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Biodegradation and osteosarcoma cell cultivation on poly(aspartic acid) based hydrogels David Juriga, Krisztina Simonné Nagy, Angela Jedlovszky-Hajdu, Katalin Perczel-Kovach, Yong Mei Chen, Gabor Varga, and Miklós Zrinyi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06489 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016

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ACS Applied Materials & Interfaces

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Biodegradation and osteosarcoma cell cultivation on poly(aspartic acid) based hydrogels Dávid Juriga1, Krisztina Nagy2, Angéla Jedlovszky-Hajdú1, Katalin Perczel-Kovách2,3, Yong Mei Chen4, Gábor Varga2, Miklós Zrínyi*1,5 1, Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Nagyvárad tér 4., H-1089 Budapest, Hungary 2, Department of Oral Biology, Semmelweis University, Nagyvárad tér 4., H-1089 Budapest, Hungary 3, Department in Community Dentistry, Semmelweis University, Üllői út 26., H-1085 Budapest, Hungary 4, State Key Laboratory for Strength and Vibration of Mechanical Structures, International Center for Applied Mechanics and School of Aerospace, Xi’an Jiaotong University, Xi’an 710049, China 5, MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary Corresponding author: *Miklós Zrínyi, E-mail: [email protected]

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2 Keywords: poly(aspartic acid) hydrogel, thiolated, RGD, MG-63, cell viability Abstract: Development of novel biodegradable and biocompatible scaffold materials with optimal characteristics is important for both preclinical and clinical applications. The aim of the present study was to analyze the biodegradability of poly(aspartic acid)-based hydrogels, and to test their usability as scaffolds for MG-63 osteoblast-like cells. Poly(aspartic acid) was fabricated from poly(succinimide) and hydrogels were prepared using natural amines as crosslinkers (diaminobutane and cystamine). Disulphide bridges were cleaved to thiol groups and the polymer backbone was further modified with RGD sequence. Biodegradability of the hydrogels was evaluated by experiments on the base of enzymes and cell culture medium. Poly(aspartic acid) hydrogels possessing only disulphide bridges as cross-links proved to be degradable by collagenase I. The MG-63 cells showed healthy, fibroblast-like morphology on the double crosslinked and RGD modified hydrogels. Thiolated poly(aspartic acid) based hydrogels provide ideal conditions for adhesion, survival, proliferation and migration of osteoblast-like cells. The highest viability was found on the thiolated PASP gels while the RGD motif had influence on compacted cluster formation of the cells. These biodegradable and biocompatible poly(aspartic acid)-based hydrogels are promising scaffolds for cell cultivation.


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Introduction: Replacement of lost, damaged, diseased tissues or organs is one of the most significant

challenges in human health care. The frequently occurring problems of organ transplantation, e.g. immunological rejections of transplanted organs or the limited availability of donors could be eliminated by the application of newly produced artificial tissues using isolated and in vitro propagated cells of the patients1,2. Cell transplantation requires appropriate scaffolds mimicking the natural environment of the cells, namely the extracellular matrix (ECM). Therefore, the preparation of native ECM-like three-dimensional artificial scaffolds has become a major field in tissue engineering and regenerative medicine in the past few decades3,4. The widely used 2D approaches in cell culturing have played a key role in understanding the fundamentals behind cell-ECM interactions. However, cells often show a non-natural behavior in artificial 2D environment, thus, their behavior cannot be thoroughly understood or extrapolated to in vivo situations4. Complex tissues and organs contain multiple cell layers therefore, it is essential to grow cells in 3D to get as close as possible to the conditions of native tissue 5,6. 3D cellular scaffolds are more than a platform for cells; they have to possess all important properties that are present in the native ECM. Hence, ideal scaffolds should not only provide an inert environment for cell proliferation, but also contain ligands to mediate cell adhesion to the surface of the scaffolds7, have appropriate mechanical and diffusion properties, be biocompatible and biodegradable, as well as facilitate cell-matrix and cell-cell communications8,9. As different cell types favor different environments, the properties of scaffolds should be tunable for various objectives10–13. Natural or artificial polymer-based hydrogels possess most of the above mentioned properties, as well as tunability. In addition, polymer hydrogels are responsive to external stimuli, such as



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4 pH, temperature, or external concentration of small bioactive molecules14–16, which could enable cell-matrix communication. Both synthetic and natural polymer-based hydrogels have become popular matrices for 3D cell proliferation17–20. All in all, ideal hydrogel scaffolds imitate natural ECM properties or even exceed them. Natural ECM mainly contains amino acid based polymers, such as collagen, elastin, fibronectin, and other proteins. It is possible to copy the composition of such structures, but their actual clinical application is rather limited because of the difficulty of their handling and formation, and also because of their structural rigidity and slow rebuilding7. Artificial protein-like polymers can be used as basic materials of the scaffolds, but the difficult and expensive synthesis diminishes their applicability in this field18. However, poly(amino acid)-based polymers that have desirable chemical, mechanical and biological properties have recently considered as promising new class of biomaterials. The important reason for the development of poly(amino acid)-based biomaterials is their virtually unlimited structural diversity21. Poly(aspartic acid) (PASP) is a very promising scaffold material since it can be synthesized easily with high molecular weight as compared to other synthetic polypeptides. It can replace the natural ECM by filling up the damaged area, but also accelerate tissue regeneration by its intelligently designed properties. PASP builds up from aspartic acid monomers that are non-essential amino acid in mammals. The preparation of hydrogels from PASP with chemical crosslinking reaction and creating special networks as potential biomaterials has got little attention so far, in spite of the many advantages of the biomedical application of such an artificial system. The synthesis and characterization of PASP-based hydrogels have been investigated by our research group for several years21–25. There are only seldom data available regarding biocompatibility of PASP and PASP-based hydrogels26– 28

. Furthermore, the effect of different mechanical properties and chemical structure of PASP

hydrogels on cell proliferation and viability have never been studied so far.


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5 Poly(succinimide) (PSI), the anhydrous form of poly(aspartic acid) can be synthesized directly from L-aspartic acid. It is worth to note that the L form of aspartic acid can directly latch on the protein synthesis circle in the living system. Since PSI reacts easily with amino group containing molecules without any catalyst, mono-functional amine molecules, such as adhesion ligands (e.g., Arg-Gly-Asp (RGD) tripeptide and its derivatives29) or growth factors30,31 can be attached to the polymer network. Furthermore, derivatives of PSI can be cross-linked with different bi- or multifunctional amines, thus, several properties (such as hydrophilicity, acid-base character of the backbone) of these gels can be altered in a wide range22. Due to the high molecular weight of the polymer, the PSI-based gels can be synthesized with different elastic modulus. PASP-based hydrogels can be formed from PSI gels with mildly alkaline hydrolysis25. According to the previously mentioned properties, the PASP-based hydrogels can be tailored regarding to the cell type, what improves their applicability during in vitro cell cultivating. Since PASP hydrogels are based on natural amino acids, they are expected to be biocompatible and also biodegradable scaffold materials32. In the present work, the applicability of PASP-based hydrogels with different chemical compositions and mechanical properties has been studied as scaffolds for in vitro cell cultivation. The biodegradability of the hydrogels has been investigated at physiological conditions ex vivo using different digestive enzymes. For in vitro experiments, MG-63 human osteoblast-like cell line was chosen, as a standard, suitable model cell line for testing biomaterials33. The ability of migration into the polymer matrix and the 3D arrangement of the cells have been investigated by 2 photon microscopy. Cell adhesion, proliferation and migration by qualitative (phase contrastand 2 photon microscopy) and quantitative (cell viability assay using cell proliferation reagent WST-1) methods have also been investigated in the paper.



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6

2

Materials and Methods:

2.1

Materials

L-aspartic acid (reagent grade, ≥98%, Sigma), o-phosphoric acid (puriss. ≥99%, Aldrich), dimethyl foramide (DMF) (anhydrous, 99.9%, VWR), dimethyl sulfoxide (DMSO) (analytical grade, 100%, VWR), Arg-Gly-Asp (RGD) (TLC, ≥97%, Sigma), dibutylamine (DBA) (≥99.5%, Aldrich), DMSO-d6 (100%, 99.96% atom% D, Aldrich), 1,4-diaminobutane (DAB) (99%, Aldrich), Cys*2HCl (purum. ≥98%, Fluka), imidazole (ACS reagent, ≥99%, Sigma-Aldrich), citric-acid*H2O (ACS reagent, ≥99.9%, VWR), sodium-chloride (99-100.5%, Sigma-Aldrich), DL-dithiothreitol (≥99%, Sigma), phosphate buffer saline (PBS) (Tablet, Sigma), sodium-azide (a.r., VWR), trypsin-EDTA (Gibco), dispase (Roche), collagenase I (Sigma), Minimum Essential Medium (MEM) (Gibco), Fetal Bovine Serum (FBS) (Gibco, USA), L-glutamine (Gibco), Non Essential Amino Acids (NEAA, Gibco), penicillin (Gibco), streptomycin (Gibco), WST-1 (Roche), Vybrant-DiD (Molecular Probes), p-formaldehyde (Sigma). All reagents and solvents were used without further purification. MG-63 human osteosarcoma cell line was purchased from Sigma-Aldrich (USA). 2.2

Synthesis of the polymer

Poly(succinimide) (PSI) was synthesized according to our previously reported method18,21,25. In short, thermal poly-condensation of L-aspartic acid in the presence of phosphoric acid has been carried out. The reaction mixture was heated up to 180 °C while the pressure was decreased to 5 mbar. After the reaction, the raw polymer was dissolved in DMF, then precipitated in water and washed till neutral pH. The polymer was dried at 40 °C and kept at room temperature after the synthesis in closed vessels. The yield of the reaction is between 95-99% and the viscosityaverage molecular mass (Mη) is 28.5 ± 3 kDa. The measurement was described in detail in our previous publication21.


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7 2.3

Functionalization of poly(succinimide) with RGD tripeptide

The primer amine group of the RGD can react with the five-member ring of succinimide. 10 mg RGD was dissolved in 1.5 mL DMSO and 9.48 µL dibutylamine (DBA) was added to adjust the pH during the reaction (solution A, Table S1 in SI). To modify PSI, concentrated PSI solution in dimethyl-sulfoxide (DMSO) (25 w%) was mixed with different amounts of RGD in a glass vial. The reaction mixture was heated up to 45 °C and was stirred for one week with a magnetic stirrer (Ika RCT Basic). RGD functionalized PSI (PSI-RGDy) was synthesized with different ratio of grafting agent to repeating units (y: 1/200(for NMR measurement) 1/300, 1/500, 1/1000, 1/5000, 1/10000), where ratio of grafting agent to repeating units represents the RGD to monomer ratio (Scheme 1., Step 1 and Table S1 in SI.). After one week, the reaction mixture was mixed with cross-linkers to synthesize polymer hydrogels. 2.4

Nuclear Magnetic Resonance (NMR) spectroscopic analysis

All NMR spectra were obtained using a JEOL SC400 spectrometer (JEOL Ltd., USA) operating at 400 MHz for 1H nucleus. Samples were prepared by dissolving 25 mg of polymer powder in 0.6 mL of DMSO-d6 in 5 mm NMR tubes. All spectra were recorded at 23.5 ± 0.5 °C with tetramethylsilane as internal standard. The pulse angle was 45o, 2 sec delay was used with 8 kHz spectral width, 16 K data points and 16 scans were collected in each measurement. 2.5 2.5.1

Synthesis of different poly(aspartic acid)-based hydrogels Cross-linking of PSI chains

Two different amines, 1,4-diaminobutane (DAB) and cystamine (CYS) were used as crosslinking agents, to prepare PSI-based gels (Scheme 1., Step 2 a). Concentrated PSI (25 w%) solution was mixed thoroughly with different amount of cross-linkers in DMSO and the mixture was loaded into a 0.75 mm thick glass frame. After one day, gel film was formed and washed with DMSO to remove the unreacted cross-linkers.



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8 The ratio of cross-linking agent to repeating units (Dcr) is defined as the mol ratio between cross-linking agents (ncr) and monomers (nm): ‫ܦ‬௖௥ =

݊௖௥ ݊௠

The samples were identified by the following symbols: cp/Dcr, in which cp is the concentration of PSI when cross-linkers were introduced and Dcr is the ratio of cross-linking agent to repeating units representing the molar ratio of cross-linkers to monomer. PSI gels were prepared with different cross-linkers (1,4-diaminobutane (PSI-DABDcr), cystamine (PSI-CYSDcr), 1:1 molar ratio of DAB and CYS (PSI-CYS-DABDcr) with different cross-linking degrees (Dcr=1/10, 1/20 and 1/40). The cp was 15 w% in each sample.

2.5.2

Cross-linking of RGD modified PSI

The RGD containing PSI gels were synthesized from the reaction mixture of PSI-RGDy (solution B) (Table S2 in SI), with CYS and DAB cross-linkers simultaneously (Scheme 1., Step 2 b). Both CYS and DAB were dissolved in DMSO and 2 mol equivalent DBA was added to the reaction mixture. The PSI-RGD solution was mixed with the cross-linkers solution, and loaded into a 0.75 mm thick glass frame and kept there for 1 day. The gel film was washed with DMSO two times for 4 days to remove unreacted molecules. RGD containing double cross-linked gels are denoted as PSI-RGDy-CYS-DAB, in which y represents the ratio of grafting agent to repeating units. We prepared PSI-RGDy-CYS-DAB1/10 gels with y=1/300, 1/500, 1/1000, 1/5000, 1/10000 (Table S2 in SI).

2.5.3

Hydrolysis of PSI-based gels

PASP-based hydrogels were created by mild alkaline hydrolysis (close to the physiological pH) of PSI gels. To avoid alkaline degradation of disulphide bonds, the reaction was carried out in


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9 imidazole buffer at pH 8 (I=0.25 M, c=0.1 M)22,24,25. The buffer solution was changed in every two days to remove the DMSO and the remains of the unreacted molecules. The gels were treated with this buffer for 7 days to hydrolyze the total amount of succinimide monomers (Scheme 1., Step 3).

2.5.4

Cleavage of disulphide bonds in PASP-CYS-DAB hydrogels by reduction

Redox agents such as dithiotreitol (DTT) can cleave the disulphide bond leading to thiolates. The redox potential of cystamine is around -230 mV 34, whereas the standard redox potential of DTT is -950 mV between pH 7 and 835. This difference indicates the ability of DTT to cleave disulphide bonds in PASP-CYS-DAB hydrogels25. To prepare thiol containing PASP-based hydrogels, PASP-CYSE-DAB (PASP-CYS-DAB1/10, PASP-CYS-DAB1/20 and PASP-RGDy-CYS-DAB1/10) hydrogel films were immersed in 0.1 M DTT / pH 8 imidazole buffer solution (Scheme 1., Step 4). After 3 days, the DTT solution was changed to pH 8 imidazole buffer and washed with it a few times to remove unreacted / reacted DTT. The pH 8 buffer was changed after one week to phosphate buffer saline (PBS, pH 7.4, I=0.15M) which is also used during cell culturing. By cleaving the disulphide bonds, the theoretical ratio of cross-linking agent to repeating units was doubled, thus PASP-CYSE-DAB1/20 and PASP-CYSE-DAB1/40 as well as PASP-RGDy-CYSE-DAB1/20 hydrogels have been obtained. The photos at the bottom of the Scheme 1. represent the diameter changes of one gel disk after hydrolysis and DTT treatment.



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Scheme 1. Reaction steps in the synthesis of thiol and RGD containing PASP hydrogels. Scale bar on the pictures: 5 mm. 2.6

Investigation of degradability of poly(aspartic acid)-based hydrogels

Two sets of degradation experiments were carried out. In the first set of experiments, enzymatic degradation of PASP-CYS and PASP-CYS-DAB hydrogels was studied by trypsin, dispase and collagenase I, according to the method described by Galler et al. in 201036. After the hydrolysis, the hydrogels were dipped in PBS (pH 7.4, I=0.15M) for 14 days to remove the imidazole buffer. Hydrogel disks were cut from hydrogel films with a round shape metal cutter, and immersed into the different enzyme solutions at 37°C (0.05 % Trypsin-EDTA, 3 mg/ml


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11 collagenase I and 4 mg/ml dispase in PBS). Six hydrogel disks were placed into each enzyme solution and two disks were kept in PBS as negative control. The weight of the hydrogel disks were measured regularly for 4 weeks with a Sartorius analytical balance with an accuracy of 0.1 mg. In the second set of experiments, hydrogel cylinders were prepared from each type of PASPbased hydrogels. The reaction mixtures (described in 2.5.1.) were loaded into cylindrical (height and width: 2 cm) molds for one day. After one day, the cylinders were immersed in DMSO and thereafter pH=8 imidazole buffer to form PASP hydrogels. After the hydrolysis, the samples were treated with PBS for 2 weeks. Later PBS was changed for MG-63 cell line specific medium, containing 10 v% fetal bovine serum and other supplements (for details we refer the reader to section 2.7), and stored at 37 °C for 17 days. Geometric parameters (height and width) and weight have been measured by a caliper and analytical balance. The elastic modulus (Instron 5942, Intesztkft. Hungary) has been determined with unidirectional stress-strain measurements on different days. The stress-strain behavior of the samples was measured at room temperature (25 °C) with 1 mm/min expanding velocity. The test was carried out until 3N compressive force or 15 % deformation. After 17 days, the samples were dried at 40 °C to determine the mass swelling degree (Qm) (Table S3 in SI), which is represent the ratio of swollen and dried gel mass. Concentration of elastically active chains (ν*) (Table S4 in SI), which is an important quantity in rubber elasticity and represent the rigidity of the samples37, was calculated by Neo-Hooken law ିଶൗ ଷ

(‫ݍ ∗ ߥܣܴܶ = ܩ‬଴

ߔ

ଵൗ ଷ ).

In the equation, R is the gas constant and T represents the temperature,

ν* is the concentration of elastically active chains in the dry network expressed in moles, and q0 denotes the so-called memory term, by which the network ‘‘remembers’’ its initial state. The molecular interpretation of q0 is controversial for networks prepared in the solution state. Finally,



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12 A is a model-dependent parameter. According to the Flory theory, A=1, while the James–Guth and Staverman theories assume A=0.538–40. For the present experiments, the Flory theory modeldependent parameter was used (detailed explanation can be found in the supporting information). 2.7

Cell culture

These osteoblast-like cells were maintained in a humidified incubator (Nuaire, USA) in T75 tissue culture flasks (Orange Scientific, Belgium) under standard culture conditions (37 °C, 5 % CO2, 100 % humidity). The culture medium was composed according to the instructions and consisted of Minimum Essential Medium supplemented with 10 v% fetal bovine serum, 2 mM Lglutamine and 1 v% Non Essential Amino Acids, as well as 100 units/ml penicillin and 100 mg/ml streptomycin. Subconfluent cultures were passaged at a ratio 1:5 using 0.05 % trypsin/EDTA solution. 2.8

Cell viability assay

To avoid any bacterial or fungal infections, the PASP hydrogel films were kept in PBS containing sodium azide for one week prior to their use in cell experiments. Disks with 6 mm diameter and 1 cm thickness were cut out from the hydrogel films with a round shape metal cutter. As a pretreatment, the hydrogel disks were incubated in the complete medium of the MG-63 cells (Section 2.7) at room temperature for 1 h with a medium change at the 30th minute. Before cell seeding, the hydrogel disks were placed into the wells of sterile 96 well plates and sterilized by UV irradiation for 1 h. Onto the surface of each hydrogel disks, 20 000 MG-63 cells were seeded in 200 µl solution and cultured at 37 °C for 1, 3 or 6 days for the different experiments, respectively (Scheme 2.). At each time point, the cell/hydrogel constructs were washed with tempered PBS (37 °C) in order to remove non-attached or loosely attached cells. Determination of cell viability was carried out by a colorimetric assay using cell proliferation reagent WST-1 [2-(4-Iodophenyl)-3-(4-


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13 nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]. After 1:20 dilution of WST-1 with PBS, 100 µl was applied in each well for 2 h at 37 °C. 90 µl of each supernatant was transferred to an unloaded 96 well plate and absorbance at 450 nm was measured using a microplate reader (Model 3550, Bio-Rad Laboratories, Japan) (Scheme 2.). The reference wavelength was 655 nm. Hydrogel disks not seeded with cells were used as background controls. Each value represents the average of minimum 4 parallel measurements.

Scheme 2. Schematic of qualitative and quantitative measurements of cells on hydrogel disks. 2.9

Phase Contrast Microscopy

Morphology of MG-63 cells growing on hydrogel surfaces have been monitored under an inverted phase contrast microscope (Nikon Eclipse TS100, Nikon, Japan). Photomicrographs were taken with 10x objective by high performance CCD camera (COHU, USA) applying Scion image software. 2.10 Two photon microscopy To visualize the cells growing into the hydrogel disks, they were labeled with the fluorescent vital dye Vybrant DiD before seeding. In these experiments, hydrogel disks with 5 mm in diameter and 24 well plates were used as well as 80 000 MG-63 cells pro well were seeded in 800



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14 µl. After 1 or 3 days these cell/hydrogel constructs were rinsed with tempered PBS (37 °C) and fixed in 4 % p-formaldehyde at room temperature for 2 h, which was followed by another wash in PBS. The fixed samples were stored in PBS at 4 °C until examination with a two photon microscope (Femto2d, Femtonics, Hungary) (Scheme 2.). Spectra Physics Deep See laser was used with 800 nm wavelength to induce the photoactive dye. Images were taken with 10x objective by the MES4.4v program. 2.11 Statistical analysis Arithmetic mean values of cell viability were calculated from at least 4 independent experiments. Statistical evaluation of the viability data was carried out by STATISTICA 10 software (Statsoft, USA) applying Kruskal-Wallis non-parametric ANOVA followed by a median test. A difference was considered as statistically significant if p < 0.05.

3

Results

3.1

Structural analysis of the PSI polymer and their relatives

To verify the structure of the RGD modified PSI, we analyzed it by 1H-NMR (Figure 1.). First, we compared the structure of PSI before and after RGD modification. The peak at 5.23 ppm shows the hydrogen connected to the methine group of succinimide rings both in Figure 1. a and Figure 1. b. The hydrogens on the methylene group appeared at 2.7 and 3.2 ppm. The chiral methine group is responsible for the separation of the peaks. The peaks at 0.85, 1.29, and 1.5 indicate the hydrogens connected to the methylene and methyl groups of DBA, which was added to adjust the pH during RGD modification (Figure 1. a). The results indicate that the chemical structure of PSI is maintained after RGD modification.


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15

Figure 1.1H-NMR spectra of PSI-RGD1/200 (a, c and e) and PSI (b, d and f) The 1H-NMR-spectra are enlarged between 3.5-5 ppm and 6.5-9 ppm (Figure 1. c, d, e, and f) to identify the specific peaks of RGD. The hydrogen peaks of the methine group of the opened succinimide rings appear at 4.49 ppm (Figure 1. c.). The peaks at 3.95, 4.07 and 4.21 ppm are the hydrogens of the methylene groups of serine and asparagine in RGD (Figure 1. c.), whereas the



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16 pure PSI spectrum does not contain peaks between 3.5-5 ppm. Considering this result, the PSI chain does not contain opened succinimide rings and peaks from other amino acids (Figure 1. d). It is clear that RGD tripeptide contains arginine and the peaks of guanidine group in arginine appeared at 6.96, 7.1 and 7.3 ppm (Figure 1. e). In the spectrum of the PSI-RGD1/200, the –NH proton in the amide groups appears at 8.01, 8.2, 8.5, and 8.8 ppm (Figure 1. e). However, the spectrum of PSI does not contain peaks between 6.5 and 9 ppm as it was expected (Figure 1. f). The above results demonstrate that the modification of PSI with RGD was successfully proven by 1

H-NMR analysis.

3.2

Biodegradability by enzymes and cell culture medium

The degradation and rebuild of native ECM is a continuous revolution regulated by progenitor cells. It is essential that the kinetics of in vivo degradation of the hydrogels is in line with the revolution of native ECM. Furthermore, the change of mechanical properties of the hydrogels derived from biodegradation has a significant impact on the cell attachment to the hydrogel surface and formation of native morphology10,13. Enzymes and substances of cell culture media can induce degradation of polymer matrices. Therefore, the degradation kinetics of the PASP hydrogel disks cross-linked with different compositions (PASP-CYS, PASP-CYS-DAB) were investigated in the presence of different enzymes involved in protein degradation (trypsin-EDTA, dispase and collagenase I) (Figure 2.) and in cell culture medium (Figure 3.). During the first two weeks, the PASP-CYS hydrogels were kept in PBS to temper the hydrogels for the physiological conditions and wash out the imidazole buffer where the hydrogels were previously stored. PBS did not induce degradation neither in the first two weeks (conditioning) nor in 42 days in case of control gels (Figure 2. a, square marking).


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18 Figure 2. Degradation of PASP-CYS hydrogel disks in the presence of trypsin-EDTA, dispase and collagenase I (a), degradation of PASP-CYS and PASP-CYS-DAB hydrogel disks in the presence of collagenase I (b) and images of hydrogel disks in different enzyme solutions in different days (c). The scale bar indicates 5mm. (

symbol indicates enzyme solution change)

In the presence of trypsin-EDTA and dispase, the mass of the hydrogel disks increased linearly (Figure 2. a, round and triangle marking). This monotone increase of mass was only small-scale during the first two weeks. After the exchange of the enzyme solutions on day 21, the enlargement of the hydrogel disks accelerated significantly in the presence of trypsin-EDTA and to a smaller extent in the case of dispase. Although the trypsin-EDTA generates a notable increase in hydrogel mass, the hydrogel disks were swollen without any observable changes in their shape during the whole experiment. The mass of the hydrogel disks increased significantly in the presence of collagenase I (Figure 2. a, quarrel marking). The PASP-CYS hydrogel disks swelled considerably during the first week (between day 14 and 16). The shape of the hydrogel disks changed to amorphous (Figure 2. c), consequently, they swelled largely and dissolved in one day after changing the enzyme solutions for the second time (day 22). It is considered that the decreasing number of network chains induces the swelling of the hydrogel matrix. To find out which bond is collagenase I sensitive in the polymer matrix, we have investigated the degradation of double (CYS-DAB) and single (CYS) cross-linked hydrogels in collagenase I solutions (Figure 2. b). In the presence of collagenase I, the mass and the volume of the double cross-linked PASP (PASP-CYS-DAB) hydrogel disks increased during the 41-day-long observation period but no deformation was observed. As in the previous experiment, the hydrogels that only contained disulphide cross-links (PASP-CYS) dissolved during the


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19 observation period, indicating that the hydrogel may dissolve due to fritter of network chains as the degradation proceeds. These results lead to the conclusion that the collagenase I enzyme cleaved the disulphide bonds in the polymer matrix rather than the polymer backbone itself. To investigate the stability of different PASP based hydrogels in cell cultivating conditions, gel cylinders of 2 cm in width and height were used (Figure 3.d). The mass swelling degree (Figure 3. c) (Table S3 in SI) and the concentration of the elastically active chains (ν*) (Figure 3. a and b) (Table S4 in SI) were analyzed as the function of degradation time, and some of the stress strain curves were attached as a supporting information (Figure S1).

Figure 3. Evolution of the concentration of elastically active chains and the relative mass swelling degree in case hydrogels with ratio of cross-linking agent to repeating units of 1/20 (a and c) and 1/40 (b and c). Photographic images of different hydrogel cylinders taken in the



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20 presence of cell culture medium at different days (d). The scale bar indicates 1 cm. (

: exchange

of cell culture medium) The results show that the various DAB cross-linked hydrogels were stable during the 17-daylong observation period in cell culture medium. The small decrease in the concentration of elastically active chains may be caused by the diffusion of small molecules from the cell medium to the polymer matrix (Figure 3. a and b black and c black and green square marking). The thiol groups containing PASP-CYSE-DAB hydrogels showed the same behavior in cell culture medium (Figure 3. b and c, blue rhombic marking). The shape and size of the hydrogel cylinders have not changed during the experiments at all (Figure 3. d). This kind of hydrogel could not been synthesized in cylinder shape with 1/20 ratio of cross-linking agent to repeating units, because it fractured during the hydrolysis. At first, the gel shrank because DMSO diffused out of the polymer gel during the hydrolysis and it was replaced by the buffer. The latter is a poor swelling agent, therefore, the gel shrank and fractured due to its the high rigidity. The shape of the cystamine cross-linked PASP based hydrogels changed after 5 days (Figure 3. d). The hydrogels deformed and swelled in the first days and later dissolved in cell culture medium (Figure 3. a and b light blue and c light blue and dark yellow triangle marking). In the case of double cross-linked PASP hydrogels, the concentration of elastically active chains (ν*) (Figure 3 a and b red round marking) (Table S4 in SI) showed a monotonous decrease while the mass swelling degree (Qm) (Figure 3. c red and magenta round marking) (Table S3 in SI) and a monotonous increase in time. The DAB containing hydrogels did not dissolve in our experiments, only swelled and deformed in the case of double cross-linked hydrogels (Figure 3. round marking and Figure 3. d). The reason of this is likely that alfa-MEM contains different anti-oxidants (such as ascorbic acid, riboflavin) and L-cysteine. In this environment a thiol-


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21 disulphide change may occur between cystamine and L-cysteine, or riboflavin can mediate the reduction of disulphide bridges, which may cause the cleavage of the disulphide bonds in the hydrogel matrix. To prove this concept, PASP-CYS-DAB gel disks were immersed into 0.1M/PBS L-cysteine solution. After 2 hours, the gels swelled and the change of the mass of the gels was similar to that in the case of 0.1M DTT solution25. The results can be found in the supporting information (Figure S2). This leads to the conclusion that not just enzymes, but also cell culture medium can degrade the disulphide bonds, which results in the change of the mechanical properties. By using DAB crosslinked PASP hydrogels where thiol groups were previously formed (PASP-CYSE-DAB), the volume change and dissolution can be hindered. 3.3 3.3.1

Cell experiments Cell culture on PASP hydrogels with different cross-linking density and chemical structure

The first experimental series with MG-63 osteoblast-like cells have been performed to test how the chemical structure and mechanical properties of PASP hydrogels influence cell adhesion and proliferation. For these investigations, single (CYS or DAB), double (CYS-DAB) cross-linked and thiol containing (CYSE-DAB) PASP hydrogel disks have been used with 2 different ratios of cross-linking agent to repeating units (1/20 and 1/40). Cell viability results on the 8 different types of PASP hydrogels after pretreatments with cell culture medium can be seen on Figure 4.



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22


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23 Figure 4. MG-63 cell viability measured 1 and 3 days after seeding on PASP hydrogel disks with different cross-linking agents and ratio (a) and phase contrast microscopic morphology (b) on PASP Dcr=1/20 2days after cell seeding. Cell viability was determined by WST-1 assay. Data are given as arithmetic mean ± SEM (standard error of the mean). *p < 0.05 compared to CYSEDAB1/20.

+

p < 0.05 compared to day 1. Each photomicrograph was taken at the same

magnification. The scale bar indicates 100 µm. One day after the cell seeding (Figure 4. a, blue columns), cell viability values were the highest on double cross-linked PASP hydrogels with ratio of cross-linking agent to repeating units of 1/20 (CYS-DAB1/20 and CYSE-DAB1/20). This suggests that double cross-linking and harder hydrogels both facilitate adhesion of osteoblast-like cells (Table S5 in SI). Similar results were found after 3 days (Figure 4. a red columns), but in this case the cell viability on CYSE-DAB1/20 hydrogels were significantly higher than on CYS-DAB1/20 hydrogels. A higher increase in cell viability between day 1 and 3 was found in the case of CYSE-DAB1/20 hydrogels as well, indicating better properties of this hydrogel type (free thiol groups were induced before the cell culture medium pretreatment) in promoting cell proliferation. It is important to note that cell culture medium can induce thiol group formation also in CYS-DAB hydrogels during the experiments (as described above in 3.2). Consequently, thiol group containing PASP based hydrogels seem to provide ideal conditions for both cell adhesion and proliferation of hard tissue generating cells, such as MG-63 cells. We originate this from the interaction between the free thiol groups in polymer matrix and free thiol groups in L-cysteine, which can be found in cell membranes. For morphological investigation of MG-63 cells adhering and growing on the surface of the PASP -based hydrogels, phase contrast and two photon microscopy have been applied (Figure 4.



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24 b and Figure 5.). On the PASP hydrogels containing only one kind of cross-linking agent (i.e. either DAB or CYS), all MG-63 cells showed round morphology, indicating that these cells were only loosely attached to the hydrogel surface and were not able to spread to obtain healthy morphology (Figure 4. b). Therefore, the cells are not able to survive and proliferate on these gels. However, most of the cells demonstrated normal fibroblast-like morphology on double cross-linked (CYS-DAB) and thiol groups containing (CYSE-DAB) hydrogels. Thus, on the surface of the double cross-linked PASP based hydrogels, MG-63 cells were able to strongly adhere and spread to reveal their typical, healthy morphology. This observation is in line with the long-term survival of the cells on these gel types. The higher amount of cells found on CYSEDAB hydrogels compared to CYS-DAB hydrogels is presumably the consequence of higher proliferative activity (Figure 4. b). These observations are in good correlation with the results of the WST-1 tests mentioned above. These phase contrast microscopic studies confirm that the thiol groups can support cell adhesion, survival and proliferation of MG-63 cells. To investigate how the cultured cells grow on the surface and keep track of potential migration into the hydrogel, two photon microscopy was utilized. Poly(aspartic acid) has autofluorescence properties, thus, the hydrogel disk can be visualized in green color, so the cells and the hydrogel matrix can easily be distinguished (Figure 5.).


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25



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26 Figure 5. Representative two photon microscopy images of Vybrant DiD-labelled MG-63 cells growing on the PASP-CYS-DAB1/20 and PASP-CYSE-DAB1/20 hydrogel disks (a) and Z-stack image of MG-63 cells growing into PASP-CYSE-DAB1/20 hydrogel 3 days after cell seeding (b). (The 2D photomicrographs were taken after 1 and 3 days. The green color is due to autofluorescence of the hydrogel while the red color indicates Vybrant DiD stained cells). The Z-stack photomicrograph was generated from a stack of laser 2-photon microscopy scans of 3µm increments from the upper 70 µm of the hydrogel disk. Under two photon microscope (Figure 5. a), clusters of healthy, fibroblast-like cells with natural morphology can be seen both in PASP-CYS-DAB1/20 and PASP-CYSE-DAB1/20 hydrogels. The thin processes connecting the members of the cell clusters to each other are also visible on the images. A strikingly larger cell population was found on day 3 compared to day 1, which is in agreement with the results of cell viability measurements. The pale green color on the pictures proves that the cells and the hydrogel parts studied are on the same level (Z-height). Two photon microscopy is a powerful technique for in-depth observation. By stacking sections of different Z-heights one can obtain the 3D structure of the samples (Figure 5. b). The representative picture was taken with 3 µm step width and in 70 µm deep from the hydrogel surface. The Z-stack image provides evidence that the MG-63 cells not only obtain the healthy morphology on the PASP hydrogel but are even capable of penetrating into the matrix. The topology of the hydrogel surface became rough, the cells formed bumps on the surface and integrated into the hydrogel by digesting it.

3.3.2

Cell culture on PASP hydrogels with different amount of RGD motif

RGD is a naturally occurring, cell adhesive amino acid sequence in the ECM, which is commonly built into scaffolds for tissue engineering to facilitate cell adhesion41,42. To further


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27 enhance cell adhesion and increase proliferation on the PASP-CYSE-DAB1/20 hydrogel, we attached different amounts of RGD sequence covalently with amide bond to the polymer backbone (Scheme 1., Step 1).The hydrogel disks were pretreated with cell line specific medium.

Figure 6. MG-63 cell viability measured 1 and 6 days after seeding by WST-1 assay (a) and phase contrast microscopic morphology after 6 days (b) on PASP-CYSE-DAB1/20 hydrogel disks with different RGD content. Data are given as arithmetic mean ± SEM (standard error of the mean). *p < 0.05 compared to control. +p < 0.05 compared to day 1. The bars indicate 100 µm. Figure 6. a shows that 24 h after cell seeding, there was no significant difference in cell viability between the hydrogels with different RGD content. According to statistical analysis significantly higher viability compared to control was only found in the case of RGD1/500 hydrogel at day 1. During the next 5 days, the cell population increased significantly on all hydrogel samples. Thus, MG-63 cells are able to adhere and proliferate on all investigated



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28 hydrogel types regardless RGD content. Based on these results, we assume that MG-63 cells do not require the RGD motif for adhesion and proliferation in the case of PASP-CYSE-DAB hydrogels. However, RGD can regulate the cells to form compact clusters, which would be important to maintain their osteoblast function43. The presence of RGD seems to slightly enhance cell adhesion and proliferation, but the statistical analysis could not show significant difference between the samples. This is likely due to the relatively high standard deviation, which is typical for biological samples. Under phase contrast microscope, healthy, fibroblast-like morphology and several connections between the cells can be observed on all hydrogel types (Figure S3 in SI and Figure 6. b). After 6 days, cell aggregation can be seen on all the studied hydrogel types regardless to the RGD content. By day 6, the cells strongly attached to each other and formed compact clusters with high cell density, which was presumably induced by the RGD motif43. This result is in agreement with cell viability data, and confirms the ability of MG-63 cells to adhere and form clusters on PASP-CYSE-DAB gels with RGD motif. Clusters of osteoblast-like cells with healthy, natural morphology and many cell-cell connections can be seen on every sample irrespectively to the RGD content (Figure 7.).


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29



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30 Figure 7. Two photon microscopic images of MG-63 cells growing on PASP-CYSE-DAB hydrogel disks with different RGD content (a) and Z-stack image of PASP-RGD1/300-CYSEDAB1/20 hydrogel, 3 days after cell seeding (b). The Z-stack photomicrograph was generated from a stack of laser 2-photon microscopy scans of 3µm increments taken from the upper 60 µm of the hydrogel disk. We assume that the only possible way for the cells to migrate into the hydrogels is if they degrade the polymer hydrogel during their penetration. Two photon micrographs also prove the cluster formation of the cells on RGD containing hydrogels. Therefore, the presence of the cells inside the hydrogel disks (Figure 7. b) provides indirect evidence for the in vitro biodegradability of the PASP-CYSE-DAB hydrogel, and for the possibility of a dynamic interaction between the cells and the matrix. However, further experiments for longer time periods are planned to prove the penetration directly.

4

Discussion: The already available sporadic data on properties of poly(aspartic acid), such as

biocompatibility, biodegradability and relatively easy modification of the polymer chain through the functional groups26,27,32, led us to study the hydrogels based on this polymer as a potential scaffold for in vitro proliferation of osteoblast-like cells. In our current work, we have investigated the effect of the mechanical property and chemical constitution (cross-linking agents, thiol groups, RGD motif) on MG-63 cell adhesion and proliferation. For chemical crosslinking of PASP hydrogels a naturally occurring diamine, the putrescine (or 1,4-diaminobutane) and an amino acid derivative, cystamine were applied (Scheme 1.).


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31 The reason for using poly(succinimide) as a starting material was that this polymer reacts easily with the molecules containing primer amino groups, such as amino acids, under mild conditions. We utilized this property to modify the polymer backbone with RGD peptide sequence. RGD (Arg-Gly-Asp) is a well-known cell recognition motif44, and is often applied to promote cell adhesion to the surface of scaffolds6,17,18,29,45. To link the RGD motif to the polymers covalently, usually an elaborate reaction in several steps is necessary45–48. In addition, it leads to unregulated amount of RGD molecules in the system. In our study, poly(succinimide) was successfully modified with the RGD motif in a one-step reaction (Scheme 1., Step 1), which was proven by 1

H-NMR spectra (Figure 1). The amount of the immobilized RGD was easily manipulated by

changing the ratio of poly(succinimide) and RGD. No further processes were required to prepare the modified polymer-based hydrogels. The degradability of scaffolds by proliferating cells is an important property of hydrogel scaffolds designed for tissue engineering17,18,49,50. Thus, we performed biodegradability experiments with differently cross-linked poly(aspartic acid) hydrogels in the presence of enzymes like trypsin, dispase and collagenase I (Figure 2.). Our hydrogels proved to be stable in trypsin and dispase solutions during the whole experimental period, but were degraded in collagenase I solution (Figure 2.). Similar results were described by Galler et al. in the case of multidomain peptide hydrogels, which were completely digested after 2-week-long incubation in collagenase I36. The poly(aspartic acid) hydrogels, having no disulphide bridges in the polymer matrix were stable for more than two weeks in cell culture medium, also in the presence of collagenase I. Hydrogels containing only disulphide bonds – depending on the amount of crosslinks – first swelled than dissolved in the presence of this particular enzyme solution, while cross-links were cleaved (Figure 2.). Thus, our results pointed out that collagenase I is not just capable of breaking



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32 the peptide bonds in collagen but it can also cleave the disulphide bonds in the polymer matrix. It is worth to note that the disulphide bridges are very sensitive to the redox environment51, but there are no reports that indicate the redox effect of collagenase I. The mechanical properties of scaffolds have great impact on cell adhesion and proliferation. The increase of concentration of elastically active chains of scaffolds can enhance adhesion and proliferation of cells originating from hard tissues4. Therefore, changes in the concentration of elastically active chains and mass swelling degree were followed in cell line specific culture medium (Figure 3.). Concentration of elastically active chains and the elastic modulus of poly(aspartic acid) based hydrogels can be changed easily in a wide range by adjusting the crosslinker to monomer ratio. PASP hydrogel cylinders were prepared with two different ratio of cross-linking agent to repeating units (1/20 and 1/40). Our results showed that this twofold change in ratio of cross-linking agent to repeating units is manifested by a change of a factor of 5-8 in the concentration of elastically active chains (Figure 3. and Table S4 in SI). The concentration of elastically active chains of disulphide containing PASP hydrogels (PASP-CYS and PASP-CYS-DAB) decreased in the presence of cell medium, which is presumably due to the cleavage of disulphide bonds in the polymer matrices. Although in PASP-CYS-DAB1/40 there were permanent DAB cross-links, after 12 days the gel dissolved in cell culture medium. After the cleavage of the disulphide bonds in the polymer matrix, there were not enough cross-links anymore in the gel, and hence the elasticity of the polymer network could not compensate the osmotic pressure of the swelling agent, which explains the dissolution of the gel. The hydrogels containing only DAB as a cross-linker (PASP-DAB and PASP-CYSE-DAB) did not undergo any degradation induced by cell culture medium, which provides indirect evidence for this explanation. It is worth noting that α-MEM contains various anti-oxidants as well as L-cysteine.


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33 As our results show (Figure S2.), the thiol-disulphide change occurred between cystamine and Lcysteine, which subsequently caused the cleavage of disulphide bonds in the hydrogel matrix in cell media. Regarding our results, the number of the cells was increased in hydrogels with higher concentration of elastically active chains (Figure 4.), which is in a good agreement with the theory mentioned above7. Our aim was to prepare a biocompatible, modified PASP scaffold for cell culturing, and hence to follow proliferation of the osteoblast-like MG-63 cells on PASP based hydrogels. For this purpose, the WST-1 cell viability tests and microscopic investigations seemed to be adequate techniques, providing both qualitative and quantitative information. Results of the WST-1 assay showed increased cell population on the thiolated polymer matrix (Figure 4. a). Although some thiolated polymers were already developed for tissue engineering purposes52–54, the effect of the free thiol groups (in scaffolds) on the proliferative abilities of the cells has not been extensively studied yet. Bae et. al found elevated viability on thiolated chitosan scaffold compared to unmodified chitosan on a mouse osteoblast cell line (MC3T3-E1), which is in line with our results53. Increased cell viability may be caused by the interaction between the free thiol groups in the polymer matrix and cell membrane proteins, as some cell membrane peptides contain free thiol groups in cysteine, which has a role in the delivery of bioactive cargoes inside the cells55. This interaction may also increase cell adhesion to hydrogel surface leading to higher proliferation potential (Figure 4. a and b). In the present study, the thiolated poly(aspartic acid) hydrogels were modified with various amounts of RGD motif in order to further enhance cell proliferation. Interestingly, the amount of RGD motif seems to have no significant effect on cell attachment and proliferation of MG-63 cells (Figure 6. a). Similar results were shown by Grigore et. al with Alginate based materials56. However, the presence of RGD facilitates compact cluster formation of MG-63 cells (Figure 6.



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34 b), in contrast with the non-modified PASP-CYSE-DAB1/20 hydrogel (Figure 4. b and 5). This behavior is crucial to maintain their osteoblast function as it was described by T. Re’em et. al in the case of RGD modified alginate based scaffolds43. By microscopic analysis, MG-63 cells can be found with native morphology on PASP hydrogels, similarly to the published data of Chien et al57. Both phase contrast (Figure 4. b and 6. b) and two photon microscopy (Figure 5. and 7.) results show unambiguous evidence that MG-63 cells can be attached to the surface of the PASP hydrogels. After 6 days (Figure 6. b) the cells organized spherical clusters, which was already published in case of another cancer cell line58. The differences between the photomicrographs taken on day 1 and 3 (Figure 5. a) provide evidence for the high cell proliferation potential on the hydrogel surface. We observed that poly(aspartic acid) exhibited green autoflourescence, a property that has never been published before. Due to this feature, the poly(aspartic acid) based hydrogels and the cells can be visualized simultaneously with two photon microscopy. The fact that, the cells and the polymer matrix are found in the same Z cross section suggests that the cells can penetrate into the hydrogel matrix. To demonstrate this phenomenon, 3D structure of the polymer hydrogels were obtained by stacking section of different Z-heights (Figure 5. b and 7. b). These investigations provided evidence that the cells can migrate into the hydrogel matrices as deep as around 30 µm, which is roughly equal to the size of one cell. As it is obvious from the Z-heights microscopic pictures, the MG-63 cells can digest the polymer matrix, and therefore these results also confirm the biodegradability of the poly(aspartic acid) based hydrogels.

5

Conclusions In our work, we successfully modified the poly(succinimide) polymer with RGD peptide

sequence, which was confirmed with 1H-NMR measurements. Polymer hydrogels could be


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35 synthesized from these polymers and after a mild hydrolysis (close to the physiological conditions) the polymers transformed to poly(aspartic acid). The prepared hydrogels contained the variation of permanent (DAB) and redox sensitive (CYS) cross-linkers to fix the structure of the scaffolds. Some of the poly(aspartic acid) hydrogels were also endowed with thiol groups on the polymer backbone (with the cleavage of disulphide bonds). The stability and biodegradation of different poly(aspartic acid)-based hydrogels were proved in the presence of different enzymes and cell culture medium by swelling and stress-strain measurements. The disulphide bonds in the polymer matrix were found to be decomposed in cell culture medium. Cell viability and morphology have been investigated by WST-1 test and different microscopic techniques (phase contrast and two photon microscopy). According to both cell viability and morphological studies, the viable population of the MG-63 osteoblast-like cells was increased in the thiolated hydrogels with higher elastic modulus. The presence of the RGD sequence was not crucial for adhesion or proliferation of these cells, however, it induced the compact cluster formation of the cells, which is important to maintain their osteoblast function. To summarize our results, the poly(aspartic acid) based hydrogels are biodegradable, biocompatible and can support survival, proliferation as well as migration of human osteoblastlike cells. Consequently, fabricated, modified poly(aspartic acid) based hydrogels seem to be ideal scaffolds for in vitro cell proliferation.

Acknowledgement:

This

research

was

supported

by

OTKA

K

115259.

Supporting Information: Tables with the entire amount of the hydrogel synthesis, mass swelling degree values, concentration of elastically active chain values and elastic modulus



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36 values with stress-strain curves are available in the Electronic Supplementary Information (ESI): This material is available free of charge via the Internet at http://pubs.acs.org.

6: References: (1)

Langer, R.; Vacanti, J. P. Tissue Engineering. Science. 1993, 260, 920–925.

(2)

Cascalho, M.; Platt, J. L. New Technologies for Organ Replacement and Augmentation. Mayo Clin. Proc. 2005, 80, 370–378.

(3)

Atala, A. Tissue Engineering of Reproductive Tissues and Organs. Fertil. Steril. 2012, 98, 21–29.

(4)

Santos, E.; Hernández, R. M.; Pedraz, J. L.; Orive, G. Novel Advances in the Design of Three-Dimensional Bio-Scaffolds to Control Cell Fate: Translation from 2D to 3D. Trends Biotechnol. 2012, 30, 331–341.

(5)

Lee, I.-C.; Wu, Y.-C. Facilitating Neural Stem/progenitor Cell Niche Calibration for Neural Lineage Differentiation by Polyelectrolyte Multilayer Films. Colloid. Surface B. 2014, 121, 54–65.

(6)

Gribova, V.; Gauthier-Rouvière, C.; Albigès-Rizo, C.; Auzely-Velty, R.; Picart, C. Effect of RGD Functionalization and Stiffness Modulation of Polyelectrolyte Multilayer Films on Muscle Cell Differentiation. Acta Biomater. 2013, 9, 6468–6480.

(7)

Discher, D. E.; Mooney, D. J.; Zandstra, P. W. Growth Factors, Matrices, and Forces Combine and Control Stem Cells. Science 2009, 324, 1673–1677.

(8)

Guilak, F.; Cohen, D. M.; Estes, B. T.; Gimble, J. M.; Liedtke, W.; Chen, C. S. Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix. Cell Stem Cell 2009, 5, 17–26.

(9)

Skotak, M.; Leonov, A. P.; Larsen, G.; Noriega, S.; Subramanian, A. Biocompatible and Biodegradable Ultrafine Fibrillar Scaffold Materials for Tissue Engineering by Facile Grafting of L-Lactide onto Chitosan. Biomacromolecules 2008, 9, 1902–1908.

(10)

Lutolf, M. P. Integration Column: Artificial ECM: Expanding the Cell Biology Toolbox in 3D. Integr. Biol. (Camb). 2009, 1, 235–241.

(11)

Cai, N.; Wong, C. C.; Gong, Y. X.; Tan, S. C. W.; Chan, V.; Liao, K. Modulating Cell Adhesion Dynamics on Carbon Nanotube Monolayer Engineered with Extracellular Matrix Proteins. ACS Appl. Mater. Interfaces 2010, 2, 1038–1047.

(12)

Yao, D.; Dong, S.; Lu, Q.; Hu, X.; Kaplan, D. L.; Zhang, B.; Zhu, H. Salt-Leached Silk Scaffolds with Tunable Mechanical Properties. Biomacromolecules 2012, 13, 3723–3729.


Page 37 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37 (13)

Kim, W.; Ferguson, V. L.; Borden, M.; Neu, C. P. Application of Elastography for the Noninvasive Assessment of Biomechanics in Engineered Biomaterials and Tissues. Ann. Biomed. Eng. 2016, 44, 1–20.

(14)

Tanaka, Y.; Gong, J. P.; Osada, Y. Novel Hydrogels with Excellent Mechanical Performance. Prog. Polym. Sci. 2005, 30, 1–9.

(15)

Lin, Z.; Cao, S.; Chen, X.; Wu, W.; Li, J. Thermoresponsive Hydrogels from Phosphorylated ABA Triblock Copolymers: A Potential Scaffold for Bone Tissue Engineering. Biomacromolecules 2013, 14, 2206–2214.

(16)

Li, L.; Gu, J.; Zhang, J.; Xie, Z.; Lu, Y.; Shen, L.; Dong, Q.; Wang, Y. Injectable and Biodegradable pH-Responsive Hydrogels for Localized and Sustained Treatment of Human Fibrosarcoma. ACS Appl. Mater. Interfaces 2015, 7, 8033–8040.

(17)

Drury, J. L.; Mooney, D. J. Hydrogels for Tissue Engineering: Scaffold Design Variables and Applications. Biomaterials 2003, 24, 4337–4351.

(18)

Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869–1880.

(19)

Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biopolymer-Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review. Biomacromolecules 2011, 12, 1387–1408.

(20)

Choi, B.; Kim, S.; Lin, B.; Wu, B. M.; Lee, M. Cartilaginous Extracellular MatrixModified Chitosan Hydrogels for Cartilage Tissue Engineering. ACS Appl. Mater. Interfaces 2014, 6, 20110–20121.

(21)

Molnar, K.; Juriga, D.; Nagy, P. M.; Sinko, K.; Jedlovszky-Hajdu, A.; Zrinyi, M. Electrospun Poly(aspartic Acid) Gel Scaffolds for Artificial Extracellular Matrix. Polym. Int. 2014, 63, 1608–1615.

(22)

Gyenes, T.; Torma, V.; Gyarmati, B.; Zrínyi, M. Synthesis and Swelling Properties of Novel pH-Sensitive Poly(aspartic Acid) Gels. Acta Biomater. 2008, 4, 733–744.

(23)

Gyenes, T.; Torma, V.; Zrínyi, M. Swelling Properties of Aspartic Acid-Based Hydrogels. Colloid. Surface A. 2008, 319, 154–158.

(24)

Varga, Z.; Molnár, K.; Torma, V.; Zrínyi, M. Kinetics of Volume Change of Poly(succinimide) Gels during Hydrolysis and Swelling. Phys. Chem. Chem. Phys. 2010, 12, 12670–12675.

(25)

Zrinyi, M.; Gyenes, T.; Juriga, D.; Kim, J.-H. Volume Change of Double Cross-Linked Poly(aspartic Acid) Hydrogels Induced by Cleavage of One of the Crosslinks. Acta Biomater. 2013, 9, 5122–5131.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 41

38 (26)

Kurland, N. E.; Ragland, R. B.; Zhang, A.; Moustafa, M. E.; Kundu, S. C.; Yadavalli, V. K. pH Responsive Poly Amino-Acid Hydrogels Formed via Silk Sericin Templating. Int. J. Biol. Macromol. 2014, 70, 565–571.

(27)

Cai, K.; Yao, K.; Hou, X.; Wang, Y.; Hou, Y.; Yang, Z.; Li, X.; Xie, H. Improvement of the Functions of Osteoblasts Seeded on Modified Poly (D,L-Lactic Acid ) with Poly (aspartic Acid). J. Biomed. Mater. Res. 2001, 62, 283–291.

(28)

Nie, J.-J.; Dou, X.-B.; Hu, H.; Yu, B.; Chen, D.-F.; Wang, R.-X.; Xu, F.-J. Poly(aspartic Acid)-Based Degradable Assemblies for Highly Efficient Gene Delivery. ACS Appl. Mater. Interfaces 2015, 7, 553–562.

(29)

Cao, F. Y.; Yin, W. N.; Fan, J. X.; Tao, L.; Qin, S. Y.; Zhuo, R. X.; Zhang, X. Z. Evaluating the Effects of Charged Oligopeptide Motifs Coupled with RGD on Osteogenic Differentiation of Mesenchymal Stem Cells. ACS Appl. Mater. Interfaces 2015, 7, 6698– 6705.

(30)

Tachibana, Y.; Kurisawa, M.; Uyama, H.; Kakuchi, T.; Kobayashi, S. Biodegradable Thermoresponsive Poly ( Amino Acid ) S. Chem. Commun. 2003, 3, 106–107.

(31)

Mammadov, R.; Mammadov, B.; Guler, M. O.; Tekinay, A. B. Growth Factor Binding on Heparin Mimetic Peptide Nanofibers. Biomacromolecules 2012, 13, 3311–3319.

(32)

Alford, D.; Wheeler, A. P.; Pettigrew, C. Biodegradation of Thermally Synthesized Polyaspartate. J. Environ. Polym. Degrad. 1994, 2, 225–236.

(33)

Groen, N.; van de Peppel, J.; Yuan, H.; van Leeuwen, J. P. T. M.; van Blitterswijk, C. a; de Boer, J. Bioinformatics-Based Selection of a Model Cell Type for in Vitro Biomaterial Testing. Biomaterials 2013, 34, 5552–5561.

(34)

Gyenes, T. Aminosav Alapú Gélek Szintézise És Duzzadási Tulajdonságainak Vizsgálata, Ph.D. Thesis, Budapest University of Technology and Economics, Budapest, Hungary 2007.

(35)

Cleland, W. W. Dithiothreitol, a New Protective Reagent for SH Groups*. Biochemistry 1964, 3, 480–482.

(36)

Galler, K. M.; Aulisa, L.; Regan, K. R.; D’Souza, R. N.; Hartgerink, J. D. Self-Assembling Multidomain Peptide Hydrogels: Designed Susceptibility to Enzymatic Cleavage Allows Enhanced Cell Migration and Spreading. J. Am. Chem. Soc. 2010, 132, 3217–3223.

(37)

Goethals, E. J. Telechelic Polymers: Synthesis and Applications; 1st ed; CRC Press: Florida, USA, 1989.

(38)

Flory, P. J.; Rehner, J. Statistical Mechanics of Cross Linked Polymer Networks I. Rubberlike Elasticity. J. Chem. Phys. 1943, 11, 512-520.


Page 39 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39 (39)

Dušek, K.; Prins, W. Structure and Elasticity of Non-Crystalline Polymer Networks. Adv. Polymer Sci. 1969, 6, 1–102.

(40)

James, M.; Burak, E. Rubberlike Elasticity: A Molecular Primer, 1st ed; John Wiley & Sons, Ltd: New York, USA, 1988.

(41)

Yoon, J. J.; Song, S. H.; Lee, D. S.; Park, T. G. Immobilization of Cell Adhesive RGD Peptide onto the Surface of Highly Porous Biodegradable Polymer Scaffolds Fabricated by a Gas Foaming/salt Leaching Method. Biomaterials 2004, 25, 5613–5620.

(42)

Williams, D. F. The Role of Short Synthetic Adhesion Peptides in Regenerative Medicine; the Debate. Biomaterials 2011, 32, 4195–4197.

(43)

Re’em, T.; Tsur-Gang, O.; Cohen, S. The Effect of Immobilized RGD Peptide in Macroporous Alginate Scaffolds on TGF??1-Induced Chondrogenesis of Human Mesenchymal Stem Cells. Biomaterials 2010, 31, 6746–6755.

(44)

Ruoslahti, E.; Pierschbacher, M. D. Arg-Gly-Asp : A Versatile Cell Recognition Signal Minireview. Cell 1986, 44, 517–518.

(45)

Hersel, U.; Dahmen, C.; Kessler, H. RGD Modified Polymers: Biomaterials for Stimulated Cell Adhesion and beyond. Biomaterials 2003, 24, 4385–4415.

(46)

Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Effects of Immobilizing Sites of RGD Peptides in Amphiphilic Block Copolymers on Efficacy of Cell Adhesion. Biomaterials 2010, 31, 7873–7882.

(47)

Panda, J. J.; Dua, R.; Mishra, A.; Mittra, B.; Chauhan, V. S. 3D Cell Growth and Proliferation on a RGD Functionalized Nanofibrillar Hydrogel Based on a Conformationally Restricted Residue Containing Dipeptide. ACS Appl. Mater. Interfaces 2010, 2, 2839–2848.

(48)

Ingavle, G. C.; Gehrke, S. H.; Detamore, M. S. The Bioactivity of Agarose-PEGDA Interpenetrating Network Hydrogels with Covalently Immobilized RGD Peptides and Physically Entrapped Aggrecan. Biomaterials 2014, 35, 3558–3570.

(49)

Lee, F.; Kurisawa, M. Formation and Stability of Interpenetrating Polymer Network Hydrogels Consisting of Fibrin and Hyaluronic Acid for Tissue Engineering. Acta Biomater. 2013, 9, 5143–5152.

(50)

Calderon, L.; Collin, E.; Velasco-Bayon, D.; Murphy, M.; O’Halloran, D.; Pandit, A. Type II Collagen-Hyaluronan Hydrogel--a Step towards a Scaffold for Intervertebral Disc Tissue Engineering. Eur. Cells. Mater. 2010, 20, 134–148.

(51)

Kühn, K. The Classical Collagens: Types I, II, and III; 1st ed; Academic Press Inc., Florida, United States, 1987.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 41

40 (52)

Kast, C. E.; Frick, W.; Losert, U.; Bernkop-Schnürch, A. Chitosan-Thioglycolic Acid Conjugate: A New Scaffold Material for Tissue Engineering? Int. J. Pharm. 2003, 256, 183–189.

(53)

Bae, I. H.; Jeong, B. C.; Kook, M. S.; Kim, S. H.; Koh, J. T. Evaluation of a Thiolated Chitosan Scaffold for Local Delivery of Bmp-2 for Osteogenic Differentiation and Ectopic Bone Formation. Biomed Res. Int. 2013, 2013, p. 10.

(54)

Fu, Y.; Xu, K.; Zheng, X.; Giacomin, A. J.; Mix, A. W.; Kao, W. J. 3D Cell Entrapment in Crosslinked Thiolated Gelatin-Poly(ethylene Glycol) Diacrylate Hydrogels. Biomaterials 2012, 33, 48–58.

(55)

Aubry, S.; Burlina, F.; Dupont, E.; Delaroche, D.; Joliot, A.; Lavielle, S.; Chassaing, G.; Sagan, S. Cell-Surface Thiols Affect Cell Entry of Disulfide-Conjugated Peptides. FASEB J. 2009, 23, 2956–2967.

(56)

Grigore, A.; Sarker, B.; Fabry, B.; Boccaccini, A. R.; Detsch, R. Behavior of Encapsulated MG-63 Cells in RGD and Gelatine-Modified Alginate Hydrogels. Tissue Eng. Part A 2014, 20, 2140–2150.

(57)

Chien, H. W.; Tan, S. F.; Wei, K. L.; Tsai, W. B. Modulation of the Functions of Osteoblast-like Cells on Poly(allylamine Hydrochloride) and Poly(acrylic Acid) Multilayer Films. Colloid. Surface B. 2011, 88, 297–303.

(58)

Deng, L.; Li, D.; Gu, W.; Liu, A.; Cheng, X. Formation of Spherical Cancer Stem-like Cell Colonies with Resistance to Chemotherapy Drugs in the Human Malignant Fibrous Histiocytoma NMFH-1 Cell Line. Oncol. Lett. 2015, 10, 3323–3331.


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