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Dextran/Albumin hydrogel sealant for Dacron® vascular prosthesis. Anna Lisman, Beata Butruk, Iga Wasiak and Tomasz Ciach J Biomater Appl published online 11 November 2013 DOI: 10.1177/0885328213509676 The online version of this article can be found at: http://jba.sagepub.com/content/early/2013/11/10/0885328213509676

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Original Article

Dextran/Albumin hydrogel sealant for DacronÕ vascular prosthesis

Journal of Biomaterials Applications 0(0) 1–11 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213509676 jba.sagepub.com

Anna Lisman, Beata Butruk, Iga Wasiak and Tomasz Ciach

Abstract In this paper, the authors describe a novel type of hydrogel coating prepared from the copolymer of human serum albumin and oxidized dextran. The material was designed as a hydrogel sealant for polyester (DacronÕ )-based vascular grafts. Dextran was chosen as a coating material due to its anti-thrombogenic properties. Prepared hydrogels were compared with similar, already known biomaterial made from gelatine with the same cross-linking agent. Obtained hydrogels, prepared from various ratios of oxidized dextran/albumin or oxidized dextran/gelatine, showed different cross-linking densities, which caused differences in swelling, degradation rate and mechanical properties. Permeability tests confirmed the complete tightness of the hydrogel-modified prosthesis. Results showed that application of the hydrogel coating provided leakage-free prosthesis and eliminated the need of pre-clotting. Keywords Albumin, dextran, dextran oxidation, hydrogel, vascular prosthesis, sealant

Introduction Development of medical technologies causes a growing need for new, cheap and safe biomaterials. One of the most promising types of the biomaterials is hydrogel, defined as high water content material with threedimensional polymeric network.1 There are numerous applications of hydrogels in medicine,2,3 especially in tissue engineering, due to their physical properties mimicking the native extracellular matrix.4,5 It is generally known that hydrophilic biomaterials characterize high biological compliance,6 mostly due to its similarity to extracellular matrix. Thus, hydrogels can be employed in vascular surgery as prosthesis’s coating and sealing material. DacronÕ vascular grafts are widely applied in order to bypass or replace a diseased or blocked blood vessel. This solution is particularly desirable when material for autograft (autologous transplantation) is not easily available. Application of artificial vessel is also less invasive than autografting, due to the lack of additional autograft vane sampling surgical procedure. Currently, the knitted polyester grafts are mainly used clinically because of their high porosity that allow tissue ingrowths (from outside of the prosthesis) and healing of the graft.7 On the other hand, the considerably high permeability of the graft fabric causes post-surgery bleeding through the graft walls, which can be dangerous. To avoid this phenomenon, standard surgical

procedure requires sealing the prosthesis with patient’s blood clot (pre-cloting).8 Blood clot sealant formed on the graft’s walls is autogenic and encourages the growth of neointima. Unfortunately, the procedure demands additional amount of patient’s blood and creates a highly thrombogenic surface. Furthermore, pre-clotting is an additional procedure that prolongs the surgery and exposes patient and prosthesis to the outer environment, which may cause local infection. According to literature review, DacronÕ grafts were pre-sealed with biodegradable coatings made of proteins (collagen, albumin (Alb) and gelatine (Gel)) cross-linked with formaldehyde or glutaraldehyde.9–11 These aldehydes are unfortunately known for their toxicity and cancerogenic properties. After implantation, coating degrades and allows tissue ingrowth between non-biodegradable polyester fibres from outside the graft. These procedures can evoke inflammatory response after implantation, as a result of contact with animal proteins or toxic cross-linkers.12,13 Moreover, morphological analysis showed that internal layer of the vascular prosthesis has not been covered with endothelial Biomedical Engineering Laboratory, Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warsaw, Poland Corresponding author: Tomasz Ciach, Faculty of Chemical and Process Engineering, Warsaw University of Technology Warynskiego Street 1, 00-645 Warsaw, Poland. Email: [email protected]

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cells, even after long time of implantation.14 Biodegradable, protein-based hydrogels have already proved their efficiency as a graft sealant,15 but a new type of hydrogel which does not produce toxic effects or inflammatory response and has anti-thrombogenic properties is needed. Dextran is a natural polysaccharide consisting predominantly of a-1,6-linked D-glucopyranose units with some degree of branching via 1,3-linkage.16 As a nontoxic, biodegradable and non-immunogenic polymer, dextran is widely applied as a macromolecular carrier for delivery of drugs or proteins. Moreover, due to its blood compatibility, dextran has been used as blood expander and thrombolytic agent.17 The classical method of functionalizing dextran is through oxidizing with periodates. This leads to opening of the glucose ring and formation of double aldehyde groups, which may react with amino groups creating Schiff base (Maillard reaction).18 There are numerous reports on cross-linking of oxidized dextran (DexOx) to form hydrogels with chitosan,19,20 8-arm polyethylene glycol amine,21 polyhydrazides,22 adipic acid dihydrazide23 or Gel.24 The novelty of the presented solution is based on the application of human serum Alb that reacts with DexOx, cross-links and forms a biocompatible hydrogel. Human serum Alb is a globular protein naturally occurring in plasma. It consists of a non-glycosylated single chain polypeptide containing 585 amino acids cross-linked by 17 disulphide bridges.25 It is generally known that native Alb limits the platelet adhesion and decreases thrombogenicity.26 Alb is applied in therapy and its solution can be purchased in hospital pharmacy. Due to the presence of free amine groups Alb is able to form hydrogels when mixed with DexOx water solution through the Maillard reaction. In this study, DexOx/ Alb hydrogels were compared with hydrogels made from DexOx and Gel. Gel is a natural fibrillar protein obtained by the degradation of collagen (most frequently from pigs) and was also tested in medical applications.26 Alb and Gel represent two different types of protein structures: globular and fibrillar, which influences the properties of obtained hydrogels. The prepared hydrogels were tested for its physicochemical properties and applied as a sealant for DacronÕ vascular prosthesis.

Materials and method Materials Dextran (from Leuconostoc mesenteroides, MW ¼ 75,000) was received from Nobilus (Kutno, Poland). Gel (from porcine skin, type A), sodium periodate, potassium iodide, standardized sodium

hydroxide solution, sodium thiosulphate solution and hydroxylamine hydrochloride were purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS), pH 7.4, was prepared by dissolving KH2PO4 (0.2 g), Na2HPO4  12H2O (2.32 g), NaCl (8.0 g) and KCl (0.2 g) in 1 L of distilled water. All reagents used to prepare PBS solution were purchased from Chempur (Piekary Slaskie, Poland). Human Alb (200 mg/mL solution) was purchased from Baxter (Poland). All reagents were of high purity, analytical grade or comply with European Pharmacopeia and have been used as received, without further purification. The knitted DacronÕ vascular grafts were received from the Polish Foundation of Cardiac Surgery Development (Zabrze, Poland). Dialysis membrane (MWCO 12-14 kDa) was purchased from BioNovo (Legnica, Poland).

Methods Synthesis of DexOx. DexOx was prepared according to the previously reported methods.27–29 Briefly, 10 g of dextran was dissolved in 200 mL distilled water, thereafter the given amount of sodium periodate was added. The mixture was stirred at room temperature (RT) for 24 h in the dark. The product solution was then dialysed against distilled water using dialysis membrane for 3 days and then water was evaporated at 40 C under atmospheric pressure. Dry DexOx samples with theoretical oxidation degree of 20% and 30% were marked as DexOx-I and DexOx-II. The amount of sodium iodate (IO3) remaining in the final products was determined by addition of iodide (KI) in acidic medium. The formed free iodine was titrated with sodium thiosulphate, as an indicator 1% starch solution was applied. The amount of sodium iodate in the final DexOx was in average 0.7 mmole IO3 (0.14 mg NaIO3) per 1 g of DexOx. DexOx was characterized by Fourier transform infrared spectroscopy-attentuated total reflectance (FTIR-ATR). The infrared spectra of DexOx were recorded using Nicolet 6700 Smart Orbit Diamond ATR (Thermo Scientific, Nankin, China). The FTIR spectra were obtained by recording 32 scans between 4000 and 400 cm1 with a resolution of 4 cm1. Determination of aldehyde groups in the DexOx. The oxidation degree (aldehyde group content) of DexOx was evaluated using the hydroxylamine hydrochloride method,30 untreated dextran samples were used as a control. Approximately 0.1 g of dried DexOx-I and DexOx-II were precisely weighed and dissolved in 25 mL of 0.25 N hydroxylamine hydrochloride solution. The mixtures were magnetic stirred for 2 h and later titrated with standardized 0.1 N sodium hydroxide solution. The number of aldehyde groups

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per 100 glucose subunits was calculated with the following formula:29

dried, weighed and the loss of dry weight was calculated.

N:Al:G: ¼ N  V  ðMW=mdex Þ  ð100=SÞ  103

Coating of DacronÕ vascular grafts with hydrogels. The solutions of 5% and 10% DexOx-I were prepared and mixed in different ratios with protein solutions. The following solutions were prepared: 5:5 DexOx-I/Alb, 8:2 DexOx-I/Alb, 5:5 DexOx-I/Gel and 8:2 DexOx-I/ Gel (according to section Hydrogel preparation). The mixtures were gently stirred for 1 min at 40 C to avoid air bubbles and thereafter 20% (w/v) of glycerine was added. Knitted DacronÕ vascular graft was cut into pieces (2.5  2 cm), the pieces of graft were dip coated 1, 2 or 3 times in the chosen DexOx/protein solution and dried at RT for 30 min each time before applying the next layer. During the coating process samples were held with tweezers and were placed upright, gently stretching. In order to prevent flowing of the coating, the covered pieces after being taken out from the mixture were turned over until the hydrogel layer solidified. After coating the final layer the materials were dried for 24 h at 40 C.

where N.Al.G. is the number of aldehyde groups, N is the concentration of standardized sodium hydroxide, V is the volume of NaOH solution used in the titration, MW is the molecular weight of dextran, mdex is the weight of dextran and S is the number of glucose subunits in dextran. Hydrogel preparation. Weighed amount of DexOx-I and Gel was dissolved in 0.01 M PBS (pH ¼ 7.4) to obtain 5% and 10% (w/v) solutions. Solution of human Alb (200 mg/mL) was diluted using PBS. The solutions were stored at 4 C. DexOx-I/Alb hydrogels were prepared by mixing solutions of DexOx-I warmed to 40 C and solution of human Alb at RT. DexOx-I/Gel was prepared by mixing solutions of DexOx and Gel warmed to 40 C. The mixtures were gently stirred for 1 min at 40 C to avoid air bubbles, poured into plastic containers and kept at 40 C for 24 h. The hydrogels prepared from 5% and 10% (w/v) solutions were coded as 5-DexOx-I/Alb, 5-DexOx-I/Gel, 10-DexOx-I/Alb and 10-DexOx-I/Gel. Morphology analysis. The morphology of lyophilized DexOx-I/Gel and DexOx-I/Alb hydrogels was analysed using Scanning Electronic Microscopy (Phenom, FEI, Eindhoven, Holland). The hydrogel samples (0.5 cm  0.5 cm) were immersed in PBS for 2 h, frozen at 20 C for 24 h lyophilized in vacuum. Fractured pieces of dried hydrogels were secured on an aluminium foil and both surface and cross-section morphologies were investigated and depictured. Swelling analysis. Swelling studies were conducted in 0.01 M PBS at 37 C. Pieces of hydrogels were dried at 50 C and weighed (Wd). Then samples were immersed in 20 mL of PBS for 24 h. Then the hydrogels were blotted with filter paper to remove excess water and weighed (Ws). The swelling ratio was calculated with the following formula:

Water permeability of coated vascular grafts. The water permeability of graft walls was examined based on the previously described method31 in the constant-flow apparatus presented in Figure 1. The water flow was forced by the pump; it circulated in the close circuit: water tank, pump, valve 3. Part of the flow goes through the measurement loop: valve 1, manometer, sample holder, water filter and valve 2. The manometer allows controlling the water pressure, which was set by valves 1 and 2. All grafts pieces were exposed to 120 mmHg water pressure. The sample of vane prosthesis was mounted in the test segment, between silicone gaskets, attached to the polypropylene tube. The area of material exposed to flow was constant for all samples and equalled 0.28 cm2. Volume of water was measured with a measuring cylinder.

EWCð%Þ ¼ ððWs  Wd Þ=Wd Þ  100% where EWC is the equilibrium water content, Ws is the weight of swollen sample and Wd is the weight of dry sample. Degradation analysis. In order to evaluate degradation ratio hydrogels were immersed in 0.01 M PBS at 37 C for 2 weeks, PBS was changed daily. After a given degradation time (t ¼ 3, 7, 10 and 14), hydrogels were

Figure 1. Diagram of the water permeation apparatus: 1,2,3: valves, M: manometer, F: filter, S: sample.

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Platelet adhesion. In order to access the thrombogenicity of the hydrogel-modified DacronÕ surfaces the pieces of materials were incubated with human platelet-rich plasma (PRP). Before the experiments, all the specimens were sterilized with ethanol and immersed in isotonic (pH 7.4) PBS for 24 h. Fresh blood, donated by healthy adult volunteer (female, age 27) was collected into K2EDTA tubes (BD Vacutainer, Poland) without using stasis, the first sample tube was rejected. Blood was centrifuged at 200 x g for 20 min to obtain PRP. PBS-hydrogenated materials were placed in 24-well culture plates and incubated with 1 mL of PRP for 1 h at 37 C. After that time, materials were gently rinsed with PBS and fixed with glutaraldehyde (2.5%/PBS, 2 h, 4 C) followed by fixation with OsO4 (1%/PBS, 1 h, 4 C). Materials were then dehydrated through graded concentrations of ethanol (50%, 60%, 70%, 80%, 90% and 100%, 5 min each), dried, coated with carbon and analysed with scanning electron microscope. Cytotoxicity analysis. In vitro cytotoxicity of hydrogel samples was determined using the direct contact method. L929 fibroblasts (ECACC, UK) were seeded in 24-well plates and cultured with Dulbecco’s Modified

Figure 2. Mechanism of dextran oxidation process.

Eagle’s Medium (DMEM) supplemented with 10% of FBS, 1% of glutamine and 1% of antibiotic at 37 C in 10% CO2. After 24 h the medium was replaced with fresh one and analysed samples (flat discs with a diameter of 3 mm, previously sterilized with 70% ethanol and hydrated in PBS) were placed in each well. Cells cultured with DMEM were used as a negative control. After 48 h, 7 days and 14 days of culture XTT assay was performed. Cell viability was calculated using the following formula: viability % ¼ ðAs=AncÞ  100 where As is the mean value of the measured absorption of the sample and Anc is the mean value of the measured absorption of the negative control.

Results and discussion Dextran oxidation Dextran was oxidized with sodium periodate. The reagent was added in a given amount that allowed obtaining the theoretical oxidation degree of 20% or 30%. The mechanism of dextran oxidation process is as follows: first, the bond between C3 and C4 breaks and two aldehyde groups are formed. In the second step, C3 is separated from the molecule as formic acid32 (Figure 2). Therefore, during the first 3 min of reaction pH decreased from 7 to 2.5. In this study, the number of aldehyde groups in the final product was determined by hydroxylamine method. As presented in Table 1, the actual number of aldehyde groups is higher than the theoretical one (based on Figure 2). Our results confirmed previously reported data,29 where the authors suggested that every molecule of periodate create about 1.7 of aldehyde groups. In our experiments, the number was similar and equalled approximately 1.6. This is due to the fact that both steps of the reaction compete for periodate ions and the kinetics of both steps of the reactions are similar. Because of that, in the described conditions,

Table 1. Contents of aldehyde groups per 100 glucose subunits in the dextran chain.

Material

Theoretical number of aldehyde groups per 100 glucose units (Oxidized glucose units)

Experimental number of aldehyde groups per 100 glucose units  SD, n ¼ 3

Experimental number of oxidized glucose units per 100

DexOx-I DexOx-II

40 (20) 60 (30)

63.4  1.6 96.5  3.0

32 48

DexOx: oxidized dextran.

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Figure 3. FTIR spectra of: A – dextran and B – oxidized dextran-I.

Figure 4. Scanning electron microscope images of lyophilized hydrogels: (a) DexOx/Alb (ratio 2:8), (b) DexOx/Gel (ratio 2:8), (c) DexOx/Alb (ratio 8:2) and (d) DexOx/Gel (ratio 8:2).

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only about 20% of periodate takes part in the second step of oxidation. It also means that about 75% of all oxidized glucose rings are the products of the first oxidation step only (Figure 2). Base on our experiments and on the available data27 one can also predict that for lower oxidation rates, lower periodate/dextran ratios, even lower fraction of

oxidized glucose units will reach the second step of oxidation. The process of dextran oxidation was monitored by the appearance of the aldehyde symmetric vibration band (carbonyl) at 1735 cm1 via FTIR. As shown in Figure 3, the FTIR spectra of Dex, DexOx-I and DexOx-II are similar and no signal from aldehyde groups was observed. It can be due to the formation of hemiacetals33,34, rather than a low degree of oxidation.35

Morphology analysis Microscopic analysis of lyophilized hydrogels revealed highly porous structure of the materials. The structures of DexOx-I/Gel and DexOx-I/Alb (DexOx:protein ratio 8:2 or 2:8) are depictured in Figure 4. There is an observable influence of the DexOx content to the networks construction and appearance. Increase in protein content resulted in more fibrous structures (Figure 4(a) and (b)). The increase in DexOx content creates more porous network (Figure 4(c) and (d)).

Swelling analysis Figure 5. Influence of hydrogel composition on swelling ratio of 10-DexOx-I/Alb and 10-DexOx-I/Gel hydrogels (MV  SD, n ¼ 3). DexOx: oxidized dextran; Alb: albumin; Gel: gelatine.

The swelling ratio was analysed as a function of DexOx contents, hydrogels with the following DexOx content were prepared: 20%, 40%, 50%, 60% and 80%.

Figure 6. Hydrolytic degradation of 10-DexOx-I/Alb hydrogels (ratios: 8:2, 2:8, 5:5), (MV  SD, n ¼ 3).. DexOx: oxidized dextran; Alb: albumin.

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Swelling studies were performed for 5-DexOx-1/Alb, 5-DexOx-1/Gel, 10-DexOx-1/Alb and 10-DexOx-1/ Gel. Swelling ratio as a function of DexOx contents for 10-DexOx-I/Alb and 10-DexOx-I/Gel is shown in

Figure 5. For 10-DexOx-I/Alb hydrogel, initially EWC decreased quickly with an increase in the DexOx contents until its minimum at 60% of DexOx. For the higher content of DexOx EWC increased up to 2.63

Figure 7. Hydrolytic degradation of 10-DexOx-I/Gel hydrogels (ratios: 8:2, 2:8, 5:5), (MV  SD, n ¼ 3). DexOx: oxidized dextran; Alb: albumin.

Figure 8. Water permeability of single-coated grafts (DexOx-I/Alb 5:5, DexOx-I/Alb 8:2, DexOx-I/Gel 5:5, DexOx-I/Gel 8:2), obtained from 5% and 10% (w/v) solutions; (MV  SD, n ¼ 3). DexOx: oxidized dextran; Alb: albumin; Gel: gelatine.

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Figure 9. Water permeability of double-coated grafts (DexOxI/Alb 8:2 and 5:5; DexOx-I/Gel 8:2 and 5:5) prepared from 10% (w/v) solutions; (MV  SD, n ¼ 3). DexOx: oxidized dextran; Alb: albumin; Gel: gelatine.

for 80% DexOx. It is known that swelling ratio is inversely proportional to the hydrogel cross-linking density. Thus, it can be concluded that for 60% of DexOx the ratio of aldehyde groups in DexOx to available amine groups in Alb (lysine, arginine) is equal to 1. This leads to the high cross-linking densities and more compact structure of the polymer. Excess of either aldehyde groups or amine groups in the preparation resulted in the lower cross-linking densities and the higher swelling ratios. In the case of the 10-DexOx-I/Gel hydrogel, the relationship between swelling ratio and DexOx content is different. EWC decreased with increase in the DexOx content from 20% to 80%. Thus, it can be inferred that Gel presents higher number of available amine groups than Alb. This can be a result of its fibrillar (linear) structure and high content of hydroxylysine, which has an additional amine group. Swelling ratios of 5-DexOx-I/Alb and 5-DexOx-I/ Gel hydrogels on DexOx content were twofold smaller than in the case of 10-DexOx-I/Alb and 10-DexOx-I/ Gel hydrogels, but the relationships between EWC and DexOx content was the same as described above (data not shown).

Degradation analysis All the examined hydrogels undergo the process of nonenzymatic hydrolysis of dextran glucose chain. Figures 6 and 7 show the results of degradation process of 10-DexOx-I/Alb and 10-DexOx-I/Gel. The weight loss was rapid in the first 3 days, after that time degradation process slowed down. As can be seen in Figures 6 and 7, the degradation ratio depended on the content of the DexOx. The higher the DexOx content, the quicker hydrogel degraded.

Figure 10. Microscopic images of DacronÕ prosthesis contacted with human platelet-rich-plasma: non-modified DacronÕ prosthesis (a), DacronÕ coated with 5-DexOx-I/Alb 2:8 (b) and DacronÕ coated with 5-DexOx-I/Alb 8:2 (c).

It is worth mentioning that the 10-DexOx-I/Alb ratio 8:2 was completely broken down after 14 days as opposed to 10-DexOx-I/Gel ratio 8:2. These differences can be a result of different structure of applied proteins (globular/fibrillar), which, in turn, determines cross-linking density and hydrolytic susceptibility of the hydrogels. Degradation profiles of 5-DexOx-I/Alb and 5-DexOx-I/Gel were very similar to those depictured in Figure 6. However, the degradation process was approximately twofold faster (data not shown). Samples of 8:2 5-DexOx-I/Alb hydrogel were completely broken after 3 days.

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Figure 11. Viability of cells cultured with hydrogel-modified materials; MV  SD, n ¼ 6. DexOx: oxidized dextran; Alb: albumin; Gel: gelatine.

Properties of hydrogel coated vascular grafts

Platelet adhesion

Sealing of DacronÕ graft by dip-coating technique demanded a quick and skilled action, because of the fast cross-linking process. Therefore, in some cases the coating was non-uniform; this was observed mostly for Gel-based hydrogels. Alb-based coatings, due to the slower reaction rate, formed smooth, evenly distributed coatings on both sides of the graft. After drying the pieces of graft were stiffer than the uncoated ones. However, after immersing in saline for few minutes they became soft and flexible again. The measurements of water permeability of the modified grafts were fundamental to access the effectiveness of the hydrogels as a sealant. The water permeation value obtained for unsealed grafts was about 1400 (mL/cm2/min). Both DexOx-I/Alb and DexOx-I/Gel showed to be effective sealants. The results of water permeation tests are shown in Figure 8. Single-layer hydrogel coating of grafts drastically decreases water permeability, and more concentrated (10%) solutions showed to be more effective. As shown in Figure 9, application of the second coating layer reduced water permeability below 1 mL/cm2/min. Both 10-DexOx-I/ Alb and 10-DexOx-I/Gel (both ratio 8:2) are the most effectively sealants.

The above-presented results of modified grafts permeability proved to be promising. However, an effective sealant for vascular prosthesis, in addition to the sealing properties, must present high haemocompatibility and do not provoke platelet adhesion. In order to access the influence of the modified surfaces on platelet activation and adhesion processes, materials have been contacted with blood and then thoroughly analysed with electron microscope. Figure 10 presents microscopic images of the non-modified DacronÕ graft (Figure 10(a)) and dextran/Alb-modified grafts, 5-DexOx-I/Alb 2:8 (Figure 10(b)) and 5-DexOx-I/Alb 8:2 (Figure 10(c)), after contact with human PRP. All analysed materials presented clear surfaces, without any visual morphological components of the blood. There was no difference in the number of adhered components between control, non-coated, surface and DexOx/Alb-modified surfaces. Thus, we assumed that the lack of platelets adhered to the hydrogelmodified surface indicates its neutral, non-irritating properties. Application of the coating does not reduce haemocompatible properties of DacronÕ surface, which confirmed good biological properties of the prepared materials.

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Cytotoxicity analysis Figure 11 presents viability of fibroblasts cultured with hydrogel-modified samples for 48 h, 7 days and 14 days. Viability values for each tested sample exceeded 70% after 24 h of culture. In the subsequent days of culture, these values were similar orincreased, reflecting the normal process of cell growth and proliferation. No significant differences in cell morphology, depending on the variant of the coating, were observed. Two materials, 5-DexOx-1 2: and 10-DexOx-I 5:5, caused a decrease in cell viability during the culture. For these materials, there was a significant swelling and degradation observed, which could impede the process of cell adhesion and cause washing them out during the medium exchange.

the blood components and Dextran – already proved to be non-thrombogenic. Water permeation studies showed that presented hydrogels are effective sealant for knitted DacronÕ grafts. Significant reduction of permeability was obtained after single coating; the lowest permeability showed grafts sealed with 80% DexOx hydrogels. The complete tightness was achieved by double coating. Obtained results showed that DexOx/Alb hydrogel coatings on DacronÕ graft provide leakage-free prosthesis without the need of preclotting procedure. Hydrogel-modified DacronÕ grafts when in contact with human PRP did not provoke platelet adhesion. In future, we plan to investigate biological properties of sealed grafts in contact with human endothelial cells, followed with experiment with animal model. Patent for presented technology is pending.

Conclusions A novel type of biodegradable hydrogel, copolymer of human Alb and DexOx, has been investigated and described. This biomaterial was compared with similar, already known hydrogel made of Gel with the same cross-linking agent. Production of the cross-linking agent, DexOx has also been investigated and presented. It was shown that only about one-fourth of oxidized glucose rings reaches the second step of oxidation. Because of that the number of aldehyde groups is about 1.6 times higher than predicted and for lower oxidation degrees, lower periodate/dextran ratios, this discrepancy will be even higher. Described hydrogels have different swelling behaviour depending on the cross-linker/protein ratio. Presented hydrogels degrade due to the simple hydrolysis and the degradation rate also depends on the hydrogel composition. Opportunity to tune the swelling properties and degradation rate of presented biomaterial can be important in medical applications. Presented hydrogel seems to be biologically safe, its degradation products, glucose, Alb and products of Alb glycation, naturally occur in blood. The potential risk associated with the application of the Maillard reaction is decomposition of the reaction products at high temperatures and formation of potentially danger acrylamides. Since the proposed modification process is conducted at mild temperature and water environment the risk is limited. It is also known that under physiological conditions human blood proteins such as serum Alb undergoes a nonenzymatic glycation by formation of Schiff base between "-amino groups of lysine (sometimes arginine) and aldehyde side of glucose molecules in blood.36 One of the possible applications of the described biomaterial, sealant for knitted vascular grafts, was investigated and described. The surfaces were haemocompatible due to application of human Alb – one of

Limitations Process of dextran oxidation is fairly long and performed in water at slightly acidic conditions, which can cause partial hydrolysis. It would be important to investigate the change of dextran molecular weight during the oxidation process. It is also possible that chosen oxidation time is too long and the reaction is finished after shorter time. However, from the graft coating perspective some minor hydrolysis of dextran does not influence its polymer forming properties, what is shown in this paper. For medical applications, DacronÕ grafts have to be stored dry in sealed disinfected package, like all implantable devices. The influence of long-term storage in dry conditions on stability of described biomaterial should be checked. However, pure dextran, according to manufacturer data, can be stored dry for at least 5 years. Obtained coated grafts are too stiff to be directly applied (when dry). Thus, coated graft need to be immersed in sterile saline directly before the application or a proper plasticizer should be applied (e.g. glycerol). Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest None declared.

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Albumin hydrogel sealant for Dacron(R) vascular prosthesis.

In this paper, the authors describe a novel type of hydrogel coating prepared from the copolymer of human serum albumin and oxidized dextran. The mate...
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