cellulose acetate fibrous scaffolds for bone tissue engineering

Materials Science and Engineering C 69 (2016) 1103–1115

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Crosslinked pullulan/cellulose acetate fibrous scaffolds for bone tissue engineering Deniz Atila a, Dilek Keskin a,b, Ayşen Tezcaner a,b,⁎ a b

Department of Engineering Sciences, Middle East Technical University, Turkey Biomaterials and Tissue Engineering Center of Excellence, Middle East Technical University, Turkey

a r t i c l e

i n f o

Article history: Received 3 December 2015 Received in revised form 23 July 2016 Accepted 7 August 2016 Available online 08 August 2016 Keywords: Pullulan Cellulose acetate Wet electrospinning Bone tissue engineering

a b s t r a c t Natural polymer based fibrous scaffolds have been explored for bone tissue engineering applications; however, their inadequate 3-dimensionality and poor mechanical properties are among the concerns for their use as bone substitutes. In this study, pullulan (P) and cellulose acetate (CA), two polysaccharides, were electrospun at various P/CA ratios (P80/CA20, P50/CA50, and P20/CA80%) to develop 3D fibrous network. The scaffolds were then crosslinked with trisodium trimetaphosphate (STMP) to improve the mechanical properties and to delay fast weight loss. The lowest weight loss was observed for the groups that were crosslinked with P/STMP 2/1 for 10 min. Fiber morphologies of P50/CA50 were more uniform without phase separation and this group was crosslinked most efficiently among groups. It was found that mechanical properties of P20/CA80 and P50/CA50 were higher than that of P80/CA20. After crosslinking strain values of P50/CA50 scaffolds were improved and these scaffolds became more stable. Unlike P80/CA20, uncrosslinked P50/CA50 and P20/CA80 were not lost in PBS. Among all groups, crosslinked P50/CA50 scaffolds had more uniform pores; therefore this group was used for bioactivity and cell culture studies. Apatite-like structures were observed on fibers after SBF incubation. Human Osteogenic Sarcoma Cell Line (Saos-2) seeded onto crosslinked P50/CA50 scaffolds adhered and proliferated. The functionality of cells was tested by measuring ALP activity of the cells and the results indicated their osteoblastic differentiation. In vitro tests showed that scaffolds were cytocompatible. To sum up, crosslinked P50/CA50 scaffolds were proposed as candidate cell carriers for bone tissue engineering applications. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Replacement of damaged or degenerated bone tissue with tissue engineered constructs is considered as alternative for conventional treatments that has clinical handicaps such as donor site scarcity, pathogen transfer risk, and immune rejection [1–5]. Among different scaffold processing techniques, electrospinning is widely used for preparing 3D-fibrous scaffolds for tissue engineering applications to provide large surface-to-volume ratio, open porous and interconnected structure mimicking natural extracellular matrix (ECM) of tissues, and suitability for physical and chemical modifications [6–10]. Polymers of synthetic and natural origin which have similar densities with tissues are widely chosen as biomaterials for tailoring the mechanical and biological properties of the scaffolds [11]. Scaffolds composed of synthetic polymers i.e., polycaprolactone, polylactic acid, etc. exhibit good mechanical properties with slow degradation rate compared to natural polymers whereas, natural polymer-based scaffolds i.e., alginate, silk fibroin, collagen, etc. meet the biocompatibility and biodegradability ⁎ Corresponding author at: Department of Engineering Sciences, Middle East Technical University, Turkey. E-mail address: [email protected] (A. Tezcaner).

http://dx.doi.org/10.1016/j.msec.2016.08.015 0928-4931/© 2016 Elsevier B.V. All rights reserved.

needs due to their inherent bioactivity [2,5,12–14]. In literature, natural polymers including proteins and polysaccharides have been used in non-load bearing bone tissue engineering purposes [15–19]. Recently, pullulan (P), a polysaccharide, receives attention for scaffolding in tissue engineering due to its non-toxic, non-immunogenic, non-mutagenic, and non-carcinogenic properties besides electrospinnability [20,21]. Furthermore, it has been demonstrated that P-containing 3D scaffolds produced by a different method, a patented gas foaming technique, exhibited osteogenic differentiation of seeded human adipose derived stem cells (hADSC) in vitro [22,23]. Additionally, these same carriers with nano-hydroxyapatite particles showed positive effect on bone formation in several preclinical models ranging from small to large animals [22,23]. Cellulose and its derivatives such as cellulose acetate are among the preferred biomaterials as polysaccharides for bone tissue engineering applications [24–26]. Cellulose acetate (CA) is specifically chosen for its electrospinnability and also for its good mechanical properties despite its lower degree of crystallinity. With these properties, use of CA based scaffolds holds promise for mimicking the properties of microenvironment of bone tissue [27,28]. Furthermore, CA (30 kDa) is appropriate for in vivo purposes since polymers having less molecular weight than 50 kDa are shown to pass through kidneys after in vivo

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degradation [29]. Moreover, CA containing scaffolds were found to support osteoblast growth in vitro and immature form of bone formation in vivo [30,31]. In our previous study, 3-D electrospun P/CA scaffolds were developed for the first time and their cytocompatibility was tested using mouse fibroblastic cell line (L929). P was intentionally used to obtain 3D structures with adjustable height. It was removed from the electrospun mesh to increase the porosity and cellular penetration. It has been observed that fiber diameter, thickness and porosity of scaffolds increased with increased P content, on the other hand, this resulted in faster degradation of scaffolds. In literature, several P-based scaffolds were studied for bone tissue engineering applications and their positive effect on osteogenic differentiation was reported; however, most of these scaffolds were hydrogels [15,32]. In the present work, it was hypothesized that crosslinking of fibrous 3-D P/CA scaffolds would improve mechanical properties to provide a suitable scaffold for bone tissue regeneration in non-load bearing regions. In this study, CA and P were used together considering their relation in nature. P is produced by an omnivorous fungi species Aureobasidium pullulans as an exopolysaccharide in order to make the organism adhere to surfaces like cellulosic tree surfaces. Hence, P acts as a binder and a stabilizer of CA as it attaches the organism to cellulose-based materials in nature [33]. Therefore, our purpose was to develop 3D electrospun P/CA scaffolds with a well-integrated texture and to characterize them for bone tissue engineering applications. The primary role of CA was to provide mechanical strength to scaffolds, as it is one of the main properties of natural bone tissue. P was mainly used for providing 3-dimensionality to the scaffold, which is also a required property of a scaffold designed for bone tissue. These matrices were cross-linked with STMP to obtain more stable structures. After characterization of structural and mechanical properties, cytocompatibility of the scaffolds was evaluated by studying the adhesion, proliferation and functionality of Human Osteogenic Sarcoma Cell Line (Saos-2) cells. 2. Experimental 2.1. Materials Pullulan (P; Mw, 200 kDa), a product of Hayashibara Inc. (Okayama, Japan) was kindly provided by Kale Kimya Group, the distributor of Turkey. Commercial cellulose acetate (CA; Mw, 30 kDa) was purchased from Aldrich (Missouri, U.S.A.). Electrospinning solvents N,N-dimethyl acetamide (DMAc) and dimethyl sulfoxide (DMSO) puriss. p.a., ≥99.5% (GC) were purchased from Sigma-Aldrich (New Jersey, U.S.A.). Trisodium trimetaphosphate (STMP) and sodium hydroxide (NaOH) were obtained from Sigma. Ethanol Absolute (Merck, Germany) was used during wet electrospinning. 2.2. Electrospinning Ratio of DMAc/DMSO was set to 55/45 (v/v) for the solvent system and 20 wt% polymer concentration was chosen as the total polymer concentration for P80/CA20, P50/CA50, and P20/CA80 (w/w)% during electrospinning. The experimental set-up for conventional and wet electrospinning experiments consisted of NE-1000 syringe pump (New Era Pump Systems, Inc., New York, USA), Gamma High Voltage Source ES30 (Gamma High Voltage Research, Inc., Florida, USA), static metal collector, rotational collector stand (Gözeler Elektronik, Ankara, Turkey), and Plexiglass cabinet (Kesit Pleksi, Ankara, Turkey). 3D-scaffold preparation via conventional electrospinning was achieved using P20/CA80 in DMAc/DMSO (55/45 v/v%) with a total polymer concentration of 20 wt% under 15 kV electrical voltage with 1.5 ml/ h flow rate at a distance of 25 cm from the collector screen. During wet electrospinning, the set-up was changed to vertical position and jet formation was enhanced by the gravitational forces along its way. The rotational collector stand was placed at the bottom of the cabinet. Solution

jet was collected in a petri dish filled with absolute ethanol (100%) placed on this collector. Experimental parameters were DMAc/DMSO ratio of 55/45 v/v% with a total polymer concentration of 20 wt% under 15 kV electrical voltages with 8 ml/h flow rate at a distance of 20 cm from the collector. Electrospun scaffolds were lyophilized via freeze-drier (Labconco Corporation, Kansas City, USA) after freezing at −80 °C. 2.3. Crosslinking of scaffolds Polymeric scaffolds are often crosslinked in order to modify their mechanical, thermal, chemical, and degradation properties [34]. Since P is a water-soluble material, it was crosslinked using trisodium trimetaphosphate (STMP). Crosslinking mechanism of P is based on the opening of cyclic structure of STMP occurring by reaction of alkoxides in strong alkaline media with phosphate groups leading to the formation of a tripolyphosphated polymer. In the second step, the addition of a new polymer chain led to the formation of a crosslinked polymer. The crosslinking reaction was accompanied by the production of pyrophosphate links. Different P-phosphates are produced by the reaction between STMP and P [35]. Amount of STMP was varied according to P content of the scaffolds ranging from 1/10th to 5/10th of P. NaOH was used as the initiator of the crosslinking reaction and was added as 1/ 10th of the amount of STMP solution. Two different crosslinking durations (2 and 10 min) were tried [36]. After crosslinking, scaffolds were washed with phosphate buffer (PBS, 0.1 M, pH 7.2) at room temperature and their wet-weights were recorded. Then, the crosslinked scaffolds were lyophilized and their dry-weights were recorded. 2.4. Degradation study For degradation studies, electrospun scaffolds (n = 4) were cut into rectangular pieces with dimensions 3 mm × 5 mm × 4 mm (length × width × height) and each was incubated in 4 ml phosphate buffered saline (PBS, 0.1 M, pH 7.2) solution at 37 °C in water bath (Nüve Bath NB 5, Turkey) for 35 days. Wet weights of the samples were determined at the end of each time period. After then, samples were washed with distilled water and dried in the vertical laminar flow hood (Faster, Italy) for dry weight measurements. Weight losspercentages were calculated according to the formula weight loss (%) = W1 / W0 ∗ 100, where W0 and W1 stand for initial weight of the sample (g) and weight of the sample at the end of each incubation period (g). 2.5. Mechanical tests Uniaxial tensile tests (n = 4) were carried out using 5 mm × 10 mm × 2 mm (length × width × thickness) electrospun specimens. Mechanical tests were performed at 4 mm/min to rupture using Lloyd LR50K mechanical tester (Lloyd Instruments, England) connected to a remote computer including Nexygen software program (Ametek, U.K.) equipped with a 10 N load cell. Four specimens were used for each group. The ultimate tensile strength (UTS), Young's modulus and strain values were determined. For compression tests all samples were cut into pieces with a dimension of 3 mm × 5 mm × 4 mm (length × width × height). They were tested with the same mechanical tester using a crosshead speed of 4 mm/min. The compressive strength, Young's modulus and strain values were determined from stress-strain curves obtained. 2.6. Bioactivity tests Crosslinked and uncrosslinked P50/CA50 scaffolds (n = 3) (5 × 2 mm diameter × height) were put in 2 ml of a simulated body fluid (SBF) at 37 °C. SBF had ion concentrations of Na+: 142.0, K+: 5.0, Mg2 +: 1.5,

D. Atila et al. / Materials Science and Engineering C 69 (2016) 1103–1115 2− 2− Ca2+: 2.5, Cl−: 147.8, HCO−4 3 : 4.2, HPO4 : 1.0, and SO4 : 0.5 mM and a pH of 7.40, nearly equal to those of human blood plasma. After 7 and 14 days of incubation periods, the specimens were washed with deionized water, freeze-dried, and their weights were recorded [37,38]. The specimens were stored in a desiccator until SEM examinations. Also, percent weight change of scaffolds was calculated and plotted.

2.7. Scanning electron microscopy Surface morphology of electrospun scaffolds and their cross-section views after fracturing in liquid nitrogen were analyzed at roughly 100 μm depth from the surface by scanning electron microscopy. Samples were prepared for analysis by coating with gold using a sputter coating device (Hummle VII, Anatech, Istanbul, Turkey). Fiber morphology of the scaffolds was examined by micro/nano SEM devices (Stereoscan S4-10, Cambridge, UK and JSM-6400 Electron Microscope, Jeol Ltd., UK), equipped with NORAN System 6 X-ray Microanalysis System & Semafore Digitizer (Thermo Fisher Scientific Inc., USA) in the Department of Metallurgical and Materials Engineering at METU (Ankara, Turkey) and Quanta 400F Field Emission SEM device (FEI, USA) in Central Laboratory at METU in Central Laboratory at METU (Ankara, Turkey). Fiber diameter distributions of electrospun scaffolds were determined by measuring diameters of 100 fibers from different regions of the samples. 2.8. Fourier transform infrared (FT-IR) spectroscopy FTIR analysis was performed to verify crosslinking of P within the scaffolds via a spectrometer of PerkinElmer L1050002 series (PerkinElmer, Inc., UK) using spectrum 100/100N software program in transmission mode. The analysis was performed within the wavelength range 400–4000 cm−1, with a resolution of 4 cm−1, and a total of 50 scans per sample were done. The spectra of samples were measured by mixing powder of electrospun scaffolds (obtained by crushing in a mortar) with sample/KBr ratio of 1. The spectra of all samples were corrected for background and atmosphere inside the FTIR. 2.9. Degree of crosslinking Before and after crosslinking P/CA scaffolds were incubated overnight with methylene blue which has optical density linearly related with STMP crosslinking density. Initial absorption of methylene blue was recorded at 665 nm (A0) preincubation and after overnight incubation (A). Results were normalized with dry preincubation (Wt) in milligrams (n = 4). A methylene blue absorption index (AIMB) was calculated based on modification of a previously defined equation; AIMB = [(A0 − A) / Wt] ∗ 1000. Scaffold AIMB was calculated by considering difference between OD of samples with or without STMP [32].

P γ θ

applied pressure (dyne), surface tension of mercury (480 dyne/cm) and, angle between pore surface and mercury (generally 140°).

In helium pycnometer measurements, volume and true density were calculated by using Archimedes' principle of fluid displacement and Boyle's gas law. Helium gas with the size 0.25 nm is suitable for deeper penetration through the pores depending on the polymer type, which should not allow the gas pass through itself [39]. 2.11. Cell culture studies Human osteogenic sarcoma cell line (Saos-2) was used for evaluation of biocompatibility of electrospun P50/CA50 scaffolds for bone tissue engineering applications. After sterilization of the scaffolds with 30 min exposure to UV and pure ethanol incubation for 2 h, the scaffolds were immersed in Dulbecco's modified Eagle medium with high glucose-glutamine (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin in the 5215 incubator (Shel Lab, USA) providing 5% carbon dioxide and 95% moisture at 37 °C for 24 h. 2.11.1. Cell viability assays Cell attachment and proliferation were tested by Alamar Blue Assay (Invitrogen, USA). Alamar Blue, or 7-hydroxy-3H-phenoxazine-3-one 10-oxetine, is a viable cell-indicator that is chemically reduced by inner metabolic activities of cells [40]. In principle, cell proliferation stabilizes reduction environment while cell growth inhibition stabilizes oxidation environment. The oxidized form of redox indicator turns from non-fluorescent blue to fluorescent red due to the reduction reaction. Cell viability analysis was performed at the end of 1, 4, 7, and 14 days of incubations [21]. During experiments, scaffolds (n = 6) were cut into discs of 9 mm in diameter and placed in 48 well plates after UV and alcohol sterilizations. Cells were seeded at an initial cell seeding density of 80,000/cm2. Cells were seeded twice from each side of the scaffolds. After each seeding on one surface, 30 min was awaited before rotating the scaffolds upside down for seeding. After seeding scaffolds were incubated in a carbon dioxide incubator (Shel Lab 5215, California, U.S.A.) for 14 days Alamar Blue cell viability assay was conducted to study the attachment and proliferation of the cells on electrospun scaffolds. For Alamar Blue assay, the medium was removed and the cells were washed with PBS (0.1 M, pH 7.22). Alamar Blue solution (10% Alamar Blue & 90% DMEM without phenol red) was added to each well and incubated at dark for 4 h. The optical densities of reduced solutions were obtained at 570 nm and 600 nm via microplate spectrophotometer (μOuantTM, Biotek Instruments Inc., USA) using the ELISA software program (Atlanta, USA) and recorded [41].

Reduction ð%Þ ¼ 2.10. Pore size and porosity measurements Pore size distribution and percent porosity of the scaffolds (n = 2) were determined by using Coremaster 60 Mercury Porosimeter (Quantachrome Corporation, Florida, USA) under low pressure (0– 50 psi for 200–4 μm diameter) using Helium Ultrapycnometer 1000 (Quantachrome Corporation, Florida, USA) in Central Laboratory at METU (Ankara, Turkey). In mercury porosimeter measurements, the relation between applied pressure and pore diameter based on Washburn equation was used given below.

D¼

D

−4γ cosθ P diameter of the pore (cm),

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εox εred A A′ λ1 λ2

ðεox Þλ2 Aλ1 −ðεox Þλ1 Aλ2 of test dilution ðεred Þλ1 A0 λ2 −ðεox Þλ2 A0 λ1 of untreated positive control  100

molar extinction coefficient of Alamar Blue oxidized form (blue), molar extinction coefficient of Alamar Blue reduced form (red), absorbance of test wells, absorbance of negative control well, 570 nm and, 600 nm.

Cells seeded on tissue culture plates (TCPS) were used as control group. The wells containing only media and scaffolds without cells were subtracted from control and cell-seeded-scaffold groups' results, respectively in calculations of percent reduction.

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Cell attachment and proliferation were also studied by quantifying DNA amounts of the cells. At different incubation periods the cells were ruptured using cell lysis buffer (150 mM NaCl, 0.1% Triton X-100, 50 mM Tris-HCl pH 8.0) and DNA amounts were determined by Hoechst staining (Protocol of supplier, Turner Biosystems). Calibration curve was constructed with calf thymus DNA standards at different concentrations (10 to 1000 ng/ml). DNA amounts were converted into cell number using the known DNA amount per cell (7.7 pg DNA/eukaryotic). The results are given in Supplementary data (Supp 2). At day 1% cell attachment was determined using the following formula. %cell attachment ¼

number of cells attached at day 1  100 initial number of cells seeded

Increase in cell number was interpreted as cell proliferation. At different incubation periods DNA amounts of cells were measured to determine the cell number on the scaffolds. 2.11.2. Cell morphology analysis Morphology of cells on electrospun scaffolds was examined with SEM (Quanta 200 FEG, The Netherlands) after 1, 4, 7, and 14 days of incubations. At the end of each period, cells were fixed in 4% paraformaldehyde solution in PBS. Fixed samples were washed with PBS, dehydrated with increasing ethanol series (20–100%), and finally dried in hexamethyldisilazane for 20 min prior to storage in desiccator. Before SEM analysis, batches were coated with ultrafine (10 nm) gold layer by precision etching coating system (682 PECS, Gatan, Inc., USA) and then imaged via Scanning Electron Microscopy (Stereoscan S4-10, Cambridge, UK and JSM-6400 Electron Microscope, Jeol Ltd., UK), equipped with NORAN System 6 X-ray Microanalysis System & Semafore Digitizer (Thermo Fisher Scientific Inc., USA). 2.11.3. Analysis of cell migration through 3D scaffolds Cell migration studies were conducted using confocal laser scanning microscopy after 7 and 14 days of incubations. Cells cultured on crosslinked scaffolds were fixed with formaldehyde at predetermined time points. Then, they were stained with Fluorescein isothiocyanate (FITC)-phalloidin, a high-affinity F-actin probe conjugated to the green fluorescent dye-FITC (Invitrogen, USA) for F-actin components and Propidium iodide-PI, a red fluorescent nuclear and chromosome counterstain (Invitrogen, USA) for nuclei. Fluorescence images of stained samples were obtained using a confocal laser scanning microscope (Zeiss LSM 510, Germany) equipped with Argon (458–477–488– 514 nm), HeNe/1 (543 nm), and HeNe/2 (633 nm) lasers. 2.11.4. Alkaline phosphatase (ALP) enzyme activity of seeded cells ALP activity was measured as the early marker of osteoblastic differentiation of cells. Saos-2 cells seeded on 3D-scaffolds were incubated in osteogenic differentiation medium (DMEM containing 10% FBS, 1% penicillin-streptomycin, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10− 8 M dexamethasone) for 7 and 14 days. At the end of each incubation period, cells on the scaffolds (n = 3 for each group) were lysed with 600 μl of 0.1% Triton X-100 in a phosphate buffer solution containing 0.1% (w/v) sodium azide and 1% protease inhibitor on ice. All cell lysates were frozen for 30 min and then thawed. Next, 20 μl of the obtained lysate was transferred into 100 μl p-nitrophenyl phosphate (pNPP) substrate solution and incubated at 37 °C for 30 min. Immediately, UV spectrophotometer was used to measure absorbance of solutions at 405 nm. A calibration curve constructed with p-nitrophenol in a concentration interval of 25–250 μM was used to determine the ALP activity of cells. Protein contents of the lysates were determined with the calibration curve obtained with bovine serum albumin (BSA) in the interval of 0–12 mg/ml via bicinchoninic acid (BCA) assay according to the manufacturer's instructions. The enzyme activity of cells was given in terms of specific enzyme activity nmol/μg protein/min [42].

2.12. Statistical analysis One way analysis of variance (ANOVA) and post-hoc Tukey tests for multiple comparisons were done to assess statistical significance of the results using SPSS Statistics and the difference was considered statistically significant at p b 0.05. 3. Results and discussions 3.1. Optimization of scaffold architecture Bone tissue engineering requires 3D-scaffolds in order to imitate bone ECM more effectively [43]. Scaffolds were obtained in 3D via conventional electrospinning on concave-shaped aluminum collector (Fig. 1a). Height of the P20/CA80 scaffold was measured as 2.3 cm after 40 min electrospinning and could be increased depending on the electrospinning duration (Fig. 1b). Discs were punched from the electrospun scaffolds using a biopsy puncher with 5 mm inner diameter. However, inner structure of 3D-scaffolds exhibited inhomogeneous patterns with separated layers (Fig. 1c). This might have resulted from (i) poor targeting of fibers leading to irregular accumulation, (ii) damaged mesh during removal from the collector surface, and (iii) wetting on the collector due to incomplete evaporation of solvents, which resulted in fusion of fibers after arriving at collector surface. Membranes produced by conventional electrospinning (Fig. 1a–c) needed to be improved since scaffolds should be produced reproducibly in terms of fiber deposition and layer density. By wet electrospinning, P/CA was electrospun into an immiscible solvent (Fig. 1d) so that; (i) targeted fibers could be homogeneously accumulated, (ii) mesh could easily be removed and shaped, and (iii) non-evaporated solvent residues could not cause fusion of fibers due to dipping into ethanol. Additionally, P80/CA20 and P50/CA50 blends could not be electrospun by using conventional electrospinning due to increased hydrophilic content (P) causing incomplete evaporation of solvents. However, 3D P80/CA20, P50/CA50, and P20/CA80 scaffolds were successfully produced by wet electrospinning. They preserved their homogeneous structure after freeze-drying and punching steps (Fig. 1e and f). 3.2. Morphological characterizations In order to understand the individual effects of P and CA on fiber morphologies and fiber diameter distributions different ratios of polymers were electrospun and examined via SEM. Fibers with various diameters were fabricated since P tends to result in thicker fibers while CA forms thinner fibers [44] (Fig. 2a and b). Unequal amounts of P and CA in P20/CA80 and P80/CA20 scaffolds resulted in phase separation preventing uniformity of fibers as the miscibility of polymers depends on some factors such as compositional mass fraction otherwise the blend undergoes phase separation [45]. In detail, P80/CA20 scaffolds showed that P clearly caused formation of curly and thick fibers, providing 3D features to the mesh (Fig. 2b). On the other hand, P50/CA50 scaffolds consisted of more homogeneous fiber size and morphology throughout the entire structure (Fig. 2c). High molecular weight polymers (i.e. Mw of P; 200 kDa N Mw of CA; 30 kDa) increase the viscosity of the solution which increases the fiber diameter. Increase in low molecular weight-CA content resulted in reduced fiber diameter [46,47]. Surprisingly, despite its intermediate combination, the thickest fiber diameter (14.19 μm) was obtained for P50/CA50 electrospun scaffolds (Fig. 2c). It was due to natural P attachment to CA and forming a coherent structure, as it is seen in nature where P adheres to woody surfaces [33]. Thus, P50/CA50 was crosslinked for further testing (Fig. 2d). The mean fiber diameter of crosslinked P50/ CA50 was found as 13.71 μm (Fig. 2e), which was similar to the fiber diameter of as-spun form. The histogram of fiber diameter distribution of crosslinked P50/CA50 scaffolds is also given in Fig. 2f. However, the distributions of P20/CA80 and P80/CA20 groups could not be determined

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Fig. 1. P20/CA80 scaffolds produced by conventional electrospinning (a), cross-sectional view of as-spun form on the collector (b), and punched version (c). P50/CA50 scaffolds produced by wet electrospinning in ethanol bath (d), after freeze-drying (e), and punched forms (f). P20/CA80, P50/CA50, and P80/CA20 scaffolds produced by wet electrospinning (g).

because of fiber aggregation. P50/CA50 was selected as the most suitable ratio group for the scaffold composition. 3.3. Determination of crosslinking degree The best conditions for crosslinking scaffolds were determined by changing P/STMP ratio and reaction time. From the degradation results P/STMP: 10/5 and 10 min reaction time was chosen as the ideal condition for crosslinking. Optimization of crosslinking parameters is critical because; (i) High solubility of the P may result in fast dissolution at sites of initial contact with aqueous crosslinking environment, i.e. surface. Thus, fusion of fibrous structures may cause formation of polymer skin layer at sites of initial contact with the crosslinker, i.e. STMP. The closure of pores at the surface may prevent the core of 3D-scaffolds from crosslinking. Hence, this phenomenon decreases cell infiltration though the pores. Additionally, the polymer skin layer also affects the water penetration through the scaffold; thereby the degradation of the scaffolds was also affected.

(ii) CA might be affected due to excess amount of NaOH, initiator molecule of crosslinking reaction, during crosslinking, which, in fact, breaks some of the hydrogen bonds that holds the cellulose structure together [48]. To conserve original CA properties within the structure, the crosslinking period of time should be shortened till crosslinking efficacy still stay within optimum range. (iii) Crosslinking may alter the mechanical properties of the scaffold. As the crosslink density increases, the fibers become more brittle [49], which would not be suitable for tissue engineering applications that require more flexible scaffolds in accordance with the tissue characteristics.

Fourier transform infrared (FTIR) spectroscopy is a simple and sensitive method to understand whether the crosslinking reaction was successful and specific bonds were formed [50]. FTIR spectra of P/CA scaffolds were studied in order to determine STMP-Na3P3O9-mediated crosslinking of P (Fig. 3a). Single bonds between hydrogen and oxygen atoms, namely H bonded O\\H stretches, are shown at 3300– 3500 cm−1 region indicating their moisture retention capacity [50,51].

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Fig. 2. SEM images of (a) P80/CA20, (b) P50/CA50, (c) P20/CA80 (magnifications: 150×) (d) crosslinked P50/CA50 (magnification: 800×) and (e) fiber diameter distribution of crosslinked P50/ CA50.

The transmittance bands of O\\C\\O stretches at 1645 cm−1 which are specific for native P in the spectra of scaffolds pointed out the presence of P before crosslinking and its disappearance after crosslinking (Fig. 3a) was due to the breakage of O\\C\\O bonds during crosslinking [35]. In addition, there were newly emerged bands at 700–800 cm−1 wavenumber interval for crosslinked scaffolds (Fig. 3a). Especially, band at 760 cm− 1 specific for Na3P3O9, demonstrated that the crosslinking had occurred [52]. Methylene blue binding was specific to STMP crosslinks, which gives quantitative information about crosslinking degree [32]. Methylene blue absorption index (MBAI) values of P/CA scaffolds before and after crosslinking showed that P50/CA50 blend weight ratio was the most effectively crosslinked group and lied within the MBAI range in the literature [32] (Fig. 3a & b). This could be due to (i) natural P attachment to CA seen in nature making the structure organized (more sites for crosslinking) and (ii) CA-mediated hydrophobic interactions leading to promotion of P organization [32,33,53]. On the other hand, P20/CA80 and P80/CA20 groups could not be crosslinked significantly due to phase separation (less active site for crosslinking) as discussed above (Fig. 3b). The schematic diagram of P-crosslinking with STMP is demonstrated in Fig. 3c. In principle, the closest polymeric P chains were crosslinked randomly with the crosslinker molecule STMP in alkali media [35]. Smaller CA chains were supposed to be enclosed within the crosslinked network. Accordingly, as P/CA ratio was increased (P80/CA20 N P50/CA50 N P20/ CA80), bands specific for STMP-bonding became sharper. However, the crosslinking intensity of crosslinked P80/CA20 scaffolds was not the highest; rather crosslinked P50/CA50 ones were more crosslinked (Fig. 3a). This could be due to the fact that P50/CA50 mixture provides a better integration between CA and P polymeric chains, keeping P more open to contact with STMP (Fig. 2c). In other words, the efficiency of the crosslinking reaction was enhanced as it was reported for different polymeric blends of P [32]. In contrast, whole P content could not

be reached during reaction for P80/CA20 mixture since formation of polymer skin layer at surface of scaffold did not allow the crosslinker to penetrate through core part of the 3D-mesh. This phenomenon has a remarkable influence on crosslinking efficacy. 3.4. Degradation studies During degradation tests, the optimum level of crosslinking was aimed to be determined since it caused a change in the fiber morphology, porosity, and mechanical properties of the scaffolds. Degradation studies were performed only with P50/CA50 scaffolds whose properties were the most suitable for tissue engineering applications; (i) fiber morphologies of P50/CA50 were more uniform without phase separation, (ii) mechanical properties of P20/CA80 and P50/CA50 were found to be satisfactory, (iii) porosities of P50/CA50 and P80/CA20 (82.90 ± 6.77% and 84.04 ± 11.93%) were higher than that of P20/CA80 (76.19 ± 5.81%), and (iv) only P50/CA50 and P20/CA80 maintained their structural integrity during degradation tests. The best condition for crosslinking scaffolds was determined by changing P/STMP ratio and reaction time. Cumulative weight loss (n = 4) of P50/CA50 scaffolds is given in Fig. 4a. The weight losses observed for scaffolds leveled-off after the third week since uncrosslinked P dissolved immediately once it was soaked in PBS. Groups that were crosslinked with P/STMP 10/5 for 10 min degraded slower than all other groups. Weight loss observed was 18.98 ± 0.77% and no significant change was recorded during the incubation. However, uncrosslinked P50/CA50 lost 56.36 ± 1.89% of their initial weights after 35 days (Fig. 4a). Morphology of crosslinked and uncrosslinked P50/CA50, scaffolds (with P/STMP 10/5 ratio for 10 min reaction times) studied by SEM showed that pore size, shape and their interconnectivity changed after 35 days (Fig. 4b–d). Pores of uncrosslinked scaffolds were more open and regular compared to the crosslinked samples, which had more compact structure. Fiber surfaces of uncrosslinked samples were

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Fig. 3. FTIR spectra of scaffolds before and after crosslinking (a). Arrows indicate disappeared and newly emerged peaks after crosslinking. *Specific for native P. **Sodium tripolyphosphate (STPP) formed after STMP crosslinking. Methylene blue absorption index (MBAI) representations of P/CA scaffolds (n = 4) (b) and schematic diagram of P-crosslinking within P/CA scaffolds (c).

significantly altered after degradation. However, a slight change in morphology of crosslinked scaffolds was observed as it is shown in the magnified images (Fig. 4b–d). This observation can be explained in terms of differences in water penetration into scaffolds, thereby the hydrolytic degradation rate and removal of P. The crosslinked scaffolds were more likely to maintain their initial shape, and porosity since the hydrophilic P component was not lost.

3.5. Bioactivity tests In order to determine mineral deposition on scaffolds as an indication of their bioactivity, crosslinked and uncrosslinked P50/CA50 were incubated in simulated body fluid (SBF) at 37 °C. Dry weights of the scaffolds were recorded [37,38] and SEM analysis was performed after 7 and 14 days of incubations (Fig. 5a–d). Uncrosslinked (as-spun) P50/

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Fig. 4. Cumulative weight loss of crosslinked and uncrosslinked P50/CA50 scaffolds (n = 4) (a); SEM images of as-spun (b), 35-days incubated in PBS (c), crosslinked and 35-days incubated in PBS (d) P50/CA50 scaffolds and their fiber surfaces (magnifications for upper images: 130×, 500×, and 200×; magnifications for lower images: 4000×).

CA50 fibers were covered with mineral after 7 and 14 days of incubations (Fig. 5a & c). In crosslinked groups, apatite-like structures with round morphology covered the surface after 14 days (Fig. 5b & d), which was reported as a continuous mineral film formation observed at late time points of SBF incubation [54]. However, no such film formation was observed in uncrosslinked groups which could have been hindered with the mass loss continued during SBF incubation period (Fig. 5c). On the other hand, crosslinked fiber surfaces were more stable for mineral deposition (Fig. 5d). Percent weight change of scaffolds was determined and plotted (Fig. 5e). Development of the apatite-like materials depends on the presence of nucleation sites [55]. Thus, the weight loss of uncrosslinked

scaffolds may alter the nucleation sites, delaying mineralization which was observed as 32% decrease after the first week (Fig. 5e). As SBF incubation was prolonged, a weight increase was observed for as-spun scaffolds at day-14 (Fig. 5e). On the other hand, crosslinked ones preserved their initial structure more which would lead to the conservation of area of active sites for nucleation as it was previously reported that nondeformed regions due to degradation or any other reason are more prone to mineral deposition [56]. Therefore, it is thought that higher deposition rate was observed compared to uncrosslinked group (Fig. 5e). Thus, the weight of as-spun scaffolds increased nearly 12% from the initial day to day 14, whereas this increase was 17% when the scaffolds were crosslinked before SBF incubation (Fig. 5e).

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Fig. 5. SEM images of SBF incubated P50/CA50 scaffolds that were uncrosslinked (a–b) and crosslinked (c–d). Scale bars: 20 μm. Percent mass change of P50/CA50 scaffolds after SBF incubation (n = 3) (e). Pore size distributions of P20/CA80 (e), P50/CA50 (f), P80/CA20 (g) and their crosslinked versions (f–k) obtained by using mercury porosimeter and helium pycnometer results.

3.6. Porosity Porosity measurements by Mercury Porosimeter and Helium Pycnometer ensured that as P amount increased, scaffolds became more porous (P20/CA80 b P50/CA50 b P80/CA20: 76.19 ± 5.81%, 82.90 ± 6.77%, and 84.04 ± 11.93%) due to total volume increase caused by P. However, once scaffolds were crosslinked, higher CA-containing scaffolds exhibited higher porosity (P20/CA80 N P50/CA50 N P80/CA20: 67.64 ± 4.89, 50.03 ± 8.87, and 41.98 ± 10.56%) (n = 2). In fact, P is the crosslinked material; thus it roughly implied that pores were closed with pronounced P amount as in P80/CA20. Histograms representing pore size distributions of scaffolds are presented in Fig. 5f–k. Pore sizes of P20/CA80 mostly decreased below 20 μm while porosity percentages increased after crosslinking (Fig. 5i). On the

other hand, pore sizes of more P-containing scaffolds (P50/CA50 and P80/ CA20) increased after crosslinking which were around 100 μm (Fig. 5j and k). In other words, when P ratio was increased (P80/CA20 and P50/ CA50), percentage of overall porosity was lowered due to closure of small pores; however, remained pores sizes were still above the CA-pronounced ones P20/CA80. Since bigger spaces between fibers were caused by thicker fibers, all large pores were not lost in P50/CA50 (Fig. 5j). Crosslinking of P80/CA20 has a more complex mechanism including two events occurring simultaneously; (i) dissolving of P making some pores greater (100–200 μm) and (ii) crosslinking of P making other pores smaller (5–80 μm). Pore size preferences of cells differ with respect to tissue types and materials used [40]. Presence of the large pores, between 100 and 400 μm, is important to enhance new bone formation and for formation

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of capillaries for successful diffusion of essential nutrients and oxygen that will increase cell survival within scaffold in time [7,57– 59]. However, it has also been stated that non-mineralized osteoid tissue grows in pores with 75–100 μm size [60]. Hence, P20/CA80 scaffolds having small pore-diameters (b or ~ 100 μm) were not very ideal for progression of osteogenesis, yet still satisfactory for ingrowth of osteoid tissue (Fig. 5i). Indeed, P50/CA50 and P80/CA20 having larger pores were considered as more preferable for bone tissue engineering applications (Fig. 5j–k). Additionally, after a period of time the scaffolds will degrade which will result in pore enlargement for newly tissue formation.

3.7. Mechanical testing Mechanical properties of P/CA scaffolds after crosslinking (P/STMP ratio: 10/5) were compared in Table 1 with the previous results for uncrosslinked P/CA reported in our previous study [61]. Compressive strength values of all groups including the uncrosslinked ones ranged between 0.43 and 7.36 MPa, which is in the interval of compressive strengths of porous biodegradable polymeric scaffold developed for tissue engineering applications [62]. Therefore, they provide required strength for several soft tissues such as cartilage and skin, besides spongy bone [62,63]. On the other hand, compressive elastic moduli of the test groups laid within 2.91 ± 0.21 and 5.50 ± 0.79 MPa which were comparable with the results reported for carbohydrate-based scaffolds developed for bone tissue engineering applications [60,64]. In tensile tests, results showed that crosslinked P makes the structure strong against tensile forces regarding to uncrosslinked scaffolds [61], especially for the highest P-containing groups (0.34 ± 0.04 MPa). In literature P scaffolds fabricated for bone tissue engineering applications were hydrogels that were prepared either by blending with other biomaterials or reinforced with fibrous meshes [15,32]. The mechanical properties of these P-scaffolds were not reported; however, CA fiber reinforced-composites have been used for construction applications [65] so that their reinforcing effect on P-scaffolds was also expected for tissue engineering applications. Additionally, strain values of P50/CA50 scaffolds were improved after crosslinking. Other P/CA scaffolds, P80/CA20 and P20/CA80 groups exhibited multiple peaks, which were considered to be due to less integrated and united fibrous structure of these groups. Unmodified P20/CA80 scaffolds had the highest Young's modulus (20.80 ± 1.46 MPa) against compressive forces while cross-linked P80/ CA20 had the highest Young's modulus (5.38 ± 0.09 MPa) against tensile forces. Compressive and tensile strengths decreased in crosslinked scaffolds containing higher CA (P20/CA80). These results showed that crosslinking process weakened scaffolds mechanically. This could be explained by breakage of some of the hydrogen bonds that holds the cellulose structure together due to NaOH exposure during crosslinking [48]. Second reason could be the fusion of P fibers due to P solubility during crosslinking in aqueous environment that damaged the fibrous structure [33], which made the scaffold weaker due to inhomogeneity. Since there is a tradeoff between low and high ratios of P use, P50/CA50 was preferred for yielding more mechanical strength besides architectural advantages of P.

3.8. Cell culture studies Morphology of cells on electrospun P50/CA50 scaffolds (crosslinked with P/STMP: 10/5 for 10 min) was examined by SEM (6a-d). SEM images of the surface and cross-section views of scaffolds showed that cells attached to the electrospun fibers of P50/CA50 and proliferated both on the surface and inside of the scaffolds (Fig. 6a–d). It was stated morphology of cells on the synthetic bone substitutes might vary and the cells would not form a homogenous monolayer on the scaffolds [66]. It was observed that most of the cells had round morphology throughout the scaffold (observed both on the surface (Fig. 6a) and interior of the scaffold (Fig. 6b)) until 4 days of incubation. Saos-2 cells continued to proliferate towards gaps and holes within the fibrous scaffolds without becoming completely flattened until day 7 (Fig. 6c). After 2-weeks-period, cells became spread and stretched from a fiber to another, exhibiting filopodia-like features among fibers (Fig. 6d). Relative cell viability percentages of Saos-2 cells on scaffolds were plotted considering the viability of cells on polystyrene culture wells (TCPS) as 100% and the graph showed that they proliferated till 14th day due to high surface area of the fibrous structure of the scaffolds (Fig. 6e). Although the proliferation rate seemed to be lower than reported doubling time (40–45 h) for Saos-2 cell line, the proliferation rate was comparable with the similar bone tissue engineering studies [67–70]. In addition to cell viability assay DNA amounts of cells on scaffolds were determined using Hoechst staining method and DNA amounts measured were converted into cell number (Supp 2). The number of cells on scaffolds and also TCPS at day 1 was calculated to determine %cell attachment. A 100% cell attachment was found on both TCPS and scaffolds showing that crosslinked electrospun P50/CA50 scaffolds provided a suitable surface for cell attachment. No change in cell number was observed on day 4 and 7 on both TCPS and scaffolds which might be due to local confluencies reached in some regions after initial seeding. At the end of 14 days, cells number increased almost two-fold with respect to initial seeding density. This finding was in agreement with the cell viability results. Alkaline Phosphatase Activity Assay was conducted to determine the ALP activity of Saos-2 cells on 3D-scaffolds incubated in osteogenic differentiation medium for 7 and 14 days (Fig. 6f). ALP activity of cells on scaffolds was statistically higher than the activity of cells seeded on TCPS. After 7 and 14 days of incubation, ALP activity of cells seeded on crosslinked scaffolds was N3 to 4 fold higher compared to control group (Fig. 6f). This could be due to 3-dimensional fibrous network of the scaffold mimicking the ECM, thereby affecting ALP activity positively. Higher ALP activity observed on scaffolds than on TCPS might be due to the number of attached cells resulting in earlier confluency in some regions or due to fibrous network of the scaffold providing an ECMlike structure, which led to expression of osteoblastic activities [71]. A decrease in ALP activity of cells at day 14 was observed which might be due to shifting of some cells to the next stage of osteogenic differentiation [72,73]. Cell proliferation was also shown via confocal laser scanning microscopy besides SEM. Saos-2 cells on P50/CA50 scaffolds stained with fluorescent probes were monitored from green channel for FITC labelled actin filaments, red channel for PI labelled nuclei, D-channel for nonlabelled fibers and combined channel for overlapped image of cells on

Table 1 Tensile & compression test results for crosslinked P/CA scaffolds (n = 4). Scaffolds

Compression test

Tension test

Compressive strength (MPa)

Elastic modulus (MPa)

Strain (%)

UTS (MPa)

Elastic modulus (MPa)

Strain (%)

P20/CA80 P50/CA50 P80/CA20

1.68 ± 0.09 0.43 ± 0.01 1.59 ± 0.36

5.50 ± 0.79 4.13 ± 0.68 2.97 ± 0.09

95.85 ± 1.56 27.64 ± 2.89 22.00 ± 4.96

0.04 ± 0.56 0.11 ± 0.02 0.34 ± 0.04

0.40 ± 0.06 1.54 ± 0.13 5.38 ± 0.09

63.85 ± 5.01 33.93 ± 2.18 48.38 ± 3.96

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Fig. 6. SEM images of Saos-2 cells on P50/CA50 scaffolds at day 1 (scale bar: 20 μm, surface) (a); 4 (scale bar: 30 μm, cross-section) (b); 7 (scale bar: 30 μm, surface) (c); and 14 (scale bar: 20 μm, cross-section) (d). Arrows: Cells adhered to the scaffolds. Proliferation (via Alamar Blue test) (n = 6) (e) ALP activity (f) of Saos-2 cells on crosslinked P50/CA50 scaffolds (n = 3). ALP activity of cells seeded on TCPS (control) was statistically lower than the activity of cells seeded on P/CA scaffolds (p b 0.05).

the scaffolds for two different depth intervals namely 0–25 and 65– 100 μm (Fig. 7a–d). It was observed that cells proliferated throughout the scaffold in agreement with the SEM results. Cells proliferated both on the surface (Fig. 7a–b), as well as within the scaffold down to 100 μm depth (Fig. 7c–d). Bone cells seemed to continue to penetrate to the inside of scaffolds down to 165 μm after 7 days of incubation and 210 μm after 14 days of incubation (Supp 1-z-stacks), which was

probably due to large porosity in the 3D structure enabling material transfer and cell penetration. 4. Conclusion Pullulan (P) and cellulose acetate (CA) scaffolds of various combinations were produced (P80/CA20, P50/CA50, and P20/CA80 w/w %) with wet

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Fig. 7. Confocal laser microscopy of Saos-2 cells on P50/CA50 scaffolds in the interval 0–25 μm from the surface at days 7 (a) and 14 (b) and in the interval 65–100 μm at days 7 (c) and 14 (d) (40× magnification). Top-right squares of each image show the channel-D representation of confocal microscope to identify only the scaffolds.

electrospinning technique and crosslinked to satisfy the needs of an engineered-tissue construct for the first time. P was useful to reach 3-dimensionality in electrospun scaffolds with poor mechanical properties. CA had good mechanical properties for enhancement of P; while increase in CA content resulted with formation of 2-dimensional membranous structures rather than intended 3-dimensional architecture. Codominance of P and CA resulted with the best blend weight ratio for adjusting mechanical properties and 3-dimensionality. Furthermore, crosslinking of P with STMP was performed to improve the structural stability of P 50 /CA50 scaffolds. SEM images showed that P50/CA50 scaffolds had the thickest fiber diameter and it was conserved after crosslinking. Porosity measurements showed that pore size distributions of P80 /CA20 and P 50 /CA50 scaffolds remained suitable for bone tissue regeneration after crosslinking. Cross-linked P50/CA50 scaffolds showed nearly 20% mass loss after 35-day of degradation with good mechanical properties compared to other groups. Cell culture studies showed that Saos-2 cells attached, spread and proliferated within crosslinked P50/CA50 scaffolds during 35 days of incubation proving the cytocompatibility of these scaffolds. It can be suggested that crosslinked P 50 /CA 50 scaffolds with adjustable thickness and adequate structural integrity might be considered as a candidate scaffold for tissue engineering applications.

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