Macroporous Biodegradable Cryogels of Synthetic Poly(Î±-amino acids

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Macroporous biodegradable cryogels of synthetic poly(#-amino acids) Tomáš Sedla#ík, Vladimír Proks, Miroslav Slouf, Miroslava Duskova-Smrckova, Hana Studenovská, and Frantisek Rypacek Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01224 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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Macroporous biodegradable cryogels of synthetic poly(α-amino acids) Tomáš Sedlačík*, Vladimír Proks, Miroslav Šlouf, Miroslava Dušková-Smrčková, Hana Studenovská, František Rypáček Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Department of Biomaterials and Bioanalogous Polymer Systems, Heyrovsky sq. 2, 162 06 – Prague 6, Czech Republic; *corresponding author: [email protected] Keywords: cryogelation, porosity, biomimetic modification, rheological properties, swelling, biodegradation

ABSTRACT. We present an investigation of the preparation of highly porous hydrogels based on biodegradable synthetic poly(α-amino acid) as potential tissue engineering scaffolds. Covalently crosslinked gels with permanent pores were formed under cryogenic conditions by free-radical copolymerization of poly[N5-(2-hydroxyethyl)-L-glutamine-stat-N5-(2-methacryloyloxy-ethyl)-L-glutamine] (PHEG-MA) with 2-hydrohyethyl methacrylate (HEMA) and, optionally, N-propargyl acrylamide (PrAAm) as minor co-monomers. The morphology of the cryogels showed interconnected polyhedral or laminar pores. The volume content of communicating water-filled pores was > 90%. The storage moduli of the swollen cryogels were in the range of 1-6 kPa, even when the water content was > 95%. The enzymatic degradation of a

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cryogel corresponded to the decrease in its storage modulus during incubation with papain, a model enzyme with specificity analogous to wound-healing enzymes. It was shown that cryogels with incorporated alkyne groups can easily be modified with short synthetic peptides using azidalkyne cycloaddition “click” chemistry, thus providing porous hydrogel scaffolds with biomimetic features.

1. INTRODUCTION The crucial role of the pore structure of implanted material (scaffolds) in tissue engineering applications has been demonstrated many times in recent years. Three-dimensional matrices with a suitable morphology and surface chemistry could provide a mechanically-stable scaffold for in vitro cell culturing by mimicking the physiological function of the extracellular matrix (ECM), which is crucial for preserving the ability of cells to differentiate into their native phenotypes1-2. Ideally, a scaffold should have an open-cell structure with different pore sizes, improving both cell proliferation and vascularization and also providing the exchange of liquids and nutrients for cell survival3. It has been shown that specific types of cells prefer certain sizes of pores for ingrowth and proliferation, and this makes specific demands on scaffolds for a desired application4. The influence of pore morphology on cell vascularization has also been demonstrated5-6. Especially for hydrogels, the most frequently used methods for forming a macroporous structure are particle-leaching and gas-blowing7-10, through which the increasing pore fraction of the resulting hydrogels usually tends to exhibit poor or inappropriate mechanical properties. Preparing porous hydrogels with an open-cell structure and suitable mechanical strength can therefore be a complicated matter. An alternative method, which is very promising in terms of the mechanical properties of the resulting porous hydrogels, and which has been


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studied inter alia for the purposes of tissue engineering in recent years, is based on cryogelation11-14. Cryogels are gels formed below the freezing point of the solvent, in most cases water. While the sample is frozen macroscopically, gelation takes place in the concentrated liquid microphase that remains in the interstitial spaces between crystals of the frozen solvent, which thus act as a porogene during the process12-14. When gelation is complete, the crystals of the solvent are easily removed by thawing, thus avoiding the use of toxic solvents that are used in many particle leaching methods. Because solvent crystals grow until they meet another one, hydrogels with an interconnected macroporous structure are formed when gelation occurs after freezing13-14. The increased concentration of reactants in the liquid microphase leads to high density of the polymeric walls of macropores. Thus cryogels with interesting properties, e.g. rapid swelling and high compressibility without damage to the structure, can be produced 15-16. Scaffolds designed for tissue engineering applications should fulfill requirements not only on suitable porous morphology, but also on biocompatibility or degradability in a biological environment1-2. After the new tissue has been formed, the biomaterial should be reabsorbed without causing any significant inflammatory response. The research in the field of tissue engineering was focused inter alia on porous hydrogel matrices, among which hydrogels made from natural (biodegradable) polymers strongly dominate. As natural polymers and their derivatives exhibit some specific drawbacks, such as dependence of properties on the source, possible immune reaction or solubility problems, here is some interest in scaffolds based on synthetic polymers allowing controllable and reproducible scaffold chemistry and properties. However, only few biodegradable cryogels based on synthetic polymers have been published: e.g., cryogel based on N-vinylcaprolactam17 that is degradable by hydrolysis, and cryogels based


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on vinylic monomers and disulfide- or peptide-crosslinker18-20. The latter cryogels are bioresorbable by enzymatic hydrolysis. Therefore, presented work is focused on using fully synthetic biodegradable materials (polypeptides), which could afford a controlled tailoring of polymeric precursors for specific demands. In previous work, we evaluated hydrogels based on synthetic poly(α-amino acid)s, PAAs, copolymers of L-glutamic acid derivatives, as a promising materials for tissue engineering applications21. Synthetic PAAs are an important type of biocompatible and biodegradable synthetic polymers, and have been studied for use in biomedical applications in many fields21-23. The use of PAAs to form degradable scaffolds provides a unique opportunity to combine the merits of synthetic degradable polymers and natural polymers. Due to their polypeptide backbone, synthetic PAAs exhibit the inherent potential to be degraded by a range of proteases and peptidases24-27. In earlier studies, we demonstrated that the rate and the enzyme specificity of poly(N5-(2-hydroxyethyl)-L-glutamine) degradation can be considerably modified by side chain modification or by the addition of minor amino acids into the polymer chain by copolymerisation28-30. Synthetic polypeptides are degradable to low-molecular-weight products, so gels made of PAAs can provide a temporary tissue support. Macroporous hydrogels considered as scaffolds for tissue engineering should contain biomimetic groups that could facilitate cell adhesion and provide stimuli for cell differentiation2. Biomaterials made from synthetic polymers do not contain these adhesive amino acid sequences (peptides). They should therefore be combined with natural proteins (by forming interpenetrating networks, by grafting, etc.)31-32, or they should be doped with synthetic peptides derived from natural glycoproteins or proteins of ECM, such as fibronectin, laminin, vitronectin or collagen33-


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34

. It has been demonstrated that these peptide motives incorporated into biomaterials have a

significant influence on the attachment of specific cells35-36. In the work presented here, biodegradable highly porous hydrogels based on synthetic poly(αamino acid)s (PAAs) have been designed as potential scaffolds for soft tissue implants. A cryogelation technique was used to form a macroporous structure in PAA hydrogels, which should allow cell migration and scaffold vascularization. An evaluation of the storage moduli of cryogels may help to predetermine the prepared scaffolds for applications in a specific branch of tissue engineering. The feasibility of PAA cryogel degradation was studied using papain, a model enzyme for wound healing. The porous scaffold to ensure cell adhesion was modified by reacting alkyne groups covalently bonded in a cryogel with the azidized RGDS peptide sequence using the “click” chemistry concept.

2. MATERIALS AND METHODS 2.1 Materials Acetonitrile, for HPLC, copper(II) sulfate, p.a., ethylendiaminetetraacetic acid disodium salt (EDTA-Na2, ≥ 98%) and glycerin, p.a. were purchased from Lach-Ner, Czech Republic. Ammonium acetate, cyclohexane and pyridine were of analytical grade, and were purchased from Lachema, Czech Republic. Ethanol (absolute), ammonium persulfate (APS, ≥ 98% for electrophoresis), Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA, ≥ 98%), Lcystein, BioUltra, papain from papaya latex (90% protein content, 21 U/mg), (+)-sodium Lascorbate (≥ 98%, crystalline), N,N,N’,N‘-tetramethylendiamine (TEMED, 99% for molecular biology)

and

iodoacetic

acid

(≥

98%)

were

purchased

from

Sigma-Aldrich.

Bis(trichlormethyl)carbonate (triphosgene, TPG, > 98%) was purchased from TCI Europe, N.V.,


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Belgium. 2-hydroxyethylmethacrylate (HEMA) was purchased from Rohm GmbH, Germany. Methacryloyl

chloride

(MA-Cl,

97%)

was

purchased

from

Fluka,

Switzerland.

Polyethyleneoxide standards (PEO) were purchased from Polymer Standard Service, Germany. All chemicals listed above were used as received. Lithium chloride (≥ 98%) was purchased from Fluka, and was dried under vacuum at 30 °C. γ-Benzyl-L-glutamate (BLG), 99% was purchased from Emmenar Bio-Tech, India, and was recrystallized from hot water. N,N-dimethylacetamide (DMA), 1,4-dioxane, chloroform, tetrahydrofurane (THF) were purchased from Lach-Ner, and were redistilled and dried over molecular sieves. 2-Aminoethanol (≥99%) was purchased from Sigma-Aldrich and was redistilled. N-propargyl acrylamide (PrAAm) was synthesized according to Macková et al.37 Azidopentanoyl-GGGRGDSGGGY-NH2 (N3-peptide) and its radiolabeled analogue N3-peptide-125I were prepared according to Proks et al.38 Deionized ultrapure water (Milli-Q®) was used in all experiments where the solvent was water.

2.2 Synthesis of polymeric precursors Polymeric precursors for the preparation of biodegradable hydrogels were synthesized as follows (see Figure 1 for the scheme). In the first step, N-carboxyanhydride of γ-benzyl-Lglutamate (NCA-BLG) was prepared by reaction of γ-benzyl-L-glutamate (BLG) with triphosgene in THF39. The product was crystalized from chloroform, and the purity of the NCA was characterized by the melting point, by elementary analysis (chlorine content below 0.6 wt.%), and by FTIR spectroscopy. In the next step, NCA-BLG was polymerized in 1,4-dioxane (0.2 mol.l-1) using sodium methanolate as the initiator, with an initial monomer-to-initiator ratio of 200:140. The resulting polymer, poly(γ-benzyl-L-glutamate), PBLG (I.), was precipitated in ethanol, dried in an oven at 60 °C, and then aminolyzed with 2-aminoethanol (50 mol of


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aminoethanol/mol BLG units) to obtain a water-soluble polymer, poly[N5-(2-hydroxyethyl)-Lglutamine], PHEG (II.). The reaction was carried out for 48 hours at 60 °C28. The resulting polymer was purified by dialysis against water (Spectrapor® 1, Cut-Off < 6,000-8,000 Da) and was freeze-dried. To obtain cross-linkable polymeric precursors, PHEG was reacted with methacryloyl chloride in DMA. Lithium chloride was added to prevent the formation of a physical gel during dissolution of PHEG in DMA, and pyridine was used as an acceptor of hydrochloride formed during reaction41. The products of this modification, i.e., statistical copolymers

poly[N5-(2-hydroxyethyl)-L-glutamine-stat-N5-(2-methacryloyl-oxy-ethyl)-L-

glutamines], PHEG-MA (III.), with different degrees of methacryloyl groups in the side chains, were stored in the freezer in bottles sealed with Parafilm® (Alcan Packaging, USA) under nitrogen.


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Figure 1. Schema of the stepwise synthesis of methacryloylated polymeric precursor, PHEGMA; n > m, x + y = 1

2.3 Preparation of cryogels and conventional gels Cryogels based on methacryloylated PHEGs were prepared by radical cross-linking copolymerization of PHEG-MA with HEMA (mole ratio to HEG units of PHEG-MA: 0.11-0.14) and, alternatively, PrAAm (mole ratio to HEG units of PHEG-MA: 0 or 0.03) as comonomers (see Figure 2 for the supposed structure of the polymer network). Polymerization was carried out in 5 ml glass ampoules 13.7 mm in diameter at -15 °C and -18 °C. The redox couple APS and TEMED was used as the initiation system. Briefly, a stock solution (15% w/w) containing the polymeric precursor (PHEG-MA) and HEMA, or HEMA and PrAAm (see Table 1 for the corresponding molar ratios of the components) was prepared and filtered (0.45 µm nylon syringe filter, Whatman, USA). Then a stock solution of TEMED (1 wt.%) was prepared. Respective amounts of these solutions and water were mixed in glass ampoules, degassed by purging with nitrogen and sealed with a rubber septum under nitrogen. Finally, 90 µl of solution of APS (1 wt.%) was injected through the rubber septum and the ampoules were immediately (within 10 seconds) immersed into a thermostated bath. The final volume of liquid in each ampoule was 3 ml, and contained a constant amount of the initiator system (0.2 mg.ml-1 TEMED, 0.3 mg.ml-1 APS). The ampoules were kept in the thermostat for 20 hours to provide sufficient gelation time. The gels that formed were removed. They were thoroughly washed with water, and were swollen in water to equilibrium.


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Figure 2. Schematic representation of the hydrogel network based on methacryloylated synthetic poly(α-amino acid), methacryloylated PHEG, and minor comonomers (HEMA and PrAAm), containing reactive alkyne groups through PrAAm units Table 1. Degree of swelling of the gel phase (SDGP), and the porosity (P%), of cryogels prepared from polymeric precursors with different degrees of methacryloylation (MA%) and under different polymerization conditions (preparative temperature, Tprep) sample ID*

MA%

Tprep

cP % (w/w)

of cryogel

of °C polymeric precursor

I II III IV V VI VII VII IX X XI XII

9 9 9 16 16 16 16 16 16 16 16 16

5 7 9 5 7 9 5 7 9 5 7 9

-15 -15 -15 -15 -15 -15 -15 -15 -15 -18 -18 -18

mol. ratio

mol. ratio

of HEMA

of PrAAm

0.13 0.13 0.13 0.14 0.14 0.14 0.11 0.11 0.11 0.14 0.14 0.14

0.03 0.03 0.03 -

SDGP

P%

g.g-1

vol. %

1.07 ± 0.09 1.03 ± 0.02 0.98 ± 0.22 0.84 ± 0.03 0.83 ± 0.04 0.87 ± 0.05 1.19 ± 0.15 1.09 ± 0.18 0.87 ± 0.16 0.86 ± 0.02 0.99 ± 0.09 1.15 ± 0.04

93.6 ± 0.6 93.2 ± 0.4 92.9 ± 0.9 94.5 ± 0.2 94.0 ± 0.2 92.7 ± 0.6 94.1 ± 0.4 93.0 ± 0.7 92.4 ± 0.7 94.6 ± 0.4 93.2 ± 0.6 91.9 ± 0.5


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XIII 25 -15 5 0.12 0.03 ND ND Control samples of conventional hydrogels made at 25 °C were coded by Arabic (samples 19); cP – concentration of all polymerizable components (PHEG-MA and minor comonomers, HEMA and PrAAm); c(APS) = 0.3 mg.ml-1); c(TEMED) = 0.2 mg.ml-1; All values are reported as the mean and standard error of the mean, n = 3 *

In addition to the cryogels, control samples of gels from solutions of the same composition were prepared at room temperature (25 °C). In this study, these gels are referred to as “conventional hydrogels“. Both types of gels are coded according to the description in Table 1. The assumed structure of the gel network modified by PrAAm is presented in Figure 2. Thermograms documenting the time course of cryogelation were measured using a type K thermocouple (with a Digi-Sense® digital meter, Cole-Palmer Instrument Company, USA). Ministat 230-CC1 (Huber, Germany) with an ethanol bath was used to freeze the samples.

2.4 Characterization of polymers The molecular weights of PBLG were determined by viscometry in dichloracetic acid. The measurements were carried out on the Vistec VS2004 viscometric system (Vistec, Czech Rep.) according to Svobodová et al.42 The viscosity averages of the molecular weights were calculated from the intrinsic viscosities by applying the relationship according Doty et al.43: 1)

[η] = 2.78×10−5 ⋅ M v 0.87 . The weight and number averages of the molecular weights of the water-soluble polymers

(PHEG and PHEG-MA) were determined by size-exclusion chromatography on a system consisting of a PolySep-GFC-P Linear column, the Knauer gradient solvent-delivery system and diode array detection (DAD) and evaporative light scattering detection (ELSD) Altech 3300 (Grace, USA). An isocratic solution of 0.03 mol.l-1 ammonium acetate and acetonitrile (80:20)


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with a flow rate of 0.3 ml.min-1 was used as a mobile phase. The system was calibrated with PEO standards, with molecular weights between 330 and 810,000 g.mol-1 (R2 = 0.9941). Clarity software (DataApex, Czech Republic) was used for the calculations. The degree of methacryloylation of the copolymers was determined by 1H NMR spectroscopy in D2O at 30 °C (Bruker Avance DPX 300) from the ratio of the peak areas of methylene (6.1 ppm) in the methacryloyl groups in the side chains of the polymer to γ-methylene (3.65 ppm) of poly(L-glutamine) units of polymer (see Figures S1a, S1b, S1c)41. The polymer samples are marked as PHEG-MAx, where x is the degree of methacryloylation in % mol.

2.5 Morphology studies SEM studies were carried out with a Quanta 200 FEG microscope (FEI, Czech Republic). Samples were observed in the frozen state using the cryo-low-vacuum SEM method (cryoLVSEM) with a secondary electron detector. To prepare a specimen for cryo-LVSEM, the hydrogel sample was immersed into liquid nitrogen and was transferred to a table conditioned at 10 °C. In the frozen state, the sample was cut with a thin blade in order to remove the upper layer and the ice crystals. In the course of scanning, the pressure in the microscope chamber was kept at a value of 80-110 Pa, and the temperature was maintained at -10 °C. The ice from the surface layers of the specimen is removed by sublimation under these conditions, and the structure of a porous material is gradually revealed without changes to its geometry. Light microscopy (LM) was performed with a Leica DM6000 M microscope (Leica, Austria), equipped with a digital camera using extra-large working distance objectives with high depth of focus. A swollen hydrogel sample, submerged in water, was cut with a razor blade to prepare a wedge-shaped specimen, which was observed at room temperature in transmitted light/bright


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field imaging (DIA/BF). The samples were placed in water between the support and the cover glass throughout the experiment.

2.6 Swelling and porosity Hydrogels were swollen in deionized water for one week or more. The water was changed frequently to wash out all the sol residues and to reach equilibrium. Then, the samples were cut into several pieces (as described in paragraph 2.6) to determine the gel yields and the water content, and to study the rheological properties and the degradation. The swelling temperature was 25 °C. The gel yield (GY%) of the cryogels was determined gravimetrically by weighing dried samples (md):

GY% =100⋅ 2)

md m0 ,

where m0 is the weight of the polymerizable components in the initial solution (of PHEG-MA and comonomers). The conventional hydrogels were extracted by standard gradual solvent exchange (ethanol-water), followed by drying under vacuum at 60 °C. The cryogels were first freeze-dried and then dried under vacuum at 60 °C. The equilibrium swelling regain of both types of gels (W) was determined gravimetrically by weighing the samples in their equilibrium-swollen state in water (msw) and after drying to a constant weight:

W= 3)

msw − md msw = −1 md md .


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The equilibrium swelling degrees of the gel phase of the cryogels (SDGP) were determined gravimetrically by weighing the freeze-dried samples and wet samples swollen to their equilibrium in water vapor (mw):

SDGP = 4)

mw − md mw = −1 md md .

Samples were incubated in a wet chamber (100% relative humidity, 25 °C) for 2 weeks. Typically they reached their equilibrium state in water vapor after approximately 8 days. The porosity (P%) of cryogels represents the volume fraction (in %) of water-filled pores in equilibrium-swollen hydrogels. The porosity was determined by a method based on water vapor adsorption44, and was calculated according to the following equation: 5a)

P% = 100 ⋅

(msw − mw ) ρ H O 2

msw ρ sw

,

where ρH 2O is density of water at 25 °C and ρ sw is density of a swollen sample. Assuming that the cryogels are very porous (P% > 90 %), i.e. , we used the following equation:

5b)

 m  SD GF + 1   P% = 100 ⋅  1 − w  = 100 ⋅  1 −  m sw  SD + 1    .

The specific surface area of the freeze-dried cryogels was determined by nitrogen adsorption (BET method, according to the ISO 9277 standard). The volume shrinking of the sample during the frieze-drying was approx. 15%.

2.7 Rheological measurements


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Rheological measurements on cryogels were performed using a Bohlin Gemini HR Nano rotational rheometer (Malvern, UK). The oscillatory shear measurements were run with the plate-plate geometry on swollen samples immersed in water during the test, using a special solvent dish, and conditioned at 25 °C. The presence of water did not influence the measured values (the additional friction of the rotating part was negligible). The corresponding conventional hydrogels were not studied, because their “fragile” character did not allow the samples to be sliced properly (see Table S1). The cryogel samples for the measurements were prepared as follows: the cryogels were swollen in an approx. 20% (v/v) solution of glycerin in water, frozen in a freezer at -19 °C, and were then placed into a special plastic holder with a guiding groove with a distance of approx. 2.5 and 5.0 mm, in order to ensure parallelism of the discs sectioned by a surgical knife. The diameter of the discs was adapted by a circular cutting tool to a uniform diameter of 14.8 mm. The change in the diameter of the samples due to re-swelling in water was negligible. The plate gap for correct rheological measurements was determined according to Karspushkin et al.45 In contrast with the results for the cited procedure used for conventional porous HEMA gels, it was found that the storage modulus of the cryogels reached a local maximum when under slight compression (meaning “searching for the correct plate gap”, see Figure S2). The maximum was chosen as a reference point for all measured cryogels following our assumption that, at this point, full contact of the measuring geometry with the sample was achieved. The initial increase in storage modulus may be related to the non-parallelism of the disc surface, while the decrease in the modulus, unusual for hydrogels and followed by a further increase in the modulus, may be associated with the behavior of the sample due to its structure and pore deformation during ongoing compression.


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Amplitude sweep tests were performed in strain-control mode for strains in the range from 10-5 to 10-3, the oscillation frequency being 1 Hz in order to find the linear viscoelasticity region of the sample. Frequency sweep tests were performed in stress-control mode. The individual stress values for each sample were selected so that the target strain was within the limits of linear viscoelasticity, as determined from amplitude sweep studies. The frequency range was typically between 0.05 and 1-5 Hz. The upper value of the frequencies was limited by the resonance of the sample and the rotating part of the rheometer system. The resonance frequency depended on the sample stiffness, as has already been documented in the literature46. All measurements were performed at least twice. Figure S3a illustrates the above described procedure. The low total harmonic distortion, along with the fairly smooth shape of the load waveforms, indicated that the measuring geometry did not slip relative to the cryogel surface during the measurement (Figure S3b)47.

2.8 Functionalization of gels for binding biomimetics Functional groups for subsequent binding of bioactive components can be conveniently introduced through copolymerization with a minor amount of functional comonomer. The cryogel with alkyne groups was therefore prepared through copolymerization of PHEG-MA (and HEMA) with PrAAm, and was used for binding a biomimetic peptide, e. g. RGDS, carrying azide groups. To this end, a cryogel from a 5% (w/w) solution of PHEG-MA25 containing 25 mol.% of methacryloylated units, HEMA and PrAAm (sample XII) was prepared at -15 °C in a 2 ml glass ampoule 10.5 mm in diameter, and was washed to remove any sol residues. The cryogel sample was flushed repeatedly with deoxygenated water directly before the Cu(I)-catalyzed


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reaction (Huisgen azid-alkyn 1,3-dipolar cycloadition48-49) was started. All the solutions and the water were deoxygenated by purging with nitrogen. The reaction mixture, consisting of 2.7 ml of water solution of N3-peptide, 0.3 ml of 20 g.l-1 (+)-sodium L-ascorbate and 12 µl of 0.05 mol.l-1 copper(II) sulphate (which is reduced to Cu(I) under these conditions) was added to the cryogel and the reaction was carried out for 20 minutes at laboratory temperature. The cryogels were intensively flushed with water in order to remove unreacted reagents, and the amount of RGDS peptide covalently bound to the cryogel-carried alkyne groups was determined by radioanalysis using an

125

I-radiolabelled peptide analogue. The radioactivities of

the solutions and samples were measured in a Bqmetr 4 ionization chamber (Empos, Czech Rep.) and with an NaI/TlSpectroAnalyzerTM scintillation detector (AccuSync Medical Research Corporation, USA).

2.9 Degradation The degradation of the cryogels was studied in the presence of papain as a model enzyme that cleaves the peptide bonds in the PHEG backbone28. Enzymatic cleavage of PHEG chains reduces the gel crosslinking density, which in turn decreases the modulus and, eventually, leads to the formation of soluble products. Cryogel discs (around 0.8 g in weight) were incubated with 0.25 mg.ml-1 papain solution in 0.05 mol.l-1 Tris-HCl buffer (pH = 7.5) with 1 mmol.l-1 of EDTA-Na2 and 5 mmol.l-1 of Lcystein as activators. The activity of the enzyme was determined by spectrophotometry, using BAPNA as a chromogenic substrate51. The activity was held constant throughout the degradation period (22.6± 1.9 U.g-1 at 25 °C) by adding the required amount of fresh enzyme and a portion of freshly prepared buffer with activators each 24 hours. Each time before the fresh enzyme was


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added, the sample was thrice washed by gentle shaking with an at least 20-times excess of deionized water. Incubation was carried out with gentle shaking (100 rpm) at 37 °C. The degradation was quenched by intensively rinsing the samples with excess of water and adding 20 ml of 1 mmol.l-1 iodoacetic acid solution in water51. The progress of the degradation was observed by determining the relative decrease in the storage modulus and the mass loss of the sample. The storage modulus of each disc sample was measured individually before starting the degradation, and then at given time intervals for a period of three days. The samples were then dried, and were weighed by the procedure described above to determine the mass loss during the degradation process.

3. RESULTS AND DISCUSSION In this study, we focused on preparing macroporous cryogel scaffolds based on poly[N5-(2hydroxyethyl)-L-glutamine (PHEG), a material which mimics natural polymers and also has the advantage that the synthetic polymer chain is biodegradable. The hydrophilic polymer (PHEG) was methacryloylated in order to obtain cross-linkable water-soluble polymeric precursors (PHEG-MA). Radical copolymerization of these copolymers with minor amount of methacrylic and with acrylic monomers (HEMA and PrAAm) produced covalently bonded gels. The PrAAm comonomer was optionally added into the copolymerization mixture in order to incorporate reactive functional groups with versatile potential to bind any bioactive substance to the cryogel surface, which could be available for specific cell receptors. The feasibility of this “click” chemistry concept has been demonstrated on 2D model surfaces38 and in 3D acrylamide cryogels52. However, the HEMA comonomer was considered to induce phase separation in conventional PHEG-based hydrogels21. HEMA was therefore added into the


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copolymerization mixture to form microporosity within the polymeric walls of macropores, which could improve the permeability of the scaffold. Moreover, the surface roughness (e.g., as a consequence of phase separation) was demonstrated to influence cell behavior53. However, polymeric walls of macropores of prepared cryogels were found to have smooth surface (Figure 3-4) meaning that HEMA does not induce phase separation in the above described system under cryogenic conditions.

3.1 Preparation of polymeric precursors NCA-BLG, an amino acid monomer, was synthesized with good yields, typically ranging from 62 to 73%. The polymerization of NCA-BLG proceeded with high yields of PBLG (89-95 %) with viscometric average molecular weights in the range of 232-246×103 g.mol-1. The yields of aminolysis products were 65-70%, and the yields of methacryloylation products were 58-71%. All polymers obtained after aminolysis and methacryloylation were soluble in water. Their molecular weight averages determined by SEC (as PEO equivalents) are presented in Table 2. The polymer chains were partially split during aminolysis as described in ref.28 The degree of methacryloylation of polymers (MA%) determined by 1H NMR spectroscopy is listed in the same table (for spectra, see Figures S1a, S1b, S1c). Table 2. Characterization of polymeric precursors sample ID

PHEG

ഥ௪ ‫ܯ‬

ഥ௡ ‫ܯ‬

g.mol-1

g.mol-1

Đ

MA% mol.%

32,300 ± 3,400 11,700 ± 400 2.82 ± 0.38

-

PHEG-MA9

26,650

11,650

2.30

9

PHEG-MA16

28,100

12,400

2.26

16


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PHEG-MA25

20,900

12,980

1.61

25

ഥ௪ , ‫ܯ‬ ഥ௡ – weight and number average of molecular weight from SEC (PEO equivalents), Đ – ‫ܯ‬ ഥ௪ /‫ܯ‬ ഥ௡ ), MA% – degree of methacryloylation dispersity (‫ܯ‬

3.2 Preparation of cryogels The cryogels were prepared by radical copolymerization of polymeric precursors (PHEG-MA) and acrylic/methacrylic comonomers (HEMA, PrAAm) with a redox initiation system: APS and TEMED. The cryogels were sponge-like, and most of them were very elastic, as demonstrated in Figure S4. The water contained in the porous structure of the cryogels can be partially squeezed out during compression (Figure S4a), and was soaked back into the gel when the applied stress was released. The compressed cryogels also very rapidly – within seconds) – recovered their original shape when placed in contact with water (Figure S4b). This behavior, which can be repeated many times, could demonstrate a communicating architecture of the pores in cryogels. By contrast, conventional hydrogels, i.e., gels prepared under ambient temperature from the reaction mixture of the same composition, were fragmented during large-strain compression (Figure S4c), and many of them were even damaged during handling, because of their poor mechanical properties. A visual-tactile assessment (“a simple comparison”) of cryogels and the corresponding conventional hydrogels is summarized in Table S1. When preparing cryogels with morphology of interconnected pores, it is important to set the polymerization condition in such a way that freezing proceeds more rapidly than gelation14. Thermograms, i.e. time-courses of temperature during freezing, were therefore recorded to determine the optimum time and freezing conditions. Thermograms of the freezing of pure water and thermograms of the cryogelation mixture are shown in Figure S5. As can be seen, the freezing point of water in the polymer solution is very close to the freezing point of pure water,


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in contrast to the solution of low-molecular-weight components, such as monomers with the same weight concentration. Moreover, in the case of polymer solutions, no supercooling occurs before crystallization. This difference in the behavior of polymeric and low-molecular-weight (monomeric) systems during freezing reflects the difference in the osmotic properties of highmolecular and low-molecular weight components, and is in accord with some earlier observations16,54. Thus, the solvent freezes almost immediately in the polymeric system, unlike in a system consisting only of low-molecular-weight components (acrylic monomers), in which supercoiling occurs. This means that the polymeric system could be more tolerant to a higher amount of the initiator, and could freeze before gelation occurs, resulting in an interconnected regular porous structure. This difference in the behavior of polymeric and monomeric solutions during freezing offers an advantage when preparing cryogels with a regular interconnected porous structure from polymeric solutions.

3.3 Morphology of cryogels The morphology of the cryogels, as revealed by cryo-LVSEM, is shown in Figure 3-4. The pores of all cryogels are tens of micrometers in size. They are interconnected and are surrounded by polymeric walls of macropores micrometers in thickness. No significant differences were observed in the structure of all cryogels prepared at -15 °C, irrespective of the different composition of the starting polymerization solutions. These cryogels show polyhedral, more or less elongated pores. By contrast, cryogels prepared at -18 °C show lamellar pores, especially when the concentration of polymerizable components in the initial solution is higher than 5% (w/w). This difference in morphology could be due to the higher viscosity of the polymer solution during freezing, which can limit the growth, the direction of growth, and the likelihood


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of encountering ice polycrystals. The presence of pore structures observed by cryo-LVSEM was confirmed by light microscopy of native water-swollen samples, as shown in Figure S6.

Figure 3. Morphology of the cross-section of cryogels prepared from PHEG-MA9 at -15 °C as revealed by cryo-LVSEM; from left to right: cP = 5, 7 and 9% (w/w)


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Figure 4. Morphology of the cross-section of cryogels prepared from PHEG-MA16 under different polymerization conditions, as revealed by cryo-LVSEM; from left to right: cP = 5, 7 and 9% (w/w); top to bottom: the effect of PrAAm addition (samples VII-IX) and different temperature (-15 °C: samples IV-VI, -18 °C samples X-XII)

3.4 Swelling and porosity of gels


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For PHEG cryogels, it was generally found that the equilibrium swelling regain decreases slightly or remains constant (approx. 25-35 g.g-1) with increasing concentration of polymerizable components in the initial solution (Figure 5a2). This occurs because cryogels prepared from a higher concentration of polymerizable components sucked more water after they were prepared than cryogels prepared from a lower concentration (data not shown), and due to slightly different conversion of the polymerizable components. The gel yield was very high for all cryogels (Figure 5a1), and decreased slightly, from approx. 90-100% to 70%, or remained constant, with an increasing concentration of polymerizable components in the initial solution. For comparison, the equilibrium swelling regain and the gel yield of conventional gels prepared from solutions of the same composition at laboratory temperature are shown in Figure 5b. The equilibrium swelling regain of conventional gels decreases dramatically with an increasing concentration of polymerizable components in the initial solution (from approx. 25 to 10 g.g-1, Figure 5d), because the gel yield of these gels increases slightly with the concentration of polymerizable components in the initial solution (Figure 5b1), and their volume after swelling is practically the same (they are close to equilibrium after gelation). It was found that all cryogels contained more water than conventional gels.


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Figure 5. Gel yield (GY) and equilibrium water regain (W) of cryogels (A1, A2) and conventional hydrogels (B1, B2); cP – concentration of all polymerizable components (PHEGMA and minor comonomers, HEMA and PrAAm); all values are reported as the mean and standard error of the mean; n = 3 The experiment based on water vapor absorption in dried cryogels allowed us to estimate the equilibrium degree of swelling only of the gel phase, and to calculate the porosity of the samples, the results of which are summarized in Table 1. It was found that the polymeric walls of macropores of all prepared cryogels show a similar degree of swelling, 0.8-1.4 grams of water per gram of dry gel (Table 1), which corresponds to 44-58% of water content. This means that


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the polymeric walls of macropores of all prepared cryogels have a similar density of the polymer chains and are highly crosslinked, so they swell only very little44. The calculated porosity of all the samples is very high (approx. 92-95%), and decreases slightly with an increasing concentration of polymerizable components in the initial solution.

3.5 Rheological behavior Our cryogels revealed a peculiar mechanical resistance to large compressive strains: when the swollen gel cylinders were compressed to approx. 10% of their initial swollen volume, they readily drained most of the water and when put back to excess of water, they quickly regained their original shape and volume, see Figure S4. This gel straining could be performed repeatedly without collapsing the gel structures. Such highly elastic and reversible behavior of cryogels was already observed for the samples of different chemical composition16,

55-57

. It seems that the

resistance of cryogels to high mechanical loading is an inherent feature of their special heterogeneous structure with interconnected pores as formed along with the freezing process during polymerization, but this special mechanism of deformation of polymeric walls of macropores on microscopic level is not fully understood. On the contrary, our conventional gels, also heterogeneous, could not withstand compressive deformation larger than approx. 40%; they completely disintegrated under the compressive load (Figure S4). The high mechanical resistance of cryogels facilitates sample handling being benefit in their application. Since the cells behavior when cultivated on artificial substrates correlates with substrate stiffness58-59, the mechanical responses of scaffolds are of researchers’ interest. We have investigated the gel mechanical responses using oscillatory shear rheometry of swollen gels. We


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have obtained their small-strain storage and loss moduli that can serve as a measure of polymer matrix stiffness. The typical dependencies of the storage and loss moduli and the loss factor on the frequency of the dynamic loading obtained with a water-swollen cryogel sample are presented in Figure 6. The higher the concentration of polymerizable components in the initial solution is, the higher the storage and loss moduli of the resulting cryogel (Figure 6a-b). The values of the storage moduli of the cryogels ranged from 1 to 6 kPa, although the portion of the gel matrix that carried the mechanical loading was below 5% (porosity higher than 95%) of the total cryogel volume (Table 1). The values of the loss moduli of the cryogels were very low, ranging from tens to hundreds pascals. While the storage moduli of the samples were within the measured frequency range practically constant (Figure 6a), the loss moduli increased with increasing frequency (Figure 6b). In the measured frequency range (limited by the resonance of the measuring system with the sample), some increase of the loss factor (from tan δ = 0.02 to tan δ = 0.06) was repeatedly observed. Similar behavior was recorded over broader frequency range for swollen gels based on PHEMA suggesting onset of the the swollen gel phase relaxation process above 110 Hz. This frequency dependent increase of storage modulus of swollen PHEMA gels was attributed to relaxation behavior due to presence of hydrophobic domains within the PHEMA structure causing gel stiffening.46


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Figure 6. Typical dependence of the storage modulus (A), the loss modulus (B) and the loss factor (C) of cryogels; series cryogels (VI-VI) based on PHEG-MA16 made of 5 (gray), 7 (white) and 9% (w/w) (black) of polymerizable components in initial solution at -18 °C The storage modulus of the swollen cryogels was influenced by several parameters (Figure 7): 1) the concentration of polymerizable components in the copolymerization mixture (as mentioned above); 2) the degree of methacryloylation of the polymeric precursor, PHEG-MA (Figure 7a: the higher the number of crosslinking sites is, the higher the storage modulus of a cryogel prepared from a solution of the same composition will be – this effect seems to be more evident when cryogels are made from more diluted solutions of a polymerizable mixture); 3) the temperature of cryogelation (Figure 7a-b: the lower the temperature is, the lower the storage modulus of a cryogel prepared from a solution of the same composition will be); 4) the composition of the copolymerization mixture (Figure 7a-b: the presence of PrAAm comonomer led to lower moduli than of cryogels made from the same concentration of polymerizable components). Note that cryogels with a storage modulus lower than 1 kPa were not selfsupporting (refer to Table S1), and so measurements of these samples could provide only approximate values.


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Figure 7. Dependence of the storage modulus of cryogels on the concentration of all polymerizable components (PHEG-MA and minor comonomers, HEMA and PrAAm); A) influence of the degree of methacryloylation of the polymer (-15 °C; samples I-III: 9%, samples IV-VI: 16%), B) effect of the addition of PrAAm (samples VII-IX) and different temperature (18 °C: samples X-XII) for a polymer with degree of methacryloylation 16%; all values are reported as the mean and standard error of the mean; n = 2-3 The effect of the cryogelation temperature on the storage moduli of the cryogels can be related to the different pore architecture: lamellar pores, rather than polyhedral pores, were formed at 18 °C (see Figure 4). The effect of the addition of PrAAm on the storage modulus of the cryogels could be caused by a decrease in the initiation efficiency of radical polymerization in the presence of this monomer with a relatively acidic acetylene group (pH1%PrAAm = 5.3; pH1%AAm = 7.7). Caglio et al.60 published that the initiation efficiency of the redox pair (ASP and TEMED) that is used can decrease significantly when pH < 7. Although the gel yields are similar for all prepared cryogels, fewer crosslinks could be formed in systems with an alkyne comonomer,


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resulting in the formation of weaker polymeric walls of macropores, leading to reduced macroscopic stiffness of the sample. The measured modulus represents the modulus of a complex system: a swollen hydrogel matrix holding communicating pores filled with water. Although cryogels retain a large amount of water (see Figure 5), which is present mainly within their permanent pores, they were very elastic and their storage modulus was tunable up to 6 kPa. Therefore, PHEG cryogels exhibit reasonably good mechanical properties (strength, elasticity) comparable to these of some soft tissues or cell cultures and they are ease to handle without damage (Figure S4). It has recently been shown that HEMA hydrogels having smaller pores within the polymeric walls of macropores (smaller pores micrometers in size and macropores tens of micrometers in size) prepared by a method combining solvent leaching and a precipitation technique exhibit similar deformation responses to that of hydrogels that have only small pores prepared in the presence of a non-solvent, although they retain twice as much water10. The mechanical performance of PHEG cryogels is similar in scale to the mechanical performance of these hydrogels, but interestingly they contain several times more water (7 vs. 25-35 g.g-1). Related to the goal of this work, a comparison of cryogels based on both synthetic poly(αamino acid)s or peptide61, and natural proteins62-70 is outlined in Table S2. With respect to material properties such as porosity, size of macropores and especially to mechanical performance, PHEG cryogels provide comparable performance to other cryogels envisaged for tissue engineering.

3.6 Degradation of cryogels


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Samples with a lower storage modulus disintegrated within a short time of degradation, which makes them inappropriate for a rheological study. A cryogel with a higher storage modulus made from PHEG-MA9 (sample III) was therefore selected for degradation studies. The influence of papain degradation on the rheological properties of the cryogel is shown in Figure 8. The storage modulus of a cryogel decreases rapidly during the first day of degradation to approx. 50% of the original value, and then degradation proceeds more slowly to approx. 35% of the original value in the course of the next two days. Degradation was accompanied by loss of mass (Figure 8) and by a negligible change in the dimensions of the sample. When degraded for more than 3 days, the samples disintegrated into several pieces during handling. No evidence of degradation was observed when samples were incubated without enzyme (Figure 8).

Figure 8. Relative change in the elastic modulus (at 1 Hz, black; blank sample is dashed) and mass loss (gray) during the degradation time for cryogel III (prepared from a 9% (w/w) solution of PHEG-MA9 and HEMA in a molar ratio of 9/1 at -15 °C), ; all values are reported as the mean and standard error of the mean, n = 3-6 The polymeric walls of macropores are very dense16, because of the relatively low swelling of the gel phase (Table 1). This prevents the enzyme penetrating inside. Thus degradation occurs only on their surface at first, and this is accompanied by a decrease in the mass of the sample.


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With ongoing degradation, papain can penetrate into the polymeric walls of macropores, where only each first cleavage between two crosslinks can affect the modulus of the cryogel. This chain splitting is not accompanied by a further relatively rapid decrease in the mass of the sample. Each further cleavage between two crosslinks does not affect the modulus, which tends to undergo a more gradual change of its rheological properties. During the late degradation, the fraction of non-cleavable poly(methacrylic) crosslinks (PHEMA) is expected to be released, which form only minor part of the hydrogel (up to 15% w/w to all solids) and should be present as linear chains. PHEMA is generally considered as material with good biocompatability71-72. Based on previous studies, we can assume that after degradation of crosslinking PAA chains only relatively short chains of methacrylic polymer remain73, which are soluble because of remaining fragments of hydrophilic polypeptide or can be absorbed by macrophages74.

3.7 Bioactivation of cryogels Cryogels based only on PHEG-MA and HEMA are expected to form a non-adhesive scaffold for cells21. PrAAm was therefore added as a comonomer into the copolymerization mixture to ensure the feasibility of modifying cryogels through alkyne groups with specific peptide motifs, and thus to make them attractive for cells. For this purpose, cryogels with incorporated alkyne groups were prepared to provide binding sites for reactive peptide derivatives, an azidized peptide containing the RGDS sequence. The gel yield of this cryogel made from PHEG-MA25 was 90±2%, and the equilibrium swelling regain was 26±2 g.g-1; n = 3. In the absence of a catalyst, only 1% of added amount of peptide was detected in the cryogels. The yield of azidealkyne cycloaddition – in the presence of a catalyst – into the cryogels was 15% (which


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corresponds to 5.1 pmol.cm-2, calculated according to BET and appropriate volume swelling of the freeze-dried sample, as an approximate estimation of the surface density of the peptide; the real density is probably lower, because reactants partially diffuse into the polymeric walls of macropores). Adjusting the reaction conditions or designing a better experimental setup to obtain higher conversion should be the topic for a further investigation. However, the specific concentration of peptide motives on the surface of the scaffold must be determined with respect to the specific cell types and with respect to the individually designed biomaterial. The concentration of RGD peptide for hydrogels is generally considered to be close to 1 pmol.cm-2 according to ref.35-36 The calculated concentration of RGDS in a PHEG-MA based cryogel was in pmol.cm-2 as indicated. Despite lower conversion, the azide-alkyne cycloaddition has therefore demonstrated the potential of this type of “click” reaction for modifying macroporous hydrogels. In addition, sponge-like cryogels could be stored in a dry state, and after rapid reswelling they could easily be modified with specific bioactive substances before a desired application. Concerning the possible toxicity of implants containing copper, we demonstrated that Cu level in cryogels is in the range of physiological concentration of Cu in blood and tissues (see Supporting Information and Figure S7) and therefore Cu click chemistry concept is suitable for modification of cryogels for medical purpose. In our previous studies it was confirmed that acrylamide cryogels treated by Cu-catalyzed “click” reaction did not show any adverse effect on cell viability52. The biological testing of PHEG cryogels is in progress.

4. CONCLUSION


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The technique for preparing biodegradable synthetic poly(α-amino acid)s-based cryogels with pendant reactive units for modification with biomimetic groups has been developed. Macroporous hydrogels were formed by radical copolymerization of multifunctional methacryloylated poly[(N5-(2-hydroxyethyl)-L-glutamine]s with 2-hydroxyethyl methacrylate and alternatively N-propargyl acrylamide (PrAAm) in an aqueous environment at -15 °C and -18 °C. Parameters such as the composition of the copolymerization mixture, the functionality of the poly(amino acid) precursors (i.e., the degree of methacryloylation), and the cryogelation temperature have been investigated, and their effects on the morphology, the swelling capacity and the mechanical responses of cryogels have been demonstrated. The equilibrium-swollen cryogels showed a structure with interconnected polyhedral or laminar pores tens of micrometers in diameter. The cryogels exhibited relatively high mechanical strength, and were able to withstand large mechanical loading although they contained more than 95% of water and showed porosities higher than 90%. A study of the rheological behavior of the cryogels revealed that their storage moduli are within the range of 1-6 kPa that falls within the relevant range of stiffness of artificial scaffolds. The benefit of poly(α-amino acid)s-based hydrogels lies in the broad range of possible modification for specific demands and, similarly to natural proteins, in the biodegradability of the main polymer chain by enzymes. The initial stage of enzymatic degradation of the cryogel was described using papain, a model enzyme. The mass loss during the incubation time was correlated with the decrease in the storage modulus. Moreover, it was confirmed that these high-porous hydrogels can be modified post-gelation, using the “click” chemistry concept, namely by Cu(I)-catalyzed azid-alkyne cycloaddition. The cryogels were modified through PrAAm added into the copolymerization mixture, thus providing alkyne groups in the gel structure capable of a catalytic reaction with some azidized peptide. The


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features of the cryogels presented here predetermine these materials for applications in soft tissue engineering. ASSOCIATED CONTENT Supporting Information Available. Supporting Information is including the 1H NMR spectra of polymeric precursors, the figure explaining the determination of correct plate gap for the shear measurements, the figures describing the rheological measurement, photos of behavior of the cryogels during long-strain compression, the table of simple comparison of cryogels and conventional gels, the table comparing PHEG cryogels with similar cryogels in the literature and the figure showing determination of copper level in cryogels and the brief description of copper determination. This material is available at free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT We are grateful for financial support from the Grant Agency of the Czech Republic P108/12/1538. We would also like to thank Jan Kučka for preparing the radio-labelled peptide, Helena Vlková for SEM imaging of cryogels, Petr Šálek for determining the specific surface area of the cryogel that was selected for bioactivation, Zdeněk Tesař for technical support and Karel Dušek for helpful comments on the manuscript. REFERENCES (1) Lee, K. Y.; Mooney, D. J. Chem. Rev. (Washington, DC, U. S.) 2001, 101, 1869-1880


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(2) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337-4351 (3) Annabi, N.; Nichol, J. W.; Zhong, X.; Ji, Ch.; Koshy, S.; Khademhosseini, A.; Dehghani, F. Tissue Eng., Part B 2010, 16, 371-383 (4) Whang, K.; Healy, K. E.; Elenz, D. R.; Nam, E. K.; Tsai, D. C.; Thomas, C. H.; Bubeger, G. W.; Glorieux, F. H.; Travers, R.; Sprague, S. M. Tissue Eng. 1999, 5, 35-51 (5) Wake, M. C.; Patrick, C. W. Jr.; Mikos, A. G. Cell Transplant. 1994, 3, 339-343 (6) Chiu, Y.-Ch.; Cheng, M.-H.; Engel, H.; Kao, S.-W.; Larson, J. C.; Gupta, S.; Brey, E. M. Biomaterials 2011, 32, 6045-6051 (7) Barbetta, A.; Rizzitelli, G.; Bedini, R.; Pecci, R.; Dentini, M. Soft Matter 2010, 6, 1785– 1792 (8) Chen, J.; Park, H.; Park, K. J. Biomed. Mat. Res. 1999, 44, 53–62 (9) Mooney, D. J.; Baldwin, D. F. ; Suh, N. P.; Vacanti, J. P.; Langer, R. Biomaterials 1996, 17, 1417-1422 (10)

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Z.; Šlouf, M.; Michálek, J. J. Polym. Res. 2014, 21, 579 (11)

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Free-radical copolymerization of biodegradable copolymer, poly[N5-(2-hydroxyethyl)-Lglutamine-stat-N5-(2-methacryloyl-oxy-ethyl)-L-glutamine], with 2-hydrohyethyl methacrylate (HEMA) and N-propargyl acrylamide (PrAAm) under cryogenic conditions produced macroporous hydrogels. These cryogels withstand large-strain compression and can be modified with biomimetics (azidized peptide) by “click” chemistry through incorporated alkyne groups.


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Macroporous Biodegradable Cryogels of Synthetic Poly(Î±-amino acids

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