-urea)urethane (PCU).

with the polymer (Phase I). Next, the EC would have to adhere to the polymer surface, preferably within a short time, to minimize the complications associated with long-term cell cultures (Phase II). These adherent EC would then need to proliferate at a steady rate (Phase III) to achieve a confluent EC monolayer (Phase IV).

MATERIALS AND METHODS Polymer Synthesis This has been described in detail elsewhere (22). In brief, polycarbonate polyol and trans-cyclohexanediolisobutyl-silsesquioxane were placed in a reaction flask equipped with stirrer and nitrogen. The mixture was heated to 125°C to dissolve the nanocage. To this, methylene di-isocyanate were added and reacted at 70 to 80°C for 90 min to form a pre-polymer. Then N,Ndimethylacetamide (DMAC) were added. Chain extension was carried out by the addition of ethylenediamine and diethylamine in DMAC. After chain extension, 1butanol and DMAC were then added slowly to the nanocomposite to complete the process. All chemicals and reagents were purchased from Aldrich Limited, Gillingham, UK.

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The Endothelialization of Nanocomposites

Primary Endothelial Cell Culture Using a previously described method (23), human umbilical vein endothelial cells (HUVEC) were extracted from umbilical cords procured within 24 h of delivery using sterile collagenase (Boehringer Mannheim; from Clostridium Haemolyticum). HUVEC were then grown in tissue culture using cell culture medium (CCM) consisting of 157 mL of M199 medium, 40 mL fetal bovine serum, 4.8 mL 7.5% sodium bicarbonate (Invitrogen, Paisley, Scotland, UK), 5 mL L-glutamine (200 mM), 1.57 mL penicillin/streptomycin (5000 units of penicillin, and 5 mg/mL streptomycin (Sigma, Poole, Dorset, UK). After incubation for 48 h at 37°C and 5% CO2, the confluent HUVEC were removed using 0.25% TrypsinEDTA (Sigma-Aldrich Company Ltd., Poole, UK) and split in a 1:4 ratio. In our experiments, confluent HUVEC from passages 4 to 5 were used.

Cell Viability Analysis DIRECT CONTACT M ETHOD A total of 1.0 × 105 fourth passage HUVEC suspensions in CCM were each placed in sterile 24-well culture plates (Helena Biosciences, Sunderland, UK) coated with POSS-PCU and incubated at 37°C and 5% CO2 for 24 h and 48 h, respectively. Alamar Blue analysis (Serotec Ltd., Kidlington, Oxford, UK) was then performed to detect the remaining viable cells after this period of culture on the polymer. Absorption was measured at 570 nm and 630 nm in accordance with the manufacturer’s guidelines, using a Multiscan MS spectrophotometer (Labsystem Multiskan MS; Labsystem, Helsinki, Finland) (23,24). All tests were repeated four times in duplicate (n = 4) with uncoated standard polystyrene tissue culture wells serving as controls.

DILUENT EXTRACTION M ETHOD POSS-PCU was pulverized using a dismembranator (Mikro-dismembranator u, B. Braun Biotech International GmbH). The powder was autoclaved, placed in CCM to constitute a 100 mg/mL solution of PCSU and then shaken at 200 rpm for 19 d at 37°C. After exposure, the mixture was sieved and centrifuged to remove all polymer from the CCM. The 24-well plates seeded with 5.0 × 104 HUVEC were then incubated at 37°C and 5% CO2 for 48 h in 25, 50, 75, and 100 mg/mL of the polymer-exposed CCM with plain CCM acting as the control. After incubation for 0, 6, 12, and 24 h, AlamarBlue analysis was performed to detect cells that remained viable after exposure; studies were conducted four times in duplicate (n = 4).

H IGH-PERFORMANCE LIQUID C HROMATOGRAPHY High-performance liquid chromatography (HPLC) analysis was performed to assess whether any compo-


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nent of POSS-PCU leached out into the media after incubation. Powdered polymer was placed in sterile phosphate-buffered saline (PBS) for 90 d to allow for leaching of the polymer, if any, before being centrifuged and filtered. The filtrated PBS was then subjected to HPLC analysis using a 30-mm gradient with the sample run at 20 cm/h through a C18 reverse plate that assessed hydrophobicity (Varian Ltd., USA). The sample was run for 30 min using buffer solutions of 0.1% tri-fluroacetic acid/TFA in water and 0.1% TFA in acetonitrile. Plain PBS controls were used as the control group.

Cell Adhesion Assay Fourth passage HUVEC were seeded at 3.5 × 105 cells/cm2 on sterile POSS-PCU sheets placed within standard 96-well plasma-treated polystyrene wells (Helena Biosciences Ltd., Sunderland, UK) (25). HUVEC were then incubated for 30, 60, 120, and 240 min at 37°C and 5% CO2. Wells were rinsed and AlamarBlue applied to wells to detect cells that had adhered to the wells over the allotted times (n = 4). As before, spectroscopic analysis at 570 and 630 nm was performed using the Multiscan MS spectrophotometer (Labsystem Multiskan MS). Plasmatreated polystyrene culture plates (control) and conventional siloxane were treated in the same way to determine differences in EC adhesion between linear siloxanes and caged SQS and to compare the endothelializing properties of SQS. As a measure of the hydrophilicity of the polymer, the water absorption index (WAI) of POSS-PCU was determined. Thin strips of ePTFE, PCU, and POSS-PCU of equal dimensions were immersed in pure distilled water (Sigma Ltd., Dorset, UK) for 1, 3, and 7 d. Their respective pre-immersion (m0) and post-immersion weights (m1) were measured and the WAI was determined using the formula: WAI =

( m1 – m0 ) × 100% m0

Cell Proliferation Assay Proliferation of HUVEC on POSS-PCU over time was determined using PicoGreen DNA quantification assay (Molecular Probes Co., OR); a vital dye that stains DNA fluorescent green. HUVEC were seeded onto glass Petri dishes at 1.0 × 103 cells/cm2 containing culture media and incubated for 1, 3, 7, 11, 14, and 21 d. The HUVEC were then extracted and reconstituted in 1 mL mixtures of 10 mM Tris-HCl with 1 mM EDTA at pH 7.5 (1X TE) to form the sample, whereas serially diluted calf thymus DNA served as the standards (26). A total of 100 µL aliquots of these solutions were then mixed with 100 µL 1:400 (v/v) dilution of PicoGreen dye in 1X TE and

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incubated for 5 min. This was then excited at 485 nm and the emissions at 538 nm were read. The amount of DNA in the sample was then determined from the standards (data not shown) and, based on the assumption that each cell contains 7.7 pg of DNA, the number of cells was calculated.

Cell Morphology LIGHT M ICROSCOPY Fifth passage HUVEC were cultured on sterile, flat sheets of the POSS-PCU for 48 h to assess cell confluence morphology. The cultured cells were then fixed with 4% paraformaldehyde for 10 min and carefully rinsed with PBS and stained with 0.1% Toluidine Blue (TB) stain (Sigma Chemical Company, Poole, Dorset, UK) for 10 min before being washed off with PBS. The sheets were then examined under a confocal microscope (Nikon D-Eclipse C1, Japan) at ×20 magnification to qualitatively assess cell attachment to the polymer.

Fig. 2. Direct contact analysis using Alamar Blue spectral absorption (ABSA), which showed no significant difference in cell viability between standard cell cultures and those on POSS-PCU (p = not significant).

E LECTRON M ICROSCOPY The morphology of the proliferating EC was also studied using electron microscopy. In a parallel experiment, fifth passage HUVEC was cultured on sterile, flat sheets of PCSU for 48 h. Thereafter, it was rinsed with PBS and fixed with glutaraldehyde solution (2.5 v/v%) in PBS for 2 h at room temperature. The samples were then dehydrated with ethanol/distilled water (10% ethanol increments) at 41°C. This was followed by freeze-drying for 48 h, attaching them to aluminium stubs, and coating them with gold using an SC500 (EM Scope) sputter coater. The parameters assessed were the presence of cell retraction, loss of filopodia, and a rounded cell appearance; indicators of impaired cell motility and morphogenesis (27).

RESULTS Cell Viability Cell viability on the polymer was assessed using both the direct contact and diluent extraction methods (27). Direct contact involved exposing HUVEC directly to the polymer, whereas the indirect technique involved using CCM exposed to the polymer to culture cells. Analysis was performed using a vital dye; Alamar Blue (AB) is metabolized from blue resazurin to pink resazurin by viable cells and the change in color was detected spectroscopically (27). Leaching from POSS-PCU into CCM was assessed using HPLC. Alamar blue detects only live cells, those that metabolize, and hence does not detect dead cells. In this respect, we can detect the


Fig. 3. Diluent extraction analysis using Alamar Blue spectral absorption (ABSA) showing no significant differences between varying doses of cell media incubated with POSS-PCU at 0, 6, 12, and 24 h (p = not significant).

metabolism of viable cells remaining after exposure to POSS-PCU.

DIRECT E FFECT

OF

POSS-PCU

Direct contact studies showed that there was no significant difference between HUVEC viability on POSS-PCU and standard tissue culture plates after measurement with AlamarBlue assay at 24 and 48 h of cell culture (two-way analysis of variance [ANOVA]; p = NS) as is shown in Fig. 2. This shows that HUVEC cultured directly onto POSS-PCU exhibited optimal cell viability for up to 48 h.

I NDIRECT E FFECT

OF

POSS-PCU

AlamarBlue analysis of the diluent extract of POSSPCU showed no significant difference (p = NS) in cell viability between the control group and cell cultures with varying concentrations of the polymer extract (0, 25, 50, 75, and 100 mg/mL) at 6, 12, and 24 h, respectively, as shown in Fig. 3. This indicates that no cytotoxic substance leaches out of POSS-PCU in vitro as EC

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can remain viable on the polymer for the prolonged in vitro culture period that is necessary for two-stage seeding procedures.

H IGH-PERFORMANCE LIQUID C HROMATOGRAPHY HPLC analysis showed here was no evidence of any substance leaching out the polymer on prolonged contact. This was in line with the results showing optimal cell viability in the diluent extraction method performed previously.

Cell Adhesion AlamarBlue is also used to detect the cell adhesion. In this case, we detect the metabolism of only the cells that remain on the POSS-PCU after the media is removed. To ensure that remaining cells have adhered and not merely settled, a wash step was carried out before adding AlamarBlue. EC adhesion to the POSS-PCU sheets showed a significant increase in adherent viable cells to the polymer (53% increase in spectral absorption over baseline values) within 30 min of contact (Student’s t-test, p < 0.05). Subsequent adhesion of viable EC to the polymer reached saturation within 2 h of cell culture on the polymer (Fig. 4A). When compared with the minimal cell adhesion seen on medical-grade siloxane these results indicate that when silicon molecules are incorporated as closed-cage SQS, they allow for improved EC adhesion to its surface and, hence, endothelialization. Two-way ANOVA analysis showed a very significant difference in EC adhesion between POSS-PCU and siloxane (p < 0.0001). There was no difference in EC adhesion between POSS-PCU and standard polystyrene cell culture plates. The WAI of POSS-PCU nanocomposites was 28.54% ± 3.216% as compared with PCU (6.194% ± 1.985%) and PTFE (3.978% ± 3.218%). Two-way ANOVA analysis (Fig. 4B) showed a statistically significant difference between these values (p < 0.0001), indicating the greater hydrophilicity of POSS-PCU nanocomposites compared with PTFE and PCU. WAI tests on medical-grade siloxane were not performed as the hydrophobicity of siloxane per se is well documented.

Fig. 4. (A) Alamar Blue (AB) assay showing significant endothelial cell adhesion to POSS-PCU within 30 min of contact (Student’s t-test, p < 0.05) and reaching saturation after 2 h of incubation (p < 0.01). These values were significantly greater than endothelial cell adhesion to medicalgrade siloxane (two-way ANOVA, p < 0.0001). (B) Water absorption index (WAI) assays that show significantly higher water absorption indices and hence hydrophilicity of POSS-PCU as compared with ePTFE and PCU (one-way ANOVA, p < 0.0001).

Cell Proliferation Quantitative analysis of HUVEC proliferation on POSS-PCU polymers studied with PicoGreen showed a significant increase in mean EC proliferation at 1, 3, 7, 11, 14, and 21 d over baseline values (one-way ANOVA, p < 0.0001). There was no significant difference found between different repeats of EC for a given time (one-way ANOVA, p = NS). The growth pattern of EC on the nanocomposite (initial seeding density of 1.0 × 103 cells per cm2) reached the exponential phase of growth beyond 1 wk in culture with a 60-fold increase in EC surface density at 14 d, reaching saturation by 21 d (Fig. 5). These


Fig. 5. PicoGreen assay showing significant increases in cell proliferation on POSS-PCU over 3 wk (one-way ANOVA, p < 0.0001).

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Fig. 6. Light microscopic analysis (×20 magnification) of Toluidine Blue (TB)-stained endothelial showing the different stages of cell confluence on POSS-PCU. (A) TBstained HUVEC on POSS-PCU at 48 h; (B) TB-stained HUVEC by d 6 showing 80 to 90% cellular confluence.

results demonstrate that EC are able to proliferate on the polymer from very low seeding densities over the prolonged periods necessary for two-stage seeding procedures.

Cell Morphology Light microscopic examination of TB-stained EC on POSS-PCU showed a reticulate pattern of confluence morphology by 48 h of culture (Fig. 6A) and confluence by d 6 of culture (Fig. 6B). These data suggest that EC are ultimately capable of reaching confluence on POSSPCU. Higher magnification of these cells with a scanning electron microscope done to assess adsorption morphology on POSS-PCU showed optimal cell motility and morphogenesis with the formation of numerous cellular filopodia as well as the absence of cell retraction and “rounded” cells (Fig. 7). As such, the development of the bio-hybrid POSS-PCU nanocomposite vascular graft is a distinct possibility in the future.


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Fig. 7. Scanning electron microscope pictures of endothelial cell adsorption morphology on POSS-PCU showing the presence of a flat, spindle-shaped cells with numerous filopodia and the absence of cell retraction. This indicates good endothelial cell motility and morphogenesis on POSS-PCU at 48 h (×320 magnification).

DISCUSSION The principal materials used in vascular prostheses at present are polyethylene terephthalate and ePTFE. Studies by Zilla and coworkers have already shown the clinical benefit of lining ePTFE vascular grafts with endothelial cells particularly in lower limb arterial bypass grafts (13). Native, uncoated polyethylene terephthalate grafts have been found to possess suboptimal endothelializing properties in vivo, shown to be a result of decreased expression of the fibronectin receptor, VLA-5 (28). This has seen the increasing use of fibronectin motifs such as RGD peptides (29), cell adhesion peptides such as P-15 (30), and even titanium coatings that promote endothelial cell confluence on the surfaces of conventional vascular prostheses (31). From the vascular prostheses perspective, the current generation of siloxane copolymers have two main limitations, namely stiffness (32) and poor endothelial cell adhesion as a result of its high hydrophobicity. One

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answer to this problem has been to coat vascular prosthetic surfaces with either gelatin-glutaraldehyde crosslinkages (33), titanium carboxonitride (31), or peptides (34,35). All these surface modifications have been shown to enhance EC seeding onto the graft surface but this involves a secondary coating procedure. An alternative is to incorporate low-adhesive silicon into higher adhesive polymers such as polyurethanes (13) in a way that would improve overall EC adhesion while maintaining its visco-elasticity. This necessitates the development of newer generation silica vascular interfaces such as POSS-PCU nanocomposites that allow both cell seeding and optimal mechanical properties. The results of these studies suggest that the improved endothelializing property of POSS-PCU is attributable to its improved hydrophilic behavior. Using these POSS-integrated PCU nanocomposites as bio-hybrid constructs would first require them to be safe in the cell culture environment (36). In all experiments performed, the cytocompatibility of these POSS-PCU nanocomposites were compared to standard cell cultures. Both direct and indirect methods of assessing toxicity showed no significant increase in toxicity with POSS-PCU. This may be explained by the inherent stability of the nanocomposite as POSS nanocages (Si8O12) that have had all of its eight reactive side groups (R) fully reacted with the PCU. In addition, the polyhedral nature of the POSS molecule ensures greater integration with both soft and hard domains of the polyurethane with lesser chances of substance degradation and leaching. All polymer sheets were cast at 70°C, well above the boiling point of DMAC. This technique thus allows for complete evaporation of the diluent, DMAC, and would explain the absence of substance leaching out into CCM. Unlike most existing silicon copolymers (32,37), we found that using a silicon pendant nanocage allowed for improved cell adhesion characteristics. Because POSS nanocages occupy minimal volumes within the polymer (38), there is a relatively greater surface area of polyurethane than is available, allowing improved endothelialization. This would also confer a greater degree of polarity to the polymer, which could explain its superior hydrophilicity to the polyurethane PCU. These experiments also indicate that once adherent to the polymer surface, EC are also capable of proliferating manifold in order to form a confluent monolayer. The PicoGreen assay showed the excellent proliferating characteristics of EC on the polymer. Light microscopy revealed how, before achieving cellular confluence, the EC aligned themselves in a reticular manner with the intervening areas being filled subsequently to achieve cellular confluence as shown in Fig. 6B. Our data also showed that the quality of cell adhesion and proliferation on the polymer was excellent.


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Qualitative data using scanning electron microscope analysis clearly visualized the presence of optimal cellpolymer interactions with the formation of numerous filopodia at its surface, flattened EC, and no rounded cells. This suggests that these EC are capable of morphogenesis and have the ability to proliferate well. This set of experiments has demonstrated POSS-PCU to be compatible with EC, including cellular attachment and proliferation. In conclusion, POSS-PCU nanocomposites are compatible for use in cardiovascular devices and cell seeding.

ACKNOWLEDGMENTS We acknowledge financial support of EPSRC and UCL BioMedica PLC, UK, for support of HJS in development of novel polymer for medical application and RMO Fellowship for support of RK. The authors are also grateful to Mr. Arnold Derbyshire for his help in the polymer synthesis.

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