Porous Heat-Treated Polyacrylonitrile Scaffolds for Bone Tissue

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Porous Heat-treated Polyacrylonitrile Scaffolds for Bone Tissue Engineering Miroslav Vetrík, Martin Parizek, Daniel Hadraba, Olivia Kukackova, Ji#í Brus, Helena Hlídková, Lucie Komankova, Jiri Hodan, Ondrej Sedlacek, Miroslav Slouf, Lucie Bacakova, and Martin Hruby ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18839 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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ACS Applied Materials & Interfaces

Porous Heat-treated Polyacrylonitrile Scaffolds

for Bone Tissue Engineering

AUTHOR NAMES Miroslav Vetrik1*, Martin Parizek2, Daniel Hadraba2, Olivia Kukackova1, Jiri Brus1, Helena Hlidkova1, Lucie Komankova1, Jiri Hodan1, Ondrej Sedlacek1, Miroslav Slouf1, Lucie Bacakova2, Martin Hruby1

AUTHOR ADDRESS 1

Institute of Macromolecular Chemistry of the Czech Academy of Sciences, Heyrovsky Sq. 2,

162 06 Prague 6, Czech Republic; *-corresponding author, e-mail: [email protected] 2

Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4,

Czech Republic KEYWORDS polyacrylonitrile, Black Orlon, 3D scaffolds, porous material, carbon-based material, human osteoblast-like cells

ACS Paragon Plus Environment

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ABSTRACT

Heat-treated polyacrylonitrile (HT-PAN), also referred to as Black Orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

1. INTRODUCTION Bones are constantly remodeling tissue with excellent, but not unlimited, self-healing ability1-2. Healing limitations occur in the elderly population due to illnesses or when large amounts of bone tissue are missing or resected3. Different approaches exist for handling such cases. One of the most common treatments is the use of bone grafts4. However, limited amounts of donor grafts are available, and they can be associated with donor morbidity, disease transmission or immune rejection5-6.


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Another approach is the use of cell therapy7. The goal of this treatment is to induce cells, usually stem cells or progenitor cells, to proliferate, migrate and differentiate into osteogenic cells. Induction of these cells can be ensured by specific factors, including ascorbic acid, vitamin D3, β-glycerolphosphate, dexamethasone or bone morphogenetic proteins8. As anchorage-dependent cells, the osteogenic cells cannot grow in suspension or in aggregates in cell culture medium, such as hematopoietic cells or cells of the immune system. Instead, these osteogenic cells require suitable surfaces in order to adhere, spread and proliferate. A surface routinely used for cell cultivation in vitro is tissue culture polystyrene (i.e., polystyrene modified by plasma or other related methods, causing material surface oxidation), which has been used in cell culture since 1965 9. However, the plating of cells on 2D surfaces is not suitable for advanced tissue engineering due to the creation of a layer of newly formed cells and the inability to create more complex 3D structures10. Therefore, tissue engineers decided to use 3D biomaterials (scaffolds) as cell support for tissue growth. The scaffolds require specific physical, chemical and morphological properties that differ depending on the type of tissue they support. These scaffolds include inorganic materials, such as titanium, stainless steel, hydroxyapatite, bioactive glasses, and bioactive ceramics. They can also be polymer-based or made from natural or synthetic non-degradable or degradable polymers, such as collagen, polyethylene or poly(lactic acid). Alternatively, hybrid scaffolds may be constructed from a combination of these materials. Recently, carbon-based materials, such as graphite or carbon nanotubes (CNTs), were shown to exhibit excellent properties for enhanced bone tissue growth 11. However, the fate of CNTs in biological environments, as well as health hazards, remains questionable. In addition, the


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formation of large 3D structures with suitable mechanical properties from CNTs is not straight for ward. Therefore, the morphology of these materials cannot fulfill the properties required for advanced scaffold applications 12-13. As an alternative to CNTs, our study used the HT-PAN, a material also known as Black Orlon (BO). The best precursor considered for this material preparation is polyacrylonitrile (PAN) that undergoes heat treatment (HT) 14. During this process, deep structural changes occur 14. PANs have been reported in biomedical applications, including the replacement of irreversibly damaged tissues. For instance, PAN has been applied for the fabrication of vascular prostheses 15-16. HT-PAN has been tested in vivo (after implantation into the right atrium of a dog heart) for its potential use as a blood-contacting material for cardiovascular applications 17. Both native PAN and HT-PAN provided support for the growth of calf aortic smooth muscle cells 18 and human bone marrow mesenchymal stem cells 19. In the present study, we focused on the new, straightforward preparation of porous structures from PAN by a newly developed cryogel technique, allowing for the preparation of large porous 3D scaffolds with an internal morphology that closely mimics bone structure. The scaffolds strongly promoted the adhesion, spreading and growth of human osteoblast-like MG 63 cells.

1. EXPERIMENTAL SECTION

Materials and methods


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All chemicals were purchased from Sigma-Aldrich (Czech Republic) and were used as received. Heat-treating of the samples was performed in a laboratory furnace LAC LE 05/11 with a controller Ht40P (LAC s.r.o., Czech Republic). Static mechanical properties were measured on an Instron model 6025/5800R (Instron Limited, UK) equipped with a 100 N load cell at room temperature with a cross-head speed of 1 mm.min−1. Cylindrically shaped specimens were tested from each system up to nominal strain (εc) 50%. Compressive modulus E and compressive strength σm (maximum stress) were evaluated. Reported values were the averages of at least five measurements. The infrared spectra were collected by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) NICOLET iS50 (Thermo Fisher Scientific) in the mid-infrared (MIR; 4,000-200 cm-1) spectral region. Solid-state NMR spectra were measured at 11.7 T using a Bruker Avance 500 US/WB NMR spectrometer (Karlsruhe, Germany, 2003) in 4mm or 3.2-mm ZrO2 rotors. The 13C cross-polarization (CP) magic angle spinning (MAS) NMR spectra were measured at a spinning frequency of 11 or 18 kHz, a nutation frequency of B1(13C) field of 62.5 kHz, and a contact time of 0.5-2 ms with a repetition delay of 4-8 s. The number of scans that provided an acceptable signal-to-noise ratio was 8 K. The 15N CP/MAS NMR spectra were measured at an MAS frequency of 11 kHz, a nutation frequency of B1 (15N) field of 42 kHz, and a contact time of 1-2 ms with a repetition delay of 4-8 s. Due to the high complexity of the system (PAN-treated at 360 ᴼC under air), the acquisition of 57 K scans was used for detection of nitrogen spectra with adequate signal-to-noise ratios. The spin-lattice relaxation time of 1H spins was measured using a simple saturation recovery experiment at 11 kHz with direct detection of 1H magnetization. The morphology of the prepared materials was studied with a scanning electron microscope (Quanta 200 FEG; FEI Company, Czech Republic). ImageJ freeware software was used for


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manual measurement of the pore diameters. Characterization of the internal pore structure of PAN and BO was performed on a mercury porosimeter Pascal 140 and 440 (Thermo Finigan, Rodano, Italy). Samples were vacuum-dried at 120 °C for 16 h before measurement. The measurement operated in two pressure intervals, 0-400 kPa and 1-400 MPa, allowing for determination of pore size in the range of 0.004-116 µm. The pore volume and most frequent pore diameter were calculated in the program Pascal by means of the Washburn’s equation using a cylindrical pore model 20. The volume of pores was evaluated as the difference between the end values on the volume/pressure curve. Porosity was obtained via the formula: p = (V × 100) / (V + 1/ρ) (%), where V was cumulative pore volume and ρ was PAN density. The specific surface area (SBET) of the samples was specified by a gas adsorption technique on a Gemini VII 2390 (Micromeritics Instruments Corp., Norcross, USA), with nitrogen as the sorbate. The surface area was calculated from the Brunauer-Emmett-Teller (BET) adsorption/desorption isotherm using Gemini software.

Porous polyacrylonitrile sample preparation Weighed amounts of PAN (molecular weight 150 000) and succinonitrile were placed in a round bottom flask equipped with a magnetic stirrer. The weighed ratio of the material was expressed as the weight percentage of PAN in succinonitrile. We used 0.5, 2, 5 and 10% PAN in succinonitrile, giving an overall weight of 5 g for each mixture. The temperature of the silicon oil bath was continuously increased, up to 139 °C. The mixtures were continually stirred and heated until both chemicals were molten, and the mixture gained liquid form with no solid presence. The melting process was typically 20 - 30 minutes. The melted liquid substance gained a slightly


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yellow color. Approximately one milliliter of molten mixture was scooped by the glass Pasteur pipette and placed in the mold in the tissue culture test plate. As a mold for PAN, tissue culture 24-well plates (diameter 16 mm) were used. Samples were allowed to cool to the ambient temperature within 12 h. Methanol was added directly to the molds of the heterogeneous mixture of PAN and SCN. Methanol was changed every 2 h for 12 h. Samples were removed from the mold and moved to the excess methanol solution (100 ml) with continuous stirring for 2 days. Methanol was changed every 12 h. Finally, samples were dried using a vacuum. The samples had a white color.

Heat treatment of porous PAN (HT-PAN preparation) Porous PAN samples were used for the HT step. Each sample was placed separately into the laboratory oven. The oven was equipped with continuous air flow (1 ml/min). The starting temperature of the oven was ca. 50 °C and was allowed to reach 360 °C within 1 h. When the heat in the oven reached 360 °C, samples were treated for 20-30 min. After the H-T samples were cooled to the ambient temperature, they were placed in methanol for 2-4 days. The methanol solution was changed every 6 h. At the end of the HT-PAN preparation, the blackcolored samples were dried in air.

Cell culture of the BO scaffolds Heat-treated scaffolds, which were prepared with initial content of 5% PAN and 10% PAN in SCN, were submitted for cell culture testing. The materials were cut into samples 10 x 10 x 2


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mm in size, sterilized with 70% ethanol for 1 h, inserted into 24-well plates (TPP, Switzerland; well diameter 1.5 cm) and seeded with human osteoblast-like MG 63 cells (30 000 cells/well or 17 000 cells/cm2). Each well contained 1.5 ml of Dulbecco’s modified Eagle's Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. D5648), supplemented with 10% fetal bovine serum (FBS; Sebak GmbH, Aidenbach, Germany) and 40 µg/ml of gentamicin (LEK, Ljubljana, Slovenia). On days 1, 3 and 7 after seeding, the cell number and morphology were evaluated. For each experimental group and time interval, four samples were used. Two samples were used for evaluating the cell number by trypsinization and counting in a Bürker hemocytometer. The remaining two samples were used for evaluating the cell morphology, including the cell shape, cell distribution on the material, and the size of the cell spreading area. The cell spreading area was evaluated only on day 1, where the spreading of individual cells was not limited by cell-cell contacts. The cells on the samples were fixed with 70% ethanol (-20 °C, 10 min) and stained with a combination of fluorescent dyes (Texas Red C2-maleimide, which stains the cell membrane and cytoplasm, and Hoechst #33342, which stains the cell nuclei). The morphology of cells was then evaluated using pictures taken with a Leica TCS SPE DH 2500 confocal microscope. Confocal microscopy was also used for analyzing the penetration of cells inside the scaffolds on vertical sections through the materials made by a razor blade and on series of parallel horizontal optical sections. As reference material, standard tissue culture polystyrene wells were used for counting cells after trypsinization, as well as measurement of the cell adhesion area. For the study of cell penetration, microscopic glass coverslips (Menzel-Gläser, Germany, diameter 12 mm) were used. This material proved to be suitable for microscopy in our earlier studies, and also provided adequate growth support for cells as PS wells.


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Evaluation of Cell Metabolic Activity by MTS Test In addition to the direct counting of cells by trypsinization after detachment, cell metabolic activity was also used as an indirect marker of cell growth, as trypsinization may not have released all cells from the porous materials. Metabolic activity was measured by a commercially available MTS kit (Promega, U.S.A.) according to the manufacturer’s protocol, which was described in more detail in an earlier study 21. Briefly, the MTS reagent solution was added to the culture medium in the ratio of 1:2 and incubated with cells at 37 °C in an atmosphere of air with 5% CO2. Metabolically active cells then converted the tetrazolium salt MTS into an orange formazan dye due to the activities of mitochondrial dehydrogenases. The resulting solution was then moved, after 4 h of incubation, into 96-well polystyrene test plates (Nunc, Denmark, well diameter 6 mm, 200 µl of solution per well), and the absorbance of the formazan dye was measured at wavelengths of 490 and 690 nm by a Versa Max Microplate Reader (Molecular Devices Corporation, Sunnyvale, California, U.S.A.). A solution from BO samples and polystyrene wells containing a medium with MTS, but without seeded cells, was used as the blank sample. For each experimental group, two samples were used, and the solution from each was divided into three parallel wells. Standard culture PS wells were used as reference material.

Statistics


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Quantitative results are presented as mean values with a standard error of the mean (mean ± S.E.M.). Statistical significance was evaluated using ANOVA and Student-Newman-Keuls methods. Values of p ≤ 0.05 were considered statistically significant.

1. RESULTS AND DISCUSSION

We prepared porous carbon-based material for potential bone tissue engineering in two phases. First, samples were weighed according to Table 1.

Table 1. Composition of mixtures for the molding procedure.

Weight ratio [%]

PAN [g]

Succinonitrile (SCN) [g]

0.5

0.025

4.975

0.5% PAN

2

0.1

4.9

2% PAN

5

0.25

4.75

5% PAN

10

0.5

4.5

10% PAN

Notation


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The mixtures were heated until both components (PAN/SCN) were dissolved into a homogenous liquid with a honey-like consistency. The molten mixture was cast into a form sample and allowed to cool to room temperature. After the cooling process, the material became a white opalescent solid. During the methanol washing process, succinonitrile is dissolved. Removing succinonitrile from the mixture is manifested by deswelling of the PAN and the samples decreased in volume. Deswelling process will depend on the overall component ratios: succinonitrile/PAN. Washed scaffold (from first phase of preparation) with higher content of succinonitrile will lose more swelling component (succinonitrile) therefore it can by manifest as higher deswelling ratio. We found that samples 10% PAN after the succinonitrile shrink in 67% and the sample with content of 5% PAN shrinks in 33%. The standard deviation across all the samples (from different batches) were not more than 5%. This volume shrinkage is advantageous, allowing for easy removal of samples from the mold. The second phase of preparation was based on the reaction induced by heat. Prepared porous PAN samples formed HT-PAN. All prepared porous samples (from the first phase) underwent the H-T process in the laboratory oven. Samples were placed in the laboratory oven preheated to 50 °C with an increasing temperature gradient (10 °C/min). After 31 min, the temperature reached 360 °C, and this temperature was retained for 30 min. During the entire process, gentle air flow through the oven was applied in order to drain gaseous by-products. Heat-treated samples are noted throughout the article by the initial weight percent content of PAN before SCN removal (e.g., 10% HT-PAN, 5% HT-PAN).


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During the H-T, we noticed at least two important factors that influenced the resulting material. The first factor was the initial temperature and gradient slope of the H-T process. A high initial temperature resulted in defective structural changes of the material and drastic deformation in shape, which gradually started to occur at the contact layers between the hot oven surface and cold PAN material. Therefore, a lower initial temperature and a slower temperature gradient must be used. With a slower temperature gradient, the samples preserved the original dimensions better than under high increases in temperature. The second important factor that affected the preparation of scaffold from porous PAN for biological purposes was the atmosphere of the H-T samples. The internal space of the oven was equipped with air flow. Airflow allows for the escape of gaseous by-products from the H-T to the funnel hood. These gaseous by-products arise during the heating process and are composed of many different substances 14. We found that without the airflow, we obtained a material that was contaminated with colored toxic pyrolysis by-products that were difficult to wash out with methanol, even after weeks of washing. During the H-T process, the material gradually changed color from white to black. The change in color could be explained by a carbonization of the polymer (i.e., leakage of non-carbon atoms), which resulted in an increase of carbon content in the material. A similar phenomenon was observed during ion implantation of synthetic polymers, which resulted in the splitting of bonds between carbon and non-carbon atoms, resulting in a loss of hydrogen or oxygen.22-23 Structural changes during the PAN and HT-PAN transition are also manifested by weight loss. The samples of 5% HT-PAN and 10% HT-PAN showed approximately 45% weight loss (46.2% ± 2.3 in 5% PAN samples and 43.6% ± 4.6 in 10% PAN samples).


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Chemical structure analysis

Figure 1. shows overall flowchart of the fabrication process. Changes in chemical structure between PAN and HT-PAN were monitored by FTIR. The polyacrylonitrile and HT-PAN spectra are shown in Figure 1. A.

Figure 1. Overall scaffold preparation flowchart. (A) FTIR spectrum of PAN (red line) before H-T and the spectrum of material after the H-T (black line). (B) I. porous PAN; II. HT-PAN.


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From the infrared spectra seen in Figure 1., it is evident that PAN changed its structure to a different material. In the case of PAN (red line), the band at 2940 cm-1 was associated with C-H stretching of the saturated aliphatic compound. Bands in the region of 2300-2000 cm-1 were associated with stretching vibration of the nitrile group, whereas the sharp band at 2241 cm-1 belongs to the non-conjugated nitrile group. A second marginal band was present at 1454 cm-1, which belongs to deformation vibrations of methylene groups. In HT-PAN (black line), the wide signal represents the amide functional group (3600-2500 cm1

). The signals belong to a fingerprint area at 1572 cm-1, 1340 cm-1 and 1226 cm-1. The band at

1572 cm-1 represents a conjugated system (-C=C-). The strong signal at 802 cm-1 may be associated with the hydrogen bonded to unsaturated carbons. Because HT-PAN is not soluble in any solvent, we used solid-state NMR for further characterization of material.

Solid-state NMR spectroscopy

This powerful method provides information regarding complex macromolecular systems and molecular arrangements in various organic and inorganic solids. In this study, we used 13C and 15

N CP/MAS NMR to follow structure evolution and final chemical composition of the PAN

product after thermal stabilization at 360 ᴼC under air flow. According to the literature, HT in air or inert atmosphere at a temperature range of 200 – 1000 ᴼC converts the linear structure of PAN to a heteroaromatic polymeric structure 24-26. These structures can vary from a polyimine cyclicladder structure to a cyclic polyenamine structure or conjugated polyenes. Moreover, it is


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possible to obtain structures with various types of nitrogen intermolecular crosslinks 27-29. Figure 2. A shows the 13C CP/MAS NMR spectra of PAN and HT-PAN after thermal treatment.

13

15

C CP/MAS NMR

A

N CP/MAS NMR

B

PAN treated at 360ᴼC


-NH2, -NH- (piperidine unit)

-N= (pyridine unit)

-CH, -CH2-

untreated PAN

-CN

untreated PAN

-CN

180

160

140

120

100

80

60

40

20

ppm

450

400

350

300

250

200

150

100

ppm

Figure 2. CP/MAS NMR spectra of PAN and HT-PAN. 13C CP/MAS NMR spectra of untreated PAN (red trace) and PAN-treated at 360 ᴼC under an air atmosphere (A). 15N CP/MAS NMR spectra of untreated PAN (red trace) and PAN-treated at 360 ᴼC under an air atmosphere (B).

From these spectra, it is clear that the molecular structure of HT-PAN is changed by heat treatment. Predominantly, the signal of aliphatic backbone units CH and CH2 resonating at 32 ppm was strongly attenuated and split into two signals (27 and 17 ppm), whereas three new signals appeared at aromatic regions of 150, 134 and 115 ppm after thermal treatment. This finding indicates that the units of PAN considerably carbonized and created some kind of polyheteroaromatic structure. Furthermore, as indicated by the decrease in T1(1H) relaxation time (from ca. 4 s for the neat PAN to ca. 2 s for the thermally treated PAN), the heteroaromatic units likely formed a system of conjugated double bonds, exhibiting limited conductivity that


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accelerates spin relaxation processes. The signal at the low-frequency region, namely, 27 ppm, corresponded to the residual CH2 units linking aromatic segments, while the signal at 17 ppm likely reflects methyl groups. From integrating the signals in aromatic and aliphatic regions (Iar = 5, Ial = 1), the aromaticity of the system was estimated to be ca. far= Iar / (Iar+Ial) = 0.83.

Complementary structural information regarding the investigated samples was derived from the measurement of 15N CP/MAS NMR spectra. However, despite a three-day acquisition of the 15N CP/MAS NMR spectra PAN systems (Figure 2. B), the resulting signal-to-noise ratio is relatively low (i.e., ca. 4-6). This prevents quantitative interpretation of the recorded spectra. Nevertheless, certain spectroscopic data that provides better insight into the molecular structure were derived. Specifically, the signal at 260 ppm (corresponding to the nitrile -C=N group) was detected in the spectrum of neat PAN only, while this resonance completely disappeared in the HT-PAN spectrum. In contrast, we identified two new signals resonating at 129 ppm and 445 ppm in the spectrum of HT-PAN. Based on data from the literature 30-34, the signal at 129 ppm can be assigned to the -NH- unit from the piperidine units and -NH2 terminal group, while the signal at 445 ppm can be interpreted as the –N= unit from pyridine structural motifs. Based on 2D 13C-13C INADEQUATE experiments performed on 13C enriched systems, the individual 13C NMR resonances unambiguously assigned to corresponding building units and structural models of PANs thermally stabilized at various conditions (nitrogen, argon or air atmosphere) were created 25-26. Following this interpretation, we performed deconvolution of the recorded 13C CP/MAS NMR spectrum of HT-PAN at 360 ᴼC under air and assigned individual resonances (Figure 3.).


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13

C CP/MAS NMR H3C

48

14

N

11

18 17

10

12

H2N

16 15

13

8

N 9

47

22

20 19 6

N H

5

N 3

7

27

N

29

1

33

50

36

34

N

CH3 37

39

32

N H

38

40 41

30

28

N

42 43

45

26

N H

44

46 25

2

4

N

24 23

21

N

NH2

35

49

31

n 20, 22, 44, 46 4, 10, 28, 34 2, 6, 26, 30

16, 40

21, 45

14, 38

19, 23, 25, 43

15, 39

17, 41 12, 36

13, 37

18, 24, 42 48, 50

Figure 3. Deconvolution of 13C CP/MAS NMR spectra of HT-PAN at 360 ᴼC under air. The alphabet specification of signals corresponds to the following numbers of carbons in the structure assignments: the signal a – 12, 36; b – 2, 6, 26, 30; c – 4, 10, 28, 34; d – 16,14; e – 20, 22, 44, 46; f – 14, 38; g – 13, 37; h – 21, 45; i – 19, 23, 25, 43; j – 15, 39; k – 17, 41; l – 18, 24, 42; m – 48, 50.

By combining these data with the results of the 15N CP/MAS NMR analysis, and also considering the relative amounts of CH2 linking units and methyl groups, we created a structural


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model of the synthesized thermally stabilized PAN system (Figure 3., upper structure). Using a simple and ideal approach, this structural model adopts a ladder-like structure in which the bicyclic heteroaromatic (pyridine-like) motif is altered with a piperidine-like motif. Two methyl groups can be expected per 3-4 repeats. The formation of a ladder-like structure, accompanied by the partial oxidation of the polymer chain that results in the formation of carbonyl groups resonating at ca. 175 ppm, is expected during thermal stabilization in an air atmosphere. However, in contrast to data in the literature 25, no carbonyl units were detected in our study. In this respect, the resulting polymer represents a product of the oxidization, which was substantially suppressed.

Porous structure characterization

The morphological structure of the materials intended for tissue engineering scaffold preparation is critical. We examined the porous structure with SEM as the main factor in our samples. SEM analysis of the prepared samples revealed different morphological structures between samples before and after H-T. The morphological structure was also different also depending on various PAN/SCN weight ratios (Figure 4.).


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Figure 4.: SEM microphotographs of porous PAN prepared from PAN/SCN mixtures: A – 0.5% PAN, B – 2% PAN, C – 5% PAN, D – 10% PAN.

The sample in Figure 4. A is composed of 0.5% PAN. Small nanofibers are also present in the created structure. As the content of PAN increased, the nanofiber structure changes were no longer present, and the creation of spherical pores was observed (Figure 4. B, C). In the sample with a content of 10% PAN, denser pores were present.


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The SCN serves as a porogen. Porogen crystals grew and formed an internal network, further converting into an interconnected network of pores, while the eutectic mixture of PAN-SCN filled gaps among the pure SCN crystals. A macroporous structure was then created by washing samples with methanol, which easily dissolves SCN, but not PAN. This process creates larger pores that remain after the dissolution of SCN crystals, as well as small pores that remain after the dissolution of the eutectic mixture PAN-SCN. The prepared scaffold samples significantly changed their inner morphological structure before and after the H-T process (Figure 4., 5.).


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Figure 5. The structure and morphology of HT-PAN. A – 0.5% HT-PAN, B – 2% HT-PAN, C – 5% HT-PAN, D – 10% HT-PAN.

Figure 5. shows that the pores in samples A and B (i.e., 0.5% HT-PAN and 2% HT-PAN, respectively) were too small to allow osteoblast ingrowth. The minimum pore diameter for adequate osteoblast penetration is considered to be 100-200 µm, which is the diameter of the Haversian systems of the concentric lamellas (i.e., osteons) in natural bone 35. The morphological structure does not allow for the migration of relatively spacious osteoblastic cells into the


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structure. Therefore, samples that previously contained 0.5% and 2% PAN were not subjected to further biological testing. Samples C and D (Figure 5.) show sufficient porous structure with interconnected pores. However, SEM images allowed us to reveal only the macroporous nature of the material. Therefore, we also used mercury intrusion porosimetry to look more closely at the structure of the pores.

Mercury intrusion porosimetry

This technique was used for further investigation of pore structure and provided values for the pore volume, pore size distribution and porosity of PAN and HT-PAN. Pore diameter (d) was determined in the area of meso-, macro- and small superpores (0.004-0.05, 0.05-1 and 1-116 µm, respectively). Due to the limitation of the measurement range, characterization of the materials was complemented with specific surface area measurement (micropores < 2 nm) 20. To cover the full range of pore sizes (large macropores), it would be appropriate to assess solvent regains and evaluate SEM micrographs 36. However, mercury intrusion porosimetry allows for the observation of pores in ranges (0-116 µm). All wider pores remain undetected by this technique. Due to this limitation, pore analysis of SEM microphotographs using ImageJ was applied. The measurement was applied to 50 different pores in each sample. Data in Table 2. shows the overall characterization (SEM/mercury int. porosimetry) of the porous structure of PAN after SCN removal and samples of final HT-PAN, as well as the mean pore size distribution using ImageJ software.


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Table 2. The characteristics of pores in PAN and HT-PAN. The results of mercury porosimetry and specific surface area (SBET). All values in Table 2. represent average values. The mean pore diameter (row 4) is data collected by the manual measurement of pores (50) using ImageJ freeware software. Pore diameter

Pore volume

Mean pore diameter

Porosity

SBET

Sample

[µm]

[ml/g]

(ImageJ) [µm]

[%]

2 [m /g]

5% PAN

12.7

9.4

16.1 ± 6.4

91.8

N.D.

5% BOa

19.5

3.1

58.4 ± 53.4

77.8

17.4

10% PAN

9.6

6.5

6.2 ± 1.6

91.1

N.D.

10% BOb

15.6

1.1

74.9 ± 50.1

53.3

0.8

a, b

Sample density ρ = 1.12 and 1 g/ml. N.D. - not determined.

The mercury porosimetry data revealed that as the PAN content increased from 5% to 10%, pore size decreased from approximately 13 to 10 µm, pore volume decreased from 9 to 6 ml/g and porosity was almost unchanged (Table 2.). Most pores varied (d = 0.4-30 µm), indicating the macroporous character of the material. After the H-T content increased from 5% to 10%, the pore size decreased from 20 to 16 µm, pore volume decreased (from 3 to 1 ml/g) and porosity decreased from 78% to 53%. Most pores varied (d = 0.4 (resp. 0.7) - 116 µm) for 5% HT-PAN and 10% HT-PAN, including 95% and 85% of the cumulative pore volume, respectively.


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Specific surface area (SBET) values referred to the fact that an increasing proportion of PAN caused a reduction of SBET (from 17 to 1 m2/g), which is related to the decrease in pore volume. These low values of SBET confirm the macroporous nature of the measured polymer.

Mechanical Properties

Heat-treated samples were evaluated for their mechanical properties where compressive modulus E and compressive strength (σm) were measured (Table 3.). Table 3. Mechanical properties of the HT-PAN (with the content of PAN 2%, 5% and 10%). The compressive modulus and compressive strength of the samples Compressive modulus E

Compressive strength σm

(MPa)

(MPa)

2% HT- PAN

1.18 ± 0.19

0.23 ± 0.05

5% HT-PAN

3.92 ± 0.80

0.55 ± 0.12

10% HT-PAN

11.7 ± 5.20

1.89 ± 0.39

Compressive modulus and compressive strength of the samples have increasing tendency as the content of PAN rise. This data are in good agreement with the increasing amount of the HT-PAN. Mechanical properties of the sample 0.5% HT-PAN was not measured due to high volume change during the HT process.


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The mechanical properties of HT-PAN in this study could be influenced by at least two factors, namely the carbonization process and the material porosity. Carbonization, i.e. procedure used for creating HT-PAN, has been reported to have controversial effects on the material mechanical properties. On one hand, during polyacrylonitrile carbonization, D-loops, i.e. a new type of structural defect in carbon fibers, were formed and had detrimental effect on mechanical properties of the material37. On the other hand, slow heating rate carbonization, also used in our study, increased the mechanical strength of polyacrylonitrile-based carbon fibers, which was explained by the increase in both the carbon sp3 bonding and the number of nitrogen atoms with quaternary bonding in the hexagonal carbon network38. The material porosity is usually inversely correlated with the mechanical strength of the scaffolds. For example, the compressive moduli of composite scaffolds made of poly(propylene fumarate) and single-wall carbon nanotubes decreased from 7.5 ± 3.1 to 0.058 ± 0.016 MPa with increasing porogen content in the material, i.e. from 7 to 90 vol.%39. In accordance with this, HT-PAN of a lower porosity, i.e. created from 10% of PAN in succinonitrile, had better mechanical properties than HT-PAN of a higher porosity, i.e. created from 10% of PAN in succinonitrile (Table 3).

Adhesion and growth of MG 63 cells on Black Orlon scaffolds

For the in vitro study, only 5% HT-PAN and 10% HT-PAN were used. The osteoconductivity, i.e. the ability of the HT-PAN samples to promote the adhesion and growth of osteoblasts, was examined in vitro using human osteoblast-like MG 63 cells. This cell line is considered an appropriate model for the study of bone cell adhesion and proliferation and has been used for the


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screening of various materials designed for bone tissue engineering 40-43. At the same time, these cells retain important markers of osteogenic differentiation, such as the activity of alkaline phosphatase and production of osteocalcin, albeit at lower levels than in primary osteoblasts 44-45. The adhesion and growth of MG-63 on 3D HT-PAN scaffolds was compared with the cell behavior in standard 2D cell culture polystyrene wells (Figure 6.)

Figure 6. Number (A), spreading area (B), growth dynamics (C) and mitochondrial activity, measured by MTS test (D), of osteoblast-like MG 63 cells on days 1, 3 and 7 after seeding on HT-PAN scaffolds based on 5% HT-PAN or 10% HT-PAN, and in control cell culture polystyrene wells (PS). The mean ± S.E.M. from 36 measurements (obtained on 2 samples; A,


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C), from 23-62 cells (B) and from 6 measurements (obtained from 2 samples; D) for each experimental group and time interval. ANOVA and Student-Newman-Keuls methods were used. Statistical significance: *, #: values significantly higher (p ≤ 0.05) in comparison with Black Orlon based on 5% PAN compared with all other groups. In addition to the direct counting of cells after detachment by trypsinization, the cell metabolic activity was used as an indirect marker of cell growth, as the trypsinization may not release all cells from the porous materials.

One day after seeding, the highest average number of initially adhered cells was found on scaffolds based on 10% HT-PAN (18 900 ± 3 300 cells/cm2). The average cell number on scaffolds based on 5% HT-PAN was lower (11 700 ± 1 100 cells/cm2). The cell number in control polystyrene (PS) wells was 16 200 ± 1 700 cells/cm2. However, the differences among these values were not statistically significant (Figure 6. A). On the other hand, the cell spreading area (i.e., the cell area projected on the material on day 1 after seeding) was significantly larger in cells on the scaffolds based on 10% HT-PAN (980 ± 40 µm2) compared with the scaffolds based on 5% HT-PAN (600 µm2), as well as control PS wells (800 µm2, Figure 6. B).

From day 1 to 3 after seeding, the cells proliferated quickly in control PS wells, manifested by a sharp slope in the growth curve (Figure 6. C). They also had the shortest cell population doubling time (Table 4.). As a result, three days after seeding, the highest cell number was obtained in control PS (40 100 ± 2 500 cells/cm2), and this value was significantly higher than those on both types of HT-PAN scaffolds. Nevertheless, on 10% HT-PAN scaffolds, the cells proliferated faster than on the 5% HT-PAN scaffolds and attained a significantly higher cell


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population density (30 000 ± 3900 cells/cm2 vs. 16 700 ± 2 000 cells/cm2) on 5% HT-PAN scaffolds (Figure 6. A). From days 3 to 7, the cells proliferated faster on both types of HT-PAN scaffolds than in PS wells (Figure 6. C). The doubling times of cells on both HT-PAN scaffolds were similar. When the doubling times were calculated throughout the experiment, the doubling times of all tested materials were similar (Table 4.). Despite this, on day 7, the cells on 10% HT-PAN scaffolds reached the highest cell population density (168 333 ± 14 150 cells/cm2), exceeding the value obtained in control PS wells (132 300 ± 11 800 cells/cm2). The lowest cell number was found on 5% HT-PAN scaffolds (100 555 ± 5 689 cells/cm2). All differences were statistically significant (Figure 6. A). These results were further supported by the MTS test of the cell mitochondrial activity. This test revealed that the absorbance of the formazan dye produced by the cells on the 10% HT-PAN scaffold materials was significantly higher than on the 5% HT-PAN scaffolds in both evaluated time intervals (days 3 and 7 after seeding). At the same time, this absorbance was similar to the values obtained from cells in standard cell culture PS wells (Figure 6. D).

Table 4. Cell population doubling time (DT, h) of MG 63 cells from days 1 to 3 (DT 1-3), days 3 to 7 (DT 3-7) and days 1 to 7 (DT 1-7) after seeding on HT-PAN scaffolds (5% HT-PAN, 10% HT-PAN) and control polystyrene wells (PS).


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Material / DT

DT 1-3

DT 3-7

DT 1-7

HT-PAN 5%

94

37

46

HT-PAN 10%

72

39

46

PS

37

56

48

The microscopic images showed that the cells on 10% HT-PAN samples were well-spread and mostly polygonal, while the shape of the cells on 5% HT-PAN samples was often rounded (Figure 7. A, C). Microscopic pictures in Figure 7. (B and D) showed that from days 1 to 3 on 5% HT-PAN scaffolds, the cell spreading improved, but the cells remained relatively sparse and less homogeneously distributed than on the 10% HT-PAN scaffolds.


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Figure 7. Morphology of human osteoblast-like MG 63 cells on HT-PAN scaffolds prepared from a mixture of 5% HT-PAN (A, B) and 10% HT-PAN (C, D) in succinonitrile. Day 1 (A, C) and day 3 (B, D) after cell seeding. Cells stained with Texas Red C2-maleimide and Hoechst #33342. Leica TCS SPE DH 2500 confocal microscope, obj. 10.0x0.30, bar = 250 µm.

Confocal microscopy analysis performed on day 7 on vertical sections through the scaffolds revealed that in both HT-PAN scaffolds, the cells mostly remained on the material surface and their penetration inside the material was limited (Figure 8. A, B). Nevertheless, horizontal


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sections showed that in scaffolds based on 10% HT-PAN, the cells were able to penetrate deeper (up to approximately 200 µm) than in the scaffolds based on 5% HT-PAN (up to 100 µm; Figure 8. C, D).

A

B

C

D

E

Figure 8. Morphology of human osteoblast-like MG 63 cells on day 7 after seeding on HT-PAN scaffolds prepared from a mixture, with an initial content of 5% PAN (A, C) or 10%


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PAN (B, D) in succinonitrile, and on the control microscopic glass coverslips (E). Cells were stained with Texas Red C2-maleimide (red fluorescence) and Hoechst #33342 (blue fluorescence). Green fluorescence represents autofluorescence of the material. Leica TCS SPE DH 2500 confocal microscope, obj. 10.0x0.30, scale bar = 100 µm. A, B: vertical sections through the material; C, D: summarization of horizontal optical sections (C: 20 sections of 4.7 µm in distance, total depth of 95 µm; D: 42 sections of 4.9 µm in distance, total depth of 205 µm).

This cellular behavior could be explained by a different surface morphology of the HT-PAN scaffolds. As is evident from Figure 5., the scaffolds based on 10% HT-PAN contained more numerous and more homogeneously distributed basin-like structures (100-200 µm in diameter). After seeding, the cells likely fell into these depressions by gravitation and spread on the bottom. Similarly, in our earlier studies performed on micropatterned fullerene C60 coatings, MG 63 cells preferred to adhere and grow in grooves among the prominences on the material surface.40-41 At the same time, the smaller basin-like structures on the surface of the 5% HT-PAN scaffolds were less colonized, and the cells remained mostly on the surface of the material (Figure 8. C, D). Thus, the superior adhesion, growth and mitochondrial activity of MG 63 cells on 10% HT-PAN scaffolds could be attributed to a more suitable surface morphology of these scaffolds, which enable higher and more homogeneous colonization of these scaffolds with cells. Another factor that may have positive effects on cellular behavior is the HT of the material, leading to a relative increase in carbon content of the material. The carbonization of polymers is often associated with the formation of double bonds between carbon atoms in the polymer chain. Carbonization can


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improve adhesion and growth of various cell types (e.g., vascular endothelial and smooth muscle cells). This improvement has been reported in synthetic polymers (e.g., polystyrene and polyethylene), modified by irradiation with N+, F+, Ar+, O+ or C+ ions 22, 46-47. Similar results were also obtained in vascular smooth muscle cells grown on polyethylene doped with carbon black 48. Another mechanism whereby carbonized PAN can increase the cell colonization is the increased adsorption of cell adhesion-mediating proteins (e.g., fibronectin) in a bioactive conformation, which facilitates binding by integrin adhesion receptors on cells. The improved protein adsorption is due to the formation of π-electron clouds, similar to carbon nanotubes 19. The positive effects of PAN carbonization on cellular behavior may be more pronounced in scaffolds based on a higher content of PAN (i.e., 10% HT-PAN). Additionally, the presence of -NH2 groups on HT-PAN, which are positively charged and polar (i.e., increasing the material wettability), could improve protein adsorption, cell adhesion and growth 49-50. Earlier studies performed on porous 3D poly(L-lactide-co-glycolide) scaffolds have shown that the optimum pore diameter for the inner colonization of the scaffolds by osteoblast and osteoblast-like cells is 400-600 µm 42-43. As mentioned above, the minimum pore diameter for adequate osteoblast penetration is considered to be at least 100 µm. Smaller pores (10-75 µm in diameter) were penetrated with fibrous tissue (for a review, see 35). In our earlier study, pores 40 µm in diameter were often spanned by MG 63 cells spread on the material, which considerably limited the migration of cells inside the scaffolds 43. The pore size in electrospun nanofibrous scaffolds based on PAN carbonized at 800 °C, which promoted ingrowth and osteogenic cell differentiation of human bone marrow mesenchymal stem cells, was in the range of 75-100 µm 19

. On the other hand, the surface of our BO scaffolds contained basin-like depressions


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approximately 100-200 µm in diameter, which were more numerous and more homogeneously distributed on the 10% HT-PAN scaffolds, which could accommodate bone cells. Another important factor influencing the cell adhesion and growth are mechanical properties of the material, particularly its rigidity and deformability, which can be very sensitively detected by cells and direct the differentiation of cells to specific phenotypes. For example, on very soft collagen-coated polyacrylamide gels (elastic modulus from 0.1 to 1.0 kPa), having mechanical characteristics similar to those of brain tissue, mesenchymal stem cells (MSCs), derived from human bone marrow, differentiated towards neurons. On stiffer gels (elastic modulus from 8 to 17 kPa), mimicking the muscle tissue, these cells differentiated towards myoblasts, and on the stiffest matrices (elastic modulus from 25 to 40 kPa), the cells differentiated towards osteoblasts51. In other words, stiff matrices can be osteoinductive, i.e. promoting the differentiation of stem cells into osteoblasts. Our study proved that the Black Orlon is also osteoconductive, i.e. able to support the adhesion, growth and enzymatic activity of osteoblasts, represented by human osteoblast-like MG 63, and its osteoconductivity increased with the increasing mechanical strength, estimated by the compressive modulus E and compressive strength σm. Supportive effect on the adhesion, proliferation and viability of MSCs was also found on a related material, i.e. carbonized polyacrylonitrile in the form of electrospun 3D scaffolds. In addition, this material acted as osteoinductive, i.e. promoting the osteogenic differentiation of MSCs, even without addition of ostegenic supplements into the cell culture medium. However, the osteoconductive and osteoinductive effects of carbonized PAN were attributed to its higher ability to adsorb specific extracellular matrix proteins, particularly fibronectin, rather than to its mechanical properties19. We conclude that 10% HT-PAN scaffolds provide better support for the adhesion and growth of human bone-derived cells than the scaffolds prepared from a matrix with a lower content of PAN. Therefore, this material is promising for the construction of scaffolds for bone tissue engineering, or the creation of layers at the bone-implant interface, in order to promote implant acceptance. However, the mechanisms underlying these effects require further investigation.


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1. CONCLUSIONS We report the preparation of porous carbon-based material by the straightforward cryogel technique. The described technique used for the preparation of scaffold allows for easy molding and shaping of the scaffolds. This is advantageous in comparison with the currently used carbon nanotube-based materials under investigation. The prepared scaffolds have a porous nature, with communicating interconnected pores that are one of the most important properties of the tissue engineered scaffolds. During the H-T process, porous material (based on PAN) significantly changed chemical structure, which was proven with the FTIR technique combined with solid-state NMR analysis. Changes in porous structure, monitored with mercury intrusion porosimetry, showed that HT-PAN had a significantly different porous structure than the untreated, starting PAN material. The mechanical properties of the HT-PAN scaffolds, evaluated by compressive modulus and compressive strength, improved with increasing content of PAN in the material. Using these methods, we were able to prepare material suitable for bone tissue engineering. The prepared materials were able to support the adhesion, spreading and proliferation of human osteoblast-like MG 63 cells on our material in a standard culture medium without additional growth-promoting factors. We found that the content of HT-PAN can influence the rate of cell spreading and proliferation, and we proved that the material prepared with an initial content of 10% PAN can induce and support the growth of cells in 3D space. Material prepared from 10% PAN positively influenced the cell proliferation, even when the pore size was not sufficient for osteoblast ingrowth. Therefore, it appears that cells can accept this material with additional tailoring (e.g., the introduction of signaling molecules or fine-tuning of pore dimensions). The structure of the material can be well utilized in advanced tissue engineering. It is also possible to use this porous material as an interface layer, increasing the


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acceptance of an artificial material by the bone tissue. In general, the porous scaffolds based on PAN provide adequate support for the adhesion and growth of human bone-derived cells, particularly when prepared from a matrix with a higher content of PAN. Therefore, this material is promising for the construction of scaffolds for bone tissue engineering. However, these conclusions require further investigation. FIGURES

Figure 1. Overall scaffold preparation flowchart. (A) FTIR spectrum of PAN (red line) before H-T and the spectrum of material after the H-T (black line). (B) I. porous PAN; II. HT-PAN


36

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13

15

C CP/MAS NMR

A

N CP/MAS NMR

B



-NH2, -NH- (piperidine unit)

-N= (pyridine unit)

-CH, -CH2-

untreated PAN

-CN

untreated PAN

-CN

180

160

140

120

100

80

60

40

20

ppm

450

400

350

300

250

200

150

100

ppm

Figure 2. CP/MAS NMR spectra of PAN and HT-PAN. 13C CP/MAS NMR spectra of untreated PAN (red trace) and PAN-treated at 360 ᴼC under an air atmosphere (A). 15N CP/MAS NMR spectra of untreated PAN (red trace) and PAN-treated at 360 ᴼC under an air atmosphere (B).


37


13

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C CP/MAS NMR H3C

48

14

N

11

18 17

10

12

H2N

16 15

13

8

N 9

47

20 19 6

N H

22 21 4

N 5

24 23 2

N 3

7

46 25 26

N H

44 45 28

N

27

42 43 30

N

29

1

40 41 32

N H

38 39 34

N

33

CH3 37

50

36

N

NH2

35

49

31

n 20, 22, 44, 46 4, 10, 28, 34 2, 6, 26, 30

16, 40

21, 45

14, 38

19, 23, 25, 43

15, 39

17, 41 12, 36

13, 37

18, 24, 42 48, 50

Figure 3. Deconvolution of 13C CP/MAS NMR spectra of HT-PAN at 360 ᴼC under air. The alphabet specification of signals corresponds to the following numbers of carbons in the structure assignments: the signal a – 12, 36; b – 2, 6, 26, 30; c – 4, 10, 28, 34; d – 16,14; e – 20, 22, 44, 46; f – 14, 38; g – 13, 37; h – 21, 45; i – 19, 23, 25, 43; j – 15, 39; k – 17, 41; l – 18, 24, 42; m – 48, 50.


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Figure 4: SEM microphotographs of porous PAN prepared from PAN/SCN mixtures: A – 0.5% PAN, B – 2% PAN, C – 5% PAN, D – 10% PAN.


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Figure 5. The structure and morphology of HT-PAN. A – 0.5% HT-PAN, B – 2% HT-PAN, C – 5% HT-PAN, D – 10% HT-PAN.


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Figure 6. Number (A), spreading area (B), growth dynamics (C) and mitochondrial activity, measured by MTS test (D), of osteoblast-like MG 63 cells on days 1, 3 and 7 after seeding on HT-PAN scaffolds based on 5% HT-PAN or 10% HT-PAN, and in control cell culture polystyrene wells (PS). The mean ± S.E.M. from 36 measurements (obtained on 2 samples; A, C), from 23-62 cells (B) and from 6 measurements (obtained from 2 samples; D) for each experimental group and time interval. ANOVA and Student-Newman-Keuls methods were used. Statistical significance: *, #: values significantly higher (p ≤ 0.05) in comparison with Black Orlon based on 5% PAN compared with all other groups. In addition to the direct counting of


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cells after detachment by trypsinization, the cell metabolic activity was used as an indirect marker of cell growth, as the trypsinization may not release all cells from the porous materials.

Figure 7. Morphology of human osteoblast-like MG 63 cells on HT-PAN scaffolds prepared from a mixture of 5% HT-PAN (A, B) and 10% HT-PAN (C, D) in succinonitrile. Day 1 (A, C) and day 3 (B, D) after cell seeding. Cells stained with Texas Red C2-maleimide and Hoechst #33342. Leica TCS SPE DH 2500 confocal microscope, obj. 10.0x0.30, bar = 250 µm.


42

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A

B

C

D

E

Figure 8. Morphology of human osteoblast-like MG 63 cells on day 7 after seeding on HT-PAN scaffolds prepared from a mixture, with an initial content of 5% PAN (A, C) or 10% PAN (B, D) in succinonitrile, and on the control microscopic glass coverslips (E). Cells were stained with Texas Red C2-maleimide (red fluorescence) and Hoechst #33342 (blue fluorescence). Green fluorescence represents autofluorescence of the material. Leica TCS SPE DH 2500 confocal microscope, obj. 10.0x0.30, scale bar = 100 µm. A, B: vertical sections through the material; C, D: summarization of horizontal optical sections (C: 20 sections of 4.7


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µm in distance, total depth of 95 µm; D: 42 sections of 4.9 µm in distance, total depth of 205 µm). TABLES Table 1. Composition of mixtures for the molding procedure.

Weight ratio [%]

PAN [g]

Succinonitrile (SCN) [g]

Notation

0.5

0.025

4.975

0.5% PAN

2

0.1

4.9

2% PAN

5

0.25

4.75

5% PAN

10

0.5

4.5

10% PAN

Table 2. The characteristics of pores in PAN and HT-PAN. The results of mercury porosimetry and specific surface area (SBET). All values in Table 3 represent average values. The mean pore diameter (row 4) is data collected by the manual measurement of pores (50) using ImageJ freeware software.

Pore diameter

Pore volume

Mean pore diameter

Porosity

SBET

Sample

[µm]

[ml/g]

(ImageJ) [µm]

[%]

2 [m /g]

5% PAN

12.7

9.4

16.1 ± 6.4

91.8

N.D.

5% BOa

19.5

3.1

58.4 ± 53.4

77.8

17.4

10% PAN

9.6

6.5

6.2 ± 1.6

91.1

N.D.

10% BOb

15.6

1.1

74.9 ± 50.1

53.3

0.8


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a, b

Sample density ρ = 1.12 and 1 g/ml. N.D. - not determined.

Table 3. shows mechanical properties of the HT-PAN (with the content of PAN 2%, 5% and 10%). The compressive modulus and compressive strength of the samples Compressive modulus E

Compressive strength σm

(MPa)

(MPa)

2% HT- PAN

1.18 ± 0.19

0.23 ± 0.05

5% HT-PAN

3.92 ± 0.80

0.55 ± 0.12

10% HT-PAN

11.7 ± 5.20

1.89 ± 0.39

Table 4. Cell population doubling time (DT, h) of MG 63 cells from days 1 to 3 (DT 1-3), days 3 to 7 (DT 3-7) and days 1 to 7 (DT 1-7) after seeding on HT-PAN scaffolds (5% HT-PAN, 10% HT-PAN) and control polystyrene wells (PS). Material / DT

DT 1-3

DT 3-7

DT 1-7

HT-PAN 5%

94

37

46

HT-PAN 10%

72

39

46

PS

37

56

48

AUTHOR INFORMATION Corresponding Author


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Miroslav Vetrik E-mail: [email protected], Phone: +420 296 809 274 Funding Sources Czech Health Research Council, Ministry of Health of the Czech Republic, grant # 15-32497A (L.B., M.P.). M.H, M.V. and O.S., Ministry of Education of the Czech Republic (grant # LM2015064 ERIC), Ministry of Health of the Czech Republic (grant # 16-30544A), Czech Grant Foundation (grant # 16-03156S).

ACKNOWLEDGMENT The biological part of the study was supported by the Czech Health Research Council, Ministry of Health of the Czech Republic, grant # 15-32497A (L.B., M.P.). M.H, M.V. and O.S. thank the Ministry of Education of the Czech Republic (grant # LM2015064 ERIC), the Ministry of Health of the Czech Republic (grant # 16-30544A) and the Czech Grant Foundation (grant # 1603156S).

REFERENCES

(1) Marsell, R.; Einhorn, T. A. The Biology of Fracture Healing. Injury 2011, 42 (6), 551-555, DOI: 10.1016/j.injury.2011.03.031. (2) Raggatt, L. J.; Partridge, N. C. Cellular and Molecular Mechanisms of Bone Remodeling. J Biol. Chem. 2010, 285 (33), 25103-8, DOI: 10.1074/jbc.R109.041087.


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SYNOPSIS The complex carbon-based material for tissue engineering and regenerative medicine applications has been prepared. The material has features of bone morphology-mimicking


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three-dimensional structure. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation and mitochondrial activity of human bone-derived cells.


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Porous Heat-Treated Polyacrylonitrile Scaffolds for Bone Tissue

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