Modified glycogen as construction material for functional biomimetic microfibers

Carbohydrate Polymers 152 (2016) 271–279

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Modified glycogen as construction material for functional biomimetic microfibers Mariia Rabyk a , Martin Hruby a,∗ , Miroslav Vetrik a , Jan Kucka a , Vladimir Proks a , Martin Parizek b , Rafal Konefal a , Pavel Krist c , David Chvatil c , Lucie Bacakova b , Miroslav Slouf a , Petr Stepanek a a

Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic Institute of Physiology AS CR, v.v.i., Videnska 1083, 14220 Prague 4, Czech Republic c Nuclear Physics Institute AS CR, v.v.i., 250 68 Rez, Czech Republic b

a r t i c l e

i n f o

Article history: Received 8 May 2016 Received in revised form 21 June 2016 Accepted 28 June 2016 Available online 2 July 2016 Chemical compounds studied in this article: Glycogen (PubChem CID 439177) Sodium hydroxide (PubChem CID 14798) Acetic acid (PubChem CID 176) Allyl bromide (PubChem CID 7841) Propargyl bromide (PubChem CID 7842)

a b s t r a c t We describe a conceptually new, microfibrous, biodegradable functional material prepared from a modified storage polysaccharide also present in humans (glycogen) showing strong potential as direct-contact dressing/interface material for wound healing. Double bonds were introduced into glycogen via allylation and were further exploited for crosslinking of the microfibers. Triple bonds were introduced by propargylation and served for further click functionalization of the microfibers with bioactive peptide. A simple solvent-free method allowing the preparation of thick layers was used to produce microfibers (diameter ca 2 ␮m) from allylated and/or propargylated glycogen. Crosslinking of the samples was performed by microtron beta-irradiation, and the irradiation dose was optimized to 2 kGy. The results from biological testing showed that these highly porous, hydrophilic, readily functionalizable materials were completely nontoxic to cells growing in their presence. The fibers were gradually degraded in the presence of cells. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Glycogen Fibers Irradiation crosslinking Wound healing dressing Tissue

1. Introduction Significant effort was recently invested into the development of materials for tissue engineering (TE) and wound healing purposes in regenerative medicine (Langer & Vacanti, 1993). The scaffolds for TE must fulfill specific requirements, such as biocompatibility, nontoxicity, controlled biodegradability, good mechanical properties and suitable surface chemistry for the successful attachment, migration, proliferation, and differentiation of the cells. In addition, the majority of native tissues have established architecture

∗ Corresponding author. E-mail addresses: [email protected] (M. Rabyk), [email protected], [email protected] (M. Hruby), [email protected] (M. Vetrik), [email protected] (J. Kucka), [email protected] (V. Proks), [email protected] (M. Parizek), [email protected] (R. Konefal), [email protected] (P. Krist), [email protected] (D. Chvatil), [email protected] (L. Bacakova), [email protected] (M. Slouf), [email protected] (P. Stepanek). http://dx.doi.org/10.1016/j.carbpol.2016.06.107 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

that ensures their specific functions or properties (West-Mays & Dwivedi, 2006 Wolinsky & Glagov, 1964), therefore, the particular scaffold must be, in fact, an artificial extracellular matrix, which will support growth of the cells to form the desired tissue (Murugan & Ramakrishna, 2007). Advanced wound dressings promoting healing processes should possess similar, yet specific properties (Lee, Jeong, Kang, Lee, & Park, 2009; Mogosanu & Grumezescu, 2014). They should be: essentially nontoxic and non-immunogenic, so as not to disturb the wound healing process; highly permeable, to allow both drainage of exudate and oxygen penetration from the outer atmosphere (open wounds) or from outer vasculature (internal use) to the lesion; hydrophilic, to maintain humidity allowing wet healing; and functionalizable, to enable controlled delivery of healing promoters. It is advantageous if these materials are slowly biodegradable and do not attract cells significantly, allowing closing of the wound space with the newly formed tissue. A fibrous nature is especially advantageous for wound healing dressings due to the

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inherently extremely high porosity and permeability given by allcommunicating pores occupying the overwhelming part of the material volume. Therefore, appropriate 3D architecture, material and technique for the preparation of biomimetic TE materials have to be selected. Various techniques have been studied and applied for the preparation of TE scaffolds, e.g., electrospinning (Li et al., 2005; Liang, Hsiao, & Chu, 2007), solvent casting/particulate leaching (Mikos et al., 1994; Xiang, Liao, Kelly, & Spector, 2006), gas foaming (Sachlos & Czernuszka, 2003), rapid prototyping (Jeong et al., 2012), and phase separation (Ma, 2008). The materials that are used for TE play a crucial role here. Most of these materials are polymer-based and are natural, synthetic or hybrid in origin. Due to biocompatibility, biodegradability, biological activity and abundance, polymers of natural origin have become increasingly recognized for application in this field. The most intensively studied natural or natural-derived materials include starch-based polymers (Gomes, Ribeiro, Malafaya, Reis, & Cunha, 2001), chitosan (Guo et al., 2006; Lee et al., 2002; Xuebing, Peixing, George, & Shuihong, 2011), alginate (Oliviera & Reis, 2011; Tilakaratne, Hunter, Andracki, Benda, & Rodgers, 2007), hyaluronic acid (Chung et al., 2006; Collins & Birkinshaw, 2013; Ji et al., 2006), dextran (Levesque & Shoichet, 2006), cellulose (Fang, Wan, Gao, Tang, & Dai, 2009; Vinatier et al., 2009), collagen (Koch et al., 2006; Xiao, Qian, Young, & Bartold, 2003; Yow, Quek, Yim, Leong, & Lim, 2009), gelatin (Holland, Tabata, & Mikos, 2005; Park, Temenoff, Holland, Tabata, & Mikos, 2005) and fibrin (Miller, Fisher, Weiss, Walker, & Campbell, 2006; Mol et al., 2005). One prospective natural polymer that has not been studied in the area of wound healing is glycogen (GG). GG is the main storage form of D-glucose in mammalian organisms, including humans. The highest concentrations in humans are present in the liver and muscles. GG is hyperbranched poly(D-glucose), where D-glucose units are connected with each other by ␣(1 → 4) bonds and branching is via ␣(1 → 6) bonds (Shearer and Graham, 2002; Tirone & Brunicardi, 2001). Recently, we have shown that GG forms different 3D nano- and microarchitecture structures depending on the initial concentration of its aqueous solution via freeze-drying from water (Vetrik et al., 2013). A mostly fibrous nature is adopted due to the highly amorphous nature and the spherical shape of the GG macromolecules. This is an organic solvent-free process that is easy to perform and allows the possibility to prepare even bulk layers of fibrous material, which is quite problematic by the widely used electrospinning technique. Nevertheless, these GG structures remain water soluble, so they cannot be considered as potential TEdedicated materials without additional modification. The presence of a high number of hydroxyl groups in a polymer backbone allows us to perform a variety of substitutions in GG to adjust the required properties. In this report, we show for the first time that modified GG may be used for the construction of biodegradable hydrophilic biomimetic microfibers with properties suitable for biomedical applications, such as wound healing. We describe the modification of GG through simultaneous alkylation of hydroxyl groups in GG with allyl bromide and propargyl bromide. The obtained GG derivatives were used to fabricate fibers and sponge-like structures followed by electron irradiation to obtain a water insoluble “pre-scaffold” crosslinked by radical polymerization of the allyl groups. The presence of triple bonds in the propargyl moieties in the structure of the obtained material provides a possibility to perform alkyne-azide click reactions to attach different biological active moieties, e.g., peptides, proliferation agents, and growing factors, which is a significant advantage compared to our previous system (Vetrik et al., 2013), which uses grafting of poly(ethyl cyanoacrylate) to the nanofibers from the vapor phase for stabilization, not allowing surface functionalization, and turning the

hydrophilic polysaccharide surface into a hydrophobic polycyanoester. Copper-catalyzed click reaction with RGD peptide (known to promote the attachment and growth of several different types of cells) (Ruoslahti, 1996) was performed to investigate the amount of available alkyne groups in the fabricated pre-scaffold. The biological behavior of the obtained materials was evaluated in in vitro cell culture and has shown that the material fulfils all criteria and is thus highly prospective for advanced wound healing dressings and/or interfaces in direct contact with bone tissue. 2. Materials and methods 2.1. Materials Sodium hydroxide (98.9%) and acetic acid (98%) were obtained from Lach-Ner Ltd. (Neratovice, Czech Republic). All other reagent grade chemicals were purchased from Sigma-Aldrich Ltd. (Prague, Czech Republic) and were used as received. Ultrapure Q-water that was ultra-filtered on a Milli-Q Gradient A10 system (Millipore, Molsheim, France) was used throughout the work. Dialysis tubing (Spectra/Por 3, molecular weight cut-off 3500 Da) was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). 2.2. Modification of glycogen In a typical experiment, glycogen (from oyster, type II, catalogue number Sigma-Aldrich G8751, weight-average molecular weight 10 MDa, 4.00 g, 22 mmol glucose units) and sodium hydroxide (1.97 g, 49 mmol) were dissolved in water (136 mL), and the solution was cooled to 0 ◦ C. Allyl bromide (640 ␮L, 7.39 mmol) and propargyl bromide (107 ␮L, 1.4 mmol) were added, and the mixture was stirred for 10 h at 0 ◦ C and then overnight at room temperature. Acetic acid (3.66 mL, 64 mmol) was added to neutralize the residual sodium hydroxide, the resulting solution was dialyzed against water using membrane tubing Spectra/Por 3 with a MWCO 3500 Da for 48 h and freeze-dried. Yield: 3.80 g (95%) of allyl propargyl glycogen (APG). 2.3. Fabrication of fibrous glycogen structures Aqueous solutions of APG (concentrations of 0.5 or 5 wt.%) were placed into crystallization dishes (forming a layer approximately 10 mm thick) and were frozen in dry ice (freezing rate was approximately 2 ◦ C min−1 ). The frozen samples (initial temperature −18 ◦ C) were then lyophilized on a Scanvac Coolsafe 110-4 Pro (MERCI Ltd., Brno, Czech Republic) freeze drier, shelf temperature −10 ◦ C, pressure 20 Pa, duration 48 h. 2.4. Irradiation of the samples The fibrous structures from APG were exposed to an electron beam (10 MeV electron energy, electron current 25 ␮A, 2 kGy/min) on a Microtron MT25 accelerator with high frequency source to cause radiation crosslinking. The dose of radiation used was in the range of 2–150 kGy. 2.5. Characterization of fibrous structures Fourier transform infrared (FT-IR) spectra were obtained on a Perkin-Elmer Paragon 1000PC spectrometer equipped with the Specac MKII Golden Gate single attenuated total reflection (ATR) system (PerkinElmer Co., U.S.A.) with a diamond crystal and angle of incidence of 45◦ . The 1 H NMR spectra were obtained on a Bruker Avance DPX-300 spectrometer (Bruker Co., Austria) at 310 K operating at 300.13 MHz. 4,4-Dimethyl-4-silapentane-1-sulfonic acid was used as an internal standard, and D2 O was used as the solvent.

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The fibrous structures were characterized by scanning electron microscopy (SEM) using a Vega Plus TS 5135 (Tescan, Brno, Czech Republic) and Quanta 200 FEG (FEI, Czech Republic) using a secondary electron (SE) detector at an accelerating voltage of 30 kV. The samples were fixed on a glass plate, supported using conductive double-adhesive carbon tape (Christine Groepl, Austria), sputtered with Pt (vacuum sputter coater, SCD 050, Balzers, Liechtenstein) and then observed by SEM/SE. Sample radioactivity after radiolabeling was measured directly using a Bqmetr 4 ionization chamber (Empos Ltd., Czech Republic) and a NaI/Tl SpectroAnalyzer, (AccuSync Medical Research Corporation, Milford CT 06460, USA). 2.6. Investigation of the optimal radiation dose (solubility test) The irradiated APG fibrous structure sample (10 mg) was stirred with water (2 mL) for 24 h, filtered and dried in vacuum over P2 O5 for 8 h. The degree of solubility (K) was calculated by K = (m1 − m2 )/m1 , where m1 and m2 are the masses of the sample before and after washing with water, respectively. 2.7. Radiolabeling of the material The irradiated APG fibrous structure (10 mg) was placed in an Eppendorf tube, and water (500 ␮L), an aqueous solution of labelled azidopentanoic-GGGRGDSGGGY(125 I)-NH2 peptide (denoted as RGD, 25 ␮L 9.852 × 10−6 g, 0.9 × 10−5 mmol, activity 0.27 MBq, prepared according to ref. (Proks et al., 2012)) and 10 ␮l of aqueous sodium ascorbate (20 mg/ml) were added. The mixture was degassed under N2 flow for 15 min. Then, 4 ␮l of 0.05 M aqueous copper(II) sulfate was added, and the mixture was bubbled with N2 for another 15 min. Then, the sample was repeatedly decanted in water (approximately 500 ␮L, aided by centrifugation) to remove all unreacted peptide until the supernatant was not radioactive (approximately 6 times). The amount of the immobilized peptides was determined directly by measuring the substrate radioactivity. 2.8. Biological investigations The APG-0.5 samples (i.e., prepared with concentration of modified glycogen material before freeze-drying 0.5 wt.%), both peptide-free and peptide-modified (8 mm diameter and 7.5 mg per well), were inserted into 24-well polystyrene (PS) plates (TPP, Switzerland; well diameter 1.5 cm) and were seeded with human osteoblast-like MG 63 cells (30,000 cells/well, i.e., 17,000 cells/cm2 ). In both cases, the cells were cultivated for 1–7 days in 1.5 ml Dulbecco´ıs Modified Eagle Minimum Essential Medium (Sigma, U.S.A.) supplemented with 10% fetal bovine serum (Sebak GmbH, Aidenbach, Germany) and 40 ␮g/ml of gentamycin (LEK, Ljubljana, Slovenia) in a cell incubator with a humidified atmosphere of 5% of CO2 in air at 37 ◦ C. For each experimental group, two samples were used. The cells on one sample for each experimental group were rinsed with phosphate-buffered saline (PBS), fixed by 70% cold ethanol (−20 ◦ C, 5 min) and stained with a combination of fluorescent membrane dye Texas Red C2 -maleimide (Molecular Probes, Invitrogen, Cat. No. T6008; 20 ng/ml in PBS) and nuclear dye Hoechst # 33342 (Sigma, U.S.A.; 5 ␮g/ml in PBS). The number and morphology of cells on the bottom of the cultivation dishes were then evaluated using images (8 for each sample, size 0.136 mm2 ) taken under an Olympus IX 50 microscope using an Olympus DP 70 digital camera. The adhesion of cells directly on the fibers was observed by a Leica TCS SPE DH 2500 confocal microscope (Germany). As control materials, standard tissue culture polystyrene dishes (PS) were used. The size of the cell spreading areas, i.e., cell areas projected on the material surface, was

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Scheme 1. Preparation of allyl and propargyl derivatives of glycogen.

measured one day after seeding using Atlas software (Tescan Ltd., Brno, CR). Between 20 and 77 cells from 4 pictures for each experimental group were evaluated. 3. Results and discussion The allylated and propargylated glycogen (denoted as APG) was prepared by alkylation of hydroxyl groups in the glycogen structure with the mixture of allyl and propargyl bromide in alkaline aqueous solution (Scheme 1). The presence of both double and triple bonds in APG provided the possibility for further functionalization (via triple bonds) and crosslinking (via double bonds). Using the equations below Eqs. (1) and (2), we calculated the degree of functionalization (number of allyl and propargyl moieties per D-glucose unit, fal and fpr , respectively) from the 1 H NMR spectrum (Supplementary data, Fig. S1). f al = S 6.01 /S 5.35

(1)

f pr = S 2.71 /S 5.35 ,

(2)

where S6.01 is the CH hydrogen nuclei integral signal in the allyl group at ␦ = 6.01 ppm, S2.71 is the CH hydrogen nuclei integral signal in the propargyl group at ␦ = 2.71 ppm and S5.35 is the acetal hydrogen nuclei integral signal in the 1 position of the D-glucose unit at ␦ = 5.35 ppm. The degree of glycogen functionalization with allyl groups should be high enough to obtain a sufficiently crosslinked, waterinsoluble material. However, according to our pilot experiments (data not shown), glycogen becomes insoluble in water when more than 36% of the hydroxyl groups per D-glucose unit are substituted with allyl. On the other hand, the degree of substitution of natural polysaccharides has a strong influence on the biodegradability, and exceeding a specific value of functionalization can lead to a significant decrease in the rate of enzymatic hydrolysis (Duncan, Gilbert, Carbajo, & Vicent, 2008). Therefore, we performed modification of the starting glycogen to obtain APG with 15% allyl and 5% propargyl group substitution per D-glucose (calculated from the 1 H NMR spectrum) unit. This glycogen derivative APG was used for the preparation of different 3D structures, depending on the initial concentration of its aqueous solution, via freeze drying (Vetrik et al., 2013). In this work we used 0.5 and 5.0 wt.% aqueous solutions of APG (denoted as APG-0.5 and APG-5, respectively) for the preparation of the scaffold. The SEM micrographs of the freeze-dried APG show microfibers and sponge-like structures fabricated from 0.5 (Fig. 1a) and 5.0 wt.% (Fig. 1b) aqueous solutions of APG. In the case of APG-0.5, fibers with an average size of approximately 2.5 ␮m in diameter were formed, and with APG-5, the formation of sponge-like structures was observed. In both cases, we obtained highly porous structures with fully communicating pores. One of the properties that scaffolds should possess for use in living organism is insolubility in water. Glycogen is highly water soluble; thus, to make it insoluble, additional modification has to be performed. For this purpose, APG was subjected to electron

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Fig. 1. SEM micrographs of the freeze-dried APG-0.5 (a) and APG-5 (b).

100

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Fig. 2. FT-IR spectra of the irradiated APG-0.5 samples (a); dependence of the solubility of the samples in water after radiation on the radiation dose (b).

irradiation, which initiated radical crosslinking of the double bonds in the allyl groups. In our work we decided to use beta- instead of UV-irradiation or thermal polymerization, since UV-irradiation can’t path through a whole layer of formed fibers and cause homogeneous cross-linking of it. In addition UV-crosslinking requires an introduction of special chemical compound such as photosensibilizator or photoinitiator (Mishra & Yagci, 1998, Chapter 7), which leads to unwanted impurities in the material. In contrast to thermally initiated crosslinking, electron beam treatment can be done at room temperature which reduces residual thermal stresses of the material (Goodman & Palmesse, 2002). To evaluate the optimal conditions for the crosslinking, different radiation doses in the range of 2–150 kGy were tested. Microtron irradiation with accelerated electrons was performed with an incident beam energy of 10 MeV, ensuring homogeneous energy transfer within the

whole thickness of the scaffold but not causing sample activation. Microtron is a more intensive beta radiation source (2 kGy/min) compared to standard gamma irradiation (where it is typically several Gy/min, sometimes even less), making radiochemical initiation of the crosslinking polymerization highly efficient. After radiation, the samples were characterized by FT-IR spectroscopy. In samples APG-0.5 radiated with 10 kGy and higher, the FT-IR spectra (Fig. 2a) show the appearance of a peak at 1725 cm−1 , which corresponds to asymmetric vibrations of the carboxyl group. With increasing radiation dose, the amount of oxidation products increases and the intensity of the peak grows. The same trend was observed for sample APG-5. Radiation dose played a significant role in the structural degradation and solubility of the material. After reaching the radiation dose of 20 kGy, the material changed its color to yellowish and became very brittle. To determine the

Fig. 3. SEM micrographs of the irradiated and refreeze-dried APG-0.5 (a) and APG-5 (b).

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optimal radiation dose, we performed the solubility test under the assumption that the sample with lowest solubility would be best crosslinked, making a compromise between radiation-induced crosslinking and radiolytic degradation. The samples that were subjected to 2 kGy radiation dose were insoluble in water (0.62 and 0.01% for APG-0.5 and APG-5, respectively). Starting from 5 kGy, the solubility of the samples increased with increasing radiation dose (Fig. 2b) due to radiodegradation. This phenomenon was attributed to the oxidation reaction and the appearance of carboxyl groups, which was detected by FT-IR spectroscopy. Therefore, the obtained data allowed us to estimate the best radiation dose for material as 2 kGy because there was

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no evidence of oxidation and increased solubility of the material. This dose is relatively low compared to typical doses used, e.g., for gamma sterilization of materials (15–25 kGy), and is actually the lowest dose deliverable in a defined and reproducible way from the microtron source due to the relatively fast delivery of the dose from the microtron (within several minutes) compared to a gamma source (within several hours) and the different characteristics of the radiation (electrons vs gamma photons). To prevent potential toxic effects of the irradiated material connected with the appearance of oxidation products, such as peroxide groups, the samples were washed with a 0.1 wt.% aqueous solution of 2-phospho-l-ascorbic acid trisodium salt and

Fig. 4. Morphology of human osteoblast-like MG 63 cells on day 1 (A, B), day 3 (C, D) and day 7 (E, F), after seeding on the control polystyrene dish (A, C, E) or on the bottom of the cultivation dish in the presence of APG-0.5 (B, D, F). Cells stained with Texas Red C2 -maleimide and Hoechst #33342. Olympus IX 50 microscope and Olympus DP 70 digital camera, obj. 10×, bar = 200 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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refreeze-dried. The resulting material was investigated by scanning electron microscopy to ensure that the structures remained the same as before irradiation and refreeze-drying. The SEM micrographs of the irradiated APG-0.5 and APG-5 samples (Fig. 3a and b) showed no significant changes in structure of the material (compared to the material before crosslinking, as shown in Fig. 1). The propargyl groups in the structures of the modified glycogen provide the possibility to introduce different biologically active

species to support cell proliferation and growth on a scaffold. This modification could be achieved via copper-catalyzed alkyne-azide cycloaddition. Using this technique, we performed labelling of the APG with azidopentanoic-GGGRGDSGGGY(125 I)-NH2 peptide (denoted as RGD-APG) to evaluate the amount of available propargyl groups on the surface of the material. In the reaction we used radiolabeled peptide in order to quantify the performed conjugation. To determine the extent of non-specifically bound peptide,

Fig. 5. Number of human osteoblast-like MG 63 cells on days 1, 3, and 7 after seeding on the bottom of polystyrene culture wells with or without the presence of the tested glycogen fibrous material (n = 8) (a). Cell spreading area on the 1st day after seeding (n = 47 and 77 for APG-0.5 and polystyrene, respectively) (b). Note: Analysis of variance, Student-Newman-Keuls method. * P ≤ 0.001 in comparison with control polystyrene.

Fig. 6. Morphology of human osteoblast-like MG 63 cells on day 1 (A, D, G), day 3 (B, E, H) and day 7 (C, F, I), after seeding on a control polystyrene dish (G, H, I) or on the bottom of a cultivation dish with RGD-APG-0.5 (A, B, C) or RGD-APG-5 (D, E, F). Cells stained with Texas Red C2 -maleimide and Hoechst #33342. Olympus IX 50 microscope and Olympus DP 70 digital camera; obj. 20×, bar = 200 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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160000

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Fig. 7. Number of human osteoblast-like MG-63 cells on days 1, 3, and 7 after seeding on polystyrene culture wells with or without RGD-modified glycogen fibrous material (n = 20) (a). Cell spreading area on the 1st day after seeding (n = 181, 152 and 46 for RGD-0.5, RGD-5 and polystyrene, respectively) (b). Note: Analysis of variance, Student-Newman-Keuls method. * P ≤ 0.001 in comparison with control PS. Table 1 Adhered cells and spreading area of human osteoblast-like MG 63 cells on the 1st, 3rd and 7th day after seeding on control polystyrene (PS), APG-0.5, APG-5, RGDAPG-0.5 and RGD-APG-5 materials; data are reported as mean ± standard deviation (n = 8). Material

PS APG-0.5 PS RGD-APG-0.5 RGD-APG-5

Adhered cells, ×102 cells/cm2 Day 1

Day 3

Day 7

101 ± 20 69 ± 17 195 ± 44 254 ± 26 230 ± 21

426 ± 30 165 ± 35 323 ± 30 435 ± 67 354 ± 49

162 ± 17 516 ± 17 1315 ± 59 1471 ± 104 1310 ± 130

non-catalyzed incubation was also performed. The calculated data based on the radioactivity measurements showed 3% nonspecifically and 28% covalently bound peptide compared to the initially added amount. After recalculation, the last value was equal to 84% conversion of the propargyl groups in APG. Initial adhesion and subsequent growth of MG 63 cells We started our biological experiments by observing cell behavior in the presence of sample APG-0.5 (Fig. 4B, D, F). Human osteoblast-like MG 63 cell line was used as a model of bone tissue. One day after seeding, a relatively high number of initially adhered cells was found in the pure control polystyrene culture dish compared to the culture dish with GG-based materials. A similar trend remained for the 3rd and 7th day of the experiment (Table 1, Fig. 5a). The cell spreading area was significantly larger on the control polystyrene dish compared to the tested glycogen sample for the whole period of investigation (Fig. 5b). The presence of the glycogen sample decreased the proliferation of osteoblast-like MG 63 cells in culture for 7 days. Nevertheless, the cells were similar in shape to those on the control polystyrene dishes, i.e., polygonal or spindle-like, and were distributed homogeneously on the bottom of the cultivation dish. The number of the cells increased continuously within the whole cultivation time, and at the end of the experiment the cells were able to create a confluent layer (Fig. 6). These results show that the cell growth in the presence of glycogen samples was physiological without noticeable signs of cell damage. The preliminary biological investigations confirmed the possibility of using such systems as wound healing dressings and stimulated further investigations. The next test was performed with the RGD-modified glycogen fibers and showed improved cell growth in the presence of the tested material. Arginine–glycine–aspartic acid (RGD) is a well-known as one of the active peptides originating from sequences of proteins of the extracellular matrix (ECM), which are

recognized by cellular integrin receptors, and thus they improve the affinity of cells and increase their adhesion (Chollet et al., 2009). Therefore, RGD-containing oligopeptides are widely used in the field of biomaterials for controlling the extent and strength of cell adhesion, and also for minimizing immune responses and infection (Wohlrab et al., 2012). Whereas in the presence of unmodified glycogen samples the cell population densities were significantly lower than in the control pure polystyrene dishes, in the presence of RGD-modified materials, they were on average higher than in pure polystyrene dishes, although these differences were not statistically significant (Figs. 6–8). A higher number of adhered cells was observed in culture dishes with RGD-APG-0.5 for the whole exposure period, compared to the polystyrene control dish (Table 1, Fig. 7a). In the case of RGD-APG-5, the number of adhered osteoblasts was comparable to the value in the control dish (Table 1, Fig. 7a). Nevertheless, the cell spreading area on the first day after seeding was significantly larger on the control polystyrene dish, with respect to the values of the RGD-APG-0.5 and RGD-APG-5 samples (Fig. 7b). The degradation of these glycogenbased fibrous scaffolds was followed microscopically in phosphate buffered saline and cell culture medium. As a result, the gradual dissolution of glycogen fibers was demonstrated by images obtained before and during the performed biological tests (Fig. S2). A negligible fraction of the cells adhered directly to the crosslinked glycogen fibers, even those modified with RGD, and this amount decreased as the fibers gradually degraded during the 7-day incubation (Fig. 8). This cell behavior is advantageous for wound dressings, which should not be infiltrated with the newly formed tissue.

4. Conclusions Glycogen was modified by alkylation with allyl and propargyl bromides. Microfibers and sponge-like structures were fabricated by freeze-drying APG aqueous solutions. These structures were characterized by 1 H NMR, FT-IR and SEM. Electron beam processing of the obtained architectures provided covalently cross-linked, water-insoluble materials. To investigate the amount of alkyne groups on the surface of the structures and to demonstrate the possibility of modification with wound healing promoters, radiolabeling with RGD-peptide was performed. Biological testing proved the nontoxicity of the prepared material and the beneficial effect of the peptide on the growth of the adjacent cells. We have shown for the first time that modified GG can be used for the construction of biodegradable hydrophilic biomimetic microfibers with properties suitable for advanced wound healing dressing construction in direct contact with the tissue in the healing process.

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Fig. 8. Morphology of human osteoblast-like MG 63 cells on day 1 (A, D), day 3 (B, E) and day 7 (C, F), after seeding on RGD-APG-0.5 (A, B, C) or RGD-APG-5 (D, E, F) fibers. The cells were stained with Texas Red C2 -maleimide and Hoechst #33342. Leica TCS SPE DH 2500 confocal microscope, obj. 10.0 × 0.30, bar = 100 ␮m, Fig. G obj. 10.0 × 0.30, zoom 4.0×, bar = 25 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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